Abstract
N 6-Methyladenosine (m6A) is a reversible RNA modification that regulates tumorigenesis. KIAA1429, a critical component of the m6A methyltransferase complex, has an unclear role in clear cell renal cell carcinoma (ccRCC). Here, we investigated the role of KIAA1429 in ccRCC tumorigenesis. The expressions of KIAA1429 and thymosin beta-10 (TMSB10) in ccRCC samples were evaluated using quantitative real-time PCR (qRT-PCR). The malignant features of ccRCC cells were assessed via CCK-8, colony formation, transwell migration, and invasion assays, as well as in vivo tumor xenograft models. The relationship between KIAA1429 and TMSB10 was verified via Pearson correlation analysis, methylated RNA immunoprecipitation, qRT-PCR, and Western blotting assays. Functional rescue experiments further confirmed their interaction. We found that KIAA1429 was highly expressed in ccRCC, and its silencing significantly suppressed cell proliferation, migration, invasion, and tumor growth in vivo, while overexpression had the opposite effect. Bioinformatics and mechanistic analyses identified TMSB10 as a downstream target of KIAA1429, whose expression was upregulated in an m6A-dependent manner. Furthermore, overexpressing TMSB10 partially reversed the inhibitory effects of KIAA1429 silencing on ccRCC cells. Moreover, TMSB10 overexpression partially reversed the inhibitory effects of KIAA1429 knockdown. Taken together, our findings demonstrate that KIAA1429 promotes ccRCC tumorigenesis by enhancing TMSB10 expression via m6A modification, suggesting it as a potential prognostic biomarker and therapeutic target. However, the lack of clinical validation limits the immediate translational impact of these findings.
Introduction
Renal cell carcinoma (RCC) is a common cancer that has shown a significant rise in occurrence, with rates increasing by 2–3% annually over the past few years (Siegel et al., 2024). The common histological types of RCC comprise clear cell RCC (ccRCC), constituting 75–80% of RCC cases and representing the most prevalent subtype, followed by papillary RCC, comprising up to 20% of cases (Gray and Harris, 2019; Gundert et al., 2021). The ccRCC arises from epithelial cells lining the proximal tubule and is marked by clear cytoplasm, with 2–3% of cases linked to hereditary von hippel-lindau tumor suppressor (VHL) gene mutations (Schiavoni et al., 2023). Interestingly, ccRCC is distinguished by increased invasiveness and a higher tendency for relapse compared to other RCC subtypes, often presenting with metastatic lesions at diagnosis or developing distant metastases (Bahadoram et al., 2022). The 5-year survival rate ranges from 50% to 69% for patients with ccRCC; however, when the tumor is advanced or has metastasized, the survival rate decreases to approximately 10% (Schiavoni et al., 2023). Despite recent advancements in the treatment of advanced ccRCC, metastatic disease remains difficult to manage and often leads to a poor prognosis (Kase et al., 2023). Hence, understanding the molecular mechanisms of ccRCC tumorigenesis is pivotal for devising strategies for its early diagnosis and effective treatment.
The N
6-methyladenosine (m6A) is an epigenetic modification where adenosine is methylated at the sixth nitrogen atom, and it serves a notable role in orchestrating RNA splicing, localization, translation, and degradation (Wang et al., 2022b). Research increasingly highlights that m6A modification is linked to tumor initiation, growth, differentiation, invasion, and metastasis (Chen et al., 2019). It is a dynamic and reversible mechanism that is primarily coordinated by a series of methyltransferases, demethylases, and methylation-recognizing proteins (Hong et al., 2022). The KIAA1429 (also known as vir-like m6A methyltransferase associated, VIRMA) serves as the largest unit within the m6A methyltransferase complex that is adept to facilitate the modification of m6A in RNA (Yue et al., 2018). Previously, in KIAA1429
In the present study, we examined the role and associated mechanisms of KIAA1429 in ccRCC. Furthermore, we examined the interactions between KIAA1429 and thymosin beta-10 (TMSB10), a gene identified through bioinformatics analysis as both an upregulated differentially expressed gene (DEG) and positively correlated with KIAA1429 in the Gene Expression Profiling Interactive Analysis (GEPIA)-ccRCC database. We anticipate that these findings will contribute to the development of new therapeutic targets and provide valuable insights into KIAA1429’s role in ccRCC, potentially advancing treatment options.
Materials and Methods
Tissue samples
The ethics committee of Hubei No.3 People’s Hospital of Jianghan University approved this study (IRB: KY2024015). We obtained ccRCC tumor tissues and adjoining non-tumor tissues from 30 patients who received surgical procedures in our hospital. The clinicopathological details of all these patients are tabulated in Supplementary Table S1. All the participants provided their informed consent in writing before the start of the investigation. The samples were rapidly frozen in dry ice and stored at −80°C for the following analyses.
Cell culture, transfection, and lentiviral infection
The two ccRCC cell lines, including 786-O (SNL-078, SUNNCELL, China) and Caki-1 (SNL-244, SUNNCELL), were cultured in RPMI-1640 (SUNNCELL) and McCoy’s 5a (SUNNCELL) media, respectively. The culturing of the normal human renal tubular epithelial cell line, HK-2 (SNL-165, SUNNCELL), was done in DMEM medium (SUNNCELL). All cells were cultured by adding 10% FBS (SUNNCELL) and incubated under the condition of 5% carbon dioxide and 37°C.
For the transfection of siRNAs and plasmids into ccRCC cell lines, Lipofectamine 3000 (Thermo Fisher Scientific, USA) was used as directed by the manufacturer. The siRNAs targeting KIAA1429 (si-KIAA1429, 5ʹ-GGAGUUGGUUACCUUGCUUTT-3ʹ) and the corresponding negative control (si-NC) were obtained from RiboBio, China. The KIAA1429 and TMSB10 overexpression vectors were also constructed by RiboBio using the pcDNA3.1 vector, while a blank vector (Vector) served as a negative control.
Quantitative real-time PCR
The RNA was isolated from ccRCC tissues and cell lines by utilizing the Trizol method by means of the Trizol reagent (Invitrogen). Next, first-strand cDNA was generated from the total RNA samples by utilizing HiScript II Q RT SuperMix for quantitative PCR (qPCR) (+gDNA Wiper) (Vazyme, Nanjing, China). Then, qPCR reaction was done using AceQ qPCR SYBR Green Master Mix (Vazyme). The data were quantitatively analyzed using the 2−ΔΔCt method, with GAPDH as an internal control. The primer sequences are shown in Table 1.
Quantitative Real-Time PCR Primers in This Study
Cell counting kit-8 assay
The transfected ccRCC cells (103 cells/well) were inoculated onto 96-well plates and incubated for the following periods: 0, 24, 48, and 72 h. Upon the completion of the specified durations, cell counting kit-8 (CCK-8) reagent (10 μL; Beyotime, China) was put into every well, and a further 2-h incubation at 37°C was done. Finally, the absorbance values (at 450 nm) were taken by employing a microplate reader.
Colony formation assay
The transfected ccRCC cells (103 cells in 2 mL culture media) were seeded in six-well plates. The cells were then allowed to grow for a period of 2 weeks. Thereafter, the colonies were stained using crystal violet, and their numbers were quantified.
Transwell migration/invasion assays
Transwell filter chambers with an 8 μm pore size (Corning, NY) were utilized for transwell migration and invasion assays, without or with Matrigel coating, respectively. To the bottom transwell chamber, culture media (600 μL) containing 20% FBS was added, while the upper transwell chamber was seeded with ccRCC cells in serum-free culture media (200 μL). After 24 h of incubation at 37°C, migrated/invaded cells were fixed in paraformaldehyde, stained in crystal violet, and examined microscopically.
Western blotting
The transfected ccRCC cells were treated with a RIPA lysis buffer (Boster, Wuhan, China) with a protease inhibitor cocktail (Roche; Sigma-Aldrich, USA). The protein amount was estimated via a BCA Protein Assay Kit (Beyotime), and equal amounts of protein were loaded onto an sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following separation, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and blocked with 5% nonfat milk. These membranes were then treated overnight at 4°C with the following primary antibodies: anti-TMSB10 (PA5-116041; Invitrogen) and anti-GAPDH (AG0122; Beyotime). Next day, these membranes were treated with the Horseradish Peroxidase-labeled secondary antibody raised in goat (A0208; Beyotime) for 1 h at room temperature, and the bands of protein were visualized with the help of BeyoECL Plus (Beyotime).
Methylated RNA immunoprecipitation assay
This assay was accomplished by employing the riboMeRIP m6A Transcriptome Profiling Kit (RiboBio). The RNA (100 μg) from ccRCC cells with empty vector (pcDNA3.1) or pcDNA3.1-KIAA1429 and si-NC or si-KIAA1429 transfections were fragmented into 100–150 bp fragments. These fragment RNAs were then treated overnight with protein A/G Sepharose beads conjugated with either anti-m6A antibody or IgG antibody (negative control). Following that, the beads were washed in RIP wash buffer, and the m6A enrichment in TMSB10 was determined through quantitative real-time PCR (qRT-PCR).
Tumor xenograft model
The ethics committee of Hubei No.3 People’s Hospital of Jianghan University granted approval to the animal experiments (IRB: 202412). Twelve male BALB/c nude mice (∼6 weeks old) were obtained from Vital River, China. These were randomly assigned to four experimental groups, each comprising three mice. The Caki-1 cells were transfected with the following lentiviral vectors: pLenti-Vector (Lv-empty) and pLenti-Lv-KIAA1429 (overexpression vector), as well as pLenti-Vector (sh-NC) and pLenti-sh-KIAA1429 (silencing vector) to modify their gene expression. After being selected with puromycin (2 μg/mL) for 2 weeks, the transfected Caki-1 cells were administered subcutaneously into the mice (right flank) of the various experimental groups. The tumor volume (½ length × width2) was recorded every week with the help of a caliper, and the mice were euthanized to collect tumors after 5 weeks. The experiments were performed three times. A post hoc power analysis was performed for using n = 3/group. For Lv-empty versus Lv-KIAA1429, the effect size (Cohen’s d) was approximately 8.66, yielding a power close to 100% (α = 0.05, two-sided). For sh-NC versus sh-KIAA1429, the effect size was approximately 6.63, with a power of approximately 99.99%. These results indicate that the sample size of three per group was sufficient to detect the observed differences.
Statistical analysis
The data analysis was carried out by GraphPad Prism 8.0 (GraphPad, USA). All data were shown as mean ± standard deviation. The t-test and analysis of variance followed by Tukey’s post hoc test were performed accordingly to compare the differences among two and multiple groups, respectively. A p value <0.05 signifies a statistically significant difference.
Results
KIAA1429 was found to be elevated in ccRCC and associated with poor survival outcomes
To examine KIAA1429 expression in ccRCC, we checked the mRNA expression profile of ccRCC tumor tissues and non-tumor tissues using data from the TNMplot database (https://tnmplot.com/analysis/). It was found that the level of KIAA1429 in ccRCC tumor tissues was considerably increased as compared with the normal tissues (Fig. 1A). Additionally, analysis using the database of GEPIA disclosed that elevated KIAA1429 expression was related to poor survival in ccRCC (Fig. 1B). The KIAA1429 mRNA expression pattern was evaluated in 30 cases of ccRCC and paired adjoining normal tissues using qRT-PCR. Consistent with the TNMplot database analysis, the results elucidated that KIAA1429 mRNA levels were elevated in ccRCC tissues (Fig. 1C). Furthermore, the mRNA levels of KIAA1429 were significantly elevated in the two ccRCC cell lines (786-O and Caki-1) in comparison to the control cell line (Fig. 1D). Collectively, the aforementioned results illustrated that KIAA1429 serves a crucial function in the tumorigenesis of ccRCC.
KIAA1429 was highly expressed in ccRCC.
Upregulation of KIAA1429 promoted the malignant properties of ccRCC cells, whereas its silencing suppressed these effects
To assess the effects of KIAA1429 on ccRCC tumorigenesis, we specifically designed overexpression constructs (Vector and KIAA1429) and siRNAs (si-NC and si-KIAA1429), which were then transfected into two ccRCC cell lines. The outcomes of the qRT-PCR assay clearly showed the effective upregulation and silencing of the relative expression of KIAA1429 in the transfected ccRCC cells (Fig. 2A). The CCK8 assay disclosed that upregulation of KIAA1429 increased cell viability in ccRCC cells, while silencing KIAA1429 reduced their viability (Fig. 2B). The colony formation assay demonstrated that upregulation of KIAA1429 promoted ccRCC cell colony formation, whereas silencing KIAA1429 inhibited their ability to form colonies (Fig. 2C). Furthermore, the transwell migration and invasion assays displayed that upregulation of KIAA1429 enhanced the migrative and invasive capabilities of ccRCC cells, while silencing KIAA1429 notably reduced these (Fig. 2D and E). Altogether, these findings imply that KIAA1429 plays an integral part in promoting the malignant behaviors of ccRCC cells, including increased viability, colony formation, migration, and invasion. All these effects can be reversed by silencing KIAA1429.
Overexpression of KIAA1429 enhanced the malignant properties of ccRCC cells, while its silencing attenuated these properties. The following transfections were performed: KIAA1429 overexpression vector (KIAA1429), empty vector control (Vector), KIAA1429-targeting siRNA (si-KIAA1429), and negative control siRNA (si-NC).
KIAA1429 upregulation promoted tumor growth of ccRCC in vivo, while its depletion restricted growth
To explore the functional role of KIAA1429 in ccRCC cell growth in vivo, tumor xenograft experiments were conducted. The harvested tumors after 5 weeks clearly demonstrated an increase in tumor size following KIAA1429 overexpression (Lv-KIAA142) and a reduction in size following KIAA1429 silencing (sh-KIAA142) (Fig. 3A). The effects on tumor growth and restriction became evident by the fourth week, as shown by an increase in tumor volume following KIAA1429 overexpression and a decrease in tumor volume following KIAA1429 silencing (Fig. 3B). These effects were clearly reflected in the harvested tumor weights, where tumors with KIAA1429 overexpression showed increased weight, while those with KIAA1429 silencing exhibited reduced weight (Fig. 3C). These observations collectively elucidate that KIAA1429 is crucial in driving ccRCC progression in vivo.
KIAA1429 overexpression enhanced the tumor growth of ccRCC in vivo, while its silencing restrained it. Lv and sh represent the lentivirus constructed overexpression or silence vectors.
KIAA1429 regulated TMSB10 as a target gene via m6A modification and correlated positively with TMSB10 expression
We next checked the downstream effectors responsible for the oncogenic roles of KIAA1429 in ccRCC. The Venny 2.1 tool (https://bioinfogp.cnb.csic.es/tools/venny/) was adopted to identify overlapping genes between the upregulated DEGs from the GEPIA-ccRCC database and KIAA1429-positive correlated genes from the GEPIA-ccRCC database. The analysis revealed TMSB10 as the only common gene (Fig. 4A). Then, the TMSB10 expression was checked in the ccRCC tissues, and the results from the qRT-PCR assay showed that TMSB10 was notably increased in the ccRCC tumor samples when compared with the control samples (Fig. 4B). In addition, the expressions of KIAA1429 and TMSB10 demonstrated a positive correlation with each other (Fig. 4C). The methylated RNA immunoprecipitation assay provided further validation of their interactions, revealing a significant enrichment of TMSB10 upon binding with anti-m6A antibodies. Moreover, upregulation of KIAA142 in ccRCC cells resulted in a marked enhancement in the enrichment of TMSB10, while the silencing of KIAA142 significantly diminished TMSB10 enrichment in these cells (Fig. 4D). In addition, we aimed to further verify the relationship between KIAA1429 and TMSB10. It was found that the overexpression of KIAA1429 caused a substantial increase in both TMSB10 mRNA and protein levels, while silencing KIAA1429 led to a marked decline in TMSB10 expression at both levels (Fig. 4E and F). Collectively, these outcomes strongly demonstrate that KIAA1429 positively modulates TMSB10 expression, potentially through m6A-mediated modification, and may have an important role in ccRCC progression.
KIAA1429 targeted TMSB10 by regulating its m6A modification and was positively associated with it.
TMSB10 addition counteracted the tumor-suppressive effects of KIAA1429 silencing in ccRCC cells in vitro
We further assessed whether KIAA1429 exerted its function in ccRCC via targeting TMSB10, and for that, in vitro rescue experiments were carried out. The TMSB10 overexpression vector (TMSB10) or control (Vector) was transfected into KIAA1429-silenced cells for rescue experiments. The CCK-8 assays displayed that cell viability that was hindered by KIAA1429 silencing was restored upon overexpression TMSB10 (Fig. 5A). The outcomes of the colony formation assay demonstrated that TMSB10 overexpression significantly counteracted the suppressive effect of KIAA1429 silencing on the colony formation capability of ccRCC cells (Fig. 5B). Similarly, the transwell migration and invasion assays elucidated that overexpression of TMSB10 partly subdued the inhibitory effect of KIAA1429 silencing on the migrative and invasive abilities of ccRCC cells, respectively (Fig. 5C and D). These findings illustrate that KIAA1429 accelerated the progression of ccRCC by interacting and upregulating TMSB10.
Addition of TMSB10 ameliorated the tumor-suppressive effects of KIAA1429 silencing in ccRCC cells. The ccRCC cells were transfected with either the TMSB10 overexpression vector (TMSB10) or a control vector (Vector) in KIAA1429-silenced (si-KIAA1429) cells for rescue experiments.
Discussion
ccRCC is known for its heightened aggressiveness, invasiveness, and metastatic potential, which contribute to its chemoresistance and radioresistance (Schiavoni et al., 2023). These characteristics are linked to the poor prognosis and elevated death rate associated with the disease (Schiavoni et al., 2023). Therefore, identifying novel biomarkers with a high level of precision and sensitivity is crucial for improving prognosis assessment and developing targeted therapies for patients with ccRCC, ultimately enhancing their outcomes. Considering the ever-growing significance of m6A modification in cancer biology, we investigated the role of KIAA1429, a component of the RNA methyltransferase complex, in ccRCC. In this work, we observed that KIAA1429 was elevated in ccRCC tissues and cell lines. Its overexpression further enhanced the malignant properties of ccRCC cells in vitro, while its silencing alleviated the malignant phenotypes. In vivo, similar results were observed, with KIAA1429 overexpression leading to increased xenograft tumor volumes and weights, whereas silencing KIAA1429 resulted in decreased tumor volumes and weights. Furthermore, we demonstrated that KIAA1429 advanced the tumorigenesis of ccRCC by upregulating TMSB10 expression, through the mediation of its m6A modification, which may enhance its stability at the mRNA level and increase protein expression.
A rising volume of studies has highlighted the pathological role of m6A dysregulation in human diseases, mainly in cancer (Yang et al., 2020). Interestingly, the m6A modification can act as either a tumor-promoter or a tumor-suppressor in cancer, depending on the cellular context (Gao et al., 2021). KIAA1429 was found to be overexpressed in various cancers and performs an oncogenic role. For instance, KIAA1429 has been documented to be upregulated in gastric cancer, lung adenocarcinoma, breast cancer, and liver cancer, where it promoted tumor progression by regulating m6A modification and stabilizing target mRNAs, correlating with poor prognosis (Guo et al., 2024; Li et al., 2023; Meng et al., 2023; Ren et al., 2024). Interestingly, bioinformatics analysis has elucidated that KIAA1429 was expressed at higher levels in kidney renal clear cell carcinoma (Chen et al., 2020; Li et al., 2019). We found upregulation of KIAA1429 in ccRCC and elucidated that KIAA1429 overexpression enhanced the malignant features of ccRCC cells and promoted tumor growth in vivo, while its silencing suppressed these effects. These outcomes implied that KIAA1429 acts as an oncogene in ccRCC and is a potential prognostic biomarker of poorer clinical outcomes.
The RNA methyltransferase KIAA1429 may regulate mRNA by m6A modification of target genes (Wang et al., 2022b). Our bioinformatics analysis identified TMSB10 as a target of KIAA1429 in ccRCC, with KIAA1429 modulating TMSB10 stability through m6A methylation. The TMSB10 gene is located on chromosome 2p11, encoding a beta-thymosin family protein that functions as a crucial G-actin-sequestering protein involved in cytoskeletal organization (Erickson-Viitanen et al., 1983). Recent studies indicate that TMSB10 is overexpressed and implicated in the progression of various human cancers. For example, serum TMSB10 levels were elevated in patients with hepatocellular carcinoma, correlated with disease stages and Alpha-Fetoprotein levels (Zahran et al., 2024). It was also overexpressed in gastric cancer, promoting tumor growth, invasion, and angiogenesis, and was linked with poor prognosis, rendering it a potential biomarker and therapeutic target (Yan et al., 2021). TMSB10 was reported to be increased in bladder cancer and related to poor prognosis, including shorter overall survival and increased invasion (Wang et al., 2019). In ccRCC also, TMSB10 was upregulated and was linked with poor clinical outcomes, thereby indicating that it could operate as a diagnostic, prognostic biomarker, and potential therapeutic target (Pan et al., 2020). Another study highlighted that TMSB10 was upregulated in ccRCC, with Jun proto-oncogene transcriptionally regulating its expression, which in turn enhanced cell proliferation and inhibited apoptosis (Wang et al., 2022a). Nonetheless, no study has yet investigated the m6A modification of TMSB10 or its potential association with KIAA1429 in any cancer type. Consistent with prior reports in ccRCC, our study also identified elevated expression of TMSB10 in the ccRCC tissue samples. Furthermore, our work is the first to disclose that KIAA1429 promotes ccRCC progression by enhancing the expression of TMSB10 by mediating its m6A modification. Altogether, we uncovered a positive correlation between KIAA1429 and TMSB10 in ccRCC, and overexpression of TMSB10 alleviated the suppressive impacts of KIAA1429 silencing on malignant phenotypes of ccRCC cells. Furthermore, KIAA1429 could possibly enhance the stability of TMSB10 by mediating its m6A modification.
There are certain limitations in this work that warrant additional examination in future research. First, the relatively smaller sample size may limit the broader applicability of the results. Expanding the sample size and including diverse cohorts will further strengthen the validity and generalizability of the findings. Additionally, while the investigation primarily focused on the interactions between KIAA1429 and TMSB10 in ccRCC, other potential pathways by which KIAA1429 could influence disease progression remain unexplored. Future research should investigate these alternative mechanisms to acquire a more thorough understanding of the disease.
Conclusion
The findings of the present work illustrated the critical role of KIAA1429 in ccRCC tumorigenesis, by promoting the malignant properties of cancer cells. KIAA1429 facilitated malignant ccRCC progression by regulating TMSB10 through m6A methylation, which enhanced its stability and expression. These outcomes demonstrated that KIAA1429 might be considered a potential target for molecular therapy of ccRCC.
Footnotes
Data Availability
All data generated or analyzed during this study are included in this article.
Ethical Approval
The Ethics Committee of Hubei No.3 People’s Hospital of Jianghan University authorized this research. Clinical tissue specimen processing was accomplished in strict observance of the ethical standards of the Declaration of Helsinki. Written consents were acquired from the patients. The Ethics Committee of Hubei No.3 People’s Hospital of Jianghan University approved this animal study, which was executed according to the ARRIVE guidelines.
Authors’ Contributions
S.J. designed this study, performed the experiments, and draft this manuscript. F.L. conducted data analysis, reviewed and edited this manuscript. All authors have read and approved this final version.
Disclosure Statement
All authors declare that there is no competing interest.
Funding Information
No funding was received for this study.
Supplementary Material
Supplementary Table S1
References
Supplementary Material
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