Fig 1.
The schematic of SOX2-positive cervical CSCs.
(a) The pSox2/EGFP reporter system containing the hSox2 promoter and transcriptional elements including the 3’ UTR, poly (A) tail, and 3’ enhancer were cloned into the pEGFP vector. The pattern of cell proliferation and division was shown in SOX2-positive cervical cancer cells isolated from SiHa and C33A. (b) The expression of SOX2 in EGFP+ and EGFP- cervical cancer cells isolated by pSox2/EGFP. (c) Sox2+ cells both in SiHa and C33A cells showed heavier tumorigencity and self-renewal in vivo than that in Sox2- cells.
Fig 2.
An integrated analysis of the SOX2 transcriptional element in cervical CSCs.
(a and b) The diagram of full promoter of SOX2 and the deletions containing the possible cis-acting elements and the SOX2 and OCT4 binding sites (c) The full promoter of SOX2 (pSOX2-4650-luc-1828) and deletions were constructed and luciferase activity relative to Renilla control was measured in SiHa-EGFP+ and SiHa-EGFP- cells. The transcriptional activity of pSOX2-mini-luc served as the negative control and the SOX2 transcriptional activity was expressed relative to pSOX2-mini-luc. Data is presented as the mean ± SD of experiments in triplicate and statistically analyzed with student’s t-test. The symbol # represent SiHa-EGFP+ vs SiHa-EGFP- and p < 0.05, *** represents a p value of < 0.001 and ns indicates no statistical difference.
Fig 3.
OCT4 partially increased SOX2 transcriptional activation.
(a) The mutations of SOX2 and OCT4 binding sites downstream of the SOX2 promoter 3’UTR. (b) The luciferase activity of mutations in SOX2 promoter was detected. (S: SOX2; O: OCT4; red dot: SOX2 binding site; blue dot: OCT4 binding site; black dot: mutant of SOX2 or OCT4 binding site). The expression level of OCT4 was detected in OCT4-overexpressing SiHa-EGFP+ and SiHa-EGFP- cells by Western blot including densitometric analysis (c and d) and immunofluorescence (e). (f and g) The luciferase activity of the SOX2 promoter deletions and mutations in OCT4-overexpressing cells. Data is presented as the mean ± SD of experiments in triplicate and statistically analyzed using student’s t-test. The symbols represent the following: *, p < 0.05; #, SiHa-EGFP+ vs SiHa-EGFP- and p < 0.05; $, SiHa-EGFP- vs SiHa-EGFP-OCT4 and p < 0.05; &, SiHa-EGFP+OCT4 vs SiHa-EGFP-OCT4 and p < 0.05; ns = no statistical difference.
Fig 4.
The NF-YA binding site CCAAT/ATTGG box was required for the transcription of SOX2 in cervical CSCs.
(a) The mutations of SOX2 and NF-Y binding sites upstream of the SOX2 promoter 5’UTR. (b) The luciferase activity of the SOX2 promoter mutations (green dot: SOX2 binding site; pink dot: NF-Y binding site; black dot: mutant of SOX2 or NF-Y binding site). (c) NF-YA protein expression was detected in SiHa and C33A cells cultured in adherent culture and tumorsphere, as well as SOX2+ and SOX- SiHa and C33A cells and densitometric analysis was performed related to GAPDH. (d) NF-YA was overexpressed in SiHa and C33A cells and the expression levels and densitometric analysis of NF-YA and SOX2 were detected by Western blot. (e) The luciferase activity of the SOX2 promoter deletions in NF-YA-overexpressing SiHa and C33A cells. Data is presented as the mean ± SD of experiments in triplicate and statistically analyzed using student’s t-test. The symbols represent the following: *, p < 0.05; ***, p < 0.001; #, NF-YA group vs GFP group and p < 0.05; ns = no statistical difference.
Fig 5.
NF-Y specifically bound to CCAAT/ATTGG box upstream of the SOX2 promoter in cervical CSCs.
(a) The luciferase activity of the SOX2 promoter mutations (green dot: SOX2 binding site; pink dot: NF-Y binding site; black dot: mutant of SOX2 or NF-Y binding site) in NF-YA-overexpressing SiHa and C33A cells. (b) The luciferase activity of the SOX2 promoter deletions in NF-YA-overexpressing SiHa and C33A cells. SOX2 was silenced by specific shRNA targeting the SOX2 CDS region in NF-YA-overexpressing C33A cells (c) and the luciferase activity of SOX2 promoter deletions was measured (d). (e) The diagram of NF-YA and SOX2 binding sites upstream of the SOX2 promoter. Data is presented as the mean ± SD of experiments in triplicate and statistically analyzed with student’s t-test. The symbols represent the following: *, p < 0.05; ***, p < 0.001; #, NF-YA group vs GFP group and p < 0.05; ns, no statistical difference.