The Expressions Of EFNB2and TM4SF1in Cervical Cancer Stem-like Cells

OBJECTIVE:Cancer stem cell (CSC) theory thinks of cancer stem cells (CSCs) as the rootcause of tumor development and recurrence and considers the targeted therapies toCSCs to be the new direction of cancer treatment. However, the absence ofrecognized cervical stem cell markers makes the studies on cervical CSCs extremelydifficult. Although some scholars have isolated cancer stem-like cells using SPmethod or serum-free suspension culture, the obtained cells neither turned out to beenough purified nor had definite surface markers. In this study, differential genedisplay technology was used to detect the cells of the study group (SP+cells andABCG2+cells) that proved to contain enriched cervical CSCs. Through comparingwith the cells of the control group that lacked cervical CSCs, diversity sequences werescreened based on the differences in the abundance of each gene expression. Ourwork provides a basis for the screening of surface markers of cervical CSCs, lays afoundation for the further separation and identification of cervical CSCs and offersclues for cervical cancer therapies that target to CSCs.METHODS:1. The specimens were collected from patients with cervical squamous cellcarcinoma Phase II, and the primary culture was performed using tissue explant,followed by cryopreservation and resuscitation; the SP sorting of established andcultured cervical cancer primary cells was conducted using flow cytometry.2. Cervical cancer Hela cells were selected for culture and the flow cytometrytechnique was adopted for ABCG2sorting.3. The total RNA of sorted SP cells, NSP cells, ABCG2+cells and ABCG2-cellswas extracted respectively and then synthesized by reverse transcription to cDNA.The differentially expressed genes between the cells from two groups were screenedusing microarray. 4. The RNA was extracted from the sorted SP cells and NSP cells ofP10-generation cells of cervical cancer for RT-PCQ detection, so as to identify theexpression level of target gene mRNA and verify differential genes.RESULTS:According to the data analysis, SP cell ratio was1.66%in cervical cancerprimary cells. In contrast, Hoechst33342-positive cells proved to be NSP. Afteraddition of ABC transporter inhibitor Verapamil, the ratio of low-Hoechst SP cellswas significantly reduced (<1%).The detection by immunofluorescence labeling flow cytometer showed that theABCG2+cell ratio was11%in cervical cancer Hela cells, with the rest as ABCG2-cell subsets.Compared with the ABCG2-group,1801upregulated genes and229downregulated genes were reported in the ABCG2+group, and compared with theNSP group,1019upregulated genes and405downregulated genes were reported inthe SP group. According to the intergroup comparison,55downregulated genes in twogroups were identified as intersection genes while12for the upregulated genes.EFNB2gene and TM4SF1gene were expressed in both SP cells and NSP cells.RT-PCR results indicated that the EFNB2gene expression level in SP cells was7.12times higher than that in the NSP cells while the TM4SF1gene expression level was7.68times higher than that in the NSP cells.CONCLUSTION:1. Similar with ABCG2+cells, SP cells isolated from cervical cancer primarycells are featured with characteristics of CSCs.2. TM4SF1and EFNB2genes are highly expressed in cervical cancer primary SPcells and can be used as candidates of specific surface markers for cervical CSCs.3. TM4SF1and EFNB2genes provide clues for the separation of cervical CSCsand offer new ideas and basis for targeted therapies to CSCs.