Studies On The Association Of Cervical Disease Progression With The Expression Level Of Candidate Hypermethylated Genes

Objective: Previous reports showed the promoter hypermethylation at CpG islands of a series of tumor suppressor genes in cervical carcinoma and suggested that this may lead to the down regulation of gene expression or silencing of the gene. The aim of this study was to investigate the association of cervical disease progression with the mRNA and protein expression level of RARβ2,DAPK1,ESR1 and CDH13 as candidate hypermethylated genes specific to cervical squamous cell carcinoma. Methods: 1. 37 cases of fresh tissue samples were collected from Uighur and Han women with cervicitis, cervical intraepithelial neoplasia (CIN) and CSCC, and after preparation of tissue RNA, the mRNA expression of RARβ2,DAPK1,ESR1 and CDH13 genes was determined by specific primer and real-time fluorescent quantitative PCR labeled with SYBR Green I, and the association of cervical disease progression with the alteration of gene expression was analyzed at the transciptonal level. 2. 180 cases of formalin fixed and paraffin-embedded cervical tissue specimens were collected from Uighur and Han women with cervical disease (cervicitis, CIN and CSCC), the protein expression level of RARβ2, DAPK1, ESR1 and CDH13 protein was detected by immunohistochemistry, and the association of the cervical disease progression with the alteration of a given protein expression was analyzed. Results:1.The results of real-time fluorescent quantitative RT-PCR analysis showed that the mRNA expression of RARβ2, DAPK1 and ESR1 was equally high in cervicitis/CIN I group ( 0.0109±0.0069 ; 3.6175±2.9584 and 0.0510±0.0441, respectively),decreased in CIN II/III group(0.0029±0.0020;0.9115±0.8639;0.0134±0.0110), very low in CSCC(0.0027±0.0019;0.2662±0.2289;0.0052±0.0052), and the difference was statistically significant(P<0.01), suggested that the cervical disease progression was accompanied by the downregulation of three genes. 2. The results of immunohistochemistry analysis showed that: (1) RARβ2 protein was mainly expressed in the nucleus of cervical epithelial cells, DAPK1 protein expression was localized into the cytoplasma of epithelial cells, ESR1 protein was expressed in the nucleus of both epithelial and stroma cells. However, the protein expression of three species was lost at various degrees with the development of CIN and CSCC.(2) The protein of RARβ2,DAPK1 and ESR1 was normally expressed in the group of cervicitis and CIN I , the loss rate was very low (9.75～22.03%) , markedly increased in the group of CIN II/III (40.00～64.00%), the highest in the group of CSCC (45.00～76.09%), the overall trend of the alteration rate was cervicitis/CIN I0.05), but both was statistically different from cervicitis/CIN I group(P<0.01). Conclusion: The down regulation at the transcriptional level (mRNA) and the loss of protein expression of RARβ2,DAPK1,ESR1and CDH13 may be early prediction marker of cervical cancer, and this study provided evidences for the feasibility of epigenetic researches related to the hypermethylation of the genes above.