LINC01857 promotes clear cell renal cell carcinoma progression by scaffolding DNMT1 to suppress WIF1 expression

Abstract

The long intergenic non-coding RNA LINC01857 has been implicated in various human malignancies; however, its specific role in clear cell renal cell carcinoma (ccRCC) remains unclear. In this study, we report that LINC01857 is markedly upregulated in ccRCC tissues and cell lines. Functionally, LINC01857 knockdown suppressed the proliferation, migration, and invasion of ccRCC cells, whereas its overexpression promoted these malignant phenotypes. Mechanistically, LINC01857 recruits the DNA methyltransferase DNMT1 to the WIF1 promoter, leading to promoter hypermethylation and transcriptional repression of WIF1. This epigenetic repression of WIF1, in turn, activates the Wnt/β-catenin pathway, which contributes to the aggressive behaviors of ccRCC cells.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-38828-6.

Introduction

Clear cell renal cell carcinoma (ccRCC) is the predominant and most aggressive form of renal cell carcinoma (RCC)^1,2. Its incidence is rising globally, partly due to an aging population and the increased incidental detection through imaging³. Alarmingly, 20–30% of patients are diagnosed with metastatic disease, which severely compromises survival^4,5. While surgery is curative for localized disease, over 30% of patients experience recurrence, often due to occult metastasis, highlighting the need for better systemic therapies^6,7. Despite advances, the molecular pathogenesis of ccRCC remains incompletely understood. A deeper exploration of its molecular drivers is therefore essential to uncover novel therapeutic targets.

Long non-coding RNAs (lncRNAs) are classified as RNA molecules longer than 200 nucleotides. Long intergenic non-coding RNAs (lincRNAs), a subset of lncRNAs, are transcribed from intergenic regions. Although the majority of lncRNAs lack protein-coding capacity, they have emerged as influential regulators involved in modulating cellular structure, function, and various aspects of physiological development. Notably, substantial research underscores the critical regulatory functions of lncRNAs in the occurrence and advancement of various human malignancies, including ccRCC^8–12. For instance, LINC00973 was found to bolster Siglec-15 expression by sequestering miR-7109, thereby contributing to immune evasion in ccRCC¹³. Similarly, lncRNA MILIP has been shown to interact with YBX1, enhancing Snail translation and the metastatic potential of ccRCC¹⁴. Furthermore, LINC01426 exacerbates the progression of ccRCC via the IGF2BP1/CTBP1/HDAC2/miR-423-5p/FOXM1 pathway¹⁵. Accordingly, identifying additional ccRCC-associated lncRNAs and elucidating their molecular mechanisms represents an important avenue for the development of novel therapeutic strategies against ccRCC.

LINC01857, located in the genomic region of human chromosome 2, is a lincRNA that has gained considerable attention in recent years for its role in various biological processes and disease pathogenesis. LINC01857 has been found to exert its oncogenic impact in diffuse large B-cell lymphoma by influencing the miR-141-3p/MAP4K4 axis¹⁶. One study revealed that LINC01857 stimulates the proliferation and migration of tumor cells by affecting CREB1 expression in breast cancer¹⁷. Additionally, LINC01857 has been shown to facilitate tumor growth, migration, and invasiveness through the miR-1281/TRIM65 pathway in glioblastoma¹⁸. Similarly, LINC01857 has been shown to drive the malignancy of endometrial carcinoma cells via the miR-19b-3p/ELAVL1/MYCN pathway¹⁹. In pancreatic cancer, LINC01857 enhances the proliferation and migration of pancreatic cancer cells by interacting with miR-450b-5p to upregulate CDC42EP3 expression²⁰. Furthermore, LINC01857, which is overexpressed in hepatocellular carcinoma, exacerbates tumor cell proliferation and inhibits apoptosis via the miR-197-3p/AGR2 axis, thereby activating the AKT and ERK signaling pathways²¹. However, the biological functions and underlying mechanisms of LINC01857 in ccRCC remain to be fully elucidated.

Given the documented roles of lncRNAs in cancer and preliminary evidence suggesting a regulatory function for LINC01857 in gene expression, we hypothesized that LINC01857 could be involved in ccRCC progression. This study was conducted to delineate the function of LINC01857 in ccRCC, with a specific focus on its interaction with DNMT1 and the subsequent effects on WIF1 expression and Wnt/β-catenin signaling. Elucidating this molecular axis may provide insights into potential therapeutic targets and contribute to the broader understanding of lncRNA functions in cancer biology.

Materials and methods

Clinical specimen

Twenty-six paired ccRCC tumors and adjacent normal kidney tissues were sourced from patients undergoing radical nephrectomy at Wuhan No. 1 Hospital between May 2020 and August 2023. All participants provided informed consent, and none had received preoperative radiotherapy, chemotherapy, immunotherapy, or targeted therapy. In accordance with the Declaration of Helsinki, ethical approval for this study was granted by the Ethics Board of Tongji Medical College, Huazhong University of Science and Technology. After tumor removal, the specimens were promptly stored in liquid nitrogen for cryopreservation before being transferred to a refrigerator set at -80 degrees Celsius for long-term storage.

Cell cultivation

The ACHN, 786-O, and A498 cell lines were acquired from the American Type Culture Collection (ATCC) and maintained in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (FBS). The SN12-PM6 cell line and the HK-2 immortalized normal renal tubular epithelial cell line were generously provided by Professor Xiaoping Zhang from Wuhan Union Hospital and were maintained in DMEM medium (Gibco) with 10% FBS. Cells were maintained in a humid incubator at a constant temperature of 37℃, with an atmosphere containing 5% carbon dioxide.

Quantitative real-time PCR (qRT-PCR)

To isolate total RNA from ccRCC tissues and cultured cells, the TRIzol reagent (Invitrogen) extraction method was employed. Subsequently, the PrimerScript One Step reverse transcription kit (Takara) was applied for reverse transcription. qRT-PCR was performed using the SYBR Premix Dimmer Eraser kit (Takara). Primer sequences for each gene are provided in Supplementary Tables 1 and were provided by Sangon Biotech (Shanghai). The 2^−ΔΔCt method was applied to assess the relative levels of gene expression, with GAPDH used for normalization in triplicate.

Subcellular fractionation assay, Fluorescence in Situ Hybridization (FISH) and Immunofluorescence (IF)

Subcellular fractionation was performed using the PARIS Kit (Invitrogen) as per manufacturer’s recommendations. FISH analysis was undertaken to ascertain the subcellular localization of LINC01857. Fluorescently labeled probes specific to the antisense strand of LINC01857 were synthesized and provided by GenePharma Company (Shanghai), with sense probes as a negative control. Immunofluorescence (IF) analysis was conducted to investigate the cellular localization of DNMT1 in renal cancer cells. An anti-DNMT1 antibody (Abcam, ab320817, 1:2000) was purchased from Abcam (USA), and CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) was prepared by Proteintech (Wuhan, China). Each experiment included three independent biological replicates (n = 3).

RNA interference and plasmid construction

Small interfering RNAs (siRNAs) targeting LINC01857, EZH2, WIF1, and DNMT1, along with a scrambled negative control siRNA, were provided by GenePharma Biological Company (Shanghai), and their sequences are detailed in Supplementary Table 1. Transient transfection was performed with Lipofectamine 2000 (Invitrogen). The LINC01857 overexpression vector (LINC01857-OE), the WIF1 expression vector (WIF1-OE), and an empty control vector were also supplied by GenePharma. Additionally, small hairpin RNAs (shRNAs) targeting LINC01857 or WIF1, along with a negative control shRNA (sh-Con), were supplied by the same company. qRT-PCR was used to verify the efficiency of knockdown or overexpression. All transfection experiments were performed in three independent biological replicates (n = 3).

Cell counting kit-8 (CCK-8)

Transfected cells were seeded in a 96-well plate at 1.0 × 10³ cells per well. Cell proliferation was monitored at 0, 24, 48, 72, and 96 h with a CCK-8 kit (Sigma) following the manufacturer’s recommendations. Absorbance measurements were taken at 450 nm with a spectrophotometer. Experiments were conducted in triplicate (n = 3).

Migration and invasion

The ccRCC cells were seeded onto chamber slides of transwells (BD Biosciences, USA) without any coating (for migration) or with a Matrigel coating (BD Biosciences, USA) (for invasion). The upper chamber contained serum-free medium, while the lower chamber was filled with medium containing 10% FBS. Following a 24-hour culture period, cells on the lower membrane were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and subsequently visualized by crystal violet staining. An inverted microscope (Olympus) was used to capture images. Three separate biological replicates were performed for each experiment (n = 3).

Western blotting analysis

The RIPA lysis buffer (Thermo Scientific) was used for total protein extraction from tissues and cells. After being subjected to 10% SDS-PAGE, proteins were transferred to PVDF membranes. Following transfer, the membranes were incubated overnight at 4 °C with the specified primary antibodies: GAPDH (CST, #5174, 1:1000), E-cadherin (CST, #3195, 1:1000), PCNA (CST, #13110, 1:1000), N-cadherin (CST, #13116, 1:1000), c-Myc (CST, #18583, 1:1000), β-catenin (CST, #9562, 1:1000), cyclin D1 (CST, #2978, 1:1000), DNMT1 (Abcam, ab320817, 1:1000), and WIF1 (Abcam, ab155101, 1:1000). Subsequently, secondary antibodies supplied by Wuhan Boster Biological Co. were applied, and band visualization was performed using an ECL substrate. All Western blot experiments were conducted with three independent biological replicates (n = 3).

Methylation-specific PCR (MSP)

Genomic DNA was extracted using a TIANGEN kit (China), followed by bisulfite conversion with a TIANGEN conversion kit. The CpG island within the WIF1 promoter region was predicted using MethPrimer (http://www.urogene.org/index.html). Methylation-specific and unmethylation-specific primers (sequences in Supplementary Table 1) were used for amplification. The MSP assay consisted of an initial heating phase at 95 °C for 5 min, followed by 35 amplification cycles (95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s), and a final extension at 72 °C for 10 min. Electrophoretic separation of the PCR amplicons was performed on a 2% agarose gel. The MSP assay was independently conducted three times (n = 3).

RNA Immunoprecipitation (RIP), Chromatin Immunoprecipitation (ChIP) and RNA pull-down

RIP assays were conducted with the EZ-Magna RIP kit (Millipore) following the manufacturer’s recommendations. After harvesting, RCC cells were lysed using RIP lysis buffer. The lysates were then immunoprecipitated with magnetic beads coated with specific antibodies or control IgG. The antibodies used included DNMT1 (Abcam, ab320817, 1:30), EZH2 (Abcam, ab307646, 1:30), SUZ12 (Abcam, ab175187, 1:30), DNMT3A (Abcam, ab307503, 1:30), and DNMT3B (Abcam, ab227883, 1:30). After immunoprecipitation, beads were subjected to proteinase K treatment, and the purified RNA was subsequently examined by qRT-PCR. ChIP assays were carried out with the EZChIP kit (Upstate Biotechnology, USA). DNA-protein cross-linking was generated by treating ccRCC cells with formaldehyde. Lysates were then subjected to sonication according to the guidelines provided by the manufacturer. Immunoprecipitation was performed using specific antibodies targeting DNMT1 (Abcam, ab320817, 10 µg) or control IgG. Precipitated chromatin DNA was checked by qRT-PCR using primers as detailed in Supplementary Table 1. RNA Pull-Down assays were performed using the Magnetic kit provided by Pierce (USA). Biotin-labeled LINC01857 probes were synthesized by RiboBio (Guangzhou). All tests included three independent biological replicates (n = 3).

In vivo animal experiment

A total of 4 × 10⁶ ACHN cells stably expressing sh-LINC01857 or sh-Con were harvested and resuspended in 100 µl of medium without serum. After subcutaneous injection of the cells, the 5-week-old nude mice (n = 5 per group) were housed in facilities that were free of specific pathogens. Tumor volume was measured as length × width²/2 every week. Following a four-week period, the mice were humanely euthanized through cervical dislocation under isoflurane-induced anesthesia, after which tumor dimensions and mass were measured. To evaluate lung metastasis, 3 × 10⁶ stably transfected ACHN cells (expressing either sh-Con, sh-LINC01857, or sh-LINC01857 + sh-WIF1) were intravenously injected into groups of 5-week-old nude mice (n = 5 per group). Six weeks later, the mice were euthanized, and their lungs were collected for further study. Approval for the animal experiments was granted by the Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. All tests were performed in compliance with relevant guidelines and regulations, including the ARRIVE guidelines.

Immunohistochemistry (IHC) and Haematoxylin-Eosin (HE) staining

Tumor and pulmonary tissues harvested from nude mice were treated with 4% paraformaldehyde, followed by paraffin embedding and sectioning. For IHC, tumor sections were stained using an anti-Ki-67 antibody, and detection was carried out using the avidin-biotin-peroxidase complex (ABC) method. Lung metastases were examined by HE staining following standard protocols. Images were captured using an optical microscope.

Statistical analysis

Graphpad Prism 9 was employed for data analysis, with results presented as mean ± standard deviation (SD). Differences between groups were assessed using Student’s t-test or one-way ANOVA. Quantification of fluorescence colocalization was performed using Pearson’s correlation coefficient. A P-value < 0.05 was considered the significance criterion.

Results

LINC01857 expression is increased in both ccRCC tissues and cell lines and localizes primarily to the nucleus

Analysis of The Cancer Genome Atlas (TCGA) data via the UALCAN platform (https://ualcan.path.uab.edu/) revealed a substantial upregulation of LINC01857 in ccRCC samples when compared with normal renal tissues (Fig. 1A). Consistent with this, we evaluated LINC01857 expression in 26 paired clinical ccRCC and adjacent normal kidney specimens, confirming its markedly higher level in tumor samples (Fig. 1B). Furthermore, LINC01857 expression was significantly elevated in the 786-O, SN12-PM6, ACHN, and A498 renal carcinoma cell lines compared to the normal renal tubular epithelial cell line HK-2. Among these cancer cell lines, ACHN cells showed the highest LINC01857 expression, while SN12-PM6 cells showed the lowest (Fig. 1C). Consequently, ACHN and SN12-PM6 cells were selected for subsequent investigations. Subcellular fractionation assays indicated that LINC01857 was distributed in both the cytoplasm and nucleus of ACHN and SN12-PM6 cells, but was primarily localized to the nucleus (Fig. 1D-E). This predominant nuclear localization was further validated by fluorescence in situ hybridization (FISH) experiments (Fig. 1F-H). Collectively, these data indicate that LINC01857 is highly expressed and predominantly localized in the nucleus of ccRCC cells, a pattern that is consistent with a potential oncogenic role in ccRCC pathogenesis.

Fig. 1.

LINC01857 is upregulated and predominantly nuclear-localized in ccRCC. (A) LINC01857 expression in ccRCC (n = 538) and normal kidney (n = 72) tissues from the TCGA dataset. (B) LINC01857 expression in 26 paired clinical ccRCC and adjacent non-tumor tissues. (C) qRT-PCR analysis of LINC01857 expression in ccRCC cell lines versus the normal renal tubular epithelial cell line HK-2 (n = 3). (D-E) Nuclear-cytoplasmic fractionation assay of LINC01857 in ACHN and SN12-PM6 cells (n = 3). GAPDH and U6 served as cytoplasmic and nuclear loading controls, respectively. (F) FISH images of LINC01857 (red) and DAPI (blue) in ACHN and SN12-PM6 cells. Scale bar, 5 μm. (G-H) Quantification of LINC01857 fluorescence intensity in ACHN and SN12-PM6 cells (n = 3). Data are reported as mean ± SD. Each set of experiments was performed in triplicate. **P < 0.01, ***P < 0.001.

LINC01857 facilitates ccRCC cell proliferation, migration, and invasion

To investigate the potential biological functions of LINC01857 in ccRCC, we first designed three siRNAs to silence its expression in ACHN cells. Based on their silencing efficiency (Fig. 2A), si-LINC01857#1 and #2 were selected for subsequent investigation. Conversely, an overexpression vector (LINC01857-OE) was transfected into SN12-PM6 cells, leading to a significant increase in LINC01857 expression (Fig. 2B). CCK-8 analysis indicated that LINC01857 knockdown significantly suppressed the proliferation of ACHN cells, whereas its overexpression markedly enhanced the proliferative activity of SN12-PM6 cells (Fig. 2C-D). Additionally, a Transwell assay was conducted to determine whether LINC01857 facilitated the migratory and invasive capacities of ccRCC cells. The data demonstrated that silencing LINC01857 substantially alleviated the migratory and invasive capabilities of ACHN cells (Fig. 2E-G), whereas overexpression of LINC01857 enhanced these abilities in SN12-PM6 cells (Fig. 2H-J). Furthermore, protein markers associated with cell proliferation, migration, and invasion were evaluated. The data demonstrated that downregulation of LINC01857 triggered a considerable rise in E-cadherin levels and a decrease in PCNA and N-cadherin levels. In contrast, upregulation of LINC01857 led to a notable reduction in E-cadherin and an elevation in PCNA and N-cadherin levels (Fig. 2K-M). These findings suggest that LINC01857 facilitates the proliferative, migratory, and invasive capacities of ccRCC cells in vitro.

Fig. 2.

Impact of LINC01857 on biological behaviors of ccRCC cells. (A) qRT-PCR analysis of LINC01857 knockdown efficiency in ACHN cells transfected with three siRNAs (n = 3). (B) qRT-PCR analysis of LINC01857 overexpression (LINC01857-OE) efficiency in SN12-PM6 cells (n = 3). (C-D) CCK-8 assays assessing proliferation of ACHN cells with LINC01857 knockdown and SN12-PM6 cells with LINC01857 overexpression (n = 3). (E-G) Transwell migration and invasion assays of ACHN cells after LINC01857 knockdown (n = 3). Scale bar, 100 μm. (H-J) Transwell migration and invasion assays of SN12-PM6 cells after LINC01857 overexpression (n = 3). Scale bar, 100 μm. (K-M) Western blot analysis (K) and quantification (L, M) of PCNA, N-cadherin, and E-cadherin expression in ccRCC cells (n = 3). Values represent the mean ± SD of triplicate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Knockdown of LINC01857 attenuates tumor growth and metastasis in vivo

To elucidate the functional role of LINC01857 in vivo, we constructed three shRNAs targeting LINC01857. Among them, sh-LINC01857#1 exhibited the highest silencing efficiency (Fig. 3A) and was selected for subsequent experiments. ACHN cells stably transfected with sh-LINC01857#1 or a negative control (sh-Con) were inoculated subcutaneously into nude mice. LINC01857 knockdown significantly impeded tumor growth in vivo (Fig. 3B), resulting in a notable decline in both tumor size and weight compared to that of the control group (Fig. 3C-D). qRT-PCR analysis confirmed the successful downregulation of LINC01857 in tumor tissues from the knockdown group (Fig. 3E). Additionally, immunohistochemistry (IHC) revealed a notable reduction in Ki-67-positive cells in the sh-LINC01857#1 group (Fig. 3F-G, Fig. S1A). In a lung metastasis model, LINC01857 knockdown group showed reduced metastatic potential compared to the control group (Fig. 3H-J). Together, these in vivo experiments demonstrate that knockdown of LINC01857 significantly suppresses tumor growth and lung metastasis in ccRCC models.

Fig. 3.

LINC01857 knockdown attenuates tumor growth and lung metastasis in vivo. (A) Knockdown efficiency of LINC01857-targeting shRNAs assessed by qRT-PCR (n = 3). (B) Representative photographs of excised subcutaneous tumors from nude mice. (C) Tumor size measurements recorded weekly over a four-week period (n = 5). (D) Tumor weight comparison between groups (n = 5). (E) qRT-PCR analysis of LINC01857 expression levels in tumor tissues from both groups (n = 5). (F) Representative images of Ki-67 immunohistochemical staining in tumor sections. Scale bar represents 50 μm. (G) Quantitative analysis of Ki-67 (n = 5). (H) Representative images of lung metastatic models and HE staining. The red arrows represent the metastatic nodules. Scale bar indicates 200 μm. (I) Number of metastatic nodules in the lungs (n = 5). (J) Quantification of metastatic area in lung sections (n = 5). Data (mean ± SD) are representative of at least three separate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

LINC01857 suppresses WIF1 expression by binding to DNMT1

To delve deeper into the underlying mechanisms of LINC01857, we focused on its predominant nuclear localization, suggesting a role in transcriptional regulation through RNA-binding proteins. Given that many nuclear lncRNAs regulate transcription via histone or DNA methylation^22,23, we hypothesized that LINC01857 might function through similar epigenetic mechanisms, potentially involving PRC2 components (EZH2, SUZ12) or DNA methyltransferases (DNMT1, DNMT3A, DNMT3B). RIP experiments identified a specific interaction between LINC01857 and DNMT1 (Fig. 4A), but not with other tested proteins (Fig. S1B-C). This direct binding was confirmed by RNA pull-down assays in both ACHN and SN12-PM6 cells (Fig. 4B-D). Furthermore, FISH combined with immunofluorescence demonstrated their nuclear colocalization (Fig. 4E-G). These findings are consistent with a model in which LINC01857 functions through its interaction with DNMT1. DNMT1 is a key mediator of DNA methylation, an epigenetic mechanism frequently involved in silencing tumor suppressor genes. We therefore examined a panel of methylation-regulated tumor suppressor genes (LAD1, VHL, PTEN, SFRP1, CDH1, FHIT, WIF1, and UCHL1) in ccRCC. qRT-PCR analysis indicated that LINC01857 knockdown increased WIF1 expression in ACHN cells, whereas LINC01857 overexpression reduced WIF1 expression in SN12-PM6 cells (Fig. 4H-I). Nonetheless, no consistent changes were observed for other candidate genes (Fig. S1D-E). Supporting its tumor-suppressive role, WIF1 mRNA expression was markedly reduced in ccRCC tissues relative to normal kidney tissues (Fig. 4J). Importantly, DNMT1 knockdown also upregulated WIF1 expression in both cell lines (Fig. 4K). Collectively, these findings suggest that nuclear LINC01857 represses the expression of the tumor suppressor gene WIF1 via its interaction with DNMT1 in ccRCC cells.

Fig. 4.

LINC01857 recruits DNMT1 to epigenetically silence WIF1. (A) Enrichment of LINC01857 in RIP assays with an anti-DNMT1 antibody (n = 3). (B) Representative western blots of RNA pull-down assays showing direct binding of LINC01857 to DNMT1 in ACHN and SN12-PM6 cells. (C-D) Quantification of DNMT1 protein levels from RNA pull-down assays in (B) (n = 3). (E) FISH assay of LINC01857 (red) combined with immunofluorescence staining of DNMT1 (green) showing their nuclear colocalization. Scale bar, 5 μm. (F-G) Assessment of the quantitative colocalization between DNMT1 and LINC01857 (n = 3). (H-I) qRT-PCR analysis of WIF1 mRNA levels after LINC01857 knockdown in ACHN cells or overexpression in SN12-PM6 cells (n = 3). (J) qRT-PCR analysis of WIF1 mRNA expression in ccRCC versus normal kidney tissues (n = 26). (K) qRT-PCR analysis of WIF1 mRNA levels in tumor cells treated with DNMT1 siRNA (n = 3). Data are mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

LINC01857 silences WIF1 transcription by recruiting DNMT1 to methylate its promoter

DNA methylation of gene promoters is a key epigenetic mechanism for transcriptional regulation. Given the interaction between LINC01857 and DNMT1, we hypothesized that LINC01857 recruits DNMT1 to methylate the WIF1 promoter, leading to its transcriptional silencing. As depicted in Fig. 5A, the CpG island localization within the WIF1 gene promoter region was predicted using http://www.urogene.org/index.html, supporting its susceptibility to methylation-mediated regulation. Subsequently, MSP assay was conducted to analyze the influence of LINC01857 on WIF1 promoter methylation. The data indicated that knockdown of LINC01857 in ACHN cells remarkably reduced WIF1 promoter methylation, while overexpression of LINC01857 in SN12-PM6 cells exhibited the opposite effect (Fig. 5B-D). Additionally, treatment with the DNMT inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) dose-dependently elevated WIF1 mRNA levels in both cell lines (Fig. 5E-F), indicating that WIF1 expression is indeed repressed by DNA methylation. To investigate whether LINC01857 is required for DNMT1 enrichment at the WIF1 promoter, we performed ChIP assays. The results showed that LINC01857 knockdown diminished DNMT1 binding to the WIF1 promoter, while its overexpression enhanced this interaction (Fig. 5G-H). Collectively, these data demonstrate that LINC01857 recruits DNMT1 to the WIF1 promoter, leading to CpG island methylation and consequent transcriptional silencing of WIF1 in ccRCC cells.

Fig. 5.

LINC01857 facilitates WIF1 gene promoter methylation by recruiting DNMT1. (A) Schematic of CpG islands in the WIF1 promoter region predicted by MethPrimer (http://www.urogene.org/index.html). (B) MSP analysis of the WIF1 promoter in ACHN (LINC01857 knockdown) and SN12-PM6 (LINC01857 overexpression) cells. (C-D) Quantification of WIF1 promoter methylation levels by MSP (n = 3). (E-F) qRT-PCR analysis of WIF1 mRNA levels in ACHN and SN12-PM6 cells treated with the DNMT inhibitor 5-aza-dC (n = 3). (G-H) ChIP assays showing DNMT1 enrichment at the WIF1 promoter region in ACHN and SN12-PM6 cells (n = 3). Data (mean ± SD) are from triplicate experiments. ** P < 0.01, *** P < 0.001.

LINC01857 promotes ccRCC malignancy via the WIF1/Wnt/β-catenin axis

Given that WIF1 is a known inhibitor of the Wnt/β-catenin pathway²⁴, we investigated whether LINC01857 modulates this oncogenic signaling axis through WIF1. Western blot analysis showed that LINC01857 knockdown in ACHN cells upregulated WIF1 and downregulated key downstream effectors of the pathway (c-myc, β-catenin, cyclin D1). Conversely, LINC01857 overexpression in SN12-PM6 cells reduced WIF1 and increased the levels of these oncogenic proteins. Critically, these effects were reversed by co-transfection with si-WIF1 or WIF1 overexpression plasmid, respectively (Fig. 6A-C). Functional rescue experiments further demonstrated that WIF1 knockdown partially rescued the anti-proliferative, anti-migratory, and anti-invasive effects caused by LINC01857 silencing. Reciprocally, WIF1 overexpression counteracted the tumor-promoting phenotypes induced by LINC01857 (Fig. 6D-K). Consistent with these findings, co-knockdown of WIF1 and LINC01857 partially restored the metastatic potential suppressed by LINC01857 silencing alone in a lung metastasis model (Fig. 6L-N). Based on these observations, we introduce a working model wherein nuclear LINC01857 recruits DNMT1 to the WIF1 promoter, inducing its hypermethylation and transcriptional silencing (Fig. 7). This suppression of WIF1 triggers the Wnt/β-catenin pathway, ultimately driving ccRCC tumor progression. These data establish that the oncogenic effects of LINC01857 are mediated, at least in part, by the epigenetic silencing of WIF1 and the subsequent activation of the Wnt/β-catenin pathway in ccRCC cells.

Fig. 6.

LINC01857 activates Wnt/β-catenin signaling and influences tumor progression through the modulation of WIF1. (A-C) Western blot analysis (A) and quantification (B, C) showing expression of WIF1, β-catenin, cyclin D1, and c-myc in ACHN and SN12-PM6 cells following modulation of LINC01857 and/or WIF1. (n = 3). (D-E) CCK-8 test assessing cell proliferation under the indicated conditions (n = 3). (F-K) Transwell migration and invasion assays of the indicated cells (n = 3). Scale bar, 100 μm. (L) Representative images of the lung metastasis model and corresponding HE staining. Red arrows indicate metastatic nodules. Scale bar, 200 μm. (M) Metastatic nodule count in the lungs (n = 5). (N) Quantitative assessment of metastatic area in lung sections (n = 5). Data are mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig. 7.

Schematic model illustrating the oncogenic mechanism of LINC01857 in ccRCC. LINC01857 recruits DNMT1 to the WIF1 promoter, thereby inducing promoter hypermethylation and transcriptional silencing of WIF1. This silencing leads to activation of the Wnt/β-catenin pathway, which drives the malignant progression of ccRCC, including enhanced proliferation, migration, invasion, and metastasis.

Discussion

Long non-coding RNAs (lncRNAs), which constitute a major fraction of the mammalian transcriptome, have garnered significant interest for their roles in regulating gene expression. In this study, we demonstrated that LINC01857 is abundantly expressed in ccRCC tissues and cell lines, and its expression is associated with the promotion of malignant phenotypes in renal cancer cells both in vitro and in vivo. These findings are consistent with previous reports documenting the oncogenic role of LINC01857 in other cancers^16–18. To elucidate its mechanism of action, we first determined the subcellular localization of LINC01857, as this is a primary determinant of lncRNA function. Nuclear lncRNAs typically regulate transcription, while cytoplasmic lncRNAs often function as competitive endogenous RNAs (ceRNA). Our data showed that LINC01857 is predominantly nuclear-localized, suggesting a role in transcriptional or epigenetic regulation. Notably, mounting evidence indicates that nuclear lncRNAs can directly modulate transcription by interacting with DNA, histones, or transcription factors at specific sites, thereby influencing oncogenes or tumor suppressor activity^25–27. A well-established mechanism involves the recruitment of epigenetic regulators, such as the Polycomb Repressive Complex 2 (PRC2), to alter the chromatin landscape^22,28,29. Specifically, the PRC2 component EZH2 functions as a histone methyltransferase that catalyzes H3K27 trimethylation (H3K27me3), leading to transcriptional repression. However, RIP assays revealed no interaction between LINC01857 and EZH2, prompting us to investigate alternative mechanisms.

DNA methylation is a critical epigenetic regulatory mechanism with a well-established role in tumorigenesis and progression. In this context, certain lncRNAs function by influencing the methylation patterns of target genes. For example, KCNQ1OT1 has been shown to promote metastasis by enhancing the promoter methylation of EIF2B5 in ovarian cancer²³. Similarly, LINC01270 promotes breast cancer progression by modulating the methylation of the LAMA2 promoter and interacting with the MAPK pathway³⁰. DNA methylation is primarily catalyzed by DNA methyltransferases (DNMTs), particularly DNMT1, DNMT3A, and DNMT3B, which establish and maintain methylation patterns at different regulatory stages^31,32. Given its role in maintaining DNA methylation patterns, DNMT1 is a common regulatory target of lncRNAs³³. For example, the knockdown of lncRNA GRIK1-AS1 was shown to increase DNMT1 enrichment at the SRFP1 promoter, thereby promoting the proliferation of endometrial stromal cells through Wnt signaling activation³⁴. In colorectal cancer, LINC01594 acts as a scaffold for DNMT1, enhancing CELF6 methylation³⁵. Furthermore, HOTAIR recruits DNMT1 to methylate the Bcl-2 promoter, which regulates apoptosis in pulmonary vascular endothelial cells in COPD³⁶. In this study, we demonstrated that LINC01857 directly binds to DNMT1 and recruits it to the WIF1 promoter. Consistent with this, methylation-specific PCR (MSP) experiments confirmed that LINC01857 enhances WIF1 promoter methylation. These findings support prior research indicating that lncRNAs can regulate gene transcription through DNA methylation^23,30.

The secreted protein Wnt inhibitory factor 1 (WIF1) is evolutionarily conserved and functions as a key Wnt antagonist³⁷. Its expression is frequently downregulated in multiple human tumors, such as bladder, gastric, lung, and breast carcinomas^38–41, as well as in RCC⁴². Consistently, our qRT-PCR analysis confirmed a marked reduction of WIF1 mRNA in ccRCC tissues in comparison to normal kidney tissues. To investigate the cause of this downregulation, we focused on promoter methylation, a well-established mechanism for transcriptional silencing when CpG islands are modified^43–45. Studies have verified that hypermethylation of the WIF1 promoter contributes to its dysregulation in human carcinomas, including RCC⁴². In line with this, bioinformatic analysis predicted a CpG island in the WIF1 promoter, suggesting its susceptibility to methylation-mediated regulation. Our MSP assays confirmed that LINC01857 promotes DNA methylation at this locus in ccRCC cells. This epigenetic silencing was functionally validated by treating cells with the DNMT inhibitor 5-aza-dC, which markedly restored WIF1 mRNA levels. These findings align with previous reports demonstrating the downregulation of WIF1 expression in ccRCC cells due to DNA methylation^42,46. Prior studies have underscored the vital role of Wnt signaling activation in the oncogenesis and progression of RCC^47–49. Notably, WIF1, a key Wnt antagonist, is frequently dysregulated in cancers, and its functional loss is tightly associated with aberrant Wnt pathway activation^38,39,50. Given the regulatory interplay between WIF1 and Wnt signaling, we conducted further experiments to investigate whether LINC01857 influences this pathway via WIF1 in renal cancer cells. The findings revealed that LINC01857 modulates the expression of Wnt pathway-associated proteins (c-myc, cyclin D1, and β-catenin), while WIF1 restoration efficiently reversed these effects. Furthermore, we demonstrated that restoring WIF1 expression substantially suppressed the LINC01857-induced proliferation, migration, and invasion of ccRCC cells, whereas WIF1 silencing had the opposite effect. These data delineate a mechanistic link in ccRCC cells, whereby the aberrant expression of LINC01857 leads to epigenetic silencing of WIF1, resulting in indirect activation of the Wnt/β-catenin pathway.

Our findings on LINC01857 exemplify the expanding repertoire of lncRNA-mediated mechanisms in cancer biology. Indeed, the role of ncRNAs in tumor progression, drug resistance, and metastasis is increasingly recognized. For instance, PIWI-interacting RNAs (piRNAs) form complexes with PIWI proteins to mediate transcriptional and post-transcriptional gene silencing, thereby impacting tumorigenesis, cancer progression, chemoresistance, and cancer stemness⁵¹. Zhang et al. demonstrated that LINC02609, regulated by HIF2α, modulates APOL1 expression by sequestering miR-149-5p in ccRCC, influencing lipid storage, endoplasmic reticulum homeostasis, and tumor progression⁵². These observations confirm the multifaceted roles of ncRNAs in cancer and their potential as therapeutic targets. Our study extends this concept by delineating a specific oncogenic axis in which LINC01857 epigenetically silences WIF1 via DNMT1 recruitment, leading to Wnt/β-catenin pathway activation. These findings highlight LINC01857 as a potential therapeutic target and suggest that its expression levels could serve as a biomarker for ccRCC. Further investigation into this axis may inform the development of novel treatment strategies for this disease.

Taken together, this study elucidates the oncogenic potential of LINC01857 in ccRCC. Specifically, LINC01857 has been shown to adversely affect WIF1 expression by interacting with DNMT1 and facilitating its recruitment to the WIF1 promoter region. This interaction induces hypermethylation of the WIF1 promoter, leading to decreased WIF1 expression and promoting tumor cell progression via the Wnt/β-catenin pathway. The findings highlight LINC01857 as a prospective therapeutic target for ccRCC. Notably, a limitation of this study is that the methylation analysis relied on MSP, which provides a semi-quantitative assessment. Future studies using quantitative bisulfite sequencing could further refine the methylation assessment with greater precision. Furthermore, although our data strongly support a model in which LINC01857 recruits DNMT1 to silence WIF1, the involvement of additional co-factors in this specific axis warrants future investigation. In addition to exploring the upstream transcription factors that regulate LINC01857 itself, examining its interplay with other non-coding RNAs could unravel more comprehensive regulatory networks in ccRCC. Therefore, future research should prioritize a more exhaustive analysis of the molecular components involved in the signaling axis, alongside relevant clinical investigations.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Jingdong Yuan conceived and designed the research project. Fuxin Zheng was responsible for tissue sample collection and data acquisition. Wei Xiang and Lei Lyu conducted both in vitro and in vivo experiments. Chuanhua Zhang performed data curation and formal analysis. Wei Xiang, Lei Lyu, and Jingdong Yuan contributed to the revision of the manuscript.

Funding

This study was funded by the National Natural Science Foundation of China (No. 82002708) and the Wuhan Municipal Health Commission Medical Scientific Research Project (WX23Z34).

Data availability

The datasets used and/or analyzed during the current study available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.