| Endometrial carcinoma is a high incidence tumor in female genital system,which seriously threatens the health of women.In recent years,the age of onset of endometrial carcinoma is gradually younger.However,the exact etiology and mechanism of endometrial carcinoma aren't yet clear.The occurrence of endometrial carcinoma is a complex process of multiple factors.Epidemiological studies have shown that obesity,diabetes and hypertension are the most important risk factors for the development of endometrial carcinoma,and are also called"Triad syndrome cancer"of endometrial cancer.In 1998,WHO defined MS?metabolic syndrome?as a clinical syndrome characterized by obesity,hypertension,hyperglycemia,hyperlipidemia and so on.Its central links is insulin resistance and secondary hyperinsulinemia.MS is closely related to the occurrence of endometrial cancer.Therefore,the study of endometrial carcinoma based on hyperinsulinemia can provide a new direction for its early prevention and treatment.On the one hand,insulin can be widely used to regulate the body's physical metabolism.On the other hand,as a growth factor,it can regulate the cell proliferation,differentiation and migration through the signal pathway PI3K/Akt and MEK/ERK.Studies have found that high levels of insulin make the PI3K/Akt signaling pathway excessively active,promote endometrial cancer cell proliferation and inhibit apoptosis,and plays a role in the development of endometrial cancer.PI3K/Akt signaling pathway can promotes cell growth and migration,inhibits cell apoptosis and promotes tumor angiogenesis.Excessive activation of this signaling pathway is involved in the development of many kinds of tumors,such as breast cancer,liver cancer and so on.IGFBP-rP1,the insulin like growth factor binding protein related protein 1,is a member of the IGFBPs family.IGFBPs.The biological function of IGFBP-rP1 has two aspects.On the one hand,it can transport and regulate the half-life of the the insulin-like growth factor?IGF?,like the traditional IGFBPs,and indirectly regulate the biological activity of IGF.Compared to the traditional IGFBP1-6,the combination of IGFBP-r P1 and IGF is low,but it can be highly combined with insulin,which can weaken the insulin sensitivity of the body and cause the disorder of glucose metabolism.On the other hand,IGFBP-rP1 can independently regulate cell proliferation,apoptosis and angiogenesis,rather than rely on IGF factors.IGFBP-rP1is used as a tumor suppressor in a variety of tumors,such as liver cancer,ovarian cancer,breast cancer and so on.Our previous study found that upregulate the IGFBP-rP1 expression in endometrial cancer cells,cell proliferation was inhibited and apoptosis increased.IGFBP-rP1 is high likely to mediate the inhibitory effect of endometrial carcinoma,but its specific mechanism is not yet clear.Modern preventive medicine model suggests that the prevention of endometrial cancer should be prevented from blocking the carcinogenesis of the endometrium to its high risk factors.Insulin resistance and hyperinsulinemia can activate the signal pathway in endometrial cancer cells to participate in the occurrence of endometrial cancer.Therefore,it is helpful to explore the effect of IGFBP-rP1 on the insulin signaling pathway,and to provide a possible scheme for the early prevention and treatment of endometrial cancer.ObjectiveThe expression of IGFBP-rP1 in endometrial carcinoma cell lines was observed.To study the effect of insulin on PI3K/Akt signaling pathway in the endometrial carcinoma cell lines with low expression of IGFBP-rP1,and to investigate the role of IGFBP-rP1 on the activation of PI3K/Akt signaling pathway induced by insulin.Materials and Methods1 MaterialsFour endometrial cancer cell lines,Ishikawa,RL95-2,HEC-1A and HEC-1B,were maintained with the corresponding medium and cultured at 37?,cell incubator containing 5%CO2.2 MethodsThe expression of IGFBP-rP1 in Ishikawa,RL95-2,HEC-1A and HEC-1B cells was detected.The endometrial carcinoma cell lines with relatively low expression of IGFBP-rP1 were selected for the follow-up subjects.The location of IGFBP-rP1 was detected by cell immunofluorescence,and the expression of IGFBP-rP1mRNA and protein was detected by RT-PCR and Western blot method.Determine the optimal time for insulin to activate the PI3K/Akt signaling pathway in HEC-1A cells.The protein expression of p-Akt and Akt were detected by Western blot methods in HEC-1A cell lines after stimulation with 10-6M insulin at different time?0?15?30?60?120?240min?.To observe the effect of IGFBP-rP1 in insulin activation of PI3K/Akt signaling pathway.The experiment was divided into blank control group and different concentrations of IGFBP-rP1 group?0?50?500?1000ng/mL?.The blank control group was no treatment,after different concentrations of IGFBP-rP1 group adding the corresponding concentration of IGFBP-rP1 effect 24h,then they were stimulated by of insulin(10-6M)for 30min.The protein expression of p-Akt and Akt in each group were detected by Western blot in HEC-1A cells.3 Statistical analysisIn this study,all the data were analyzed by SPSS20.0 software,and the results were expressed as the?mean±SD?.One-way ANOVA was used to compare the data of multiple groups,and LSD-t test was used for the comparison between multiple groups.A?=0.05 was considered statistically significant.Results1 Expression of IGFBP-rP1 in four endometrial carcinoma cell linesThe immunofluorescence staining showed that IGFBP-rP1 protein was mainly expressed in cytoplasm in four endometrial cancer cell lines.RT-PCR showed that the mRNA expression of IGFBP-rP1 in HEC-1A cells was lower than that in the other three cell lines?P<0.05?.The results of Western blot showed that the relative expression of IGFBP-rP1 protein in Ishikawa,RL95-2,HEC-1A and HEC-1B cells were 0.586±0.065,0.367±0.042,0.047±0.025 and 0.132±0.028 respectively.The expression of IGFBP-rP1 protein in HEC-1A cells was the lowest,and the difference was statistically significant compared with the other three cell lines?P<0.05?.2 Activation of PI3K/Akt signaling pathway in HEC-1A cells by insulinAfter the stimulation of 10-6 mol/L insulin on HEC-1A cells at different time,the activation level of Akt protein increased significantly at 15min?P<0.05?.The activation level of Akt protein reached the peak at 30 min,and the difference was statistically significant compared with the blank control group?P<0.05?.Therefore,30min was selected for the follow-up experiment.3 The effect of exogenous IGFBP-rP1 protein in PI3K/Akt signaling pathway activated by insulin in HEC-1A cellsWhen different concentrations of IGFBP-rP1 were used,compared with 0ng/ml IGFBP-rP1 group,the relative expression of p-Akt/Akt ratio in the50ng/mLIGFBP-rP1 group was 0.906±0.030,and the difference was not statistically significant?P>0.05?.Compared with the 0ng/mLIGFBP-rP1 group,the expression of p-Akt/Akt ratio in the 500ng/mLIGFBP-rP1 group and the 1000ng/mLIGFBP-rP1group was 0.677±0.052 and 0.404±0.071,and the activation level of Akt protein decreased,the difference was statistically significant?P<0.05?.ConclusionsInsulin can active the PI3K/Akt signaling pathway in endometrial carcinoma cells,which might be suppressed by exogenous IGFBP-rP1. |
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