The Underlying Mechanisms Of MiRNA-34a/KLF4in X-ray Induced Apoptosis Of Liver Cells

Objective:To investigate the underlying mechanisms of miRNA-34a/KLF4in X-ray induced apoptosis of liver cells. These results can provide experimental basis for further research on mechanism of miR-34a regulating radiation induced damage, and promote the exploration of new means of radiation damage as a target for treatment.Methods:1. The dual luciferase assay analyzes the relationship of miR-34a and KLF4.Mouse cells were transiently transfected with miR-34a mimics or miR-34a inhibitor to detect the level of miR-34a and KLF4by real time PCR and to detect the level of KLF4by western blot.2. Mouse liver cells were exposed to10Gy of X-ray, and then respectively cultured at different time. Flow cytometry was applied to detect the changes of liver cells’ apoptosis. Real-Time PCR assay was applied to detect the expression of miR-34a. Cells transfected with miR-34a mimics or miR-34a inhibitor were exposed to10Gy of X-ray and then were analyzed for cell apoptosis. After the virus interference of KLF4, flow cytometry detect10Gy X-ray irradiated mice hepatocyte apoptotic changes.Results:1. KLF4was predicted to have a putative miR-34a binding site within its3’UTR, using computational prediction via Target Scan.In order to detect whether miR-34a regulates KLF4through direct3’UTR interaction, the3’UTR of KLF4was cloned into a reporter plasmid downstream from luciferase and reporter assays were performed in293cells. As a result, miR-34a obviously repressed the activity of luciferase fused to KLF43’UTR, which indicated a direct interaction between miR-34a and KLF4in293cells. To assess whether miR-34a had a functional role in the down-regulation of KLF4expression, BNL CL.2and NCTC1469cells were transfected with miR-34a mimics and miR-34a inhibitor. Expectedly, KLF4expression was decreased by miR-34a mimics at both mRNA and protein level. KLF4expression was increased by miR-34a inhibitor at both mRNA and protein level. Taken together, these results showed that miR-34a suppressed KLF4expression in liver cells through direct interaction with KLF43’UTR.2. Mouse liver cell line BNL CL.2was exposed to lOGy X-ray for indicated lengths of time and then analyzed for miR-34a expression using real-time PCR. The results showed that miR-34a was upregulated after radiation. These results suggest that radiation could upregulate miR-34a expression in liver cells. Since miR-34a was upregulated after radiation, we next investigated its role in irradiated liver cells. BNL CL.2cells were transiently transfected with miR-34a mimics or miR-34a inhibitor to overexpress or knockdown the level of this miRNA. Cells transfected with miR-34a mimics or miR-34a inhibitor were then analyzed for the apoptosis of irradiated cells by flow cytometry assay. As a result, miR-34a mimics induced a significant promotion in apoptosis after radiation exposure compared to the control. MiR-34a inhibitor, in contrast, inhibited the radiation-induced apoptosis in liver cells. Aiming to assess whether the regulation of KLF4by miR-34a accounted for its positive role in apoptosis; experiments of knockdown of KLF4were carried out subsequently. Real time PCR analysis confirmed that expression of KLF4in BNL CL.2cells was effectively inhibited by KLF4virus interference. The knockdown of KLF4promoted irradiated cells apoptosis, resembling the suppressive effect of miR-34a overexpression, which indicated that miR-34a might exert its pro-apoptotic role through targeting KLF4.Conclusions:1. KLF4is a direct target gene of miR-34.2. miR-34a/KLF4signaling positively promotes apoptosis of10Gy X-ray irradiated liver cells.