1. HNF1α is increased in radio-resistant human CC tissues and CC cells.
To identify the role of HNF1α in the radio-resistance of CC, we explored the level of HNF1α in radio-sensitive and radio-resistant CC tissues. As shown in Fig. 1A and 1B, the protein level of HNF1α was significant up-regulated in radio-resistant CC tissues compared with in radio-sensitive CC tissues by using IHC and western blot analysis. Additionally, the radio-resistant CC tissues had a higher mRNA level of HNF1α than radio-sensitive CC tissues (Fig. 1C). To demonstrate the expression level HNF1α in different CC cell lines, we performed the colony formation assay to observe to evaluate the sensitivity to radiotherapy in different CC cell lines. As shown in Fig. 1D, CC cells were treated with 0, 2, 4, and 8 Gy of irradiation. Cell proliferation was markedly decreased after 4 Gy of IR in 4 CC cell lines. Importantly, the survival percentage of SiHa cell line was much higher than other cell lines after treated with different different doses of irradiation considering that the SiHa cell line as the radio-resistant cell line. While the ME180 cells has a lower survival percentage after IR considering the ME180 cell line as the radio-sensitive cell line. Then, we detected the mRNA and protein levels of HNF1α in CC cell lines by using qRT-PCR and western blot analysis. The results revealed that the the mRNA and protein levels of HNF1α cell line were lowest in ME180 than in other CC cell lines; therefore, this cell line was selected for gain-of-function experiments. While the SiHa cell line which had a higher level of HNF1α was chose for loss-of-function experiments (Fig. 1E and 1F). Together, these findings suggest that the higher expression level of HNF1α may contribute to radio-resistance in CC cells.
2. HNF1α plays an essential role in CC cell radio-resistance
In order to clarify the effect of HNF1α on CC radio-resistance, some loss-and-gain experiments were performed. First, to knock down of HNF1α level in CC cell, SiHa cells were transfected with specific shRNA. Western blot and qRT-PCR were used to detect the level of HNF1α. As the results showed in Fig. 2A and 2B, the mRNA and protein levels of HNF1α were significantly supp shRNA-transfected SiHa cells. Colony formation analysis showed that depletion of HNF1α in SiHa cells suppressed the cell proliferation after IR compared with the control group, suggesting that downregulated HNF1α level promoted cell radio-sensitivity in CC cells (Fig. 2C). On the contrast, transfection of overexpression plasmid of HNF1α significantly enhanced the mRNA and protein levels in Me180 cells (Fig. 2D and 2E). Colony formation analysis result revealed that enforced HNF1α expression in ME180 cells significantly increased cell radio-resistance in ME180 cells (Fig. 2F). These findings suggests that knockdown of HNF1 reduces while overexpression of HNF1α induces the radio-resistance in CC cells.
3. HNF1α positively regulates RAD51D protein expression in CC cells
HNF1α is known to be involved in chemotherapeutic and radiotherapeutic resistance of cancer cells by directly or indirectly regulating the expression of multiple proteins. To determine the mechanism by which HNF1α regulated radio-resistance of CC cells, we detected the protein levels of the genes that have previously been shown to be regulated by HNF1α and that had shown a correlation with radiotherapeutic resistance. As shown in Fig. 3A and 3B, the radio-resistant CC cell line-SiHa showed dramatic upregulation of the protein expression of DNA repair protein homolog 4 (RAD51D); however, there was no significant difference at the mRNA level. Western blot result showed that increase of HNF1α level dramatically reduced the protein expression in ME180 cells (Fig. 3C). However, the mRNA level of RAD51D was not influenced by the increase in HNF1α (Fig. 3D). On the contrary, knockdown of HNF1α level in SiHa cells suppressed the expression of RAD51D at the protein level, but not at the mRNA level; these findings indicated that HNF1α regulated the RAD51D level at the post-transcriptional level (Fig. 3E and 3F). Subsequently, we detected the expression level of RAD51D in different kinds of CC tissues. Western blot results showed higher protein expressions of RAD51D in radio-resistant CC tissues compared to that in radio-sensitive CC tissues (Fig. 3G). However, the mRNA expressions of RAD51D were not significantly different between the radio-resistant and radio-sensitive CC tissues (Fig. 3H); these findings suggested that the higher protein level of RAD51D may be involved in radio-resistance of CC cells. To further identify the role of RAD51D in conferring CC cell radio-resistance, we transfected the ME180 cells with overexpression plasmid of RAD51D. The colony formation assay result showed that overexpression of RAD51D could increase the resistance to radiotherapy. Co-transfection of overexpression plasmid of RAD51D with shHNF1α in ME180 cells partly abrogated the induced-effect of RAD51D on cell proliferation (Fig. 3I). Collectively, these findings suggested a positive correlation between HNF1α and RAD51D protein level, which is involved in radio-resistance of CC cell.
4.Upregulation of HNF1α increases the expression of circHNF1α in radio-resistant CC cells
HNF1α regulates the gene and non-coding RNA expressions at the transcriptional level. In CC cells, we found upregulation of hsa_circ_0028940 (named circHNF1α), which is formed from the HNF1α exon 2–7 (1392 bp length), in radio-resistant CC cell line (Fig. 4A and B). Interestingly, upregulation of HNF1α in ME180 cells increased the expression level of circHNF1α, while knockdown of HNF1α in SiHa cell decreased the expression level of circHNF1α (Fig. 4C and D); these findings indicated that HNF1α may positively regulate the expression of circHNF1α. Furthermore, colony formation assay result performed that overexpression of circHNF1α increased the radio-resistance of CC cells compared with the control group. However, this increase was reversed by concomitant depletion of HNF1α (Fig. 4E). These findings suggest that HNF1α positively regulates circHNF1α expression, which participates in radio-resistance of CC cells.
Next, we determined whether HNF1α affected the expression of circHNF1α at the transcriptional level. First, PROMO prediction software was used to identify the putative transcriptional factor of HNF1α and circHNF1α; The results showed that promoter region of HNF1α and circHNF1α has 2 putative binding sites of HNF1α. ChIP-PCR analysis confirmed that HNF1α could directly bind to the region of the HNF1α and circHNF1α promoter (Fig. 4F). Luciferase activity assay further revealed that overexpression of HNF1α could significantly increase the luciferase activity of the plasmid which containing the promoter region of HNF1α and circHNF1α (Fig. 4G). These findings indicated that HNF1α regulates the expression level of circHNF1α at the transcriptional level.
5. circHNF1α elevated the expression of RAD51D by sponging miR-204-3p
As described above, we sought to investigate the involvement of circHNF1α in mediating the correlation between HNF1α and RAD51D. Previous studies have shown that circRNAs function as ceRNAs by sponging microRNAs (miRNAs) to regulate the downstream gene expression in the post-transcriptional level. We first analyzed the potential binding microRNAs of circHNF1α different target prediction programs, miRanda, RNA22, and Rnahdrid. The Venn diagram showed that 11 miRNAs contained the binding-sequences circHNF1α, including miR-33b-3p, miR-184-3p, miR-193a-3p, miR-204-3p, miR-323-3p, miR-363-5p, miR-367-5p, miR-373, miR-483-3p, miR-650, miR-1285-3p (Fig. 5A). Subsequently, we used the biotin-labeled circHNF1α probe pull-down assay to explore the expressions of miRNAs in the circHNF1α-overexpressed ME180 cells. qRT-PCR and agarose gel electrophoresis of PCR products showed significant enhancement of circHNF1α in circHNF1-overexpressed ME180 cells by using biotin-labeled circHNF1α probes (Fig. 5B and 5C). Subsequently, RT-qPCR was performed to detect the expressions of the candidate miRNAs in the precipitates. The results showed enrichment of miR-184-3p, miR-650, and miR-204-3p in the circHNF1α-overexpressed precipitates (Fig. 5D). Furthermore, luciferase assays showed that co-transfection with circHNF1-luciferase-reporter vector and miR-204-3p mimics, but not miR-650 mimics or miR-184-3p mimics, significantly decreased the luciferase activity (Fig. 5E). These findings suggested that circHNF1α can sponge miR-204-3p in CC cells.
To clarify the role of miR-204-3p in mediating the relationship between HNF1α and RAD51D, we overexpressed miR-204-3p or suppressed miR-204-3p in CC cells. The results showed that overexpression of miR-204-3p in CC cells reduced the protein level of RAD51D while silencing of miR-204-3p increased the protein level of RAD51D (Fig. 5F). Next, we found that the RAD51D 3′-UTR has a putative miR-203-3p binding site by using TargetScan. Subsequently, we co-transfected ME180 cells with wild-type (WT) or mut RAD51A 3′-UTR-luciferase reporter and miR-204-3p mimic. Results of luciferase assay result revealed that miR-204-3p mimic markedly reduced luciferase activity of WT RAD51D 3′-UTR but not in the mutation type(Fig. 5H). Additionally, we transfected miR-204-3p mimics or circHNF1α overexpression plasmid in ME180 cells, respectively, or co-transfected them together. Western blot analysis showed that the increased protein level of RAD51D induced by miR-204-mimic was reversed by enforced circHNF1α expression (Fig. 5I). Collectively, these findings indicated that circHNF1α regulates RAD51D protein level by sponging miR-204-3p.
6. Hnf1α/circhnf1α/mir-204-3p/rad51d Axis Regulates The Radio-resistance Of Cc Cells
In order to explore the role of the HNF1α/circHNF1α/miR-204-3p/RAD51D axis in CC cell radio-resistance, some rescue experiments were performed. Firstly, we transfec the shHNF1α or miR-204-3p inhibitor in ME180 cells, respectively, or transfected them together. The protein level of RAD51D was detected by western blot. As shown in Fig. 1A, suppression of miR-204-3p increased the protein level of RAD51D. However, their co-transfection reversed miR-204-3p-induced RAD51D increase alone. Then, colony formation assay showed that downregulated of miR-204-3p in ME180 cells markedly decreased cell sensitivity to irradiation and this inhibitory effect was reversed by concomitant knockdown of HNF1α (Fig. 6B). Similarly, we circHNF1α overexpression plasmid or shRAD51D in Hep2 cells, respectively, or transfected them together. Western blot result showed that overexpression of circHNF1α increased the protein level of RAD51D (Fig. 6C). However, co-transfection of shRAD51D almost reversed the circHNF1α-induced increase in RAD51D. Colony formation assay also showed that overexpression of circHNF1α in CC cells significantly increased their resistance to irradiation and this effect was reversed by depletion of RAD51D (Fig. 6D). These findings further confirmed that the HNF1α/circHNF1α/miR-204-3p/RAD51D axis regulates CC cell radio-resistance.
7. Hnf1α Is Involved In Cc Radio-resistance In Vivo
To determine the pathophysiological effect of HNF1α on CC radio-resistance, we established a CC xenograft model of nude mouse. First, the stable-knockdown-HNF1α SiHa cells or normal SiHa cells were implanted into nude mice. After 14 days, the mice were administered 16 Gy of irradiation. As shown in Fig. 7A and Fig. 7B, the suppression of HNF1α group had a smaller tumor volume compared with that in the control group. Consistently, knockdown of HNF1α resulted in decreased level of RAD51D protein. The results of qRT-PCR showed decreased expression of circHNF1α in the HNF1α-suppression group compared with the control group (Fig. 7D). These results suggested that knockdown of HNF1α increases the radio-sensitivity of CC in vivo.