Research On Biological Functions And Target Genes Of MiR-146a-5p In The Progression Of Prostate Cancer

BackgroundPCa(prostate cancer)is an important public health problem,it affects an estimated 10 million men worldwide,and kills more than 400,000 people a year.As life expectancy increases and the population ages,and the reduction in medical resources in recent years due to the COVID-19 pandemic,the incidence of PCa will inevitably increase.Distant metastases of tumors account for 90%of cancer-related mortality,bone metastases are the most common manifestation of advanced prostate cancer,it often leads to serious bone-related diseases and adverse clinical outcomes,that will be helpful to find new therapeutic targets for cancer to better understand the molecular mechanisms of prostate cancer metastasis.MicroRNA(miRNA)is a small non-coding single stranded RNA,it will induce post-transcriptional regulation of genes,and identify target messenger RNA(miRNA)by base complementary pairing.miRNA is a key regulatory molecule involved in regulating a variety of basic cellular processes,such as proliferation,apoptosis,differentiation,migration and invasiveness.Abnormal expression of miRNA was observed in a variety of pathological events,more important thing is regulation of miRNA and downstream genetic alterations are closely related to the pathogenesis of most human cancers.Depending on the nature of the regulation of downstream target genes,different miRNAs can exhibit tumor-promoting or tumor-suppressing effects.Among them miR-146a-5p has been reported to be abnormally expressed in a variety of different cancers,such as non-small cell lung cancer,gastric cancer,pancreatic cancer,and prostate cancer.Functionally,miR-146a-5p often acts as a tumor suppressor gene that inhibits cell growth,migration,and invasion in certain types of cancer.However,there is less research on the role of miR-146a-5p in prostate cancer initiation and metastasis,by comparing multiple databases,we predicted STAT1 as a downstream target gene for miR-146a-5p.The STAT protein family(signal transduction and transcriptional activators)is transferred into the nucleus after phosphorylation activation through the classical signal transduction pathway JAK/STAT pathway.The nuclear STAT complex binds to the GAS or ISRE elements of DNA,recruits co-modulators and RNA polymerase Ⅱ,triggers its target gene expression,and regulates cell proliferation,differentiation,and apoptosis from multiple aspects.As a member of the STAT family,STAT1 has been reported to be closely related to the occurrence and development of malignant tumors,and is involved in cell phenotypes of cell cycle,apoptosis and proliferation.In this research,the biological function and synergy of miR-146a-5p in prostate cancer was preliminarily verified through a series of basic experiments by exploring the "targeted regulation of miR-146a-5p on STAT1" and the relationship between STAT1 and paved the way for subsequent research on the specific mechanism of miRNA-146a-5p and STAT1 in prostate cancer.Purpose of the research1.Determine the expression level of miR-14 6a-5p in different prostate cancer cell lines2.Explore the role of miR-146a-5p in the proliferation and invasion of prostate cancer3.Verify the targeted regulation of MIR-146a-5p on STAT1 in prostate cancerResearch methods1.RT-qPCR was used to detect the expression level of miR-146a-5p in different prostate cancer cell lines;2.The biological function of prostate cancer cell line after miR-146a-5p mimic and inhibitor intervention was used.The effect of CCK8 on proliferation was detected,the stem nature of cells was detected by clonal formation test,and its effect on invasion was detected by Transwell and Wound Healing tests.3.Detect the expression difference of miR-146a-5p overexpression and STAT1mRNA after knockdown,and verify miR-14 6a-5p by dual-luciferase reporter gene experiment with STAT1;Results1.qRT-PCR detected the expression of miR146a-5p and STAT1 mRNA in different prostate cancer cell lines,and RWPE-1 cells were used as controls.The results showed that compared with normal prostate epithelial cells,the expression of miR146a-5p in various prostate cancer cells was significantly reduced,regardless of hormone sensitivity or not,and the difference was statistically significant(p<0.05).2.Transfection of overexpressed miR-146a-5p in prostate cancer cell lines significantly reduced cell proliferative activity,stemness and migration invasion ability,while the results were opposite after knockdown,suggesting that miR-146a-5p plays a role in tumor suppression in the progression of prostate cancer.3.The expression of STAT1 mRNA after transfection of miR-146a-5p mimic and inhibitor was detected by qRT-PCR.The results showed that the expression level of STAT1 mRNA in the mimic group was significantly lower than that in the control group.Conversely,inhibitor group had elevated expression of STAT1 mRNA compared to controls.The differences were statistically significant(p<0.05).This suggests that miR-146a-5p has a reverse regulatory effect on ST AT1,as the dual-luciferase labeling gene shows,when wild-type ST ATI After the plasmid was co-transfected with miR-146a-5p mimic,it could be seen that luciferase activity was significantly inhibited,and when the binding site was mutated,the relative luciferase activity increased again,indicating that miR146a-5p and STAT1 were significantly inhibited 3’UTR binds,causing STAT1 degradation,thereby reducing luciferase activity.ConclusionThe expression of miR-146a-5p in prostate cancer cell lines was significantly lower than that of normal prostate epithelial cells.After overexpression of miR-146a-5p,the migration invasion,proliferation ability and stem ability of prostate cancer cells were significantly inhibited,but the results were reversed after knocking down miR-146a-5p.Overexpression of miR-146a-5p simultaneously inhibited the expression of STAT1 in prostate cancer cells,and wild-type STAT1 plasmid could bind to miR-146a-5p.It suggests that there may be targeted regulation between the two.