| Objective:To investigate the effects of microRNA-148a(miR-148a)on the stemness properties and malignant phenotypes of pancreatic cancer cells and its pote(ntial regulatory mechanisms.Methods:1、Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression levels of miR-148 a in 61 cases of pancreatic cancer tissues,five kinds of pancreatic cancer cell lines(Panc-1,SW1990,MiaPaca-2,BxPC-3 和Canpan-2)and normal human pancreatic ductal epithelial cell line(HPDE).According to the median expression of miR-148 a in pancreatic cancer tissues,the patients were divided into high and low expression groups,and survival analysis was carried out.At the same time,the relationship between the expression level of miR-148 a and the prognosis of patients was analyzed by an online database(http://kmplot.com/analysis).2、The corresponding lentiviral vectors that overexpressed or interfered with miR-148 a were transfected into pancreatic cancer cells(Capan-2,BxPC-3,and MiaPaca-2)by Lipofectamine 3000.The experiment cells included 4 groups: miRcon group(transfection overexpression empty vector),miR-148 a overexpression group(transfection miR-148 a overexpression vector),anti-miR-con group(transfe-ction interference empty vector),anti-miR-148 a group(transfection interference miR-148 a expression vector).The stable cell lines were screened by puromycin,and the expression of miR-148 a was assessed by qRT-PCR.CCK-8 was used to detect cell proliferation ability,colony formation assay to detect cell clone formation ability,and transwell cell invasion and migration assays were used to detect the ability of invasion and migration.The expressions of stemness markers(Nanog,Oct4 and Sox2)and(Epithelial mesenchymal transition)markers(E-cadherin,Vimentin and N-cadherin)were detected by Western blot.3、Double luciferase reporter gene assay was used to verify whether miR-148 a could be combined with Wnt1-3′UTR.QRT-PCR detected the expression levels of miR-148 a and Wnt1 in pancreatic cancer tissues,and analyzed their correlation.The expression levels of Wnt1 and Wnt/β-catenin signaling pathway related genes(β-catenin,MMP-9 and cyclin D1)were detected by qRT-PCR and Western blot,respectively.Results:1、The expression level of miR-148 a in pancreatic cancer tissues was significantly lower than that of corresponding paracancerous tissues(P<0.001).Compared with HPDE cells,the expressions of miR-148 a in pancreatic cancer cell lines were significantly downregulated(P<0.01).The low expression of miR-148 a was associated with poor prognosis in patients with pancreatic cancer(P<0.05).2、Compared with the corresponding control group,the cell proliferation,clonal formation,invasion and migration abilities of pancreatic cancer cells were significantly inhibited in the miR-148 a overexpression group(P<0.05),while in the anti-miR-148 a group,the cell proliferation,clonal formation,invasion and migration abilities were significantly increased(P<0.05).Meanwhile,miR-148 a overexpression increased the expression of epithelial marker E-cadherin(P<0.05),accompanying with decreased mesenchymal markers(N-cadherin and Vimentin)and stemnessassociated markers(Nanog,Oct4 and Sox2)expression(P<0.01).Conversely,the expression level of mesenchymal markers(N-cadherin and Vimentin)and stemness-associated markers(Nanog,Oct4 and Sox2)were significantly upregulated(P<0.05),whereas E-cadherin was downregulated(P<0.001)in anti-miR-148 a group.3、The directly combination of miR-148 a with Wnt1-3′UTR was verified by dual luciferase reporter assay.Up-regulation of miR-148 a could inhabit the expres-sion of Wnt1,β-catenin,cyclin D1 and MMP-9(P<0.05).Whereas,down-regulation of miR-148 a could increase the expression of Wnt1,β-catenin,cyclin D1 and MMP-9(P<0.05).In addition,there was a negative correlation between the expression of Wnt1 and miR-148 a in pancreatic cancer tissues(r=-0.3393,P<0.01).Conclusions:MiR-148 a was downregulated in pancreatic cancer tissues and pancreatic cancer cells,and low expression of miR-148 a was associated with poorer prognosis in patients with pancreatic cancer.MiR-148 a could suppress the stemness properties and malignant phenotype of pancreatic cancer cells.Its mechanisme was related to miR-148 a inhibiting epithelial-mesenchymal transition through negatively regulating Wnt/β-catenin signaling pathway by targeting Wnt1. |
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