Study On The Role Of EGF-EGFR-ERK1/2Path On The Estrogen Regulation And The Target Therapy Of EGFR-siRNA Lentivirus In Endometriosis

Objection:To explore wether EGF-EGFR-ERK1/2path participates to the mechanism of estrogen regulation in endometriosis, and to approach wether EGFR-siRNA could be a new target for the therapy of endometriosis.Method:Part1:1.In vitro experiments:To investigate the effect of various kinds treatment on the viability and the expression levels of EGF,EGFR,p-ERK1/2of ectopic endometrial cells by MTT and westernblot methods under estrogen stimulation and deprivation conditions, respectively.2.In vivo experiments:64female nude mice were randomly divided into control and castration groups(both n=32). The mice in castration group were castrated by bilateral oophorectomy on3weeks after the endometriosis model was established. The levels of EGF, EGFR, p-ERK1/2protein expressed in ectopic lesions of both groups were monitored on4,6,8,10weeks after the endometriosis model was established by westernblot.Part2:Established lentivirus vector pSIHl-H1-GFP/EGFRsiRNA carring EGFR-siRNA and then infected ectopic endometrial cells. The ectopic endometrial cells were fell into9groups according to the estrogen’s level(simple cultivation, estrogen stimulatioin, estrogen deprivation)and wether the cells were infected by interferon lentivirus(blank, blank virus, EGFR-siRNA lentivirus). The effect of pSIHl-H1-GFP/EGFRsiRNA on the mRNA and protein expression levels of EGF, EGFR, p-ERKl/2, Ki67in ectopic endometrial cells were investigated by RT-PCR and westerblot and the viability and apoptosis rate of ectopic endometrial cells were detected by MTT and Annexin V Apoptosis Kit.Part3:30female nude mice were randomly divided into5groups(each n=6) according to different treatmeats which began on3weeks after the endometriosis model was established(the first exploration). They were:control group, blank virus group, castration group(C group),EGFR-siRNA group(E group), castration+EGFR-siRNA group(E+C group). The size of the endometriosis lesions was messured and the level of EGFR, p-ERKl/2, Ki67in the lesions were detected by westernblot on the time of10weeks after the endometriosis model was established(the second exploration).Result:Part1:In vitro experiments:An concentrations depentent incresment of the viability of ectopic endometrial cells and the expression of EGF,EGFR,p-ERKl/2were detected after the sitmulation of E2, E2-BSA or EGF (under estrogen deprivation).The proliferation of ectopic endometrial cells stimulated by E2or EGF could be inhibited by PD98059, which stimulated by E2-BSA could be blocked by ICI182780along with the decent of the expression of p-ERKl/2.2. In vivo experiments:The expression level of EGF,EGFR,p-ERK1/2in ectopic lesions in castration group fell initially and then rised and finally up to the level equaled to control group. Part2:The pSIHl-H1-GFP/EGFRsiRNA Lentivirus vector resulted in a decent of viability and the expression level of EGF, EGFR, p-ERKl/2Ki67and an elevation of apoptosis rate of ectopic endometrial cells no matter there was estrogen stimulation or not. Part3:The lesion volums in C, E and C+E groups were lessen than in control and blank virus groups on the second exploration. The turn was E group>C group>E+C group, with statistical difference(P<0.05) when compared with each other. The expression of EGFR、 p-ERK1/2in E and E+C groups were lower than the other three groups(P＜0.05).The level of Ki67in C, E and C+E groups were lower than the other two groups(P＜0.05).Conclusion:Our study showed that EGF-EGFR-ERK1/2signaling system was implicated in the mechnism of estrogen regulation in endometriosis. It played a role in the effects of estrogen by amplifying the cell survival and proliferate signal when estrogen existed. When estrogen was absent, it mimiced estrogen stimulation by activating ER in a ligands independent manner resulting estrogen like effect. The ivtro and in vivo experiments showed that EGFR-siRNA induced by Lentivirus could inhibit the viability of ectopic endometrial cells and facilitate apoptosis via ERK1/2path which result in a synergistic effect with anti-estrogen therapy.