Studies Of Cervical Cancer Specific Downregulated Candidate Genes At Transcription And Promoter Methylation Level

Objectives:The cervical cancer has occurred in ethnic Uyghur women with realtively high morbidity and mortality in Xinjiang,and may effectively be treated at early detection.Many research groups in and outside of China have been engaged in the transcriptomics studies of cervical cancer for the identification of differentially expressed genes and the establishment of profiles for early diagnosis of cervical cancer.However,the upregulated genes have mainly been in focus of previous studies,while very little was reported about the studies upon downregulated genes so far.At the basis of previous transcriptomics studies of cervical cancer by our research team,this study aimed at the investigation of the mechanisms of cervical carcinogenesis and the identification of early diagnostic markers of cervical cancer by analysis of downregulated genes at transcriptional and promoter methylation level.Methods:1)Collect the fresh tissue specimen of Uyghur cervical cancer patients,among them are 25 cases of cervical squamous cell carcinoma(CSCC),22 cases of cervical intraepithelial neoplasia(CIN)and 25 cases of cervicitis,and extract total RNA from the tissue samples.Design the specific mRNA primers for ABI3BP,FOS,KLF4,RHOB,ID4 and IER2 with professional software,and validate the expression level of above candidate genes by quantitative RT-PCR;2)Choose 12 cervical cancer specific down-regulated genes based on previous study.Acquire their sequence information through searching the online human genome database.Scan the promoter and flanking sequence of the candidate genes,identify the CpG-rich target fragments(CpG islands),and design the bisulfate-modified sequencing primers with professional software.Bisulfate-modify the DNA of SiHa cervical cancer cells,amplify,clone,and sequence the target CpG fragment of every candidate genes.Analyze the methylation status of target fragments by aligning the sequencing result with the original sequence;3)Select 4 cases of cervical cancer tissue DNA samples,and 4 cases of cervicitis tissue DNA as control,amplify the target CpG fragments after bisulfate modification of the DNA samples.Choose 10 clones of the BSP product for each sample and each gene to sequence.Analyze cervical cancer tissue specific variation of promoter methylation level of candidate genes.Results:1)By quantitative RT-PCR detection of the mRNA expression level of 6 candidate genes,we found that,compared to cervicitis,the transcription level of ABI3BP,FOS,KLF4,RHOB,ID4 and IER2 was significantly decreased in cervical cancer(P<0.05),only KLF4 and FOS was significantly decreased in CIN tissue(P<0.05).In addition,the expression level of ABI3BP,FOS,KLF4,ID4 and IER2 was significantly decreased in cervical cancer compared to CIN(P<0.05);2)By scanning analysis using a professional software(Methyl Primer Express v1.0),we has identified CpG islands in the promoter region of FOSB,EGR1,ISL1,CFD,FOS,KLF4,RHOB,ID4 and IER2 genes,while no CpG islands were found for the other genes;3)By bisulfite sequencing of target CpG fragments of SiHa cervical cancer cell DNA,we found that methylation occurred in ISL1 and ID4,and confirmed the methylation of 29 out of 33 CpG sites in ISL1 gene and 47 out of 55 CpG sites in ID4 gene at the promoter,respectively,but no methylation was found for other genes;4)The hypermethylated clone percentage of ISL1 in cervical cancer tissue is 41.94%,significantly higher than that in cervicitis which is 11.76%.Comparing every single CpG sites in hypermethylated clones of cervical cancer and cervicitis,we found that CpG-4,CpG-5,CpG-8,CpG-11,CpG-21,CpG-26 and CpG-27 showed high level of tumor specificity.However,the CpG fragment of ID4 gene is methylated both in cervical cancer and normal control tissue,with no significant difference.Conclusion:1)The occurence and development of cervical cancer is closely related with transcriptional down-regulation of ABI3BP,FOS,KLF4,RHOB,ID4 and IER2.KLF4 and FOS exhibits significant difference between CIN and normal control,thus these 2 genes could serve as early diagnosis marker for cervical cancer and precancerous lesion;2)Among cervical cancer specific down-regulated genes,ISL1 exhibits cervical cancer tissue specific promoter methylation,which could be the epigenetic regulation mechanism of cervical cancerigenesis.