MiR-34b-5p Regulates Cattleyak Sertoli Cells Proliferation And Apoptosis By Targeting PPP1R11

Cattleyaks,the crossbred offspring between yak(Bos grunniens)and cattle(Bos taurus),exhibits markedly higher performance in milk and meat production than yak,and equivalent adaptability on Qinghai-Tibetan Plateau as yak.However,the F1 males of cattleyak are infertile resulted from spermatogenic arrest,which greatly restricts the breeding process of cattleyak and the effective utilization of heterosis.Sertoli cells play an important role in male reproduction.Meanwhile,the proliferation and activity of cattleyak SCs is significantly lower than the counterparts of yak.Previous studies showed that miR-34b-5p is differently expressed in adult cattleyak and yak testis tissues.Up to now,it’s still a tremendous challenge to reveal the mechanisms of cattleyak male sterility.In this study,cattleyak and yak SCs were used as experimental materials.RT-PCR,q RT-PCR,Western blot,immunohistochemical staining,immunofluorescence staining,Ed U staining,TUNEL staining,vector construction,dualluciferase reporter gene reporter system,RNA interference,overexpression and other molecular and cellular biological methods were used to preliminarily explored molecular mechanism of miR-34b-5p and PPP1R11 on the proliferation and apoptosis of cattleyak SCs.The results are as follows:(1)The effects of miR-34b-5p on cattleyak SCs.The SCs of cattleyak and yak(N=3)were isolated in vitro,and identified by GATA4 and Vimentin antibodies.The miR-34b-5p expression in cattleyak SCs was significantly higher than that of yak(P<0.01).Overexpression of miR-34b-5p inhibited SCs proliferation and promoted apoptosis,and the m RNA expression of PCNA,CCND1,and CCNE1 were significantly down-regulated(P<0.05),while the m RNA expression of BAX,CASP8,and CASP9 were significantly up-regulated(P<0.05).On the contrary,knockdown of miR-34b-5p promoted the proliferation and inhibited apoptosis of cattleyak SCs,and the expressions of PCNA,CCNB1,CCND1,CDK4 and BCL-2 were significantly increased(P<0.05),but CASP9 m RNA expression was significantly downregulated(P<0.05).(2)Identification the targeted relationship between miR-34b-5p and PPP1R11.The miR-34b-5p targeted PPP1R11 was predicted by bioinformatics software.Overexpression and knockdown of miR-34b-5p respectively inhibited and promoted the expression of PPP1R11 in cattleyak SCs.The dual-luciferase reporter gene experiment showed that the luciferase activity of WT and MUTA1 vector groups were decreased extremely significantly after overexpression of miR-34b-5p(P<0.01),but the luciferase activity of pmir GLO,MUTA2 and MUTA1&2vector groups were no significant difference.All above results indicated that miR-34b-5p inhibits PPP1R11 expression by targeting PPP1R11 3’UTR 393-400 nucleotide bases.(3)Expression pattern of PPP1R11 in cattleyak testis.The PPP1R11 gene encoding 107 amino acids,and the PPP1R11 protein has high homology in mammals.PPP1R11 is widely expressed in adult cattleyak,and the expression level in testis is significantly higher than other tissues(P<0.01).Furthermore,there was significant difference(P<0.01)in testis at juvenile and adult stage of cattleyak compared with the counterpart of yak.In addition,IHC staining showed that PPP1R11 protein was significantly lower expressed in cattleyak spermatogonia and SCs compared with that of yak.The expression levels of PPP1R11 m RNA and protein in cattleyak SCs were also significantly lower than that of yak(P<0.01).(4)The effects of overexpression and knockdown PPP1R11 on cattleyak SCs.Overexpression of PPP1R11 promoted cattleyak SCs proliferation and inhibited apoptosis,and the expression levels of PCNA and CCND1 were significantly up-regulated(P<0.05),while the expression levels of CASP8 and CASP9 were significantly down-regulated(P<0.05).In contrast,knocking down the expression of PPP1R11 inhibited cattleyak SCs proliferation and promoted apoptosis,and PCNA,CCNB1,CCND1 m RNA levels were significantly downregulated(P<0.05),but BAX,CASP8,CASP9 m RNA levels were significantly decreased(P<0.05).In conclusion,miR-34b-5p targets PPP1R11 to regulate cattleyak SCs proliferation and apoptosis.It suggesting that male cattleyak infertility may be related to the abnormally expression of miR-34b-5p and PPP1R11 in SCs.This study preliminarily revealed that miR-34b-5p has play an important role in cattleyak SCs,and provided a reference for further research on molecular mechanism of male cattleyak infertility.