The Role Of MiR-29c Targeting MMP2 In Regulating Metastasis Of Pancreatic Cancer

Objective:To investigate the role of mi R-29 c on metastasis of pancreatic cancer.Methods:1.The expression of the miR-29 family members as determined by RT–PCR in five pancreatic cancer cell lines with different tumor grades; MMP2 expression and activity in five pancreatic cancer cell lines were determined by RT –PCR, western blotting and zymography analyses. The correlation between the expression of miR-29 c and MMP2 was analyzed by Spearman analysis.2. Effects of transient expression of mi RNA-29 s mimic and its inhibitors were studied in the pancreatic cancer cell lines; Cell proliferation was measured using the Cell Counting Kit-8; We transfected pancreatic cancer cells with miR-29 c mimics and negative control to determine whether the upregulation of miR-29 s suppresses migration and invasion of pancreatic cancer cells by Transwell Migration and Invasion Assays.3. We combined the prediction algorithmthe and dual-luciferase reporter system to identify whether mi R-29 c regulates MMP2 expression by directly binding to MMP2 3’UTR.4. The pancreatic cancer cells Hs766 t were labeled with luciferase and injected into the pancreases of nude mice. We then isolated pancreatic cancer cells with different metastatic potentials(Hs766t, Hs766t-L1 and Hs766t-L2) by the orthotopic model of pancreatic cancer spontaneous liver metastases. The expression of mi R-29 c determined by RT-PCR, MMP2 expression in this these pancreatic cancer cell lines was determined by RT –PCR and western blotting; We overexpressed mi R-29 c in Hs766t-L2 and determined the effect on liver metastasis of pancreatic cancer in vivo.5. Clinically, the expression of mi R-29 c and MMP2 were determined by RT-PCR and ICH in pancreatic cancer tissues and corresponding paracancerous tissues, respectively. And we analyzed the relationship between mi R-29 c and MMP2. We then analyzed association of miR-29 c and MMP2 expression with clinicopathological characteristics of pancreatic cancer patients by χ2 tests. Eventually, the Kaplan–Meier method was used to compare overall survival among the different patient groups.Results:1. The expression of mi R-29 c was inversely correlated with the expression of MMP2 in pancreatic cancer cells.2. Re-expression of miR-29 c had no effect on pancreatic cancer cell proliferation but substantially suppressed the migration and invasion of pancreatic cancer cells. Inhibition of miR-29 c significantly increased cell migration.3. There is one miR-29 c binding site in the 3’UTR of MMP2 m RNA. Overexpressing miR-29 c significantly decreased MMP2 expression and activity compared with the mimic negative control; MMP2 expression was significantly increased when Capan1 cells were transiently transfected with mi R-29 c inhibitors. Luciferase assay showed that miR-29 c could reduce luciferase activity when the reporter gene was fused with the wild-type 3’UTR but not with the mutated one.4. We successfully isolated pancreatic cancer cells with different metastatic potentials in orthotopic model of pancreatic cancer. The expression of miR-29 c and MMP2 were involved in the metastatic potential of pancreatic cancer cells. Re-expression of mi R-29 c significantly reduced liver metastasis in pancreatic cancer cells compared to the negative control.5. The expression of mi R-29 c was significantly lower in pancreatic cancer tissues than in pair-matched adjacent paracancerous tissues; the expression of MMP2 was frequently overexpressed in pancreatic cancer tissue compared with paracancerous tissues. The miR-29 c expression was also inversely associated with MMP2 expression in pancreatic cancer tissues. Downexpression of mi R-29 c was significantly correlated with lymph node metastasis, tumor differentiation and TNM stage in pancreatic cancer. Meanwhile, low expression of mi R-29 c was significantly associated with poor overall survival rate for pancreatic cancer.Conclusion:1.mi R-29 c suppress pancreatic cancer liver metastasis via targeting MMP2 in vivo.2.mi R-29 c plays an important role in regulating the expression of MMP2 to suppress pancreatic cancer metastasis, but there may be other mechanisms that regulate MMP2 expression.3.mi R-29 c targeting MMP2 may serve as a novel marker of pancreatic cancer metastasis and possibly as a therapeutic target to treat pancreatic cancer liver metastasis.