The Effect Of Metformin By Regulating MiRNA-133b On Human Granulosa Cells’ Proliferation And Apoptosis

ObjectiveThe incidence rate of Polycystic ovary syndrome(PCOS)has up to 5%-11%,The main feature of PCOS is rare ovulation or anovulation,hyperandrogenism and ultrasound ovarian polycystic changes,now PCOS has seriously affected women Reproductive and health,but the pathogenesis of the disease is not yet clear,it is said that the etiology of PCOS is concerned with insulin resistance or hyperinsulinemia,the imbalance of immune factors,the changing environmental and psychological factors and so on.It is known that follicular granulosa cells play an important role in follicles’ development and maturation,the follicles’ ovulation or maturation are one of the important causes of infertility in PCOS patients.In recent years the researchers have founded that metformin in improving PCOS patients with insulin resistance can also be effective in improving PCOS patients with ovulation.Some scholars have found that the PCOS patients serum microRNA-133 b expression decreased.In recent years,our teams have been committed to miRNA-133 b research,we have found that miRNA-133 b was overexpressed in human MI oocytes.In mouse oocytes we found miRNA-133 b could affect follicular development.The miRNA-133 b could promote the proliferation of granulosa cells and inhibit the apoptosis of granulosa cells,and the expression of miRNA-133 b and caspase-3 in human granulosa cells showed the opposite trend.However,the expression of miRNA-133 b and caspase-3 in the human granulosa cells of PCOS patients was not studied.Further,how is the effect of metformin in vivo or in vitro in human granulosa cells of PCOS patients about the expression of miRNA-133 b and caspase-3.Since the mechanism of metformin affects follicular growth and development is not clear,it is important to explore whether metformin affects the proliferation and apoptosis of granulosa cells by interfering with the expression of miRNA-133 b in granulosa cells of PCOS patients,and it is important to explore the pathogenesis and drug therapy of PCOS significance.MethodCollected from 20-40 years old,from September 2016 to 2017 in April at the Third Affiliated Hospital of Guangzhou Medical University Reproductive Medicine Center,with IVF-ET treatment of non-PCOS,PCOS and oral metformin treatment for more than 3 months PCOS female infertility patients’ follicular fluid,separated and extracted granulosa cells in vitro and doing the following study:(1)The growth of human granulosa cells was observed by optical microscope,and the cell viability was detected by CCK-8 method.(2)The expression of microRNA-133 b and caspase-3 in granulosa cells was detected by fluorescence quantitative PCR.(3)Observed the activity and growth status and detected the expression of miRNA-133 b and caspase-3 in the granulosa cells when intervened granulosa cells by metformin in vitro.Results1,The granulosa cells’ growth status,activity and the expression of miRNA-133 b and caspase-3 in three groups under normal circumstances(without metformin intervention)(1)Observed by optical microscope,three groups of granulosa cells’ growth status in 0h-192 h in vitro culture process are similar,there are no significant difference.(2)CCK-8 method was used to detect the cell activity.From the whole,the growth trend of the three groups were the same,which was similar to the optical microscope.The activity of PCOS group was significantly lower than that of non-PCOS group at 72 h and 96h(p <0.05).(3)Q-PCR results showed that the expression of miRNA-133 b and caspase-3 in the three groups of granulosa cells was significantly different(p <0.05).On the whole,the expression of miRNA-133 b and caspase-3 was opposite.2,The growth status,drug toxicity and the expression of miRNA-133 b and caspase-3 in three groups of granulosa cells treated with different concentrations of metformin(1)Observed under the optical microscope: metformin concentration ≥ 50 mmol / l can be lethal to the cells;metformin concentration of 10 mmol / l-50 mmol / l can affect cell growth,cells can be seen small particles like vacuoles;metformin concentration of 0mmol / l-5mmol / l granulosa cells grow normally.(2)CCK-8 method showed that granulosa cells’ activity significantly increased when metformin concentration was 1mmol/l-20mmol/l(p<0.01),but when metformin concentration was 30-50mmol/l,it significantly decreased(p <0.01).(3)Q-PCR results showed that different concentrations of metformin had significant effects on the expression of miRNA-133 b and caspase-3 in three groups granulosa cells,and there is no law we can find.3,When intervened granulosa cells with 1 mmol / l metformin,the growth status,activity and the expression of miRNA-133 b and caspase-3 in the three groups of granulosa cells.(1)Under the observation of optical microscope,the apoptosis of PCOS granulosa cells significantly alleviated at 6d-8d after intervened at 1 mmol/l metformin concentration.(2)CCK-8 showed that the activity of granulosa cells in PCOS group was no difference between non-PCOS cells in 72h-96h(p>0.05),but in late period of cell culture the cell viability of PCOS patients was still lower than that of non-PCOS group(p<0.05).(3)Q-PCR results: on the whole,the expression of miRNA-133 b and caspase-3 in the three groups were opposite.Specifically,the expression of miRNA-133 b and caspase-3 in the non-PCOS group was similar than no metformin,but the expression of miRNA-133 b in PCOS and PCOS+metformin groups granulosa cells decreased at d8 days.Conclution1,In vitro culture process,human follicular granulosa cells’ growth cycle is only 8 days,the growth state of the best at 72 h,which has nothing to do with the patient’s disease.2,MicroRNA-133 b may participate in the pathogenesis of PCOS.3,Metformin may regulate the expression of miRNA-133 b and thus affect the proliferation and apoptosis of granulosa cells.4,The effect of metformin on the proliferation and apoptosis of cumulus granulosa cells in PCOS patients was more obvious.