The Expression Of R-smads Phosphatase In TGF-? And Its Effects On Mechanism During The Development And Progression Of Cervical Cancer

Transforming growth factor-?(TGF-?)is a growth factor family that exerts its biological effects via the TGF-?/Smads signaling pathway and plays an important role particularly in the development and progression of tumors.Abnormality in any elements in the TGF-?/Smads pathway may cause disturbance in the signaling transduction,which diminishes the biological functions of TGF-? and its inhibition on cell growth,leading to the occurrence and development of tumors.The key genes in the TGF-?/Smads signaling pathway are not exactly the same in different tumors.Epidemiological statistics have shown that cervical cancer ranks the second place in gynecologic malignancies worldwide,with a relatively high incidence in developing countries.However,the specific pathogenesis of cervical cancer is not very clear to date.Therefore,the present study is designed mainly to explicate the mechanism of TGF-?/Smads pathway in the pathogenesis of cervical cancer to provide a molecular basis for the comprehensive explanation of the pathogenesis of this disease.In the present study,expression levels of TGF-?1,two TGF-?1 receptors(TGFBR1and TGFBR2)and Smad2,Smad3,Smad4 and Smad7 in cervical cancer tissue were determined and clinical parameters were observed;distribution of gene mutation frequency of loci 509,800,and 988 in the promoter region and loci 869,915 and 788 in exons of TGF-?1,locus 875 in TGFBR2,and loci Int7G24 A and *6A in TGFBR1 were analyzed in cervical cancer tissue to explore the relationship between gene polymorphism and the susceptibility of cervical cancer;and the methylation level of TGFBR1,TGFBR2 and Smad4 genes in cervical cancer and intraepithelial neoplasia were detected to further explore the association of m RNA and protein levels with methylation level.Part I: Expression of key genes in the TGF-?/Smads signaling pathway in cervical cancerObjective: To investigate the expression of key genes in the TGF-?/Smads pathway in cervical cancer.Methods: Expression levels of TGF-?1,TGFBR1,TGFBR2,Smad2,Smad3,Smad4 and Smad7 m RNAs in cervical cancer tissue were determined by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR)and those of TGF-?1,TGFBR1,TGFBR2,Smad2/3,Smad4 and Smad7 proteins were measured by immunohistochemical method.Results: Compared with the normal tissue,the expression levels TGFBR1,TGFBR2,Smad2,Smad3 and Smad4 m RNAs were significantly reduced and those of TGF-?1and Smad7 m RNAs were significantly increased in cervical cancer tissue;The expression levels of TGFBR1,TGFBR2,Smad2,Smad3 and Smad4 proteins were also significantly decreased and those of TGF-?1 and Smad7 proteins were significantly increased compared to the normal tissue,and the expression levels of these proteins were associated with cervical cancer stages.Conclusions: Invasion and metastasis of cervical cancer may be associated with the overexpression of TGF-?1 and Smad7.A positive correlation between the expression of TGFBR1 and TGFBR2,p-Smad2/3and Smad4,TGF-?1 and Smad7,and TGFBR1 and p-Smad2/3,respectively,and a negative correlation between TGF-?1 and TGFBR1,TGF-?1 and p-Smad2/3,and TGFBR1 and Smad7,respectively,and no significant correlation between p-Smad2/3and Smad7 were found in cervical cancer.These results suggested that any changes in TGF-? receptors,receptor-regulated Smad(R-Smad)and Smad4 genes may abolish the sensitivity of tumor cells to TGF-?1 and cause abnormal cell proliferation.Part II: Polymorphisms in TGF-?1,TGFBR1 and TGFBR2 genes and susceptibility to cervical cancerObjective: To investigate the correlation of polymorphisms in TGF-?1,TGFBR1 and TGFBR2 genes with susceptibility to cervical cancer in a case-control study.Methods: Polymerase chain reaction-primer introduced restriction analysis(PCR-PIRA),polymerase chain reaction-restriction fragment length polymorphism(PCR-RELP),PCR product size analysis,and polymerase chain reaction-amplification refractory mutation system(PCR-ARMS)analysis were performed,respectively,on the related genes.Expression level of plasma TGF-?1 was determined by enzyme linked immunosorbent assay(ELISA)in cervical cancer patients and healthy control subjects.Apoptosis of tumor infiltrating lymphocytes were detected using immunohistochemistry and TUNEL double labeling method in cervical cancer tissue.Results:Mutation frequency of loci 509 and 869 in TGF-?1 gene in the cervical cancer group was significantly higher than that in the healthy group(p < 0.01).Genetic polymorphism in Int7G24 A and *6A loci of TGFBR1 gene was observed in patients with cervical cancer,which was not directly correlated with the expression of TGFBR1.Genetic polymorphism in locus 875 of TGFBR2 gene was observed in patients with cervical cancer,which was not directly correlated with the expression of TGFBR2.Conclusion: There is a correlation between the genetic polymorphism and the occurrence of cervical cancer,i.e.,T allele on TGF-?1-509 is associated with high risk of cervical cancer;A allele on Int7G24 A locus of TGFBR1 gene is associated with high risk of cervical cancer;and A allele on locus 875 of TGFBR2 gene is associated with low risk of cervical cancer.Part III: Analysis of the methylation level of TGFBR1,TGFBR2 and Smad4 genes of in cervical cancerObjective: To explore the methylation level of TGFBR1,TGFBR2 and Smad4 genes in cervical cancer and its correlation with the expression of the m RNAs and proteins.Methods: Methylation level of TGFBR1,TGFBR2 and Smad4 genes in cervical cancer tissues was detected by methylation specific polymerase chain reaction(MSP),and its correlation with clinical indicators was analyzed.Results : Expression of TGFBR1 gene increased significantly with the increase of cervical cancer stage.Expression of TGFBR2 gene increased significantly with the increase of cervical cancer stage,and its methylation level increased with a lower degree of differentiation of the cancer.Conclusion: Methylation of TGFBR1 and TGFBR2 genes is significantly correlated with deficient m RNA and protein expression in cervical cancer.Detection of the methylation of TGFBR1,TGFBR2 and Smad4 genes is of great significance in the evaluation of prognosis and progression of cervical cancer.