Tim-3 and galectin-9 expression were increased due to genes methylation level decreased in cervical cancer
Using the Oncomine databases (https://www.oncomine.org/), we compared the mRNA expression of HAVCR2 and LGALS9 between cervical cancer and normal cervical samples. The results indicated that the expression levels of HAVCR2 and LGALS9 were all higher in cancer than in normal cervical samples (Fig. 1a, b). Furthermore, HAVCR2 was positively corrected with LGALS9 (R = 0.26, p <0.05) based on GEPIA (Gene Expression Profiling Interactive Analysis) dataset (http://gepia.cancer-pku.cn/) (Fig. 1c). Hence HAVCR2 has a positive correlation with LGALS9 in cervical cancer. The expression of Tim-3 and galectin-9 protein in cancer tissues were higher than in normal cervix tissues (Fig. 1d, e). The detail data of patients’ clinicopathological is shown in table 3.
The online software “MethPrimer” (http://www.urogene.org/methprimer/) profiled CpG island in the region that was located from -2000 to -200 bp upstream from ATG, the transcription starts site (TSS) in the HAVCR2 and LGALS9 promoters respectively (Fig. 1f). One pair of primers was designed to amplify the genes promoter regions respectively. HAVCR2 and LGALS9 promoters in cervical cancer tissues displayed hypermethylation status in normal cervical tissues (Fig. 1g, h), possibly leading to the inhibition of its gene expression in normal cervical tissues. Immunohistochemistry results revealed that Tim-3 and galectin-9 expressed in tumor cells of cervical cancer tissues (Fig. 1i).
Tim-3 and galectin-9 expression were reversed by alter the methylation status in the promoter regions of HAVCR2 and LGALS9 in cervical cancer cells
It showed that HAVCR2 and LGALS9 promoter regions from ATG in SiHa, HeLa and C33A cells were all partially methylated (Fig. 2a). The Tim-3 and galectin-9 expressed in SiHa, HeLa and C33A cells (Fig. 2b). The mRNA expression level of HAVCR2 and LGALS9 in SiHa, HeLa and C33A cell lines after treatment with DNA demethylation reagent 5-aza-2'-deoxycytidine (5-Aza-CdR) to identify whether the methylation status in the promoter regions regulate the expression of these genes at the transcription level. The results suggested that the expression of HAVCR2 and LGALS9 mRNA in SiHa, HeLa and C33A cells increased in dose-dependent manner after cellular DNA demethylation (Fig. 2c, d). It illustrated the Tim-3 and galectin-9 expression were reversed by 5-Aza-CdR, which promoted the expression of these genes at the transcriptional level. What’s more, immunofluorescence assay showed that the SiHa and HeLa cells all staining cytoplasm and nuclear Tim-3 and galectin-9 (Fig. 2e).
Tim-3 and galectin-9 expression were repressed by DNMT3A through mediated DNA methylation
We found that knocking-down DNMT3A activated Tim-3 and galectin-9 expression (Fig. 3a, b), and was accompanied by decreased DNA methylation level of the gene’s promoter in SiHa and HeLa cells (Fig. 3c, d). It was suggested that DNMT3A may play an important role in regulating Tim-3 and galectin-9 expression. The ChIP analysis revealed enhanced binding of DNMT3A (Fig. 3f, h) to the HAVCR2 and LGALS9 promoter regions (Fig. 3e, g) in SiHa and HeLa cells. Altogether, these results suggest that DNMT3A-mediated DNA methylation contributes to the transcriptional silencing of HAVCR2 and LGALS9.
SUV39H1 mediated DNMT3A expression through up-regulating H3K9me3 in cervical cancer cells
SUV39H1 is the histone methyltransferase (HMTase) of histone H3 lysine 9 trimethylation (H3K9me3) [17]. SUV39H1 recognizes trimethylated H3K9 (H3K9me3) via its chromodomain (CD), and enriched H3K9me3 afterwards [18]. The H3K9me3 and DNMT3A expressed in SiHa, HeLa and C33A cells (Fig. 4e).
As shown in Fig. 4b, Overexpressed SUV39H1 in SiHa and HeLa cells (Fig. 4a) significantly increased H3K9me3 and DNMT3A expression. Fig. 4c showed that knocking-down SUV39H1 expression in SiHa and HeLa cells displayed dramatically down-regulation in H3K9me3 and DNMT3A protein expression (Fig. 4d). Collectively, these results indicated that SUV39H1 participate in regulation of DNMT3A through changing H3K9me3 expression in cervical cancer cells.
In a screening for epigenetic mechanisms that regulating DNMT3A expression, ChIP analysis revealed that the SiHa and HeLa cells exhibit the highest H3K9me3 level in a region upstream of the transcription initiation region (Fig. 4f). H3K9me3 regulated the expression of DNMT3A by acting on the -1000 to +1 region of the promoter region of DNMT3A (Fig. 4g, h). Taken together, above results revealed that SUV39H1 regulated the expression of DNMT3A through elevating H3K9me3 level on the DNMT3A promoter in cervical cancer cells.
The methylation status of HAVCR2 and LGALS9 affected by SUV39H1 in cervical cancer cells
We have determined the baseline levels of DNA methylation on HAVCR2 and LGALS9 promoters among cervical cancer cell lines and then evaluated whether SUV39H1 mediated DNA methylation through DNMT3A is required for HAVCR2 and LGALS9 transcription. The results showed that SUV39H1 overexpression increased methylation levels at the HAVCR2 and LGALS9 promoters (Fig. 5a, b), these changed methylation level contributed to the decrease of Tim-3 and galectin-9 expression among overexpressed SUV39H1 cell lines (Fig. 5e). SUV39H1-knockdown cells showed the opposite results (Fig. 5c, d, f).
These results indicating that changes in histone modification precede the changes in DNA methylation level of HAVCR2 and LGALS9. Consistent with SUV39H1 affecting the expression of DNMT3A.
SUV39H1 mediated Tim-3 and galectin-9 expression through DNA methylation in vivo
For the purpose of investigating SUV39H1 mediated the costimulatory factors Tim-3 and galectin-9 expression through DNA methylation in vivo, we generated SiHa-SUV39H1 and HeLa-SUV39H1 tumor xenografts in nude mice. As shown in Fig. 6a, tumors formed from the SiHa-mock and HeLa-mock cells (Fig. 6b, c) grew faster than those formed from the SiHa-SUV39H1 and HeLa-SUV39H1 cells, respectively. All these data indicated that up-regulating SUV39H1 may inhibited the tumor growth in SiHa and HeLa cells.
To determine SUV39H1 mediated Tim-3 and galectin-9 expression through DNA methylation in vivo, the levels of H3K9me3, DNMT3A and Tim-3 and galectin-9 in the tumor xenograft tissues were examined by western blotting. As shown in Fig. 6d-f, the expression of DNMT3A increased significantly when SUV39H1 overexpressed, followed by the down-regulation of Tim-3 and galectin-9 in SiHa-SUV39H1 and HeLa-SUV39H1 cells derived tumors. SUV39H1 overexpression significantly up-regulated the methylation level of HAVCR2 and LGALS9 in tumor tissues (Fig. 6g). ChIP analysis revealed that H3K9me3 regulated the expression of DNMT3A by acting on the -1000 to +1 region of the promoter region of DNMT3A (Fig. 6h, i) in tumor tissues. H3K9me3 directly regulate the expression of DNMT3A in vivo, these results indicating that SUV39H1 precede the changes in DNA methylation. All the results in xenograft tissues were consistent with those in vitro, indicating that similar SUV39H1 mediated Tim-3 and galectin-9 expression through DNA methylation in vivo.
H3K9me3 expression was independent from HR-HPV oncogenes
As persistent HR-HPV infection contributes to almost all cervical cancer cases [19], we attempted to explore whether HR-HPV oncogenes E6 and E7 participated in SUV39H1 mediated DNA methylation in SiHa and HeLa cell lines. We found that there was no difference in the expression level of H3K9me3 in cervical cancer cells SiHa, HeLa and C33A (Fig. 4e). Expression of HPV16/18 E6 and E7 oncogenes were detected by western blotting in overexpression or knockdown SUV39H1 SiHa and HeLa cells respectively. As show in Fig. 7i and j, the expression level of HR-HPV oncogenes E6 and E7 were no difference between overexpressed or knockdown SUV39H1 SiHa and HeLa cells with control. In the following studies we transiently overexpressed or knocked-down HPV16/18 E6 and E7 in cervical cancer cells for further illustration (Fig. 7a-d). The results showed that, the level of H3K9me3 was not changed in over-expressed or knocked-down HPV16/18 E6 and E7 cells compared with control (Fig. 7e-h).
Taken together, our data suggested that H3K9me3 expression was independent from HR-HPV oncogene E6 and E7 in cervical cancer.