摘要
口腔鳞癌是口腔部位及周边最为常见的恶性肿瘤,其发病率占到口腔部位肿瘤总发病率的80%以上,是值得引起人们高度关注和重视的恶性疾病之一。虽然在欧美等发达国家口腔鳞癌的发病率有所下降,但在全世界范围内发病率仍然呈现上升趋势。近年来随着医疗水平的不断提高,许多恶性肿瘤的治疗方面取得了不小的进步,通过治疗提高了患者的5年生存率。但在口腔鳞癌的治疗方面,由于此疾病的发生早期不易被患者察觉等特殊性,致使该疾病的治疗滞后,癌症晚期的治疗难度加大。因而有必要加大对口腔鳞癌治疗的关注度,寻找出口腔鳞癌的更好的治疗方法。
口腔鳞癌的治疗方面,常规的治疗方法包括手术治疗、放射治疗、化学治疗、免疫治疗等。这些治疗方法虽然在一定程度上会抑制肿瘤的发生发展,但又存在较大的治疗弊端。例如放疗、化疗会不可避免的引起严重副反应而手术治疗存在操作不易进行等特点,单纯手术治疗无法根治肿瘤等方面的缺陷。免疫治疗的疗效并不明显。而基因治疗和分子靶向治疗为近年来新兴的肿瘤治疗手段,由于其副反应小、疗效确切等方面的优势得到了科研工作者的青睐。基因治疗中的RNA干涉技术为近年来新兴的疾病诊疗技术,找到疾病致病的相关靶基因,对其进行干预可有效的阻止疾病的进程,尤其在肿瘤治疗中越来越多的引起人们的关注。
STAT3基因为信号转导活化因子3的编码基因,其常被视为原癌基因。STAT3信号传导通路的下游通路可影响许多推进肿瘤进程的相关因子的表达,诸如cyclinD1、Bcl-xl、survivin、c-myc等因子。此外STAT3还参与了肿瘤的免疫逃逸过程,因而将STAT3基因作为肿瘤基因治疗的靶点,对其进行相应的干预,可成为肿瘤治疗的有效手段。
本研究正是利用了基因治疗中的RNA干涉疗法,将肿瘤相关的癌基因STAT3作为治疗靶点,设计了针对STAT3基因位点的RNAi质粒,将质粒导入口腔鳞癌细胞,首先在体外观察该质粒的RNA干涉效应对口腔鳞癌进程的影响,之后利用口腔鳞癌动物模型进行相关的动物体内实验,验证RNAi对STAT3基因进行干预后所产生的肿瘤抑制效果。
本研究对口腔鳞癌的治疗方法进行了全新的探索,有望通过本研究开辟出口腔鳞癌治疗的新途径。
目的:
探讨通过RNA干涉技术对STAT3基因进行敲除对口腔鳞癌细胞造成的体外抑制以及对口腔鳞癌动物模型的抑瘤效果。
方法:
(1)应用免疫组化方法检测正常口腔黏膜组织、不典型增生组织及口腔鳞癌组织中STAT3、EGF及EGFR蛋白的表达。
(2)应用基因重组技术构建出STAT3基因的干扰质粒,即psilencer1.0- U6-stat3-siRNA质粒。
(3)应用基因工程下游技术,诸如Western blot、RT-PCR、免疫组化等方法在体外和体内对不同实验分组的口腔鳞癌肿瘤细胞的STAT3 mRNA表达水平及相关蛋白的表达情况进行检测,判断RNAi的抑瘤效果。
(4)体外实验:针对RNAi的干扰位点设计STAT3的siRNA模板,在体外成功设计并合成出该模板,对质粒载体进行酶切,使其线性化并进行回收。之后对样本进行核算定量。利用T4连接酶将线性质粒载体同干扰片段进行连接。之后进行感受态细胞的制备,再将重组质粒同感受态细胞共同孵育,进行质粒对感受态细胞的转化。要进行重组质粒的提取与鉴定。之后进行重组质粒对细胞的转染。转染要按照如下的方式进行实验分组,实验分为四组,包括阳性重组质粒实验组;阴性重组质粒对照组;空白质粒对照组;空白溶剂对照组。实验分组的相应实验完成后,进行相关体外实验对各组STAT3相关蛋白表达的抑制情况进行检测。制备SDS-聚丙烯酰胺凝胶电泳,经过蛋白质的电转移,封闭,抗体的结合,显色照相等步骤完成免疫印迹实验;对转染细胞的总RNA进行提取,经RNA定量,cDNA的获取及逆转录反应,引物合成,聚合酶链式反应,PCR产物的检测等步骤对STAT3 mRNA的表达情况进行检测;利用流式细胞仪对实验细胞的凋亡情况进行检测;配置比色试剂,对细胞进行胰蛋白酶的酶解,进行显色、比色等步骤对细胞凋亡情况进行MTT法的相关检测。
(5)体内实验:将人Tca8113舌鳞癌细胞重悬培养后对裸鼠进行背部的接种,建立人舌鳞癌的裸鼠动物模型,待肿瘤体积增长到50mm3后模型建立成功。之后利用电转染的方法将psilencer1.0-U6-stat3-siRNA重组质粒及空白质粒、对照质粒转入肿瘤组织的癌细胞内。定期对肿瘤的生长情况进行检测,定期检测各组裸鼠肿瘤体积的增长速度并在实验结束后取下肿瘤,对肿瘤的重量进行测定。利用RT-PCR方法检测STAT3基因的mRNA表达情况,利用western blot方法检测STAT3及其相关基因MMP2、VEGF的蛋白表达情况,利用免疫组化方法检测STAT3及其相关基因BAX、BCL-2的表达情况。
结果:
(1)免疫组化研究结果显示:口腔鳞癌癌变过程中STAT3蛋白表达在正常黏膜组织、不典型增生组织及癌组织中的表达率依次增高,分别为13.3%(8/60)、30.0%(9/30)、71.2%(43/60)组间比较差异有统计学意义(P<0.05);而EGF蛋白在正常口腔黏膜组织、不典型增生组织及口腔鳞癌组织中的表达率分别为25.0%(15/60)、46.7%(14/30)、76.7%(46/60),组间比较差异有统计学意义(P<0.05);EGFR在正常黏膜组织、不典型增生组织及癌组织中的表达率分别为15.0%(9/60)、36.7%(11/30)、70.0%(42/60),组间比较差异有统计学意义(P<0.05),三者呈正相关关系。口腔鳞癌组织中STAT3蛋白表达与癌的组织学分级、浸润深度及淋巴结转移密切相关(P<0.05); EGF及EGFR蛋白表达均与癌的组织浸润深度及淋巴结转移密切相关(P<0.05)。
(2)经相关的酶切检定,检测出了psilencer1.0-U6-stat3-siRNA质粒构建成功。
(3)体外实验的实验结果:研究结果表明,实验组重组质粒对舌鳞癌细胞具备较强的促细胞凋亡和增殖抑制作用;Western blot的实验结果和逆转录PCR的实验结果均显示重组质粒对细胞的增殖产生抑制效果,对细胞的凋亡产生促进效果,为了验证重组质粒的促凋亡、抑制增殖的分子机制,特进行了MTT实验和流式细胞仪检测细胞周期实验。MTT实验的结果显示重组质粒可明显的抑制细胞的增殖,而流式细胞仪实验则显示重组质粒将细胞周期阻断在G0/G1分期,在正常2倍体峰之前出现了亚二倍体峰,具备较高的凋亡率。
(4)体内实验的实验结果:成功的构建出口腔鳞癌裸鼠皮下移植瘤动物模型,实验组动物的肿瘤体积增长速度明显小于其他对照组肿瘤体积的生长速度(p<0.05)。实验组动物肿瘤的最终体积要明显小于各对照组动物肿瘤的最终体积(P<0.05)。RT-PCR检测STAT3基因mRNA的表达情况显示实验组的STAT3 mRNA的表达水平要明显高于对照组(P<0.05),western blot方法检测STAT3及其相关基因MMP2、VEGF的蛋白表达情况显示实验组的蛋白表达量要明显的低于对照组(P<0.05),免疫组化方法检测STAT3及其相关基因BAX、BCL-2的表达情况显示实验组的相关蛋白表达量要明显低于对照组(P<0.05)。
结论:
(1)STAT3、EGF及EGFR在口腔鳞癌的浸润、转移及粘膜上皮癌变过程中起着重要作用,提示联合检测STAT3、EGF及EGFR可望成为口腔鳞癌早期诊断和判断预后的客观指标之一。
(2)涉及STAT3的siRNA基因阻断质粒,成功进行细胞转染后,可有效的抑制口腔鳞癌细胞中STAT3信号传导通路的激活和信息传递。信号传导通路的抑制又使肿瘤细胞的细胞周期被阻断,诱导口腔鳞癌细胞的体外凋亡。该结论提示可将STAT3 siRNA作为口腔鳞癌的治疗分子。
(3)体内实验的实验结果表明,成功的转染STAT3 siRNA质粒进入实验模型的肿瘤部位后可有效的抑制肿瘤体积的增长。对STAT3基因表达的抑制可致使该信号通路下游的MMP-2及VEGF分子的表达被抑制,这提示对STAT3信号通路抑制,可间接抑制MMP-2、VEGF等肿瘤相关基因的表达,进而抑制肿瘤的发生与发展。
As the most common malignant oral or perioral tumor, oral squamous cell cancer(OSCC) accounts for over 80% of the total incidence of oral tumors, making it a malignant disease deserving much attention. Despite its declining incidence in the developed world, OSCC incidence is on the raise globally. In recent years, although considerable progresses have been made in the treatment of a variety of malignant tumors, resulting in improved 5-year survivals, the treatment for OSCC has been lagged behind with particular difficulty in late-stage treatment due to the imperceptible features at early stages. Therefore greater attention should be paid to the treatment of OSCC in the effort to seek better treatment options.
Routine treatment options for OSCC include surgical therapy, radiotherapy, chemotherapy, and immunotherapy, etc., all of which have substantial shortcomings in spite of the certain efficacy achieved in inhibiting the genesis and development of tumors. For instance, radio and chemotherapies would inevitably result in serious side effects, while in surgical treatment, there are difficulties in practice and eradication of tumor is impossible,and immunotherapy showed no significant efficacy. Given this, gene therapy and molecular-targeted therapy, which has newly emerged for the treatment of tumors, have won the favor of the research field with less adverse reactions and accurate efficacy. RNA interference (RNAi) technology, one of the novel gene therapies for disease diagnosis and treatment, can effectively hinder disease progression by identifying and interfering with the pathogenic target genes. Recently it has drawn increasing attention in the treatment of tumors.
STATS,the coding gene of signal transduction activating factor 3, is often considered as the proto-oncogene. The downstream pathway of STAT3 signal transduction can affect the expression of many factors, including cyclinD1, Bcl-xl,surviving and c-myc, etc, that are related to the promotion of tumor progression. Besides, STAT3 plays a role in the immune escape process in tumors. Therefore STAT3 gene has been selected as the target to be subject to appropriate interference for effective gene therapy of tumors.
In this study, RNAi therapy, one of the gene therapies, was applied, using the tumor-related proto-oncogene STAT3 as the treatment target. RNAi plasmid specific for the STAT3 gene locus was designed and introduced to the OSCC cells. The influence of the RNA interference effect of this plasmid on OSCC progression was firstly observed in vitro, and then tested in vivo in animal models of OSCC, in order to verify the tumor inhibition effect of RNAi with STAT3 gene.
In this study, the treatment method for OSCC was explored from an entirely new perspective, in the hope to provide a new paradigm for the treatment of OSCC.
Objective:
To investigate the in-vitro inhibition of OSCC cells and in-vivo tumor inhibition effect in OSCC animal models by knocking out STAT3 gene using RNA interference technology.
Method:
(1)Immunohistochemical assay was using in detecting normal oral mucosa, atypical hyperplasia and patients with oral squamous cell carcinoma of STAT3, EGF and EGFR protein expression.
(2) The STAT3 gene interfering plasmid, i.e., psilencer1.0-U6-stat3-siRNA plasmid, was constructed.
(3)The expression levels of STATmRNA and the related proteins in OSCC cells were determined in different in-vitro and in-vivo study groups using downstream gene engineering technologies such as Western blot, RT-PCR, and IHC methods, so as to assess the tumor inhibiting effect of RNAi.
(4)In-vitro test:The siRNA template specific for STAT3 RNAi locus was designed and synthesized in vitro. The plasmid vector was then subject to enzyme cutting for linearization and collected. The samples then underwent quantification of nucleic acids. The linearized plasmid vector was connected to the interference fragment by T4 ligase. Subsequently the competent cells were prepared and co-incubated with the recombinant plasmids for transformation of competent cells by plasmids. Extraction and identification of recombinant plasmid were also required. Transfection of cells by the recombinant plasmid was then performed for the four test groups, namely, positive recombinant plasmid group, negative recombinant plasmid control group, blank plasmid control group, and blank vehicle control group. After completion of the tests for each group, the appropriate in-vitro tests were performed for each group to determined the inhibition on the expression of STAT3-related proteins. SDS-PGE was prepared for the immunoblotting test by the process of protein electrotransfer,blockage, binding with antibody, colorization, and photographing ,etc.; the expression of STAT3mRNA was determined by extracting total RNA of the transfected cells followed by RNA quantification, cDNA collection and reverse transcription reaction, primed synthesis,polymerase chain reaction, and determination of PCR products, etc; apoptosis of the tested cells were determined using flow cytometer; colorimetric reagent was prepared, cells were subject to trypsin enzymolysis, and cell apoptosis was tested as appropriated using MTT method by the process of colorization and color comparison, etc.
(5)In-vivo test:Athymic mice were inoculated with human Tca8113 tongue squamous cell resuspension culture to establish the animal model of human tongue squamous cell carcinoma in ahtymic mice. Modeling was completed when the tumor had grown to the size of 50mm3. Psilencer1.0-U6-stat3-siRNA recombinant plasmid, blank plasmid and control plasmid were then transferred into the tumor cells using electroblot. The status of tumor growth and the increase rate of tumor size were regularly monitored, and tumor tissues were collected at the end of test for weighing. mRNA expression of STAT3 gene was determined by RT-PCR method, the expression of proteins MMP2 and VEGF from STAT3 and the related genes was monitored by western blot method, and the expression of BAX and BCL-2 by IHC method.
Results:
(1) oral squamous cell carcinoma carcinogenesis STAT3 in protein expression in normal mucosa, dysplasia and carcinoma rates were higher, respectively, 13.3% (8 / 60), 30.0% (9 / 30), 71.2% (43/60) group had a significant difference (P <0.05), and EGF protein in normal oral mucosa, dysplasia and oral squamous cell carcinoma rates were 25.0% (15/60) , 46.7% (14/30), 76.7% (46/60), the group had a significant difference (P <0.05); EGFR protein in normal mucosa, dysplasia and carcinoma rate 15.0% (9 / 60), 36.7% (11/30), 70.0% (42/60), the group had a significant difference (P <0.05), the three were positively correlated. In oral squamous cell carcinoma of STAT3 protein expression and the histological grade cancer, the depth of invasion and lymph node metastasis (P <0.05); EGF expression and depth of cancer invasion and lymph node metastasis (P <0.05); EGFR protein expression and depth of cancer invasion and lymph node metastasis (P <0.05).
(2) Psilencer1.0-U6-stat3-siRNA plasmid was found to be successfully constructed as determined by enzyme cutting.
(3)The results of the in-vitro test suggest that recombinant plasmid exhibited relatively strong pro-apoptosis and proliferation inhibition effect on the tongue squamous carcinoma cells in the test group; the results of both Western blot test and reserve transcription PCR test suggest that recombinant plasmid produced inhibition on cell proliferation and promoted apoptosis. In order to verify the molecular mechanism of the pro-apoptosis and proliferation inhibition effect of recombinant plasmid, MTT test and cell cycle test by flow cytometer were performed. The results of MTT test suggest that the recombinant plasmid significantly inhibited cell proliferation, and flow cytometer test suggests that cell cycle was blocked by recombinant plasmid at the GO/G1 phase, with hypodiploid peak present before normal diploid peak. The apoptosis rate was high.
(4)The results of the in-vivo test suggest that animal model of subcutaneous implantation of OSCC was successfully constructed in athymic mice; the growth rate of tumor size was significantly lower in the test group than in all the control groups(p<0.05); the ultimate tumor size was significantly smaller in the test group than in all the control groups(P<0.05); mRNA expression levels of STAT3 gene determined by RT-PCR suggest that STAT3mRNA expression level was significantly higher in the test group than in the control groups(P<0.05); expression of proteins MMP2 and VEGF from STAT3 and the related genes monitored by western blot method suggests that protein expression level was significantly lower in the test group than in the control groups (P<0.05); and expression of BAX and BCL-2 from STAT3 and the related genes monitored by IHC method suggests that expression level of the related proteins was significantly lower in the test group than in the control groups(P<0.05).
Conclusions:
(1)STAT3, EGF and EGFR in oral squamous cell carcinoma invasion, metastasis and mucosal epithelial carcinogenesis process plays an important role, suggesting that the detection of STAT3, EGF and EGFR is expected to become oral squamous cell carcinoma early diagnosis and prognosis of the objective indicators .
(2)After successful cell transfection, siRNA gene blocking plasmid involving STAT3 effectively inhibited the activation of and information transfer by STAT3 signal transduction pathway, which further resulted in blocking of tumor cell cycle, inducing in-vitro apoptosis of OSCC cells. This indicates that STAT3siRN may be used as a therapeutic molecule for OSCC.
(3)The results of the in-vivo test suggest that successful transfection of STAT3 siRNA plasmid into the tumor sites of the test models effectively inhibited the increase of tumor size. Inhibition of STAT3 expression inhibited the expression of MMP-2 and VEGF at the downstream of the signal pathway, indicating that inhibition of STAT3 signal pathway could indirectly inhibit the expression of the tumor-related genes of MMP-2 and VEGF, etc., thus inhibiting the genesis and development of tumor.
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