Studies Of Genes Down-regulated In,and Specific To,Cervical Cancer

Objective:Screening of downregulated genes by studying the differentially expressed gene profile specific to the tissues of cervical squamous cell carcinoma and its precursors by human genome-wide expression chip analysis,and establishing markers for early prognosis of cervical cancer by quantitative assessment of the specifity and sensitivity of candidate downregulated genes.Methods:We collected seventy two cases of fresh tissue specimens from Uyghur women with cervical lesions,including twenty five cases of cervical squamous cell carcinoma(CSCC),twenty two cases of cervical intraepithelial neoplasia(CIN)and twenty five cases of cervicitis or normal cervix(NC).Twenty cases of high quality tissue specimens(seven cases of CSCC,six cases of CIN Ⅲ and seven cases of NC)were selected for the screening of candidate downregulated genes specific to cervical carcinoma and its precursor by extraction tissue RNA and subsequent analysis with human genome-wide expression profile chip followed by bioinformatics investigation.Four candidate downregulated genes were selected for the design of mRNA sequence specific primers and subsequent detection of candidate gene transcription in seventy two cases of tissue RNA by semi-quantitative RT-PCR,and to assess the significancy of differential expression of candidate genes by statistical analysis.To further confirm the data of semi-quantitative RT-PCR and the specificity and sensitivity of candidate gene detection for the early diagnosis of cervical cancer,we designed specific primers for quantitative PCR and analyzed tissue RNAs of cervical lesions by fluorescence quantitative RT-PCR.Results:By analysis of 20 cases of cervical tissue RNA by human genome expression profile chip,and setting up at least two times of difference in gene expression as quantitative standard for comparative analysis,we identified 86 or 23 genes downregulated in CSCC tissues,respectively,compared to CINⅢ or NC,whereas only 3 genes were downregulated in CIN Ⅲ in relation to NC.Based on the data of gene expression profile analysis and the enquiry of literature,we selected 4 candidate genes downregulated in cervical cancer including FOSB,DNASE1L3,SCARA5 and EGR1 for the screening analysis of 72 cases of tissue RNAs by semi-quantitative RT-PCR and statistical softwares,and the results have shown that the expression of all genes above was downregualted with the development of cervical lesions,with significant differences in downregulation of 4 genes at transcriptional level,when CSCC and NC was compared(P<0.05),whereas the downregulation of only FOSB and SCARA5 in CSCC compared to CIN or of genes DNASE1L3 and EGR1 in CIN to NC has statistical difference(P<0.01).Further analysis of 4 candidate expression by fluorescent quantitiative RT-PCR showed that the candidate gene expression was downregulated with the development of cervical lesions,with a high level expression in NC,decrease in CIN and very low in CSCC,with statistical significance in expression of 4 genes in CSCC or CIN compared to NC(P<0.05).The statistical analysis of the correlation of gene expression between different candidates has shown that there was a moderate for EGR1 in double combination with FOSB,DNASE1L3 or SCARA5(0.4<r≦0.7,P<.0.05),a low correlation for SCARA5 in combination with FOSB or DNASE1L3(0.25≦r≦0.4,P<0.05),but no correlation was found for FOSB and DNASE1L3(r=0.016,P>0.05).The analysis of specifity,sensitivity and accuracy by detection of candidate gene expression for the diagnosis of cervical cancer and the ROC curve have shown that the detection FOSB,DNASE1L3 and EGR1 expression has a relatively high specifity and sensitivity(82.6 and 80%;82.6 and 66.0%;81.8 and 73.3%),and a high accuracy based on the assessment of area under curve(AUC)which was bigger than 0.70 by ROC curve analysis,suggested that the detection of the 3 genes have an impact in early diagnosis of cervical cancer.Conclusion:The results of this study confirmed previous reports again that the development of cervical cancer,in this case the cervical cancer in Uyghur women,is accompanied with the downregulation of multiple genes.The changes in EGR1,FOSB and DNASE1L3 expression may be,to some extent,representative markers of cervical cancer in Uyghur women,and the detection of EGR1 in combination with other genes may have a relatively high impact in the prognosis of cervical cancer.