| ObjectiveTo study the effect of cadmium on the activation of PERK-eIF2α-ATF4 pathway in ovarian granulosa cells induced by endoplasmic reticulum stress and its possible mechanisms.The aim was to provide scientific basis for further study on the damage of ovarian granulosa cells induced by cadmium.Methods1 Expressions of BIP,p-PERK and p-eIF2α in rat ovarian granulosa cells exposed to cadmium21 days old 50-60 g female SD rat ovarian granulosa cells were cultured in vitro for36 h,cultured with CdCl2 at dose of 5 μM,10 μM,and 20 μM CdCl2 for 12 h,and the negative control group(cultured with no cadmium)was set up.Extracting total RNA and total protein,and detecting the expressions of BIP,p-PERK and p-eIF2α by RT-PCR and Western Blot respectively.2 Expressions of BIP,p-PERK and p-eIF2α in COV434 cells exposed to cadmium after BIP knockdownUsing COV434 cells as a tool,using RNA interference technology to construct BIP gene knockdown cell line COV434-BIPshRNA,dose group were set up as 0μM CdCl2COV434,20 μM CdCl2 COV434,20 μM CdCl2 COV434-Control,20 μM CdCl2COV434-BIPshRNA.After treatment for 12 h,extracting total RNA and total protein,and detecting the expressions of BIP,p-PERK and p-eIF2α by RT-PCR and Western Blot respectively.3 MicroRNAs expressions of BIP gene in rat granulosa cells exposed to cadmiumThrough the miRNA chip technology to screen differential expression of miRNA in ovarian granulosa cells exposured to cadmium(Fold,change≥ 1.33,or ≤0.77 and P <0.05),to use online database predict miRNA gene “http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk/ predicted mirna gene.php”,and through the database miRDB,microRNA.org,Target Scan compared by miRNA score,selecting endoplasmic reticulum stress related miRNA to detect;21 day old female 50-60 g SD rat granulosa cells were cultured in vitro for 12 h,5 μM、10 μM、20 μM CdCl2 and the negative control group(culture medium with no cadmium)were set up.Extracting total RNA,and detecting the expressions of miRNAs by RT-PCR.Results1 The expressions of BIP,p-PERK and p-eIF2α in rat ovarian granulosa cells exposed to cadmium(1)Compared with the control group,the expression of BIP mRNA of 5 μM CdCl2 was downregulated,expressions of 10 μM CdCl2、20 μM CdCl2 BIP mRNA and protein were upregulated,the difference was statistically significant(P < 0.05).(2)Compared with the control group,the expressions of p-PERK protein of 5 μM CdCl2,10 μM CdCl2,20 μM CdCl2 were upregulated,the differences were statistically significant(P < 0.05).(3)Compared with the control group,the expressions of p-eIF2α protein of 5 μM CdCl2 10 μM CdCl2,20 μM CdCl2 were upregulated,the differences were statistically significant(P < 0.05).2 Expressions of BIP,p-PERK and p-eIF2α in COV434 cells exposed to cadmium after BIP gene silencing(1)Compared with the control group,the expressions of BIP mRNA and protein of20 μM CdCl2 COV434 was incresed,the difference were statistically significant(P<0.01),the expressions of BIP mRNA and protein of 20 μM CdCl2 COV434 were incresed,the differences were statistically significant(P < 0.01);(2)Compared with 20 μM CdCl2 COV434,the expression of p-PERK protein of 20μM CdCl2 COV434-BIPshRNA showed no significant difference(P > 0.05);the expression of p-eIF2α protein of 20 μM CdCl2 COV434-BIPshRNA was declined,the difference were statistically significant(P<0.05);(3)There was no significant difference in the expressions of BIP,p-eIF2α protein and p-PERK protein in COV434-C compared with 20 μM CdCl2 COV434(P > 0.05).3 Changes of microRNAs related to BIP gene expressed in rat granulosa cells exposed to cadmium(1)5 miRNAs were selected: rno-miR-199a-5p、rno-miR-181a-5p、rno-miR-495、rno-miR-30b-3p and rno-miR-150-5p;(2)Compared with the control group,the expressions of rno-miR-181a-5p,rno-miR-30b-3p,rno-miR-150-5p,rno-miR-199-5p,rno-miR-495 showed no significant difference compared with control group(P > 0.05).Conclusions Under this experimental condition:1 Cadmium induced the damage of ovarian granulosa cells through endoplasmic reticulum stress.2 When the rat ovarian granulosa cells exposed to high dose(20 μM)cadmium,rno-miR-181a-5p,rno-miR-30b-3p,rno-miR-150-5p,rno-miR-199-5p,rno-miR-495 were not likely involved in the activation of BIP. |
