Studies On The Association Of Cervical Carcinogenesis And HPV Infection With Regulation Of Cancer Stem Cell Marker Expression

Objective: Study the association of cervical cancer and precancerous lesions in Uyghur women with the expression of cancer stem cell markers, and its dependence on human papillomavirus(HPV) infection to elucidate the mechanism of cervical carcinogenesis and early prognosis. Methods: 1) We collected 47 cases of fresh tissue specimens from Uyghur women with cervical lesions, including 16 cases of cervical squamous cell carcinoma(CSCC), 15 cases of cervical intraepithelial neoplasia(CINⅡ-Ⅲ) and 16 cases of cervicitis or normal cervix(NC). We focused on several cancer stem cell markers previously reported, and analyzed expression of these genes by fluorescent quantitative RT-PCR. The specificity, sensitivity and accuracy of the detection profile of candidate genes were evaluated by statistical analysis. 2) The HPV infection and subtypes were detected by standard method using quantitative PCR for analysis of the dependence of candidate cancer stem cell markers expression on HPV infection. 3) The expression of ALDH1A1 was analyzed in plasma samples from 85 cases of patients by enzyme-linked immunosorbent assay(ELISA). Results:1) Based on literature enquiry, we focused on genes ALDH1A1, OCT4, NANOG, SOX2 and TWIST1 as tumor stem cell markers, and transcription of these genes was detected in 47 biopsies by quantitative RT-PCR. The results showed that, the expression of all genes above was up regulated with the development of cervical lesions, and significant differences were found for all genes between CSCC and cervicitis(P﹤0.05). A significant difference was found for ALDH1A1 and OCT4 genes when CSCC and CIN was compared(P<0.05), and ALDH1A1, OCT4 and NANOG genes in the case of CIN and NC(P<0.05). These results indicated that detection of ALDH1A1, OCT4 and NANOG genes may become markers for early prognosis of cervical carcinogenesis. 2) Eight different HPV genotypes were detected in cervical lesions, such as HPV16, 18, 45, and 31. The HPV positivity were 93.7, 73.3 and 31.3% for cases of CSCC, CIN and cervicitis, respectively, among which HPV16 infection were 62.5, 66.6 and 31.3%. 3) Analysis based on overall HPV infection: ①Although mRNA expression of 5 candidate genes was remarkably higher in HPV-positive CSCC cases than in CINⅡ-Ⅲ cases positive for HPV, and cervicitis cases negative for HPV, but a significant difference was found for only OCT4 between CSCC and CIN(P<0.05). ②Significant differences were found for the expression of ALDH1A1, OCT4 and NANOG genes when all cases positive and negative for HPV were compared(P<0.05); Analysis based on HPV16 infection: ①Although mRNA expression of 5 candidate genes, ALDH1A1, OCT4, NANOG, SOX2 and TWIST1, was remarkably higher in HPV16-positive CSCC cases than in CIN cases positive for HPV16, and cervicitis cases negative for HPV, and but a significant difference was found for only OCT4 between CSCC and CIN(P<0.05), similar to results concerning overall HPV infection above. ② Significant differences were found for the expression of OCT4, NANOG and SOX2 genes when all cases positive and negative for HPV16 were compared(P<0.05); 4) The analysis of changes the expression of cancer stem cell markers during cervical cancer development by Pearson correlation showed that OCT4 expression was moderately correlated with NANOG or ALDH1A1(0.4﹤r≦0.7, P<0.05). The analysis of specificity and sensitivity in the detection of candidate gene expression showed that the detection of ALDH1A1, OCT4, NANOG and TWIST1 has a high accuracy based on AUC which was bigger than 0.70, suggested that the detection of four genes have an impact in early prognosis of cervical cancer. 5) ELISA results showed that CSCC development was associated with the significant increase in plasma ALDH1A1 compared to CIN or cervicitis, but no difference was found when CIN and cervicitis were compared(P>0.05). Conclusion: 1) The development of cervical cancer is closely associated with the upregulation of 5 genes, ALDH1A1, OCT4, NANOG, SOX2 and TWIST1, among which changes in ALDH1A1, OCT4 and NANOG may serve as molecular marker for diagnosis of cervical precancerous lesions or early prognosis of cervical cancer; 2) The upregulation of ALDH1A1, OCT4, NANOG and SOX2 gene expression, especially the expression of OCT4, is associated with HPV infection. In addition, OCT4 gene expression may depend on HPV16 infection, and may serve as auxiliary diagnostic marker for HPV screening in cervical cancer. 3) The change in plasma levels of cancer stem cell markers may become predictive marker for cervical cancer.