H12、RS13和RL6在miR-199b-5p敲除小鼠卵巢中的表达研究

Expression of H12,RS13 and RL6 in ovarian tissues of miR-199b-5p knockout mouse

目的探讨组蛋白H1变体(H12)、核糖体蛋白S13(RS13)和核糖体蛋白L6(RL6)在miR-199b-5p敲除小鼠卵巢中的表达变化。方法(1)自行繁育miR-199b-5p敲除(Knockout,KO)和野生型(Wild-type,WT)C57BL/6小鼠,分别记录两组雌鼠产仔情况(2)基因型鉴定验证分组,取性成熟期小鼠(鼠周龄为11~13周)卵巢用于验证实验。(3)用实时荧光定量PCR(qPCR)检测每组小鼠(n=10)卵巢组织中H12、RS13和RL6的mRNA表达水平变化。(4)用蛋白免疫印迹法(WB)检测每组小鼠(n=3)卵巢组织中H12、RS13和RL6的蛋白表达水平变化。结果(1)共统计半年内两组雌鼠产仔情况:WT组生产27窝,平均每窝产仔数为(8.15±0.41)只;KO组生产28窝,平均每窝产仔数为6.43±0.32只,与WT组比较,KO组雌鼠产仔数显著下降(P=0.004),差异有统计学意义。(2)qPCR结果示:与WT组比较,KO组卵巢组织中H12(P=0.038)、RS13(P=0.011)和RL6(P=0.009)的mRNA表达水平显著升高,差异均有统计学意义。(3)WB结果示:与WT组比较,KO组小鼠卵巢组织中H12(P=0.014)蛋白表达水平显著升高,而RS13(P=0.005)和RL6(P=0.009)蛋白表达水平显著下降,差异均有统计学意义。结论敲除miR-199b-5p可能影响H12、RL6和RS13基因的表达使小鼠卵巢储备能力下降。

Objective To investigate the influence of miR-199b-5p knockout on ovarian reproductive capacity in mice,and to verify the expression changes of candidate genes which are histone H1 variants(H12),ribosomal protein S13(RS13)and ribosomal proteins L6(RL6)in the ovaries of miR-199b-5p knockout mouse.Methods The miR-199b-5p knockout(KO)and Wild-type(WT)C57BL/6 mice were self-bred,and the litter size of female mice was recorded respectively.(2)Genotype identification to verify the group,the ovaries of sexually mature mice(11-13 weeks old were taken for verification experiments.(3)The mRNA expression levels of H12,RS13 and RL6 genes in each group mouse of ovarian tissues(n=10)were detected by real-time quantitative PCR(qPCR).The protein expression levels of H12,RS13 and RL6 in each group mouse of ovarian tissues(n=3)were detected by western blot(WB).Results(1)We observed 27 litters of WT group and 28 litters of KO group in six months,respectively its average litter size were(8.15±0.41)and(6.43±0.32).Interestingly,the litter size of KO group females was significantly reduced(P=0.004).(2)Compared with WT group,the mRNA expression levels of H12(P=0.038),RS13(P=0.011)and RL6(P=0.009)were significantly increased by qPCR.(3)The protein expression level of H12(P=0.014)was visibly increased,while RS13(P=0.005)and RL6(P=0.009)were distinctly declined by WB.Conclusions The study demonstrates miR-199b-5p knockout may affect the expression of H12,RL6 and RS13 to induce the decrease of the ovarian reproductive capacity in mice.