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. 2024 Nov 18;14(1):szae087. doi: 10.1093/stcltm/szae087

Human umbilical cord mesenchymal stem cells small extracellular vesicles-derived miR-370-3p inhibits cervical precancerous lesions by targeting DHCR24

Weizhao Li 1,2,3,#, Chi Zhang 4,#, Tianshun Gao 5,#, Yazhou Sun 6, Huan Yang 7, Lixiang Liu 8, Ming Shi 9, Lu Ding 10, Changlin Zhang 11,12,✉, David Y B Deng 13,✉, Tian Li 14,15,✉
PMCID: PMC11825698  PMID: 39552565

Abstract

Background

Cervical cancer is often caused by persistent high-risk human papillomavirus (HPV) infection, causing precancerous lesions. Human umbilical cord mesenchymal stem cells-derived small extracellular vesicles (hucMSC-sEV) exhibit diverse effects on tumors. This study investigates hucMSC-sEV, the impact and mechanisms on HPV-positive cervical precancerous lesion cells to provide new treatment insights.

Materials and Methods

We previously obtained hucMSC and hucMSC-sEV. In vitro experiments evaluated hucMSC-sEV effects on the proliferation and migration of S12 cells (derived from cervical precancerous lesions). Bioinformatics identified key microRNA components, and their impact on S12 cell proliferation and migration was investigated. The target gene of the microRNA component was predicted and confirmed via bioinformatics and dual-luciferase reporter assays. Lentiviral systems overexpressed target gene in S12 cells to examine the effects on microRNA impacts. SH-42 inhibitor was used to investigate target gene treatment potential. Immunohistochemistry assessed target gene expression in cervical precancerous lesions tissue.

Results

hucMSC-sEV significantly inhibited S12 cell proliferation and migration. Bioinformatics identified miR-370-3p as an effective cargo, which also suppressed S12 cell proliferation and migration. miR-370-3p was confirmed targeting DHCR24 (24-Dehydrocholesterol Reductase). DHCR24 overexpression reversed miR-370-3p’s inhibitory effects, while SH-42 counteracted DHCR24 overexpression’s promoting effects. Clinical specimen analysis supported these findings, demonstrating a positive correlation between DHCR24 protein expression and cervical precancerous lesions’ progression.

Conclusions

hucMSC-sEV inhibits S12 cell proliferation and migration, mediated by miR-370-3p targeting DHCR24 to regulate cellular cholesterol content. DHCR24 inhibition reduces the cholesterol level and cell functions, suggesting its potential as a therapeutic target in cervical precancerous lesions.

Keywords: cervical intraepithelial neoplasia, mesenchymal stem cells, exosome, microRNA, cholesterol

Graphical Abstract

Graphical Abstract.

Graphical Abstract

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Significance statement.

The cervical precancerous lesion is a window to prevent cervical cancer but lacks effective treatment methods. Mesenchymal stem cells-derived small extracellular vesicles are well known in cell-free stem cell therapy, but the effect and mechanism on cervical precancerous lesions are still unknown. miR-370-3p is the key cargo, which could inhibit S12 cells proliferation and migration through targeting DHCR24. DHCR24 is a potential target, which is positively correlated with cervical precancerous lesion progression, and the DHCR24 inhibitor could effectively inhibit S12 cells. These results show a potential cell-free therapy method for cervical precancerous lesions, which could also be a prevention therapy for cervical cancer.

Introduction

Cervical cancer ranks fourth among women’s cancer types, presenting a significant health concern.1 Almost all cases of cervical cancer are linked to human papillomavirus (HPV) infection, which infiltrates basal cells through small breaks in the cervix.2 Persistent high-risk HPV infection can lead to the progression of cervical lesions to cervical precancerous lesions (also known as cervical intraepithelial neoplasia, CIN) and ultimately cervical cancer.3 While preventive HPV vaccines target specific high-risk types of HPV, they do not provide therapeutic benefits for existing infections. Current treatments such as drug, laser therapy, and excisional treatment often fail to completely eradicate HPV-infected cells. In a cohort study, the researchers found 2074 patients who were diagnosed with CIN II or worse among whom had received the HPV vaccine. After conization, 82 patients experienced a recurrence and were diagnosed with CIN II or worse within a year.4 A meta-analysis found that the risk of CIN recurrence after local excision could be reduced by HPV vaccination, although some recurrence still occurred.5 Therefore, it is urgent to develop novel therapeutic strategies for cervical precancerous lesions.6,7

Recent research has focused on the functional role of HPV-encoded proteins, particularly E6 and E7, which drive cervical cancer progression.8,9 Additionally, attention is shifting to HPV-induced noncoding RNA changes, particularly microRNAs. Dysregulated microRNAs have been implicated in the progression of cervical precancerous lesions.10 He et al summarized that as cervical precancerous lesions progress to cervical cancer, the number of dysregulated microRNAs increases: in CIN I, 5 microRNAs are upregulated and 7 are downregulated, while in cervical cancer, 42 microRNAs are upregulated and 21 are downregulated.11 Choi et al summarized that microRNAs could regulate HPV viral proteins, while HPV also causes changes in microRNAs.12 However, it remains uncertain whether restoring dysregulated microRNAs could reverse cervical precancerous lesions.

Mesenchymal stem cells-derived small extracellular vesicles (MSC-sEV) are a type of extracellular vesicle secreted by mesenchymal stem cells, typically ranging in diameter from 30 to 150 nm.13 MSC-sEV are rich in cargo such as microRNAs, proteins, and lipids, capable of modulating recipient cell functions.14 MSC-sEV have shown regulatory effects in various cancers, including cervical cancer, through the transfer of cargo.15 Our previous research revealed that MSC-sEV carrying the Jagged1 protein promotes squamous differentiation of cervical cancer cells via NOTCH pathway activation, inhibiting proliferation and migration.16 Similarly, MSC-sEV was found to transport miR-23b, inducing dormancy in breast cancer cells and consequently reducing proliferation, invasion, and sensitivity to docetaxel.17 However, the impact and underlying mechanisms of MSC-sEV on cervical precancerous lesions remain unclear.

This study seeks to assess the effects of human umbilical cord mesenchymal stem cells-derived small extracellular vesicles (hucMSC-sEV) on cervical precancerous lesions. Through bioinformatics analysis, we aim to identify key microRNAs contained within hucMSC-sEV, thereby elucidating their target genes and therapeutic potential. Our goal is to provide novel insights into the treatment of cervical precancerous lesions.

Methods

Cell culture and reagents

S12 cells were generously provided by Professor Hui Wang at the Huazhong University of Science and Technology Union Hospital and cultured in S12 culture medium (Supplementary Table S1). HEK293T cells were obtained from the American Type Culture Collection and confirmed to be free of mycoplasma contamination (D101-02, Vazyme, China). HEK293T cells were cultured in complete DMEM medium (containing 10% fetal bovine serum, 100-U/mL penicillin, and 100-μg/mL streptomycin). All cells were cultured in a cell culture incubator at 37 °C with 5% CO2. SH-42 (HY-143228, MCE, USA) was purchased from MedChemExpress.

S12 cells uptake Dil-labeled hucMSC-sEV

To evaluate hucMSC-sEV uptake by S12 cells, we labeled hucMSC-sEV with Dil dye (red fluorescence). Dil dye (1-mg/mL, dissolved in DMSO) (D3911, Thermo Fisher Scientific, USA) was combined with hucMSC-sEV at a 1:50 ratio to produce Dil-hucMSC-sEV. Dil-hucMSC-sEV was introduced into the S12 cell culture medium. The presence of red fluorescence on the surface of S12 cells would indicate hucMSC-sEV uptaking. S12 cells were seeded at a density of 20 000 cells per well in a 12-well plate, and Dil-hucMSC-sEV was added. After 24 hours, the supernatant was removed, and cells were washed twice with 1× PBS. Fluorescence images were captured using a fluorescence inverted microscope (DMi8, Leica, Germany).

Cell counting kit-8

To determine relative cell viability using the cell counting kit-8 (CCK8) method, 5000 S12 cells were seeded into individual wells of a 96-well plate and incubated for 72 hours. The CCK8 working solution was prepared by diluting CCK8 reagent (K1018, APExBIO, USA) with complete S12 cells culture medium at a ratio of 1:10. Add 100 μL of the CCK8 working solution to each well, followed by incubation in a cell culture incubator at 37 °C with 5% CO2 for 1 hour. Absorbance readings at 450 nm, obtained using a microplate reader (Synergy H1M, BioTek, USA).

Wound healing assay

For the wound healing assay, once the S12 cells reach 100% density in a 6-well plate, make a scratch with a 200-μL pipette tip. Subsequently, images were captured using a 4× objective on an inverted microscope. Five random fields were chosen for photography. The average wound area across the 5 fields was determined as the wound area at each time point. The wound area was automatically quantified using the MRI_Wound_Healing_Tool plugin in ImageJ software. The healing rate at time point X hours was calculated using the following formula:

X hours healing rate (%) = [(0 hours average wound area − X hours average wound area)/0 hours average wound area] × 100%.

Transwell

To evaluate the migration ability of S12 cells using Transwell migration assay. S12 cells were resuspended in serum-free DMEM/F12 medium and counted. Subsequently, 100 000 cells were added to the upper chamber of Transwell inserts (3422, Corning, USA) with an 8.0-μm pore size, while S12 culture medium (500 μL) was added to the lower chamber. Following 24 hours of incubation, cells were fixed and stained with 0.1% crystal violet solution (dissolved in methanol) at room temperature for 15 minutes. Nonmigrated cells on the upper side of the membrane were removed. Five random fields were chosen for cell counting with a microscope. The average number of migrated cells per well was counted.

Colony formation assay

To assess the proliferation ability of S12 cells using the colony formation assay, 200 S12 cells were seeded into each well of a 6-well plate. The medium was changed every 3 days, and after 7-8 days, the cells were fixed and stained with 0.1% crystal violet solution (dissolved in methanol) for 15 minutes. The colonies were visualized using an imaging analysis system (ChemiDoc, Bio-Rad, United States). The number of colonies containing more than 50 cells was counted.

Construction and transfection of plasmid and microRNA mimics

We obtained the mature sequences of miR-370-3p and miR-193b-3p from miRBase and synthesized microRNA mimics through GenePharma (Shanghai, China). The lentiviral packaging plasmids pMD2.G and psPAX2 were purchased from GeneChem (Shanghai, China). The coding sequence of DHCR24 was retrieved from NCBI, and plasmid construction was performed by the Public Protein/Plasmid Library (Nanjing, China). Specifically, we constructed the plasmids pLenti-CMV-DHCR24-Flag-RFP-BDS, pLenti-RFP-BDS vector, pmirGLO-DHCR24 3ʹ UTR (WT), and pmirGLO-DHCR24 3ʹ UTR (MUT). Hieff Trans Liposomal Transfection Reagent (40802ES, Yeasen, China) was used for the transfection of microRNAs and plasmids, following the manufacturer’s instructions.

Lentiviral transduction for stable cell lines

Transfect packaging plasmids (pMD2.G and psPAX2) along with the plasmid carrying the DHCR24 gene into HEK293T cells to produce lentivirus. Harvest the lentivirus-containing culture supernatant and infect S12 cells with the lentivirus, supplemented with polybrene. Perform continuous screening of S12 cells with 30 μg/mL of blasticidin S for at least 1 week until all S12 cells not infected with lentivirus are dead.

RNA extraction and quantitative real-time PCR assay

microRNA was extracted using the MolPure Cell/Tissue miRNA Kit (19331ES, Yeasen, China), and reverse transcription of microRNA was performed using the Hifair Ⅲ 1st Strand cDNA Synthesis Kit (11139ES, Yeasen, China). While mRNA was extracted using the RNA-Quick Purification Kit (RN001, ESScience, China), reverse transcription was also carried out using the Fast All-in-One RT Kit (RT001, ESScience). Quantitative real-time polymerase chain reaction (RT-qPCR) detection was conducted using Taq Pro Universal SYBR qPCR Master Mix (Q712-02, Vazyme, China) in a Bio-Rad CFX96 instrument. The primers used in this study are detailed in Supplementary Table S2.

Western blot

Proteins were detected using the standard Western blot method according to our prior research.16 DHCR24 antibody (DF12472, Affinity, USA), GAPDH antibody (GB15002, Servicebio, China), and HRP-conjugated secondary antibody (SA00001-2, ProteinTech, China) were used as the handbook provided by the company.

Dual-luciferase reporter assay

To explore the targeting relationship between miR-370-3p and the 3'UTR region of DHCR24 via a dual-luciferase reporter gene assay, HEK293T cells were seeded in a 96-well plate and transfected with 0.1 μg of dual-luciferase reporter gene plasmid and 10 nM of microRNA mimics. The experimental groups were organized as follows, each with 4 replicate wells: (1) Blank group, (2) DHCR24 3'UTR (WT) + NC mimics, (3) DHCR24 3'UTR (WT) + miR-370-3p mimics, (4) DHCR24 3'UTR (MUT) + NC mimics, and (5) DHCR24 3'UTR (MUT) + miR-370-3p mimics. After 36 hours of transfection, the dual-luciferase reporter gene assay was conducted using the Dual-Luciferase Reporter Gene Assay Kit (11402ES, Yeasen), following the manufacturer’s instructions.

Detection of cell total cholesterol

The total cholesterol content in S12 cells was measured using the Cholesterol Assay Kit (MAK043, Sigma, Germany), following the manufacturer’s instructions. The relative cholesterol content of each group was calculated using the average OD value of the control group as a reference.

Immunohistochemistry

To detect the expression of DHCR24 in normal cervical epithelial tissues, cervical precancerous lesions tissues, and cervical cancer tissues, 31 relevant pathological tissue sections from the Seventh Affiliated Hospital of Sun Yat-sen University were collected. Immunohistochemistry (IHC) staining of DHCR24 protein was performed to assess the relative expression level of DHCR24. Obtaining of human specimens was approved by the Ethics Committee of the Seventh Affiliated Hospital of Sun Yat-sen University (Approval No: KY-2022-060-01). A total of 8 specimens of normal cervical tissue, 7 cases of low-grade squamous intraepithelial lesions (LSIL), 7 cases of high-grade squamous intraepithelial lesions (HSIL), and 9 cases of squamous cell carcinoma (SCC) were selected. Standardized immunohistochemical methods were used for staining. The results were captured using a slide scanner (VS200, OLYMPUS, Japan) and reviewed by a pathologist. Statistical analysis was then conducted. To analyze the staining intensity using ImageJ 9.0 and the IHC Profiler plugin (downloaded from https://github.com/ethanzhao9/ImageJ-Tutorial/blob/master/IHC_Profiler.zip). The relative expression intensity of DHCR24 in each group was calculated by multiplying the proportion of different intensity areas by their respective scores and summing them up.

Bioinformatics

We obtained microRNA expression data from cervical tissues (GSE30656), mRNA expression data (GSE75132), and microRNA expression data from hucMSC-sEV (GSE159814) from the GEO datasets.18 Expression matrices were standardized using the DESeq2 package, and differentially expressed microRNAs were analyzed. Volcano plots were generated using the ggplot2 package, and heatmaps were created using the heatmap package. Additionally, we used the ENCORI database to predict the targets of miR-370-3p. Transcriptome data for cervical cancer and normal cervical tissues were obtained from the TCGA database (https://portal.gdc.cancer.gov/) and identified genes associated with poor prognosis in cervical cancer. Lastly, Venn diagrams were generated using the bioinformatics website (https://www.bioinformatics.com.cn/) to visualize the results.

Statistics

All experiments were conducted with a minimum of 3 replicates, and statistical analysis was performed using GraphPad Prism 9.0. Quantitative data are presented as mean ± SD. For comparisons between 2 groups of data that followed a normal distribution and exhibited equal variances in nonpaired designs, an unpaired t-test was used. Differences among multiple groups were analyzed using one-way analysis of variance (ANOVA) followed by Student’s t test for comparisons between 2 groups. Statistical significance was defined as P < .05.

Results

hucMSC-sEV inhibits the proliferation and migration of S12 cells

To investigate the impact of hucMSC-sEV on cervical precancerous lesions, we used S12 cells, a cell line from CIN Ⅰ. Previous research has confirmed that hucMSC and hucMSC-sEV meet international standards.16,19 Firstly, we investigated whether S12 cells could uptake hucMSC-sEV. After co-incubated Dil-hucMSC-sEV with S12 cells for 24 hours, S12 cells exhibited red fluorescence, indicating uptake of Dil-hucMSC-sEV by S12 cells (Figure 1A). Subsequently, we evaluated hucMSC-sEV effects on S12 cell proliferation and migration using CCK8, wound healing, Transwell, and colony formation assay. In Figure 1B, both concentrations of 30- and 50-μg/mL hucMSC-sEV inhibited S12 cells’ viability, with stronger inhibition at 50 μg/mL. In Figure 1C, after 36 hours, both concentrations significantly reduced wound healing rates. Figure 1D shows both concentrations significantly decreased the migration ability of S12 cells. In Figure 1E, both concentrations significantly suppressed S12 cells’ colony formation in the assay. In summary, hucMSC-sEV inhibits the proliferation and migration of S12 cells.

Figure 1.

Figure 1.

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hucMSC-sEV inhibit proliferation and migration ability of S12 cells. (A) Immunofluorescence assay showed S12 cell uptake Dil-MSC-sEV (scale bar = 200 μm). (B) Effects of hucMSC-sEV on the viability of S12 cells. (C) Effects of hucMSC-sEV on the wound healing ability of S12 cells. (D) Effects of hucMSC-sEV on the migration ability of S12 cells. (E) Effects of hucMSC-sEV on the colony formation ability of S12 cells (*P < .05, **P < .01, ***P < .001, ns = no significance).

hsa-miR-370-3p is identified as the key cargo within hucMSC-sEV that inhibits S12 cells

The microRNAs within hucMSC-sEV are considered key cargo. We hypothesize that hucMSC-sEV delivers microRNAs to S12 cells. Initially, we obtained microRNA expression datasets from the GEO database (GSE30656) for CIN and normal cervical tissues and analyzed differentially expressed microRNAs. Figure 2A and B shows 5 downregulated microRNAs in CIN tissues (P < .05, |log2FoldChange| > 1). Subsequently, we obtained a hucMSC-sEV microRNA expression level from the GEO database (GSE159814) (Figure 2C). Through intersection analysis, 2 microRNAs were identified: miR-370-3p and miR-193b-3p (Figure 2D). Synthetic microRNA mimics were transfected into S12 cells, and after 72 hours, cell viability assessment revealed stronger inhibition by miR-370-3p (Figure 2E). Considering the microRNA expression and inhibitory effects, we chose to further investigate miR-370-3p.

Figure 2.

Figure 2.

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Identify miR-370-3p as a key component in hucMSC-sEV inhibiting S12 cells. (A and B) Volcano plot and heatmap of differentially expressed microRNAs in cervical intraepithelial neoplasia tissues compared with normal cervical tissues. (C) microRNA expression profile in hucMSC-sEV. (D) Venn diagram showing the intersection. (E) Effects of microRNA mimics on the viability of S12 cells (**P < .01, ***P < .001).

miR-370-3p inhibits the proliferation and migration of S12 cells

Initially, we evaluated the transfection efficiency of miR-370-3p mimics in S12 cells. As shown in Figure 3A, a significant increase in miR-370-3p expression was observed posttransfection compared with the control group. Subsequently, we studied the impact of miR-370-3p mimics on S12 cell proliferation and migration using CCK8, wound healing, Transwell, and colony formation assay. In Figure 3B, miR-370-3p mimics reduced S12 cell viability. Figure 3C demonstrates that miR-370-3p significantly reduced wound healing rates at both 18 and 36 hours. Figure 3D illustrates that miR-370-3p inhibited cell migration. Furthermore, the colony formation assay in Figure 3E reveals that miR-370-3p suppressed S12 cell colony formation ability. In conclusion, miR-370-3p significantly inhibits the proliferation and migration of S12 cells.

Figure 3.

Figure 3.

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miR-370-3p inhibits the proliferation and migration ability of S12 cells. (A) Relative miR-370-3p level after transfection miR-370-3p mimics. (B) Effects of miR-370-3p on the viability of S12 cells. (C) Effects of miR-370-3p on the wound healing ability of S12 cells. (D) Effects of miR-370-3p on the migration ability of S12 cells. (E) Effects of miR-370-3p on the colony formation ability of S12 cells (**P < .01, ***P < .001).

miR-370-3p targets 3' UTR of DHCR24

microRNAs exert regulatory effects by binding to the 3ʹ untranslated region (UTR) of target mRNAs, thereby inhibiting mRNA translation. We acquired transcriptome data from the GEO database (GSE75132) for normal cervical tissues and LSIL tissues, identifying 600 upregulated genes (Figure 4A). Subsequently, utilizing the ENCORI (starBase) database predicted potential targets of miR-370-3p, revealing 2810 potential target genes. Furthermore, from the TCGA database, we identified 1637 genes related to poor prognosis in cervical cancer. Intersection analysis of these datasets led to the identification of the DHCR24 (24-Dehydrocholesterol Reductase) as the target gene (Figure 4B). To validate whether miR-370-3p targets DHCR24 and identifies the binding site, dual-luciferase reporter gene assay was conducted. The TargetScan database predicted a specific binding site of miR-370-3p targeting the 3' UTR of DHCR24. As shown in Figure 4C, the luciferase reporter gene assay revealed miR-370-3p significantly downregulated the relative firefly luciferase activity of WT-DHCR24 compared with NC mimics. In contrast, miR-370-3p did not downregulate the relative firefly luciferase activity of MUT-DHCR24. This result indicates miR-370-3p targets the 3' UTR region of DHCR24.

Figure 4.

Figure 4.

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Identify DHCR24 as a target of miR-370-3p. (A) Volcano plot of differentially expressed mRNAs in low-grade cervical intraepithelial neoplasia. (B) Venn diagram showing the intersection result. (C) Prediction results of miR-370-3p targeting DHCR24 sites in TargetScan database and dual-luciferase reporter gene assay results (***P < .001, ns = no significance).

DHCR24 overexpression counteracts the inhibitory effects of miR-370-3p

To investigate whether overexpression DHCR24 could reverse the inhibitory effects of miR-370-3p on S12 cell proliferation and migration, we transfected microRNA mimics and overexpression DHCR24 in S12 cells. The details for each group are shown in Figure 5A. Results show a significant upregulation of miR-370-3p after transfection (Figure 5B). Additionally, the mRNA expression level of DHCR24 was significantly upregulated in the oeDHCR24 group and significantly downregulated in the miR-370-3p group (Figure 5C). Furthermore, the DHCR24 protein expression level was downregulated in the miR-370-3p group and upregulated in the oeDHCR24 group (Figure 5D). This indicates successful overexpression of miR-370-3p and DHCR24, with miR-370-3p overexpression leading to downregulation of DHCR24 expression.

Figure 5.

Figure 5.

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Overexpression DHCR24 reverse the inhibitory effect of miR-370-3p on proliferation and migration of S12 cells. (A) Group flag in this figure. (B) Effects of miR-370-3p and DHCR24 on cellular total cholesterol content. (C) Effects of miR-370-3p and DHCR24 on cell viability. (D) Effects of miR-370-3p and DHCR24 on wound healing ability of S12 cells. (E) Effects of miR-370-3p and DHCR24 on migration ability of S12 cells. (F) Effects of miR-370-3p and DHCR24 on colony formation ability of S12 cells (*P < .05, **P < .01, ***P < .001).

DHCR24 is an important enzyme in cholesterol synthesis, Figure 5E demonstrated that miR-370-3p mimics significantly reduced total cholesterol content, while overexpression DHCR24 significantly increased it, and DHCR24 overexpression reversed the downregulation caused by miR-370-3p. Moreover, the CCK8 assay revealed that miR-370-3p significantly inhibited cell viability, and DHCR24 overexpression reversed the inhibition (Figure 5F). The wound healing assay shows that miR-370-3p significantly inhibited cell wound healing ability, and DHCR24 overexpression reversed the inhibition (Figure 5G). Transwell assay indicated miR-370-3p significantly inhibited cell migration ability, and DHCR24 overexpression reversed the inhibition of miR-370-3p (Figure 5H). Additionally, the colony formation assay demonstrated that miR-370-3p significantly inhibited cell colony formation ability, and DHCR24 overexpression reversed this inhibition (Figure 5I). These findings suggest that overexpression of DHCR24 could reverse the inhibitory effects of miR-370-3p on S12 cell proliferation, migration, and total cholesterol content.

SH-42 inhibits the proliferation and migration abilities of S12 cells

To investigate whether DHCR24 could serve as a therapeutic target for cervical precancerous lesions, the DHCR24 inhibitor SH-42 was used. The experimental design is illustrated in Figure 6A. In Figure 6B, it is shown that SH-42 significantly reduced cellular total cholesterol content and reversed the upregulation induced by DHCR24 overexpression. Figure 6C demonstrated that SH-42 significantly inhibited cell viability and reversed the promotion induced by DHCR24 overexpression. In the wound healing assay (Figure 6D), SH-42 significantly inhibited cell wound healing ability and reversed the promotion induced by DHCR24 overexpression. As depicted in Figure 6E, SH-42 significantly inhibited cell migration ability and reversed the promotion induced by DHCR24 overexpression. In the colony formation assay (Figure 6F), SH-42 significantly inhibited cell colony formation ability and reversed the promotion induced by DHCR24 overexpression. These results suggest that SH-42 significantly inhibits the promoting effects of DHCR24 on S12 cell proliferation, migration, and total cholesterol content.

Figure 6.

Figure 6.

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SH-42 reverses the promotion effect of overexpression DHCR24 on proliferation and migration of S12. (A) Group flag in this figure. (B) Effects of DHCR24 and SH-42 on cellular total cholesterol levels. (C) Effects of DHCR24 and SH-42 on cell viability. (D) Effects of DHCR24 and SH-42 on wound healing ability of S12 cells. (E) Effects of DHCR24 and SH-42 on migration ability of S12 cells. (F) Effects of DHCR24 and SH-42 on colony formation ability of S12 cells. (*P < .05, **P < .01, ***P < .001).

DHCR24 expression level positively correlates with the progression of cervical precancerous lesions

To further validate the relationship between DHCR24 and the progression of cervical precancerous lesions, an IHC assay was conducted to evaluate DHCR24 protein expression levels in normal cervix tissues, LSIL, HSIL, and SCC tissues. The baseline information for the patient specimens is provided in Supplementary Table S3. As depicted in Figure 7A and B, DHCR24 expression was higher in LSIL tissues than in normal cervical tissues, and it was further elevated in HSIL tissues compared with LSIL tissues. However, no significant difference was observed between HSIL tissues and SCC tissues. These findings suggest a positive correlation between DHCR24 expression levels and cervical precancerous lesions progression, indicating the potential role of DHCR24 in cervical precancerous lesions progression and its candidacy as a therapeutic target.

Figure 7.

Figure 7.

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Analysis of DHCR24 expression in clinical specimens. (A) Immunohistochemical detection of DHCR24 expression in specimens of normal cervical tissue, low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and squamous cell carcinoma tissue. (B) Statistical analysis of DHCR24 expression in clinical specimens (*P < .05, ***P < .001, ns = no significance).

Discussion

The period from HPV infection to precancerous lesions presents a critical window for halting cervical cancer progression.20 However, existing treatments for cervical precancerous lesions have limited efficacy. This study demonstrates that hucMSC-sEV inhibits the proliferation and migration of cervical precancerous cells in vitro. Analysis reveals that miR-370-3p in hucMSC-sEV plays a pivotal role in inhibiting S12 cell proliferation and migration. DHCR24 is identified as a target of miR-370-3p. Overexpression of DHCR24 reverses the inhibitory effects of miR-370-3p, while the DHCR24 inhibitor SH-42 suppresses the proliferation and migration of S12 cells. Clinical specimen analysis confirms a positive correlation between DHCR24 expression and progression of cervical precancerous lesions. This study provides new insights into cervical precancerous lesions treatment and identifies a potential therapeutic target.

MicroRNAs exhibit dysregulation during HPV infection and cervical precancerous lesions, with their numbers increasing as the disease progresses.21 For instance, HPV16 E6 upregulates miR-20a, targeting PDCD6 to enhance cervical cancer cell growth.22 HPV16 E6/E7 downregulates miR-142-5p, leading to PD-L1 upregulation and promoting immune evasion in cervical cancer.23 miR-34a downregulates HPV E6, subsequently inhibiting cervical cancer cells by downregulating Cdt2.24 Moreover, miR-21 targets RASA1, promoting lymph node metastasis in cervical cancer.25 In this study, we identified 5 downregulated microRNAs in cervical precancerous tissues, and miR-370-3p inhibited the proliferation and migration capabilities of S12 cells by targeting DHCR24. This research enhances our understanding of the interplay between microRNAs and HPV-related disease progression and offers a potential microRNA-based therapy for cervical precancerous lesions.

miR-370-3p plays various roles in different cancers, including cervical cancer, but its role in cervical precancerous lesions remains unexplored. In the serum of cervical cancer patients, the miR-370-3p levels are reduced.26 CircAGFG1 sponge miR-370-3p, downregulating RAF1 and enhancing RAF/MEK/ERK pathway activity, promoted cervical cancer cell proliferation and migration.27 CircEPSTI1 sponges miR-370-3p, upregulating MSH2, and advancing cervical cancer progression and cisplatin resistance.28 Circ_0025033 sequesters miR-370-3p, upregulating SLC1A5 and promoting ovarian cancer progression.29 miR-370-3p also impacts breast cancer,30 gliomas,31 colorectal cancer,32 osteosarcoma,33 and other tumors, inhibiting tumorigenesis, progression, and metastasis. Furthermore, the noncoding RNAs also play a significant role in a variety of cancers. CircKIF4A binds to miR-637 and increases the expression of STAT3 to promote metastasis of triple-negative breast cancer to the brain.34 The long noncoding RNA of HDAC4 (LOC85009) targets USP5/USF1 axis to inhibit docetaxel resistance in lung adenocarcinoma.35 CircMYBL2 promotes hepatocellular carcinoma through circMYBL2/miR-1205/E2F1 axis.36 This study found that miR-370-3p is downregulated in cervical precancerous lesions. And miR-370-3p inhibited the proliferation and migration of S12 cells through targeting DHCR24. These findings offer new insights about miR-370-3p.

DHCR24 promotes progression in various solid tumors. In diffuse large B-cell lymphoma, SOX9 promotes DHCR24 expression, facilitating cholesterol synthesis, cancer cell proliferation, and inhibiting apoptosis.37 In metastatic bladder cancer, DHCR24 upregulates TBK1 expression, activating the PI3K/AKT pathway and promoting lymphangiogenesis and lymph node metastasis.38 Genkwadaphnin inhibits DHCR24 expression, suppressing invasion and migration in hepatocellular carcinoma.39 This study revealed elevated DHCR24 expression in cervical precancerous lesions tissues, correlating with poor prognosis in cervical cancer. DHCR24 overexpression promotes cholesterol synthesis, proliferation, and migration of S12 cells, while targeting DHCR24 inhibits these abilities. This study investigated the role of DHCR24 in S12 cells and identified DHCR24 as a novel therapeutic target.

DHCR24 is an important enzyme of cholesterol synthesis, which is related to infection of virus, including hepatitis C virus (HCV) and herpes simplex virus (HSV). Cholesterol is an important component of lipid rafts, which has a significant role in virus entry, membrane fusion, and pathology.40,41 Comparing with the human immunodeficiency virus (HIV) infection patient, the patients who are coinfected with HPV and HIV have higher low-density lipoprotein cholesterol level, and 46.3% of them have dyslipidemia.42 Statin, which can lower cholesterol, could provide prevention for HPV-positive head and neck squamous cell carcinoma.43 HCV upregulates DHCR24, inhibiting p53 acetylation in the nucleus and dampening the p53 stress response.44 HSV requires cholesterol deacylation for viral particle fusion with the cell membrane, and DHCR24 knockdown impairs HSV entry efficiency.45 This study reveals miR-370-3p in hucMSC-sEV targeting DHCR24, restraining the proliferation and migration between HPV-positive S12 cells. However, further investigation is needed to elucidate the relationship between DHCR24 and HPV.

In this study, there are still some limitations that need more investigation. First, sequencing data from clinical samples is essential, and methods’ improvements lead to the discovery of new microRNAs. Therefore, there is a need for higher-quality sequencing data on microRNA expression in cervical precancerous lesion tissues. Another challenge is the absence of animal models for an HPV-positive cervical precancerous lesion. Currently, K14-HPV16 transgenic mice could be induced cervical cancer, but simulating HPV infection is still challenging.46 Organoid is a promising method for tumor research. Hu et al recently established organoid models of HSIL organoid, providing new insights into the study of cervical precancerous lesions.47 Therefore, it is necessary to further investigate the mechanism in vivo and preclinical research. Additionally, DHCR24 converts desmosterol to cholesterol in the final step, inhibiting that DHCR24 could elevate desmosterol levels.48 Thus, whether miR-370-3p increases desmosterol by reducing DHCR24 expression, the effect of desmosterol on cervical precancerous lesions needs further investigation in vivo and in vitro.

The cervical precancerous lesion is a crucial window for cervical cancer prevention and novel treatments are urgently needed. This study found that hucMSC-sEV-derived miR-370-3p inhibits the proliferation and migration of S12 cells by targeting DHCR24. Additionally, DHCR24 is a potential target for cervical precancerous lesions, and DHCR24 protein expression is positively correlated with cervical precancerous lesions progression. This study presents a promising treatment approach for cervical precancerous lesions. However, the effects of hucMSC-sEV and cholesterol effect on HPV, and developing new drug targets such as DHCR24 still require more evidence both in vivo and in vitro.

Conclusion

This study elucidated that hucMSC-sEV-derived miR-370-3p could effectively inhibit the proliferation, migration, and total cholesterol content of S12 cells by targeting DHCR24. The protein expression levels of DHCR24 positively correlate with the progression of cervical precancerous lesions, highlighting DHCR24 as a potential target for the treatment of cervical precancerous lesions.

Supplementary Material

szae087_suppl_Supplementary_Tables
szae087_suppl_supplementary_tables.docx (16KB, docx)

Acknowledgments

We thank Shenzhen Wingor Bio-technology Co., Ltd., for the assistance in isolation of hucMSC-sEV.

Contributor Information

Weizhao Li, Department of Gynecology, Pelvic Floor disorders Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518107, People’s Republic of China; Shenzhen Key Laboratory of Chinese Medicine Active substance screening and Translational Research, Shenzhen 518107, People’s Republic of China; Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Kowloon, Hong Kong 999077, People’s Republic of China.

Chi Zhang, Department of Gynecology, Pelvic Floor disorders Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518107, People’s Republic of China.

Tianshun Gao, Department of Gynecology, Pelvic Floor disorders Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518107, People’s Republic of China.

Yazhou Sun, Department of Gynecology, Pelvic Floor disorders Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518107, People’s Republic of China.

Huan Yang, Department of Gynecology, Pelvic Floor disorders Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518107, People’s Republic of China.

Lixiang Liu, Department of Gynecology, Pelvic Floor disorders Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518107, People’s Republic of China.

Ming Shi, Department of Gynecology, Pelvic Floor disorders Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518107, People’s Republic of China.

Lu Ding, Department of Gynecology, Pelvic Floor disorders Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518107, People’s Republic of China.

Changlin Zhang, Department of Gynecology, Pelvic Floor disorders Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518107, People’s Republic of China; Shenzhen Key Laboratory of Chinese Medicine Active substance screening and Translational Research, Shenzhen 518107, People’s Republic of China.

David Y B Deng, Department of Gynecology, Pelvic Floor disorders Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518107, People’s Republic of China.

Tian Li, Department of Gynecology, Pelvic Floor disorders Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518107, People’s Republic of China; Shenzhen Key Laboratory of Chinese Medicine Active substance screening and Translational Research, Shenzhen 518107, People’s Republic of China.

Author contributions

W.L.: conception and design, collection and assembly of data, and writing manuscript; C.Z. and T.G.: collection and assembly of data, data analysis, and interpretation; Y.S.: bioinformatics; H.Y.: financial support and data analysis and interpretation; L.L., M.S., and L.D.: provision of study material. D.Y.B.D.: administrative support and supervision of manuscript writing. T.L. and C.Z.: conceptualization and design, administration support, financial support, and full responsibility of the study. All authors critically reviewed the article and final approval of manuscript.

Funding

This work was supported by the funds from the National Natural Science Foundation of China (grant no. 82172883 and 82002899), Guangdong Basic and Applied Basic Research Foundation (grant no. 2022A1515012444 and 2023A1515012662), Open Funds of State Key Laboratory of Oncology in South China (grant no. HN2023-01 to C.Z.), Shenzhen Medical Research Funds (grant no. A2302019 to Y.H.), and Shenzhen Key Laboratory of Chinese Medicine Active Substance Screening and Translational Research (grant no. ZDSYS20220606100801003 to T.L.).

Conflict of Interest

The authors declared no potential conflicts of interest.

Ethics approval and consent to participate

Ethical approval was obtained from the Ethical Committee of Sun Yat-sen University Affiliated Seventh Hospital (KY-2022-060-01).

Data availability

All data generated or analyzed in this study are included in this published article. Any other data will be available on reasonable request from corresponding author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

szae087_suppl_Supplementary_Tables
szae087_suppl_supplementary_tables.docx (16KB, docx)

Data Availability Statement

All data generated or analyzed in this study are included in this published article. Any other data will be available on reasonable request from corresponding author.

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