RESUMO
Objective To explore the effect of endoplasmic reticulum stress activating transcription factor 6(ATF6)on the expression of reproduction related gene heat shock protein family A member 1 like(HSPA1L)and preliminari-ly clarify its regulatory molecular mechanism.Methods The ATF6 over-expression plasmid was transfected into HEK-293T cells and the over-expression efficiency was verified by RT-qPCR and Western blot.The transcriptome sequen-cing information of testis tissue of male ATF6 knockout mice was used to screen five reproduction related genes down-stream of ATF6.The dual luciferase reporter assay selected the downstream genes with high promoter activity and de-tected the effect of over-expression of ATF6 on the promoter activity of downstream genes.The possible binding sites of ATF6 and downstream gene promoters were predicted by gene-regulation.RT-qPCR and Western blot were used to detect the effect of over-expression of ATF6 on downstream gene expression in HEK-293T cells.Whether ATF6 binds to downstream gene promoters was determined by electrophoretic mobility shift assay(EMSA).Results The expres-sion of ATF6 mRNA(P＜0.001)and protein(P＜0.001 and P＜0.05)in HEK-293T cells was significantly increased after transfection.HSPA1L(P＜0.001 and P＜0.05),a reproductive related gene downstream of ATF6 was screened by transcriptome sequencing and dual luciferase reporter assay.ATF6 promoted the truncated promoter activity of HSPA1L(P＜0.001).After over-expression of ATF6,the expression of HSPA1L was significantly increased(P＜0.001).The differences were statistically significant.ATF6 protein could bind to the aagtcgtcac DNA sequence of HSPA1L promoter.Conclusions ATF6,a key molecule of endoplasmic reticulum stress,regulates the expression of reproduction related gene HSPA1L by binding to the promoter of HSPA1L.This result will lay a foundation for further research on the prevention or treatment of endoplasmic reticulum stress(ERS)related male infertility.
RESUMO
Objective To investigate the effect of ADAR2 on the expression of MAVS and its mechanism.MethodsThe RT-qRCR was used to detect the expression of ADAR gene family, the expression level of ADAR2 and MAVS in cells.The effect of ADAR2 on the 3'UTR region of MAVS was detected by Pyrosequencing.To detect the expression of luciferase by dual luciferase reporter plasmid assay;The expression of ADAR2 and MAVS were detected by Western blot.Results In the ADAR family, the abundance of ADAR1 was the highest, followed by ADAR2, but the expression of ADAR3 was poor, which was almost impossible to detect(P<0.05).ADAR2 played a critical role in RNA editing of chr20:3869744 sites on the 3'UTR region of MAVS(P<0.001).On the 3'UTR editing site of MAVS, the luciferase activity of the edited G allele was significantly lower than that of the normal A allele(P<0.01).At the level of transcription and translation, ADAR2 significantly inhibited the expression of MAVS(P<0.05).Conclusions ADAR2 by editing MAVS` 3'UTR on the chr20:3869744 site, which makes the occurrence of A to G replacement, inhibits the expression of MAVS.