Hsa＿circ＿0070512 Promotes Prostate Cancer Progression By Regulating The MiR-338-3p/hedgehog Signaling Pathway

Objective:Circ RNAs are a special class of noncoding RNAs,and a growing number of studies have shown that circ RNAs can promote the development and progression of a variety of tumors.However,circ RNAs that regulate prostate cancer progression are still largely unknown and deserve further exploration.The aim of this study was to investigate the effect of circ RNA hsa＿circ＿0070512 on the biological function of prostate cancer cells and to further elucidate the specific molecular mechanisms underlying its function in prostate cancer.Methods:(1)By analyzing the high-throughput sequencing results of three pairs of prostate cancer tissues and the corresponding paracancer tissues,the circ RNA hsa＿circ＿0070512 was screened for significant high expression in prostate cancer tissues.q RT-PCR was used to verify the expression of hsa＿circ＿0070512 in prostate cancer tissues and cell lines.The back-splicing junction site of hsa＿circ＿0070512 was verified by Sanger sequencing.RNase R and Act D assays were performed to verify the stability of hsa＿circ＿0070512.The expression of hsa＿circ＿0070512 in c DNA and g DNA of prostate cancer cells was detected by agarose gel electrophoresis.The localization of hsa＿circ＿0070512 in prostate cancer cells was verified by FISH and nucleoplasm separation experiments.Further,the expression of hsa＿circ＿0070512was detected on human prostate cancer tissue microarrays by ISH assay,and the association between the expression level of hsa＿circ＿0070512 and the clinicopathological features of prostate cancer patients was analyzed.(2)The effects of overexpression or knockdown of hsa＿circ＿0070512 on the proliferation ability of prostate cancer cells were examined by cck-8 and Edu assays,the effects of overexpression or knockdown of hsa＿circ＿0070512 on the migration ability of prostate cancer cells were examined by scratch and transwell assays,and the effects of hsa＿circ＿0070512 on the cell cycle progression of prostate cancer cells were examined by flow cytometry.(3)Screening of mi RNA molecules with potential binding sites to hsa＿circ＿0070512 by bioinformatics,a dual luciferase reporter gene assay to verify the interaction between hsa＿circ＿0070512 and mi RNA,and FISH to verify the co-localization of hsa＿circ＿0070512 and mi RNA in prostate cancer cells.The effect of elevated or decreased mi RNA on the proliferation and migration of prostate cancer cells was examined by cck-8,Edu,scratch,transwell,and flow cytometry,and the rescue assay verified whether the effect of hsa＿circ＿0070512 on the biological function of prostate cancer cells was mi RNA-dependent.Further,prostate cancer cell lines stably overexpressing hsa＿circ＿0070512 were constructed,and the downstream target genes co-regulated by hsa＿circ＿0070512 and mi RNA molecules were screened by high-throughput sequencing and bioinformatics methods.q RT-PCR and western blot assays were performed to verify the effects of hsa＿circ＿0070512 and mi RNA on the expression levels of downstream target genes.Rescue assays were performed to verify whether the effect of hsa＿circ＿0070512 on the biological function of prostate cancer cells was dependent on the downstream target genes.(4)Subcutaneous tumorigenesis assay in naked mice to confirm the effect of hsa＿circ＿0070512 on prostate cell proliferation in vivo.The morphology of subcutaneous tumor tissue in naked mice was analyzed by H&E staining.The expression levels of hsa＿circ＿0070512,mi RNA,and downstream target genes in subcutaneous tumor tissues of nude mice were detected by q RT-PCR or western blot assay.The expression levels of downstream target genes and KI67 in subcutaneous tumor tissues of nude mice were detected by IHC assay.Results:(1)hsa＿circ＿0070512 was highly expressed in prostate cancer tissues and cells compared to paraneoplastic tissues and normal epithelial cells of the prostate.Compared with its homologous linear m RNA,PPP3 CA,hsa＿circ＿0070512 is more resistant to RNase R and Act D and has better stability.hsa＿circ＿0070512 can only be amplified in the c DNA of prostate cancer cells and not in g NDA.hsa＿circ＿0070512 is mainly distributed in the cytoplasm of prostate cancer cells.Prostate cancer patients with high expression of hsa＿circ＿0070512 had a higher tumor T-stage,Gleason score,Gleason grade,and lymph node metastasis rate.(2)In terms of function,overexpression of hsa＿circ＿0070512 promoted the proliferation and migration of prostate cancer cells,while knockdown of hsa＿circ＿0070512 inhibited the proliferation and migration of prostate cancer cells.(3)Mechanistically,hsa＿circ＿0070512 could sponge mi R-338-3p and down-regulate mi R-338-3p expression.The results of the dual luciferase reporter gene assay showed that hsa＿circ＿0070512 and mi R-338-3p have direct binding sites.FISH assay results showed that hsa＿circ＿0070512 and mi R-338-3p co-localized in the prostate cancer cytoplasm.mi R-338-3p was lowly expressed in prostate cancer tissues and cells and inhibited prostate cancer cell proliferation and migration.Elevated mi R-338-3p blocked to some extent the promotion of hsa＿circ＿0070512 on prostate cancer cell proliferation and migration.GLI2 is a central regulator of the hedgehog signaling pathway,and overexpression of hsa＿circ＿0070512 promoted the expression level of GLI2,while overexpression of mi R-338-3p inhibited the expression level of GLI2.The rescue assay showed that knockdown of GLI2 could inhibit the pro-proliferative and migratory effects of hsa＿circ＿0070512 on prostate cancer cells.(4)The results of the nude mice experiment showed that PC3 cells stably overexpressing hsa＿circ＿0070512 significantly promoted subcutaneous tumorigenesis in nude mice.Compared with the control nude mouse tumor tissues,the expression levels of hsa＿circ＿0070512 and GLI2 were significantly higher in hsa＿circ＿0070512overexpressing nude mouse tumor tissues,while the expression level of mi R-338-3p was significantly lower.Conclusion:This study reveals that hsa＿circ＿0070512,which is highly expressed in prostate cancer,promotes the proliferation and migration of prostate cancer cells through regulating the mi R-338-3p/hedgehog signaling pathway,providing new targets and directions for the diagnosis and treatment of prostate cancer.