C21orf2 as a potential regulator of JAK2/STAT3 signaling in prostate cancer cell proliferation and apoptosis: an exploratory study

ShiHao Jiang; Shuai Su; Delin Wang

doi:10.1186/s12885-026-15637-8

. 2026 Jan 29;26:305. doi: 10.1186/s12885-026-15637-8

C21orf2 as a potential regulator of JAK2/STAT3 signaling in prostate cancer cell proliferation and apoptosis: an exploratory study

ShiHao Jiang ^1,^#, Shuai Su ^1,^#, Delin Wang ^1,^✉

PMCID: PMC12930579 PMID: 41612237

Abstract

Background

Prostate cancer (PCa) is a major health concern for men worldwide, and its incidence is rising rapidly, so identifying key molecules that regulate the malignant biological behavior of PCa is crucial for early diagnosis and targeted therapy. This study aimed to elucidate the regulatory role of chromosome 21 open reading frame 2 (C21orf2) in the biological functions of prostate cancer cells and further explore the molecular mechanisms by which C21orf2 regulates cell proliferation and apoptosis via the JAK2/STAT3 signaling pathway.

Materials and methods

In vitro, C21orf2’s effects on cell proliferation, migration, invasion, and apoptosis were tested using CCK-8, scratch assays, Transwell assays, TUNEL staining, and electron microscopy. Western blotting assessed the impact of C21orf2 on JAK2/STAT3 proteins and related markers. Co-immunoprecipitation and immunofluorescence explored interactions with downstream targets. In vivo, a subcutaneous xenograft model in nude mice validated C21orf2’s role in tumor growth and apoptosis.

Results

C21orf2 was upregulated in prostate cancer tissues and regulated the malignant biological behavior of prostate cancer cells, and C21orf2 showed a negative correlation with KCTD5. High expression of C21orf2 promoted the activation of the JAK2/STAT3 signaling pathway and upregulated its downstream target genes, including c-MYC, Cyclin A1, Bcl-2, and Cleaved caspase-3, thereby promoting cell proliferation and inhibiting apoptosis.

Conclusions

Our study indicates that C21orf2 is a potential biomarker for the diagnosis and treatment of prostate cancer and highlights its potential as a therapeutic target through the JAK2/STAT3 signaling pathway in prostate cancer.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12885-026-15637-8.

Keywords: Prostate cancer, C21orf2, Proliferation, Apoptosis, JAK2/STAT3 signaling pathway

Background

Prostate cancer (PCa) is one of the most common malignancies in men worldwide and poses a significant threat to male health [1]. According to the World Health Organization, PCa ranks second in the incidence of newly diagnosed cancers among men globally [2]. In Asia, particularly in China, although the incidence and mortality rates of prostate cancer are lower, but are rising at a faster rate compared to those in Western countries [3]. Therefore, identifying key molecules that regulate the malignant biological behavior of PCa and elucidating their mechanisms of action in the development and progression of the disease will significantly contribute to early diagnosis and targeted therapy of PCa.

C21orf2 is a novel gene located on chromosome 21 and is closely associated with human genetic diseases [4]. However, its specific biological functions remain unclear. C21orf2 has been recently identified as a susceptibility gene for amyotrophic lateral sclerosis (ALS), harboring rare missense mutations that cause this fatal disease [5]. Additionally, mutations in C21orf2 have been found in certain ciliopathies, suggesting its potential role in common signaling pathways [6]. Our research team previously discovered, through proteomic sequencing of benign prostatic hyperplasi(BPH) a and PCa tissues, that C21orf2 is highly expressed in PCa tissues. Furthermore, in the genomic sequencing results of autosomal dominant clear cell renal cell carcinoma, a high mutation rate of C21orf2 was observed, which is closely related to the malignant progression of renal clear cell carcinoma. C21orf2 is a potential therapeutic target for urological tumors, yet its role in PCa remains unexplored. Elucidating its molecular mechanisms is critical for advancing early diagnosis and targeted therapy of PCa.

Materials and methods

Bioinformatics analysis

Gene expression and clinical data for The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) were retrieved from the TCGA database (https://portal.gdc.cancer.gov/). Further organization and analysis of the data were performed using the UALCAN online platform (https://ualcan.path.uab.edu/index.html). RNA sequencing (RNA-seq) data and corresponding clinical information were searched and downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). Additionally, RNA-seq data were retrieved and downloaded from the TIMER database (https://cistrome.shinyapps.io/timer/).

Clinical tissue samples and cell lines

Tissue samples: Clinical tissue samples were collected from postoperative specimens at the Department of Urology, First Affiliated Hospital of Chongqing Medical University, between September 2014 and December 2021. The collection included 121 PCa specimens following radical prostatectomy and 29 BPH specimens following transurethral resection of the prostate. All samples were confirmed by pathological diagnosis and stored at -80℃ and in liquid nitrogen.

Cell lines: PCa cell lines and normal prostate epithelial cell lines were purchased from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, and authenticated by STR profiling. The cell lines and their culture conditions were as follows: PC3: F12K (Gibco; 21127030) + 10% FBS (Gibco; A5670701) + 1% P/S (Gibco; 15140148); LNCaP: RPMI 1640 (Gibco; A4192301) + 10% FBS + 1% P/S; 22Rv1: RPMI 1640 + 10% FBS + 1% P/S; RWPE-1: K-SFM (Gibco; 17005042) + 10% FBS + 1% P/S. All cells were cultured in an incubator at 37℃ with 5% CO₂.

Lentivirus infection and plasmids

The lentivirus (Genechem, China) was based on the GV248 plasmid vector, with the specific sequence provided in Supplementary Material, Table 1. The efficiency of lentivirus infection was assessed by observing fluorescence expression in target cells under a fluorescence microscope. Puromycin (Amresco Inc., J593) was used to select stably infected PCa cells.The C21orf2 and KCTD5 overexpression plasmids and its control plasmid were purchased from Genechem Company (Shanghai, China).

Quantitative real-time PCR (qRT-PCR)

RNA was extracted from clinical samples of BPH and PCa tissues using TRIzol reagent (Invitrogen, 12183555) and the purity was qualified (A260/A280 ratio of 1.8–2.1, A260/A230 ≥ 2.0). Reverse transcription to synthesize cDNA was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, RR037A). The primers used for synthesis were designed by Tsingke Biotechnology Company (Beijing, China), with the specific sequences listed in Supplementary Material, Table 2.

Western blot (WB)

Cells were lysed in ice-cold RIPA lysis buffer (20–188, Sigma Aldrich) and subsequently sonicated. The concentration of proteins in the supernatant was measured using a NanoDrop One (Thermo Scientific, CA, USA). Protein samples were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% skim milk and incubated with specific primary and secondary antibodies. Finally, ECL substrate (Invitrogen, WP20005) was prepared, and the membranes were scanned using an Image Lab system (Bio-Rad Laboratories, Hercules, CA, USA).The related reagents, and antibodies are provided in Supplementary Material, Table 3.

Immunohistochemical(IHC) staining

Paraffin-embedded sections of PCa and BPH tissues were obtained from our hospital’s pathology department. IHC staining was performed according to the instructions of the IHC staining kit (Boster Bio, SA1022), which included 3% H2O2, 5% BSA, rabbit secondary antibody, SABC, and DAB (Abcam, 59718), as well as specific antibodies against C21orf2 (Proteintech, 27609-1-AP) and Ki67 (Abcam, ab16667). The staining intensity and positive area were scored accordingly.

Animal experiment

Male nude mice (4–6 weeks old) were purchased from SPF Company (Beijing, China) and maintained under the supervision of the Animal Ethics Committee. Mice were housed in individually ventilated cages under pathogen-free conditions. Tumor length (L) and width (W) were regularly monitored, and tumor volume was calculated using the formula V = π/6×L×W^2 (where V represents tumor volume, π/6 ≈ 0.52, L is the longest diameter, and W is the shortest diameter). After 4 weeks, the mice were euthanized, and tumors were excised and weighed. Mice were euthanized after deep anesthesia using Tribromoethyl alcohol (75-80-9, MedChem Express, diluted to 2.5% concentration with PBS, and the injection dose was 15–20 µL/g) by intraperitoneal injection.

CCK-8 experiment

Cells were seeded at a density of 2000 cells per well in a 96-well plate, with three replicates per group. After cell attachment, the plate was incubated in a cell culture incubator (37℃, 5% CO2) for 24 h. CCK-8 solution (HY-K0301, MedChem Express) was added to each well and incubated for 2 h. The absorbance at 450 nm was measured using a microplate reader.

Wound healing

Marking the six-well plate: Using a marker pen, draw three horizontal lines on the back of each well in a six-well plate, with a spacing of approximately 0.5 cm between parallel lines. Cells were seeded into 6-well plates at 5 × 10⁵ cells/well, when the cell density reached 90%, a scratch was made using a 10 µl pipette tip. Photos were taken under a microscope as the 0-hour control. The cells were then incubated in a cell culture incubator (37℃, 5% CO2). After 24 h of incubation, the cells were photographed again under the microscope.

Transwell assay

Cell suspensions were prepared for the invasion assay by diluting Matrigel matrix and applying 80 µL evenly onto the upper chamber of each Transwell insert. For the migration assay, this step was omitted. The Transwell inserts were then placed into wells, with 100 µL of cell suspension added to the upper chamber (2 × 10⁴cells were added in migration assay, 5 × 10⁴ cells were added in invasion assay) and 600 µL of complete medium added to the lower chamber. The inserts were incubated in a cell culture incubator (37℃, 5% CO₂) for 24 h. After incubation, the inserts were removed, the medium was aspirated, and the lower surface was fixed in 70% methanol for 30 min, followed by staining with crystal violet or trypan blue for 30 min. After air-drying, the cells were observed and photographed under a microscope.

TUNEL experiment

Cell suspensions were prepared, and cells were fixed in 4% paraformaldehyde for 30 min. The cells were then resuspended in PBS and incubated at room temperature for 5 min. TUNEL detection solution (HY-K1091, MedChem Express) was added, and the cells were incubated at 37℃ in the dark for 1 h. After washing with PBS, the cells were photographed under a fluorescence microscope.

Transmission electron microscopy (TEM)

Cells were mixed with electron microscopy fixative and fixed for 2–4 h at 4℃. The cells were then washed multiple times with phosphate-buffered saline (PBS) to remove residual fixative. The cells were dehydrated using a series of increasing concentrations of ethanol or acetone. After embedding in resin, the cells were transferred to embedding molds for polymerization. Ultrathin Sects. (50–90 nm) were cut from the polymerized resin blocks using an ultramicrotome. The sections were stained and washed before observation under the electron microscope.

Flow cytometry

Apoptosis analysis was conducted with an Annexin VFITC Apoptosis Detection Kit (KeyGen Biotech, China) according to the manufacturer’s protocol. The percentage of apoptotic cells was determined using a FACS flow cytometer equipped with software (BD).

Immunofluorescence(IF) experiment

PC3 and 22Rv1 cells were fixed with 4% paraformaldehyde at room temperature for 20 min, permeabilized for 10 min, and then blocked for 30 min. Primary antibodies specific to C21orf2 (1:2000; 27609-1-AP; Proteintech) and KCTD5 (1:2000; 15553-1-AP; Proteintech) were added and incubated overnight at 4℃. The next day, the cells were warmed at room temperature for 20 min, followed by the addition of secondary antibodies conjugated with fluorophores, matching the species of the primary antibodies, diluted to the appropriate concentration, and incubated overnight at 4℃. The cells were then warmed at room temperature for 20 min, washed three times with PBST for 5 min each, and stained with DAPI (HY-D0814, MedChem Express) for nuclear staining for 5 min.

Co-immunoprecipitation assay

IP Lysis Buffer (Beyotime Institute of Biotechnology, Shanghai, China) was added to the protein samples, and the mixture was agitated on a rocker for 20 min to fully lyse and disrupt the cells. The lysate was then centrifuged at 1200 rpm for 10 min at 4℃ to remove unlysed cell debris, and the supernatant was transferred to a new EP tube. Using a magnetic rack and a large-bore pipette tip, magnetic beads (Bimake, Houston, Texas, USA) were added to the supernatant. The mixture was agitated on a rocker at 4℃ for 4 h to allow the interaction between the FLAG-tagged KCTD5 (1:2000; 15553-1-AP; Proteintech) and HA-tagged C21orf2 (1:2000; 27609-1-AP; Proteintech) transfected into PC3 cells. Non-specifically bound proteins were removed, and the protein complexes were eluted. The eluted proteins were boiled for 10 min and stored at -20℃ for subsequent protein detection.

Detection of ubiquitination

Protein samples were treated with MG132 (HY-13259, MedChem Express) at a concentration of 10 µM for 8 h. An appropriate amount of IP Lysis Buffer (Beyotime Institute of Biotechnology, Shanghai, China) was added, and the mixture was incubated on ice for 30 min. The lysate was then centrifuged at 1200 rpm for 10 min at 4℃. The subsequent steps were as described in the Co-immunoprecipitation section. WB analysis was performed to detect the ubiquitination levels of the substrate proteins.

Statistic analysis

Quantitative experimental data are presented as mean ± standard deviation. Comparisons between two groups were analyzed using independent samples t-tests, while comparisons among three or more groups were analyzed using one-way ANOVA. A p-value of ≤ 0.05 was considered statistically significant. Survival curves were plotted using the Kaplan-Meier method, and the log-rank test was employed to evaluate the impact of C21orf2 expression on overall survival (OS) in PCa patients. Statistical analysis and graphing were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA).

Results

Upregulation of C21orf2 in PCa tissues

Using the TIMER database, we retrieved and analyzed the differential expression of C21orf2 across various cancers. The results showed that C21orf2 was significantly upregulated in PCa (TCGA-PRAD) and renal clear cell carcinoma (TCGA-KIRC) among urological tumors, as shown in Fig. 1A. In TCGA-PRAD and GSE46602, C21orf2 expression was higher in PCa tissues compared to normal prostate tissues, as shown in Fig. 1B and C. Using RNAseq data from the TCGA-PRAD dataset and its corresponding clinical information, we further analyzed the correlation between C21orf2 expression and the clinical pathological features of PCa. We found that C21orf2 expression was significantly associated with the Gleason score and T stage of PCa, as shown in Fig. 1D.

Fig. 1 — High expression of C21orf2 in prostate cancer tissues. A Pan-cancer analysis of C21orf2 expression. B C21orf2 is upregulated in prostate cancer in the TCGA-PRAD dataset, P = 0.00005. C C21orf2 is upregulated in prostate cancer in the GSE46602 dataset, P = 0.0017. D Correlation between C21orf2 expression and clinicopathological characteristics in TCGA-PRAD dataset, C21orf2 expression was significantly upregulated in PCa tissues with lower Gleason scores. C21orf2 expression increased in PCa tissues with earlier T stages. No statistically significant difference in C21orf2 expression was observed between N0 and N1 PCa patients. E Volcano plot. F Expression of C21orf2 in common prostate cancer cell lines in CCLE database. G qRT-PCR showing higher expression of C21orf2 in prostate cancer tissues at the RNA level. Error bars represent mean ± standard error (n = 25). P values were calculated by t-test. H Immunohistochemical staining of C21orf2 in BPH (left) and PCa (right) tissues across various stages. I Association of C21orf2 expression with overall survival probability. ns P>0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

In our previous study, we performed 4D label-free proteomics sequencing on three PCa and three BPH tissues. The results indicated that C21orf2 was upregulated in PCa tissues, as shown in Fig. 1E.

In CCLE database(https://sites.broadinstitute.org/ccle/datasets), the expression of C21orf2 in three prostate cancer cell lines (PC3, LNCaP,22Rv1) was significantly elevated compared to normal prostate epithelial cells (RWPE-1)(Fig. 1F).

To further validate the expression of C21orf2 in PCa tissues, frozen tissues from 25 PCa and 25 BPH cases were subjected to qRT-PCR. The results showed that C21orf2 expression was higher in PCa tissues at the RNA level, as shown in Fig. 1G. IHC staining of 29 BPH and 121 PCa paraffin sections was scored amd the results demonstrated that C21orf2 expression was significantly higher in PCa tissues than in BPH tissues(Fig. 1H, Supplementary Material, Tables 4 and 5). Subsequently, we conducted survival analysis to evaluate the prognostic significance of C21orf2 expression in PCa. It was found that patients with high C21orf2 expression exhibited significantly reduced overall survival (P = 0.001), as shown in Fig. 1I.

Regulation of PCa cell biology by C21orf2 in vitro

C21orf2 was relatively highly expressed in PC3 and LNCaP cells. To investigate the regulatory effects of C21orf2 on PCa cell biology, we used lentivirus to infect cells and constructed three knockdown sites. After continuous puromycin selection and validation of C21orf2 knockdown efficiency by WB, we selected the lentivirus with the highest knockdown efficiency for subsequent experiments (Fig. 2A). We successfully established stable C21orf2-knockdown cell lines.

CCK-8 assay results showed that C21orf2 knockdown inhibited the proliferation of PCa cells (Fig. 2B). Scratch assay results indicated that C21orf2 knockdown suppressed the migration of PCa cells (Fig. 2C). Transwell assay results demonstrated that C21orf2 knockdown inhibited both the migration and invasion of PCa cells (Fig. 2D). TUNEL staining revealed a significant increase in TUNEL-positive cells in PC3 and LNCaP PCa cells after C21orf2 knockdown (Fig. 2E).

We used TEM to observe the ultrastructural changes in PCa cells. PC3 and LNCaP cells were selected as the subjects, with shNC as the control group and shC21orf2 as the experimental group. The results showed that the control group cells had intact structures with clear edges, normal-sized mitochondria with distinct cristae, and no dilated endoplasmic reticulum or apoptotic bodies. In contrast, the experimental group cells exhibited disordered structures with unclear edges, enlarged mitochondria with vacuolization and disrupted or absent cristae, dilated and fragmented endoplasmic reticulum pools, and apoptotic bodies budding from the cell membrane (Fig. 2F). These findings suggest that C21orf2 knockdown induces apoptosis in PCa cells.

Effects of C21orf2 on PCa cell proliferation and apoptosis in vivo

In vitro, knockdown of C21orf2 inhibits the proliferation of PCa cells. To further confirm the effects of C21orf2 on PCa cell proliferation in vivo, we conducted a subcutaneous tumor formation experiment in nude mice. Ten nude mice were divided into control and experimental groups, with five mice injected with PC3 shNC cells as the control group and another five injected with PC3 shC21orf2 cells as the experimental group. Tumor volumes were measured regularly. After 4 weeks, the mice were euthanized under anesthesia, and the prostate tumors were excised and weighed. As shown in Fig. 3A-D. The expression level of Ki-67 and TUNEL staining were shown in Supplementary Fig. 1.These findings indicate that C21orf2 knockdown induces apoptosis in PCa cells in vivo.

Fig. 3 — Effects of C21orf2 on prostate cancer cell proliferation and apoptosis in vivo. A, C Subcutaneous tumor formation in nude mice after 4 weeks, showing that C21orf2 knockdown inhibits tumor formation in vivo. B Growth curve of subcutaneous tumor volume. D Statistical analysis of subcutaneous tumor weight, **P < 0.01, ***P < 0.001

C21orf2 regulates PCa cell proliferation and apoptosis via the JAK2/STAT3 signaling pathway

Based on the TCGA-PRAD dataset, samples were divided into high-expression (≥ 50%) and low-expression (< 50%) groups according to C21orf2 expression levels. GSEA analysis was performed to evaluate the biological functions and related molecular mechanisms of C21orf2. As shown in Fig. 4A, high expression of C21orf2 was positively correlated with the ubiquitin-proteasome pathway, cell cycle, PCa, and the JAK/STAT signaling pathway (NES > 1, FDR < 0.25).

Fig. 4 — C21orf2 regulates prostate cancer cell proliferation and apoptosis via the JAK2/STAT3 signaling pathway. A GSEA analysis of C21orf2 expression in the TCGA-PRAD dataset. B CCK−8 assay showing the effect of C21orf2 on prostate cancer cell proliferation regulated by the JAK2/STAT3 signaling pathway. Compared with the Vector control group, the proliferation of 22Rv1 cells was significantly increased in the oeC21orf2 group, while it was significantly decreased in the Vector + AG490 group and the oeC21orf2 + AG490 group, P < 0.01. C Flow cytometry showing the effect of C21orf2 on prostate cancer cell cycle regulated by the JAK2/STAT3 signaling pathway. Knockdown of C21orf2 induces G2 phase cell cycle arrest in PC3 cells, while overexpression of C21orf2 in 22Rv1 cells promoted G2 phase progression. AG490 rescued the cell cycle phenotype induced by C21orf2 overexpression. D Flow cytometry showing the effect of C21orf2 on prostate cancer cell apoptosis regulated by the JAK2/STAT3 signaling pathway. Knockdown of C21orf2 in PC3 cells induced apoptosis, while overexpression of C21orf2 in 22Rv1 cells inhibited apoptosis. AG490 rescued the apoptotic phenotype induced by C21orf2 overexpression. WB analysis of C21orf2 regulation of the JAK2/STAT3 signaling pathway. E Knockdown of C21orf2 inhibits the JAK2/STAT3 signaling pathway in PC3 cells. F Decreased phosphorylation levels of JAK2 and STAT3 proteins in PC3 cells. G Reduced expression ofc-MYC, Cyclin A1, and Bcl−2 proteins, and increased expression of Cyclin B1, CDC2 and Cleaved caspase3 protein in PC3 cells. *P < 0.05, **P < 0.01, ***P < 0.001

AG490 is a selective JAK tyrosine kinase inhibitor that blocks JAK2 activity and affects STAT3 activation, thereby inhibiting the JAK2/STAT3 signaling pathway. After treating PC3 and 22Rv1 cells with AG490 (20 µM) for 24 h, CCK-8 assay results showed that overexpression of C21orf2 enhanced the proliferation of PCa cells. Inhibition of the JAK2/STAT3 signaling pathway using AG490 not only reduced the proliferative capacity of PCa cells but also rescued the proliferative phenotype induced by C21orf2 overexpression, as shown in Fig. 4B.

Flow cytometry results indicated that C21orf2 knockdown induced G2 phase cell cycle arrest in PC3 cells, while C21orf2 overexpression promoted G2 phase progression in 22Rv1 cells. Inhibition of the JAK2/STAT3 signaling pathway with AG490 induced G2 phase arrest and rescued the cell cycle phenotype induced by C21orf2 overexpression, as shown in Fig. 4C.

C21orf2 knockdown induced apoptosis in PC3 cells, while C21orf2 overexpression inhibited apoptosis in 22Rv1 cells. Inhibition of the JAK2/STAT3 signaling pathway with AG490 induced apoptosis in PCa cells and rescued the apoptotic phenotype induced by C21orf2 overexpression, as shown in Fig. 4D.

To elucidate the mechanism by which C21orf2 regulates the JAK2/STAT3 signaling pathway, we used WB to detect the effects of C21orf2 expression on the proteins in the JAK2/STAT3 signaling pathway and its downstream target genes. Dysregulation of the cell cycle is one of the causes of tumorigenesis, and the cell cycle regulatory genes Cyclin B1, CDC2 protein, c-MYC and Cyclin A1 are downstream target genes of the JAK2/STAT3 signaling pathway. Inhibition of apoptosis is also one of the causes of tumorigenesis, and the mitochondrial pathway is a classical pathway of apoptosis. C21orf2 encodes four different isoforms, three of which are mitochondrial proteins. Bcl-2, an anti-apoptotic protein, and Cleaved caspase3, a pro-apoptotic protein, are key proteins in the mitochondrial pathway.WB results showed that knockdown of C21orf2 inhibits the JAK2/STAT3 signaling pathway, with decreased phosphorylation levels of JAK2 and STAT3 proteins. The expression levels of downstream target genes c-MYC, Cyclin A1, and Bcl-2 were reduced, while the expression of Cyclin B1, CDC2 and Cleaved caspase3 was increased. This induced cell cycle arrest, inhibited cell proliferation, and promoted apoptosis, as shown in Fig. 4E-G).

Interaction of C21orf2 with the E3 ubiquitin ligase KCTD5

We downloaded RNA-Seq data for PCa (including 499 cases) from the TCGA website. Based on the median expression level of the C21orf2 gene, samples were divided into low-expression (n = 250) and high-expression (n = 249) groups, as shown in Fig. 5A-B. Differential expression analysis was performed, and the related genes were clustered. KEGG and GO analyses were conducted to explore the relationship between C21orf2 expression and gene characteristics. The results showed that C21orf2 is closely related to ubiquitination, cell cycle, autophagy, homologous recombination repair of genes, and the biosynthesis of amino acids and glycans, as shown in Fig. 5C-D.

Using the BioGRID database (https://thebiogrid.org/), we constructed a protein-protein interaction (PPI) network related to C21orf2, as shown in Fig. 5E. A total of 45 related genes were included, among which KCTD5 and SKP1 overlapped with 34 representative E3 ubiquitin ligases, as shown in Fig. 5F. To further verify their correlation, we analyzed the data using the GEPIA2 database (http://gepia.cancer-pku.cn/). The results showed that in PCa, C21orf2 is negatively correlated with KCTD5 (R=-0.37, P = 2.7e-17) and SKP1 (R=-0.25, P = 1.3e-08). Given the stronger correlation coefficient between C21orf2 and KCTD5, we selected KCTD5 for further experiments, as shown in Fig. 5G.

Negative correlation between C21orf2 and KCTD5 in PCa cells

To confirm the correlation between C21orf2 and KCTD5, we used WB to detect the effect of C21orf2 expression on KCTD5 expression levels. The results showed that in PCa cells, C21orf2 and KCTD5 are negatively correlated, as shown in Fig. 6A. Immunofluorescence experiments indicated that in PC3 and 22Rv1 cells, C21orf2 and the E3 ubiquitin ligase KCTD5 are co-expressed in the cytoplasm, as shown in Fig. 6B. Co-immunoprecipitation experiments showed that when KCTD5 was immunoprecipitated with an anti-KCTD5 antibody, the endogenous C21orf2 protein bound to KCTD5 was also precipitated, as shown in Fig. 6C. We overexpressed KCTD5 in 22Rv1 cells and performed immunoprecipitation with an anti-C21orf2 antibody, followed by WB analysis with an anti-Ub antibody. The results suggested that overexpression of KCTD5 significantly increased the ubiquitination level of C21orf2, as shown in Fig. 6D. KCTD5 downregulates C21orf2 expression by promoting its ubiquitination.

Fig. 6 — Negative correlation between C21orf2 and KCTD5 in prostate cancer cells. A WB showing the effect of C21orf2 expression on KCTD5 expression levels. In PC3 cells, KCTD5 protein expression was increased in the shC21orf2 group compared to the shNC control group; conversely, in 22Rv1 cells, KCTD5 protein expression was decreased in the oeC21orf2 group compared to the Vector control group. B Immunofluorescence showing co-expression of C21orf2 and KCTD5 in the cytoplasm of PC3 and 22Rv1 cells. Scale bar, 50 μm. C Co-immunoprecipitation showing the interaction between C21orf2 and KCTD5 in PC3 cells. D Co-immunoprecipitation showing increased ubiquitination of C21orf2 protein after overexpression of KCTD5 in 22Rv1 cells. E WB showing that overexpression of KCTD5 significantly reduces C21orf2 protein levels in 22Rv1 cells. After treatment with MG132 (10 µM) and CQ (10 µM) for 24 h, C21orf2 protein levels were significantly higher in the MG132-treated group compared to the CQ-treated group. F WB showing that overexpression of KCTD5 inhibits the JAK2/STAT3 signaling pathway in 22Rv1 cells. The ratio of phosphorylated JAK2 to total JAK2 (p-JAK2/JAK2) and phosphorylated STAT3 to total STAT3 (p-STAT3/STAT3) was decreased in the oeKCTD5 group compared to the Vector control group. G CCK-8 assay showing the effect of KCTD5 on the proliferation of 22Rv1 prostate cancer cells. H TEM showing ultrastructural changes in prostate cancer cells. *P < 0.05, **P < 0.01, ***P < 0.001

Proteins are primarily degraded via the autophagy-lysosome pathway and the ubiquitin-proteasome pathway. We used the autophagy-lysosome inhibitor CQ and the ubiquitin-proteasome inhibitor MG132 to investigate which pathway is involved in C21orf2 degradation. After treating KCTD5-overexpressing 22Rv1 cells with CQ (10 µM) and MG132 (10 µM) for 24 h, we detected the effect of KCTD5 expression on C21orf2 levels using WB. The results showed that the E3 ubiquitin ligase KCTD5 mediates the degradation of C21orf2 via the ubiquitin-proteasome pathway, as shown in Fig. 6E.

We used the BioGRID Interaction Database (https://thebiogrid.org) and the GPS-SUMO software (https://sumo.biocuckoo.cn/index.php/) to predict potential ubiquitination sites of C21orf2. The results indicated that eight sites (K2, K6, K13, K22, K84, K120, K156, and K192) of C21orf2 may be ubiquitinated, with higher scores indicating a higher likelihood of ubiquitination. Among them, the K13 site has the highest probability of ubiquitination, as shown in Supplementary Material, Table 6.

To explore whether KCTD5-mediated ubiquitination of C21orf2 acts through the JAK2/STAT3 signaling pathway, we used WB to detect the expression of related proteins in KCTD5-overexpressing 22Rv1 cells. The results showed that KCTD5 overexpression induces C21orf2 ubiquitination and inhibits the JAK2/STAT3 signaling pathway, as shown in Fig. 6F.

We used the CCK-8 assay to measure the proliferative capacity of PCa cells in different treatment groups. The results showed that overexpression of C21orf2 enhances the proliferation of PCa cells, while overexpression of KCTD5 not only reduces the proliferative capacity of PCa cells but also rescues the proliferative phenotype induced by C21orf2 overexpression, as shown in Fig. 6G. We used TEM to observe the ultrastructural changes in PCa cells, as shown in Fig. 6H. Overexpression of KCTD5 induces apoptosis in PCa cells.

Discussion

PCa is one of the most common malignancies among men globally [7]. Although its incidence in our country is lower than the average level in Western countries, it is on an annual upward trend [8]. Identifying new key molecules and exploring specific therapeutic methods are of great significance for the early diagnosis and targeted treatment of PCa [9]. C21orf2 is a novel gene located on chromosome 21, primarily functioning in regulating cell morphology and cytoskeleton organization, participating in DNA damage repair, and post-translational modification of proteins. It is closely related to human genetic diseases, yet its specific biological functions remain unclear [10, 11]. Our team’s previous research identified C21orf2 as a potential therapeutic target for urological tumors, but the potential role and molecular mechanisms of C21orf2 in PCa remain unknown.

We have discovered that C21orf2 is highly expressed in PCa tissues. In both in vitro and in vivo experiments, we have clarified that C21orf2 regulates the malignant biological behavior of PCa cells. C21orf2 promotes the proliferation of PCa cells and inhibits their apoptosis, thereby playing an oncogenic role in PCa. The abnormal activation of proliferation signals and the inhibition of apoptosis pathways jointly promote the growth and survival of tumor cells [12, 13]. Exploring the regulatory mechanisms of cell proliferation and apoptosis, inhibiting the proliferative capacity of tumor cells, and restoring the apoptotic capacity of tumor cells are of great significance for suppressing the malignant progression of PCa and developing new therapeutic strategies [14, 15].

We successfully identified that C21orf2 functions via the JAK2/STAT3 signaling pathway, regulating downstream target genes of the JAK2/STAT3 pathway, including cell cycle regulatory genes Cyclin B1, CDC2 protein, c-MYC and Cyclin A1, and apoptosis-related genes Bcl-2 and Cleaved caspase3. The JAK/STAT signaling pathway is an important node involved in cross-talk between pathways and is a hotspot in tumor research [16, 17]. Studies have found that the JAK2/STAT3 signaling pathway is highly expressed in PCa tissues and plays a key role in the occurrence, development, proliferation, and apoptosis of PCa cells [18]. Sennoside A can promote autophagic death of PCa cells by inhibiting JAK2/STAT3 signaling and upregulating autophagy- and apoptosis-related protein levels. Inhibiting the JAK2/STAT3 signaling pathway can promote apoptosis in PCa cells [19]. Additionally, asiatic acid can induce mitochondrial apoptosis in PCa cells by inhibiting the JAK2/STAT3 signaling pathway, thereby exerting anti-PCa effects. Activating the JAK2/STAT3 signaling pathway in tumor cells can promote tumor cell proliferation and regulate the cell cycle [20]. Dysregulation of the cell cycle is one of the causes of tumorigenesis in cancer cells [21], and cell cycle regulatory genes c-MYC and Cyclin A1 are downstream target genes of the JAK2/STAT3 signaling pathway [22, 23]. Inhibition of apoptosis is also one of the causes of tumorigenesis in cancer cells, and the mitochondrial pathway is a classical pathway of apoptosis [24, 25]. C21orf2 encodes four different isoforms, three of which are mitochondrial proteins. Bcl-2, an anti-apoptotic protein [26], and Cleaved caspase3, a pro-apoptotic protein [27], are key proteins in the mitochondrial pathway [25]. Inhibition of the JAK2/STAT3 signaling pathway suppresses the proliferation and survival of PCa cells and promotes apoptosis in PCa cells, providing new strategies and directions for drug development in PCa treatment [28, 29].

By mining RNA-Seq data from TCGA-PRAD, we performed clustering analysis on differentially expressed related genes and found that C21orf2 is closely related to protein ubiquitination. Within cells, 80% to 90% of proteins are degraded via the ubiquitination pathway [30]. The ubiquitin-proteasome system [31, 32] not only clears erroneous proteins but also regulates cell growth cycles and DNA damage repair [33]. It is significant for tumorigenesis and development [34], degradation of oncogenes [35], regulation of apoptosis proteins [36], and promotion of tumor invasion and metastasis [37]. In the C21orf2-related PPI network, C21orf2 interacts with the E3 ubiquitin ligase KCTD5. KCTD5 is a protein composed of 234 amino acids [38], with a relatively conserved N-terminal domain and variable C-terminal [39, 40]. Experimental results revealed that KCTD5 is negatively correlated with the C21orf2 protein and inhibits the JAK2/STAT3 pathway, participating in the proliferation and apoptosis of PCa cells. We also predicted potential ubiquitination sites of C21orf2 and found that eight sites (K2, K6, K13, K22, K84, K120, K156, and K192) may be ubiquitinated, with the K13 site having the highest likelihood of ubiquitination. C21orf2 has many K (lysine) sites, suggesting that C21orf2 is likely to be modified by polyubiquitination. Types of ubiquitin chains include K6, K11, K27, K29, K33, K48, K63, and M1. Different types of ubiquitin chains have different biological functions [41, 42]. For example, K48-linked polyubiquitin chains mainly mediate protein degradation [43], while K63-linked polyubiquitin chains are involved in DNA damage response and repair [44]. However, the type of ubiquitination linkage for C21orf2 has not yet been further clarified and requires more experimental validation. Based on this study, we will further investigate the specific molecular mechanisms of KCTD5 ubiquitination of C21orf2 and the regulation of the JAK2/STAT3 signaling pathway in the occurrence and development of PCa.

Conclusions

In summary, C21orf2 is upregulated in PCa tissues and exerts oncogenic effects. It regulates the malignant biological functions of PCa cells and interacts with the E3 ubiquitin ligase KCTD5 to modulate proliferation and apoptosis via the JAK2/STAT3 signaling pathway. For the first time, we have comprehensively elucidated the role of C21orf2 and its underlying molecular mechanisms in PCa, as illustrated in Fig. 6I. By activating the JAK2/STAT3 signaling pathway, C21orf2 promotes the proliferation of PCa cells and inhibits their apoptosis. C21orf2 not only serves as a potential biomarker for the diagnosis and treatment of PCa but also provides valuable insights into targeting the JAK2/STAT3 signaling pathway as a therapeutic strategy for PCa.

Supplementary Information

Supplementary Material 1^{(1.8MB, docx)}

Supplementary Material 2^{(35.5MB, zip)}

Supplementary Material 3^{(35.4MB, zip)}

Acknowledgements

We acknowledge the support received from the Natural Science Foundation of Chongqing Province (Grants 41021300060463 and 41021300060605). We appreciate the great help from Molecular Medicine and Oncology Research Center, The First Affiliated Hospital of Chongqing Medical University.

Abbreviations

BPH: Benign prostatic hyperplasia
C21orf2: Chromosome 21 open reading frame 2
CRPC: Castration resistant prostate cancer
CQ: Chloroquine
DAPI: 4’,6-diamidino-2-phenylindole
DEPC: Diethyl pyrocarbonate
DMSO: Dimethyl Sulfoxide
DNA: Deoxyribonucleic acid
E3s: E3 ubiquitination ligases
GAPDH: Glyceraldehyde-3-phosphate dehydrogenase
GSEA: Gene set enrichment analysis
HE: Hematoxylin and eosin
HR: Homologous recombination
IB: Immunoblotting
IF: Immunofluorescence
IHC: Immunohistochemical
IP: Immunoprecipitation
KEGG: Kyoto encyclopedia of genes and genomes
JAK/STAT: Janus kinase/signal transducer and activator of tran-ions
MOI: Multiplicity of infection
NC: Negative control
OD: Optical density
OE: Overexpression
PBS: Phosphate-buffered solution
PCa: Prostate cancer
PFI: Progression free interval
PI: Propidium Iodide
PPI: Protein-protein interaction
PMSF: Phenylmethyl sulfonyl fluoride
PTM: Post-translational modification
qRT-PCR: Quantitative real-time polymerase chain reaction
RNA: Ribonucleic acid
RPM: Rotate per minute
TCGA: The cancer genome altas
TEM: Transmission electron microscope
TUNEL: Terminal-deoxynucleotidyl transferase mediated nick end labeling
Ub: Ubiquitin
WB: Western Blot

Authors’ contributions

ShiHao Jiang.,Conceptualization, Methodology, Formal analysis, Investigation, Writing Original Draft. Shuai Su.,Software, Data Curation, Visualization, Writing & Review. Delin Wang.,Validation, Resources, Supervision, Project Administration, Funding acquisition, Review & Editing. All authors contributed to the article and approved the final manuscript.

Funding

The Natural Science Foundation of Chongqing Province (Grants 41021300060463 and 41021300060605).

Data availability

The datasets generated the current study are openly available in TCGA-PRAD database (https://portal.gdc.cancer.gov) and GEO database GSE46602 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE46602), performed by UALCAN (https://ualcan.path.uab.edu/index.html), TIMER (https://cistrome.shinyapps.io/timer) and g: Profiler (https://biit.cs.ut.ee/gprofiler/gost). All data created and analyzed during this current work are involved in this published paper (and its supplementary information files).

Declarations

Ethics approval and consent to participate

All experiments related to clinical specimens were approved by Institutional Animal Care and Use Committee of Chongqing Medical University, Chongqing, China (approval NO. 2022-051) and was conducted with informed consent from the patients. This study involving human data was performed in accordance with the Declaration of Helsinki (https://www.wma.net/policies-post/wma-declaration-of-helsinki/).All animal experiments were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University, Chongqing, China (approval NO.IACUC-CQMU-2022-K234).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

ShiHao Jiang and Shuai Su contributed equally to this work.

References

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Associated Data

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

Supplementary Materials

Supplementary Material 1^{(1.8MB, docx)}

Supplementary Material 2^{(35.5MB, zip)}

Supplementary Material 3^{(35.4MB, zip)}

Data Availability Statement

[CR1] 1.Luo L-S, Huang J, Luan H-H, Mubarik S, Zhong Q, Zeng X-T. Estimating disparities of prostate cancer burden and its attributable risk factors for males across the BRICS-plus, 1990–2019: A comparable study of key nations with emerging economies. Prostate. 2024;84:570–83. [DOI] [PubMed] [Google Scholar]

[CR2] 2.Stone BV, Labban M, Beatrici E, Filipas DK, D’Amico AV, Lipsitz SR, et al. The association of County-level prostate-specific antigen screening with metastatic prostate cancer and prostate cancer mortality. Eur Urol Oncol. 2024;7(3):563–9. [DOI] [PubMed] [Google Scholar]

[CR3] 3.Abbasi-Kangevari M, Saeedi Moghaddam S, Ghamari SH, Azangou-Khyavy M, Malekpour MR, Rezaei N, et al. The burden of prostate cancer in North Africa and middle East, 1990–2019: findings from the global burden of disease study. Front Oncol. 2022;12:961086. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR4] 4.Shim KS, Bergelson JM, Furuse M, Ovod V, Krude T, Lubec G. Reduction of chromatin assembly factor 1 p60 and C21orf2 protein, encoded on chromosome 21, in down syndrome brain. J Neural Transm Suppl. 2003;(67):117–28. [DOI] [PubMed]

[CR5] 5.Gregorczyk M, Pastore G, Muñoz I, Carroll T, Streubel J, Munro M, et al. Functional characterization of C21ORF2 association with the NEK1 kinase mutated in human in diseases. Life Sci Alliance. 2023;6(7):e202201740. [DOI] [PMC free article] [PubMed]

[CR6] 6.Watanabe Y, Nakagawa T, Akiyama T, Nakagawa M, Suzuki N, Warita H, et al. An amyotrophic lateral Sclerosis-Associated mutant of C21ORF2 is stabilized by NEK1-Mediated hyperphosphorylation and the inability to bind FBXO3. iScience. 2020;23(9):101491. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR7] 7.Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. Cancer J Clin. 2024;74(1):12–49. [DOI] [PubMed] [Google Scholar]

[CR8] 8.Xia C, Dong X, Li H, Cao M, Sun D, He S, et al. Cancer statistics in China and united States, 2022: profiles, trends, and determinants. Chin Med J. 2022;135(5):584–90. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR9] 9.Wang H, Yang L, Liu M, Luo J. Protein post-translational modifications in the regulation of cancer hallmarks. Cancer Gene Ther. 2023;30(4):529–47. [DOI] [PubMed] [Google Scholar]

[CR10] 10.Khan AO, Eisenberger T, Nagel-Wolfrum K, Wolfrum U, Bolz HJ. C21orf2 is mutated in recessive early-onset retinal dystrophy with macular Staphyloma and encodes a protein that localises to the photoreceptor primary cilium. Br J Ophthalmol. 2015;99(12):1725–31. [DOI] [PubMed] [Google Scholar]

[CR11] 11.Iyer S, Acharya KR, Subramanian V. Prediction of structural consequences for disease causing variants in C21orf2 protein using computational approaches. J Biomol Struct Dyn. 2019;37(2):465–80. [DOI] [PubMed] [Google Scholar]

[CR12] 12.Alshanqiti KH, Alomar SF, Alzoman N, Almomen A. Irisin induces apoptosis in metastatic prostate cancer cells and inhibits tumor growth in vivo. Cancers. 2023;15(15):4000–4000. [DOI] [PMC free article] [PubMed]

[CR13] 13.Kar S, Niharika, Roy A, Patra SK. Overexpression of SOX2 gene by histone modifications: SOX2 enhances human prostate and breast cancer progression by prevention of apoptosis and enhancing cell proliferation. Oncology. 2023;101(9):591–608. [DOI] [PubMed] [Google Scholar]

[CR14] 14.Karelia DN, Kim S, Plano D, Sharma AK, Jiang C, Lu J. Seleno-aspirin compound AS-10 promotes histone acetylation ahead of suppressing androgen receptor transcription, G1 arrest, and apoptosis of prostate cancer cells. Prostate. 2023;83(1):16–29. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR15] 15.Haustrate A, Shapovalov G, Spriet C, Cordier C, Kondratskyi A, Noyer L, et al. Lehen’kyi V: TRPV6 Calcium Channel Targeting by Antibodies Raised against Extracellular Epitopes Induces Prostate Cancer Cell Apoptosis. Cancers. 2023;15(6):1825–1825. [DOI] [PMC free article] [PubMed]

[CR16] 16.Park H, Lee S, Lee J, Moon H, Ro SW. Exploring the JAK/STAT signaling pathway in hepatocellular carcinoma: unraveling signaling complexity and therapeutic implications. Int J Mol Sci. 2023;24(18):13764–13764. [DOI] [PMC free article] [PubMed]

[CR17] 17.Gravina AG, Pellegrino R, Esposito A, Cipullo M, Romeo M, Palladino G, et al. The JAK-STAT pathway as a therapeutic strategy in cancer patients with immune checkpoint Inhibitor-Induced colitis: A narrative review. Cancers. 2024;16(3):611–611. [DOI] [PMC free article] [PubMed]

[CR18] 18.Kazan O, Kir G, Culpan M, Cecikoglu GE, Atis G, Yildirim A. The association between PI3K, JAK/STAT pathways with the PDL-1 expression in prostate cancer. Andrologia. 2022;54(10):e14541. [DOI] [PubMed] [Google Scholar]

[CR19] 19.Qiao S, Zhang W, Jiang Y, Su Y. Sennoside A induces autophagic death of prostate cancer via inactivation of PI3K/AKT/mTOR axis. J Mol Histol. 2023;54(6):645–54. [DOI] [PubMed] [Google Scholar]

[CR20] 20.Alafnan A, Hussain T, Rizvi SMD, Moin A, Alamri A. Prostate apoptotic induction and NFκB suppression by dammarolic acid: mechanistic insight into Onco-Therapeutic action of an aglycone Asiaticoside. Curr Issues Mol Biol. 2021;43(2):932–40. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR21] 21.Qin X, Zhong J, Wang L, Chen Z, Liu X. LncRNA LNC-565686 Promotes Proliferation of Prostate Cancer by Inhibiting Apoptosis through Stabilizing SND1. Biomedicines. 2023;11(10):2627–2627. [DOI] [PMC free article] [PubMed]

[CR22] 22.Ke F, Wang Z, Song X, Ma Q, Hu Y, Jiang L, et al. Cryptotanshinone induces cell cycle arrest and apoptosis through the JAK2/STAT3 and PI3K/Akt/NFκB pathways in cholangiocarcinoma cells. Drug Des Devel Ther. 2017;11:1753–66. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR23] 23.Qin R, Wang T, He W, Wei W, Liu S, Gao M, et al. Jak2/STAT6/c-Myc pathway is vital to the pathogenicity of Philadelphia-positive acute lymphoblastic leukemia caused by P190(BCR-ABL). Cell Communication Signaling: CCS. 2023;21(1):27. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

C21orf2 as a potential regulator of JAK2/STAT3 signaling in prostate cancer cell proliferation and apoptosis: an exploratory study

ShiHao Jiang

Shuai Su

Delin Wang

Abstract

Background

Materials and methods

Results

Conclusions

Supplementary Information

Background

Materials and methods

Bioinformatics analysis

Clinical tissue samples and cell lines

Lentivirus infection and plasmids

Quantitative real-time PCR (qRT-PCR)

Western blot (WB)

Immunohistochemical(IHC) staining

Animal experiment

CCK-8 experiment

Wound healing

Transwell assay

TUNEL experiment

Transmission electron microscopy (TEM)

Flow cytometry

Immunofluorescence(IF) experiment

Co-immunoprecipitation assay

Detection of ubiquitination

Statistic analysis

Results

Upregulation of C21orf2 in PCa tissues

Fig. 1.

Regulation of PCa cell biology by C21orf2 in vitro

Fig. 2.

Effects of C21orf2 on PCa cell proliferation and apoptosis in vivo

Fig. 3.

C21orf2 regulates PCa cell proliferation and apoptosis via the JAK2/STAT3 signaling pathway

Fig. 4.

Interaction of C21orf2 with the E3 ubiquitin ligase KCTD5

Fig. 5.

Negative correlation between C21orf2 and KCTD5 in PCa cells

Fig. 6.

Discussion

Conclusions

Supplementary Information

Acknowledgements

Abbreviations

Authors’ contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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