USF1 regulated circPRDM4 modulates tumorigenesis and immune escape in chemoresistant cervical cancer

Abstract Cervical cancer (CC) represents a major global health concern, characterized by chemoresistance and immune evasion mechanisms. Circular RNAs (circRNAs), which play a crucial role in cancer pathogenesis, particularly in the case of CC, have gained significant attention. The primary objective of this study was to investigate the functional significance of circRNAs in chemoresistant CC. A significant upregulation of circPRDM4 expression in chemoresistant CC cells. To investigate the functional consequences, we conducted circPRDM4 knockdown experiments, which resulted in the effective blockade of immune escape mechanisms employed by chemoresistant CC cells. Furthermore, circPRDM4 knockdown demonstrated a significant suppression of tumorigenesis in CC cells, highlighting its contribution to the oncogenic potential of CC. Investigating the regulatory mechanisms involved, we found that the transcriptional factor upstream stimulatory factor 1 (USF1) acts as an inducer of circPRDM4 expression. Remarkably, USF1 was found to effectively modulate CC cell immune escape via its interaction with circPRDM4. Moreover, our results revealed that USF1 is intricately involved in CC cell tumorigenesis through the regulation of circPRDM4. Collectively, our study elucidates the significant roles of circPRDM4 and its upstream regulator USF1 in chemoresistant CC cells. These findings underscore the importance of circRNAs in CC pathogenesis and provide valuable insights into the mechanisms underlying immune escape and tumorigenesis.

impede the progression of distant metastases. 4As a first-line agent for CC chemotherapy, cisplatin (DDP) currently plays a prominent role.However, the curative impact of this treatment is restricted due to the emergence of chemo-resistance in a significant subset of patients. 5Hence, the identification of new prognostic biomarkers assumes significance as it would furnish clinicians with potential therapeutic targets for the implementation of personalized treatment regimens.
Circular RNAs (circRNAs) are a distinct class of RNA molecules that arise from precursor-mRNAs through a process called backsplicing.Unlike linear RNAs, circRNAs possess a covalently closed loop structure without a specific 5′-3′ polarity or a polyadenylated tail.1][12] Extensive research has unveiled the critical pathological roles played by cir-cRNAs, establishing their close association with the development and progression of various cancers.Dysregulation of circRNAs has emerged as a significant contributing factor in cancer pathogenesis. 10,13Chen et al. 14 made a noteworthy discovery regarding the role of circMTO1 in the chemoresistance of CC.Their findings demonstrated that circMTO1 acts as a miRNA sponge, exacerbating the chemoresistance of CC cells. 14Through their study, Guo et al. provided compelling evidence regarding the significant involvement of hsa_circ_0023404 in the progression of CC.Their findings revealed that hsa_circ_0023404 plays a crucial role by reducing the sensitivity of CC cells to cisplatin treatment. 15The involvement of circRNAs in the development of chemoresistance in CC is of paramount importance.However, the precise underlying mechanisms by which circRNAs regulate CC chemoresistance remain poorly elucidated.
Our study aims to explore a novel functional circRNA in CC chemoresistance.First, we utilized circRNA microarray analysis on two pairs of CC chemoresistance (DDP) cell lines.It was found that circPRDM4 was found to be a most significantly upregulated gene in both DDP-resistant CC cells.By performing a serial experiment, it was found that circPRDM4 knockdown markedly promoted the sensitivity of CC cells to cisplatin treatment and blocked immune escape of CC cells.Additionally, circPRDM4 knockdown significantly inhibited tumorigenesis of CC cells.Mechanically, upstream stimulatory factor 1 (USF1) was found to transcriptionally regulate circPRDM4 expression and modulate chemoresistance and immune escape of CC cells through inducing circPRDM4.Our findings revealed a novel USF1/circPRDM4 axis in the progression of CC cell chemoresistance and immune escape, which underscores the importance of circRNAs in CC pathogenesis and provide valuable insights into the mechanisms underlying immune escape and tumorigenesis.

| Cell treatment
The HeLa, SiHa, HeLa/DDP and SiHa/DDP cell lines were procured from Procell Life Science and Technology in Wuhan, China.
These cell lines were cultured in Dulbecco′s modified eagle medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.The cells were maintained at a temperature of 37°C in a humidified atmosphere containing 5% CO 2 .To establish drug resistance, a concentration of 500 ng/mL Dox (Sigma-Aldrich) was added to the medium for the culture of Dox-resistant cells.However, the Dox maintenance treatment for the resistant cells was discontinued 3 days prior to the experiment.
For the experiments, the mock plasmid pcDNA3.1,small hairpin RNAs (shRNAs) targeting circRNA, and non-specific negative control oligos (sh-NC) were obtained from GenePharma.Lentivirus was purchased from GeneChem.The cells were seeded in six-well plates 24 h before transfection.Transfections were carried out using Lipofectamine 3000 (Invitrogen) as per the manufacturer's instructions.The effects of knockdown or overexpression were assessed using reverse transcription quantitative polymerase chain reaction (RT-qPCR), with RNA extraction performed 48 h after transfection.

| CirRNA microarray analysis
Total RNA was isolated using TRIzol reagent (Life Technologies), and its concentration was determined using a NanoDrop ND-1000 spectrophotometer.To selectively enrich circular RNA (circRNA) while removing linear RNA, Rnase R treatment (Epicentre) was performed.The enriched circRNA was then subjected to amplification and fluorescent labeling using the Super RNA Labeling kit (Arraystar), following the manufacturer's instructions.The labelled complementary RNA (cRNA) was hybridized to the Arraystar Human circRNA Array V2 (8 × 15 K).Subsequently, the slides were washed and scanned using an Agilent Scanner G2505C.The acquired images were analysed using the Agilent Feature Extraction software (version 11.0.1.1).For data processing, including quantile normalization and analysis, the limma package in R was utilized.Differential expression of circRNAs and their expression patterns were identified through fold change filtering and hierarchical clustering techniques.

| Cell Counting Kit-8 assay
For the assessment of cell proliferation rate, a Cell Counting Kit-8 (CCK-8) assay was employed.The cells were seeded in 96-well plates and cultured for 24 h until reaching a density of 3000 cells per well.
Subsequently, a mixture comprising 90 μL of serum-free medium and 10 μL of CCK-8 reagent (Abxin) was added to each well.After incubation with the CCK-8 mixture for 2 h, the absorbance was measured at 450 nm using a microplate reader.This measurement provided quantitative data on cell proliferation.

| Detection of peripheral blood mononuclear cell proliferation
Peripheral blood mononuclear cells (PBMCs) were subjected to a series of steps for immunostaining.First, the cells were fixed using 4% paraformaldehyde, followed by permeabilization with 0.1% Triton X-100 and subsequent blocking with 3% bovine serum albumin.For Ki-67 staining, the cells were incubated overnight at 4°C with the primary antibody, anti-Ki-67 (ab15580, Abcam) and then with the secondary antibody, IgG (Alexa Fluor 647) (ab150075, Abcam).To visualize the nuclei, 4′,6-diamidino-2-phenylindole (Sigma-Aldrich) was used for counterstaining.After applying a sealing solution supplemented with an antifluorescence quenching agent, the slides were captured and analysed using a fluorescence microscope (Olympus) and ImageJ software.

| Western blotting
About 50 μg of total proteins was loaded onto an SDS-PAGE gel and transferred into a PVDF membrane.The membrane was blocked using 5% non-fat milk and incubated overnight at 4°C with primary antibodies.The next day, the membrane was incubated with secondary antibodies.Following that, the membrane was exposed to an ECL solution and imaged to detect the protein bands.The antibodies used in this study were anti-IgG (Abcam), anti-USF1 (Abcam), and anti-GAPDH (Cell Signaling Technology).

| Cell colony assay
Cell proliferation in CC was evaluated by plating cells at a density of 100 cells per well.Following a 10-day culture period, colonies were immobilized using methanol and subjected to staining with a haematoxylin solution.The enumeration of colonies containing at least 50 cells was performed under a light microscope.To investigate cell chemoresistance, CC cells were exposed to cisplatin or carboplatin at specified concentrations for a duration of 6 h prior to seeding at a concentration of 500 cells per well.After an incubation period of 10 days, the colonies were fixed, stained, and captured through photography to facilitate subsequent analysis.

| Transwell assay
To assess the migratory and invasive potential of CC cells, Transwell assays were employed.A cell suspension was introduced into the upper chamber of Transwell plates, which were either coated with Matrigel (BD Biosciences) for invasion assays or left non-coated for migration assays.The lower chambers were supplemented with DMEM containing 10% FBS.Following incubation, the cells that migrated or invaded through the chambers were immobilized, stained and captured through photography using a light microscope.

| Chromatin immunoprecipitation
The chromatin immunoprecipitation (ChIP) assay was performed using the EZ ChIP™ Chromatin Immunoprecipitation Kit (Millipore).HCC cells were first fixed with 1% formaldehyde and then subjected to sonication to generate chromatin fragments.Chromatin immunoprecipitation was carried out using an anti-USF1 antibody (Abcam) and an IgG antibody (Abcam) as a control.The immunoprecipitated DNA was subsequently extracted for PCR analysis.

| Luciferase reporter gene assay
The wild-type PRDM4 promoter region was amplified using PCR and subsequently inserted into the pGL3-basic dual-luciferase

| Statistical analysis
Statistical analysis was conducted using the SPSS software (SPSS).
The two-tailed Student's t-test was utilized for comparing two independent groups.For comparisons involving three or more groups, one-way anova was employed.A p < 0.05 was considered statistically significant.

| Upregulation of circPRDM4 in chemoresistant CC cells
To have a better understanding of chemoresistance-related cir-cRNA in CC progression, circRNA microarray analysis was explored to investigate the circRNA profile of chemoresistant SiHa and HeLa to cisplatin (Figure 1A), the top 10 different expressed circRNAs in two pairs of cell lines are shown (Figure 1B), where circPRDM4 is the most significantly upregulated circRNA.A markedly upregulation of circPRDM4 in DDP-chemoresistant SiHa and HeLa cells had been observed (Figure 1C).A noticeable decrease of circPRDM4 expression upon treatment with oligo (dT)18 primers, indicating the absence of a poly-A tail in its structure, treatment with random hexamer primers did not affect circPRDM4 expression level (Figure 1D).
The transcription inhibitor actinomycin D treatment illustrated that circPRDM4 displayed a longer half-life than PRDM4 mRNA (Fig- ure 1E).Furthermore, circPRDM4 exhibited significant resistance to RNase R digestion when compared to PRDM4 mRNA, as depicted in Figure 1F,G.Results of cellular RNA fractionation indicated that circPRDM4 was abundantly distributed in the cytoplasm of SiHa and HeLa cells (Figure 1H).

| CircPRDM4 knockdown blocked immune escape of chemoresistant CC cells
To explore the effect of circPRDM4 on CC chemoresistance, circPRDM4 knockdown models were constructed using DDPresistant SiHa and HeLa cells (Figure 2A). the viability of DDPresistant SiHa and HeLa cells treated with circPRDM4 shRNAs were detected, circPRDM4 knockdown resulted in a lower IC 50 value compared with normal control group (Figure 2B).CircPRDM4 has been reported that modulates immune escape phenomena. 16Hence, DDP-resistant SiHa and HeLa cells were used to co-culture with PBMCs, the proliferation level of PBMCs were significantly promoted (Figure 2C).Furthermore, the expression of TNFα, IFNγ, IL-10 and TGFβ in the supernatant were also detected.The expression of TNFα and IFNγ were significantly reduced in the supernatant from PBMCs co-cultured with DDP-resistant SiHa and HeLa cells compared with non-co-cultured PBMCs, while these phenomena were reversed by circPRDM4 knockdown (Figure 2E,F,I,J).Moreover, IL-10 and TGFβ expression were markedly increased in the supernatant from PBMCs co-cultured with DDP-resistant SiHa and HeLa cells but reduced upon circPRDM4 knockdown (Figure 2G,H,K,L).

| CircPRDM4 knockdown suppressed tumorigenesis of CC cells
In addition, circPRDM4 knockdown cell models were also generated using normal SiHa and HeLa cells to explore the biological functions of circPRDM4 in CC (Figure 3A).Cell colony results suggested that circPRDM4 knockdown significantly decreased the proliferation ability of CC cells (Figure 3B,C).Furthermore, Transwell assays were also utilized to determine cell migration and invasion ability.It was observed that circPRDM4 knockdown markedly inhibited cell migration (Figure 3D,E) and invasion (Figure 3F,G) level.

| Transcriptional factor USF1 induced circPRDM4 expression
8][19] To reveal the upregulation of circPRDM4 in CC, JASPAR database was utilized, USF1 was found as a potential transcriptional factor of circPRDM4.
Next, it was found that PRDM4 mRNA expression could be positively regulated by USF1 in CC cells (Figure 4A).USF1 has two binding sites on the upstream region of PRDM4 mRNA transcript (Figure 4B) and the binding sequence of USF1 was also obtained (Figure 4C).Luciferase reporter gene assay elucidated that USF1 enhanced the luciferase activity of USF1 + PRDM4 WT1/2, but not USF1 + PRDM4 Mut1/2, indicating that USF1 bound to PRDM4 promoter two regions (Figure 4D).ChIP assay showed that PRDM4 significantly enriched by anti-USF1 (Figure 4E).As indicated in Figure 4F, circPRDM4 expression in CC cells could be positively regulated by USF1.

| USF1 participated in the progression of CC cell immune escape via circPRDM4
To elucidate the USF1/circPRDM4 axis in CC chemoresistance, DDP-resistant CC cell models were generated through stably transfecting USF1 and sh-circ-1.USF1 overexpression resulted in a higher IC50 value compared with normal control group, but this effect reversed by circPRDM4 knockdown (Figure 5A,B).
Regarding to the immune escape detection, PBMC proliferation ability was markedly inhibited by USF1 overexpression but following been promoted by circPRDM4 knockdown (Figure 5C).
Subsequently, the expression of TNFα and IFNγ were significantly induced in the supernatant from PBMCs co-cultured with USF1 overexpression DDP-resistant cells and were inhibited by circPRDM4 knockdown (Figure 5D,E,H,I).IL-10 and TGFβ expression were markedly increased but reduced upon circPRDM4 knockdown (Figure 5F,G,J,K).

| USF1 involved in the tumorigenesis of CC cells through regulating circPRDM4
The tumorigenesis functions of USF1/circPRDM4 axis were also explored.The expression of USF1 and circPRDM1 were detected by western blot and qRT-PCR (Figure 6A,B).The proliferation level

| DISCUSS ION
Cisplatin is regarded as the most efficacious drug for neoadjuvant and salvage treatment of CC. 20 However, the therapeutic efficacy of cisplatin-based chemotherapy is significantly compromised by the inherent resistance to cisplatin as well as the development of acquired resistance. 1,5The intricate mechanism responsible for the inherent reporter plasmid from Promega.This resulted in the generation of the pGL3-PRDM4 reporter construct.The CC cells were transfected with this reporter construct.The cells were seeded in 96well plates at a density of 1.5 × 10 4 cells per well.The Attractene Transfection Reagent (catalogue number 301005, QIAGEN) was utilized for the transfection process.The activity of firefly luciferase was measured and normalized by using Renilla luciferase as an internal control.F I G U R E 1 Upregulation of circPRDM4 in chemoresistant cervical cancer (CC) cells.(A) circRNA microarray analysis utilized to reveal the circRNA expression profile in DDP-resistant CC cells.(B) The top significantly upregulated circRNAs in both DDP-resistant CC cells.(C) CircPRDM4 expression in SiHa/SiHa-DDP and HeLa/HeLa-DDP cells was measured by qRT-PCR.(D) Reverse transcription assays were conducted using either random hexamer or oligo (dT)18 primers.(E) RT-PCR was performed to measure the relative RNA levels after treatment with actinomycin D at the specified time points.(F, G) RT-qPCR was utilized to examine the relative RNA levels after treatment with RNase R or a mock treatment.(H) The expression of circPRDM4 in the nucleus and cytoplasm was analysed, and the results are presented as mean ± SD.All experiments were repeated three times.*p < 0.05, **p < 0.05, ***p < 0.001.

F I G U R E 2
CircPRDM4 knockdown blocked immune escape of chemoresistant cervical cancer (CC) cells.(A) CircPRDM4 knockdown cell models were generated by transfecting sh-NC, sh-circ-1, and sh-circ-2 into SiHa/SiHa-DDP and HeLa/HeLa-DDP cells, circPRDM4 and PRDM4 mRNA expression were detected by qRT-PCR.(B) Cell viability were detected by CCK-8.(C, D) Ki-67 staining was used to measure the proliferation level of peripheral blood mononuclear cells (PBMCs) after co-cultured with DDP-resistant CC cells.(E-H) Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression of TNFα, IFNγ, IL-10, and TGFβ in the supernatant of PBMCs cocultured with DDP-resistant SiHa cells.(I-L) ELISA assay was used to measure the expression of TNFα, IFNγ, IL-10, and TGFβ in the supernatant of PBMCs co-cultured with DDP-resistant HeLa cells.The results are presented as mean ± SD.All experiments were repeated three times.*p < 0.05, **p < 0.05, ***p < 0.001.

F I G U R E 3
CircPDM4 knockdown suppressed tumorigenesis of cervical cancer (CC) cells.(A) CircPRMD4 knockdown cell models were constructed by stably transfecting sh-NC, sh-circ-1, and sh-circ-2 into SiHa and HeLa cells, results were verified by qRT-PCR.(B, C) The proliferation level of CC cells was detected by cell colony assay.(D, E) The migration level was measured by Transwell migration assay.(F, G) The invasion level was detected by Transwell invasion assay.The results are presented as mean ± SD.All experiments were repeated three times.**p < 0.05, ***p < 0.001.

F I G U R E 4
Transcriptional factor USF1 induced circPRDM4 expression.(A) Relative expression of PRDM4 mRNA in USF1 overexpression or knockdown cell models were detected by qRT-PCR.(B, C) The graphical illustration of the binding sites between USF1 and PRDM4 promoter.(D) Luciferase reporter gene assay was performed to evaluate the binding fact between USF1 and PRDM4 promoter.(E) ChIP assay was utilized using anti-IgG and anti-USF1, results were detected by qRT-PCR.(F) CircPRDM4 expression in USF1 overexpression or knockdown cell models were detected by qRT-PCR.The results are presented as mean ± SD.All experiments were repeated three times.**p < 0.05, ***p < 0.001. of CC cells was significantly promoted by USF1 overexpression and attenuated by circPRDM4 knockdown (Figure 6C).The same phenomena were also observed in Transwell migration (Figure 6E) and Transwell invasion (Figure 6E) assays.Those findings suggested that USF1 modulates immune escape and tumorigenesis in chemoresistant CC cells.
insensitivity and acquired resistance to cisplatin is complex and not yet fully comprehended.Hence, uncover the underlying mechanisms of chemoresistance of CC in-depth is urgently needed.By conducting circRNA microarray analysis on two DDP-chemoresistant CC cell lines, circPRDM4 was identified as a most significantly upregulated circRNA.CircPRDM4 knockdown markedly re-sensitive CC cells to cisplatin treatment and blocked immune escape of CC cells.In the past few years, significant emphasis has been placed on understanding the impact of immune evasion in the development of CC.Cellular immunity, particularly involving T lymphocytes, dendritic cells, natural killer cells and macrophages, plays a crucial role in anti-tumour immune responses.Nevertheless, tumours can persist and metastasize despite the presence of a functioning immune F I G U R E 5 USF1 blocked cervical cancer (CC) cell immune escape via circPRDM4.(A, B) Cell viability were detected by CCK-8.(C) Ki-67 staining was used to measure the proliferation level of peripheral blood mononuclear cells (PBMCs) after co-cultured with DDP-resistant CC cells.(D-G) Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression of TNFα, IFNγ, IL-10 and TGFβ in the supernatant of PBMCs co-cultured with DDP-resistant SiHa cells.(H-K) ELISA assay was used to measure the expression of TNFα, IFNγ, IL-10, and TGFβ in the supernatant of PBMCs co-cultured with DDP-resistant HeLa cells.The results are presented as mean ± SD.All experiments were repeated three times.*p < 0.05, **p < 0.05, ***p < 0.001.