Hsa-microRNA-138-5p Inhibits Pancreatic Cancer Cell Proliferation, Invasion And Metastasis Through Targeting FOXC1 And Vimentin

Part I Hsa-micro RNA-138-5p expression and clinical significance in pancreatic cancerObjective: To identify mi RNAs differentially expressed in pancreatic cancer and corresponding normal pancreatic tissues adjacent to the cancer, and expand the number of samples for validating the results, then define the research objective for subsequent experiments. Methods: To identify the expression of mi RNAs in three pancreatic cancer and corresponding normal pancreatic tissues adjacent to the cancer by Gene Chip mi RNA 4.0. Quantitative real-time reverse transcription polymerase chain reaction(q RT-PCR)detected the expression levels of hsa-mi R-138-5p(mi R-138-5p)in eighteen pancreatic cancer and corresponding normal pancreatic tissues adjacent to the cancer, the pancreatic cancer cells and the normal epithelial cells of the pancreas;To collect the clinical information and analyze the difference between the expression of mi R-138-5p and the clinical pathological features of pancreatic cancer. Results: There are eighty mi RNAs differentiation expressed in three pancreatic cancer and corresponding normal pancreatic tissues adjacent to the cancer, sixty-eight mi RNAs were overexpressed and twelve mi RNAs were underexpressed, among them, mi R-138-5p is generally lower expressed in three pancreatic cancer, but the corresponding tissue adjacent to carcinoma high expressed, difference ratio of 3.64 times. Expanded samples show mi R-138-5p expression in pancreatic cancer tissue is lower than that in corresponding tissue adjacent to the cancer(P<0.05), the expression level associated with pancreatic cancer pathologic differentiation degree and the incidence of distant metastases. mi R-138-5p respectively expressed in 8 pancreatic cancer cell lines is the 1~10 times lower than the normal epithelial cells of the pancreas, the expression level of mi R-138-5p in the pancreatic cancer cell with diffetent proliferation and invasive ability is different.Conclusion: mi R-138-5p in primary pancreatic cancer tissue and pancreatic cancer cell lines is in the low expression level, associated with pancreatic cancer pathologic differentiation degree and the incidence of distant metastases, which can be used as the follow-up study.Part II The effects of hsa-mi R-138-5p on the proliferation of pancreatic cancer cellsObjective: To explore the mi R-138-5p effects on proliferation of pancreatic cancer cell in vivo and in vitro. Methods: To construct overexpression and knockdown lentiviral vector of mi R-138-5p, respectively, and infecte pancreatic cancer cell lines, q RT-PCR detects efficiency of infection; CCK-8, colony formation assay, Ed U incorporation assays detect mi R-138-5p effects on the proliferation of pancreatic cancer cells; Flow cytometry(FCM) detects the effect of mi R-138-5p on pancreatic cancer cell cycle; Using a xenograft model in nude mice to detect mi R-138-5p effect on the ability of pancreatic cancer cells tumorigenicity. Results: Successfully construct a lentiviral vector of mi R-138-5p, CCK-8、colony formation assay、Ed U incorporation assays have the same results, Up-regulate the expression levels of mi R-138-5p can significantly decrease the proliferation ability of pancreatic cancer cells, while down-regulate the expression levels of mi R-138-5p the proliferation ability of pancreatic cancer cells improved remarkably,compared with the negative control group(P<0.05). FCM results show that up-regulate the expression levels of mi R-138-5p can obstruct cell cycle in G0/G1 phase. The xenograft model in nude mice shows that mi R-138-5p can inhibit pancreatic cancer cells tumorigenicity. Conclusion: mi R-138-5p has obviously inhibited pancreatic cancer cell proliferation and tumorigenicity, its effect at least partly because of obstructing cells in G0/G1 phase.Part III The effects of hsa-mi R-138-5p on the invasion and chemotherapy sensitivity of pancreatic cancer cellsObjective: To explore the mi R-138-5p effects on pancreatic cancer cell invasion and chemotherapy sensitivity. Methods: Wound-healing assays detects the effect of mi R-138-5p on pancreatic cancer cell migration ability; Transwell invasion assays detects the effect of mi R-138-5p on invasion ability of pancreatic cancer cells; CCK-8 detects the effect of mi R-138-5p on pancreatic cancer cell chemotherapy sensitivity. We established a liver metastasis model of pancreatic cancer to detect mi R-138-5p for the influence of pancreatic cancer cell invasion and metastasis ability in vive. Results: Up-regulate the expression levels of mi R-138-5p significantly decreased the metastasis and invasion ability of pancreatic cancer cells, while down-regulate the expression levels of mi R-138-5p the metastasis and invasion ability of pancreatic cancer cells improved remarkably, compared with the negative control group(P<0.05). mi R-138-5p can increase the chemotherapy sensitivity of pancreatic cancer cells to 5- fluorouracil(5-FU), compared with negative control group, the IC50 of two groups 24 h, 48 h and 72 h(P<0.05); liver metastasis model of pancreatic cancer shows mi R-138-5p can inhibit the distant metastasis of PANC-1 cells(P<0.05). Conclusion: mi R-138-5p can inhibit cancer cell migration, distant metastasis and increased chemosensitivity to 5-FU.Part IV The research of target genes and mechanism of hsa-mi R-138-5p on proliferation, invasion and metastasis of pancreatic cancer cellsObjective: To identify target genes and mechanism of mi R-138-5p on proliferation, invasion and metastasis of pancreatic cancer cells and clarify its biological functions. Methods: mi R-138-5p target genes were predicted by the use of Target Scan, mi Randa and Micro Cosm, we got two potential target genes associated with the cellular proliferation and migration of cancer cells, which are fork head box C1(FOXC1) and Vimentin(VIM), with luciferase reporter assay validation ofmi R-138-5p respectively with FOXC1 and VIM 3’untranslated region(3’UTR) we can determine their targeted regulation relationship directly, q RT-PCR and western blot were used for further validation of mi R-138-5p on pancreatic cancer cell FOXC1 and VIM gene m RNA and protein expression level influence. To detect FOXC1 and VIM for the effect on cell biology of pancreatic cancer by using small interfering RNA(si RNA). Finally, preliminary exploration related pathways proteins by western blot and immunofluorescence. Results: We successfully constructed the target gene 3’UTR luciferase reporter vectorsand and confirmed target genes FOXC1 and VIM by the dual luciferase reporter assay. q RT-PCR and western blot were also observed that mi R-138-5p can obviously inhibit FOXC1 and VIM gene m RNA and protein expression, compared with the negative control group(P<0.05). FOXC1 and VIM were significantly lower expression in pancreatic cancer tissue; We confirmed that FOXC1 si RNA(analog of mi R-138-5p the role) can decrease the proliferation of pancreatic cancer cells, and that VIM si RNA(simulation of mi R-138-5p the role) can decrease pancreatic cancer cell invasion capacity. To further investigate whether the proliferation and invasion induced by mi R-138-5p overexpression in pancreatic cancer cells was due to inhibited FOXC1 and VIM expression, Capan-2 cells were co-transfected with FOXC1 si RNA and mi R-138-5p inhibitor. CCK-8 showed that transfection of mi R-138-5p inhibtor promoted pancreatic cancer cells proliferation, whereas transfection of FOXC1 si RNA decreased the number of pancreatic cancer cells, however, after co-transfection, the proliferation cells number induced by mi R-138-5p inhibitor were reversed(P<0.05). These results suggest that mi R-138-5p inhibits pancreatic cancer cells proliferation at least partly through regulation of FOXC1. To investigate whether the invasion induced by mi R-138-5p overexpression in pancreatic cancer cells was due to inhibited VIM expression, PANC-1 cells were co-transfected with VIM si RNA and mi R-138-5p inhibitor. Transwell showed that transfection of mi R-138-5p inhibtor promoted pancreatic cancer cells invasion ability, whereas transfection of VIM si RNA decreased the number of invasion cells, however, after co-transfection, the invasion cells number induced by mi R-138-5p inhibitor were reversed(P<0.05). These results suggest that mi R-138-5p inhibits pancreatic cancer cells invasion at least partly through regulation of VIM. mi R-138-5p downstream genes are discussed in this paper, the results show that the expression of cell cycle protein CNND1 significantly reduced and E-cadherin significantly increased.Conclusion: mi R-138-5p inhibits pancreatic cancer cells proliferation byregulating the expression of FOXC1, maybe at least partly by inhibiting the expression of CNND1; mi R-138-5p inhibits pancreatic cancer cells invasion by regulating the expression of VIM, maybe partially through regulating Epithelial-mesenchymal transition(EMT) process.