PiR-16 Regulates The Proliferation And Migration Of Vascular Smooth Muscle Cells By Targeting HnRNPK

Objective: In this study,we investigated the physiological function of piRNA in vascular smooth muscle cell(VSMC).This study may provide a new potential target for clinical targeting therapy of atherosclerosis.Methods:(1)APOE-/-mice were fed with high fat for 12 weeks to establish atherosclerosis(AS)mice model.The target gene PIWI-interacting RNA(piRNA)was screened by gene chip,RT-q PCR,reverse transcription of low deoxyribonucleoside triphosphate(RTLP).The expression of the target gene piRNA in many cell lines was detected to find most suitable cell line.(2)To synthesize the target piRNA to overexpress RNA and inhibit the expression of RNA,and to verify whether it is successfully expressed in cells.RNA synthesized by VSMC transfection was detected by CCK8,Ed U,transwell and wound healing.(3)Predict the target protein in the downstream of piRNA,and detect its expression changes by RT-q PCR and western blot.(4)Synthesizing gene silencing of target protein and verifying whether the gene is successfully expressed in cells.CCK8,Ed U,transwell and wound healing were used to detect the effect of target protein on cell biological function.(5)Co-transfecting the synthesized RNA that inhibits the expression of piRNA and the gene silenced target protein,CCK8,Ed U,transwell and wound healing to detect whether piRNA affects the biological function of cells by regulating the target protein.(6)Sulfite treatment method is used to verify whether piRNA regulates the expression of target protein through methylation.(7)The mice were divided into NC group,AS group and piR-16 group.The aorta of the mice was taken and stained with oil red to observe the plaque size.Fluorescence in situ hybridization(FISH)experiment was used to observe the expression of piR-16 in aorta.(8)RNA was extracted from serum samples of AS patients and the expression of piRNA of interest was detected by RT-q PCR.Results:(1)piR-16 was identified as the target gene by gene chip,RT-q PCR and RTLP.HA-VSMC was selected for the experiment.(2)RT-q PCR results showed that piR-16 mimic and piR-16 inhibitor was synthesized by transfection.The results of CCK8,Ed U,transwell and wound healing showed that the proliferation and migration rate of piR-16 group were significantly lower than NC group(p < 0.05).(3)Western blotting and RTq PCR showed that the m RNA level and protein expression level of hn RNPK in piR-16 mimic group were down-regulated compared with NC group(p < 0.05).(4)The m RNA and protein expression of hn RNPK in si-hn RNPK group were down-regulated compared with NC group(p < 0.05).(5)The results of CCK8,Ed U,transwell and wound healing showed that the proliferation and migration rate of cells in piR-16 inhibitor+si-hn RNPK group were significantly increased compared with si-hnrnpk group(p < 0.05).(6)Sulfite experiments show that piR-16 may silence hn RNPK expression by promoting methylation of Cp G islands in the promoter region of hn RNPK.(7)AS group mice had obvious plaques in aortic arch and larger area,while piR-16 group had plaques in aortic arch and smaller area than AS group mice.(8)In blood samples of clinical patients,piR-16 expression in serum of AS patients was significantly lower than that of healthy people.Conclusion: PiR-16 expression was down-regulated in AS mouse aorta and patient blood samples.PiR-16 can regulate cell proliferation and migration by methylation targeting hn RNPK in HA-VSMC.Therefore,piR-16 plays an important role in atherosclerosis and may provide a new target site for clinical treatment of atherosclerosis.