The Expression Of Glyoxalase â…  And It's Roles On Proliferation And Apoptosis Of Endometrial Carcinoma

Endometrial carcinoma is a common gynecological malignancy and epidemiological investigations had found that diabetes mellitus is one of the high risk factors in carcinogenesis of endometrial carcinoma.However, since the pathogenesis of endometrial carcinoma remains unclear as yet,the role of diabetes mellitus in the development and progression of endometrial carcinoma cannot be confirmed,which has hampered the development of an effective prevention strategy.Further study on the pathogenesis of endometrial carcinoma will help us to make effective prevention and improve its prognosis.Glyoxalase I(GLO-I) is an important enzyme in cell metabolism,especially in glycolysis,playing a role as cell growth promotor. Recent basic research showed that GLO-I is very important to the regulation of cell proliferation and apoptosis.And as the metabolic substrate of GLO-I, methylglyoxal(MG) is an important factor for controlling cell proliferation and apoptosis,playing a key role as growth inhibitor in cell metabolism.More and more evidences showed that the role of GLO-I and MG in cell metabolism can directly affact the development of various tumors,especially on the regulation step of cell proliferation and apoptosis.But little is known about the relationship between GLO-I and endometrial carcinogenesis,neither the roles of GLO-I on proliferation and apoptosis of endometrial carcinoma cell.Our experiments began with the investigation of the expression and enzyme activity of GLO-I in normal endometrium and endometrial carcinoma. RNA interference technique was used,which could silence GLO-I expression in endometrial carcinoma cell.Subsequently,RT-PCR,Western blot,flow cytometric assessment,MTT were applied to explore the roles of GLO-I on proliferation,apoptosis in endometrial carcinoma cell.The experiments were divided into 3 steps,as following:①Expression of GLO-I in normal endometrium and endometrial carcinoma;②GLO-I activity in fresh normal endometrium and endometrial carcinoma and its differences;③The roles of GLO-I on proliferation and apoptosis in endometrial carcinoma cell. SectionⅠ.Expression of GLO-I in endometrial carcinomaObjective To examine the expression of GLO-I in normal endometrium,endometrial carcinoma tissues and cell lines and then explore the relationship between the aberrant expression of GLO-I and endometrial carcinoma.Methods Immunohistochemistry,RT-PCR and Western blot were used to investigate the expression of GLO-I mRNA and protein in normal endometrium and endometrial carcinoma tissues,and in normal endometrial stromal cell(ESC) and Ishikawa cell lines,then analyze the data statistically.Results(1) GLO-I mRNA was present in endometrial carcinoma cell and ESC.The level of GLO-I mRNA was significantly higher in endometrial carcinoma cell than those in ESC(P＜0.01).(2)There were significant differences of GLO-I expression between normal endometrium and endometrial carcinoma tissues,p＜0.01.No GLO-I protein was stained in normal endometrium by immunohistochemistry.But GLO-I expressed in 75.86%of endometrial carcinoma samples.(3)After RNAi,the expression level of GLO-I in endometrial carcinoma cell is significantly lower than the non-interference group and the negtive-control group(P＜0.01),and is as high as that of ESC.GLO-I expression was located in the cytoplasm of endometrial carcinoma cell.Conclusions Overexpression of GLO-I existed in endometrial carcinoma and only tiny expression in normal endometrium.Abnormal high expression of GLO-I might be relatd to the carcinogenesis of endometrial carcinoma.SectionⅡ.Enzyme activity of GLO-I in endometrial carcinomaObjective To explore GLO-I activity in fresh endometrial carcinoma,adjacent tissue and normal endometrial tissue.Methods Fresh samples of endometrium were all from hysterectomy or diagnostic curettage for endometrial carcinoma,uterine myoma,endometrial polyp or dysfunctional uterine bleeding,including normal endometrium, endometrial carcinoma and adjacent tissues.Quantitative analysis of GLO-I protein was based on Bradford methods after necessary treatment of samples. To detect GLO-I activity in endometrial samples with spectrophotometer.The absorbance(A) of 240nm was measured by ultraviolet spectrophotometer (UVS).Calculation Formula of the enzyme activity of GLO-I was as follow:GLO-I activity(IU/mg) =ΔA240/ε(Extinction Coefficient,mM^-1cm^-1)×V(Reaction Volume,mL)÷1(cm)×k(Dilution Multiple) +Protein Concentration-Sample Volume Note:ε=3310;V=1mL;k=20 OR 400;Sample Volume=1UI.Results(1) GLO-I activity could be detected with spectrophotometer in normal endometrium,endometrial carcinoma and adjacent tissues.(2) Enzyme activity of GLO-I in fresh normal endometrium and endometrium around carcinomous lesions was weak,both of which were lower than 2.0IU/mg;the differences of GLO-I activity between these two groups were not significant(p＞0.05)(3) GLO-I activity in fresh endometrial carcinoma samples was highly-expressed,with the average level up to 92.30208 IU/mg.There existed significant difference from that of normal endometrial tissue(P＜0.01).Conclusions GLO-I activity in endometrial carcinoma is significantly increased;there was only traces of GLO-I activity existed in fresh normal endometrial tissues.SectionⅢ.The role of GLO-I on proliferation and apoptosis of endometrial carcinoma cellObjective To investigate the role and the related mechanism of GLO-I on proliferation and apoptosis in endometrial carcinoma cell.Methods According to the sequence of GLO-I mRNA,three GLO-I specific siRNAs and one non-target siRNA were designed,and then transfected into Ishikawa cell lines through lipofectin.RNA interference was used to silence the GLO-I expression.Inhibition of GLO-I expression by RNAi was assessed by RT-PCR and Western blot.Before and after RNAi procedure,proliferation and apoptosis of Ishikawa cell were analyzed by the MTT and flow cytometry, respectively.The preliminary analysis on the regulation mechanism of GLO-I on cell proliferation and apoptosis was discussed.Results(1)The relative value of GLO-I mRNA in Ishikawa cell transfected with GLO-I siRNA,negative control group,blank control group and ESC group were 0.25±0.06,0.93±0.10,1.0 and 0.33±0.05,respectively.The difference of GLO-I mRNA expression between RNAi groups and negative control group was significant(P＜0.01).Compared with negative control and blank control group, the level of GLO-I mRNA in Ishikawa cell transfected with GLO-I siRNA was significantly lower.There was no significant differences in the expression of GLO-I mRNA between negative control and blank control group(P＞0.05).(2) Compared with negative control group and blank control group,the level of GLO-I protein in Ishikawa cell transfected with GLO-I siRNA was significantly lower(P＜0.01).The degree of strip gray was analyzed by software IPP5.1,and the ratio of GLO-I over GAPDH represent the relative level of GLO-I protein. Setting the relative value of GLO-I protein in blank control group to be 1 and then the value would be 0 in ESC cell group;the level of GLO-I protein in negative control group was 0.94±0.13,while that of Ishikawa cells transfected with GLO-I siRNA was 0.38±0.06,lower than that of negativel control group and blank control group.The difference between RNAi group and negative control group was significant(P＜0.01).And the difference between negative control group and blank control group was not significant(P＞0.05).(3) Proliferation in Ishikawa cell was significantly inhibited after silencing RNA expression of GLO-I.(4) Apoptosis in Ishikawa cell was significantly enhanced after RNA interference;the apoptosis rates of Ishikawa cell increased from 5.31±1.15%(negative control group) to 50.9±2.83%(RNAi group).The apoptosis rate of cell transfected with GLO-I siRNA was significantly different from that of negative control group and blank control group(4.76±1.37%) (p＜0.01).Conclusions RNA interference can effectively inhibit GLO-I expression in endometrial carcinoma cell.GLO-I can promote proliferation of endometrial carcinoma cell and inhibit their apoptosis.The possible mechanism may be that overexpression of GLO-1 could lead to over-enzymolysis of MG,which was main metabolic substrates of GLO-I and playing a key role as growth inhibitor in cell metabolism and promote apoptpsis in cell;hence apoptosis in endometrial carcinoma cell was inhibited and abnormal proliferation would be promoted subsequently.