Aberrant DNA Hypermethylation-Mediated Silencing Of LncRNA GAS5 Aggravates Prostate Cancer Via Sponging MiR-223-3p

Objective:This study aims to clarify the function and mechanism of long-chain non-coding RNA GAS5 in the progression of prostate cancer,and the regulation of GAS5 expression by DNA methylation.Method:(1)The GEO database was used to analyzes the expression level of lncRNA GAS5 in prostate cancer tissues,and qRT-PCR was applied to detect the expression of GAS5 in prostate cancer clinical samples and cell lines.(2)The expression plasmid or siRNA of GAS5 was transiently transfected into prostate cancer cells DU-145 and PC-3 to upregulated and knockdown GAS5,the effect of abnormal expression of GAS5 on the biological behavior of prostate cancer was determined.CCK8 and clone formation experiment was used to analyze the proliferation ability of prostate cancer.Wound healing was used to analyze the migration ability of prostate cancer cells.Transwell was used to analyze cell migration and invasion ability.(3)Construct a DU-145 cell line with stable overexpression or knockdown of GAS5,and use a nude mouse subcutaneous tumor model to determine the effect of GAS5 on prostate cancer cell tumor formation and growth.HE staining was used for pathological analysis of tumor tissues.(4)Western blot was applied to detec he expression of key DNA methylation modification proteins-DNMT1 and DNMT3B in human normal prostate epithelial cells WPMY-1 and prostate cancer cells DU-145 and PC-3.Methyl Primer Express v1.0 software analyzes the distribution of CpG islands in the GAS5 promoter region,and MSP was used to detect the methylation level of the GAS5 promoter in prostate cancer tissues and cells.Prostate cancer cells treated with DNA methylation inhibitor 5-Azacytidine were used to detect the expression of GAS5 to determine the effect of DNA methylation on the expression of GAS5 in prostate cancer.(5)Using online database starBase and miRCancer to predict the potential target gene of GAS5-miR-223-3p.The luciferase reporter gene experiment verified the targeted binding of GAS5 and miR-223-3p.Functional replenishment experiments verified that miR-223-3p mediates the tumor suppressor effect of GAS5.CCK8,clone formation,scratch and transwell were used to analyze the biological phenotype of prostate cancer cells.Results:(1)The expression of lncRNA GAS5 in prostate cancer tissues and cell lines was significantly decreased.(2)Increased expression of lncRNA GAS5 inhibits the proliferation,migration and invasion of prostate cancer cells.(3)LncRNA GAS5 silencing promotes the proliferation,migration and invasion of prostate cancer cells.(4)LncRNA GAS5 inhibits prostate cancer tumor growth.(5)The hypermethylation of the lncRNA GAS5 promoter leads to a decrease in its expression in prostate cancer.(6)LncRNA GAS5 regulates the occurrence and development of prostate cancer by targeting miR-223-3p.Conclusion:The expression of lncRNA GAS5 is significantly reduced in prostate cancer tissues and cell lines,and this expression inhibition is caused by hypermeth lation in the promoter region of GAS5.High expression of lncRNA GAS5 inhibits the proliferation,migration and invasion of prostate cancer cells,while GAS5 silencing promotes the malignant progression of prostate cancer.LncRNA GAS5 negatively regulates its expression through targeted binding of miR-223-3p,and exerts the function of inhibiting the progression of prostate cancer.The above results suggest that GAS5 plays an important role in the occurrence and development of prostate cancer,and may become an effective target in the treatment of prostate cancer in the future,and is expected to become a biomarker for the diagnosis and prognosis of prostate cancer patients.