Epigenetic Regulation Mechanisms Of ZCCHC13 And PLCXD3 Genes In Azoospermia And Hepatocellular Carcinoma Revealed By Integrated Microarrays

Highly expressed genes within testicular tissues not only play essential roles for biological process of germ cells,but also have strong relationship with various tumors.Cancer-testis gene is the most representative example.miRNA and DNA methylation are two main ways of epigenetic regulation.The objective of this study was to illustrate the epigenetic mechanisms of highly expressed genes in testicular tissues.Testis biopsies of patients with non-obstructive azoospermia(NOA)and obstructive azoospermia OA(with normal spermatogenesis)were evaluated by microarray analysis to examine the profiles of DNA methylation,mRNA and miRNA expression.Some of the most interesting results were validated by real time PCR using samples from the same cohort.Two bioinformatic approaches:miRNA-mRNA integrated analysis and mRNA methylation integrated analysis were built and applied to screen microarray data and biological information.I mmunohistochemical results showed positive stainings of PLCXD3 and ZCCHC13 in OA testes while negative stainings in NOA testes.According to the microarray data,targeted miRNAs and DNA methylated CpG sites were further studied in the germ and tumor cell lines using the molecular biological methods.The main results were listed as following:1.mRNA,miRNA and DNA methylation profiles were built and compared using the testis biopsies of NOA and OA,showing two types of testicular tissues have totally different patterns in either transcription or methylation.2.Differentially expressed genes and miRNAs were integrated analyzed and then constructed a miRNA-gene network.Both software prediction and experiment results revealed two pairs with negative relationship:PLCXD3-miR-34c-3p,and ZCCHC13-miR-369-3p.3.We detected the distributions of PLCXD3 and ZCCHC13 in testis of mice,OA patients and NOA patients.Positively i mmunological signals were showed in the testes of mice and OA(normal spermatogenesis)instead of NOA.Besides,mRNA and protein expressions of ZCCHC13 in normal liver tissues were extremely low.4.Differentially expressed genes and DNA methylated probes were integrated analyzed,showing hypermethylated modifications in the promoter regions of ZCCHC13.The hypomethylating agent resulted in a dose-dependent increase of levels of ZCCHC13 protein and mRNA.5.Hypomethylation is the epigenetic factor causing high expressions of ZCCHC13 protein and mRNA in hepatocellular carcinoma(HCC)tissues.Mechanistic investigations revealed that ZCCHC13 upregulated CDK1 and CDK4 expression,accompanying by the activation of AKT/ERK/c-MYC pathway.Overexpression of ZCCHC13 protein facilitates the G1-S cell cycle transition.Taken together,this study established an integrative analysis of microarray data using human testicular biopsies and identified two unknown genes PLCXD3 and ZCCHC13.For two genes,we made a deep investigation on targeted miRNA and DNA methylation in germ cells and tumor cells.Our results provided vital experimental evidence and clues for exploring the pathological mechanisms of azoospermia and cancer in futures.