MDH2 Promoted The Proliferation And Invasion Of Endometrial Carcinoma Through The Inhibition Of PTEN

Endometrial carcinoma is one of the three female genital tract tumors.In North America and Northern Europe,endometrial carcinoma is the most common malignancy of female genital tract,accounting for 6%in new cases and 3%in death cases every year.The distribution of the disease showed obvious regional characteristics.The highest incidence of endometrial carcinoma is observed in North America and Northern Europe,next is Eastern Europe and Latin America,and the lowest incidence in Asia and Africa.The overall survival rate of early endometrial carcinoma is 74-91%but patients with advanced tumor only have the survival rate of 20-26%.According to clinical manifestation,pathological characteristics and prognosis,endometrial carcinoma is usually divided into two types.Type Ⅰ was generally referred to endometrioid carcinoma,which is often associated with endometrial hyperplasia.Patients with Type Ⅰ endometrial carcinoma are mainly in reproductive and postmenopausal age.The process from endometrial hyperplasia to endometrioid carcinoma is commonly associated with unopposed estrogen stimulation.Type Ⅰ endometrial carcinoma usually has a long-term prognosis.The most common histological tumor type of Type Ⅱendometrial carcinoma is serous or even clear cell tumors,which develops from atrophic endometrium.Most women presenting with Type Ⅱ endometrial carcinoma are in postmenopausal age.This type of tumor is estrogen-independent with poor prognosis.As is known to all,estrogen plays an important role in the pathogenesis of endometrial carcinoma.Unopposed exposure of estrogen has been confirmed as a major risk factor of this tumor.One study has suggested that the content of estrogen in tumor tissue is higher than that in normal tissue both in type I and typeⅡ endometrial carcinoma.In traditional theory,estrogen diffuses into the cytoplasm and then combines with ER in nucleus to stimulate transcriptional factors and downstream signaling pathway triggering a series of physiological responses.The process takes several hours.In fact,apart from ER,estrogen can also bind with G-protein transmembrane receptor GPR30 and activate a rapid pathway which only takes several minutes even several seconds.Our previous investigation has demonstrated that estrogen promotes the proliferation and invasion of endometrial carcinoma through GPR30-related MAPK pathway.The mutation and inactivation of PTEN,PIK3CA,K-RAS,P53,P16 and the microsatellite instability have been considered as major factors in the tumorgenesis of endometrial carcinoma.Many investigations have showed that the pathway of PI3K-AKT-mTOR,Wnt-β-catenin are involved in the survival and proliferation of the tumor.Mutation and inactivation of tumor suppressor PTEN has been detected in almost 80%endometrioid carcinoma and 55%hyperplasia endometrium.Many data show that mutations of PTEN is a major factor in early stage of endometrial carcinoma.Warburg Effect opened a new era of tumor metabolism research eighty years ago.Warburg Effect pointed out that proliferating tumor cells prefer glycolysis even in oxygen-rich conditions.It is widely known that glycose can be completely converted into C02 and H2O via aerobic oxidation,with 34 molecules ATP produced.However,glycolysis can only produce 2 molecules ATP.The reason why tumor cells select glycolysis,a way of low energy productivity,arouses scientists’ reflection.Warburg thought that it was the mitochondrial defect that inhibited the tumor cells from oxidizing glucose into CO2.Yet a large amount of data demonstrated that in most of tumor cells no damage of mitochondria was detected.Furthermore,mitochondrial reprogramming has been found.The theory of metabolic reprogramming holds that altered metabolism is the major characteristic in the formation and development of tumors.Activated oncogenes and inactivated tumor suppressors promote the proliferation and invasion of tumor cells through the regulation of the metabolism.Metabolic enzymes play a critical part in metabolic reprogramming via catalysis,gene mutations,inhibition of cell death and so on to enhance the level of metabolism of tumor cells,promoting proliferation and invasion.Recently it has been revealed that some metabolic enzymes play as transcriptional factors in the regulation of tumor suppressors.Malate dehydrogenase is an important metabolic enzyme in TCA.MDHs catalyze the interconversion of malate and oxaloacetate.In eukaryote,there are two types of MDH,MDH1 and MDH2.MDH2 is usually involved in TCA,while MDH1 takes part in malate-aspartate shuttle.MDHs are overexpressed in many types of tumors,for example poorly differentiated gastrointestinal cancer,malignant paraganglioma and prostatic cancer.The expression of MDHs is upregulated and the activity is increased in tumor tissues,which enhance the level of tumor cells metabolism and promote the proliferation and invasion of cancer cells.Interestingly,one study on the relationship between MDH1 and tumor suppressor p53 showed that without glucose MDH1 plays as a transcriptional factor by binding to the promoter of p53-downstream genes and regulate p53 related cell-cycle arrest and cell death,ultimately leading to the formation and development of tumor.Our study aimed to explore the correlation between MDH2 and PTEN,to discuss the part GPR30 played in the regulation of estrogen on MDH2 and PTEN,so as to pave the way for the further research on the metabolism of endometrial carcinoma.In the first part,we used IHC to detect the expression of MDH2 in both endometrial carcinoma tissues and normal tissues.Then we analyzed the relationship between MDH2 expression and clinicopathological data,for instance age,stage,grade and lymphatic metastasis,to reveal the significance of the expression of MDH2 in endometrial carcinoma.In the second part,Western-blot,Realtime PCR,RNA interference,plasmid transfection were used to explore the relationship between MDH2 and PTEN.Immunofluorescence was performed to reveal the expression site of MDH2 and PTEN and whether they were co-expressed in endometrial carcinoma cells.In the third part,we used CCK8,flow cytometry,Transwell,apoptosis analysis,RNA interference and plasmid transfection to study the impact of MDH2 and PTEN on the proliferation,invasion and migration of tumor cells.In the last part,we used Western-blot and Realtime PCR to investigate the impact of estrogen,G1(agonist of GPR30),G15(antagonist of GPR30)on the expression of MDH2 and PTEN.Part 1 The expression of MDH2 in the tissue of endometrial carcinoma and the clinicopathological significanceWe used immunohistochemistry to reveal the expression of MDH2 in the tissue chip with 60 poles.The immunohistochemical staining showed that MDH2 overexpressed in the tissue of endometrial carcinoma and MDH2 expressed in the cytoplasm.Next,we analyzed the clinicopathological data of 34 cases of endometrial carcinoma.The results demonstrated that MDH2 was related to the grade of this tumor,which implied that MDH2 might play an important role in the formation and growth of endometrial carcinoma.Part 2 The relationship between MDH2 and PTENWe used RNA interference to knockdown the gene MDH2 and detected the expression of PTEN by Western-blot and Realtime PCR respectively.The results showed that siRNA-MDH2 upregulated the expression of PTEN.Next,we transfected the overexpressed plasmid GV219-MDH2 into endometrial carcinoma cell line,Western-blot and Realtime PCR demonstrated the decrease of the expression of PTEN.Similarly we detected that siRNA-PTEN upregulated the expression of MDH2 and the overexpression of PTEN decreased the expression of MDH2.Therefore we came to the conclusion that MDH2 and PTEN regulated each other.What’s more,the results of immunofluorescence displayed that MDH2 and PTEN co-expressed in the cytoplasm of endometrial carcinoma cells.We supposed that MDH2 combined with PTEN directly to regulate the biological behavior of tumor cells.Of course the hypothesis requires further investigation.Part 3 The impact of MDH2 and PTEN on the biological behavior of endometrial carcinomaTo investigate the effect of MDH2 on the biological behavior of Endometrial carcinoma,siRNA-MDH2 and overexpression plasmid GV219-MDH2 were transfected respectively to HEC-1-A and AN3CA.The results showed that knockdown of MDH2 inhibited the proliferation,invasion and migration of the cancer cells,but promoted the apoptosis.However,the overexpression of MDH2 promoted the proliferation,invasion and migration,but inhibited the apoptosis.Similarly,we detected siRNA-PTEN promoted the proliferation,invasion and migration,but inhibited the apoptosis.On the other hand,the overexpression of PTEN inhibited the proliferation,invasion and migration,but promoted the apoptosis.What’s more,the inhibition of proliferation,invasion and migration,and the upregulation of apoptosis resulted from overexpression of PTEN were reversed by the addition of overexpressed MDH2.Hence,we reached the conclusion MDH2 enhanced the proliferation,invasion and migration but inhibited the apoptosis of endometrial carcinoma cell line through the suppression of PTEN.Part 4 The impact of estrogen on the expression of MDH2 and PTENTo investigate the impact of estrogen on the expression of MDH2 and PTEN,we used estrogen,G1(GPR30 agonist)and G15(GPR30 antagonist)to stimulate the endometrial carcinoma cell lines HEC-1-A(ER+,GPR30+)and AN3CA(ER-,GPR30+)respectively,and then performed Western-blot and Realtime PCR to detect the expression of MDH2 and PTEN.The results demonstrated that estrogen and G1 both upregulated the expression of MDH2 and downregulated PTEN.And G15 inhibited the expression of MDH2 but enhanced the level of PTEN,In conclusion,estrogen might upregulate the level of MDH2 but downregulate the level of PTEN by GPR30 related pathway.To sum up,estrogen might promote the proliferation and invasion of endometrial carcinoma by inhibiting the expression of PTEN through GPR30-related pathway.The exploration of the relationship between MDH2 and PTEN paved the way for the research on cell metabolism of endometrial carcinoma.Further investigation is required to reveal the important role MDH2 plays in the pathogenesis of the tumor.MDH2 might become a new target for the therapy of endometrial carcinoma in the clinical practice.