Expression And Role Of MiR-331-3p In Pancreatic Cancer

ObjectiveTo investigate the expression of miR-331-3p in pancreatic cancer(PC)cells,tissue and plasma of patients and clarify the potential molecular mechanism and effect of miR-331-3p in the proliferation,migration and invasion of PC cells.Methods1.Plasma samples of 44 PC patients and 23 healthy individuals,17 pairs of PC and corresponding adjacent non-cancerous tissue samples,5 kinds of PC cell lines and the human normal pancreatic ductal epithelial cell line were recruited.miRNA microarray was used to analyze the miRNA expression profile between the plasma of PC patients and that of healthy control.qRT-PCR and in situ hybridization(ISH)were used to detect the expression of miR-331-3p in the clinical specimens and PC cells.2.CFSE labeling assay,colony formation assay,wound-healing assay and transwell assay were performed to measure the proliferation,migration and invasion of PANC-1 and MIAPaCa-2 cells transfected with the miR-331-3p inhibitor or inhibitor control.Western blotting analysis of E-cadherin,N-cadherin and vimentin expression in PANC-1 cells transfected with the miR-331-3p inhibitor or inhibitor control.3.Bioinformatics database was used to predict the candidate target genes of miR-331-3p,luciferase reporter assay was performed to verify the potential relationship between miR-331-3p and ST7L.qRT-PCR,western blotting and immunohistochemisitry(IHC)were used to detect the expression of ST7L.4.CFSE labeling assay,colony formation assay,wound-healing assay and transwell assay were performed to measure the proliferation,migration and invasion of PANC-1 cells co-transfected with the miR-331-3p mimic and ST7L expression plasmids.Western blotting analysis of the protein levels of ST7L,E-cadherin,N-cadherin and vimentin in PANC-1 cells post transfection.Result1.According to the miRNA microarray analysis,we found that miR-331-3p was one of the upregulated miRNAs in the plasma of PC patients.In five PC cell lines(PANC-1,MIAPaCa-2,AsPC-1,BxPC-3,SW1990),miR-331-3p was expressed at a higher level(2.53±0.38,3.14±0.74,2.75±0.54,3.20±0.49,2.69±0.95)than in a human normal pancreatic ductal epithelial cell line.The plasma levels of miR-331-3p from PC patients(7.15±0.95)were significantly higher than healthy donors(3.61±0.72).We analyzed miR-331-3p expression level in the plasma of PC patients before(8.36±1.66)and after(3.75±0.72)surgical removal of the tumors.The miR-331-3p level in the plasma was significantly reduced after the operation.The expression of miR-331-3p was observably higher in fresh cancer tissues(7.40±5.18)than in the adjacent non-cancerous tissues(3.41±3.82).ISH analysis showed that the expression of miR-331-3p was higher in cancer tissues(6.6±1.85)than in the adjacent non-cancerous tissues(4.83±2.29).High miR-331-3p expression was associated with advanced PC stage and distant metastasis.2.CFSE labeling assay indicated that both PANC-1 and MIAPaCa-2 cells transfected with the miR-331-3p inhibitor(9.37±0.43,10.51±1.22)had a decrease in proliferation index compared to the control(14.56±1.74,17.49±0.81).Colony formation experiment illustrated that the propagation ability of these two cell lines transfected with the miR-33 1-3p inhibitor(0.63±0.04,0.46±0.07)was significantly decreased in comparison with the control.Wound healing assay showed that reduced levels of miR-331-3p weakened the capacity of cell migration in both PANC1 and PaCa-2 cells(0.40±0.10,0.45±0.05).Transwell migration and invasion assays revealed that decreased expression of miR-33 1-3p significantly reduced the capacity of PANC-1 and MIAPaCa-2 cells migration(272±33,149±23)and invasion(125±31,84± 12)compared to the control(554±68,368±43)(230±37,196±22).These results indicated that inhibition of miR-331-3p suppressed the cell proliferation,migration and invasion of both PANC-1 and MIAPaCa-2 cells.3.ST7L is a direct target of miR-33 1-3p in PC.qRT-PCR,western blotting and IHC analysis indicated that the expression of ST7L was negatively related to miR-33 1-3p.4.ST7L re-expression attenuated the effects of miR-33 1-3p on cell proliferation,migration and invasion in PANC-1 cells.ConclusionOur researches demonstrates that miR-33 1-3p is upregulated in PC.miR-33 1-3p facilitates the proliferation,migration and invasion by suppressing ST7L in PC cells.miR-33 1-3p may serve as a potential non-invasive diagnostic biomarker for PC and provide new insight for clinical treatment.