The Effect Of Inhibition Of Expression Of MIR-31and The Preliminary Exploration Of The Mechanisms

Objiective Employ the sequence-sepcific siRNA inhibiting the expression ofmicroRNA-31and to observe the effection of inhibition of the expression of miR-31on thebiological properties of panc-1cells,and to investigate the miR-31regulating mechanismsby predicting and detecting the target protein of miR-31in panc-1cells for the purpose ofproviding a theoretical basis for further research of effection of miR-31and PancreaticCancer,expected helpful for the clinical applications of PC.Methods1. Structure of miR-31inhibitors was analysed by software to selecttargeted interference sites. The miR-31inhibitors was transfected into pancreaticcarcinoma line Panc-1by positive ion liposome Lipofectamine2000;2.Transfectefficiency was assessed by real-time RT-PCR, verify the effect of siRNA interference.Prepare the positive control group at the same time；3.The cell growth inhibition wasassesses by MTT，apoptosis were measured by flow cytometry, invasion and migrationwere assaied by transwell invasion and migration assay for the functional consequencesrespectively;4. Target proteins were predicted through anthoritatively target geneprediction websites.Selected and assaied the express level of these proteins by western blot.According the results, we conjectured the regulating mechanism of miR-31in Panc-1cells.Results1.Negative control FAM-siRNA was successfully transfected into humanpancreatic cancer cell line Panc-1.The best transfection efficiency(90.6%) was detected byFCM with cell density at1.5×105/well and siRNA concentration with the proportion of5ul:5ul in6well play;2.After miR-31inhibitors were successfully transfected into panc-1cells24h, detected by RT-PCR，miR-31expression level in cells was significantly inhibited,contrasted with negative control and blank control (p<0.05);3. Inhibition of expression ofmiR-31could effectively inhibit the invasion (p<0.05) and migration (p<0.05) of panc-1cells, but coult not affect proliferation and apoptosis in vitro (p>0.05);4Of all theproteins selected, the expression level of RhoBTB1was enhanced most significantly(p<0.05). Conclution:1. Inhibition of expression of miR-31could inhibite the abilities ofinvasion and migration in panc-1cells;2. Inhibition of expression of miR-31could notaffect proliferation and apoptosis in vitro;3. The expression of RhoBTB1plays animportant role at the mechanism of miR-31regulating the abilities of invasion andmigration in panc-1cells.