Estrogen Promoting Endometrial Cancer Ishikawa Cell Proliferation Associated Analysis Of MAPK Pathway And Proteomics

Part ? Effect of RNA interference mediated MAPK gene down regulation on estradiol activation in human endometrial cancer ishikawa cell1 MAPK shRNA lentiviral vector's constructed and established Ishikawa stable transfected cell line[objective] Based on the previous experiment,we found estradiol can induced the production endometrial carcinom via a mechanism involving activity of MAPK signaling pathway. To clear the relationship between MAPK signaling pathway and estradiol activation in endometrial carcinoma, in this part,MAPK gene downregulation expression in Ishikawa by using RNA interference,and then established Ishikawa stable transfected cell line to lay the foundation for the research of the influence on estradiol activation of Ishikawa cells.[Method](1)4 groups of siRNA sequences specific for MAPK gene were designed,and plasmid vector with MAPK shRNA was constructed and infected of Ishikawa.(2)The MAPK gene's expression was confirmed by RT-PCR and western blot, and the best target effect on silence was chosed.(3)Slow virus was packed, application of RT-PCR and Western-blot showed the best target effect on silence to infection to Ishikawa cell.(4)The stably silenced expression of MAPK cell was selected by flow sorting technology and the gene silencing efficiency of these recombinants was confirmed by RT-PCR and Western blot.[Results ](1)The best interference target was identified by RT- PCR and Western blot,the sequence was GGACTGTCTCTGGTAGAGATGGCGGTTGG.(2)The best interference target of LV-shRNA-MAPKlentiviral vector was successfully constructed, and packed with high virus of 3×108 TU/ml, observed under inverted fluorescence microscopy, transduction rate was approximately 90%, the lentiviral shRNA was successfully constructed and transduced into the Ishikawa cells.(3)The stably silenced expression of MAPK cell was selected by flow sorting technology and the gene silencing efficiency of these recombinants was confirmed by RT-PCR andWestern blot.[Conclusion]RNA interference can inhibit MAPK expression effectively on endometrial carcinoma cell line Ishikawa, which laid a foundation for the subsequent experiment.2 Effect of RNA interference mediated MAPK gene downregulation on Estradiol Activation in human endometrial cell line Ishikawa[objective] In order to clear the correlation between MAPK signaling pathway and estradiol activation in endometrial carcinoma , in this part, MAPK gene downregulation expression in Ishikawa by using RNA interference, and then observed cell proliferation, cell cycle and apoptosis of Ishikawa cells which were silenced by MAPK, in order to get better understand the relationship between MAPK signaling pathway and estradiol activation in endometrial carcinoma.[Method](1)The MAPK-shRNA-transfected cell and untransfected cell was treated by E2.The effects on VEGF?bFGF were analyzed through RT-PCR and western blot.(2)The effects on cell survival of the MAPK-shRNA-transfected were examined by MTT assay, to analysis the effect on cell survival rate of these cells after silencing the MAPK gene.(3)The effects on migration of the MAPK-shRNA-transfected were examined by transwell assay, to analysis the effect on migration index of these cells after silencing the MAPK gene.(4)The cell cycle and apoptosis of each group were treated by E2 were measured by FCM, which was used to study the effect on cell cycle and apoptosis of these cells after silenced MAPK gene of Ishikawa cells.[Results](1)The abilities of proliferation in Ishikawa cell were increased after estradiolstimulation(p<0.05).There was no difference of abilities of proliferation in non-transfected group and NC-transfected group(p>0.05) The abilities of proliferation in MAPK-RNAi-LV group was significantly lower than the other two groups (p<0.05).(2)The abilities of migration in Ishikawa cell were increased after estradiol stimulation(p<0.05).The a migration index in MAPK-RNAi-LV group was significantly lower than the other two groups(p<0.05).(3)The apoptosis rate in Ishikawa cell were increased after estradiol stimulation. There was no difference of apoptosis rate in non-transfected group and NC-transfected group(p<0.05).The apoptosis rate in MAPK-RNAi-LV group was significantly higher than the other two groups(p<0.05).(4)The G0/G1-phase cell population of Ishikawa cell were decreased after estradiol stimulation. The G0/G1-phase cell population in MAPK-RNAi-LVgroup was significantly increased than the other two groups,while the S-phase cell population and the G2-phase cell population were decreased(p<0.05).[Conclusion]RNA interference can inhibit MAPK expression inhibit cell proliferation and promote cell apoptosis. Our findings implied that MAPK could serve as a potential target for EC therapy.3 Establishment of a endometrial carcinoma nude mouse xenograft model with low expression of MAPK gene[objective] To observed the effect on the transplanted tumors growth of endometrial carcinoma nude mouse xenograft model with low expression of MAPK gene, and further explore the relationship between MAPK gene and endometrial carcinoma, which to provide experimental evidence for the gene therapy of endometrial carcinoma.[Method] Nude mice were randomly divided into three groups, each group has 6 nude mice. Ishikawa cells infected with MAPK shRNA lentivirus(MAPK-RNAi-LV group), scrambled shRNA-transfected group (NC group) and non-transfected group (Control group) were respectively inoculated to subcutaneous of nude mices's back. We observed the growth of transplanted tumors, determined time of tumor formation. After these transplanted tumors were formatted, the long diameter (a) and short diameter (b) of tumor were measured every 3 days. According to the formula: tumor volume V= 0.5×a×b2 to calculate the volume of tumor and draw the curve of tumor growth. Expression of p-MEK?p-ERK activity in xenograft tumors of each groups assessed by Western blot.[Results] The model transplanted tumors of nude mouse of endometrial carcinoma was successfully constructed. Compared with the other two groups,the tumor growth of MAPK gene silent group is slow, the average tumor volume less then the Ishikawa group and the NC group(p<0.05).There was no difference in p-MEK?p-ERK protien expression between NC group and Ishikawa group.Compared with the other two groups, the t expression of p-MEK?p-ERK protein in MAPK gene silent group was decreased(p<0.05).[Conclusion] Inhibition the expression of MAPK gene can effectively inhibit the growth of transplanted tumors of nude mouse of endometrial carcinoma. MAPk gene may become the molecular targets of EC.Part ? Proteins research of estrogen Ishikawa cells based on TMT labeling and LC-M S/MS technology[objective ]Using proteomics technology to find and identify human endometrial cancer cell line ishikawa protein expression changes after estrogen treatment,to clarify the mechanism that estrogen causes Endometrial cancer.[Method] Experiments were divided into 2 groups: control group and 10-6mol/L E2 group.The experiment using SDS-polyacrylamide gel electrophoresis technology,used based on TMT protein group learn technology analysis different group of Ishikawa cell differences protein, selected differences expression of protein for liquid phase color spectrum instrument joint quality spectrum analysis, with biological informatics method filter out has important value of protein, Mascot software search Uniprot database identification differences protein, used In Cell ELISA technology validation its expression of law.[Results ](1)By SDS-polyacrylamide gel electrophoresis and mass spectrometry analysis : according to Ratio>1.5 (raised of differences protein) or Ratio<0.67(down of differences protein) of standard, E2 group and control group compared total filter out 92 difference proteins, which raised protein 80 , down protein 12 ;(2)Differences in protein subcellular localization is concentrated in the nucleus;(3)Differences protein participation main metabolism ten pathways:mTOR signaling pathway( protein: RPS6KA1)?RNA Transport pathway (protein:NCBP1; RPP30; EIF3B;Pinin;ACIN1) and mRNA surveillance pathway(protein:NCBP1;Pinin; ACIN1);(4)Using bioinformatics methods for comprehensive analysis of 92 different proteins,and to validate the differences in protein RPS6KA1 In Cell ELISA technology,express trends consistent with MS.[Conclusion](1)This study proves that TMT marked combine with LC-MS/MS technology can be used as screening effective method of differential protein in endometrial carcinoma;(2)RPS6KA1 expression of estrogen group increased significantly,speculated that estrogen by mTOR signaling pathway affecting the development of the endometrium.