Studies On The Role And Mechanisms Of COL11A1 In Human Gastric Cancer

With the development of the diagnosis and treatment,the morbidity and mortality of gastric cancer have declined around the world.However,gastric cancer is still one of the most common malignant tumors worldwide,especially in China.So far,the development and progression of gastric cancer are believed to correlate with the accumulation of activated oncogenes and silenced tumor-suppressor genes.Thus,identification and validation of new oncogenes will not only contribute to the pathogenetic understanding and target interventions of gastric cancer,but also help establish new tumor markers for early diagnosis and monitoring.As a member of type XI collagen family,COL11A1(type XI collagen)has been previously considered as a candidate oncogene in gastric cancer by cDNA microarray analysis.COL11A1 belongs to the fibrillar collagen family,and plays an important role in the maintenance of appropriate fiber diameter.Recent studies have revealed the relationship between COL11A1 and cancers,and COL11A1 plays a role in cell proliferation and invasion.The abnormal expression of COL11A1 has been reported in ovarian cancer,head and neck squamous cell carcinoma,thyroid cancer,colon cancer and non-small cell lung cancer.However,no deep investigation of the role of COL11A1 in the development and progression of gastric cancer has been studied.In this study,we firstly determined the COL11A1 mRNA expression of paired gastric cancer tissue and non-tumor gastric tissue by quantitative real-time PCR(qRT-PCR)and analyzed the relationship between COL11A1 expression and clinicopathological parameters of patients with gastric cancer;Then we investigated the biological effects of COL11A1 knockdown on gastric cancer cells(cell proliferation,migration,invasion,cell cycle and apoptosis);In addition,we testify the potential downstream genes of COL11A1 by cDNA microarray and qRT-PCR to investigate the potential molecular mechanisms of COL11A1 in gastric cancer.Materials and Methods1.The expression of COL11A1 in gastric cancer tissue and cells and the relationship between COL11A1 expression and clinicopathological parameters of patients with gastric cancer? The expression of COL11A1 mRNA in 57 paired gastric cancer samples and normal gastric tissue was determined by qRT-PCR;? The expression levels of COL11A1 in a panel of gastric cancer cell lines(n = seven)and one GES-lwere examined by qRT-PCR and Western Blot analysis.? The relationship between COL11A1 mRNA expression level and clinicopathological parameters of patients with gastric cancer was analyzed.2.The biological effects of COL11A1 in gastric cancer cells? The gastric cancer cell line with stable COL11A1 knockdown was established.? The cell proliferation by COL11A1 knockdown was confirmed by plate colony formation and MTS assay;? Cell apoptosis and cell cycle by COL11A1 knockdown were assessed by flow cytometry and the expression of cell cycle and apoptosis-related protein was determined by Western Blot;? Cell migration and invasion by COL11A1 knockdown were investigated by transwell cell migration and collagen-based cell invasion system.3.Screening and validation of the possible downstream genes of COL11A1? cDNA microarray analysis was performed in cells with COL11A1 knockdown or vector.? We further validated seven candidate downstream genes in cells with knockdown of COL11A1 or vector by qRT-PCR analysis.Results1.COL11A1 mRNA expression was significantly up-regulated in gastric cancer tissue,but its expression was only high in HGC-27 cells.Up-regulation of COL11A1 was related to age,tumor size,invasion depth and lymph node metastasis.? The results of qRT-PCR showed that COL11A1 mRNA expression was significantly upregulated in cancer tissue compared to normal gastric tissue(p<0.0001,Wilcoxon paired test).? Compared to GES-1,the COL11A1 expression in HGC-27 cells was significantly high,while the COL11A1 expression in the other six gastric cancer cells(SGC-7901,BGC-823,MGC-803,AGS,MKN-28,MKN-45)were significantly low.? COL11A1 mRNA expression was related to gastric cancer age,tumor size,depth of invasion and lymph node metastasis,but was not correlated with gender and and degree of differentiation.2.COL11A1 knockdown significantly inhibited proliferation,promoted cell apoptosis,induced cell cycle arrest and impaired migration and invasion of HGC-27 gastric cancer cells in vitro? HGC-27 gastric cancer cell line with stable knockdown of COL11A1 was successfully established;? The number of surviving colonies formed on the plates was significantly reduced in HGC-27 cells with stable knockdown of COL11A1 when compared to the control vector transfectants(p<0.01),while the significant reduction of surviving cells in HGC-27 cells with stable knockdown of COL11A1 was confirmed by MTS assay(p<0.01);? We observed that stable knockdown of COL11A1 significantly induced apoptois of gastric cancer cells and accumulated G1 phase populations compared to control vector transfected cells.Additionally,we observed the increased expression of p21 and cleaved caspase 3 proteins and decreased expression of cyclin Di and CDK6 proteins by Western Blot;? Furthermore,stable knockdown of COL11A1 significantly suppressed HGC-27 cell migration(p<0.01)and cell invasion(p<0.01)by transwell assays.3.Several possible downstream genes of COL11A1 were screened and validated? Our results of cDNA microarray screened 614 upregulated downstream genes and 423 downregulated downstream genes.These genes mainly participated in cell proliferation,apoptosis,cell migration,adhesion and signal transduction,and other important life activities of cells;? The results of qRT-PCR in HGC-27 cells with COL11A1 knockdown or vector confirmed that CDK6,TIAM1,ITGB8,WNT5A and XIAP mRNA expression levels were significantly downregulated,while RGS2 and NEFL mRNA expression levels were significantly increased.These results were in consistent with cDNA microarray analysis.Conclusions1.COL11A1 mRNA expression was significantly upregulated in gastric cancer tissue and positively correlated with age,tumor size,depth of invasion and lymph node metastasis;2.Stable knockdown of COL11A1 significantly inhibited proliferation,impaired cell migration and invasion in vitro;3.COL11A1 may regulate cyclin D1,CDK6,p21 and casapase 3 to promote cell apoptosis and induce cell cycle arrest of HGC-27 gastric cancer cells in vitro.4.COL11A1 may regulate the development and progression of gastric cancer through several possible downstream genes involved in biological processes such as cell proliferation,apoptosis,metastasis and signal transduction.