MDH2 Stimulated by Estrogen-GPR30 Pathway Down-Regulated PTEN Expression Promoting the Proliferation and Invasion of Cells in Endometrial Cancer Yan.

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MDH2 Stimulated by Estrogen-GPR30 Pathway Down-Regulated PTEN Expression Promoting the Proliferation and Invasion of Cells in Endometrial Cancer Yan Zhuang, Jiangdong Xiang, Wei Bao, Ya Sun, Lina Wang, Meihua Tan, Yinyan He, Xiaowei Xi Translational Oncology Volume 10, Issue 2, Pages 203-210 (April 2017) DOI: 10.1016/j.tranon.2017.01.009 Copyright © 2017 The Authors Terms and Conditions

Figure 1 PTEN inhibited the proliferation, invasion and migration but promoted the apoptosis of endometrial cancer cell line. It suppressed the expression of MDH2. To investigate the impact of PTEN on the biological behavior of endometrial cancer cell line, we respectively transfected endometrial cancer cell line HEC-1-A with siRNA-PTEN (siPTEN) and overexpression plasmid GV219-PTEN, with which PTEN was overexpressed (OE-PTEN). Proliferation, invasion, migration and apoptosis were analyzed to determine the difference among normal group(Normal), negative control(NC) group and siPTEN group or OE-PTEN group. (a-c): siRNA-PTEN to knockdown of PTEN promoted the proliferation, invasion and migration of HEC-1-A but inhibited the apoptosis. (d-f): the overexpression of PTEN inhibited the proliferation, invasion and migration of HEC-1-A but promoted the apoptosis. To detect the impact of PTEN on the expression of MDH2, western blot and real-time PCR were carried out in the endometrial carcinoma cell line HEC-1-A transfected with siPTEN and overexpression plasmid GV219-PTEN. (g and h): siRNA-PTEN upregulated the expression of MDH2. (i and j): The overexpression of PTEN downregulated the expression of MDH2.*P<.05, One-way ANOVA. Translational Oncology 2017 10, 203-210DOI: (10.1016/j.tranon.2017.01.009) Copyright © 2017 The Authors Terms and Conditions

Figure 2 MDH2 protein was overexpressed in endometrial carcinoma tissue. The expression of MDH2 protein was detected in normal endometrial tissue and endometrial carcinoma tissue with immunohistochemical staining. MDH2 was expressed in cytoplasm. Magnification,×200. Translational Oncology 2017 10, 203-210DOI: (10.1016/j.tranon.2017.01.009) Copyright © 2017 The Authors Terms and Conditions

Figure 3 MDH2 inhibited the expression of PTEN and was co-localized with PTEN. Western-blot and real-time PCR were used to explore the relationship between MDH2 and PTEN on the level of protein and mRNA in endometrial cancer line HEC-1-A. Immunofluorescent staining was performed to investigate the localization of MDH2 and PTEN. (a and b): siRNA-MDH2 to knockdown of MDH2 upregulated the expression of PTEN. (c and d): The overexpression of MDH2 downregulated the expression of PTEN. (e and f) The overexpression of PTEN was inhibited by MDH2. (g) MDH2 was co-localized with PTEN in endometrial cell line HEC-1-A. *P<.05, One-way ANOVA. Translational Oncology 2017 10, 203-210DOI: (10.1016/j.tranon.2017.01.009) Copyright © 2017 The Authors Terms and Conditions

Figure 4 MDH2 enhanced the proliferation, migration and invasion but inhibited the apoptosis of endometrial cancer cell line through suppressing PTEN. To explore the role of MDH2 in biological behavior of endometrial cancer cell line, MDH2 RNAi (siMDH2) and overexpression plasmid of MDH2-plasmid GV219-MDH2 (OE-MDH2) was transfected into HEC-1-A and AN3CA respectively. (a-c): siRNA-MDH2 to knockdown of MDH2 inhibited the proliferation, migration and invasion but promoted the apoptosis of HEC-1-A. (d-f): The overexpression of MDH2 promoted the proliferation, migration and invasion but suppressed the apoptosis of AN3CA. (g-i) The suppression of cell proliferation, migration, invasion and the enhancement of apoptosis resulted from the overexpression of PTEN were inhibited by addition of overexpressed MDH2. * P<.05, One-way ANOVA. Translational Oncology 2017 10, 203-210DOI: (10.1016/j.tranon.2017.01.009) Copyright © 2017 The Authors Terms and Conditions

Figure 5 E2 might inhibit the expression level of PTEN but enhanced MDH2 through GPR30-related pathway. (a) The stimulation of estrogen and G1, an agonist of GPR30, downregulated the mRNA level of PTEN, while the stimulation of G15, an antagonist of GPR30, upregulated the mRNA level of PTEN in both HEC-1-A (ER positive, GPR30 positive) and AN3CA (ER negative, GPR30 positive). (b) The stimulation of estrogen and G1, upregulated the mRNA level of MDH2, but G15 downregulated the mRNA level of MDH2 in both HEC-1-A and AN3CA. One-way ANOVA was used. *P<.05 versus Normal; #P<.05 versus E2; &P>.05 versus E2. Translational Oncology 2017 10, 203-210DOI: (10.1016/j.tranon.2017.01.009) Copyright © 2017 The Authors Terms and Conditions

Figure 6 E2 might modulate PTEN and MDH2 in endometrial carcinoma via GPR30-dependent mechanisms. Our previous data in 2009 has demonstrated that E2 stimulated the proliferation of endometrial carcinoma cells via MEK/ERK/MAPK pathway. In the present study, we revealed that E2 might promote the proliferation, migration and invasion but inhibited apoptosis in endometrial carcinoma through upregulating the expression of MDH2 and downregulating the expression of PTEN via GPR30-dependent pathway. There might be a complicated regulation network between MDH2 and PTEN. Translational Oncology 2017 10, 203-210DOI: (10.1016/j.tranon.2017.01.009) Copyright © 2017 The Authors Terms and Conditions