Significance Of Vascular Endothelial Growth Factor C Promoter Methyiation Status And Protein Expression In Endometrial Carcinoma

BackgroundEndometrial carcinoma is the third gynecological malignancy besides cervical carcinoma and ovarian carcinoma, which possibly occurred in reproductive age or post-menopause. It’s the most common carcinoma in America, and hold half of the female reproductive system malignancies. In China, there are almost 200 thousand neopatients aged 50-69 every year and the mortality occupies the second place. In spite of the endometrial cancer morbility is increasing year by year domestic and abroad, and has been harming female health severely, the pathogenesis wasn’t illuminate sufficiently.Most of the endometrial cancer is estrogen-dependent endometrioid adenocarcinoma, high performance progestogen can suppress the tumor recurrence when estrogen and progestogen acceptor were positive. Furthermore, anti-oncogene mutation, gene silencing, oncogene hyperexpression and microsatellite instability may be the possible moleculer mechanism during the carcinogenesis.The primary mortiferous cause of tumor patients was invasion and metastasis, and vascular system including blood vessel and lymphatic vessel played an important role. Direct pervasion and lymphatic metastasis were major metastasis route in endometrial carcinoma, and prognosis poorly when metastasis occurred. Studies indicated that lymph-vascular proliferation and spread was detected in tumor groundsubstance with lymphatic metastasis, moreover, that would be the significant reason to result in poor therapeutic efficacy even failed. Lymph-vascular hyperplasia and expansion has been frequently discovered in tumor groundmass with lymphatic metastasis.Angiogenesis plays a key role in tumor growth and metastasis. Blood vessel provides nutrition for tumor, so it can only grow to 1-2 mm3 and can’t occur metastasis before angiopoiesis. Oppositely, tumor cell doubling increases after angiopoiesis and along with which the metastasis opportunity increasing. Vascular endothelial growth factor and its receptors is the most important factor in angiopoiesis although it is regulated by multiple genes.VEGF has six isotypes, including VEGF-A,-B(VEGF-B167 and VEGF-B186),-C,-D,-E, varied between 35 and 44kDa. Every isotype combines with three definite grouping receptor (VEGFR-1,-2,-3)specially. The latest study discovered that the influence of VEGF-C,-D for lymphatic vessel was more important than blood vessel, it combines with VEGFR-3 which major locus on lymphatic vessel endothelial cell and induces VEGFR-3 PTK phosphorylation to result in lymph-vascular production, hyperplasia and expansion.Among which, VEGF-C is the key gene for tumor invasion and metastasis in lymph-vascular tumor, which was also be proved in some experiments abroad. Poorly differentiated tumor secretes VEGF-C and activates its receptor to generate new lymphatic vessel and induces lymph-vascular metastasis.Da et al. reported that VEGF-C overexpression was not only related with lymph-vascular metastasis and invasion, but also may be a factor of implying early lymph-vascular metastasis.Cyclooxygenase-2 hypso-expression correlated with endometrial carcinoma intimately, and showed an synergistic effect with VEGF in some malignancies lymph-vascular metastasis. Fujiwaki et al.reported that COX-2 expressed on endometrial cancer epithelial cell but not interstitial cell, and concerned with microvessel density, which mediated microvessel production by inducing VEGF to participate tumor metastasis.Glucose transporter-1 as an important transmembrane transport carrier provides elementary glyco requirement for histiocyte. It generally expresses in mammal embryo and mature tissues and expresses up-regulation in part cancer and atypical hyperplasia tissues. The absorption and utilization increasing when microenvironment hypoxia in cell to satisfy the energy demand. Hypoxia inducible factor-la through mediating hypoxia reaction to up-regulate Glut-1 expression to accelerate glycometabolism.Deoxyribonucleic acid methylation is the major pattern of genome apparent genestics, and is an important mechnism to regulate gene function, meanwhile, it is also the only natural covalent modification form in vertebrate. DNA methylation is the third mechanism to induce anti-oncogene inactivated besides gene mutation and heterozygosity deletion, which may induce production and development of many kinds of disesses especially malignancies.The CpG island is non-methylation in normal cell, and anticancer capacitation will be deprived when anti-oncogene CpG island occurs methylation. Another study reported that hypomethylation plays an important role in tumor development and carcinogenesis, who increases the genome instability and frequency of gene mutation. Duan et al.detected VEGF-C promoter hypomethylation in gastric carcinoma. Because VEGF hypo-expression in endometrial carcinoma, we suspect the correlation of VEGF-C promoter methylation, especially hypomethylation or nor-methylation is concerned with endometrial carcinoma intimately. To resist the production of tumor vessel and lymphatic vessel may be one of the most expect tumor therapy approach and in favor of so many sufferers. So far, the research of VEGF methylation stastus regard to endometrial carcinoma invasion and metastasis still not be reported, we envolve our experiments based on the consideration.In this study, we choose normal endometrium, simple hyperplasia endometrium, complex hyperplasia endometrium, atypical hyperplasia endometrium to detect the VEGF-C promoter methylation status and analyze the correlation between VEGF-C anomaly methylation and endometrial carcinoma. Moreover, we unite detect the protein expression of VEGF, COX-2, GLUT-1 and HIF-1α for first time focus on the endometrial carcinoma invasion and metastasis. The endometrial cancer cell lines also were treated with the methyltransferse inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) to detect mRNA and investigate the promoter methylation status of VEGF-C in carcinogenesis, invasion and metastais of endometrial cancer.Part ⅠThe detection of VEGF-C promoter methylation status in endometrial carcinomaObjectiveTo investigate the expression and promoter hypermethylation status of VEGF-C gene in endometrial carcinoma, simple hyperplasia endometrium, complex hyperplasia endometrium, atypical hyperplasia endometrium and normal endometrium. To explore the correlation between the anomaly VEGF-C promoter methylation and the development in endometrial carcinoma.MethodsRT-PCR was used to examine the expression of VEGF-C in endometrial carcinoma, simple hyperplasia endometrium, complex hyperplasia endometrium, atypical hyperplasia endometrium and normal endometrium tissues. The promoter methylation of VEGF-C was evaluated with MSP method.Results1. Expressions of VEGF-C mRNA in all types endometriums The expressions of VEGF mRNA in endometrial carcinoma and atypical hyperplasia endometrium were higher than that in complex hyperplasia endometrium, simple hyperplasia endometrium and normal endometrium tissues. The expression frequency was 91.7%(55/60),83.3%(20/24),28.6%(4/14),4.5%(1/22) and 1.7%(1/60), respectively. The expression level of VEGF mRNA was significantly higher than that in NE, SH and CH(P<0.01), while had no statistically significance compared with AH(x2=1.244, P>0.05). The positive rate of VEGF-C expression in AH was significantly higher than that in CH(x2=11.396, P<0.01), SH and NE(P<0.01). Moreover, there was statistically significance compared CH with SH, NE(x2=4.129,13.042, P<0.05,0.01), but no difference between SH and NE(x2=0.561, P>0.05). Figure 1;Table 1,3; Graph 12. Investigation of VEGF-C promoter methylation in all types endometriums We detected VEGF-C promoter methylation in NE, SH, CH, AH and EC tissues were step down by turns. The positive rate of VEGF-C methylation was 83.3%(50/60), 81.8%(18/22) in EC and SH, respectively. But only 15%(9/60) in EC tissues. There was no significant difference for NE compared with SH and CH(x2=0.026,1.049, P>0.05), but oppositely to compare with AH and EC(x2=33.185,56.049, P<0.01). No difference existed between SH and CH(x2=0.534, P>0.05), while there was significant distinction compared SH with AH and EC(x2=19.526,32.542, P<0.01). Methylation of VEGF-C in CH was higher than that in AH and EC distinctly (x2=11.396,18.941, P<0.01). No difference existed between SH and EC (x2=0.036, P>0.05). Figure 2,3;Table 2,3; Graph 23. Correlation of VEGF-C methylation status with clinic-pathological characteristic of endometrial carcinomaIn our study, the samples with lymphatic metastasis exhibited an increased promoter methylation frequency for VEGF-C(x2=9.804, P<0.05). There was no significant association between the methylation frequency for VEGF-C and age, clinical stage, histological grade. Table 44. Correlation of VEGF-C mRNA expressions with promoter methylation in all types endometriumsThe positive rate of VEGF mRNA expression steped up but the promoter methylation degree steped down by turns in NE/SH, CH, AH and EC tissues. There was no obviously correlation between them(r=-0.104, P>0.05).Conclusion1.The methylation rate was usually lower in VEGF-C mRNA hypso-expression tissues, otherwise, the methylation rate was higher, they presented opposite tendency, but no directly correlation.2.The normethylation or hypomethylation of VEGF-C promoter CpG island may increase instability of genome and play an important role in EC development.3.VEGF-C promoter methylation may play an important role in the lymphatic metastasis of EC.4.The VEGF-C presented highly normethylation in EC, so regulation and intervention the normethylation status for VEGF-C may be the new therapy direction in EC.4.The VEGF-C presented highly normethylation in EC, so regulation and intervention the normethylation status for VEGF-C may be the new therapy direction in EC.Part IIInvestigation of VEGF and relevant gene protein expression in endometrial carcinomaObjectiveTo investigate the protein expression of VEGF-C, COX-2, GLUT-1 and HIF-1α gene in endometrial carcinoma, simple hyperplasia endometrium, complex hyperplasia endometrium, atypical hyperplasia endometrium and normal endometrium. To analyze the relationship with pathological characteristics of endometrial carcinoma.MethodsImmunohistochemical method was applied to detect the protein expression in above-mentioned tissues, in order to check the relevance one another and with endometrial carcinoma.Results1. Protein expressions of the four genes in all types endometriums The protein expression of VEGF% COX-2、GLUT-1 and HIF-la were advanced by turns in NE, SH, CH, AH and EC tissues. NE and SH groups has no obviously difference one another(x2=2.517,0.067,0.752,2.761, P>0.05), but there was statistilcally significance compared NE to CH, AH and EC groups(x2=13.042,24.102,52.004; x2=2.663,23.671,38.763; x2=2.663,30.199,57.416; x2=13.400,42.000,91.765; P<0.01). SH and CH groups has no obviously difference one another(x2=2.338,1.063,3.328,2.469; P>0.05), but there was statistilcally significance compared SH to AH and EC groups(x2=7.111,20.790; x2=10.148,16.884; x2=16.611,31.570; x2=15.112,47.492; P<0.01). The protein expression of VEGF in CH was similar to that in AH(x2=0.652, P>0.05), but obviously lower than that in EC(x2=5.589, P<0.05). The protein expressions of COX-2, GLUT-1 and HIF-1α in CH has statistically significance compared with AH and EC(x2=3.91,7.549; x2=5.886,14.617; x2=4.871,25.316; P<0.05). The protein expressions of VEGF, COX-2 and GLUT-1 were similar between AH and EC(x2=3.286, 0.578,1.901, P>0.05), while oppositely for HIF-1α(x2=8.174, P<0.01). NE and CH group has not exhibited association one another(P>0.05), but it was oppositely compared to AH and EC group(P<0.05). Table 1, Graph 12. Protein expressions of the four genes in CH, AH and EC groups Compared CH, AH and EC groups, the protein expression of VEGF、COX-2, GLUT-1 and HIF-1α has statistically significance(x2=7.164,7.571,14.689,25.412, P<0.05). Table23. Correlation of the four protein expressions with clinic-pathological characteristic of endometrial carcinomaThere were no significant associations between the protein expression of VEGF, COX-2, GLUT-1, HIF-1α and age, but has obviously associations with pathological stage, histological grade and metastasis(P<0.01 or<0.05). Moreover, the positive rate increased along with the poorer pathological stage and histological grade as well as with metastasis. Table 34. Correlation of the four protein expressionsThe protein expression of VEGF、COX-2、GLUT-1 and HIF-la were presented positive correlation compared one to another.Conclusion1. The positive expressions of VEGF、COX-2、GLUT-1 and HIF-la protein implied the correlation between the four genes with endometrial carcinoma.2.VEGF、COX-2、GLUT-1 and HIF-1α may have coordinated or promoted the occurrence and development for endometrial carcinoma.3.Maybe experiment error exsisted or indicated that not all the four genes has direct correlation with endometrial carcinoma from our experiment, but coordinated one another obviously.4. VEGF、COX-2、GLUT-1 and HIF-1α may have played an important role in the invasion and metastasis of endometrial carcinoma. So produce the gene-inhibitors will probably a new method to improve patient’s prognosis. obviously.4. VEGF、COX-2、GLUT-1 and HIF-1α may have played an important role in the invasion and metastasis of endometrial carcinoma. So produce the gene-inhibitors will probably a new method to improve patient’s prognosis.PartⅢAnalysis of the status of VEGF-C promoter methylation in endometrial cancer cell linesObjectiveTo detect mRNA, protein expression and promoter methylation of VEGF-C in endometrial cancer cell lines (HEC-1A, Ishikawa, RL-952, HHUA) with MSP after 5-aza-dC treatment. To explore the possible mechanism of endometrial carcinoma. MethodsCell culture first. RT-PCR was used to examine the expression of VEGF-C in endometrial cancer cell lines before and after 5-aza-dC treatment. The promoter methylation and protein expression of VEGF-C was detected with MSP and Western blot methods, respectively. Meanwhile we detect the changes of biological behavior of endometrial cancer cells with MTT and flow cytometry.Results1. Expressions of VEGF-C mRNA was detected in all four endometrial caner cell lines The endometrial cancer cell lines HEC-1A, Ishikawa, RL-952 and HHUA were cultivatied to the logarithmic growth phase in our experiment, and then make RNA and DNA extraction. Normal endometrium as the contrast. VEGF-C mRNA were detected in all four endometrial cancer cell lines via RT-PCR, but not in normal endometium. Figure12. VEGF-C promoter methylations were investigated in part of the four endometrial cancer cell linesVEGF-C promoter methylations were detected in HEC-IA、Ishikawa and RL-952 cell lines by MSP, but HHUA not, which showed that amplification product only existed in USP-primer, but not in MSP-primer. Among the total, HEC-1A occurred exhaustive methylation, Ishikawa and RL-952 occurred partial methylation, which showed that amplification product only existed in MSP-primer, or existed in both MSP-primer and USP-primer, respectively. Figure23. Expressions of VEGF-C mRNA in endometrial cancer cell lines were inhibited result in demethylation after 5-aza-dC effectedSelecting endometrial cancer cell lines in logarithmic growth phase which were inoculated in six-well plates when the density was 1×105 and establishing the control group (without drug treatment) and blank control group (broth instead of the drug) and experimental group (10μM 5-aza-dC drug treatment). Collecting cells and extracting the total RNA and genomic DNA of cells after 96h. The expressions of VEGF-C mRNA were detected by RT-PCR method to compare the expression changes before or after drug effection. The results showed that the VEGF-C mRNA expressions were inhibited after demethylation drug treatment. HEC-1A cells became partially methylated, Ishikaw cells still partially methylated, while RL-952 cells changed into nor-methylation status. Figure34. Expressions of VEGF-C protein in endometrial cancer cell lines were decreased after 5-aza-dC effectedWestern Blot was adopted to detect the protein expressions of VEGF-C in four endometrial cancer cell lines. The results showed that the expressions of VEGF-C protein were detected in four cell lines which decreased after demethylation drug 5-aza-dC treated, and has significant difference to compared with the control groups(P<0.01). Whereas there was no significant difference between the two control groups(P>0.05), indicating that the protein expressions decreased result in the transcription function was inhibited after drug treated.5. Assay of the proliferation changes in HEC-1A cell after 5-aza-dC effection Detecting the time-effect relationship of HEC-1A cell with demethylation drug 5-aza-dC.The results showed that the cell proliferation activity was inhibited by 10μM 5-aza-dC treated for 24h,48h,72h,96h, respectively, and the reaction is more obviously with time extended.6. Detection of cell apoptosis by FCM after demethylation drug treatment Apoptosis was significantly increased in endometrial cancer cell line HEC-1A, increased from 3.81% to 27.04%, after treated by 5-aza-dC. The ability of cells to resist apoptosis was significantly inhibited.Conclusion1. The hypomethylation status of the VEGF-C gene promoter in endometrial cancer cells were detected and the expression of mRNA descended after 5-aza-dC treatment, which indicated that VEGF-C promoter hypomethylation destroyed the stability of genome and was one of the important reasons for carcinogenesis.2.The expression of VEGF-C protein was decreased after 5-aza-dC treatment for endometrial cancer cells, which may be owing to the genetic transcription was suppressed by medicine.3.The methylation status might be a potential therapeutic target for the treatment of endometrial cancer.