Construction Of LncRNA-miRNA-mRNA Regulatory Network And Related Mechanism In Nickel-Induced Steroidogenesis Disturbance In Rat Leydig Cells

Objectives:To screen significantly differentially expressed long non-coding RNAs?lncRNAs?,microRNA?miRNAs?and mRNAs,construct the competitive endogenous RNA?ceRNA?network and explore the role of phosphoinositide 3-kinase?PI3K?/protein kinase B?Akt?signaling pathways.Our findings provided a better understanding of the potential molecular mechanism in steroidogenesis disturbance induced by NiSO₄ in rat Leydig cells.Methods:1)The peeled testes were digested with 0.25%collagenase IV,and the Leydig cells were then purified by gradient density centrifugation with percoll at concentrations of 5,30,58 and 70%as well as culturing for 24 h.2)The morphology of the Leydig cells treated with different concentrations NiSO₄?0,250,500 and 1000?mol/L?for 24 h was observed using an inverted microscope.3)The cell survival rate was assayed by MTT method.4)Testosterone production of the Leydig cells in response to different doses of NiSO₄?0,250,500,1000?mol/L?for 24 h in the presence of 1 IU/mL human chorionic gonadotropin?hCG?was measured using enzyme-linked immunosorbent assay?ELISA?.5)Detection of steroidogenic acute regulatory protein?StAR?and cytochrome P450 cholesterol side chain cleavage enzyme?CYP11A1?was performed using Western Blot.6)To investigate the differentially expressed lncRNAs,miRNAs and mRNAs?DEMs?in rat Leydig cells exposed to 0 or 1000?mol/L NiSO₄ for 24 h by RNA sequencing?RNA-Seq?based on a high-throughput platform.7)The Gene ontology?GO?as well as Kyoto Encyclopedia of Genes and Genomes?KEGG?biological pathway analysis for DEMs were performed to infer potential functions in biological processes?BP?and pathway.8)The ceRNA network of lncRNA-miRNA-mRNA associated with steroid production was constructed based on the negative target relationship pairs between lncRNA-miRNA and miRNA-mRNA by using MiRanda and PITA.9)RT-qPCR was performed to verify the expression levels of the ceRNA network related genes and their dose dependence relationship under different doses of NiSO₄.10)RT-qPCR was performed to quantify the relative mRNA expression of Pik3r1 and Akt1.Detection of relative protein expression and phosphorylated protein levels of PI3K and Akt were performed using Western Blot.Results:1)An intense occurrence of vacuoles was found in Leydig cells treatment with 1000?mol/L NiSO₄ in comparison with the control group?P<0.05?.The cell viability was decreased with the increase of NiSO₄ concentration?0,250,500and 1000?mol/L?.2)The levels of testosterone in the culture medium of each treatment group were obviously lower in a dose-dependent manner than that in the control?P<0.05?.In addition,the protein expression of StAR and CYP11A1 in each treatment group were decreased with the increase of NiSO₄ concentration?P<0.05?.3)A total of 372 lncRNAs and 27 miRNAs?FC>2,P<0.05?and 3666 mRNAs?FC>2,P<0.01,FDR<0.01?were identified to be greatly differentially expressed between the NiSO₄ group and control group.The GO terms in BP and pathways associated with steroidogenesis were significantly enriched,including the“MAPK signaling pathway”,“steroid biosynthetic process”,“PI3K/Akt signaling pathway”,etc.CeRNA network of lncRNA-miRNA-mRNA related to steroidogenesis was constructed between 75lncRNAs,16 miRNAs,and 15 mRNAs in NiSO₄-treated rat Leydig cells.4)Among the 18 verified RNAs,15 RNAs expression levels were in line with the high-throughput sequencing results.Meanwhile,LOC102549726/miR-760-3p/Atf6,LOC102549726/miR-760-3p/Ets1,LOC102549726/miR-760-3p/Sik1,as well as AABR07037489.1/miR-708-5p/Mapk14 ceRNA networks were confirmed.With the exception of Mapk14,the expression of the genes involved in the four ceRNA networks were altered with increasing concentration of NiSO₄ and were consistent with the expression trend of RNA-Seq and previous validation.5)Compared with the control group,the mRNA expression level of Pik3r1 was increased in each group,and the mRNA expression levels of Akt1 treatment with 500 and 1000?mol/L NiSO₄ were decreased?P<0.05?.The protein expression of PI3K and Akt in each group did not change?P>0.05?.The expression level of p-PI3K p85 was decreased with the increase of NiSO₄ concentration while there was no significant difference?P>0.05?.The p-Akt protein expression was significantly lower in 1000?mol/L groups than that in the control?P<0.05?.The ratio of p-PI3K p85/PI3K p85 in each group and p-Akt Ser473/Akt in 1000?mol/L NiSO₄ group were significantly decreased?P<0.05?.Conclusions:1)The ceRNA networks?LOC102549726/miR-760-3p/Atf6,LOC102549726/miR-760-3p/Ets1,LOC102549726/miR-760-3p/Sik1,as well as AABR07037489.1/miR-708-5p/Mapk14?may involve in NiSO₄-induced steroid synthesis disorder in rat Leydig cells.2)The PI3K/Akt signaling pathway was inhibited in rat Leydig cells exposed to NiSO₄,which may affect testosterone synthesis.