| Objectives:After optimize the method for purification of miRNAs in seminal plasma,To investigate the expression of miR-106b in seminal plasma of patients with different types of male infertility,and to explore the possibility of miR-106b as a molecular target for the diagnosis of male infertility.The testes of mice at different developmental stages and the spermatogonia(GC-1),spermatogon(GC-2)cell lines were used as materials to study the effect of miR-106b on the function of spermatogenic cells and its molecular mechanism.Methods:1.Optimization and verification of purification methods of miRNA in human seminal plasma:Collecting semen samples from National Health and Family Planning of Reproductive Health Service Center.The semen sample were divided into four groups according to different RNA extraction methods:Trizol LS group,protease K+ Trizol LS group,miRNeasy Micro kit group and Trizol LS + miRNeasy Micro kit group(modified group).The purity and quality of purified miRNAs were detected by Agilent2100 bioanalyzer.The melt curves of miR-106b and miR-9 were compared by real-time PCR.2.Expression of miR-106b in seminal plasma of male infertility:Collecting 15 cases of normal semen samples(Semen parameters are normal and the spouse is pregnant within one year and normal delivery),30 cases of semen samples which were clinically diagnosed as non-obstructive azoospermia,10 cases of semen samples which were clinically diagnosed as asthenospermia,all semen samples were from Technical service center of family planning and reproductive health.Real-time PCR was used to detect the expression of miR-106b in normal group(n=5)and non-obstructive azoospermia group(n = 10),then expanded sample size as nonnal group(in = 10),the non-obstructive azoospermia(n = 20)and the asthenospermia group(n = 10)were used to detect the expression of miR-106b.3.The effect of miR-106b on the function of spermatogenic cells and its molecular mechanism(1)Real-time PCR was used to detect the expression level of miR-106b in testes of mice at different developmental stages(2)Lentivirus packaging miR-106b mimics,miR-106b inhibitor miR-106b mimics control and miR-106b inhibitor control infect the mouse spermatogonia(GC-1),spermatogon(GC-2)cell line to format stable infection of cell lines.MTS method was used to detect the proliferation of GC-1 and GC-2 after infection with lentivirus.The effect of miR-106 on the apoptosis and cycle of spermatogonia and spermatocytes was detected by flow cytometry(FCM).(3)The target gene of miR-106b was predicted by bioinformatics analysis software,and the target genes related to proliferation and apoptosis of spermatogenic cells were screened.The correlation between miR-106b and predicted target gene was detected by Real-time PCR.Results:(1)The miRNA OD250/280 of modified group[(1.90 ± 0.03)]was significantly higher than that of the other three groups(P<0.05).(2)The purified miRNAs of modified group were detected by Aglient 2100 bioanalysis system,the chromatographic position was specific,the peak was clear and the quality was satisfactory,which could meet the requirements of the follow-up experiment.(3)Real-time PCR detection of miR-106b showed that the peaks of the first three groups were not single,non-specific amplification significantly,but the melt curve of the modified group was quite correctly,the shape of the peaks were narrow and sharp.Real-time PCR detection of miR-9 showed that the first two groups were still not a single peak,with non-specific amplification,although the third group had a single peak,but the rise curve compared to the modified group,was not smooth enough.2.Real-time PCR was used to validate the expression of miR-106b in the small sample size of normal seminal plasma(n=5)and seminal plasma from non-obstructive azoospermia(n=10).The results showed that the expression of miR-106b in seminal plasma of patients with non-obstructive azoospermia was significantly down-regulated(P<0.05).Expand the sample size to detect the expression of miR-106b in seminal plasma samples from normal people(n = 10),the patient of non-obstructive azoospermia(n = 20)and asthenospermia(n = 10).The result showed that compared with the normal group,the expression ofmiR-106b in the patients with non-obstructive azoospermia and the asthenospermia group was significantly lower(P<0.01).Compared with the non-obstructive azoospermia group,the expression of miR-106b in the asthenospennia group was increased(P<0.05).3(1)Real-time PCR was used to detect the expression of miR-106b in the testis of mice at different developmental stages.It was found that the expression level of miR-106b was the highest at 7th day.Between 7-35day,the expression of miR-106b gradually decreased with the development of mouse testes(P<0.001).The expression of miR-106b in mouse testis was not significantly changed from 35 days to 64 days.(2)Lentivirus infected GC-1 and GC-2,and the green fluorescence intensity was very high under the confocal microscopy.The infection rate was as high as 85%.Real-time PCR results showed that after lentivirus infected GC-1 and GC-2,The expression of miR-106b in miR-106b mimics group was significantly higher than its control(/P<0.01),and the expression of miR-106b in miR-106b inhibitor was significantly lower than its control(P<0.01)(3)miR-106b mimics infected GC-1,compared with its control group,cell proliferation ability has increased significantly(P<0.05),while the trend of miR-106b inhibitor is just the opposite of miR-106b mimics,after inhibit the expression of miR-106b,the proliferation of GC-1 was weakened,compared with the control group,the difference was significant(P<0.01).After miR-106b mimic infected GC-2,cell proliferation ability has increased significantly,compared with the control group,especially at day 2 and day 4(P<0.01).While the proliferation ability of GC-2 was decreased when inhibite the expression of miR-106b.Compared with the control group,the difference was significant at day 3 and day 4.(4)Flow cytometry showed that the apoptotic rate of miR-106b mimics was significantly lower than its control,and the apoptotic rate of miR-106b inhibitor was significantly higher than its control.That explain miR-106b inhibit germ cell apoptosis;In the study of the regulation of miR-106b on GC-1 and GC-2 cycles,after lentivirus infected GC-1,GC-2,miR-106b mimics group compared with miR-106b inhibitor group,the percentage of cells in GO/G1 phase was significantly reduced,and the percentage of cells in S phase was increased significantly(P<0.05)(5)Target gene prediction and verification results showed that promoting the expression of miR-106b in GC-1,GC-2,significantly reduced the expression of STAT3,whereas inhibited the expression of miR-106b can upregulate the expression of STAT3.In GC-1,the expression of miR-106b was significantly decreased(P<0.05),while the expression of miR-106b inhibitor was not significantly different from that of the control.In GC-2,promoting the expression of miR-106b that made the expression of SOCS3 decreased(P<0.05),while the expression of SOCS3 was significantly decreased in the miR-106b inhibitor group(P<0.05).In GC-1,up-regulated the expression of miR-106b that made the expression of SMAD5 down.Down-regulated the expression of miR-106b that made the expression of SMAD5 up(P>0.05).In GC-2,promoting the expression of miR-106b can increase in the expression of SMAD5,while the expression levels of SMAD5 in miR-106b inhibitor and its control were below miR-106b mimics and its control(P>0.05).Conclusion:1.Trizol LS combined with miRNeasy Micro kit for purifying seminal plasma miRNA is the best way to meet the follow-up real-time quantitative PCR,and other experimental requirements.2.Compared with the normal group,miR-106b was significantly down-regulated in seminal plasma of patients with non-obstructive azoospermia and asthenospernia.Compared with non-obstructive azoospermia group,the expression level of miR-106b was significantly increased,all of which were statistically significant.3.The expression of miR-106b gradually decreased with the development of mouse testes,and the expression level of miR-106b became stable after 35 days.MiR-106b can promote the proliferation of GC-1 and GC-2 and inhibit its apoptosis,and promote the transition from G1 phase to S phase.The inhibition of miR-106b expression can induce GC-1,GC-2 cells cycle arrest in the G1 phase.The predicted target gene STAT3,SOCS3 have a negative relationship with miR-106b,and the interaction between smad5 and miR-106b was not significant,but whether they were target genes for miR-106b,further validation was needed. |
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