Identification Of Suitable Reference Genes For MRNA And MicroRNA Normalization In Androgen-treated Human Prostate Cancer Cell Lines

Real-time quantitative PCR(RT-qPCR)is a powerful technique for studying gene expression and it is widely used to analyze changes in gene expression due to its high sensitivity.To obtain accurateqPCR results,it is necessary to select suitable reference genes for normalisation.Indeed,a large number studies have shown that an ideal reference gene for all samples and conditions does not exist,and biased normalisation can cause inaccurate quantification and incorrect conclusions.Prostate cancer is a common malignancy in the male genitourinary system.Previous studies have indicated that androgen plays an important role in prostate cancer.Because by androgen receptor(AR)androgen can regulate the expression of many genes including mRNAs and miRNAs.Therefore,identification of androgen-regulated genes as therapeutic targets in prostate cancer is of considerable importance for understandingthe mechanisms of prostate cancer development and progression.However,there is currently no study about selection of the most suitable reference genes in prostate cancer cell lines with differ androgen treatment.In this study,one human prostate epithelial cell and five human prostate cancer cell lines were used to evaluate the expression stability of ten mRNA and six npcRNA reference genes by using various dihydrotestosterone(DHT)treatments.We examined the effects of DHT-treatments on the test sample by Western blotexperiments.Reference gene expression stability was calculated using three programs include geNorm,NormFinder and Bestkeeper,and the recommended comprehensive ranking was provided.Our results show that TBP and ACTB,miR-16 and miR-1228-3p are the most suitable reference gene combinations for mRNA and miRNA analysis in all prostate cancer cell linesrespectively.Considering prostate cancer cell types,YWHAZ/ACTB and miR-16/miR-1228-3p are the most suitable reference gene combinations for mRNA and miRNA analysis in AR+ cell lines,when TBP/GAPDH and RNU43/RNU6-2 are the optimal combination of genes in AR-cell lines.In addition,when the target genes PCA3 and miR-141 were normalized using reference genes with different stability,the expression level of the target gene showed a significant difference.This suggests that the importance of selecting the appropriate reference gene for standardization in the RT-qPCR gene expression analysis.To our knowledge,this is the first report on validation of reference genes under different DHT treatments in prostate cancer cells.This study is helpful in discovering more accurate and reliable DHT-regulate genes in prostate cancer cell lines.