| BackgroundPancreatic cancer is an important global health problem.Particularly in China,pancreatic cancer is one of the malignant tumors with the highest mortality rate.Metastasis and recurrence of pancreatic cancer seriously affect the curative effect and quality of life of patients,which is the main cause of death of pancreatic cancer patients.Despite considerable progress in surgical and adjuvant therapies,the mortality rate of pancreatic cancer has remained high in the past decade.An important reason is that most patients present with advanced stages and distant metastasis.Hence,studies wich further investigate mechanisms related to pancreatic cancer metastasis and early diagnosis and potential treatment targets is particularly important.The aim of this study was to investigate the role and clinical significance of collagen VI alphal(COL6A1)in promoting metastasis of pancreatic cancer.MethodsUsing pancreatic cancer cell line BxPC-3 as parent,a new high metastasis cell model of pancreatic cancer BxPC-M8 was established by repeated passage of parent cell lines eight times through Transwell chamber.The ability of BxPC-3 and BxPC-M8 cells to migrate and invade in vitro was compared by scratch,migration and invasion experiments.Genome sequencing and real-time quantitative PCR(qRT-PCR)were used to identify COL6A1,a potential regulatory gene of pancreatic cancer metastasis.The effect of COL6A1 on metastasis of pancreatic cancer cells was studied by siRNA knockdown assay.Meanwhile,WB was used to detect the effect of knocking down COL6A1 on the expression of epithelial-mesenchymal transition(EMT)related proteins in pancreatic cancer cells.Finally,the correlation between abnormal expression of COL6A1 and clinicopathological characteristics of pancreatic cancer patients was studied by tissue microarray.Results:Compared with parent BxPC-3,BxPC-M8 showed no difference in proliferation and sensitivity to gemcitabine.However,BxPC-M8 cells showed EMT phenotype,and significantly enhanced migration and invasion ability in vitro.Genome sequencing and qRT-PCR results showed that COL6A1 was overexpressed in BxPC-M8 cells.Further protein detection showed that COL6A1,Vimentin,Snail and MMP-9 expressions were significantly higher in BxPC-M8 compared to BxPC-3 cells(p<0.001).The down-regulation of COL6A1 expression by siRNA inhibited the migration and invasion ability of BxPC-M8 in vitro,and significantly down-regulated the expression of Vimentin(p<0.05)and Snail and(p<0.05),while up-regulating the expression of E-cadherin(p<0.001).In pancreatic cancer clinical samples,the expression of COL6A1 in tumor tissue was significantly higher than that of the adjacent tissues(p=0.001).In addition,we found that the median OS of COL6A1?and COL6A1-patients were 8+4 and 14+ 7 months,respectively(p=0.021).The positive expression of COL6A1 in tissue samples was significantly correlated with shorter OS(p=0.001).Univariate and multivariate analysis showed that COL6A1 positive expression was an independent predictor of overall survival(OS),which was significantly associated with poor prognosis.Conclusion:BxPC-M8,the new highly metastatic pancreatic cancer cell,has the same biological characteristics as its parent BxPC-3,such as proliferation rate.It only differs in the migratory and invasive characteristics.This makes BxPC-M8 an excellent cell model for studying the metastasis of pancreatic cancer.COL6A1 is highly expressed in BxPC-M8.Inhibition of COL6A1 expression can significantly inhibit the EMT and metastasis characteristics of BxPC-M8 cells,suggesting that COL6A1 promotes the metastasis of pancreatic cancer cells by regulating EMT.Clinical studies have found that COL6A1 is highly expressed in pancreatic cancer tissues,and its positive expression is significantly associated with poor prognosis of patients.The development of this study supports the assertion that COL6A1 is a potential biomarker and target for diagnosis and treatment of pancreatic cancer metastasis,and provides a theoretical basis for the development of new strategies for effective diagnosis and treatment of pancreatic cancer metastasis. |
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