胶原在胃癌发病中的作用及机制探讨
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摘要
胃癌是全球高发的恶性肿瘤之一,其相关疾病死亡率仅次于肺癌,在全球范围内排名第二位。胃癌预后直接取决于疾病发现的时期。局限于粘膜层或粘膜下层的早期胃癌是唯一可以治愈的胃癌,早期发现并手术切除其5年生存率往往超过90%。但是,胃癌早期缺乏特异性的症状,大多数患者就诊时已到晚期,失去最佳手术时机,常因广泛的侵袭转移或复发而死亡。而以现有的诊断手段,一般早期胃癌的检出率仅15%-20%,远远不能满足临床需要。
胃癌区别于正常组织的特征就是其细胞的异型性以及组织浸润性。然而,细胞形态学的异型性可见于胃炎性组织修复、胃粘膜肠上皮化生和异型增生或瘤样变,唯有见到异型细胞的组织浸润才能确认为癌。因此单纯通过病理组织形态学水平早期诊断胃癌变得较为困难,常常难以确认,这也成为早期胃癌检出率低的重要原因之一。按照慢性胃炎-萎缩性胃炎-肠上皮化生-不典型增生-胃癌的胃癌变假说来推理,如果对主要的胃癌前病变实施动态监测,从寻找肿瘤浸润的特征性生物学行为着手,研究癌前病变癌变的分子生物学标志物,查找异型细胞浸润组织的分子证据,就能够准确地判别癌前病灶癌变,在病理形态学改变前甄别出早期胃癌,达到早期诊断胃癌目的,为早期治疗和治愈胃癌奠定坚实的基础。
基于肿瘤分子水平的诊断及其机制的研究逐渐成为热点,并已经显示出肿瘤早期诊断及分子干预特别优势。例如,利用芯片技术建立了胃癌早期诊断的基因预警系统和采用小RNA干扰技术抑制癌变。我们以前的相关基因芯片筛查结果显示,胶原系列的基因表达在胃癌中存在明显的异常表达。本课题作为深入研究,将基因芯片筛查得到的胶原系列基因进行进一步的定量Real-time PCR技术检测,从中筛选可能用于胃癌早期诊断的胶原标记,建立胃癌前病变癌变判别和胃癌早期分子诊断的模型,同时采用现代细胞与分子生物学技术,通过体外胃癌细胞模型研究胃癌中高表达I型间质胶原在胃癌发病中的作用机制,应用RNA干扰技术对胃癌细胞I型胶原实施干预试验,研究COL1A1基因对细胞生物学行为的影响及可能机制,为胃癌早期诊断和分子治疗提供科学的依据。
一材料和方法：
1收集手术确诊的胃癌患者,取同一个体不同区域胃粘膜组织样本(癌组织,癌旁癌前病灶及癌切除远端近似正常的胃组织),通过定量Real-time PCR分三阶段检测8个胶原基因(COL1A1, COL1A2, COL3A1, COL4A6, COL6A3, COL8A1, COL10A1, COL11A1) mRNA在这些组织中的表达情况。使用单因素方差分析或非参数检验逐一分析各个胶原基因在胃癌,癌前病灶及相对正常组织中的组间表达差异。将有判癌价值的胶原系列基因在相对正常,癌前病灶,早期胃癌及进展期胃癌不同个体同类样本中的表达情况进行Binary Logistic回归分析(癌与非癌),采用Forward:conditional法,作胃癌与非癌判别,以建立胃癌早期诊断的分子生物信息学判别公式。进一步将判别公式在不同个体、不同类别样本中进行验证并作修正,以获得高敏感性、高特异性的判癌公式。
2通过体外细胞模型,探讨经PCR验证在胃癌组织中明显高表达的I型胶原作为细胞生长的微环境,对胃癌细胞生物学行为(形态学,细胞骨架,迁徙及增殖)的影响,通过检测相关通路蛋白及基因的改变探讨其可能的作用机制。
3使用COL1A1-shRNA表达载体转染BGC-823胃癌细胞株,干扰癌细胞I型胶原主要基因COL1A1的表达,通过MTT及Transwell检测研究COL1A1RNA干扰对胃癌细胞增殖及迁移的影响。
二主要结果：
1①第一阶段收集22个胃癌手术患者男性19例,女性3例,包括肿瘤部位,癌前病变部位及远端正常部位组织的同个体配对病理样本66个。8个胶原基因定量Real-time PCR结果发现：与癌旁癌前病变组织配对的样本比较,除COL10A1外,肿瘤组织中有6个胶原基因(COL1A1, COL1A2, COL3A1, COL6A3, COL8A1, COL11A1)表达明显上调(P<0.05)。一个胶原基因(COL4A6)表达下调(P<0.05)。
②第二阶段收集33个不同疾病状态患者包括癌前病变11例,早期胃癌11例,进展期胃癌11例不同个体的共33个配对病理样本入组。各组均为男性8例,女性3例。7个基因定量Real-time PCR结果发现：与癌前病变组相比,COL11A1在胃癌中表达明显上调,尤其在早期胃癌中更明显(P<0.05),而COL4A6在胃癌中的表达则明显下调(P<0.05)。根据系列胶原基因在胃癌及癌前期病变组织中的相对表达值,采用Binary Logistic回归分析,作癌与非癌判别,结果有3个胶原基因(COL11A1,COL8A1,COL4A6)进入回归方程,初步建立了基于胶原系列基因PCR表达水平的胃癌早期诊断的分子生物信息学判别公式。
③第三阶段共收集106个不同患者,男性61例,女性45例,采取不同类别的样本共106个,包括胃镜检查基本正常的20例,癌前病变28例,胃癌58例。4个胶原基因在胃癌及癌前病灶组织表达存在统计学差异,其中COL1A1,1A2,11A1在胃癌组织高表达(P<0.05),而COL4A6则低表达(P<0.05)。根据系列胶原基因在胃癌及癌前期病变组织中的相对表达值,对第二阶段提出的公式进行验证,再次经过Binary logistic回归分析修正了基于胶原系列基因PCR表达水平的胃癌癌变预测公式,修正后公式的敏感性和特异性分别达91.4%和91.7%。
2①体外细胞实验发现,常规培养三株不同分化程度的胃癌细胞株,在胶原刺激后细胞变形,胞体伸展,表面突起增多,呈现明显增多的丝状伪足和片状伪足,细胞内微丝明显拉长增粗。
②Transwell细胞迁移试验表明,在外膜涂有I型胶原的小室内的胃癌细胞通过膜的细胞较未涂胶原组明显增多,提示I型胶原可以明显提高胃癌细胞的迁移能力,这与胶原刺激后细胞形态学改变相一致。
③相关通路研究提示：胶原刺激主要使E-cadherin/catenin粘附复合体减少,可溶性的E-cadherin,β-catenin也有轻度下降。提示胶原促进E-cadherin/catenin粘附复合体解聚,破坏细胞间的粘附连接,这些结果可能与FAK磷酸化,β-catenin酪氨酸磷酸化及与β-catenin结合的PTEN下调有关。
④MTT试验表明：胃癌细胞在铺有胶原的平板内生长速度明显高于未铺胶原组,提示胶原可促进胃癌细胞增殖。胶原刺激不仅使胃癌细胞P-catenin酪氨酸磷酸化,E-cadherin/catenin复合体解聚,而且使细胞核内β-catenin表达上调,促使其核转位。且3株分化程度不同的细胞株在胶原刺激后,cyclinD1表达均明显上调,尤其是NCI-N87上调最明显,达对照组的3倍。
3①我们将已构建并经测序鉴定的COL1A1-shRNA质粒载体进行细胞转染,G418筛选,获得3个稳定转染COL1A1-shRNA细胞(COL1A1-shRNA-1, COL1A1-shRNA-2, COL1A1-shRNA-3)及阴性载体对照细胞(Negative control)。转染后见部分细胞边缘圆,细胞核浓缩,细胞中颗粒增多,细胞碎片增多,细胞增殖减慢且易脱落。
②Real-time PCR结果示相对于Blank control组,COL1A1-shRNA-1, COL1A1-shRNA-2, COL1A1-shRNA-3转染组细胞COL1A1 mRNA的表达明显下调,尤以COL1A1-shRNA-1转染组效果明显,被抑制了8±0.2倍(P<0.01)。
③MTT试验表明：COL1A1-shRNA-1转染组细胞增殖减慢,在各实验点其A值均明显低于Negative control及Blank control (P< 0.05).
④Transwell迁移试验表明：COL1A1-shRNA-1转染组细胞明显少于Negative control及Blank control,其细胞数量减少超过3倍以上(P<0.05),提示COLlAl干扰可明显抑制胃癌BGC-823细胞迁移。
三结论：
1发现2个胶原基因(COL4A6, COL11A1)可能作为胃癌前病变早期癌变监测指标,COL11A1可能是更为敏感的监测指标。
2初步建立起了基于胶原系列基因表达水平的胃癌前期病灶癌变判别公式,对临床早期胃癌的生物学诊断提出新的诊断模式。
3Ⅰ型胶原可诱导胃癌细胞骨架重组,促进细胞增殖及迁移,主要与β-catenin酪氨酸磷酸化及核转位有关。
4成功建立和筛选体外稳定转染COL1A1-shRNA的胃癌BGC-823细胞株,COL1A1-shRNA转染可抑制胃癌细胞增殖迁移,有望成为胃癌治疗新的基因靶标。
Gastric cancer is a common malignant tumor worldwide. It remains the second most common cause of cancer death. Early detection and resection improve the long-term survival, as gastric cancer could be more curable if diagnosed at earlier stage. The prognosis of advanced cancer remains poor due to high recurrence and metastasis. Most patients are asymptomatic in the early stages of GC. With current histopathological diagnostic approaches, only about 15% to 20% of GC patients are eligibly detected in early stages. With no reliable marker or diagnostic model available for the early diagnosis of GC, it is imperative to identify and develop new biomarkers and/or diagnostic model to improve the early detection of GC.
The malignancy is evidenced by the presence of the morphological atypia and biological invasive behavior of the cell. Cell atypia is also observed in the inflammation, premalignant lesions, such as metaplasia or dysplasia gastric epithelium, which is one of the contributions to low diagnostic rate of early gastric cancer (EGC). It is widely accepted that atrophy-metaplasia-dysplasia-carcinoma is sequential development of GC carcinogenesis. Atrophic gastritis, intestinal metaplasia and/or dysplasia are believed to be precancerous or predisposing conditions for GC and the majority of GC results from precancerous lesions. The traditional histopathological classification systems of GC, on the basis of cell morphological atypia, are difficult to efficiently predict the risk of sequential development from precancerous lesions. From molecular mechanism of cancer cell invasion, we can search for more molecular information so as to earlier discriminate EGC from premalignant lesions than traditional histopathological level.
Molecular mechanisms of neoplastic transformation have been under the thorough investigation. Novel techniques and new genes involved into the gastric cancer development have been indentified and showed promising prospect. For example, Cui and his colleagues developed a microarray-based prewarning system for early detection of gastric cancer and precancerous lesions. Yu found several novel genes associated with early gastric carcinogenesis and development by microarray analysis. Our previous microarray analysis has shown the aberrant expression profile of collagens in GC, suggesting that collagens have a great potential to be used as a diagnostic and prognostic marker in GC and maybe become biomarkers of atypical cell invasion. To identify potential biomarkers or diagnostic model for the early detection of GC, we compared the expression of collagen genes from different group patients'gastric tissues including normal group (chronic superficial gastritis), premalignant lesions, EGC and advanced gastric cancer (AGC). In addition, we investigated the effects of collagen type I that upregulated in gastric cancer tissues on gastric carcinoma cell lines as tumor microenvironment and by siRNA technology to silence COL1A1 gene.
Material and Methods
1 To identify potential biomarkers or diagnostic model for the early detection of GC, we compared the expression levels of collagen genes among different group patients' gastric tissues including normal group (chronic superficial gastritis), premalignant lesions, EGC and advanced gastric cancer (AGC) by three sequential phase. Gastric tissues samples were obtained from 106 patients who underwent gastrectomy or gastroscopy. Eight collagen genes were further determined by our microarray analysis and related literature. The expression levels of eight genes in clinical tissue samples of stomach were detected by real-time quantitative reverse transcription-PCR (qRT-PCR). The significance of eight collagens mRNA levels among different groups (phaseⅠ:normal, premalignant and GC; phaseⅡ:Premalignant, EGC and AGC; phaseⅢ:normal, premalignant and GC) was determined by ANOVA or Kruskal-Wallis Test where appropriate. Binary logistic regression analysis was performed to predict the probability that a sample was from a GC patient (disease) or not (control), and to propose the possible cancerous predictive formula for early diagnosis of gastric cancer.
2 To investigate the role of collagen typeⅠon invasion and metastasis of gastric cancer, we emplyed three gastric cancer cell lines originated from various differentiation stages to explore the effects of collagen typeⅠon gastric cancer cell behavior including cell morphology, cytoskeleton organization, migration and proliferation as tumor microenvironment.In addition, the possible molecular mechanism was explored by detecting the expression levels of related signal protein and gene.
3 COL1A1 in BGC-823 cell lines was knocked down by small interference RNA. MTT assay and Transwell migration assay were performed to investigate the effects of COL1A1 silencing on cell proliferation and migration.
Results
1①PhaseⅠ:66 matched tissue samples from different regions of resected stomach of 22 surgery patients (22 malignant lesions,22 premalignant lesions and 22 normal tissues) were chosen for qRT-PCR analysis by 8 collagen genes. Seven genes were statistically significantly different between pair-matched malignant and premalignant groups (P<0.05). Six of these genes (COL1A1, COL1A2, COL3A1, COL6A3, COL8A1 and COL11A1) were significantly overexpressed and only one (COL4A6) were under-expressed in GC patients as compared with premalignant controls.
②PhaseⅡ:33 samples from different patients (11 premalignant lesions,11 EGC and 11 AGC) were chosen for further qRT-PCR analysis by 7 collagen genes. COL11A1 was up-regulated in GC patients than those premalignant group control, especially up-regulated in EGC patients(P< 0.05); COL4A6 were significantly down-regulated in GC patients than those premalignant group control (P< 0.05). Binary logistic regression analysis resulted in a predictive formula with COL11A1, COL8A1 and COL4A6 to be the best grouping as variables that predicted GC with an overall correct classification of 100%.
③PhaseⅢ:106 from different patients (20 normal tissues,28 premalignant lesions, 58 GC) were chosen for further qRT-PCR analysis by 7 collagen genes. Four genes were statistically significantly different between premalignant group and GC groups (P＜0.05). Three of these genes (COL1A1, COL1A2 and COL11A1) were overexpressed and COL4A6 was under-expressed in GC patients. On the basis of the relative expression values of collagen genes in the tissues of GC patients and premalignant lesions, the model from phase II was validated and Binary logistic regression analysis resulted in a new predictive formula with COL1A1, COL1A2, COL3A1, COL4A6, COL6A3 and COL11A1 to be the best grouping as variables that predicted GC with an overall correct classification of 91.5%.
2①Gastric carcinoma cell lines BGC-823 and SGC-7901 showed apparent changes in cell morphology after 24 h collagen type I stimulation. Cells cultured on collagen type I exhibited elongated or enlarged shape with a higher cytoplasm/nucleus ratio as compared with the cells directly cultured on dishes. They groveled on the collagen type I substrate with spreading of pseudopodia and a loss of cell-cell contacts typically observed in mesenchymal cells. Immunofluorescence microscopy demonstrated that cells grown on collagen type I were scattered with a loss of cell-cell contacts but more lamellipodia and filopodia, characteristic of a great motility.
②Transwell migration assays showed that BGC-823 and SGC-7901 cells showed eightfold increase in cell migration towards the collagen type I-coated outer-side porous membranes compared with uncoated ones, which indicated collagen type I induced cell migration in the similar way as chemotactic factors, corresponding to cell morphology change and cytoskeleton reorganization.
③Related signal pathway study showed that the E-cadherin/catenin adhesion complex associated with the actin cytoskeleton were reduced after collagen type I stimulation, Whereas, the amount of E-cadherin remained constant in NCI-N87 and BGC-823 cells after collagen type I stimulation; the amount ofβ-catenin was slightly reduced in BGC-823 cells or remained constant in NCI-N87 cells after 2 days of collagen type I treatment. These results suggested that collagen type I treatment causes disruption of the E-cadherin/catenin adhesion complex and dissociation from the actin cytoskeleton in gastric carcinoma cells, subsequently resulted in a decrease in cell-cell adhesion. These results maybe associated with the phosphorylation of FAK, tyrosine phosphorylation ofβ-catenin and the dissociation of endogenous PTEN andβ-catenin.
④MTT assays showed the proliferation rates of BGC-823 cells and SGC-7901 cells grown on collagen type I were significantly increased compared with the controls on dishes, which was related withβ-catenin nuclear translocation and the activation ofβ-catenin-LEF/TCF pathway.
3①Three recombinant plasmids targeting COL1A1 using pSilencerTM 4.1-CMV neo siRNA expression vector were constructed successfully and were transfected into gastric cancer BGC-823 cells.
②The expression of COL1A1 mRNA and protein in BGC-823 cells was significantly inhibited by small interfering RNAs (siRNA) transfectants, especially by COL1A1-shRNA-1 transfectant.
③MTT assays showed that the proliferation rate of BGC-823 cells with COL1A1-shRNA-1 trasfected was significantly decreased compared with the parental cells and the negative control cells.
④Transwell migration assays showed that the migration capacity of BGC-823 cells with COL1A1-shRNA-1 trasfected was decreased compared with the parental cells and the negative control cells.
Conclusions
1 COL4A6 and COL11A1 can be potential mRNA markers for early gastric cancer, and COL11A1 was more sensitive than COL4A6.
2 The possible cancerous predictive formula was developed and showed remarkerble discriminatory power for the diagnosis of early gastric cancer. This provided new biological diagnose model for clinical diagnosis of early gastric cancer.
3 Collagen type I not only induced gastric carcinoma cells scattering and cytoskeleton remodeling, but also prompted cell migration and proliferation,which was related to tyrosine phosphorylation and nuclear translocation ofβ-catenin.
4 Gastric cancer BGC-823 cell transfected with COL1A1-shRNA was successfully established, whose COL1A1 expression was clear decreased and resulted in a clear reduction of cell proliferation and migration. This maybe provided potent therapy target for gastric cancer.
胃癌区别于正常组织的特征就是其细胞的异型性以及组织浸润性。然而,细胞形态学的异型性可见于胃炎性组织修复、胃粘膜肠上皮化生和异型增生或瘤样变,唯有见到异型细胞的组织浸润才能确认为癌。因此单纯通过病理组织形态学水平早期诊断胃癌变得较为困难,常常难以确认,这也成为早期胃癌检出率低的重要原因之一。按照慢性胃炎-萎缩性胃炎-肠上皮化生-不典型增生-胃癌的胃癌变假说来推理,如果对主要的胃癌前病变实施动态监测,从寻找肿瘤浸润的特征性生物学行为着手,研究癌前病变癌变的分子生物学标志物,查找异型细胞浸润组织的分子证据,就能够准确地判别癌前病灶癌变,在病理形态学改变前甄别出早期胃癌,达到早期诊断胃癌目的,为早期治疗和治愈胃癌奠定坚实的基础。
基于肿瘤分子水平的诊断及其机制的研究逐渐成为热点,并已经显示出肿瘤早期诊断及分子干预特别优势。例如,利用芯片技术建立了胃癌早期诊断的基因预警系统和采用小RNA干扰技术抑制癌变。我们以前的相关基因芯片筛查结果显示,胶原系列的基因表达在胃癌中存在明显的异常表达。本课题作为深入研究,将基因芯片筛查得到的胶原系列基因进行进一步的定量Real-time PCR技术检测,从中筛选可能用于胃癌早期诊断的胶原标记,建立胃癌前病变癌变判别和胃癌早期分子诊断的模型,同时采用现代细胞与分子生物学技术,通过体外胃癌细胞模型研究胃癌中高表达I型间质胶原在胃癌发病中的作用机制,应用RNA干扰技术对胃癌细胞I型胶原实施干预试验,研究COL1A1基因对细胞生物学行为的影响及可能机制,为胃癌早期诊断和分子治疗提供科学的依据。
一材料和方法：
1收集手术确诊的胃癌患者,取同一个体不同区域胃粘膜组织样本(癌组织,癌旁癌前病灶及癌切除远端近似正常的胃组织),通过定量Real-time PCR分三阶段检测8个胶原基因(COL1A1, COL1A2, COL3A1, COL4A6, COL6A3, COL8A1, COL10A1, COL11A1) mRNA在这些组织中的表达情况。使用单因素方差分析或非参数检验逐一分析各个胶原基因在胃癌,癌前病灶及相对正常组织中的组间表达差异。将有判癌价值的胶原系列基因在相对正常,癌前病灶,早期胃癌及进展期胃癌不同个体同类样本中的表达情况进行Binary Logistic回归分析(癌与非癌),采用Forward:conditional法,作胃癌与非癌判别,以建立胃癌早期诊断的分子生物信息学判别公式。进一步将判别公式在不同个体、不同类别样本中进行验证并作修正,以获得高敏感性、高特异性的判癌公式。
2通过体外细胞模型,探讨经PCR验证在胃癌组织中明显高表达的I型胶原作为细胞生长的微环境,对胃癌细胞生物学行为(形态学,细胞骨架,迁徙及增殖)的影响,通过检测相关通路蛋白及基因的改变探讨其可能的作用机制。
3使用COL1A1-shRNA表达载体转染BGC-823胃癌细胞株,干扰癌细胞I型胶原主要基因COL1A1的表达,通过MTT及Transwell检测研究COL1A1RNA干扰对胃癌细胞增殖及迁移的影响。
二主要结果：
1①第一阶段收集22个胃癌手术患者男性19例,女性3例,包括肿瘤部位,癌前病变部位及远端正常部位组织的同个体配对病理样本66个。8个胶原基因定量Real-time PCR结果发现：与癌旁癌前病变组织配对的样本比较,除COL10A1外,肿瘤组织中有6个胶原基因(COL1A1, COL1A2, COL3A1, COL6A3, COL8A1, COL11A1)表达明显上调(P<0.05)。一个胶原基因(COL4A6)表达下调(P<0.05)。
②第二阶段收集33个不同疾病状态患者包括癌前病变11例,早期胃癌11例,进展期胃癌11例不同个体的共33个配对病理样本入组。各组均为男性8例,女性3例。7个基因定量Real-time PCR结果发现：与癌前病变组相比,COL11A1在胃癌中表达明显上调,尤其在早期胃癌中更明显(P<0.05),而COL4A6在胃癌中的表达则明显下调(P<0.05)。根据系列胶原基因在胃癌及癌前期病变组织中的相对表达值,采用Binary Logistic回归分析,作癌与非癌判别,结果有3个胶原基因(COL11A1,COL8A1,COL4A6)进入回归方程,初步建立了基于胶原系列基因PCR表达水平的胃癌早期诊断的分子生物信息学判别公式。
③第三阶段共收集106个不同患者,男性61例,女性45例,采取不同类别的样本共106个,包括胃镜检查基本正常的20例,癌前病变28例,胃癌58例。4个胶原基因在胃癌及癌前病灶组织表达存在统计学差异,其中COL1A1,1A2,11A1在胃癌组织高表达(P<0.05),而COL4A6则低表达(P<0.05)。根据系列胶原基因在胃癌及癌前期病变组织中的相对表达值,对第二阶段提出的公式进行验证,再次经过Binary logistic回归分析修正了基于胶原系列基因PCR表达水平的胃癌癌变预测公式,修正后公式的敏感性和特异性分别达91.4%和91.7%。
2①体外细胞实验发现,常规培养三株不同分化程度的胃癌细胞株,在胶原刺激后细胞变形,胞体伸展,表面突起增多,呈现明显增多的丝状伪足和片状伪足,细胞内微丝明显拉长增粗。
②Transwell细胞迁移试验表明,在外膜涂有I型胶原的小室内的胃癌细胞通过膜的细胞较未涂胶原组明显增多,提示I型胶原可以明显提高胃癌细胞的迁移能力,这与胶原刺激后细胞形态学改变相一致。
③相关通路研究提示：胶原刺激主要使E-cadherin/catenin粘附复合体减少,可溶性的E-cadherin,β-catenin也有轻度下降。提示胶原促进E-cadherin/catenin粘附复合体解聚,破坏细胞间的粘附连接,这些结果可能与FAK磷酸化,β-catenin酪氨酸磷酸化及与β-catenin结合的PTEN下调有关。
④MTT试验表明：胃癌细胞在铺有胶原的平板内生长速度明显高于未铺胶原组,提示胶原可促进胃癌细胞增殖。胶原刺激不仅使胃癌细胞P-catenin酪氨酸磷酸化,E-cadherin/catenin复合体解聚,而且使细胞核内β-catenin表达上调,促使其核转位。且3株分化程度不同的细胞株在胶原刺激后,cyclinD1表达均明显上调,尤其是NCI-N87上调最明显,达对照组的3倍。
3①我们将已构建并经测序鉴定的COL1A1-shRNA质粒载体进行细胞转染,G418筛选,获得3个稳定转染COL1A1-shRNA细胞(COL1A1-shRNA-1, COL1A1-shRNA-2, COL1A1-shRNA-3)及阴性载体对照细胞(Negative control)。转染后见部分细胞边缘圆,细胞核浓缩,细胞中颗粒增多,细胞碎片增多,细胞增殖减慢且易脱落。
②Real-time PCR结果示相对于Blank control组,COL1A1-shRNA-1, COL1A1-shRNA-2, COL1A1-shRNA-3转染组细胞COL1A1 mRNA的表达明显下调,尤以COL1A1-shRNA-1转染组效果明显,被抑制了8±0.2倍(P<0.01)。
③MTT试验表明：COL1A1-shRNA-1转染组细胞增殖减慢,在各实验点其A值均明显低于Negative control及Blank control (P< 0.05).
④Transwell迁移试验表明：COL1A1-shRNA-1转染组细胞明显少于Negative control及Blank control,其细胞数量减少超过3倍以上(P<0.05),提示COLlAl干扰可明显抑制胃癌BGC-823细胞迁移。
三结论：
1发现2个胶原基因(COL4A6, COL11A1)可能作为胃癌前病变早期癌变监测指标,COL11A1可能是更为敏感的监测指标。
2初步建立起了基于胶原系列基因表达水平的胃癌前期病灶癌变判别公式,对临床早期胃癌的生物学诊断提出新的诊断模式。
3Ⅰ型胶原可诱导胃癌细胞骨架重组,促进细胞增殖及迁移,主要与β-catenin酪氨酸磷酸化及核转位有关。
4成功建立和筛选体外稳定转染COL1A1-shRNA的胃癌BGC-823细胞株,COL1A1-shRNA转染可抑制胃癌细胞增殖迁移,有望成为胃癌治疗新的基因靶标。
Gastric cancer is a common malignant tumor worldwide. It remains the second most common cause of cancer death. Early detection and resection improve the long-term survival, as gastric cancer could be more curable if diagnosed at earlier stage. The prognosis of advanced cancer remains poor due to high recurrence and metastasis. Most patients are asymptomatic in the early stages of GC. With current histopathological diagnostic approaches, only about 15% to 20% of GC patients are eligibly detected in early stages. With no reliable marker or diagnostic model available for the early diagnosis of GC, it is imperative to identify and develop new biomarkers and/or diagnostic model to improve the early detection of GC.
The malignancy is evidenced by the presence of the morphological atypia and biological invasive behavior of the cell. Cell atypia is also observed in the inflammation, premalignant lesions, such as metaplasia or dysplasia gastric epithelium, which is one of the contributions to low diagnostic rate of early gastric cancer (EGC). It is widely accepted that atrophy-metaplasia-dysplasia-carcinoma is sequential development of GC carcinogenesis. Atrophic gastritis, intestinal metaplasia and/or dysplasia are believed to be precancerous or predisposing conditions for GC and the majority of GC results from precancerous lesions. The traditional histopathological classification systems of GC, on the basis of cell morphological atypia, are difficult to efficiently predict the risk of sequential development from precancerous lesions. From molecular mechanism of cancer cell invasion, we can search for more molecular information so as to earlier discriminate EGC from premalignant lesions than traditional histopathological level.
Molecular mechanisms of neoplastic transformation have been under the thorough investigation. Novel techniques and new genes involved into the gastric cancer development have been indentified and showed promising prospect. For example, Cui and his colleagues developed a microarray-based prewarning system for early detection of gastric cancer and precancerous lesions. Yu found several novel genes associated with early gastric carcinogenesis and development by microarray analysis. Our previous microarray analysis has shown the aberrant expression profile of collagens in GC, suggesting that collagens have a great potential to be used as a diagnostic and prognostic marker in GC and maybe become biomarkers of atypical cell invasion. To identify potential biomarkers or diagnostic model for the early detection of GC, we compared the expression of collagen genes from different group patients'gastric tissues including normal group (chronic superficial gastritis), premalignant lesions, EGC and advanced gastric cancer (AGC). In addition, we investigated the effects of collagen type I that upregulated in gastric cancer tissues on gastric carcinoma cell lines as tumor microenvironment and by siRNA technology to silence COL1A1 gene.
Material and Methods
1 To identify potential biomarkers or diagnostic model for the early detection of GC, we compared the expression levels of collagen genes among different group patients' gastric tissues including normal group (chronic superficial gastritis), premalignant lesions, EGC and advanced gastric cancer (AGC) by three sequential phase. Gastric tissues samples were obtained from 106 patients who underwent gastrectomy or gastroscopy. Eight collagen genes were further determined by our microarray analysis and related literature. The expression levels of eight genes in clinical tissue samples of stomach were detected by real-time quantitative reverse transcription-PCR (qRT-PCR). The significance of eight collagens mRNA levels among different groups (phaseⅠ:normal, premalignant and GC; phaseⅡ:Premalignant, EGC and AGC; phaseⅢ:normal, premalignant and GC) was determined by ANOVA or Kruskal-Wallis Test where appropriate. Binary logistic regression analysis was performed to predict the probability that a sample was from a GC patient (disease) or not (control), and to propose the possible cancerous predictive formula for early diagnosis of gastric cancer.
2 To investigate the role of collagen typeⅠon invasion and metastasis of gastric cancer, we emplyed three gastric cancer cell lines originated from various differentiation stages to explore the effects of collagen typeⅠon gastric cancer cell behavior including cell morphology, cytoskeleton organization, migration and proliferation as tumor microenvironment.In addition, the possible molecular mechanism was explored by detecting the expression levels of related signal protein and gene.
3 COL1A1 in BGC-823 cell lines was knocked down by small interference RNA. MTT assay and Transwell migration assay were performed to investigate the effects of COL1A1 silencing on cell proliferation and migration.
Results
1①PhaseⅠ:66 matched tissue samples from different regions of resected stomach of 22 surgery patients (22 malignant lesions,22 premalignant lesions and 22 normal tissues) were chosen for qRT-PCR analysis by 8 collagen genes. Seven genes were statistically significantly different between pair-matched malignant and premalignant groups (P<0.05). Six of these genes (COL1A1, COL1A2, COL3A1, COL6A3, COL8A1 and COL11A1) were significantly overexpressed and only one (COL4A6) were under-expressed in GC patients as compared with premalignant controls.
②PhaseⅡ:33 samples from different patients (11 premalignant lesions,11 EGC and 11 AGC) were chosen for further qRT-PCR analysis by 7 collagen genes. COL11A1 was up-regulated in GC patients than those premalignant group control, especially up-regulated in EGC patients(P< 0.05); COL4A6 were significantly down-regulated in GC patients than those premalignant group control (P< 0.05). Binary logistic regression analysis resulted in a predictive formula with COL11A1, COL8A1 and COL4A6 to be the best grouping as variables that predicted GC with an overall correct classification of 100%.
③PhaseⅢ:106 from different patients (20 normal tissues,28 premalignant lesions, 58 GC) were chosen for further qRT-PCR analysis by 7 collagen genes. Four genes were statistically significantly different between premalignant group and GC groups (P＜0.05). Three of these genes (COL1A1, COL1A2 and COL11A1) were overexpressed and COL4A6 was under-expressed in GC patients. On the basis of the relative expression values of collagen genes in the tissues of GC patients and premalignant lesions, the model from phase II was validated and Binary logistic regression analysis resulted in a new predictive formula with COL1A1, COL1A2, COL3A1, COL4A6, COL6A3 and COL11A1 to be the best grouping as variables that predicted GC with an overall correct classification of 91.5%.
2①Gastric carcinoma cell lines BGC-823 and SGC-7901 showed apparent changes in cell morphology after 24 h collagen type I stimulation. Cells cultured on collagen type I exhibited elongated or enlarged shape with a higher cytoplasm/nucleus ratio as compared with the cells directly cultured on dishes. They groveled on the collagen type I substrate with spreading of pseudopodia and a loss of cell-cell contacts typically observed in mesenchymal cells. Immunofluorescence microscopy demonstrated that cells grown on collagen type I were scattered with a loss of cell-cell contacts but more lamellipodia and filopodia, characteristic of a great motility.
②Transwell migration assays showed that BGC-823 and SGC-7901 cells showed eightfold increase in cell migration towards the collagen type I-coated outer-side porous membranes compared with uncoated ones, which indicated collagen type I induced cell migration in the similar way as chemotactic factors, corresponding to cell morphology change and cytoskeleton reorganization.
③Related signal pathway study showed that the E-cadherin/catenin adhesion complex associated with the actin cytoskeleton were reduced after collagen type I stimulation, Whereas, the amount of E-cadherin remained constant in NCI-N87 and BGC-823 cells after collagen type I stimulation; the amount ofβ-catenin was slightly reduced in BGC-823 cells or remained constant in NCI-N87 cells after 2 days of collagen type I treatment. These results suggested that collagen type I treatment causes disruption of the E-cadherin/catenin adhesion complex and dissociation from the actin cytoskeleton in gastric carcinoma cells, subsequently resulted in a decrease in cell-cell adhesion. These results maybe associated with the phosphorylation of FAK, tyrosine phosphorylation ofβ-catenin and the dissociation of endogenous PTEN andβ-catenin.
④MTT assays showed the proliferation rates of BGC-823 cells and SGC-7901 cells grown on collagen type I were significantly increased compared with the controls on dishes, which was related withβ-catenin nuclear translocation and the activation ofβ-catenin-LEF/TCF pathway.
3①Three recombinant plasmids targeting COL1A1 using pSilencerTM 4.1-CMV neo siRNA expression vector were constructed successfully and were transfected into gastric cancer BGC-823 cells.
②The expression of COL1A1 mRNA and protein in BGC-823 cells was significantly inhibited by small interfering RNAs (siRNA) transfectants, especially by COL1A1-shRNA-1 transfectant.
③MTT assays showed that the proliferation rate of BGC-823 cells with COL1A1-shRNA-1 trasfected was significantly decreased compared with the parental cells and the negative control cells.
④Transwell migration assays showed that the migration capacity of BGC-823 cells with COL1A1-shRNA-1 trasfected was decreased compared with the parental cells and the negative control cells.
Conclusions
1 COL4A6 and COL11A1 can be potential mRNA markers for early gastric cancer, and COL11A1 was more sensitive than COL4A6.
2 The possible cancerous predictive formula was developed and showed remarkerble discriminatory power for the diagnosis of early gastric cancer. This provided new biological diagnose model for clinical diagnosis of early gastric cancer.
3 Collagen type I not only induced gastric carcinoma cells scattering and cytoskeleton remodeling, but also prompted cell migration and proliferation,which was related to tyrosine phosphorylation and nuclear translocation ofβ-catenin.
4 Gastric cancer BGC-823 cell transfected with COL1A1-shRNA was successfully established, whose COL1A1 expression was clear decreased and resulted in a clear reduction of cell proliferation and migration. This maybe provided potent therapy target for gastric cancer.
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