Study On The Mechanism Of Circ＿0001178’s Tumor Suppressor Effect In Prostate Cancer By Mediating The MiR-587/CTSB Molecular Axis

Background:Prostate cancer(PCa)is one of the most common cancers in men worldwide,and its incidence rate ranks second in the world.In recent years,the incidence of PCa in my country has been increasing at an average rate of 12%.In addition,the proportion of patients with high-risk progressive and metastatic prostate cancer(mPC)in China is relatively high,and the 5-year survival rate is only 29%.Therefore,we need to deeply study the molecular mechanism of prostate cancer occurrence and metastasis,and the development of new diagnostic markers and therapeutic targets is the focus of current prostate cancer research.Autophagy is the main biological process of programmed cell death.Recently,some studies have reported that mutations in autophagy-related genes play an important role in the occurrence of neurodegenerative diseases,inflammation and tumors.Exploring autophagy-related mechanisms is of great significance for the occurrence and development of prostate cancer.Recent studies have demonstrated that a closed-loop RNA molecule,called circular RNA(circRNA),plays a key role in gene regulation,neurodevelopment and carcinogenesis,and has certain tissue and cell specificity.It shows great potential in terms of prognosis and treatment.However,there are few studies on the role of circRNAs in prostate cancer.Therefore,studying the expression and function of circRNA in the occurrence and metastasis of prostate cancer and its mechanism of action can provide a new idea for the diagnosis and treatment of prostate cancer.Through data mining technology and verification,our group found that the autophagy-related gene CTSB was under-expressed in prostate cancer.The previous circRNA chip detection results identified 163 up-regulated circRNAs and 145 down-regulated circRNAs,of which circ＿0001178 was significantly down-regulated.The molecular axis of circ＿0001178/miR-587/CTSB was determined by analysis.This topic will explore the regulatory mechanism of circ＿0001178 on CTSB,in order to find new targets for prostate cancer treatment,and provide a theoretical basis for the clinical application of autophagy inhibition.Objectives:(1)To screen the prostate cancer autophagy-related gene CTSB from the database.(2)To explore the effect of circ＿0001178 on prostate cancer progression in vitro.(3)To explore the tumor suppressor effect of circ＿0001178 in prostate cancer by mediating the miR-587/CTSB molecular axis at the animal level.Methods:(1)Genomic information was obtained from the TCGA database,GEO database,and HADb database.The GSE61790 dataset was screened to identify differentially expressed autophagy-related genes.Based on the analysis results,a protein interaction network was constructed,and GO enrichment and KEGG pathway analysis were performed.(2)Verify the mRNA and protein levels of differentially expressed autophagy-related genes in clinical samples.(3)CircRNA chips were used to screen differentially expressed circRNAs in prostate cancer tissues,and to verify gene expression differences in cell experiments.(4)The circRNA lentivirus overexpression vector was constructed and transfected into LNCap cells.The experimental method of CCK8,scratch assay,flow cytometry,and Transwell assay were used to detect cell proliferation,migration,apoptosis,and invasion abilities;Western Blotting,RT-qPCR assay were detected the changes of apoptosis and proliferation-related mRNA and protein levels.(5)The circRNA＿0001178-miR587,miR587-CTSB targeting regulation relationship were detected by the Dual-Luciferase Reporter Assay.To inhibit miR-587 in prostate cancer cells,the experimental method of CCK8,scratch assay,flow cytometry,and transwell assay were used to detect changes in cell proliferation,migration,apoptosis,and invasion;The experimental method of western Blotting,RT-qPCR assays were used to detect the changes of mRNA and protein levels of apoptosis and proliferation.The reverse experiment verified that circ＿0001178 could target and regulate miR-587 through ceRNA mechanism to affect cell proliferation,migration,apoptosis and invasion.(6)Construct circ＿0001178 plasmid and empty vector plasmid to stably transform LNCaP cells to establish prostate cancer xenograft mouse model.The weight and volume of the tumor were measured;Western Blotting and PCR were used to detect the expression levels of related proteins and mRNAs in the tumor tissue.Results(1)Twenty-six co-expressed differentially autophagy-related genes(ARGs)were identified between TCGA and HADb data,of which 7 genes were up-regulated and 19 genes were down-regulated.Through differential expression validation on the GSE61790 dataset,5 ARGs were identified,of which 4 were up-regulated and 1 was down-regulated,including BIRC5,TP63,FOS,NFE2L2 and CTSB.(2)RT-qPCR and Western Blot verification results in 3 pairs of prostate cancer and adjacent tissue samples showed that CTSB mRNA and protein were lowly expressed in prostate cancer tissue,and had statistical significance.(3)The circRNA chip detection results identified 163 up-regulated circRNAs and 145 down-regulated circRNAs.Nucleocytoplasmic separation experiments confirmed that circ＿0001178 was located in the cytoplasm,and sanger sequencing confirmed the ring structure of circ＿0001178.(4)Overexpression of circ＿0001178 significantly inhibited the proliferation,invasion and apoptosis of LNCaP cells in vitro,but did not affect the migration of LNCaP cells.(5)The dual-luciferase reporter gene assay confirmed that circ＿0001178 targeted to hsa-miR-587 and hsa-miR-587 targeted to CTSB.Inhibition of hsa-miR-587 can significantly inhibit the proliferation,migration and invasion of LNCaP cells,and promote their apoptosis.Over-expression of hsa-miR-587 attenuated the inhibitory effect of over-expression of circ＿0001178 on the proliferation and invasion of LNCaP cells.(6)Animal experiments verified that over-expression of circ＿0001178 could inhibit the expression level of hsa-miR-587,up-regulate the expression of CTSB in tumor tissue,thereby inhibiting tumor growth.Conclusions:(1)CTSB is lowly expressed in prostate cancer and can be used as a target for diagnosis and treatment.(2)The low expression of circ＿0001178 in prostate cancer and clarified the antitumor effect of circ＿0001178.Determined the molecular mechanism of circ＿0001178 inhibiting the development of prostate cancer through the hsa-miR-587/CTSB molecular axis.Circ＿0001178 is a potential role as clinical diagnostic and therapeutic target for prostate cancer.