The Role Of Research GPR30 In The Autophagy Of Estrogen Induced Ishikawa Cells

Objective:To investigate the role of GPR30 mediated AMPK/mTOR signaling pathways of estrogen induced autophagy increasing viability in Ishikawa cells.Methods: Cell Culture:Cells adhered to the bottom in monolayer and were chosen to be used in the test at logarithmic growth phase.Ishikawa cells treat with0, 25, 50, 100, 200ng/ml estrogen for 24 h or 100ng/ml estrogen treat with Ishikawa cells for 0, 1.5, 3, 6 and 12 h, cell viability measure by MTT. The expression of p62, LC3,beclin1, AMPK, p-AMPK, mTOR and p-mTOR in the cells of each group detect by Western blot method. Then transfect GPR30 si RNA and added AMPK pathway inhibitors compound C pretreatment of cells.The protein expression of LC3 was observed by fluorescence microsco-e. The effect of autophagy induced by estrogen through X-ray electron microscope in Ishikawa cells, and Western blot was used to detect p62, LC3 ?AMPK?p-AMPK?mTOR?p-mTOR and beclin-1 protein expression.Results: 1?Estrogen promoted autophagy and Ishikawa cell viability Different concentrations of E2(0,25, 50, 100, and 200 ng/ml) were used to treat Ishikawa cells for 0, 1.5, 3, 6, and 12 h. OD value was measured and the viability were calculated. The results suggested that E2 effectively promoted the Ishikawa cells viability by a dose and time-dependent manner. 2. GPR30 was involved in estrogen-induced autophagy and Ishikawa cell viability To verify our speculation, we transfected GPR30 small interfering RNA(si RNA) in Ishikawa cells, and then detected the change of autophagy induced by estrogen. The result Shows estrogen significantly induced conversion of LC3 I into LC3 II and beclin-1 expression but reduced p62 expression.In addition, we also detected the change of AMPK signal pathway. Estrogen also induced AMPK phosphorylation and inhibited mTOR phosphorylation,Opposite results were shown by GPR30 si RNA. Thus, estrogen induced Ishikawa cell autophagy and cell viability through GPR30, and AMPK-mTOR pathway is possibly involved in this process. 3.Estrogen induced autophagy and viability in Ishikawa cells via the AMPK/mTOR pathway Estrogen activated AMPK,however GPR30 expression inhibited AMPK activation as demonstrated above. To determine whether AMPK contributes to the effect of estrogen on autophagy and proliferation, we treated the cells with the compound C(AMPK inhibitor)2h and add estrogen100ng/ml then To observe the changes of autophagy and autophagy marker protein by Western blot and transmission electron microscope. Result show that estrogen significantly increased p62 expression and induced LC3 I to LC3 II conversion, but reduced p62 expression. These results indicated that AMPK was involved in estrogen-induced Ishikawa cell autophagy and viability. Moreover we detected mTOR expression using Western blot. The result show that estrogen reduced MTOR activation and addition of compound C reversed this effect. The estrogen induced Ishikawa cell autophagy and cell viability via the AMPK/mTOR pathway. 4. The activation of AMPK/mTOR signaling pathway induced by blocking estrogen after GPR30 was inhibited In order to clarify whether GPR30 mediates the activation of the AMPK/mTOR pathway induced by estrogen. Transfected GPR30 si RNA in cells silencing the expression of GPR30. The effect of estrogen on AMPK/mTOR pathway was observed, These results showed that the activation of AMPK by estrogen was significantly inhibited, andmTOR(the downstream factor of AMPK) activity was recovered by Comp C. 5. Effect of estrogen on cell viability of Ishikawa cells treated with autophagy inhibitor Basing on the results of the investigations presented above, we further investigated the relationship between the estrogen-induced Ishikawa cell autophagy and proliferation. We pretreated the Ishikawa cells with 3-MA(25 ?M), an autophagy inhibitor, followed by estrogen treatment. Cell viability was detected using MTT assay. Resulte shows that the estrogen-induced proliferation slightly changed after addition of the autophagy inhibitor 3-MA, and no further change was observed. Similar results were obtained when we employed a genetic approach involving knockdown of the transient ATG5(Fig. 4B). These results indicated that estrogen-induced o autophagy enhance cell viability.Conclusion: Estrogen induced autophagy and cell viability in dose- and time-dependent manner in Ishikawa cells; Inhibited the expression of GPR30 could be blocked autophagy and viability induced by estrogen,and also blocked AMPK/mTOR pathway. These results indicated that GPR30 mediated estrogen induced autophagy enhanced viability via AMPK/mTOR signaling pathway in Ishikawa cells.