miR-9-5p/HMMR regulates the tumorigenesis and progression of clear cell renal cell carcinoma through EMT and JAK1/STAT1 signaling pathway

doi:10.21203/rs.3.rs-4856915/v1

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Research Article

miR-9-5p/HMMR regulates the tumorigenesis and progression of clear cell renal cell carcinoma through EMT and JAK1/STAT1 signaling pathway

https://doi.org/10.21203/rs.3.rs-4856915/v1

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Background

The most common type of kidney cancer that easily metastasizes is clear cell renal cell carcinoma (ccRCC). The expression levels of hyaluronan-mediated motility receptor (HMMR) in many tumor types are significantly enhanced. HMMR is closely associated with tumor-related progression, treatment resistance, and discouraging prognosis, has yet to be fully investigated in terms of its expression patterns and molecular mechanisms of action in ccRCC. Further research is imperative to elucidate these aspects.

Methods

We used The Cancer Genome Atlas (TCGA) database to preliminarily investigate HMMR expression and function in ccRCC. We assessed the differential expression level of HMMR between ccRCC cancerous tissues and their matched non-tumor tissues. Subsequently, a series of in vivo and in vitro experiments were designed to elucidate the biological function of HMMR in ccRCC, including Transwell migration assays, CCK-8 assays, clone formation assays and subcutaneous xenograft experiments in nude mice. Through bioinformatics analysis, we identified potential microRNAs (miRNAs) that may regulate HMMR, as well as the possible signaling pathways involved. Finally, we conducted a series of cellular functional experiments to validate our hypotheses regarding the HMMR axis.

Results

HMMR expression was observably up-regulated in tumor tissues of ccRCC patients, and elevated HMMR expression level showed a strong correlation with ccRCC progression and adverse prognoses of patients. Knocking down HMMR inhibited the proliferative and migratory abilities of ccRCC cells, while its overexpression amplified these oncogenic properties. In nude mouse model, reduced HMMR expression inhibited ccRCC tumor proliferation in vivo. Furthermore, overexpression of an upstream transcriptional regulator, miR-9-5p, effectively downregulated HMMR expression and thus impeded ccRCC cells proliferation and migration. In addition, HMMR might influence ccRCC growth via the Epithelial-Mesenchymal Transition (EMT) pathway and the Janus Kinase1/Signal Transducer and Activator of Transcription1 (JAK1/STAT1) pathway.

Conclusions

HMMR is overexpressed in ccRCC, and there is a significant link between high HMMR expression and tumor progression, as well as poor patient prognosis. Specifically, HMMR could be targeted and inhibited by miR-9-5p and might modulate the tumorigenesis and progression of ccRCC through both EMT and JAK1/STAT1 signaling pathway.

ccRCC

HMMR

miR-9-5p

EMT

JAK1/STAT1

Renal cell carcinoma (RCC) makes up roughly 90 percent of all kidney cancers and constitutes around 3 percent of all adult malignancies, and the global incidence is steadily increasing, with an estimated annual tally of around 403,000 newly diagnosed cases [1, 2]. ccRCC, the most prevalent histological variant, makes up about 70% of RCC diagnoses [3]. Due to the inconspicuous characteristic of early clinical symptoms in patients with ccRCC, a significant portion of patients are firstly diagnosed when the cancer has already progressed to advanced or metastatic stages [4]. Currently, the precise mechanisms underlying the initiation, metastasis, and acquisition of drug resistance in ccRCC remain elusive. Thus, elucidating the specific molecular mechanisms driving the progression of ccRCC is imperative for facilitating early diagnosis and implementing standardized treatments.

HMMR, also known as CD168, was initially identified in the conditioned media of fibroblasts and later cloned from murine 3T3 mesenchymal cells [5, 6]. The HMMR gene resides on human chromosome 5q33.2, consisting of two start codons and 18 exons [7]. Characterized by complex biological functions and diverse subcellular localization, HMMR is a versatile protein [8–10]. The membrane-bound HMMR has been shown to activate signaling pathways associated with cell motility, with its function identified in cells harboring Ras mutations [11]. HMMR is crucial for numerous cellular processes, including cell mitosis and proliferation [12, 13], and its absence might disrupt cell cycle [14]. Previous research has implicated HMMR in the invasion of several tumors. For instance, HMMR has the regulatory ability to affect the expression of cell-cycle regulators in prostate cancer, thereby promoting the EMT process [15]. Moreover, HMMR induces resistance to 5-fluorouracil chemotherapy in gastric cancer [16], highlighting its multifaceted impact on cancer therapeutic response. What^, s more, HMMR overexpression has been documented across various tumor types, making it one of the prognostic markers in gastric, colorectal, multiple myeloma, and cancers of pancreas [17–20]. In a prior investigation, it was observed that HMMR exhibited significant upregulation in RCC, qualifying as an independent prognostic factor [21]; however, the precise mechanistic details of its role in ccRCC have remained elusive. Given its involvement in cancer biology, clarifying the exact functions and regulatory mechanisms of HMMR in ccRCC could offer invaluable insights into cancer progression and potential therapeutic targets.

miRNAs can attach to the 3' untranslated region (3’-UTR) of target mRNAs to regulate gene expression, which is crucial for cell proliferation, differentiation, apoptosis, and cancer progression [22]. There is evidence that miRNAs participate in the genesis and progression of multiple types of tumors, including ccRCC [23]. Among these, miR-9-5p exerts its influence on tumor proliferation and metastasis by regulating diverse cellular functions and signaling pathways. One study revealed that miR-9-5p facilitated the growth and migration of non-small cell lung cancer cells by downregulating TGFBR2 [24]; it could also inhibit PDK4 to modulate the physiological function of mitochondria in hepatocellular carcinoma (HCC) cells, illustrating its regulatory role in energy metabolism [25]. In our study, we found a special differentially expressed miRNA, miR-9-5p, in ccRCC, and HMMR expression may be regulated by miR-9-5p.

Clinical tissue samples

The expression levels of HMMR in primary kidney tumors and matched adjacent tissues from ccRCC patients were determined through Western blot assay. Pathological specimens from ten ccRCC patients at the First Affiliated Hospital, Zhejiang University School of Medicine (FAHZU) were collected. Each specimen consisted of both tumor tissue and adjacent non-tumor tissue. All sample collections were conducted with the patients' informed consent and obtained approval from our hospital's ethics board, adhering to ethical guidelines and ensuring patient confidentiality and rights.

Cell lines and culture

The study utilized the human renal proximal tubule epithelial cell line (HK-2), the human renal cell carcinoma cell lines (769-P, ACHN, 786-O, and Caki-1), and the stable packaging cell line (HEK-293T). All aforementioned cell lines were procured from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and were cultured according to the ENCODE cell culture standards.

Nude mice and animal experiments:

Ten male BALB/c nude mice, aged four weeks, were obtained and maintained at the Experimental Animal Center of Hangzhou Medical College. The subcutaneous implanted tumor models were established by injecting 786-O cells (2*10⁶ cells per mouse) stably expressing shHMMR or a negative control (shNC) under the armpit of nude mice. All animal procedures were conducted in compliance with institutional guidelines approved by FAHZU.

RNA and Quantitative real-time PCR (RT-qPCR)

The methods and steps of RNA isolation and RT-qPCR have been detailed extensively in prior studies [25, 26]. All the primers utilized in this study are listed in Supplementary table 1. The PCR primers were synthesized and ordered from Beijing Tsingke Biotech Co., Ltd. (Hangzhou, China).

Dual-luciferase reporter assay

The HMMR mutant (Mut) and wild-type (WT) sequences, which had been pre-designed, were inserted into the pmirGLO double luciferase reporter vector (Promega, USA). The aforementioned luciferase vectors were separately transfected into 293T cells. Following 48 hours of cell culture, the relative luciferase activity in each cell group was assessed using a dual luciferase reporter assay kit (Promega, Madison, WI, USA), as per the provided protocol.

Western blot assay

The methods and steps of Western Blotting were described detailedly in an earlier study [26]. The antibodies used in this study and their detailed information are displayed at the Supplementary table 2.

Transwell migration assay and other assays in vitro experiments

Transwell migration assay, CCK-8 and colony formation assay were described detailedly as previously reported [27].

Bioinformatics Analysis:

(1) Data Acquisition: We downloaded the raw mRNA expression data for ccRCC from the TCGA database, encompassing a total of 614 samples—normal (n = 72) and tumor (n = 542). Additionally, for single-cell analysis, data for 19 ccRCC patients were extracted from the NCBI GEO public database (GSE207493).

(2) Gene Set Enrichment Analysis (GSEA): We downloaded annotated gene sets version 7.0 from the MsigDB database for pathway enrichment analysis between subtypes. The patients were artificially categorized into a high expression group and a low expression group based on their levels of HMMR expression, and GSEA was performed to identify differentially enriched pathways.

(3) Drug Sensitivity Analysis: The Genomics of Drug Sensitivity in Cancer (GDSC) database was used, along with the "pRRophetic" package, to predict drug sensitivity for each tumor sample. IC₅₀ values for specific drug treatments were estimated using regression methods and validated through 10-fold cross-validation.

(4) Nomogram Model Construction: A multivariate regression model was built to calculate prediction scores based on the contribution of each influencing factor (size of regression coefficients). Scores were assigned to each level of each factor, summed to obtain a total score, and used to compute predicted outcomes.

(5) Single-Cell Analysis: We used the Seurat package to process data, and t-SNE algorithm was applied to visualize the spatial relationships between clusters. We annotated the clusters by using the celldex package to associate them with cell types relevant to tumor biology.

Statistical Analysis

The statistical analyses of the acquired data were performed using GraphPad Prism 8.0 software and the R programming language (version 4.3.0) for both graphical presentation and the assessment of statistical significance. To evaluate the differences between two groups and to analyze correlations, Student's t-test and Pearson correlation analysis were applied, respectively, with a threshold of p < 0.05 indicating statistical significance.

Enhanced expression of HMMR in ccRCC is linked to the cancer progression and adverse clinical outcomes.

According to the TCGA database, we downloaded and integrated raw mRNA expression datasets of ccRCC, comprising a total of 614 samples, with 72 normal samples and 542 tumor samples. The findings indicated that there are notable differences in the expression levels of HMMR between normal and cancerous tissues, with a marked elevation observed in the latter (Fig. 1A1-2). The survival analysis, encompassing overall survival, disease-specific survival, and progression-free interval, revealed that the low expression group had a significantly longer survival duration compared to the high expression group (Fig. 1B1-3). HMMR expression also had satisfactory specificity and sensitivity in the ccRCC diagnosis wth AUC = 0.867 (Fig. 1B4). Combined with clinical data and HMMR expression levels, univariate and multivariate Cox regression models were established. Forest maps showed that HMMR was a risk factor for ccRCC patients in both univariate and multivariate analyses (Fig. 1C). Moreover, we found significant correlations between HMMR expression and patient characteristics such as gender, pathological grade, clinical stage, T stage, and M stage with HMMR expression increasing in tandem with advanced stages (Fig. 1D). This indicates a strong association between high HMMR expression and poor clinical outcomes. Additionally, Western blot analysis of 10 ccRCC samples from our center confirmed that cancerous tissues had significantly higher HMMR protein levels than non-cancerous tissues (Fig. 1E).

In summary, HMMR expression is up-regulated in the cancerous tissues of ccRCC patients, and this abnormal expression shows a positive correlation with the progression of ccRCC and predicts unfavorable clinical outcomes.

HMMR plays a significant role in the progression of ccRCC.

Through regression analysis of HMMR expression across all our samples, we discovered that different clinical indicators in ccRCC contribute to varying extents to the overall scoring process (Fig. 2A). Additionally, predictive analyses were conducted for 1-year and 3-year OS models (Fig. 2B), revealing a close correlation between the predicted OS and the observed OS, thereby validating the nomogram model's robust predictive performance. Expanding our investigation, we leveraged drug sensitivity data from the GDSC database to forecast chemotherapy sensitivity. This exploration further illuminated a significant relationship between HMMR and the sensitivity to several common anticancer drugs, including Nutlin-3a, AG-014699, AICAR, AZD-0530, AZD-2281, and A-443654 (Fig. 2C), suggesting HMMR may act as a potential factor influencing drug resistance.

Considering the tumor microenvironment (TME) has an important impact on aspects such as cancer diagnosis, patient survival, and responsiveness to clinical treatment, our study delved into the relationship between HMMR expression levels and the extent of immune cell infiltration within TCGA dataset. Our findings revealed a remarkable positive association between HMMR and certain immune cell populations integral to antitumor immunity, including cytotoxic CD8 + T cells and T follicular helper cells (Fig. 2D). Of note, a particularly striking finding was the positive correlation between heightened HMMR expression and an increased infiltration of T follicular helper cells, with a correlation coefficient (r) of 0.186 (Fig. 2E). This suggests that elevated HMMR levels coincide with an enrichment of helper T cells, which are vital for effective antibody responses and fostering an immunologically active TME. Such correlations provide some basis for further understanding the complex interaction between HMMR expression and the composition of the immune infiltrate, potentially informing future strategies for immunomodulatory therapies in cancer treatment.

To explore the mechanism of HMMR on tumor development, we also performed single-cell analysis of HMMR. Downloading data from GSE207493, we included 19 samples and utilized the t-SNE algorithm to cluster the cells, resulting in 21 distinct subtypes (Fig. 2F). Applying the R package SingleR for cell type annotation, we annotated these 21 clusters into nine cell categories, such as T cells, tissue stem cells, neurons, among others (Fig. 2G). The expression pattern of HMMR across these nine cell types was further depicted in Fig. 2H and 2I, shedding light on its cellular heterogeneity and potential roles in different cell populations.

HMMR regulates tumor growth in vivo and biological activities in vitro of ccRCC.

By conducting RT-qPCR and Western blot analyses on several RCC cell lines and HK-2, we found that both the transcriptional and protein levels of HMMR were notably higher in 786-O and 769-P cells than in normal cells (Fig. 3A). To reduce HMMR expression, we transfected these cells with small interfering RNAs targeting HMMR (siHMMR-1 and siHMMR-2) and confirmed a substantial decrease in HMMR expression at both mRNA and protein levels in 786-O and 769-P cells (Fig. 3B). Following HMMR knockdown, the migratory capability (Fig. 3C1), cellular viability (Fig. 3C2), and the efficiency of colony formation (Fig. 3C3) for these cells were notably reduced in vitro. To investigate the oncogenic potential of HMMR further, we established xenograft tumor models by subcutaneously injecting 786-O cells that stably expressed shHMMR or a negative control (shNC) into the axillary region of nude mice. The experimental results showed that HMMR knockdown significantly diminished the weight of 786-O xenograft tumors (Fig. 3D, E).

Moreover, we used an overexpression plasmid to overexpress HMMR in cells, confirming the efficiency of overexpression (Fig. 3F). Cells with HMMR overexpression exhibited significantly enhanced migratory ability (Fig. 3G1), proliferation (Fig. 3G2), and colony formation (Fig. 3G3) in vitro.

Collectively, these findings suggest that HMMR positively regulates the migration and proliferation of ccRCC, highlighting its crucial role in ccRCC progression.

miR-9-5p is postulated to regulate HMMR expression and influence ccRCC cells proliferation and migration.

Available literature underscores the key regulatory functions of miRNAs in the onset and advancement of diverse malignancies including RCC [23]. Consequently, we hypothesized that the oncogenic function of HMMR in ccRCC might be modulated by upstream miRNAs. To test this, we retrieved the miRNA expression profiles for ccRCC from TCGA database and performed differential expression analysis using three R packages: DEseq2, limma, and edgeR, to identify miRNAs that are under-expressed in tumor tissues. Integrating these findings with the predictive interactions from the ENCORI online tool, we constructed a Venn diagram, which led us to select 2 candidate miRNAs, miR-509-3p and miR-9-5p (Fig. 4A). Notably, miR-9-5p was expressed at a lower level than HK-2 in the RCC cell lines we used, with a particularly pronounced downregulation observed in 786-O and 769-P cells (Fig. 4B).

Subsequently, we introduced miR-9-5p mimics or negative controls (NCs) into 786-O and 769-P cells. Through RT-qPCR and Western Blotting, we verified that the transfection of miR-9-5p mimics markedly decreased HMMR expression compared to the NC group (Fig. C).

For further study of the carcinogenic potential of miR-9-5p in ccRCC cells, we carried out additional cellular assays after the transfection with negative controls or miR-9-5p mimics. Transwell migration assays demonstrated that there was a notable decrease in the migration capability of both 786-O and 769-P cells (Fig. 4D1). CCK-8 assays showed that cell proliferation was significantly inhibited in cells miR-9-5p mimics group compared to control group (Fig. 4D2). Colony formation assays also showed that the cloning efficiency of cells decreased significantly after overexpression of miR-9-5p (Fig. 4D3). In conclusion, the above functional assays imply that miR-9-5p may exert an antitumor effect in ccRCC by suppressing ccRCC cells proliferation and migration.

miR-9-5p exhibits targeted interaction with HMMR to mediate biological activity of ccRCC.

We had uncovered the oncogenic potential of HMMR and the significant tumor-suppressive effect exerted by miR-9-5p. To explore the intrinsic mechanism, a series of rescue experiments involving HMMR and miR-9-5p were conducted. HMMR overexpression could partially restore the decreased HMMR protein expression induced by miR-9-5p mimics, as confirmed by Western blot analysis (Fig. 5A). Functional assays following this setup, including colony formation assays (Fig. 5B) and CCK-8 assays (Fig. 5C), suggested that elevated HMMR expression could partially reverse the reduction in proliferation caused by miR-9-5p. Similarly, Transwell migration assays (Fig. 5D) indicated that HMMR overexpression could also rescue the migratory capacity decrease induced by miR-9-5p.

To conclusively validate whether HMMR is a direct target of miR-9-5p, the WT and Mut 3’-UTRs of HMMR mRNA were separately cloned into the pmirGLO vector (depicted in Fig. 5E). The outcome revealed a significant decrease in the relative luciferase activity of the WT group after miR-9-5p was overexpressed in 293T cells (Fig. 5F), confirming the direct interaction and regulation of HMMR by miR-9-5p.

HMMR facilitates the progression of ccRCC via the activation of both EMT and JAK/STAT signaling pathway.

EMT is a critical process involved in the acquisition of invasive properties by cancer cells, affecting their motility and migratory abilities [28]. By knocking down HMMR expression, at the protein level, we observed a decrease in N-cadherin, Slug, and Vimentin alongside an increase in E-cadherin, suggesting a potential involvement of HMMR in the EMT process of ccRCC cells. Matrix metalloproteinases (MMPs), closely tied to the EMT process and responsible for degrading extracellular matrix components to facilitate cancer cell invasion and migration, were also found to be downregulated, specifically MMP9 (Fig. 6A). These results indicate that HMMR might promote the progression of ccRCC by enhancing EMT process.

Furthermore, we employed GSEA to further elucidate the specific signaling pathways implicated by HMMR, thereby delving into the underlying molecular mechanisms by which HMMR influences tumor initiation and progression. Our findings indicated a notable enrichment of the JAK/STAT signaling pathway in association with HMMR (Fig. 6B). Recent study has shown that JAK1/STAT1 is involved in the progression and immunotherapy evasion of ccRCC, thus the pathway was tested in this research [29]. By transfecting 786-O and 769-P cells with either HMMR-overexpressing plasmids or empty vectors, and subsequently assessing the expression levels of JAK1, STAT1, and phosphorylated STAT1 (p-STAT1) proteins, we found that the overexpression of HMMR led to increased expression of both JAK1 and p-STAT1 proteins in these cells (Fig. 6C). This indicates that HMMR upregulates key components of the JAK/STAT signaling pathway. To further validate whether HMMR promotes the progression of ccRCC via the JAK/STAT signaling pathway, we cultured these cells in media containing Ruxolitinib, a selective inhibitor of JAK. The outcomes demonstrated that Ruxolitinib effectively suppressed the migratory (Fig. 6D) and proliferative capacities (Fig. 6E) of cells that had been induced by HMMR overexpression.

In summary, these findings suggested that HMMR facilitated the progression of ccRCC through the activation of both EMT and JAK1/STAT1 signaling pathway.

Recent researches have increasingly highlighted the significant role of HMMR in the field of oncology. It has been reported that HMMR is overexpressed in numerous cancer types, including breast cancer, bladder cancer, prostate cancer, and head and neck squamous cell carcinoma, among others and this overexpression is intimately linked with disease progression, therapeutic resistance, and adverse prognoses [15, 30–32]. Bioinformatics has always played a key role in the analysis of key genes and signaling pathways. In the preliminary phase of our study, leveraging the vast repository of TCGA database, we performed bioinformatics analysis on the expression pattern of HMMR in ccRCC. Our analysis demonstrated that ccRCC cancerous tissues had significantly higher HMMR protein levels than non-cancerous tissues, aligning with previous research findings and suggesting that HMMR could be used as a promising molecular biomarker for cancer diagnosis. Moreover, we observed a close association between HMMR expression levels and important clinical features such as histological grading and clinical staging. By incorporating survival analysis alongside Cox proportional-hazards model, we uncovered a significant relationship between high HMMR expression and poor patient outcomes, identifying it as an independent risk factor for ccRCC patients. These findings carry substantial clinical implications, emphasizing the importance of HMMR in prognosis prediction and potentially guiding tailored therapeutic strategies.

The tumor immune microenvironment plays a vital part in regulating tumor growth and consists of immune cells, tumor cells, and other components [33]. A prior study has identified HMMR as a key gene associated with tumor-infiltrating lymphocytes in acute myeloid leukemia [34]. In lung adenocarcinoma, HMMR has been shown to positively correlate with infiltration levels of immune cells [35]. The degree of immune infiltration in ccRCC is relatively high. Nevertheless, the mechanism of its immune microenvironment is not fully understood. With this in mind, we proceeded to explore the connection between the expression level of HMMR and immune infiltration in the TCGA dataset. Our findings revealed a remarkable positive association between HMMR and certain immune cell populations, including cytotoxic CD8 + T cells and T follicular helper cells. These findings suggest that HMMR is intricately involved in shaping the immune landscape, potentially influencing the efficacy of immunotherapeutic strategies.

ccRCC is typically resistant to chemotherapy but may show greater sensitivity to novel therapies, including targeted therapy and immunotherapy [36]. In recent years, there have been numerous advancements in cancer pharmacotherapy. Nutlin3a, an inhibitor of MDM2 protein, prevents p53 proteasomal degradation and thereby promoting tumor cell apoptosis [37]. Rucaparib (AG014699), a PARP (PolyADP-ribose polymerase) inhibitor specifically targeting BRCA mutations, has been approved by the FDA for treating adult patients with metastatic, castration-resistant prostate cancer, who have BRCA mutations of germline and/or somatic origin [38]. Similarly, Olaparib (AZD-2281) is approved for recurrent ovarian cancer in BRCA mutation carriers [39]. AICAr, an AMP-activated protein kinase activator, inhibits cancer cell proliferation and induces apoptosis [40]. Src inhibitor saracatinib (AZD-0530) reduces cancer cell migration and, when combined with tamoxifen, inhibits proliferation in breast cancer cells [41], and has been shown to suppress bone metastasis formation in an in situ prostate cancer model [42]. A-443654, an AKT inhibitor, interferes with mitotic progression and remains in early stages of investigation [43]. Over the past decade, combination therapy with immune checkpoint inhibitors has become a frontline treatment option for metastatic ccRCC [44]. In the study, leveraging bioinformatic analysis, we explored the potential sensitivity of common novel anti-tumor drugs in relation to the expression patterns of HMMR in ccRCC. These findings indicated a significant correlation between HMMR expression and sensitivity to Nutlin3a, Rucaparib, saracatinib, AICAR, Olaparib, and A-443654, suggesting that HMMR expression level might be a determinant of drug responsiveness. This implicates HMMR as a potential target for personalized medicine.

We noted that miRNAs can regulat target genes' expression to perform their biological functions [45]. Extensive literature documents the pivotal role of miRNAs in the pathogenesis of ccRCC [46]. To identify miRNAs that potentially interact with the HMMR gene, we employed the RNCORI online database and intersected the results with those from screening the TCGA-KIRC database for miRNAs that are downregulated when HMMR is highly expressed. This approach helped us narrow down candidate miRNAs, whose predictive accuracy was then validated through RT-qPCR and Western blot analysis. Ultimately, miR-9-5p was confirmed as an upstream regulator of HMMR.

Previous studies have demonstrated either oncogenic or tumor-suppressive roles of miR-9-5p in various tumors. In prostate cancer, it suppresses NUMB expression by direct binding, affecting the growth and invasive potential of prostate cancer stem cells [47]. Conversely, for breast and ovarian cancers, miR-9-5p enhances tumor cells motility and invasiveness by reducing E-cadherin expression via CDH1 targeting and stimulates angiogenesis by activating VEGF expression [48, 49]. We propose a novel mechanism in which miR-9-5p binds to the 3’-UTR of the HMMR gene, reducing its expression and thereby inhibiting the tumorigenesis and progression of ccRCC. Our findings indicate low expression of miR-9-5p in ccRCC cells, suggesting its potential to be a tumor suppressor. Experiments involving miR-9-5p overexpression and rescue assays confirmed the interactive relationship between miR-9-5p and HMMR. Consistent with our findings, one study has reported that miR-9-5p suppresses tumor growth by downregulating androgen receptor expression, slowing down cell proliferation rates [50].

To gain insights into HMMR's oncogenic activities, we employed GSEA to identify signaling pathways associated with HMMR. These analyses revealed connections between HMMR and several pathways, including the JAK/STAT pathways. Subsequently, we investigated how the expressions of proteins related to EMT and JAK/STAT pathways change following manipulation of HMMR expression levels. Additionally, we employed the JAK-specific inhibitor Ruxolitinib in functional assays to further elucidate HMMR's role. Our results showed that altered HMMR expression indeed modified the expression of proteins associated with both EMT and JAK1/STAT1 pathways, and Ruxolitinib effectively reduced cell migration and proliferation promoted by HMMR. These findings suggested that HMMR, through engagement with diverse signaling pathways and interactions with various proteins, facilitated tumor cell proliferation and invasion.

Despite these, our study has limitations. While we explored HMMR's oncogenic role in databases and in vitro/in vivo models, we did not thoroughly examine its effect on in vivo invasion capabilities. Secondly, although we clarified the role of the miR-9-5p/HMMR axis in ccRCC. However, it is undeniable that our understanding of the precise mechanisms by which HMMR promotes tumor formation and invasion remains incomplete. Further study of HMMR in ccRCC is needed.

HMMR showed significantly high expression in ccRCC tissues and cells, and was associated with pathological grade and stage and patient prognosis. The elevated expression of HMMR could enhance the proliferative and migratory capabilities of ccRCC cells. As the upstream regulator of HMMR, miR-9-5p could inhibit the carcinogenic effect induced by HMMR to a certain extent. HMMR might be implicated in the proliferation and metastasis of ccRCC through EMT and JAK1/STAT1 signaling pathways. These findings suggest that HMMR has the potential as a biomarker for ccRCC in the future.

RCC	renal cell carcinoma
ccRCC	clear cell RCC
HMMR	hyaluronan-mediated motility receptor
EMT	epithelial-mesenchymal transition
3’-UTR	3’-untranslated regions
miRNA/miR	microRNA
RT-qPCR	real-time quantitative PCR
cDNA	complementary DNA
mRNA	messenger RNA
CCK-8	Cell Counting Kit-8
MMP	matrix metalloprotein
TCGA	The cancer genome atlas
GSEA	Gene Set Enrichment Analysis
TME	tumor immune microenvironment
PARP	poly ADP-ribose polymerase
VEGF	vascular endothelial growth factor
JAK/STAT	Janus Kinase/Signal Transducer and Activator of Transcription

Acknowledgement

We are grateful for the supports from Jie Fang in all animal experiments.

Author contribution

Ben Liu and Dingheng Lu designed the study. Xinyang Niu carried out the experiments and drafted the manuscript. Xinyang Niu, Weitao Zhan, Jiazhu Sun, Yuxiao Li, Yuchen Shi, Kai Yu, Suyuelin Huang and Xiaoyan Liu performed data analysis. Ben Liu, Dingheng Lu and Xueyou Ma helped in the experimental design and manuscript writing. Ben Liu revised the manuscript. All authors have seen and approved the final version of the manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (82273132, 82072848), Key R&D Program of Zhejiang (No.2023C03073), the Medical and Health Research Project of Zhejiang Province (2024KY065), China Postdoctoral Science Foundation Grant (2023M743022).

Data availability

Data will be made available on request. The data supporting this study's findings are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

Written informed consent was gained from all participants based on the guidelines of the Declaration of Helsinki. The collection of human samples and operation of animals in our study were approved by the Medical Ethics Com mittee of the First Affiliated Hospital, Zhejiang University School of Medicine.

Consent for publication

The authors consent for publication.

Competing interests

The authors declare there are no competing interests, and all authors consent to publish the data.

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Editorial decision: Major revision
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miR-9-5p/HMMR regulates the tumorigenesis and progression of clear cell renal cell carcinoma through EMT and JAK1/STAT1 signaling pathway

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