| Objective:Through using RT-PCR and immunohistochemistry, theexpression situation of LKB1and mTOR in normal ovarian tissue, benignepithelial ovarian tumors and epithelial ovarian cancer were examined. Thecorrelation of LKB1and mTOR and their relation with clinicopathologicalparameters of epithelial ovarian cancer were analyzed. In order to provide anew theoretical basis for the early diagnosis, targeted therapy and prognosis ofepithelial ovarian cancer, we further explore the mechanism of them in theoccurrence and development of epithelial ovarian tumor.Methods:1Research objectThe63cases with epithelial ovarian tumors from the Fouth Hospital ofHe Bei Medical University, were confirmed by pathology following the surgery,including20cases benign epithelial ovarian tumors and43cases epithelialovarian cancer. Another20normal ovarian tissues from patients underwentsurgery because of other diseases, were selected as control. All the patients didnot receive chemotherapy and radiotherapy before surgery.2RT-PCRTotal RNA was extracted and then was reverse transcribed to synthesizecDNA. The PCR was carried out in a complex mixture, initial denaturation at95℃for8mins, denaturation at95℃for45s, annealing at55℃or58℃for35s, extension at72℃for1min,38or35circulations, and then72℃for7min,terminating reaction at4℃. After amplification, each sample was analyzed by1%agarose gel electrophoresis for30mins and visualized by ethidium bromidestaining. The fragment for glyceraldehydes-3-phosphate dehydrogenase(GAPDH) cDNA gene was used as an internal control and the relativeexpression was quantitated as standardization on GAPDH mRNA content by Gel Pro Analysizer3.1densitometric image analyzer.3ImmunohistochemistryThe protein expression of LKB1and mTOR were detected withimmunohistochemistry SP method. The immunohistochemistry procession wasalso carried out according to manufacturer’s instructions. Then DABstaining,hematoxylin staining, grad alcohol dehydration, dimethyl benzenetransparence, neutral gum mounting, observed under light microscope.Negative control was carried out in identical condition excluding PBSinsteading antibody, and the known positive result was used as the positivecontrol.4Statistics analysisThe experimental results were analyzed by SPSS13.0statistical package.The measurement data were expressed asx±SD. x2test, one-way ANOVA,SNK, linear correlation and Spearman rank correlation were used fornumeration and measurement data respectively. α=0.05, P≤0.05indicatingsignificant difference.Results:1The expression of LKB1in epithelial ovarian tumor1.1The expression of LKB1mRNA and its relation with clinicopathologicalparameters of epithelial ovarian cancerThe result of RT-PCR showed that the expression levels of LKB1mRNAin normal ovarian tissue and benign epithelial ovarian tumors were0.55±0.04and0.48±0.03respectively, much higher than that in epithelial ovarian cancer,the difference was significant (P<0.05). But the statistical difference was notfound between normal ovarian tissue and benign epithelial ovarian tumors. Theexpression levels of LKB1mRNA in Stage Ⅰ and Ⅱ was much higher thanthat in stage Ⅲ and Ⅳ, P<0.05. In different tumor histological grade, theexpression levels of high and moderate differentiation group (0.22±0.04) washigher than that of poor differentiation group (0.04±0.01)(P<0.05). Theexpression of LKB1mRNA in different pathological types and different agegroups was no difference (P>0.05). 1.2The expression of LKB1protein and its relation with clinicopathologicalparameters of epithelial ovarian cancerThe positive expression rate of LKB1protein in ovarian cancer tissuewas30.2%(13/43), significantly lower than that in normal ovarian tissue90.0%(18/20) and benign tumors75.0%(15/20)(P<0.05). The positiveexpression rate of LKB1protein of ovarian cancer patients was related to thepathological grade and clinical stage (P<0.05). However, it doesn’t have anyconnection with patients’age or the type of tumor pathology (P>0.05).2The expression of mTOR in epithelial ovarian tumor2.1The expression of mTOR mRNA and its relation with clinicopathologicalparameters of epithelial ovarian cancerThe expression levels of mTOR mRNA in normal ovarian tissue andbenign epithelial ovarian tumors were0.14±0.03and0.19±0.04respectively,much lower than that in epithelial ovarian cancer, the difference was significant(P<0.05). There was no significant difference of mTOR mRNA expressionlevel among different age groups and different pathological types (P>0.05). Butthe expression levels of mTOR mRNA was concerned with the differentiationof tumor and clinical stage. The expression levels of mTOR mRNA in poorlydifferentiated group was significantly higher than that in well and moderatelydifferentiated group, the expression levels of mTOR mRNA in Ⅲ-Ⅳ periodwas significantly higher than that in Ⅰ-Ⅱ period, the difference has statisticalsignificance (P<0.05).2.2The expression of mTOR protein and its relation with clinicopathologicalparameters of epithelial ovarian cancerThe positive expression rates of mTOR protein in epithelial ovariancancer was81.4%(35/43), significantly higher than that in normal ovariantissue20.0%(4/20) and benign epithelial ovarian tumors40.0%(8/20)(P<0.05). The positive expression rate of mTOR protein in poorlydifferentiated cancer was higher than that in well and moderatelydifferentiated cancer, the difference was significant (P<0.05). The expressionof mTOR protein in early cancer (Ⅰ,Ⅱstage ovarian cancer) was significantly lower than that in advanced cancer (Ⅲ, Ⅳstage ovarian cancer)(P<0.05). Indifferent age groups and different pathological types, mTOR expression was noobvious difference (P>0.05).3Correlation of LKB1and mTOR expression3.1Correlation of LKB1and mTOR mRNA expressionIn the level of mRNA, there was negative correlation between LKB1andmTOR expression (r=-0.946, P=0.000, P<0.01).3.2Correlation of LKB1and mTOR protein expressionIn immunohistochemical protein expression level, statistical analysisshowed that there was negative correlation between LKB1and mTORexpression (r_s=-0.726, P=0.000, P<0.01).Conclusions:1The mRNA expression levels of LKB1gene in epithelial ovariancancer were significantly lower than that in normal ovarian tissue and benignepithelial ovarian tumors. This shows that the absent expression of LKB1,which is a tumor suppressor gene, might promote the occurrence anddevelopment of epithelial ovarian cancer. And the expression of mTOR mRNAin epithelial ovarian cancer displayed a upward trend, which indicates that theover expression of mTOR is closely related to the formation of epithelialovarian cancer.2Compared with normal ovarian tissue and benign epithelial ovariantumors, the positive expression rate of LKB1protein in ovarian cancer tissuewas lower. This suggests that LKB1, to some extent, could inhibit theoncogenesis of epithelial ovarian cancer.The positive expression rate of mTORprotein in ovarian cancer tissue was obviously higher than that in the others.This shows that mTOR may play a important role in the process of malignanttransformation of ovarian tumor.3In the level of mRNA and protein, LKB1and mTOR was closelyrelated to the clinical stage and differentiation of tumor, suggesting that LKB1and mTOR may be involved in the invasion and metastasis of epithelial ovariancancer, which has important value to the clinical diagnosis and prognosis. 4The results of this study showed that the expression of LKB1andmTOR present a negative correlationship. This is to say, in the sameorganization, the expression of LKB1was lower, while with the higherexpression of mTOR. Thus we learn that they have the opposite effect on theoccurrence and development of ovarian cancer. The loss of LKB1expressioncould cause the up-regulation expression of mTOR, which stimulated theformation of tumor.5By studying LKB1and mTOR, we may be able to learn more of thepathogenesis of epithelial ovarian cancer. This study provides theoretical basisfor early diagnosis and potential targets and new ideas for therapy of ovariancancer, in order to improve the prognosis and survival rate of patients withovarian cancer. |
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