Inhibition And Mechanisms Of Hsa-miR-365a On Breast Cancer Cells MCF-7

BackgroundmicroRNA (miRNA) was found to induce the mRNA degredation and translational repression by specific binding to the3’-UTR of target genes. As an important cancer related transcription factor, c-Myc involved in a variety of biological processes, such as cell proliferation, differentiation, transformation and apoptosis. Resent study indicate that c-Myc may participate in the development of cancer by mediating the expression of miRNAs. In our study, we use bioinformatics analysis predicted abundance miRNAs regulated by c-Myc through known experimental data. Among these result, there are110miRNAs located in the intron and107miRNAs located between different genes. According to the screening condition-high expression level (reads>4), containg c-Myc binding site(motif>0), close distance between E-box and miRNAs(TSS<5kb), and large regulation strength of transcription factor to genes (FDR value<0.05)and so on-we set, we chose5miRNAs located in and16miRNAs between the genes. By the last, we determined the has-miR-365a as the final study object after realtime-PCR verification and target analysis, and systematic investigated the effect to the breast cancer cell line MCF-7proliferation and the molecular mechanism.Result1. C-Myc specific siRNA could significant inhibit the protein expression of C-Myc in breast cancer cell line MCF-7, and the repression effect to mRNA could be up to80%. The result of Serum dependent cell growth experiment and saturation density detection showed, C-Myc specific siRNA mediated C-Myc repression could attenuate the MCF-7proliferation. At the same time, our result showed, the C-Myc repression could significantly increase the endogenous has-miR-365a gene level.2. In our Serum dependent cell growth experiment, saturation density detection and CCK8cell activity detection results, Has-miR-365a mimic could significantly repress the malignant proliferation of MCF-7, while the inhibitor could completely avoid the effect of has-miR-365a. 3. Has-miR-365a mimic and inhibitor could significant alternatively repress or avoid the expression of target gene HSPA8(coding protein Hsc70). Luciferase report assay proved that Has-miR-365a mimic could specific binding to the3’UTR of HSPA8, and then inhibit the target gene translation.Discussion and conclusionc-Myc participating to the cancer development through miRNA regulation pathway has cause great concern. HSPA8is a member of HSP70family, coding73kDa heat shock cognate protein70, which involved in an important part to the protein folding and maintain the normal construction and function. Our previous work indicate that Hsc70played an important role in maintain protein homeostasis (protein folding, translocation, assemble and degradation) in both normal and stress condition. Our study result showed, c-Myc regulated has-mir-365a has an great significance on the breast cancer cell line MCF-7proliferation, what’s more, the repression effect to MCF-7mediated by has-mir-365a was coursed by regulation its target gene HSPA8(Hsc70). So we get the conclusion:The regulatory pathway formed by c-Myc, hsa-miR-365a and Hsc70may play an important role in the regulation of proliferation of MCF-7.