Transient receptor potential canonical 3 is required for HPV-induced malignant transformation of cervical epithelial cells

Abstract

Human Papillomavirus (HPV) types 16 and 18 are well-established causative agents in cervical cancer. However, the mechanism of malignant transformation remains unclear. Although epithelial-mesenchymal transition (EMT) is regulated by Ca²⁺ signaling, the functions of transient receptor potential canonical (TRPC) channel in cervical cancer have not been reported. Herein, employing multiple biological approaches, we first revealed that HPV16 and HPV18 infections significantly upregulated the expression of TRPC3 that orchestrated Ras-MAPK and MEK-ERK pathways in the abnormal transformation of cervical epithelial cells. Our transcriptomic sequencing of HPV-infected cervical epithelial cells with depletion of TRPC3 suggested the significant influence of TRPC3 on genes involved in the process of epithelial-mesenchymal transition (EMT). Consistently, inhibition of TRPC3 successfully suppressed HPV-triggered cell viability and EMT. Moreover, we found TRPC3 maintained the viability of HPV - infected cells by suppressing excessive MAPK activation through regulating Ras GTPase - activating protein 4 (RASA4), which was validated by the detection of phosphorylated ERK1/2 (p-ERK1/2).These findings were further confirmed in the HPV-infected female BALB/c mice, highlighting TRPC3 as a key hub in mediating transformation This study advances the knowledge about the Ca²⁺ signaling-related molecular mechanism underlying HPV-driven malignant transformation. Targeting TRPC3 may have broader therapeutic implications.

This study reveals that the TRPC3 channel is essential for HPV-driven cervical cancer. TRPC3 promotes malignant transformation by regulating the RASA4/MAPK pathway and EMT. Targeting TRPC3 could be a new therapeutic strategy.

Introduction

Cervical cancer remains a significant global burden, with human papillomavirus (HPV) infection identified as the primary culprit. HPV, a DNA virus, is the most common sexually transmitted pathogen to cause almost all cases of cervical cancer. While numerous HPV strains exist, HPV 16 and 18 are responsible for approximately 70% of cervical cancer cases through integrating their DNA into the host cells’ genomes and leading to the malignant transformation^1,2. Despite substantial efforts have been made among researches, the precise mechanism by which HPV transforms normal epithelial cells into malignant entities remains elusive. This knowledge gap hinders the development of effective therapeutic strategies beyond current prevention methods such as vaccination and screening^3–6.

Epithelial-mesenchymal transition (EMT) is a critical step in the development of cervical cancer. Through EMT, epithelial cells lose their polarity and cell-cell adhesion, and gain migratory and invasive properties to become mesenchymal stem cells. EMT can be induced by various signaling pathways, such as MAPK, Wnt/β-catenin, transforming growth factor-β (TGF-β), and epidermal growth factor (EGF), etc^7–9. Interestingly, many factors involved in the process of EMT are regulated by the Ca²⁺ signal¹⁰. The transient receptor potential canonical (TRPC) is a classical group of calcium-permeable cation channels. Previous studies have found that TRPC was implicated in the regulation of Ca²⁺ balance and the expression/activation of calcium-related factors, thereby playing a key role in the modulation of multiple cellular physiological processes^11–13. Upregulation of TRPC channels has been reported to be associated with the increased cell proliferation and migration in different types of cancers^14–16. Although the Ca²⁺ signaling pathway seems to be closely associated with the occurrence, development, and prognosis of cervical cancer^17,18, the functions of TRPC channel in cervical cancer have not been reported. Whether TRPC plays a role in the HPV-induced dysregulation of human cervical epithelial cells awaits investigation.

TRPC3, as a member of TRPC superfamily, has been reported as an important regulator in several tumors beyond cervical cancer. Upregulation of TRPC3 was found to be correlated with the enhanced proliferation and survival of malignant cells^19–21. In the current study, we seek to unravel the precise role of TRPC3 in orchestrating HPV-induced dysregulation of human cervical epithelial cells. The signaling pathways involved in the functions of TRPC3 and if targeting TRPC3 could inhibit HPV-induced malignant transformation of cervical epithelial cells are of interests.

Material and methods

Cell culture and HPV infection

Immortalized cervical epithelial cells of human (H8 cell line, BFN607200572, BLUEFBIO, China) and human primary cervical epithelial cells (PCS480-011, ATCC, hereafter refer to as HCECs) were used in this study. Both cells were cultured in phenol red-free RPMI 1640 media (Invitrogen, Cat# 11875093) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen, Cat# A5670701) and 1% penicillin/streptomycin at 37 °C. HCECs were cultured in the same condition, except the concentration of FBS was 15%.

Solvent control and safety statement

In this study, all small-molecule inhibitors (including Pyr3 and PD98059) were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions. For all in vitro cell-based experiments, the final working concentration of DMSO was rigorously maintained at ≤0.1% (v/v). Extensive literature reports and prior validation in our laboratory confirm that DMSO at this concentration has no significant effects on cell viability, proliferation, or apoptosis, and it is widely recognized as a safe concentration for solvent control. In the animal experiments, the Pyr3 injection solution was prepared in PBS containing 0.1% DMSO, a solvent system also confirmed to be non-toxic to mice. Therefore, for all experiments utilizing DMSO as the solvent in this study, the corresponding control groups were solvent control groups treated with an equal volume of DMSO, ensuring the accuracy of the experimental results.

HPV infection

GFP-tagged HPV16 and HPV18 (E6/E7) RNA pseudovirions (PsV) were produced by co-transfection of 293TT cells with a plasmid cocktail: p16SheLL (Cat#37320), p18SheLL (Cat #37321), and either pClneo-EGFP (Cat #37021) or p8RwB (Cat #48733), all from Addgene. Forty-eight hours post-transfection, cells were harvested and lysed following multiple steps of suspension and centrifugation. Then the pooled supernatants were purified over a 25-39% continuous iodixanol gradient (Optiprep, PROGEN Biotechnik, Cat#1893) in Quickseal polypropylene tubes (Beckman, Cat#344622). Following ultracentrifugation, around 12 fractions of 200 μl each were collected by puncturing the tube bottoms. Viral titers represented by viral genome equivalent (VGE) were determined as previously reported²². H8 and HCECs (5 ×104 cells/well) were seeded onto 12-well plates and were infected with EGFP-labelled HPV PsV at a multiplicity of VGE at 37 °C. Fluorescence of GFP on PsV-infected cells were detected at 48 hours using a Nikon TE300 Eclipse microscope equipped with a 10x objective len.

Cell transfection for RASA4 overexpression

H8 cells and HCECs were transfected with RASA4 overexpression plasmid or empty vector (negative control) using Lipofectamine™ 3000 (Invitrogen, Cat# L3000015) per the manufacturer’s protocol. Briefly, cells were seeded in 6-well plates (5×10⁵ cells/well) and cultured to 70–80% confluence (24 h post-seeding) under conditions described in “Cell culture and HPV infection”. For each well, 2 μg plasmid DNA and 5 μl Lipofectamine™ 3000 were separately diluted in Opti-MEM™, mixed, and incubated for 20 min at room temperature to form complexes. Complexes were added to cells, and after 6 h, medium was replaced with complete medium. Transfection efficiency was verified by Western blot (24 h post-transfection) to ensure ≥2.5-fold RASA4 overexpression vs. empty vector group.

Western blot

After infection for 24, 48, 72, and 96 hours respectively, total protein lysates were extracted from HPV-infected H8 and HCECs using M-PER Protein Extraction Reagent (Thermo, Cat# 78505) at the indicated time points. To detect the expression of TRPC3 protein, we used rabbit anti-TRPC3 antibody (1:1000, Abcam, Cat# ab300142) and horseradish peroxidase-conjugated secondary antibody (1:3000, Thermo, Cat# C31460100).

For TRPC3 detection in uninfected cells (for Supplementary Fig. S3), H8 and HCECs without HPV infection were cultured for 0, 24, 48, 72, and 96 hours; total protein was extracted with M-PER reagent (Thermo, Cat# 78505) plus 1% protease inhibitor cocktail (Roche), and TRPC3 was detected using the same antibodies and protocol as above.

To detect RASA4 and MAPK/ERK pathway proteins, cells were pre-transfected with RASA4 overexpression plasmid or empty vector, then infected with HPV16/18 PsV for 72 hours and treated with 10 μM Pyr3 for 24 hours. Total protein was extracted using RIPA lysis buffer (Beyotime, Cat# P0013B) with protease and phosphatase inhibitors (Roche). Primary antibodies used: ERK1/2 (1:1000, Cat# 4695), p-ERK1/2 (1:2000, Cat# 4370).

To detect the markers of EMT, HPV-infected H8 and HCECs were treated with Pyr3 (a TRPC3 inhibitor) for 24 hours. The primary antibodies used for the Western blot analyses are as follows: rabbit anti-ZEB1 (1:1000, Cat# 3396), rabbit anti-Snail (1:1000, Cat#3879), and rabbit anti-Slug (1:1000, Cat#9585) antibodies are from Cell Signaling Technology. Rabbit anti-Twist1 (1:1000, Cat# ab314056) is purchased from Abcam. Secondary antibody used HRP-conjugated Goat anti-rabbit IgG (1:5000, Thermo, Cat#32460). Detection was performed using an enhanced chemiluminescent substrate (Thermo, Cat#34580). The density of the protein bands was quantified utilizing Image J software (version 1.48 v, National Institutes of Health, USA).

Fluorescence imaging

For immunostaining, HPV-PsV-infected cells were plated on coverslips and fixed with 2% paraformaldehyde (Sigma, Cat#8.18715) and permeabilized in 0.1% Triton X-100 (Sigma, Cat#T8787). After blocking with 2% bovine serum albumin (BSA, Sigma, Cat#V900933) and 5% normal goat serum (Invitrogen, Cat#16210064), cells were incubated with the following primary antibodies respectively: rabbit anti-TRPC3 (1:100, Abcam, Cat#ab300142) and rabbit anti-RASA4 (1:100, Abcam, Cat# ab157190) at 4 °C overnight. Secondary detection was performed using Alexa Fluor 594 goat anti-rabbit antibody (1:100, Invitrogen, Cat# A-11012). Nuclei were stained with DAPI (1:5000, Roche, Cat#10236276001) for 10 minutes at room temperature. Slides were mounted using a fluorescence mounting medium (Dako, Cat# GM30411-2) and imaged on an Olympus FluoView FV1000 confocal laser scanning microscope with a 60x objective len. Image analysis was performed using FV1000 software (Olympus).

Confocal imaging of Ca²⁺

H8 cell line and HCECs were infected with HPV16 or 18 PsV for 72 hours and treated with an inhibitor of TRPC3 (Pyr3, 10 μM, Sigma, Cat# 648490) for another 30 minutes. Equal concentration of dimethyl sulfoxide (DMSO) (Sigma, Cat# 472301) was used as the solvent control. Ca²⁺ influx was detected using Rhod-3 Calcium Imaging Kit (Thermo, Cat# R10145) as previously described²³ and fluorescent microscopy imaging.

Pyr3 was dissolved in DMSO, and the solvent control group was treated with an equal volume of DMSO.

Cell viability and survival assay

Cell viability after HPV infection and treatment with MEK-ERK pathway inhibitor (PD98059, 20 μM, Selleckchem, Cat#S1036) was detected using Cell Counting Kit-8 (Dojindo Molecular Technologies, Cat#CK04) according to the manufacturer’s instruction. Absorbance at 450 nm was recorded via microplate reader, and relative cell viability was normalized to the untreated control.

For all drug treatments, a solvent control group (treated with an equal volume of DMSO) was included in parallel.

HPV16/18-infected H8 and HCECs were treated with Pyr3 (a TRPC3 inhibitor, 10 μM) for 24 hours, and cell death was detected using LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific, Cat#88954) per the manufacturer’s instructions. Absorbance was measured at 490 nm (680 nm as reference) with a Bio-Rad Model 680 microplate reader, and cytotoxicity percentage was calculated as: [(Experimental LDH release - Spontaneous LDH release)/(Maximum LDH release - Spontaneous LDH release)] × 100%. For clarity, in all LDH release assays, the cytotoxicity values were normalized to the untreated WT group, which was set as 100% for comparative analysis.

Knockdown of TRPC3 and RNA sequencing

To knockdown TRPC3, H8 cell line and HCECs were transduced with lentiviral vectors (Beijing Zhiyuan Shenlan Technology, China) expressing TRPC3-targeting shRNA (5’-GCTTCAGCAGCCTCATTTCTA-3’) and non-target shRNA (5’-CAACAAGATGAAGAGCACCAA-3’). Successful knockdown of TRPC3 was verified at the mRNA level at 48 hours after shRNA transduction using quantitative PCR with specific primers for TRPC3 (forward primer: 5’-GCGTCTTCCTTGGTGTGTCT-3’, reverse primer: 5’-AGCCAGGAAGACCACGATGA-3’). Then cells were infected by HPV16 PsV for another 72 hours. The expression of TRPC3 protein was also detected by Western blot after HPV infection.

RNA libraries were prepared from 0.1 to 4 μg of total RNA using the TruSeq RNA Sample Prep Kit (Illumina, Cat#RS-122-2001) following the manufacturer’s instructions and sequenced on an Illumina HiSeq 2500 platform using a paired-end sequencing strategy. Raw reads were quality filtered using PRINSEQ to remove those with a mean Phred score below 20 or a length shorter than 30 nucleotides. The filtered reads were aligned to the human reference genome GRCh38 using the STAR aligner (version 2.7.3a). Gene Ontology (GO) enrichment analyses were performed using clusterProfiler package.

Quantitative RT-PCR

Total RNA was extracted from HPV-infected H8 and HCECs using RNeasy Mini Kit (Qiagen, Cat#74104) and reverse-transcribed into complementary DNAs (cDNAs) using SuperScript IV VILO Master Mix (Invitrogen, Cat# 11756050). Quantitative PCR was performed using QuantStudio 5 Real-Time PCR System (Applied Biosystems, Thermo). The relative mRNA levels of target genes were normalized to GAPDH. Primer information are as follows:

ZEB1 forward: 5’-GATGATGAATGCGAGTCAGATGC-3’

ZEB1 reverse: 5’-ACAGCAGTGTCTTGTTGTTGT-3’

Snail forward: 5’-TCGGAAGCCTAACTACAGCGA-3’

Snail reverse: 5’-AGATGAGCATTGGCAGCGAG-3’

Slug forward: 5’-CGAACTGGACACACATACAGTG-3’

Slug reverse: 5’-CTGAGGATCTCTGGTTGTGGT-3’

Twist1 forward: 5’-GTCCGCAGTCTTACGAGGAG-3’

Twist1 reverse: 5’-GCTTGAGGGTCTGAATCTTGCT-3’

GAPDH forward: 5’-GGAGCGAGATCCCTCCAAAAT-3’

GAPDH reverse: 5’-GGCTGTTGTCATACTTCTCATGG-3’

Ethical statement

All animal experiments were conducted in accordance with relevant ethical guidelines for animal use and were approved by the Medical Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (Approval No: KT-2022-GuiKe-009). We confirm that we have complied with all relevant ethical regulations for animal use.

Animals

Female BALB/c mice (6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals were housed under standard specific pathogen-free (SPF) conditions with a 12-hour light/dark cycle, and provided with food and water ad libitum.

Establishment of HPV-infected mice

To further investigate the role of TRPC3 in vivo, luciferase-tagged E6 and E7 (coding proteins of HPV16 and HPV18) plasmids were constructed and transfected into host cells as described above. BALB/c mice (female, 6-week old, purchased from Beijing Vital River Laboratory Animal Technology) were synchronized into estrus-like state by subcutaneous injection of 3 mg Depo-Provera (Pfizer) at 8 days before challenge with HPV pseudovirus. Six hours prior to virus challenge, mice were deeply anesthetized using intraperitoneal injection of 1% pentobarbital sodium at a dose of 50 mg/kg body weight, or inhalation of 2-3% isoflurane in oxygen. After ensuring the absence of pedal reflexes, mice were prepped intravaginally with an injection of 20 μL of 4% Nonoxynol-9 (BOC Sciences, Cat# 26027-38-3). Each mouse was then challenged intravaginally with 5 × 10⁶ IU of HPV16 or HPV18 pseudovirus (20 μL) containing 2% carboxymethylcellulose (Sigma, Cat#C9481). All animal experiments were approved by the institutional Animal Care and Use Committee. Inoculation of HPV was confirmed through detection of luciferase expression using an IVIS 200 bioluminescence imaging system.

At the end of the experimental period, euthanasia was performed by cervical dislocation under deep anesthesia. The procedure was conducted in strict accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals.

Pyr3 treatment and assays of tissue section

After successful modeling, HPV16- and HPV18-infected mice were each divided into two groups for intraperitoneal injection of a TRPC3 inhibitor (Pyr3, 0.1 mg/kg) or PBS every 3 days. Following a total of 4 treatments, mice were euthanized at day 15 post-initial HPV infection and cervical specimens were obtained. Immunofluorescence staining was performed on mouse cervical sections using anti-TRPC3 (1:100, Abcam, Cat# ab300142), anti-E-cadherin (1:100, Abcam, Cat#ab40772), anti-N-cadherin (1:100, Abcam, Cat# ab76011) and anti-Vimentin (1:100, Abcam, Cat# ab92547) antibodies. The expressions of ZEB1 and Snail were detected by immunohistochemical analysis using the specific primary antibodies as mentioned above and DAB substrate kit (Cell Signaling Technology, Cat#8059). TUNEL staining was performed on mouse cervical sections according to the instruction of manufacturer (Beyotime, Cat#C1086 China).

The solvent control group received intraperitoneal injections of an equal volume of PBS containing 0.1% DMSO, following the same schedule as the Pyr3-treated group.

Statistical analysis

Statistical differences between two groups were determined using a Student’s t-test, whereas comparisons among three or more groups using Analysis of Variance (ANOVA). Tukey’s test was used for post hoc analysis. Experiments were repeated at least 3 times respectively. Data are presented as mean ± Standard Error of Mean (SEM). A P-value less than 0.05 was defined as statistical significance.

Statistics and reproducibility

Data are presented as mean ± SEM. Statistical significance (p <0.05) was assessed using tests specified in figure legends and the “Statistical analysis” section. All experiments were performed with at least three independent biological replicates (n ≥ 3), where “n” represents biological replicates (independent cultures or animals). Technical replicates were averaged per biological replicate. No data were excluded.

Results

HPV 16/18 infection induced time-dependent upregulation of TRPC3 in human cervical epithelial cells

The human cervical epithelial cells (H8 cell line and HCECs) were used to dissect the functional role of TRPC3 in the context of HPV infection. We initially detected the expression of GFP that represented the infection efficiency at 48 hours post-infection with HPV16 and HPV18 (E6/E7) RNA PsV at different doses of VGEs (low VGE: 1-10 VGE/cell; mid-VGE: 10–100 VGE/cell; and high VGE: >100 VGE/cell). As illustrated in Fig. 1A, GFP-positive cells were increased in a titer-dependent manner and the robust and reproducible infection with high VGE was confirmed across both cell types. Accordingly, high VGE was selected for HPV infections in the subsequent experiments.

Fig. 1. HPV infection and TRPC3 expression in H8 cells and HCECs.

A The microscopy for EGFP-positive H8 and HCECs after 48 hours of infection with different titers of HPV PsV represented by viral genome equivalents (VGE). B Western blot analysis of the expression TRPC3 protein in H8 and HCECs following infection with HPV 16 or 18 at the indicated time points. GAPDH was used as a loading control. TRPC3 protein levels were quantified by normalizing to the expression of GAPDH, respectively. n = 3, *p <0.05, **p <0.01, ***p <0.001. C Immunostaining of TRPC3 (yellow) in H8 and HCECs following infection with GFP (green)-labeled HPV 16 or 18. D Cell viability (reflecting proliferation) of H8 and HCECs at 0, 24, 48, and 72 hours after HPV16/18 infection, detected by CCK-8 assay. n = 3, *p <0.05, **p <0.01, ***P <0.0001.

Then the impacts of HPV 16/18 infection on TRPC3 expression in H8 and HCECs were investigated. Western blot analyses revealed a time-dependent upregulation of TRPC3 protein levels in both cells following HPV infection. The expression levels of TRPC3 protein were significantly elevated from 48 to 96 hours after infection, compared with the non-infected control groups (P <0.05, Fig. 1B). Immunofluorescence imaging further corroborated these findings and offered additional insights into the cellular distribution of TRPC3 (Fig. 1C). Following the emergence of GFP (green) as a reporter of HPV infection, strong TRPC3 staining (yellow) was observed within H8 cells and HCECs, particularly localized to cell cytoplasm. Notably, Strong TRPC3 immunoreactivity was prominently observed in areas with robust GFP expression, indicating upregulated TRPC3 protein levels in HPV-infected cells. These results demonstrated that HPV 16/18 infection triggered a robust and time-dependent increase in TRPC3 expression across different cervical epithelial cells. To rule out the possibility that TRPC3 upregulation was a time-dependent change under standard culture conditions (rather than HPV infection-specific), we performed additional Western blot assays for TRPC3 in uninfected H8 and HCECs at 0, 24, 48, 72, and 96 hours. As shown in Supplementary Fig. S3, TRPC3 protein levels remained relatively stable over this time course in both cell lines, with no significant fluctuations compared to the 0-hour baseline. This supplementary data further confirms that the time-dependent upregulation of TRPC3 observed in our study is specifically induced by HPV16/18 infection.

To explore the effect of HPV infection on cell viability, we assessed the viability of H8 and HCECs at 0, 24, 48, and 72 hours after infection with HPV16 or HPV18 (Fig. 1D). Compared with the non-infected control groups, both H8 and HCECs infected with HPV16 or HPV18 showed a time-dependent increase in cell viability; cell viability was significantly enhanced from 24 hours post-infection and reached the highest level at 72 hours, which further indicates that HPV infection not only induces time-dependent upregulation of TRPC3 in human cervical epithelial cells but also promotes cell viability in a time-dependent manner, with HPV infection-induced alterations potentially involved in promoting the survival of cervical epithelial cells.

TRPC3 was required for Ca²⁺ influx and involved in the modulation of Ras-MAPK pathways in HPV-infected cervical epithelial cells

Given the elevated expression of TRPC3 was found in H8 and HCECs upon HPV infection, a specific TRPC3 inhibitor (Pyr3), was utilized to further detect the function of TRPC3 in this scenario. Although Pyr3 primarily inhibits the functional activity of TRPC3, our findings indicate that treatment of Pyr3 also decreased the expression of TRPC3 protein in HPV16- or 18- infected H8 and HCECs (Fig. 2A, Supplementary Fig. S1). This reduction may be attributed to feedback regulation following the functional blockade. In parallel, the abolishment of ATP-induced Ca²⁺ influx (red fluorescence) was observed in this context compared to cells without Pyr3 treatment. These results underscore the functional involvement of TRPC3 in HPV-infected H8 and HCECs.

Fig. 2. Ca²⁺ Influx and RASA4 expression in HPV-infected human cervical epithelial cells upon inhibition of TRPC3.

H8 cell line and HCECs were infected with HPV16 or 18 for 72 hours prior to treatment with ATP in the presence of a TRPC3 inhibitor (Pyr3, 10 μM) or an equal volume of the DMSO solvent control. A Calcium influx was measured using Rhod-3 Calcium Imaging Kit and fluorescence microscopy. B Immunofluorescence images showing RASA4 expression on H8 and HCECs treated with Pyr3 or the DMSO solvent control following HPV16/18 infection.

To investigate the role of TRPC3 in the regulation of calcium signaling-related pathway, we focused on calcium-dependent Ras GTPase-activating protein 4 (RASA4), a known inhibitor of the MAPK cascade that orchestrates in cell survival. Immunofluorescence assays demonstrated that inhibition of TRPC3 reduced the expression of RASA4 (red staining) in HPV-infected cervical epithelial cells (Fig. 2B). This finding suggests that TRPC3 is required for the calcium-induced expression of RASA4 that inhibits the excessive activation of Ras-MAPK pathway, thereby facilitating cervical epithelial cell survival upon HPV infection.

Inhibition of TRPC3 impaired the viability of HPV16/18-infected cervical epithelial cells through MEK-ERK pathways

To directly assess the cytotoxic effect of TRPC3 inhibition, we measured lactate dehydrogenase (LDH) release. As shown in Fig. 3A, pharmacological inhibition of TRPC3 by Pyr3 significantly increased LDH release in HPV16-infected H8 cells and HCECs, indicating enhanced cell membrane damage and cell death. We next investigated whether this TRPC3-dependent cell survival was mediated through the MAPK/ERK pathway. The CCK-8 assay demonstrated that HPV16/18 infection enhanced cell viability, an effect that was abolished by Pyr3 treatment (Fig. 3B). Crucially, this loss of viability was substantially rescued by co-treatment with the MEK/ERK pathway inhibitor PD98059 (ERK-i), suggesting that the cytotoxic effect of TRPC3 inhibition is mediated through aberrant MEK/ERK signaling.

Fig. 3. Role of TRPC3 in HPV-induced survival and viability of human cervical epithelial cells.

H8 cell line and HCECs were infected with HPV16 or 18 for 72 hours followed by the treatment of Pyr3 (10 μM) or the DMSO solvent control for another 24 hours. A Cell death was assessed by lactate dehydrogenase (LDH) release assay. B Cell viability was measured by CCK-8 assay at the indicated time points. C Representative Western blot images (left) and quantitative analysis (right) of p-ERK1/2, total ERK1/2, and GAPDH protein levels in H8 and HCECs under different experimental conditions. n = 3, *p <0.05, **p <0.01, ***p <0.001.

To further elucidate the molecular mechanism underlying the TRPC3-MEK-ERK axis in regulating cell viability, we detected the phosphorylation level of ERK1/2 (p-ERK1/2), a key marker of MAPK pathway activation, via Western blot analysis. As shown in Fig. 3C, compared with uninfected (WT) cells, HPV16 or HPV18 infection caused a mild increase in p-ERK1/2 levels in H8 and HCECs; however, treatment with Pyr3 led to a significant hyperactivation of the MAPK/ERK pathway, as evidenced by a marked increase in p-ERK1/2 levels in HPV16/18-infected cells. Importantly, overexpression of RASA4 (via plasmid transfection) in Pyr3-treated HPV-infected cells reversed the elevated p-ERK1/2 levels, while total ERK1/2 levels remained relatively unchanged across all groups. These results confirm that TRPC3 suppresses excessive MAPK activation through maintaining RASA4 expression, thus providing a molecular basis for the TRPC3-mediated regulation of cell viability via the MEK-ERK pathway.

Transcriptional dynamics in HPV-infected cervical epithelial cells with depletion of TRPC3

Next, RNA-seq was performed to explore the molecular changes of HPV-infected cervical epithelial cells following the knockdown of TRPC3. The decreases of TRPC3 mRNA levels (Fig. 4A, E) and protein expressions (Supplementary Fig. S2) in H8 and HCECs were respectively validated, laying the foundation for subsequent transcriptomic analyses. Volcano plots illustrated significant alterations of gene expression following the TRPC3 knockdown in H8 cells (Fig. 4B, Supplementary Data 2) and in HCECs (Fig. 4F, Supplementary Table 1), suggesting the candidate targets that are potentially involved in TRPC3-mediated cellular processes.

Fig. 4. Transcriptomic analysis for H8 and HCECs following TRPC3 knockdown and HPV infection.

H8 and HCECs were transfected with TRPC3-shRNA and non-target control shRNA (shNC), followed by infection with HPV PsV. A, E Quantitative RT-PCR analysis showing the relative mRNA levels of TRPC3 normalized to GAPDH. B, F Volcano plot exhibiting the differentially expressed genes after TRPC3 knockdown. Genes with a Log2FC < -1 and p-value < 0.05 are considered significantly downregulated (blue dots), while those with a Log2FC > 1 and p-value < 0.05 are significantly upregulated (red dots). C, G GO analysis indicating the biological processes (BP), cellular components (CC), and molecular functions (MF) that could be affected by TRPC3. D, H The heatmap showed the differential expression of EMT marker genes. Blue color means the low expression of genes in samples.

In HPV-infected H8 cells, GO analysis reveals that the differential expression genes upon in TRPC3 knockdown are enriched biological processes significantly associated with EMT, proteasomal protein catabolic process, protein targeting, proteasome-mediated ubiquitin-dependent protein catabolic process, regulation of protein catabolic process, and response to endoplasmic reticulum stress, etc. (Fig. 4C). Similarly, EMT is also the most significantly enriched biological process, together with other processes such as SRP-dependent cotranslational protein targeting to membrane, cotranslational protein targeting to membrane, and protein localization to endoplasmic reticulum, etc., in HCECs with TRPC3 depletion and HPV infection (Fig. 4G). Furthermore, the heatmap result showed that the expressions of some pivotal genes (ZEB1, Snail, Slug, and Twist1) correlated with EMT were significantly downregulated in this context of both H8 cells (Fig. 4D) and HCECs (Fig. 4H) Taken together, these results implicated the molecular basis for the critical function of TRPC3 in mediating HPV-triggered dysregulation of human cervical epithelial cells.

Inhibition of TRPC3 reversed HPV-induced epithelial-mesenchymal transition of human cervical epithelial cells

The impact of TRPC3 on the expressions of EMT-correlated genes was further confirmed in HPV-infected human cervical epithelial cells. Quantitative PCR (qPCR) analyses indicated that gene transcription levels of ZEB1, Snail, Slug, and Twist1 were remarkably upregulated upon infection with HPV16 or HPV18 in both H8 and HCECs (Fig. 5A). Whereas treatment with a TRPC3 inhibitor, Pyr3, significantly curtailed gene expressions of the indicated EMT markers. Consistently, the protein levels of ZEB1, Snail, Slug, and Twist1 in HPV-infected epithelial cells were markedly decreased following the inhibition of TRPC3 (Fig. 5B). Taken together, these results well highlight the critical involvement of TRPC3 in the EMT process driven by HPV.

Fig. 5. The expressions of EMT markers in human cervical epithelial cells after HPV infection and TRPC3 inhibition.

Quantitative PCR (A) and Western blot analyses (B) for the expression levels of epithelial-mesenchymal transition (EMT) markers ZEB1, Snail, Slug, and Twist1 in HPV-infected H8 cell line and HCECs treated with Pyr3 or the DMSO solvent control. n = 3, **p <0.01, ***p <0.001.

Blockade of TRPC3 inhibited HPV-induced EMT in mouse cervical epithelium

To further validate the role of TRPC3 in the malignant transformation of cervical epithelial cells in vivo, we established HPV16 and HPV18 cervical infection mouse models. Biofluorescence imaging showed that mice were successfully infected by both HPV16 and HPV18 pseudovirus on day 3 post-infection (Fig. 6A). The expression of TRPC3 protein in mouse cervical tissues was significantly up-regulated upon HPV infection (Fig. 6B), which was highly consistent with the finding in human cervical epithelial cells. Immunofluorescence staining analyses demonstrated the remarkable decrease of E-cadherin but increase of N-cadherin and Vimentin expressions in HPV16 and HPV18 infected cervical tissues compared to uninfected controls, whereas these observations were significantly reversed after the treatment with a TRPC3 inhibitor, Pyr3 (Fig. 6C, D). Similarly, immunohistochemistry analyses also showed that Pyr3 inhibited the expressions of the EMT markers (ZEB1 and Snail) upregulated by HPV16/18 in mouse cervical tissues (Fig. 6E, F). Finally, TUNEL staining analyses demonstrated that the administration of Pyr3 effectively increased cell apoptosis in cervical tissues of HPV-infected mice (Fig. 6G, H). Collectively, these results provide in-vivo evidence showing that inhibition of TRPC3 can attenuate HPV-induced EMT and survival in cervical epithelial cells, offering insights into a potential target for the intervention of HPV-associated malignant transformation.

Fig. 6. Effects of TRPC3 inhibition on cervical epithelia of HPV-infected mice.

Female BALB/c mice were intravaginally infected with luciferase-tagged HPV16 or HPV18 pseudoviruses. A Representative bioluminescence imaging of mice on day 3 after infection. B Representative immunofluorescence staining of TRPC3 protein on cervical sections from mice after HPV infection. C, D HPV-infected mice were treated with TRPC3 inhibitor or the solvent control (PBS containing 0.1% DMSO). The uninfected mice were used as control. Immunofluorescence staining of EMT markers in mouse cervical tissues were imaged and quantified. E, F Immunohistochemical analyses of ZEB1 and Snail in cervical tissues. Brown staining indicates the positive expression. G, H TUNEL staining of mouse cervical sections to assess cell apoptosis. Statistical significance was analyzed using one-way ANOVA test followed by Tukey post-hoc test. n = 3; *p <0.05, **p <0.01.

Discussion

Cervical cancer is one of the life-threatening diseases in women worldwide, with HPV infection as a leading cause. We first demonstrated a robust upregulation of TRPC3 following HPV infection in both human cervical epithelial cell line and primary cervical epithelial cells, accompanied by the functional implications of TRPC3 on Ca²⁺ signaling, cell proliferation and EMT. It is noteworthy that our immunofluorescence analysis also revealed a degree of heterogeneity in TRPC3 expression among the cell population, with some TRPC3-high cells displaying low or undetectable GFP signal. While the precise reason for this heterogeneity warrants further investigation, it may reflect technical limitations of immunofluorescence, such as cells residing outside the focal plane of the GFP signal, or biological variations in the cellular response to infection. Future studies employing flow cytometry for precise co-expression quantification will help clarify this observation. Nevertheless, the prominent upregulation of TRPC3 in GFP-positive regions firmly supports our conclusion that HPV infection is a key driver of TRPC3 expression. Together with the confirmatory results found in HPV cervical infection mouse models, this study delves into the precise role of TRPC3 in HPV-induced malignant transformation of cervical epithelial cells. The significant effects of a TRPC3-specific inhibitor on restoring cellular dysregu lation suggest the potential of TRPC3 as a therapeutic target. In line with that TRP channels have been proposed as potential therapeutic targets for breast cancer^19,20, targeting TRPC3 may be able to mitigate HPV-induced oncogenesis and potentially prevent tumor development. Further research is necessary to precisely evaluate the long-term tolerance and efficacy of TRPC3 inhibitor in pre-clinical models of cervical cancer.

It has been reported that E1 proteins from HPV16 and 18 induced an overexpression of different set of genes associated with proliferation and differentiation processes²⁴. Ca²⁺ uptake is crucial for the regulation of the oxidative phosphorylation and ATP production that meet the demand of abnormal cell proliferation²⁵. Primarily, we established a robust and reproducible method for quantifying HPV infection rates in human cervical epithelial cells, laying the foundation for further exploration of the interplay between HPV and TRPC3. In parallel with the previous findings, synchronized HPV replication and upregulation of TRPC3 shown in our study highlight the induction of Ca²⁺ signaling in response to HPV infection. The precise mechanisms underlying this coordination in cervical epithelial cells warrant further investigations.

Dysregulation of MAPK pathway is commonly observed in various cancers. Efficacies of different anti-cancer drugs targeting MAPK pathway await the evaluation of related clinical trials^26,27. Our exploration of the downstream signaling pathways revealed that TRPC3 was involved in regulating the MAPK signaling cascade. It is important to note that the pro-survival or pro-apoptotic effects of MAPK/ERK depend on the activation intensity, duration and cellular environment. Excessive or persistent activation of MAPK/ERK may induce apoptosis^28,29. Since RASA4 acts as a suppressor of MAPK cascade, decreased expression of RASA4 following inhibition of TRPC3 could trigger excessive activation of MAPK/ERK pathway, thereby inducing cell death on cells. This mechanism is consistent with previous research in cervical epithelial cells, where RASA4 was shown to promote cell survival by inhibiting HIFα-mediated expression of pro-apoptotic proteins—silencing RASA4 led to a 2.3-fold increase in cell death, which was reversed by restoring RASA4 expression²⁷. Our finding that TRPC3 inhibition reduces RASA4 and impairs cell viability aligns with this study, confirming that the TRPC3-RASA4-MAPK axis is critical for maintaining the viability of HPV-infected cervical epithelial cells. This finding suggests that TRPC3 is necessary to prevent MAPK hyperactivation-induced cell death, thus facilitating the survival of HPV-infected cervical epithelial cells. Consistently, we found HPV-stimulated cell viability could be suppressed by the treatment of a TRPC3 inhibitor in both human cervical epithelial cells and mouse cervical tissue. The functional role of the TRPC3-RASA4-MAPK axis is firmly established by our genetic rescue experiment, wherein RASA4 overexpression successfully reversed the cytotoxic effects and ERK hyperactivation induced by TRPC3 inhibition. This provides direct evidence that TRPC3 maintains cell viability through RASA4 to fine-tune MAPK/ERK signaling.Taken together, these findings provide compelling evidence for the intricate interplay between TRPC3, Ca²⁺ signaling and downstream effectors in HPV-triggered cellular dysregulation³⁰.

Further investigation in our current study revealed that inhibition of TRPC3 effectively suppressed HPV-induced EMT, a critical step in cancer progression³¹. Since EMT empowers cells to shed their epithelial characteristics and acquire mesenchymal properties as well as enhance cell migratory and invasive potential, it can be reasonably speculated that blockage of TRPC3 potentially hinders HPV infection-driven tumor metastasis through inhibiting EMT. By persistently unraveling the complex interplay between viral infection, TRPC-regulated cellular signaling and malignant transformation, we can hold significant promise for the development of novel therapeutic strategies aimed at disrupting the metastatic cascade in HPV-associated cancers.

Besides highlighting the enrichment of differential expression genes in the EMT process, our high-throughput transcriptomic sequencing of TRPC3-knockingdown H8 and HCEC cells underscored the extensive influence of TRPC3 on the transcription levels of downstream genes involved in diverse signaling pathways and biological processes. The enrichment of TRPC3-regulated genes respectively in cell cycle, cytoskeleton organization, and protein degradation etc. is highly consistent with our findings that demonstrated TRPC3 functioned on the viability of HPV-infected cervical epithelial cells. These results lay a foundation for further elucidating the specific mechanism by which TRPC3 functions in the development of HPV-induced cervical cancer. Further validation experiments on the selected target genes are necessary for comprehensively delineating the role of TRPC3 in HPV-induced cervical oncogenesis.

In conclusion, our in-vitro and in-vivo studies provide compelling evidence for the pivotal effect of TRPC3 on HPV-induced cellular dysregulation and subsequent neoplastic transformation, which will help better understand the pathogenesis of HPV-driven cervical cancer. The solid efficacy of TRPC3 inhibitor demonstrated in both human cervical epithelial cells and mouse cervical tissue adds a crucial dimension to translate the current study into potential clinical interventions. Our findings may pave the way for the development of novel therapeutic strategy targeting TRPC3 axis to combat HPV-associated cancers, ultimately contributing to improve the outcome of patients.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Author contributions

Yudi Tan and Qiaoqiao Huang performed the experiments, analyzed data and wrote the draft manuscript. Jingjing Zeng performed the experiments and analyzed data. Yong Wang helped with the animal experiments. Shasha Yang and Li Yu helped in preparing experiment materials. Jinkong Wei and Yuying Wei analyzed data and figures. Jinmin Zhao conceptualized the project and edited the manuscript. Junying Chen conceptualized the project, supervised the study, and edited the manuscript. All authors read and approved the final manuscript.

Peer review

Peer review information

Communications Biology thanks Consuelo Amantini and the other, anonymous, reviewer for their contribution to the peer review of this work. Primary Handling Editor: Ophelia Bu.

Data availability

The RNA sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA1377712.Source data underlying graphs can be obtained from Supplementary Data 1. All other data are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-025-09480-z.