HNF1α Contributes to Radio-Resistance in Cervical Cancer Via Regulating the CircHNF1α/miR-204-3p/RAD51D Regulatory Axis

Background: Radiotherapy as an important primary treatment has effectively improved the survival of patients with cervical cancer (CC). However, some patients do not show optimal benets of radiotherapy due to radio-resistance. Therefore, identication of biomarkers of radio-resistance and unraveling the underlying mechanisms of radio-resistance is a key imperative for these patients. Methods: The expression levels of circHNF1α, miRNAs, and mRNA in tissues and cell lines were detect by qRT-PCR analysis. The levels of proteins were analyzed by western blot. Cell proliferation ability was measured by colony formation assay. RNA pull-down and uciferase reporter assay analysis were performed to identify the sponging microRNAs of circHNF1α. The target gene of miR-204-3p was determined by luciferase reporter assay. Chromatin immunoprecipitation (ChIP) analysis and luciferase reporter assay were performed to identify the transcription factor of circHNF1α. Results: We found signicant upregulation of HNF1α expression in radio-resistant cervical cancer tissues and cell lines. Depletion of HNF1α reduced whereas overexpression of HNF1α promoted the resistance of CC cells to irradiation in vitro and in vivo. HNF1α positively regulated RAD51D at the protein level but not at the mRNA level, thus attenuating radio-resistance of CC cells. Mechanistically, upregulation of HNF1α enhanced circHNF1α transcription and promoted circHNF1α biogenesis, which in turn sponged miR-204-3p and thus relieved their repression of the RAD51D expression. The HFN1α/circHNF1α/miR-204-3p/RAD51D regulatory axis was found to play a critical role in conferring radio-resistance of CC cells. Conclusions: Dysregulation of the HFN1α/circHNF1α/miR-204-3p/RAD51D axis may promote the radioresistance of CC cells. Blocking this pathway may provide therapeutic benets against CC radioresistance. 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-Page

3p/RAD51D regulatory axis was found to play a critical role in conferring radio-resistance of CC cells.
Conclusions: Dysregulation of the HFN1α/circHNF1α/miR-204-3p/RAD51D axis may promote the radioresistance of CC cells. Blocking this pathway may provide therapeutic bene ts against CC radioresistance.

Background
Globally, cervical cancer (CC) is the 4th most frequently cancer and the second lethal malignancy in females in females. In the United States, there were an estimated 570,000 new cases of CC and 311,000 deaths in 2018 [1,2]. Recent advances in diagnostic technology, surgical methods, and radiotherapy have helped improve the 5-year OS and PFS of patients with CC. However, more than 25% patients develop local or distant relapse due to radio-resistance, leading to poor 5-year PFS rates (< 20%) [3,4]. Therefore, characterization of the underlying molecular mechanisms of CC radio-resistance and development of alternative therapeutic targets is a key imperative. Circular RNAs (circRNAs) is a kind of new endogenous non-coding RNA which has a covalently closed loops structure with neither poly-adenylated tail in 3'ends nor the cap structure at 5'end [5,6]. Accumulate evidence has con rmed that circRNAs may regulate gene expression and pathways by sponging miRNAs, interacting with RNPs and moderating transcription [7][8][9]. Recent studies have unraveled a wide range of functions of circRNAs; this has led to increasing recognition of the role of circRNAs in diseases, especially human cancers [10]. Some studies have con rmed the effects of circRNAs on cervical cell tumorigenesis and tumor progression. For example, circSLC26A4 was upregulation in CC tissues and cell lines.
Overexpression of circSLC26A4 had a relationship with poor survival [11]. Similarly, CircCLK3 was shown to be upregulated in CC tissues and cell lines. Knockdown of circCLK3 inhibited CC cell growth, invasion and migration. Additionally, studies have documented the involvement of circAMOTL1, circMOT1, and circ_0023404 in cell viability, chemo-resistance, and autophagy of CC [12][13][14]. However, the potential role of circRNAs in CC radio-resistance has not been explored.
HNF1α is a key member of the hepatocyte nuclear factor 1 (HNF-1) transcription factor family; it has been identi ed as one of the critical transcription factors in the early development of liver, pancreas, and kidney [15,16]. An increasing body of evidence has implicated mutant of HNF1α in the causation of diabetes, including maturity onset diabetes of the young and type 2 diabetes [17,18]. Recent studies have shown that HNF1α may affect survival of cells by moderating cell growth and metastasis in different human cancers [19,20]. For example, HNF1α as an oncogene, was upregulated in pancreatic ductal adenocarcinoma (PDA) cells. Overexpression of HNF1α in PDA cells was shown to induce cell proliferation in vivo and in vitro [21]. Knockdown of HNF1α was found to promote epithelialmesenchymal transition (EMT) in liver cancer cell lines via decreasing the protein level of E-cadherin and increasing the level of TGFβ1 [22]. Of note, HNF1α functions as an oncogene in CC and has involvements in CC cell survival and EMT, all of which enhance cell motility and metastasis [23]. However, the effect of HNF1α in radio-resistance of CC cells is not clear.
In this study, we revealed that the expression of HNF1α was increased in radio-resistant CC tissues and cell lines. Overexpression of HNF1 in CC cells increased cell resistance to irradiation, in vitro and in vivo.
Furthermore, HNF1α positively regulated RAD51D protein level at the post-transcriptional level. HNF1α, as a transcription factor, was found to regulate circHNF1 level in CC cells. circHNF1α induced RAD51D protein level by sponging miR-204-3p, which directly targeted RAD51D in CC cells. The HNF1α/circHNF1α/miR-204-3p/RAD51D axis was performed to play an important role in the radioresistance of CC and represents a novel therapeutic target to overcome the resistance of CC to radiotherapy.

Methods
The detailed procedures cell proliferation assays, chromatin immunoprecipitation-qPCR, luciferase reporter assay, analyses of apoptosis, RNA synthesis and biotin pull-down as well as key reagents are described in Supplementary Experimental Procedures.

Patients And Tumor Samples
Ten pairs of cervical carcinoma radio-sensitive tissues and cervical carcinoma radio-resistance tissues were obtained from the fourth Hospital of the Hebei Medical University between July 2012 to June 2017.
The effectiveness of radiotherapy was evaluated as previously described [24]. All patients had a con rmed histopathological diagnosis of CC (stage IIIB) and had not been treated with chemotherapy or radiotherapy.

Cell Culture And Transfection
Human CC cell lines (Hela, ME180, SiHa, and MS751) were obtained from our laboratory. All cell lines were cultured in high-glucose DMEM medium containing 10% FBS and 100 units/mL of penicillin and streptomycin at 37 °C in an atmosphere of 5% CO 2 .
Lipofectamine 2000 (Invitrogen) was used for cell transfection according to the manufacturer's protocols. The siRNA, miRNA mimic and anti-miRNA, and negative controls were designed by GenePharma Co. Ltd.
(Shanghai, China). The overexpression plasmids and luciferase assay plasmids were purchased from the Genewiz Company (Suzhou, China).

Statistical analysis
All data are presented as mean ± standard error of the mean. Student's t test was used to assess betweengroup differences; for multiple comparisons or repeated measurements, ANOVA or repeated ANOVA followed by Tukey's post hoc test was used. P values < 0.05 were considered indicative of statistical signi cance. All statistical analyses were performed using the Graphpad Prism 7 software (GraphPad Software, San Diego, CA, USA).

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 radiosensitive and radio-resistant CC tissues. As shown in Fig. 1A and 1B, the protein level of HNF1α was signi cant 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 radiosensitive 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-offunction 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 ndings suggest that the higher expression level of HNF1α may contribute to radio-resistance in CC cells.

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 speci c 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 signi cantly 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 signi cantly 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 signi cantly increased cell radio-resistance in ME180 cells (Fig. 2F). These ndings suggests that knockdown of HNF1 reduces while overexpression of HNF1α induces the radio-resistance in CC cells.

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 [25]. 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 signi cant 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 in uenced 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 ndings 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 signi cantly different between the radio-resistant and radio-sensitive CC tissues (Fig. 3H); these ndings 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 ndings 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 ndings 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 ndings 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 con rmed 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 signi cantly increase the luciferase activity of the plasmid which containing the promoter region of HNF1α and circHNF1α (Fig. 4G). These ndings indicated that HNF1α regulates the expression level of circHNF1α at the transcriptional level.
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 ndings indicated that circHNF1α regulates RAD51D protein level by sponging miR-204-3p.

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 circHNF1αinduced increase in RAD51D. Colony formation assay also showed that overexpression of circHNF1α in CC cells signi cantly increased their resistance to irradiation and this effect was reversed by depletion of RAD51D (Fig. 6D). These ndings further con rmed that the HNF1α/circHNF1α/miR-204-3p/RAD51D axis regulates CC cell radio-resistance.

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.

Discussion
The main ndings of this study were: 1) HNF1α was up-regulated in radio-resistant CC tissues compared with radio-sensitive CC tissues;2) Overexpression of HNF1α promoted CC cell radio-resistance, while depletion of HNF1α reduced CC cell radio-resistance in vivo and in vitro 3) HNF1α was found to regulate circHNF1α expression at the transcriptional level which could sponge of miR-204-3p in CC cells; 4) HNF1α was found to positively regulate RAD51D protein expression in CC cells via the circHNF1α/miR-204-3p axis. These ndings suggest a critical role of the HNF1α/circHNF1α/miR-204-3p/RAD51D axis in CC cell radio-resistance.
Radiotherapy is the primary treatment for CC that confers survival bene t. However, failure of radiotherapy due to radio-resistance contributes to poor clinical outcomes and high morality in these patients. Previous studies have found that abnormal expression of gene may be associated with radioresistance. For example, Yao reported that higher level of SOX2 was responsible for CC cell radioresistance. Overexpression of SOX2 was observed to be closely related to cell growth, suvival, and cell cycle changes by regulating the Hedgehog pathway [26]. Suzuki also revealed that GRP94 may serve as one of the molecular targets for combating radio-resistance in CC cells. Silencing of GRP94 in CC cells induced increased sensitivity to radiotherapy [27]. Besides, Zou found that depletion of HMGB3 signi cantly enhanced the radio-sensitivity of CC cells, while overexpression of HMGB3 had the opposite effect; this effect was mediated via transcriptional upregulation of hTERT [28]. The present study found a correlation between high level of HNF1α and radio-resistance of CC tissues. Importantly, knockdown of HNF1α markedly induced radio-sensitivity of CC cells while overexpression of HNF1α had the opposite effect. These ndings suggest that HNF1α may serve as a biomarker for predicting the sensitivity of CC cells to radiotherapy.
An increasing body of evidence has implicated the abnormal expression of non-coding RNAs (including miRNA, lncRNA, and circRNA) in the development and radio-resistance of CC cells. For example, studies have demonstrated reduced expression of LncRNA GAS5 in the radio-resistant human CC tissues and cell lines. Overexpression of GAS5 was found to enhance the radio-sensitivity of CC cells by inhibiting the function of miR-106 [29]. Additionally, high level of LncRNA HOTAIR showed an association with radioresistance. Depletion of HOTAIR in CC cells led to upregulation of apoptosis and increased the sensitivity to radiotherapy [30]. Moreover, LncRNA UCA1, and LncRNA DANCR have been indicated to play an important role in radio-resistance in CC cells [31,32]. Furthermore, miR-181a, miR-4429, and miR-16-5p were found to be involved in radio-resistance of CC cells [24,33,34]. Despite the recent advances regarding lncRNAs and miRNA, the biogenesis of circRNAs and the molecular mechanisms underlying CC cell radio-resistance are not well characterized. In the present study, we identi ed for the rst time the upregulation of hsa_circ_0028940 (which we named as circHNF1α), formed from the exon 2-7 of HNF1α, in radio-resistant CC cell line and tissues. Overexpression of circHNF1α signi cantly decreased the radio-sensitivity of CC cells. According to a previous study, circular RNAs may be generated along with the transcription of its parental gene [35]. Similarly, in the present study, we demonstrated that HNF1α as transcription factor can bind to the promoter region of circHNF1α and positively regulate the expression of circHNF1α. However, further research is required to determine whether the circHNF1α in turn regulates the expression of its parental gene.
Increasing evidence has suggested that the most important function and mechanism of action of circRNAs is their role as miRNA sponge molecules. In the present study, we found that circHNF1α may directly sponge miR-204-3p, which targeted and repressed the expression of RAD51D. According to a previous study, the correlation between HNF1α and RAD51D is likely mediated by circHNF1α. These results suggests that the knockdown of HNF1B may play a role in drug resistance in ovarian cancer by directly relating with these drug resistance-related proteins including RAD51 (as determined by bioinformatics analyses). However, the underlying mechanism of the relationship between HNF1α and RAD51 was not elaborated. In the present study, we found that HNF1α regulates circHNF1α at the transcriptional level. Upregulation of HNF1α enhances circHNF1α transcription and promotes circHNF1α biogenesis, which in turn sponges miR-204-3p and thus relieves their repression of the RAD51D expression. In other words, on one hand, HNF1α may function as a transcriptional factor and directly regulate the downstream gene expression. On the other hand, the TF may also regulate the protein level indirectly by affecting the expression of non-coding RNAs.

Conclusion
In summary, our ndings indicate that HNF1α upregulation in CC cells led to the formation of the HNF1α/circHNF1α/miR-204-3p/RAD51D regulatory axis. These results highlight the importance of the HNF1α/circHNF1α/miR-204-3p/RAD51D axis in conferring radio-resistance in CC. The identi ed regulatory axis may serve as a potential therapeutic target against radio-resistance of CC.