MicroRNA-34a Influences Cutaneous Squamous Cell Carcinoma By Targeting SIRT6

Objective: To investigate the functions of micro RNA-34 a on proliferation,migration,invasion and apoptosis of CSCC A431 cells,and its association with silent signal regulatory protein 6(sirtuin 6).Methods: Cutaneous squamous cell carcinoma A431 cell line and human normal keratinocyte cell line HaCaT were routinely cell cultured,and the expression levels of miR-34 a in A431 and HaCaT were determined by q PCR(quantitative real-time PCR,real-time PCR),followed by in vitro transfection of A431 cells,The cells were grouped according to the transfectants as follows: control(no transfection,control),enhanced transfection(transfected with miR-34 a mimic)and its negative control(transfected with miR-34 a mimic NC),repressed transfection(transfected with miR-34 a inhibitor)and its negative control(transfected with miR-34 a inhibitor NC).The transfection efficiency of miR-34 a was validated using q PCR,and further cell function experiments were subsequently performed.Cell proliferation was estimated by CCK-8;Transwell was used to detect migration and invasion ability of A431;Cell apoptosis was estimated by flow cytometry.Starbase was used to predicte the target genes SIRT6 of miR-34 a,a target gene related to cancer,was selected and the expression of SIRT6 in HaCaT cells,using q PCR and Western blot to check A431 cells,respectively;The expression levels of SIRT6 in the cells of each group after transfection were detected by Western blot.Finally,dual luciferase assay was used to verb the targeting relationship between miR-34 a and SIRT6 gene.Statistical analysis was performed using SPSS 24.0Results:(1)QPCR results showed that miR-34 a expression was significantly reduced in A431 cells compared to HaCaT cells(P < 0.001);(2)QPCR results after transfection showed that miR-34 a expression was significantly upregulated(P < 0.001)in the miR-34 a mimic group,comparing with the blank group and the corresponding NC group,whereas miR-34 a inhibitor expression was significantly downregulated(P < 0.001);(3)CCK8 assays revealed that cell proliferation and viability were significantly downregulated(P < 0.001)in the miR-34 a mimic group,but upregulated(P < 0.001)in the miR-34 a inhibitor group,compared to the control and corresponding NC groups;(4)The results of Transwell cell migration assay exhibited that the miR-34 a mimic group was remarkablely decreased cell migration ability(P< 0.001)compared with the control and corresponding NC groups,but inhibitor group was a significant upwards(P < 0.01);(5)Cell invasion assay demonstrated that the ability was significantly decline(P < 0.001)in the miR-34 a mimic group compared with the control and corresponding NC groups,but it was remarkably rised(P < 0.05)in the miR-34 a inhibitors group;(6)Flow cytometry revealed that the miR-34 a mimic group exhibited a significant increase(P < 0.001)but not the miR-34 a inhibitor group(P < 0.001)in apoptosis compared to the control and corresponding NC groups;(7)QPCR and Western blot assays showed that SIRT6 was significantly increased in A431 compared to HaCaT cells at both transcript and protein levels(P < 0.001,P < 0.01,respectively;(8)The effect of miR-34 a on SIRT6 expression was assessed by Western blot,which showed that compared with the control and corresponding NC groups,the expression of SIRT6 was significantly downregulated(P <0.001)in the miR-34 a mimic group,but upregulated(P < 0.001)in the miR-34 a inhibitors group;(9)Dual luciferase assays showed that luciferase activity was significantly(P < 0.001)decreased in the former by CO transfection of miR-34 a mimic with SIRT6 wild-type and mutant 3 ’-terminal untranslated region(3’UTR)plasmids.However,there has none obvious fluctuation in luciferase activity(P >0.05).Conclusion: The expression of miR-34 a was lower in A431 cells and SIRT6 was higher in A431 cells;Upregulation of miR-34 a levels can inhibit CSCC cell proliferation,migration and invasion and induce apoptosis,which may play a tumor suppressor role by negatively regulating the target gene SIRT6,with some promise in CSCC targeted therapy.