Hsa-miR-622靶向调控RB1抑制中晚期低位直肠癌放疗敏感性的初步研究
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摘要
研究背景和目的：
我国结直肠癌的流行病学特点提示直肠癌尤其是低位直肠癌仍是影响群众健康的重要疾病。其流行病学有着自身的特点：一是发病年龄偏低,我国大肠癌的中位发病年龄大约是45岁,比欧美国家提前12-18年；二是直肠癌特别是中下段直肠癌多见——约70%左右的大肠癌为直肠癌,而70%的直肠癌位于距肛缘8cm以下；三是患者就诊时多属中晚期,极大地影响了治疗效果,5年生存率一直在低水平徘徊。术前新辅助放疗成为中晚期低位直肠癌的重要治疗手段,但其有效性只有70%左右,而目前缺少有效的预测患者放疗敏感性的生物学指标。因此寻找有价值的放疗敏感性预测指标是本研究的重点。
术前新辅助放疗业已成为进展期中低位直肠癌治疗的标准方案,寻找预测放疗敏感性的分子标记值得深入研究。近年来,随着欧美几宗大样本随机对照研究的完成,提示进展期直肠癌的辅助放疗更倾向于术前实施。主要理由是：术前肿瘤周围区域血运未被破坏、血供好、氧合程度高,放疗效果确切；且于术前行新辅助放疗后肿瘤降期明显、切缘阴性率高、保肛概率大；术前行辅助放疗对肿瘤的局部控制率优于术后实施；同时,术前实施的不良反应明显低于术后。更有文献报道,术前放疗可提高病人的长期生存率。但是,直肠癌是一种腺癌,其整体对单纯放射治疗不如鳞癌、恶性淋巴瘤等敏感,对放疗有明显效果、部分效果和效果不明显的病人各占约1/3左右,总有效率约为70%,近30%患者从新辅助放疗中获益较少,甚至只增加了治疗毒性反应,反而有延误病情之虞。因此,寻找有关直肠癌放射敏感性的分子标记,更好地对病人进行个性化筛选应是当前研究的重点。许多研究试图找到有关直肠癌放疗敏感性的生物学标志物以更好的对病人进行选择,包括P53和P21等,但在临床上尚难以得到广泛的应用。目前主要的方法还是依据肿瘤的临床特征,包括肿瘤的大小、活动度,肿瘤分化程度以及影像学检查提示的肿瘤分期等进行筛选,而这些临床指标均缺乏与肿瘤放疗敏感的相关性。因而预测放疗敏感性的新型分子标记值得探寻。
microRNA几乎参与了肿瘤发生发展的全过程,在外周血和组织中都可稳定存在并可通过现有手段进行相对定量,有望成为肿瘤诊断的新型分子标记。Giuseppina Della Vittoria Scarpati等人选取了38例中晚期地位直肠癌患者的放疗前标本,对标本进行microRNA芯片筛选并用荧光实时定量PCR进行验证,并结合超声内镜对患者放疗前后肿瘤缓解程度的评价,发现hsa-miR-622和hsa-miR-630预测中晚期低位直肠癌放疗敏感与否,其敏感性和特异性都达到100%。经过筛选,我们选择了hsa-miR-622作为研究对象。Hsa-miR-622是由位于染色体13q31.3的基因编码,据PNAS报道,hsa-miR-622与结直肠癌的发生发展有重要关系。
研究方法：
一、hsa-miR-622在细胞和组织水平的差异表达及其与放疗敏感性的关系
1、荧光实时定量PCR检测hsa-miR-622在SW480、HT29、HCT116、LS174.T、 SW837、HR8348细胞中的内源性表达。对SW480、HT29、HCT116、LS174.T、 SW837、HR8348分别给予2、3、4、5Gy的放疗剂量进行处理,利用CCK-8和平板克隆形成实验观测放疗后细胞增殖情况,摸索适合的放疗剂量并对比不同细胞株的放疗敏感性差异。
2、分别将六株细胞注射到裸鼠皮下,待其成瘤后,取荷瘤裸鼠肿瘤组织,通过荧光实时定量PCR检测荷瘤裸鼠肿瘤组织中hsa-miR-622的表达。肿瘤最大直径至10mm后,对荷瘤裸鼠按临床治疗剂量进行放疗处理,用游标卡尺记录放疗组和未放疗组肿瘤的大小变化情况。以肿瘤缩减率(未放疗组肿瘤最大直径-放疗组肿瘤最大直径/未放疗组肿瘤最大直径)作为评价肿瘤的放疗敏感性的指标,对比内源性hsa-miR-622的表达与荷瘤裸鼠放疗敏感性的关系。
3.收集进展期中低位直肠癌患者的放疗前肿瘤组织活检标本,共17例。荧光实时定量PCR检测17例进展期中低位直肠癌患者放疗前活检标本中hsa-miR-622的表达。根据南方医院病历管理系统中所记录的放疗前后CT、肠镜以及术后病理报告对比患者放疗敏感性的差异,利用Mandard肿瘤缓解程度分级的标准对患者放疗后肿瘤缓解程度进行分级。
二、沉默和过表达hsa-miR-622对结直肠癌细胞及荷瘤裸鼠敏感性的影响
1.利用细胞瞬时转染实验,选择SW480和HCT116两株细胞进行瞬时转染,其中SW480对hsa-miR-622进行干扰,而HCT116则转入mimics模拟过表达hsa-miR-622,荧光实时定量PCR检测细胞瞬时转染效率。对细胞进行放疗处理,利用PI染色后进行流式细胞实验对比细胞的放疗敏感性变化。
通过慢病毒转染技术获得稳定过表达和沉默hsa-miR-622的细胞株,荧光实时定量PCR检测四株细胞慢病毒转染效率,利用细胞增殖实验(CCK-8和平板克隆形成实验)对比在转染慢病毒后细胞放疗敏感性的变化。
2、将稳定过表达组、沉默组细胞和N.C组分别注射到裸鼠皮下。待荷瘤裸鼠肿瘤最大直径约10mm左右后,对荷瘤裸鼠按临床治疗剂量进行放疗处理,用游标卡尺记录放疗组和未放疗组肿瘤的大小变化情况。以肿瘤缩减率(未放疗组肿瘤最大直径-放疗组肿瘤最大直径/未放疗组肿瘤最大直径)作为评价肿瘤的放疗敏感性的指标,将对照组和沉默组、对照组和过表达组进行对比。对比荷瘤裸鼠在沉默和过表达hsa-miR-622后放疗敏感性的变化。
三、hsa-miR-622的靶基因的预测及表达特性分析
1、以hsa-miR-622为检索词,检索microRNA.org、TargetScan两个数据库进行靶基因的预测。将2个数据库的预测结果取交集后,对所得靶基因进行GO分类,同时利用DAVID数据库对分类结果进验证,最后根据microRNA.org数据库对基因作用的注解及评分选取目的靶基因。
2、构建预测所得靶基因的3'UTR表达载体以及将hsa-miR-622结合位点突变后的3'UTR的突变载体。在工具细胞293FT中利用双萤光素酶报告系统验证hsa-miR-622是否能够与预测所得基因的3'UTR区相结合并对预测所得靶基因的表达产生抑制,并在直肠癌细胞株SW837中验证。
3、western-blot检测裸鼠皮下肿瘤组织内源性及在沉默和过表达后RB1基因的差异表达,对比荷瘤裸鼠放疗敏感性与RB1表达强弱的关系,分析RB1的表达与放疗敏感性的关系。
4、免疫组化染色检测中晚期直肠癌患者的放疗前活检石蜡标本中RB1的表达,对比个体间RB1的表达差异与患者放疗敏感性的关系
5、western-blot检测直肠癌细胞株HCT116中RB1、E2F1、E2F8三个蛋白在放疗后1-8h的表达情况,探讨三个蛋白间的相互作用。
研究结果：
一、hsa-miR-622在细胞水平和组织水平的差异表达及其与放疗敏感性的关系
1、荧光实时定量PCR检测hsa-miR-622在SW480、HT29、HCT116、LS174.T、 SW837、HR8348细胞中的表达。以Ls174.T细胞的表达为参照组,对结果通过单因素方差分析显示：5种细胞株中hsa-miR-622的表达量之间差异具有显著性(F=118.335,P<0.001)。方差齐性检验显示方差齐性,LSD两两比较后发现：HR8348细胞当中hsa-miR-622的表达量最高,且高于SW480(P<0.001)、 HT29(P<0.001)、HCT116(P<0.001)、SW837(P<0.001)细胞中的表达：hsa-miR-622表达较高的是SW480细胞,且高于HT29(P<0.001)、HCT116(P<0.001)、 SW837(P<0.001)细胞中的表达；hsa-miR-622在HT29中的表达高于HCT116(P=0.032)、SW837(P=0.018)。而hsa-miR-622表达较低的两株细胞之间无统计学差异SW837与HCT116(P=0.741)。hsa-miR-622在HR8348、SW480、 HT29、HCT116、SW837、LS174.T中依次降低。在HR8348、SW480、HT29表达相对较高,在HCT116、SW837、LS174.T表达相对较低,对SW480、HT29、 HCT116、LS174.T、SW837、HR8348分别给予2、3、4、5Gy的放疗剂量进行处理后,CCK-8、平板克隆形成实验以检测细胞放疗敏感性显示SW480、HT29、 HR8348对放疗不敏感,而HCT116、Ls174.T、SW837对放疗敏感。我们发现hsa-miR-622的内源性表达与细胞的放疗敏感性负相关。
2、荧光定量PCR检测荷瘤裸鼠肿瘤组织中hsa-miR-622的表达,以Ls174.T为参照组,将其他5个实验组进行单因素方差分析结果显示：五株细胞株在裸鼠皮下成瘤后,检测肿瘤组织hsa-miR-622表达结果有统计学差异(F=315.155P<0.001)。方差齐性检验显示方差不齐,Dunnett T3两两比较后发现：HR8348细胞当中hsa-miR-622的表达量最高,且高于SW480(P=0.011)、HT29(P=0.002). HCT116(P=0.004)、SW837(P=0.003)细胞中的表达；hsa-miR-622表达较高的是SW480细胞,且高于HT29(P=0.066)、HCT116(P=0.022)、SW837(P=0.016)细胞中的表达；hsa-miR-622在HT29中的表达比HCT116(P=0.008)、 SW837(P=0.005)细胞中的表达。而hsa-miR-622表达较低的两株细胞中HCT116的表达要高于SW837(P<0.001)的表达。HR8348、SW480、HT29、HCT116、 LS174.T、SW837在裸鼠皮下成瘤后,肿瘤组织中hsa-miR-622的表达依次降低,在HR8348、SW480、HT29表达相对较高,在HCT116、SW837、LS174.T表达相对较低。对比荷瘤裸鼠在注射不同结直肠癌细胞株的放疗敏感性差异,结果显示：不同细胞株在裸鼠皮下成瘤后,肿瘤组织放疗敏感性有统计学差异(F=6.141P=0.005)。LSD两两比较结果显示,注射HT29细胞的荷瘤裸鼠放疗敏感性要低于HCT116(P=0.002)、Ls174.T (P=0.008)和SW837(P=0.024),与HR8348(P=0.829)和SW480(P=0.984)无统计学差异。注射SW480的荷瘤裸鼠放疗敏感性要低于HCT116(P=0.004)、Ls174.T (P=0.012)和SW837(P=0.036),与HR8348(P=0.845)相比无统计学差异。注射HR8348的荷瘤裸鼠放疗敏感性要低于HCT116(P=0.002)、Ls174.T (P=0.008)和SW837(P=0.025)。HCT116与Ls174.T (P=0.522)、Ls174.T与SW837(P=0.562)、 SW837与HCT116(P=0.233)之间放疗敏感性无统计学差异。我们发现hsa-miR-622在荷瘤裸鼠肿瘤组织中的内源性表达与其放疗敏感性负相关
3、收集17例进展期中低位直肠癌患者的放疗前瘤组织标本,检测其中hsa-miR-622的表达。并利用南方医院病历管理系统中记录的CT、肠镜以及术后病理报告,对比临床患者的放疗敏感性差异情况,利用Mandard肿瘤缓解程度分级的标准对患者进行分级。17例标本中,5例患者经过Mandard肿瘤缓解程度分级为TRG4,其hsa-miR-622的表达水平较高,而在相对最低表达的3例患者均达到完全缓解。
二、沉默和过表达hsa-miR-622对结直肠癌细胞及荷瘤裸鼠放疗敏感性的影响
1、荧光实时定量PCR检测细胞转染效率,对结果进行t检验显示hsa-miR-622在SW480N.C中的表达要高于SW480IN中的表达(t=13.313,P=0.006),结果具有统计学差异而HCT116N.C中的表达要低于HCT116hsa-miR-622中的表达(t=13.313,P=0.008)。说明瞬时转染对hsa-miR-622的表达产生影响。对细胞进行放疗处理,利用PI染色后进行流式细胞实验对比细胞的放疗敏感性变化。发现mimics转染的细胞株与对照组相比,放疗敏感性下降(t=-16.186,P<0.001),而inhibitor转染后的细胞株与对照组相比,放疗敏感性增强(t=-13.556,P<0.001)。荧光实时定量PCR检测四株细胞慢病毒转染效率,采用单样本t检验对结果进行分析,在干扰组中SW480IN的hsa-miR-622的表达与SW480N.C组相比无统计学差异(t=4.285,P=0.050)；而HT29IN的hsa-miR-622表达要高于HT29N.C (t=5.387,P=0.033);在过表达组中HCT116hsa-miR-622的表达与HCT116N.C组相比无统计学差异(t=2.853, P=0.104); SW837hsa-miR-622的表达要高于SW837N.C组的表达(t=5.541,P=0.031)。同时利用细胞增殖实验(CCK-8和平板克隆形成实验)对比在转染慢病毒后细胞放疗敏感性的变化,结果显示：与对照组相比,沉默组细胞株放疗敏感性增强,而在过表达组中,细胞放疗敏感性减弱。
2、对荷瘤裸鼠在沉默和过表达hsa-miR-622后放疗敏感性的变化。结果显示：注射SW480IN的荷瘤裸鼠比注射SW480N.C的荷瘤裸鼠的肿瘤缩减率明显(t=5.572,P=0.005)。注射HCT116N.C的荷瘤裸鼠比注射HCT116hsa-miR-622的荷瘤裸鼠的肿瘤缩减率明显(t=3.283,P=0.030)。注射SW837N.C的荷瘤裸鼠比SW837hsa-miR-622的荷瘤裸鼠肿瘤缩减率明显(t=2.917,P=0.043)。
三、hsa-miR-622的靶基因预测及表达特性分析
1、通过软件预测分析并查阅相关文献,最终得到hsa-miR-622的靶向调控基因RB1。
2、在工具细胞293FT中,双荧光素酶报告系统结果显示,在转染克隆有RB1基因3'UTR质粒的实验组中,单因素方差分析结果显示：组间存在统计学差异(F=139.970,P<0.001)。LSD进行两两比较：hsa-miR-622组与blank组相比萤光素酶活性降低(P<0.001),hsa-miR-622组与N.C组相比萤光素酶活性降低(P<0.001)。blank组和N.C组相比荧光素酶活性未见明显变化(P=0.838),说明对照转染序列未对荧光素酶活性产生影响。Hsa-miR-622inhibitor与N.C inhibitor组(P=0.168)和blank组(P=0.231)相比,萤光素酶活性均差异无统计学意义。在转染克隆有MutRB1基因3'UTR质粒的实验组中,单因素方差分析结果显示各组间荧光素酶活性无统计学差异(F=2.397,P=0.120)。证明hsa-miR-622能与RB1基因3'UTR结合,从而抑制了萤光素酶的活性。这以结果也在SW837细胞中得到验证。
3、不同细胞在裸鼠皮下成瘤后,western-blot检测肿瘤组织内源性RB1的表达,对比裸鼠放疗敏感性与RB1表达强弱的关系,发现在RB1高表达的组织中,荷瘤裸鼠对放疗更敏感。同时检测注射稳定转染细胞的裸鼠肿瘤组织中的RB1表达,分析其与放疗敏感性的关系。结果显示：发现在转染hsa-miR-622过表达慢病毒的细胞在皮下成瘤后的肿瘤组织中RB1的表达较N.C组降低,而在干扰组中RB1表达升高。在放疗敏感性方面,与N.C组相比,过表达组荷瘤裸鼠放疗敏感性降低,抑制组放疗敏感性增加。
4、免疫组化染色检测中晚期直肠癌患者的放疗前活检石蜡标本中RB1的表达,对比个体间RBl的表达情况与患者对放疗敏感性的关系,结果显示RB1在27例患者中均呈强阳性表达,个体间无统计学差异。
5、western-blot检测RB1、E2F1、E2F8三个蛋白在放疗后1-8h的直肠癌细胞株中的表达情况。结果显示：三个白蛋白在放疗后1-8h都有先降低再升高最后降低的变化过程。他们之间的关系有待进一步的研究。
结论
Hsa-miR-622的差异表达结直肠癌细胞的放疗敏感性负相关
Hsa-miR-622的差异表达与荷瘤裸鼠放疗敏感性负相关
RB1是hsa-miR-622的直接作用靶基因
RB1的差异表达与结直肠癌细胞放疗敏感性正相关
RB1的差异表达与荷瘤裸鼠放疗敏感性正相关
Background and purpose
Epidemiological characteristics of colorectal cancer in China suggest that colorectal cancer especially low rectal cancer important disease is an important disease which affects the health. With the development of economy, the rapid change in lifestyle, diet structure, the incidence of colorectal cancer in China are increasing, the incidence is trending gradually to the level of Europe and the United States closely. But compared with western developed countries, the epidemiology of colorectal cancer in China has its own characteristics; first, the age of patients is younger, median age of onset of colorectal cancer at the age of45, than the patients in Europe and the United States about12to18years. Second, middle and low rectal cancer is more frequency detected. Rectal cancer accout for about70%of the colorectal cancer and70%of colorectal cancer locates in8cm from the anal verge. Third, large numbers of advanced patients greatly influences the treatment the effect. As a consequence, the5year survival rate has remained at a low level.
Preoperative neoadjuvant radiotherapy has become the standard scheme for the treatment of advanced lower rectal cancer, worthy of further research on molecular markers for predicting the radiosensitivity. In recent years, with a few big sample randomized controlled study completion in Europe and the United States, adjuvant radiotherapy showed preoperative chemoradiotherapy has more andvantages compared with afteroderative for the treatment of advanced rectal cancer, first, the surrounding area of tumor blood supplication did not destroy, so the tumor has good blood supply and high oxygenayion, result in the exact effect of preoperative radiotherapy. Second, the tumor degradation significantly after neoadjuvant radiotherapy compared with high negative margin rate and doubled anus preserving operation rate. Third, preoperative radiotherapy will increase tumor local control rate than that after the implementation. Last but not least, the adverse reaction was significantly lower than the afteroperative postoperative. More reports, preoperative radiotherapy can improve the patient's long-term survival rate. However, rectal cancer a kind of adenocarcinoma, the whole efficiency of radiotherapy is not as sensitive as squamous cell carcinoma and malignant lymphoma. Patients with obvious degraion, part degration and no degration each account for about1of/3, and the total efficiency is about70%, the latter benefited less from neoadjuvant radiotherapy. Apparently, patients with invalid treatment cannot benefit from preoperative radiotherapy, even only added to the toxic effects of treatment, even a delay of the treatment. Therefore, looking for molecular markers related to radiosensitivity of rectal cancer, making a pefect choice of patients should be the focused in the current study. Many studies trying to find biological markers to predict the radiosensitivity in rectal carcinoma to get abetter patients selection, including P53and P21, but it is difficult to be widely applied. At present, we are mainly depend on clinical features of tumors, including tumor size, the activity of the tumor, the degree of tumor differentiation and imaging examination. Unfortutinary, those kinds of clinical indexes are lack of correlation with tumor radiosensitivity. Therefore, the search for new molecular markers related to radiosensitivity of rectal cancer is worth looking forward to.
MicroRNA is expected to become a new molecular diagnostic marker, which almost involved in the whole process of the development of tumor. While the Giuseppina Della Vittoria Scarpati et al. Selected the miRNAs to predict to sensitivity of patients to radiotherapy in38cases of advanced low rectal cancer patients before neoadjuvent therapy by microarray assay that was verified by RT-Q-PCR. Combined with clinical data, it showed that hsa-miR-622and hsa-miR-630are perfect the sensitivity to radiotherapy with both100%sensitivity and specificity. After screening and Preliminary experiments, we chose hsa-miR-622as the object of study.
Methods
Relationship between the expression of hsa-miR-622and radiation sensitivity.
1. Expression of hsa-miR-622in SW480, HT29, HCT116, LS174.T, SW837, HR8348cells was fluorescence quantitative PCR detection. Then SW480, HT29, HCT116, LS174.T, SW837, HR8348were given radiotherapy dose of2,3,4,5Gy processing, cell proliferation after radiotherapy was observed by CCK-8and plate colony forming experiment.
2. Expression of hsa-miR-622detected by fluorescence quantitative PCR tumor-bearing nude mice tumor tissue. Comparison of the sensitivity of tumor-bearing nude mice.
3.17turner tissue specimens before radiotherapy were collected status in rectal cancer patients. The expression of hsa-miR-622was detected in17cases with advanced status in rectal cancer patients before radiotherapy. After a comparison of CT and colonoscopy results of the patients before and after radiation, the tumor regression grade was classified by the standard of Mandard tumor remission degree.
The effect of silence and overexpression of hsa-miR-622on colorectal cancer cells and tumor-bearing nude mice to radiosensitivity
1. SW480and HCT116were selected for the transient transfection. Hsa-miR-622was interfered in SW480, while HCT116was transfected with mimics to overexpress hsa-miR-622. Efficiency of cell transfection was detected by RT-Q-PCR. The apoptosis of the different group after radiotherapy was detected flow cytometry experiment with PI staining.Then we use lentiviral to transfect cells to overexpress or silence of hsa-miR-622stablily. Lentiviral transfection efficiency was detected by RT-Q-PCR in four cell lines. Cell proliferation assay (CCK-8and plate colony formation assay) was used to compare the varience of sensitivity of the cell to radiotherapy.
2、Cells transfected with letiviral were injected subcutaneously into athymic nude mice. The nude mice were treat with radiotherapy after the diameter of the tumor was more than1cm according to clinical therapeutic dose. The tumor size was recorded every three days by vernier caliper in both with radiotherapy group and without radiotherapy group. Tumor regression rate (radiotherapy group/without radiotherapy group), an index to evaluate the radiation sensitivity, was used to compared the control group and the interference group, control group and the overexpression group.
Characteristic analysis and a prediction of target gene of hsa-miR-622
1、Taking hsa-miR-622as the search term, we tried to predict the target genes of hsa-miR-622in microRNA.org, TargetScan. Intersection the two datebase was classified by GO classification, and further verification was made in the DAVID database. According to the annotations and score of gene in microRNA.org database, three the target gene is closely related with hsa-miR-622.
2、RB1gene3'UTR expression vector and hsa-miR-622binding site mutated MutRB1gene3'UTR expression vector was constructed. We tried to verify whether hsa-miR-622can combine with RB13'UTR and inhibit RBI gene expression in posttranscriptional level through the dual luciferase system in293FT cell. The result was convinced in the SW837cell line.
3、Edogenous expression of RBI in tissues of nude mice subcutaneous tumor was detected by western-blot. Then we take a contrast of the relationship of the radiosensitivity and RB1expression in nude mice bearing human tumor.
4、Paraffin specimens of the patients with advanced rectal cancer before radiotherapy were immunohistochemically stained. Relationship to the expression of RBI and radiosensitivity were compared among individual.
5、The expression of RB1, E2F1, and E2F8in first to eighth hour was detected by western-blot in the cells that were treated with radiotherapy.
Results
Relationship between the expression of hsa-miR-622and radiation sensitivity.
1. Expression of hsa-miR-622in SW480, HT29, HCT116, LS174.T, SW837, HR8348cells was fluorescence quantitative PCR detection. When the conference group was disputed, there was significant difference between five cell lines (F=118.335, P<0.001). The results showed that hsa-miR-622was the highest expressed in HR8348. It was higher than that of SW480(P<0.001), HT29(P<0.001), HCT116(P<0.001), SW837(P<0.001) expression in the cells, there was significant difference. Hsa-miR-622expression was higher in SW480cells, and higher than that of HT29(P<0.001), HCT116(P<0.001), SW837(P<0.001) expression in the cells, there was significant difference. Hsa-miR-622expression in HT29higher than that in HCT116(P=0.032), SW837(P=0.018) expression in the cells, there were statistical differences. But the expression of hsa-miR-622in the left two cell lines showed no significant difference between SW837and HCT116(P=0.741). The expression of hsa-miR-622decreased in HR8348, SW480, HT29, HCT116, SW837, and LS174.T. It relatively high expressed in HR8348, SW480, and HT29. All of the cell lines were given radiotherapy of2,3,4,5Gy. The results of CCK-8and plate clone formation assay showed that SW480, HT29, HR8348are not sensitive to radiotherapy, while HCT116, Ls174.T, SW837is sensitive to radiotherapy. We found that endogenous hsa-miR-622expression correlated with the radiosensitivity of cells negatively.
2. Expression of hsa-miR-622detected by fluorescence quantitative PCR tumor-bearing nude mice tumor tissue. Results showed that hsa-miR-622expression were statistically significant in subcutaneous tumors formed by five cell lines in nude mice when the conference group was disputed (F=315.155, P<0.001). Dunnett T3comparison found:HR8348cells and the expression of hsa-miR-622was the highest in the tumor tissue formed by HR8348, and it was higher than that of SW480(P=0.011), HT29(P=0.002), HCT116(P=0.004), SW837(P=0.003) expression in the cells, there was significant difference. Hsa-miR-622expression was higher in SW480cells, and it was higher than that of HT29(P=0.066), HCT116(P=0.022), SW837(P=0.016) expression in the cells. The expression of hsa-miR-622in HT29was higher than in HCT116(P=0.008), SW837(P=0.005) expression in the cells, there was statistical differences. Expression of hsa-miR-622in HCT116cell was higher than that expressed in SW837(P<0.001). Comparison of the sensitivity of tumor-bearing nude mice showed that:there was significant difference in radiosensitivity of subcutaneous tumor in nude mice formed by different cell lines (F=6.141P=0.005). Homogeneity of variance test showed there was no variance of homogeneity, LSD comparison showed that:radiosensitivity of subcutaneous tumor formed by HT29is lower than that of HCT116(P=0.002), Ls174.T (P=0.008) and SW837(P=0.024), while there was no significant difference compared with that of HR8348(P=0.829) and SW480(P=0.984). Radiosensitivity of subcutaneous tumor formed by SW480is lower than that of HCT116(P=0.004), Ls174.T (P=0.012) and SW837(P=0.036), while there was no significant difference compared with that of HR8348(P=0.845). Radiosensitivity of subcutaneous tumor formed by HR8348is lower than that of HCT116(P=0.002), Ls174.T (P=0.008) and SW837(P=0.025). There was no difference between HCT116and Ls174.T (P=0.522), Ls174.T and SW837(P=0.562), SW837and HCT116(P=0.233) in sensitivity to radiotherapy. We found that endogenous hsa-miR-622expression in the tissue of mude mice hearing human tumor correlated with the radiosensitivity of cells negatively.
3.17turner tissue specimens before radiotherapy were collected status in rectal cancer patients. The expression of hsa-miR-622was detected in17cases with advanced status in rectal cancer patients before radiotherapy. After a comparison of CT and colonoscopy results of the patients before and after radiation, the tumor regression grade was classified by the standard of Mandard tumor remission degree. By contrast,5cases were classified as TRG4, the hsa-miR-622expression level was higher, and the lowest expression in3patients achieved complete remission.
The effect of silence and overexpression of hsa-miR-622on colorectal cancer cells and tumor-bearing nude mice to radiosensitivity
1. SW480and HCT116were selected for the transient transfection. Hsa-miR-622was interfered in SW480, while HCT116was transfected with mimics to overexpress hsa-miR-622. Efficiency of cell transfection was detected by RT-Q-PCR. The t test of the results showed that hsa-miR-622was higher expressed in SW480N.C than the expression in SW480IN (t=13.313, P=0.006), the difference was statistically significant. The expression of hsa-miR-622was lower in the HCT116N.C than in the HCT116expression (t=13.313, P=0.008), the difference was statistically significant. It indicated that hsa-miR-622expression was infected by the transient transfection. The apoptosis of the different group after radiotherapy was detected flow cytometry experiment with PI staining. The sensitivity of the cell was decreased in mimics transfected cell line compared with the control group (t=16.186, P<0.001), while the sensitivity was enhenced in the inhibitor transfected cell line compared with the control group (t=13.556, P<0.001). Then we use lentiviral to transfect cells to overexpress or silence of hsa-miR-622stablily. Lentiviral transfection efficiency was detected by RT-Q-PCR in four cell lines, single sample t test was usd to analyze the results, expression of hsa-miR-622was no difference between the SW480IN and SW480N.C (t=4.285, P=0.050), HCT116-miR622and HCT116N.C showed no significant difference (t=2.853,P=0.104); The expression of hsa-miR-622in HT29IN was higher than that of HT29N.C (t=5.387,P=0.033); The expression of hsa-miR-622in SW837miR-622was higher than that of SW837N.C (t=5.541,P=0.031). Cell proliferation assay (CCK-8and plate colony formation assay) was used to compare the varience of sensitivity of the cell to radiotherapy. Compared with the control group, the sensitivity of the interference group was enhenced, while it was decreased in the overexpression group of cell radiosensitivity.
2. Cells transfected with letiviral were injected subcutaneously into athymic nude mice. The nude mice were treat with radiotherapy after the diameter of the tumor was more than1cm according to clinical therapeutic dose. The tumor size was recorded every three days by vernier caliper in both with radiotherapy group and without radiotherapy group. Tumor regression rate (radiotherapy group/without radiotherapy group), an index to evaluate the radiation sensitivity, was used to compared the control group and the interference group, control group and the overexpression group. The results showed:tumor reduction rate was more obvious in SW480IN than thatin SW480N.C (t=5.572, P=0.005), the results were statistically difference. Nude mice injected with HCT116N.C was more significantly regressed than that with HCT116miR-622(t=3.283,P=0.030), the results were statistically difference. It wais also verified in SW837(t=2.917, P=0.043).
Characteristic analysis and prediction of target gene of hsa-miR-622
1. Taking hsa-miR-622as the search term, we tried to predict the target genes of hsa-miR-622in microRNA.org, TargetScan. Intersection the two datebase was classified by GO classification, and further verification was made in the DAVID database. According to the annotations and score of gene in microRNA.org database, three the target gene is closely related with hsa-miR-622. According to the literature, there were important relations between tumor occurrence and development and RB1. In addition, RBI was play an important role in the regulation of cell cycle and influencing on apoptosis.
2. RB1gene3'UTR expression vector and hsa-miR-622binding site mutated MutRB1gene3'UTR expression vector was constructed. We tried to verify whether hsa-miR-622can combine with RBI3'UTR and inhibit RB1gene expression in posttranscriptional level through the dual luciferase system in293FT cell.combined with the translation of the RBI level. Results in293FT cell transfected with RBI gene3'UTR plasmid analysied by single factor variance analysis showed that the difference between groups was significant (F=139.970, P<0.001), test of homogeneity of variance by Levene homogeneity of variance show there was homogeneity of variance (F=1.103, P=0.407). LSD two two comparisons showed: luciferase activity was inhibited in the group transfected with hsa-miR-622compared with blank (P<0.001) and N.C (P<0.001). There was statistically difference. It showed that hsa-miR-622can combine with RBI gene3'UTR, leading to the inhibition of luciferase activity. There was no significant difference between blank group and N.C group (P=0.838), the control sequence had no effect on luciferase activity. Luciferase activity has no difference in the group transfected with hsa-miR-622inhibitor compared with blank (P=0.231) and N.C inhibitor (P=0.168). The results of single factor analysis of variance based on the plasmid of MutRB1gene showed no significant difference between groups (F=2.397, P=0.120). The results are also verified in SW837cells.
3. Edogenous expression of RBI in tissues of nude mice subcutaneous tumor was detected by western-blot. With a contrast of the relationship of the radiosensitivity and RB1expression in nude mice bearing human tumor, we found the higher expression of RB1in the tumor tissues, the more sensitive to radiotherapy of the nude mice. Using western-blot to detect the expression of RB1in tissues from the nude mice injected with stably transfected cell and analyzed its relationship with radiotherapy sensitivity. The results showed that RB1expressed lower in the tumor tissue which was formed by the cells transfected with overexpression lentiviral compared with N.C group. And the RBI expressed higher in the inhibitor group compared with N.C group.
4. Paraffin specimens of the patients with advanced rectal cancer before radiotherapy were immunohistochemically stained. Relationship to the expression of RBI and radiosensitivity were compared among individual. We found that RBI expressions were all strongly positive in27patients, no statistically significant differences between individuals.
5. The expression of RB1, E2F1, and E2F8in first to eighth hour was detected by western-blot in the cells that were treated with radiotherapy. The result showed that three albumin has the same trend after radiotherapy in1-8h. They were all decreased first and then increased, but dreased again at last. The relationship between them needs to be studied further
Conclusion
1. The relationship between differential expressions of Hsa-miR-622and radiotherapy sensitivity of colorectal cancer cell was negative correlation
2. Radiosensitivity of the nude mice bearing human tumor associated negatively with hsa-miR-622expression.
3. RB1is a direct target gene of hsa-miR-622
4. The relationship between differential expressions of Rbl and radiotherapy sensitivity of colorectal cancer cell was positive correlation
5. Radiosensitivity of the nude mice bearing human tumor associated positively with RB1expression
我国结直肠癌的流行病学特点提示直肠癌尤其是低位直肠癌仍是影响群众健康的重要疾病。其流行病学有着自身的特点：一是发病年龄偏低,我国大肠癌的中位发病年龄大约是45岁,比欧美国家提前12-18年；二是直肠癌特别是中下段直肠癌多见——约70%左右的大肠癌为直肠癌,而70%的直肠癌位于距肛缘8cm以下；三是患者就诊时多属中晚期,极大地影响了治疗效果,5年生存率一直在低水平徘徊。术前新辅助放疗成为中晚期低位直肠癌的重要治疗手段,但其有效性只有70%左右,而目前缺少有效的预测患者放疗敏感性的生物学指标。因此寻找有价值的放疗敏感性预测指标是本研究的重点。
术前新辅助放疗业已成为进展期中低位直肠癌治疗的标准方案,寻找预测放疗敏感性的分子标记值得深入研究。近年来,随着欧美几宗大样本随机对照研究的完成,提示进展期直肠癌的辅助放疗更倾向于术前实施。主要理由是：术前肿瘤周围区域血运未被破坏、血供好、氧合程度高,放疗效果确切；且于术前行新辅助放疗后肿瘤降期明显、切缘阴性率高、保肛概率大；术前行辅助放疗对肿瘤的局部控制率优于术后实施；同时,术前实施的不良反应明显低于术后。更有文献报道,术前放疗可提高病人的长期生存率。但是,直肠癌是一种腺癌,其整体对单纯放射治疗不如鳞癌、恶性淋巴瘤等敏感,对放疗有明显效果、部分效果和效果不明显的病人各占约1/3左右,总有效率约为70%,近30%患者从新辅助放疗中获益较少,甚至只增加了治疗毒性反应,反而有延误病情之虞。因此,寻找有关直肠癌放射敏感性的分子标记,更好地对病人进行个性化筛选应是当前研究的重点。许多研究试图找到有关直肠癌放疗敏感性的生物学标志物以更好的对病人进行选择,包括P53和P21等,但在临床上尚难以得到广泛的应用。目前主要的方法还是依据肿瘤的临床特征,包括肿瘤的大小、活动度,肿瘤分化程度以及影像学检查提示的肿瘤分期等进行筛选,而这些临床指标均缺乏与肿瘤放疗敏感的相关性。因而预测放疗敏感性的新型分子标记值得探寻。
microRNA几乎参与了肿瘤发生发展的全过程,在外周血和组织中都可稳定存在并可通过现有手段进行相对定量,有望成为肿瘤诊断的新型分子标记。Giuseppina Della Vittoria Scarpati等人选取了38例中晚期地位直肠癌患者的放疗前标本,对标本进行microRNA芯片筛选并用荧光实时定量PCR进行验证,并结合超声内镜对患者放疗前后肿瘤缓解程度的评价,发现hsa-miR-622和hsa-miR-630预测中晚期低位直肠癌放疗敏感与否,其敏感性和特异性都达到100%。经过筛选,我们选择了hsa-miR-622作为研究对象。Hsa-miR-622是由位于染色体13q31.3的基因编码,据PNAS报道,hsa-miR-622与结直肠癌的发生发展有重要关系。
研究方法：
一、hsa-miR-622在细胞和组织水平的差异表达及其与放疗敏感性的关系
1、荧光实时定量PCR检测hsa-miR-622在SW480、HT29、HCT116、LS174.T、 SW837、HR8348细胞中的内源性表达。对SW480、HT29、HCT116、LS174.T、 SW837、HR8348分别给予2、3、4、5Gy的放疗剂量进行处理,利用CCK-8和平板克隆形成实验观测放疗后细胞增殖情况,摸索适合的放疗剂量并对比不同细胞株的放疗敏感性差异。
2、分别将六株细胞注射到裸鼠皮下,待其成瘤后,取荷瘤裸鼠肿瘤组织,通过荧光实时定量PCR检测荷瘤裸鼠肿瘤组织中hsa-miR-622的表达。肿瘤最大直径至10mm后,对荷瘤裸鼠按临床治疗剂量进行放疗处理,用游标卡尺记录放疗组和未放疗组肿瘤的大小变化情况。以肿瘤缩减率(未放疗组肿瘤最大直径-放疗组肿瘤最大直径/未放疗组肿瘤最大直径)作为评价肿瘤的放疗敏感性的指标,对比内源性hsa-miR-622的表达与荷瘤裸鼠放疗敏感性的关系。
3.收集进展期中低位直肠癌患者的放疗前肿瘤组织活检标本,共17例。荧光实时定量PCR检测17例进展期中低位直肠癌患者放疗前活检标本中hsa-miR-622的表达。根据南方医院病历管理系统中所记录的放疗前后CT、肠镜以及术后病理报告对比患者放疗敏感性的差异,利用Mandard肿瘤缓解程度分级的标准对患者放疗后肿瘤缓解程度进行分级。
二、沉默和过表达hsa-miR-622对结直肠癌细胞及荷瘤裸鼠敏感性的影响
1.利用细胞瞬时转染实验,选择SW480和HCT116两株细胞进行瞬时转染,其中SW480对hsa-miR-622进行干扰,而HCT116则转入mimics模拟过表达hsa-miR-622,荧光实时定量PCR检测细胞瞬时转染效率。对细胞进行放疗处理,利用PI染色后进行流式细胞实验对比细胞的放疗敏感性变化。
通过慢病毒转染技术获得稳定过表达和沉默hsa-miR-622的细胞株,荧光实时定量PCR检测四株细胞慢病毒转染效率,利用细胞增殖实验(CCK-8和平板克隆形成实验)对比在转染慢病毒后细胞放疗敏感性的变化。
2、将稳定过表达组、沉默组细胞和N.C组分别注射到裸鼠皮下。待荷瘤裸鼠肿瘤最大直径约10mm左右后,对荷瘤裸鼠按临床治疗剂量进行放疗处理,用游标卡尺记录放疗组和未放疗组肿瘤的大小变化情况。以肿瘤缩减率(未放疗组肿瘤最大直径-放疗组肿瘤最大直径/未放疗组肿瘤最大直径)作为评价肿瘤的放疗敏感性的指标,将对照组和沉默组、对照组和过表达组进行对比。对比荷瘤裸鼠在沉默和过表达hsa-miR-622后放疗敏感性的变化。
三、hsa-miR-622的靶基因的预测及表达特性分析
1、以hsa-miR-622为检索词,检索microRNA.org、TargetScan两个数据库进行靶基因的预测。将2个数据库的预测结果取交集后,对所得靶基因进行GO分类,同时利用DAVID数据库对分类结果进验证,最后根据microRNA.org数据库对基因作用的注解及评分选取目的靶基因。
2、构建预测所得靶基因的3'UTR表达载体以及将hsa-miR-622结合位点突变后的3'UTR的突变载体。在工具细胞293FT中利用双萤光素酶报告系统验证hsa-miR-622是否能够与预测所得基因的3'UTR区相结合并对预测所得靶基因的表达产生抑制,并在直肠癌细胞株SW837中验证。
3、western-blot检测裸鼠皮下肿瘤组织内源性及在沉默和过表达后RB1基因的差异表达,对比荷瘤裸鼠放疗敏感性与RB1表达强弱的关系,分析RB1的表达与放疗敏感性的关系。
4、免疫组化染色检测中晚期直肠癌患者的放疗前活检石蜡标本中RB1的表达,对比个体间RB1的表达差异与患者放疗敏感性的关系
5、western-blot检测直肠癌细胞株HCT116中RB1、E2F1、E2F8三个蛋白在放疗后1-8h的表达情况,探讨三个蛋白间的相互作用。
研究结果：
一、hsa-miR-622在细胞水平和组织水平的差异表达及其与放疗敏感性的关系
1、荧光实时定量PCR检测hsa-miR-622在SW480、HT29、HCT116、LS174.T、 SW837、HR8348细胞中的表达。以Ls174.T细胞的表达为参照组,对结果通过单因素方差分析显示：5种细胞株中hsa-miR-622的表达量之间差异具有显著性(F=118.335,P<0.001)。方差齐性检验显示方差齐性,LSD两两比较后发现：HR8348细胞当中hsa-miR-622的表达量最高,且高于SW480(P<0.001)、 HT29(P<0.001)、HCT116(P<0.001)、SW837(P<0.001)细胞中的表达：hsa-miR-622表达较高的是SW480细胞,且高于HT29(P<0.001)、HCT116(P<0.001)、 SW837(P<0.001)细胞中的表达；hsa-miR-622在HT29中的表达高于HCT116(P=0.032)、SW837(P=0.018)。而hsa-miR-622表达较低的两株细胞之间无统计学差异SW837与HCT116(P=0.741)。hsa-miR-622在HR8348、SW480、 HT29、HCT116、SW837、LS174.T中依次降低。在HR8348、SW480、HT29表达相对较高,在HCT116、SW837、LS174.T表达相对较低,对SW480、HT29、 HCT116、LS174.T、SW837、HR8348分别给予2、3、4、5Gy的放疗剂量进行处理后,CCK-8、平板克隆形成实验以检测细胞放疗敏感性显示SW480、HT29、 HR8348对放疗不敏感,而HCT116、Ls174.T、SW837对放疗敏感。我们发现hsa-miR-622的内源性表达与细胞的放疗敏感性负相关。
2、荧光定量PCR检测荷瘤裸鼠肿瘤组织中hsa-miR-622的表达,以Ls174.T为参照组,将其他5个实验组进行单因素方差分析结果显示：五株细胞株在裸鼠皮下成瘤后,检测肿瘤组织hsa-miR-622表达结果有统计学差异(F=315.155P<0.001)。方差齐性检验显示方差不齐,Dunnett T3两两比较后发现：HR8348细胞当中hsa-miR-622的表达量最高,且高于SW480(P=0.011)、HT29(P=0.002). HCT116(P=0.004)、SW837(P=0.003)细胞中的表达；hsa-miR-622表达较高的是SW480细胞,且高于HT29(P=0.066)、HCT116(P=0.022)、SW837(P=0.016)细胞中的表达；hsa-miR-622在HT29中的表达比HCT116(P=0.008)、 SW837(P=0.005)细胞中的表达。而hsa-miR-622表达较低的两株细胞中HCT116的表达要高于SW837(P<0.001)的表达。HR8348、SW480、HT29、HCT116、 LS174.T、SW837在裸鼠皮下成瘤后,肿瘤组织中hsa-miR-622的表达依次降低,在HR8348、SW480、HT29表达相对较高,在HCT116、SW837、LS174.T表达相对较低。对比荷瘤裸鼠在注射不同结直肠癌细胞株的放疗敏感性差异,结果显示：不同细胞株在裸鼠皮下成瘤后,肿瘤组织放疗敏感性有统计学差异(F=6.141P=0.005)。LSD两两比较结果显示,注射HT29细胞的荷瘤裸鼠放疗敏感性要低于HCT116(P=0.002)、Ls174.T (P=0.008)和SW837(P=0.024),与HR8348(P=0.829)和SW480(P=0.984)无统计学差异。注射SW480的荷瘤裸鼠放疗敏感性要低于HCT116(P=0.004)、Ls174.T (P=0.012)和SW837(P=0.036),与HR8348(P=0.845)相比无统计学差异。注射HR8348的荷瘤裸鼠放疗敏感性要低于HCT116(P=0.002)、Ls174.T (P=0.008)和SW837(P=0.025)。HCT116与Ls174.T (P=0.522)、Ls174.T与SW837(P=0.562)、 SW837与HCT116(P=0.233)之间放疗敏感性无统计学差异。我们发现hsa-miR-622在荷瘤裸鼠肿瘤组织中的内源性表达与其放疗敏感性负相关
3、收集17例进展期中低位直肠癌患者的放疗前瘤组织标本,检测其中hsa-miR-622的表达。并利用南方医院病历管理系统中记录的CT、肠镜以及术后病理报告,对比临床患者的放疗敏感性差异情况,利用Mandard肿瘤缓解程度分级的标准对患者进行分级。17例标本中,5例患者经过Mandard肿瘤缓解程度分级为TRG4,其hsa-miR-622的表达水平较高,而在相对最低表达的3例患者均达到完全缓解。
二、沉默和过表达hsa-miR-622对结直肠癌细胞及荷瘤裸鼠放疗敏感性的影响
1、荧光实时定量PCR检测细胞转染效率,对结果进行t检验显示hsa-miR-622在SW480N.C中的表达要高于SW480IN中的表达(t=13.313,P=0.006),结果具有统计学差异而HCT116N.C中的表达要低于HCT116hsa-miR-622中的表达(t=13.313,P=0.008)。说明瞬时转染对hsa-miR-622的表达产生影响。对细胞进行放疗处理,利用PI染色后进行流式细胞实验对比细胞的放疗敏感性变化。发现mimics转染的细胞株与对照组相比,放疗敏感性下降(t=-16.186,P<0.001),而inhibitor转染后的细胞株与对照组相比,放疗敏感性增强(t=-13.556,P<0.001)。荧光实时定量PCR检测四株细胞慢病毒转染效率,采用单样本t检验对结果进行分析,在干扰组中SW480IN的hsa-miR-622的表达与SW480N.C组相比无统计学差异(t=4.285,P=0.050)；而HT29IN的hsa-miR-622表达要高于HT29N.C (t=5.387,P=0.033);在过表达组中HCT116hsa-miR-622的表达与HCT116N.C组相比无统计学差异(t=2.853, P=0.104); SW837hsa-miR-622的表达要高于SW837N.C组的表达(t=5.541,P=0.031)。同时利用细胞增殖实验(CCK-8和平板克隆形成实验)对比在转染慢病毒后细胞放疗敏感性的变化,结果显示：与对照组相比,沉默组细胞株放疗敏感性增强,而在过表达组中,细胞放疗敏感性减弱。
2、对荷瘤裸鼠在沉默和过表达hsa-miR-622后放疗敏感性的变化。结果显示：注射SW480IN的荷瘤裸鼠比注射SW480N.C的荷瘤裸鼠的肿瘤缩减率明显(t=5.572,P=0.005)。注射HCT116N.C的荷瘤裸鼠比注射HCT116hsa-miR-622的荷瘤裸鼠的肿瘤缩减率明显(t=3.283,P=0.030)。注射SW837N.C的荷瘤裸鼠比SW837hsa-miR-622的荷瘤裸鼠肿瘤缩减率明显(t=2.917,P=0.043)。
三、hsa-miR-622的靶基因预测及表达特性分析
1、通过软件预测分析并查阅相关文献,最终得到hsa-miR-622的靶向调控基因RB1。
2、在工具细胞293FT中,双荧光素酶报告系统结果显示,在转染克隆有RB1基因3'UTR质粒的实验组中,单因素方差分析结果显示：组间存在统计学差异(F=139.970,P<0.001)。LSD进行两两比较：hsa-miR-622组与blank组相比萤光素酶活性降低(P<0.001),hsa-miR-622组与N.C组相比萤光素酶活性降低(P<0.001)。blank组和N.C组相比荧光素酶活性未见明显变化(P=0.838),说明对照转染序列未对荧光素酶活性产生影响。Hsa-miR-622inhibitor与N.C inhibitor组(P=0.168)和blank组(P=0.231)相比,萤光素酶活性均差异无统计学意义。在转染克隆有MutRB1基因3'UTR质粒的实验组中,单因素方差分析结果显示各组间荧光素酶活性无统计学差异(F=2.397,P=0.120)。证明hsa-miR-622能与RB1基因3'UTR结合,从而抑制了萤光素酶的活性。这以结果也在SW837细胞中得到验证。
3、不同细胞在裸鼠皮下成瘤后,western-blot检测肿瘤组织内源性RB1的表达,对比裸鼠放疗敏感性与RB1表达强弱的关系,发现在RB1高表达的组织中,荷瘤裸鼠对放疗更敏感。同时检测注射稳定转染细胞的裸鼠肿瘤组织中的RB1表达,分析其与放疗敏感性的关系。结果显示：发现在转染hsa-miR-622过表达慢病毒的细胞在皮下成瘤后的肿瘤组织中RB1的表达较N.C组降低,而在干扰组中RB1表达升高。在放疗敏感性方面,与N.C组相比,过表达组荷瘤裸鼠放疗敏感性降低,抑制组放疗敏感性增加。
4、免疫组化染色检测中晚期直肠癌患者的放疗前活检石蜡标本中RB1的表达,对比个体间RBl的表达情况与患者对放疗敏感性的关系,结果显示RB1在27例患者中均呈强阳性表达,个体间无统计学差异。
5、western-blot检测RB1、E2F1、E2F8三个蛋白在放疗后1-8h的直肠癌细胞株中的表达情况。结果显示：三个白蛋白在放疗后1-8h都有先降低再升高最后降低的变化过程。他们之间的关系有待进一步的研究。
结论
Hsa-miR-622的差异表达结直肠癌细胞的放疗敏感性负相关
Hsa-miR-622的差异表达与荷瘤裸鼠放疗敏感性负相关
RB1是hsa-miR-622的直接作用靶基因
RB1的差异表达与结直肠癌细胞放疗敏感性正相关
RB1的差异表达与荷瘤裸鼠放疗敏感性正相关
Background and purpose
Epidemiological characteristics of colorectal cancer in China suggest that colorectal cancer especially low rectal cancer important disease is an important disease which affects the health. With the development of economy, the rapid change in lifestyle, diet structure, the incidence of colorectal cancer in China are increasing, the incidence is trending gradually to the level of Europe and the United States closely. But compared with western developed countries, the epidemiology of colorectal cancer in China has its own characteristics; first, the age of patients is younger, median age of onset of colorectal cancer at the age of45, than the patients in Europe and the United States about12to18years. Second, middle and low rectal cancer is more frequency detected. Rectal cancer accout for about70%of the colorectal cancer and70%of colorectal cancer locates in8cm from the anal verge. Third, large numbers of advanced patients greatly influences the treatment the effect. As a consequence, the5year survival rate has remained at a low level.
Preoperative neoadjuvant radiotherapy has become the standard scheme for the treatment of advanced lower rectal cancer, worthy of further research on molecular markers for predicting the radiosensitivity. In recent years, with a few big sample randomized controlled study completion in Europe and the United States, adjuvant radiotherapy showed preoperative chemoradiotherapy has more andvantages compared with afteroderative for the treatment of advanced rectal cancer, first, the surrounding area of tumor blood supplication did not destroy, so the tumor has good blood supply and high oxygenayion, result in the exact effect of preoperative radiotherapy. Second, the tumor degradation significantly after neoadjuvant radiotherapy compared with high negative margin rate and doubled anus preserving operation rate. Third, preoperative radiotherapy will increase tumor local control rate than that after the implementation. Last but not least, the adverse reaction was significantly lower than the afteroperative postoperative. More reports, preoperative radiotherapy can improve the patient's long-term survival rate. However, rectal cancer a kind of adenocarcinoma, the whole efficiency of radiotherapy is not as sensitive as squamous cell carcinoma and malignant lymphoma. Patients with obvious degraion, part degration and no degration each account for about1of/3, and the total efficiency is about70%, the latter benefited less from neoadjuvant radiotherapy. Apparently, patients with invalid treatment cannot benefit from preoperative radiotherapy, even only added to the toxic effects of treatment, even a delay of the treatment. Therefore, looking for molecular markers related to radiosensitivity of rectal cancer, making a pefect choice of patients should be the focused in the current study. Many studies trying to find biological markers to predict the radiosensitivity in rectal carcinoma to get abetter patients selection, including P53and P21, but it is difficult to be widely applied. At present, we are mainly depend on clinical features of tumors, including tumor size, the activity of the tumor, the degree of tumor differentiation and imaging examination. Unfortutinary, those kinds of clinical indexes are lack of correlation with tumor radiosensitivity. Therefore, the search for new molecular markers related to radiosensitivity of rectal cancer is worth looking forward to.
MicroRNA is expected to become a new molecular diagnostic marker, which almost involved in the whole process of the development of tumor. While the Giuseppina Della Vittoria Scarpati et al. Selected the miRNAs to predict to sensitivity of patients to radiotherapy in38cases of advanced low rectal cancer patients before neoadjuvent therapy by microarray assay that was verified by RT-Q-PCR. Combined with clinical data, it showed that hsa-miR-622and hsa-miR-630are perfect the sensitivity to radiotherapy with both100%sensitivity and specificity. After screening and Preliminary experiments, we chose hsa-miR-622as the object of study.
Methods
Relationship between the expression of hsa-miR-622and radiation sensitivity.
1. Expression of hsa-miR-622in SW480, HT29, HCT116, LS174.T, SW837, HR8348cells was fluorescence quantitative PCR detection. Then SW480, HT29, HCT116, LS174.T, SW837, HR8348were given radiotherapy dose of2,3,4,5Gy processing, cell proliferation after radiotherapy was observed by CCK-8and plate colony forming experiment.
2. Expression of hsa-miR-622detected by fluorescence quantitative PCR tumor-bearing nude mice tumor tissue. Comparison of the sensitivity of tumor-bearing nude mice.
3.17turner tissue specimens before radiotherapy were collected status in rectal cancer patients. The expression of hsa-miR-622was detected in17cases with advanced status in rectal cancer patients before radiotherapy. After a comparison of CT and colonoscopy results of the patients before and after radiation, the tumor regression grade was classified by the standard of Mandard tumor remission degree.
The effect of silence and overexpression of hsa-miR-622on colorectal cancer cells and tumor-bearing nude mice to radiosensitivity
1. SW480and HCT116were selected for the transient transfection. Hsa-miR-622was interfered in SW480, while HCT116was transfected with mimics to overexpress hsa-miR-622. Efficiency of cell transfection was detected by RT-Q-PCR. The apoptosis of the different group after radiotherapy was detected flow cytometry experiment with PI staining.Then we use lentiviral to transfect cells to overexpress or silence of hsa-miR-622stablily. Lentiviral transfection efficiency was detected by RT-Q-PCR in four cell lines. Cell proliferation assay (CCK-8and plate colony formation assay) was used to compare the varience of sensitivity of the cell to radiotherapy.
2、Cells transfected with letiviral were injected subcutaneously into athymic nude mice. The nude mice were treat with radiotherapy after the diameter of the tumor was more than1cm according to clinical therapeutic dose. The tumor size was recorded every three days by vernier caliper in both with radiotherapy group and without radiotherapy group. Tumor regression rate (radiotherapy group/without radiotherapy group), an index to evaluate the radiation sensitivity, was used to compared the control group and the interference group, control group and the overexpression group.
Characteristic analysis and a prediction of target gene of hsa-miR-622
1、Taking hsa-miR-622as the search term, we tried to predict the target genes of hsa-miR-622in microRNA.org, TargetScan. Intersection the two datebase was classified by GO classification, and further verification was made in the DAVID database. According to the annotations and score of gene in microRNA.org database, three the target gene is closely related with hsa-miR-622.
2、RB1gene3'UTR expression vector and hsa-miR-622binding site mutated MutRB1gene3'UTR expression vector was constructed. We tried to verify whether hsa-miR-622can combine with RB13'UTR and inhibit RBI gene expression in posttranscriptional level through the dual luciferase system in293FT cell. The result was convinced in the SW837cell line.
3、Edogenous expression of RBI in tissues of nude mice subcutaneous tumor was detected by western-blot. Then we take a contrast of the relationship of the radiosensitivity and RB1expression in nude mice bearing human tumor.
4、Paraffin specimens of the patients with advanced rectal cancer before radiotherapy were immunohistochemically stained. Relationship to the expression of RBI and radiosensitivity were compared among individual.
5、The expression of RB1, E2F1, and E2F8in first to eighth hour was detected by western-blot in the cells that were treated with radiotherapy.
Results
Relationship between the expression of hsa-miR-622and radiation sensitivity.
1. Expression of hsa-miR-622in SW480, HT29, HCT116, LS174.T, SW837, HR8348cells was fluorescence quantitative PCR detection. When the conference group was disputed, there was significant difference between five cell lines (F=118.335, P<0.001). The results showed that hsa-miR-622was the highest expressed in HR8348. It was higher than that of SW480(P<0.001), HT29(P<0.001), HCT116(P<0.001), SW837(P<0.001) expression in the cells, there was significant difference. Hsa-miR-622expression was higher in SW480cells, and higher than that of HT29(P<0.001), HCT116(P<0.001), SW837(P<0.001) expression in the cells, there was significant difference. Hsa-miR-622expression in HT29higher than that in HCT116(P=0.032), SW837(P=0.018) expression in the cells, there were statistical differences. But the expression of hsa-miR-622in the left two cell lines showed no significant difference between SW837and HCT116(P=0.741). The expression of hsa-miR-622decreased in HR8348, SW480, HT29, HCT116, SW837, and LS174.T. It relatively high expressed in HR8348, SW480, and HT29. All of the cell lines were given radiotherapy of2,3,4,5Gy. The results of CCK-8and plate clone formation assay showed that SW480, HT29, HR8348are not sensitive to radiotherapy, while HCT116, Ls174.T, SW837is sensitive to radiotherapy. We found that endogenous hsa-miR-622expression correlated with the radiosensitivity of cells negatively.
2. Expression of hsa-miR-622detected by fluorescence quantitative PCR tumor-bearing nude mice tumor tissue. Results showed that hsa-miR-622expression were statistically significant in subcutaneous tumors formed by five cell lines in nude mice when the conference group was disputed (F=315.155, P<0.001). Dunnett T3comparison found:HR8348cells and the expression of hsa-miR-622was the highest in the tumor tissue formed by HR8348, and it was higher than that of SW480(P=0.011), HT29(P=0.002), HCT116(P=0.004), SW837(P=0.003) expression in the cells, there was significant difference. Hsa-miR-622expression was higher in SW480cells, and it was higher than that of HT29(P=0.066), HCT116(P=0.022), SW837(P=0.016) expression in the cells. The expression of hsa-miR-622in HT29was higher than in HCT116(P=0.008), SW837(P=0.005) expression in the cells, there was statistical differences. Expression of hsa-miR-622in HCT116cell was higher than that expressed in SW837(P<0.001). Comparison of the sensitivity of tumor-bearing nude mice showed that:there was significant difference in radiosensitivity of subcutaneous tumor in nude mice formed by different cell lines (F=6.141P=0.005). Homogeneity of variance test showed there was no variance of homogeneity, LSD comparison showed that:radiosensitivity of subcutaneous tumor formed by HT29is lower than that of HCT116(P=0.002), Ls174.T (P=0.008) and SW837(P=0.024), while there was no significant difference compared with that of HR8348(P=0.829) and SW480(P=0.984). Radiosensitivity of subcutaneous tumor formed by SW480is lower than that of HCT116(P=0.004), Ls174.T (P=0.012) and SW837(P=0.036), while there was no significant difference compared with that of HR8348(P=0.845). Radiosensitivity of subcutaneous tumor formed by HR8348is lower than that of HCT116(P=0.002), Ls174.T (P=0.008) and SW837(P=0.025). There was no difference between HCT116and Ls174.T (P=0.522), Ls174.T and SW837(P=0.562), SW837and HCT116(P=0.233) in sensitivity to radiotherapy. We found that endogenous hsa-miR-622expression in the tissue of mude mice hearing human tumor correlated with the radiosensitivity of cells negatively.
3.17turner tissue specimens before radiotherapy were collected status in rectal cancer patients. The expression of hsa-miR-622was detected in17cases with advanced status in rectal cancer patients before radiotherapy. After a comparison of CT and colonoscopy results of the patients before and after radiation, the tumor regression grade was classified by the standard of Mandard tumor remission degree. By contrast,5cases were classified as TRG4, the hsa-miR-622expression level was higher, and the lowest expression in3patients achieved complete remission.
The effect of silence and overexpression of hsa-miR-622on colorectal cancer cells and tumor-bearing nude mice to radiosensitivity
1. SW480and HCT116were selected for the transient transfection. Hsa-miR-622was interfered in SW480, while HCT116was transfected with mimics to overexpress hsa-miR-622. Efficiency of cell transfection was detected by RT-Q-PCR. The t test of the results showed that hsa-miR-622was higher expressed in SW480N.C than the expression in SW480IN (t=13.313, P=0.006), the difference was statistically significant. The expression of hsa-miR-622was lower in the HCT116N.C than in the HCT116expression (t=13.313, P=0.008), the difference was statistically significant. It indicated that hsa-miR-622expression was infected by the transient transfection. The apoptosis of the different group after radiotherapy was detected flow cytometry experiment with PI staining. The sensitivity of the cell was decreased in mimics transfected cell line compared with the control group (t=16.186, P<0.001), while the sensitivity was enhenced in the inhibitor transfected cell line compared with the control group (t=13.556, P<0.001). Then we use lentiviral to transfect cells to overexpress or silence of hsa-miR-622stablily. Lentiviral transfection efficiency was detected by RT-Q-PCR in four cell lines, single sample t test was usd to analyze the results, expression of hsa-miR-622was no difference between the SW480IN and SW480N.C (t=4.285, P=0.050), HCT116-miR622and HCT116N.C showed no significant difference (t=2.853,P=0.104); The expression of hsa-miR-622in HT29IN was higher than that of HT29N.C (t=5.387,P=0.033); The expression of hsa-miR-622in SW837miR-622was higher than that of SW837N.C (t=5.541,P=0.031). Cell proliferation assay (CCK-8and plate colony formation assay) was used to compare the varience of sensitivity of the cell to radiotherapy. Compared with the control group, the sensitivity of the interference group was enhenced, while it was decreased in the overexpression group of cell radiosensitivity.
2. Cells transfected with letiviral were injected subcutaneously into athymic nude mice. The nude mice were treat with radiotherapy after the diameter of the tumor was more than1cm according to clinical therapeutic dose. The tumor size was recorded every three days by vernier caliper in both with radiotherapy group and without radiotherapy group. Tumor regression rate (radiotherapy group/without radiotherapy group), an index to evaluate the radiation sensitivity, was used to compared the control group and the interference group, control group and the overexpression group. The results showed:tumor reduction rate was more obvious in SW480IN than thatin SW480N.C (t=5.572, P=0.005), the results were statistically difference. Nude mice injected with HCT116N.C was more significantly regressed than that with HCT116miR-622(t=3.283,P=0.030), the results were statistically difference. It wais also verified in SW837(t=2.917, P=0.043).
Characteristic analysis and prediction of target gene of hsa-miR-622
1. Taking hsa-miR-622as the search term, we tried to predict the target genes of hsa-miR-622in microRNA.org, TargetScan. Intersection the two datebase was classified by GO classification, and further verification was made in the DAVID database. According to the annotations and score of gene in microRNA.org database, three the target gene is closely related with hsa-miR-622. According to the literature, there were important relations between tumor occurrence and development and RB1. In addition, RBI was play an important role in the regulation of cell cycle and influencing on apoptosis.
2. RB1gene3'UTR expression vector and hsa-miR-622binding site mutated MutRB1gene3'UTR expression vector was constructed. We tried to verify whether hsa-miR-622can combine with RBI3'UTR and inhibit RB1gene expression in posttranscriptional level through the dual luciferase system in293FT cell.combined with the translation of the RBI level. Results in293FT cell transfected with RBI gene3'UTR plasmid analysied by single factor variance analysis showed that the difference between groups was significant (F=139.970, P<0.001), test of homogeneity of variance by Levene homogeneity of variance show there was homogeneity of variance (F=1.103, P=0.407). LSD two two comparisons showed: luciferase activity was inhibited in the group transfected with hsa-miR-622compared with blank (P<0.001) and N.C (P<0.001). There was statistically difference. It showed that hsa-miR-622can combine with RBI gene3'UTR, leading to the inhibition of luciferase activity. There was no significant difference between blank group and N.C group (P=0.838), the control sequence had no effect on luciferase activity. Luciferase activity has no difference in the group transfected with hsa-miR-622inhibitor compared with blank (P=0.231) and N.C inhibitor (P=0.168). The results of single factor analysis of variance based on the plasmid of MutRB1gene showed no significant difference between groups (F=2.397, P=0.120). The results are also verified in SW837cells.
3. Edogenous expression of RBI in tissues of nude mice subcutaneous tumor was detected by western-blot. With a contrast of the relationship of the radiosensitivity and RB1expression in nude mice bearing human tumor, we found the higher expression of RB1in the tumor tissues, the more sensitive to radiotherapy of the nude mice. Using western-blot to detect the expression of RB1in tissues from the nude mice injected with stably transfected cell and analyzed its relationship with radiotherapy sensitivity. The results showed that RB1expressed lower in the tumor tissue which was formed by the cells transfected with overexpression lentiviral compared with N.C group. And the RBI expressed higher in the inhibitor group compared with N.C group.
4. Paraffin specimens of the patients with advanced rectal cancer before radiotherapy were immunohistochemically stained. Relationship to the expression of RBI and radiosensitivity were compared among individual. We found that RBI expressions were all strongly positive in27patients, no statistically significant differences between individuals.
5. The expression of RB1, E2F1, and E2F8in first to eighth hour was detected by western-blot in the cells that were treated with radiotherapy. The result showed that three albumin has the same trend after radiotherapy in1-8h. They were all decreased first and then increased, but dreased again at last. The relationship between them needs to be studied further
Conclusion
1. The relationship between differential expressions of Hsa-miR-622and radiotherapy sensitivity of colorectal cancer cell was negative correlation
2. Radiosensitivity of the nude mice bearing human tumor associated negatively with hsa-miR-622expression.
3. RB1is a direct target gene of hsa-miR-622
4. The relationship between differential expressions of Rbl and radiotherapy sensitivity of colorectal cancer cell was positive correlation
5. Radiosensitivity of the nude mice bearing human tumor associated positively with RB1expression
引文
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[4]Yang, Z., et al., Clinicopathological characteristics and long-term outcomes of colorectal cancer in elderly Chinese patients undergoing potentially curative surgery. Surg Today,2013.
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[13]Kelley, S.T., et al., Tumor response to neoadjuvant chemoradiation therapy for rectal adenocarcinoma is mediated by p53-dependent and caspase 8-dependent apoptotic pathways. Clin Colorectal Cancer,2005.5(2):p.114-8.
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[16]Kobunai, T., T. Watanabe and T. Fukusato, REG4, NEIL2, and BIRC5 gene expression correlates with gamma-radiation sensitivity in patients with rectal cancer receiving radiotherapy. Anticancer Res,2011.31(12):p.4147-53.
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[18]Li, Y., et al., MicroRNA-21 targets LRRFIP1 and contributes to VM-26 resistance in glioblastoma multiforme. Brain Res,2009.1286:p.13-8.
[19]Schetter, A.J., et al., MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma. JAMA,2008.299(4):p. 425-36.
[20]Sotiropoulou, G., et al., Emerging roles of microRNAs as molecular switches in the integrated circuit of the cancer cell. RNA,2009.15(8):p.1443-61.
[21]Rosenfeld, N., et al., MicroRNAs accurately identify cancer tissue origin. Nat Biotechnol,2008.26(4):p.462-9.
[22]Malumbres, R., et al., Differentiation stage-specific expression of microRNAs in B lymphocytes and diffuse large B-cell lymphomas. Blood,2009.113(16):p. 3754-64.
[23]Yan, L.X., et al., MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis. RNA,2008.14(11):p.2348-60.
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[26]Chen, X., et al., Characterization of microRNAs in serum:a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res,2008.18(10):p. 997-1006.
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[28]Jacobsen, N., D. Andreasen and P. Mouritzen, Profiling microRNAs by real-time PCR. Methods Mol Biol,2011.732:p.39-54.
[29]Sicard, F., et al., Targeting miR-21 for the Therapy of Pancreatic Cancer. Mol Ther,2013.
[30]Mayne, G.C., D.J. Hussey and D.I. Watson, Can miRNA profiling allow us to determine which patients with esophageal cancer will respond to chemoradiotherapy? Expert Rev Anticancer Ther,2013.13(3):p.271-3.
[31]Calin, G.A. and C.M. Croce, MicroRNA signatures in human cancers. Nat Rev Cancer,2006.6(11):p.857-66.
[32]Lebanony, D., et al., Diagnostic assay based on hsa-miR-205 expression distinguishes squamous from nonsquamous non-small-cell lung carcinoma. J Clin Oncol,2009.27(12):p.2030-7.
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[35]Ng, E.K., et al., Differential expression of microRNAs in plasma of patients with colorectal cancer:a potential marker for colorectal cancer screening. Gut, 2009.58(10):p.1375-81.
[36]Xie, Y., et al., Altered miRNA expression in sputum for diagnosis of non-small cell lung cancer. Lung Cancer,2010.67(2):p.170-6.
[37]Mitchell, P.S., et al., Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A,2008.105(30):p.10513-8.
[38]Chen, X., et al., Characterization of microRNAs in serum:a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res,2008.18(10):p. 997-1006.
[39]Gilad, S., et al., Serum microRNAs are promising novel biomarkers. PLoS One, 2008.3(9):p. e3148.
[40]Cai, B., et al., miRNA-181b increases the sensitivity of pancreatic ductal adenocarcinoma cells to gemcitabine in vitro and in nude mice by targeting BCL-2. Oncol Rep,2013.
[41]Golubovskaya, V.M., et al., MiR-138 and MiR-135 Directly Target Focal Adhesion Kinase, Inhibit Cell Invasion, and Increase Sensitivity to Chemotherapy in Cancer Cells. Anticancer Agents Med Chem,2013.
[42]Hu, H., et al., The overexpression of hypomethylated miR-663 induces chemotherapy resistance in human breast cancer cells by targeting heparin sulphate proteoglycan 2 (HSPG2). J Biol Chem,2013.
[43]Zhang, J., et al., Genome-wide analysis of miRNA signature differentially expressed in doxorubicin-resistant and parental human hepatocellular carcinoma cell lines. PLoS One,2013.8(1):p. e54111.
[44]Svoboda, M., et al., Micro-RNAs miR125b and miR137 are frequently upregulated in response to capecitabine chemoradiotherapy of rectal cancer. Int J Oncol,2008.33(3):p.541-7.
[45]Hummel, R., D.J. Hussey and J. Haier, MicroRNAs:predictors and modifiers of chemo- and radiotherapy in different tumour types. Eur J Cancer,2010.46(2):p. 298-311.
[46]Wouters, M.D., et al., MicroRNAs, the DNA damage response and cancer. Mutat Res,2011.717(1-2):p.54-66.
[47]Zhao, L., et al., MiRNA expression analysis of cancer-associated fibroblasts and normal fibroblasts in breast cancer. Int J Biochem Cell Biol,2012.44(11): p.2051-9.
[48]Salim, H., et al., miRNA-214 modulates radiotherapy response of non-small cell lung cancer cells through regulation of p38MAPK, apoptosis and senescence. Br J Cancer,2012.107(8):p.1361-73.
[49]Wu, Y., et al., MicroRNA-148b enhances the radiosensitivity of non-Hodgkin's Lymphoma cells by promoting radiation-induced apoptosis. J Radiat Res,2012. 53(4):p.516-25.
[50]Zhao, L., et al., Regulatory mechanisms and clinical perspectives of miRNA in tumor radiosensitivity. Carcinogenesis,2012.33(11):p.2220-7.
[51]Balca-Silva, J., et al., Effect of miR-34b overexpression on the radiosensitivity of non-small cell lung cancer cell lines. Anticancer Res,2012.32(5):p.1603-9.
[52]Ghawanmeh, T., et al., miR-34a expression, cell cycle arrest and cell death of malignant mesothelioma cells upon treatment with radiation, docetaxel or combination treatment. Oncology,2011.81(5-6):p.330-5.
[53]Della, V.S.G., et al., A specific miRNA signature correlates with complete pathological response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer. Int J Radiat Oncol Biol Phys,2012.83(4):p.1113-9.
[54]Cummins, J.M., et al., The colorectal microRNAome. Proc Natl Acad Sci U S A,2006.103(10):p.3687-92.
[55]Chen, C., et al., Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res,2005.33(20):p. e179.
[56]Lagos-Quintana, M., et al., Identification of novel genes coding for small expressed RNAs. Science,2001.294(5543):p.853-8.
[57]Lee, R.C. and V. Ambros, An extensive class of small RNAs in Caenorhabditis elegans. Science,2001.294(5543):p.862-4.
[58]Brennecke, J., et al., Principles of microRNA-target recognition. PLoS Biol, 2005.3(3):p. e85.
[59]Sethupathy, P., M. Megraw and A.G. Hatzigeorgiou, A guide through present computational approaches for the identification of mammalian microRNA targets. Nat Methods,2006.3(11):p.881-6.
[60]Enright, A.J., et al., MicroRNA targets in Drosophila. Genome Biol,2003.5(1): p.R1.
[61]Lewis, B.P., et al., Prediction of mammalian microRNA targets. Cell,2003. 115(7):p.787-98.
[62]Fukuda, Y., H. Kawasaki and K. Taira, Exploration of human miRNA target genes in neuronal differentiation. Nucleic Acids Symp Ser (Oxf),2005(49):p. 341-2.
[63]McGee, T.L., D.W. Yandell and T.P. Dryja, Structure and partial genomic sequence of the human retinoblastoma susceptibility gene. Gene,1989.80(1):p. 119-28.
[64]Xu, H.J., et al., The retinoblastoma susceptibility gene product:a characteristic pattern in normal cells and abnormal expression in malignant cells. Oncogene, 1989.4(6):p.807-12.
[65]Zhu, X., et al., Mechanisms of loss of heterozygosity in retinoblastoma. Cytogenet Cell Genet,1992.59(4):p.248-52.
[66]Sherr, C.J., Cancer cell cycles. Science,1996.274(5293):p.1672-7.
[67]Sun, H., et al., An E2F binding-deficient Rb1 protein partially rescues developmental defects associated with Rbl nullizygosity. Mol Cell Biol,2006. 26(4):p.1527-37.
[68]Zalmas, L.P., et al., DNA-damage response control of E2F7 and E2F8. EMBO Rep,2008.9(3):p.252-9.
[2]Li, J.N., et al., [The trends in clinical characteristics of colon cancer in last two decades]. Zhonghua Nei Ke Za Zhi,2010.49(3):p.226-9.
[3]Li, M. and J. Gu, Changing patterns of colorectal cancer in China over a period of 20 years. World J Gastroenterol,2005.11(30):p.4685-8.
[4]Yang, Z., et al., Clinicopathological characteristics and long-term outcomes of colorectal cancer in elderly Chinese patients undergoing potentially curative surgery. Surg Today,2013.
[5]Kapiteijn, E., et al., Preoperative radiotherapy combined with total mesorectal excision for resectable rectal cancer. N Engl J Med,2001.345(9):p.638-46.
[6]Faerden, A.E., et al., Total mesorectal excision for rectal cancer:difference in outcome for low and high rectal cancer. Dis Colon Rectum,2005.48(12):p. 2224-31.
[7]Camma, C., et al., Preoperative radiotherapy for resectable rectal cancer:A meta-analysis. JAMA,2000.284(8):p.1008-15.
[8]Penopoulos, V., et al., A short course of preoperative radiotherapy improves prognosis of operable rectal carcinoma:a case control study. Hepatogastroenterology,2008.55(85):p.1280-7.
[9]Sebag-Montefiore, D., et al., Preoperative radiotherapy versus selective postoperative chemoradiotherapy in patients with rectal cancer (MRC CR07 and NCIC-CTG C016):a multicentre, randomised trial. Lancet,2009. 373(9666):p.811-20.
[10]Sauer, R., et al., Preoperative versus postoperative chemoradiotherapy for rectal cancer. N Engl J Med,2004.351(17):p.1731-40.
[11]Improved survival with preoperative radiotherapy in resectable rectal cancer. Swedish Rectal Cancer Trial. N Engl J Med,1997.336(14):p.980-7.
[12]Biondo, S., et al., Response to neoadjuvant therapy for rectal cancer:influence on long-term results. Colorectal Dis,2005.7(5):p.472-9.
[13]Kelley, S.T., et al., Tumor response to neoadjuvant chemoradiation therapy for rectal adenocarcinoma is mediated by p53-dependent and caspase 8-dependent apoptotic pathways. Clin Colorectal Cancer,2005.5(2):p.114-8.
[14]Kim, N.K., et al., p53, BCL-2, and Ki-67 expression according to tumor response after concurrent chemoradiotherapy for advanced rectal cancer. Ann Surg Oncol,2001.8(5):p.418-24.
[15]Komuro, Y., et al., Evaluating the combination of molecular prognostic factors in tumor radiosensitivity in rectal cancer. Hepatogastroenterology,2005.52(63): p.666-71.
[16]Kobunai, T., T. Watanabe and T. Fukusato, REG4, NEIL2, and BIRC5 gene expression correlates with gamma-radiation sensitivity in patients with rectal cancer receiving radiotherapy. Anticancer Res,2011.31(12):p.4147-53.
[17]Meng, F., et al., Involvement of human micro-RNA in growth and response to chemotherapy in human cholangiocarcinoma cell lines. Gastroenterology,2006. 130(7):p.2113-29.
[18]Li, Y., et al., MicroRNA-21 targets LRRFIP1 and contributes to VM-26 resistance in glioblastoma multiforme. Brain Res,2009.1286:p.13-8.
[19]Schetter, A.J., et al., MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma. JAMA,2008.299(4):p. 425-36.
[20]Sotiropoulou, G., et al., Emerging roles of microRNAs as molecular switches in the integrated circuit of the cancer cell. RNA,2009.15(8):p.1443-61.
[21]Rosenfeld, N., et al., MicroRNAs accurately identify cancer tissue origin. Nat Biotechnol,2008.26(4):p.462-9.
[22]Malumbres, R., et al., Differentiation stage-specific expression of microRNAs in B lymphocytes and diffuse large B-cell lymphomas. Blood,2009.113(16):p. 3754-64.
[23]Yan, L.X., et al., MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis. RNA,2008.14(11):p.2348-60.
[24]Xi, Y., et al., Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples. RNA,2007. 13(10):p.1668-74.
[25]Mitchell, P.S., et al., Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A,2008.105(30):p.10513-8.
[26]Chen, X., et al., Characterization of microRNAs in serum:a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res,2008.18(10):p. 997-1006.
[27]Meyer, S.U., et al., Profound effect of profiling platform and normalization strategy on detection of differentially expressed microRNAs--a comparative study. PLoS One,2012.7(6):p. e38946.
[28]Jacobsen, N., D. Andreasen and P. Mouritzen, Profiling microRNAs by real-time PCR. Methods Mol Biol,2011.732:p.39-54.
[29]Sicard, F., et al., Targeting miR-21 for the Therapy of Pancreatic Cancer. Mol Ther,2013.
[30]Mayne, G.C., D.J. Hussey and D.I. Watson, Can miRNA profiling allow us to determine which patients with esophageal cancer will respond to chemoradiotherapy? Expert Rev Anticancer Ther,2013.13(3):p.271-3.
[31]Calin, G.A. and C.M. Croce, MicroRNA signatures in human cancers. Nat Rev Cancer,2006.6(11):p.857-66.
[32]Lebanony, D., et al., Diagnostic assay based on hsa-miR-205 expression distinguishes squamous from nonsquamous non-small-cell lung carcinoma. J Clin Oncol,2009.27(12):p.2030-7.
[33]Li, J., et al., Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells. BMC Biotechnol,2007.7:p.36.
[34]Mitchell, P.S., et al., Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A,2008.105(30):p.10513-8.
[35]Ng, E.K., et al., Differential expression of microRNAs in plasma of patients with colorectal cancer:a potential marker for colorectal cancer screening. Gut, 2009.58(10):p.1375-81.
[36]Xie, Y., et al., Altered miRNA expression in sputum for diagnosis of non-small cell lung cancer. Lung Cancer,2010.67(2):p.170-6.
[37]Mitchell, P.S., et al., Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A,2008.105(30):p.10513-8.
[38]Chen, X., et al., Characterization of microRNAs in serum:a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res,2008.18(10):p. 997-1006.
[39]Gilad, S., et al., Serum microRNAs are promising novel biomarkers. PLoS One, 2008.3(9):p. e3148.
[40]Cai, B., et al., miRNA-181b increases the sensitivity of pancreatic ductal adenocarcinoma cells to gemcitabine in vitro and in nude mice by targeting BCL-2. Oncol Rep,2013.
[41]Golubovskaya, V.M., et al., MiR-138 and MiR-135 Directly Target Focal Adhesion Kinase, Inhibit Cell Invasion, and Increase Sensitivity to Chemotherapy in Cancer Cells. Anticancer Agents Med Chem,2013.
[42]Hu, H., et al., The overexpression of hypomethylated miR-663 induces chemotherapy resistance in human breast cancer cells by targeting heparin sulphate proteoglycan 2 (HSPG2). J Biol Chem,2013.
[43]Zhang, J., et al., Genome-wide analysis of miRNA signature differentially expressed in doxorubicin-resistant and parental human hepatocellular carcinoma cell lines. PLoS One,2013.8(1):p. e54111.
[44]Svoboda, M., et al., Micro-RNAs miR125b and miR137 are frequently upregulated in response to capecitabine chemoradiotherapy of rectal cancer. Int J Oncol,2008.33(3):p.541-7.
[45]Hummel, R., D.J. Hussey and J. Haier, MicroRNAs:predictors and modifiers of chemo- and radiotherapy in different tumour types. Eur J Cancer,2010.46(2):p. 298-311.
[46]Wouters, M.D., et al., MicroRNAs, the DNA damage response and cancer. Mutat Res,2011.717(1-2):p.54-66.
[47]Zhao, L., et al., MiRNA expression analysis of cancer-associated fibroblasts and normal fibroblasts in breast cancer. Int J Biochem Cell Biol,2012.44(11): p.2051-9.
[48]Salim, H., et al., miRNA-214 modulates radiotherapy response of non-small cell lung cancer cells through regulation of p38MAPK, apoptosis and senescence. Br J Cancer,2012.107(8):p.1361-73.
[49]Wu, Y., et al., MicroRNA-148b enhances the radiosensitivity of non-Hodgkin's Lymphoma cells by promoting radiation-induced apoptosis. J Radiat Res,2012. 53(4):p.516-25.
[50]Zhao, L., et al., Regulatory mechanisms and clinical perspectives of miRNA in tumor radiosensitivity. Carcinogenesis,2012.33(11):p.2220-7.
[51]Balca-Silva, J., et al., Effect of miR-34b overexpression on the radiosensitivity of non-small cell lung cancer cell lines. Anticancer Res,2012.32(5):p.1603-9.
[52]Ghawanmeh, T., et al., miR-34a expression, cell cycle arrest and cell death of malignant mesothelioma cells upon treatment with radiation, docetaxel or combination treatment. Oncology,2011.81(5-6):p.330-5.
[53]Della, V.S.G., et al., A specific miRNA signature correlates with complete pathological response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer. Int J Radiat Oncol Biol Phys,2012.83(4):p.1113-9.
[54]Cummins, J.M., et al., The colorectal microRNAome. Proc Natl Acad Sci U S A,2006.103(10):p.3687-92.
[55]Chen, C., et al., Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res,2005.33(20):p. e179.
[56]Lagos-Quintana, M., et al., Identification of novel genes coding for small expressed RNAs. Science,2001.294(5543):p.853-8.
[57]Lee, R.C. and V. Ambros, An extensive class of small RNAs in Caenorhabditis elegans. Science,2001.294(5543):p.862-4.
[58]Brennecke, J., et al., Principles of microRNA-target recognition. PLoS Biol, 2005.3(3):p. e85.
[59]Sethupathy, P., M. Megraw and A.G. Hatzigeorgiou, A guide through present computational approaches for the identification of mammalian microRNA targets. Nat Methods,2006.3(11):p.881-6.
[60]Enright, A.J., et al., MicroRNA targets in Drosophila. Genome Biol,2003.5(1): p.R1.
[61]Lewis, B.P., et al., Prediction of mammalian microRNA targets. Cell,2003. 115(7):p.787-98.
[62]Fukuda, Y., H. Kawasaki and K. Taira, Exploration of human miRNA target genes in neuronal differentiation. Nucleic Acids Symp Ser (Oxf),2005(49):p. 341-2.
[63]McGee, T.L., D.W. Yandell and T.P. Dryja, Structure and partial genomic sequence of the human retinoblastoma susceptibility gene. Gene,1989.80(1):p. 119-28.
[64]Xu, H.J., et al., The retinoblastoma susceptibility gene product:a characteristic pattern in normal cells and abnormal expression in malignant cells. Oncogene, 1989.4(6):p.807-12.
[65]Zhu, X., et al., Mechanisms of loss of heterozygosity in retinoblastoma. Cytogenet Cell Genet,1992.59(4):p.248-52.
[66]Sherr, C.J., Cancer cell cycles. Science,1996.274(5293):p.1672-7.
[67]Sun, H., et al., An E2F binding-deficient Rb1 protein partially rescues developmental defects associated with Rbl nullizygosity. Mol Cell Biol,2006. 26(4):p.1527-37.
[68]Zalmas, L.P., et al., DNA-damage response control of E2F7 and E2F8. EMBO Rep,2008.9(3):p.252-9.