Abstract
Hypoxia is an adverse factor in cervical cancer, and hypoxia-related gene expression could be a powerful biomarker for identifying the aggressive hypoxic tumors. Reverse transcription quantitative PCR (RT-qPCR) is a valuable method for gene expression studies, but suitable reference genes for data normalization that are independent of hypoxia status and clinical parameters of cervical tumors are lacking. In the present work, we aimed to identify reference genes for RT-qPCR studies of hypoxia in squamous cervical cancer. From 422 candidate reference genes selected from the literature, we used Illumina array-based expression profiles to identify 182 genes not affected by hypoxia in cervical cancer, i.e. genes regulated by hypoxia in eight cervical cancer cell lines or correlating with the hypoxia-associated dynamic contrast-enhanced magnetic resonance imaging parameter ABrix in 42 patients, were excluded. Among the 182 genes, nine candidates (CHCHD1, GNB2L1, IPO8, LASP1, RPL27A, RPS12, SOD1, SRSF9, TMBIM6) that were not associated with tumor volume, stage, lymph node involvement or disease progression in array data of 150 patients, were selected for further testing by RT-qPCR. geNorm and NormFinder analyses of RT-qPCR data of 74 patients identified CHCHD1, SRSF9 and TMBIM6 as the optimal set of reference genes, with stable expression both overall and across patient subgroups with different hypoxia status (ABrix) and clinical parameters. The suitability of the three reference genes were validated in studies of the hypoxia-induced genes DDIT3, ERO1A, and STC2. After normalization, the RT-qPCR data of these genes showed a significant correlation with Illumina expression (P<0.001, n = 74) and ABrix (P<0.05, n = 32), and the STC2 data were associated with clinical outcome, in accordance with the Illumina data. Thus, CHCHD1, SRSF9 and TMBIM6 seem to be suitable reference genes for studying hypoxia-related gene expression in squamous cervical cancer samples by RT-qPCR. Moreover, STC2 is a promising prognostic hypoxia biomarker in cervical cancer.
Highlights
Tumor hypoxia is a major factor leading to radiotherapy resistance, metastasis and poor prognosis for many malignant diseases including cervical cancers [1,2,3,4,5]
The most likely candidates among the 410 genes were identified by utilizing Illumina arraybased expression profiles of 150 cervical cancer patients and eight cervical cancer cell lines grown under normoxia and hypoxia
reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses were performed for three genes that we have previously reported to be induced by hypoxia in cervical cancer cell lines and associated with across patient subgroups with different hypoxia status (ABrix) in cervical cancer [25], i.e. DDIT3, ERO1A and STC2
Summary
Tumor hypoxia is a major factor leading to radiotherapy resistance, metastasis and poor prognosis for many malignant diseases including cervical cancers [1,2,3,4,5]. In RT-qPCR analysis, it is important to choose optimal reference genes for data normalization to remove non-biological, experimentally induced variation from the data. Previous work on reference genes in the uterine cervix has focused on different stages of cervical carcinogenesis by evaluating candidate genes across human papillomavirus (HPV) negative and positive lesions [10], and across normal, precancerous and cancerous samples [11,12,13]. Suitable reference genes for studying hypoxia-associated gene expression in cervical cancer biopsies have not been reported. Tumor-site specific evaluation is required [9,15] and should include associations to clinical features and outcome in addition to hypoxia status
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