Abstract Clear cell renal cell carcinoma (ccRCC) is a subtype of renal cancer primarily originating from renal tubular epithelial cells. The absence of effective treatments has contributed to its poor prognosis. PDLIM7 is a protein containing PDZ and LIM structural domains, which performs varied functions through interactions with different proteins. To elucidate the role of PDLIM7 in ccRCC, we aimed to investigate its potential functions in relation to drug sensitivity. We analyzed multiple databases to assess the relationship between PDLIM7 expression and clinicopathological features, and conducted survival prognosis analysis. The expression level of PDLIM7 was validated through Western blotting and immunohistochemistry (IHC). We determined the effects and mechanisms of PDLIM7 on cancer cell proliferation, migration, and invasion through cloning assays, scratch assays, and Transwell assays. The functions and potential mechanisms of action of PDLIM7 were analyzed using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses. Its relationship with drug sensitivity was predicted utilizing the CellMiner database. Our study demonstrated that elevated PDLIM7 expression in ccRCC tissues and cell lines is significantly associated with the prognosis of patients with ccRCC. The knockdown of PDLIM7 markedly reduced the activity of ccRCC cells. KEGG and GO functional enrichment analyses indicated that PDLIM7 is implicated in various biological functions and signaling pathways. Higher levels of PDLIM7 are associated with increased sensitivity to a range of therapeutic agents. In conclusion, PDLIM7 is highly expressed in ccRCC and functions as an oncogene; its knockdown significantly inhibits the activity of ccRCC cells. PDLIM7 may serve as a prognostic marker for ccRCC and represents a potential therapeutic target. Keywords: PDLIM7, CcRCC, Migration, Drug sensitivity Subject terms: Renal cancer, Renal cell carcinoma Introduction As the global population continues to age, the incidence of renal cancer is rising, currently comprising approximately 3% of all cancers, with ccRCC accounting for 80% to 90% of these cases^[28]1. Numerous studies have demonstrated that the prognosis for early-diagnosed and surgically resected localized ccRCC is generally favorable; however, approximately 30% of patients develop distant metastasis due to the absence of effective early diagnostic markers and treatments. Furthermore, about 12% of patients experience metastasis post-surgery, leading to a relatively poor prognosis^[29]2. The complex cellular composition and high degree of heterogeneity in ccRCC tumors, which frequently display aberrant gene expression and molecular regulatory mechanisms, have led to a growing number of studies focusing on potential therapeutic targets. However, the lack of relevant clinical trials has hindered the advancement of these protocols into clinical practice^[30]3. Consequently, there is a pressing need for better molecular markers to enable early diagnosis and treatment of ccRCC^[31]4. PDZ-LIM family proteins possess multiple functional structural domains, with all members containing at least one PDZ and one LIM domain. They are involved in regulating various functions during biological development, including cytoskeletal organization, cell differentiation, organ development, neural signaling, and tumorigenesis^[32]5–[33]8. For instance, blocking PDLIM7-mediated nuclear translocation of YAP1 in prostate cancer inhibits the progression of the disease^[34]9. Another study demonstrated that knockdown of PDLIM7 not only inhibited cell proliferation, migration, and angiogenesis in desmoplasia-resistant prostate cancer but also enhanced sensitivity to docetaxel chemotherapy. Mechanistically, PDLIM7 has long been recognized as a scaffold for binding actin-related proteins and various signaling proteins^[35]10. In a recent study, it was observed that the protein expression of PDLIM7 decreased following the knockdown of CNN1, while mRNA expression remained unchanged. This finding suggests that CNN1 may promote chemoresistance in gastric by influencing the post-translational modification of the PDLIM7 protein, which subsequently affects the rigidity of the extracellular matrix^[36]11. However, the role of PDLIM7 in ccRCC remains unclear, making it particularly important to investigate its effects on ccRCC and its prognostic value. To explore and elucidate the impact of PDLIM7 expression on ccRCC cells and its correlation with patient prognosis, we analyzed PDLIM7 expression in the GEO and TCGA databases. We conducted clinical correlation analysis, survival analysis, and drug sensitivity analysis using the expression profile data from these public databases. Finally, we validated the effect of PDLIM7 on ccRCC cells through in vitro experiments, aiming to provide theoretical support for understanding the mechanisms underlying ccRCC development. Materials and methods PDLIM7 differential expression and survival analysis The microarray sequencing dataset [37]GSE46699 for renal clear cell carcinoma was obtained from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus database ([38]https://www.ncbi.nlm.nih.gov/geo/, GEO). Perl software was used for data matching, and gene differential expression analysis was performed using the limma package in R^[39]12. PDLIM7 expression data were obtained from the Cancer Cell Line Encyclopedia (CCLE) database ([40]https://sites.broadinstitute.org/ccle), and the R package ggplot2 was employed to visualize PDLIM7 expression in renal cancer cell lines. The relationship between PDLIM7 differential expression in ccRCC and patient survival prognosis was analyzed using an online database ([41]https://guolab.wchscu.cn/GSCA/#/). Analysis of PDLIM7 expression and clinicopathologic correlation The raw data of renal clear cell carcinoma RNA-seq and clinical information were obtained from the TCGA([42]https://portal.gdc.cancer.gov/) database using the GDC data transfer tool^[43]13, comprising 614 samples, including 542 tumor samples and 72 normal tissues. The expression data were merged with the clinical data using the R merge function, and normal tissues and duplicate samples were removed, resulting in 521 tumor samples. These samples were categorized into high and low expression groups based on the median PDLIM7 expression, and a chi-square test was conducted to analyze the relationship between PDLIM7 expression and clinicopathological features. Immunohistochemical staining PDLIM7 primary antibody(1:100,finetest,China), a DAB-enhanced chromogenic solution, and a rabbit two-step assay kit were obtained from Beijing Zhongsui Jinqiao Company. Paraffin sections were preheated in an oven at 65 °C for 30 min until the paraffin wax melted. The sections were then deparaffinized, sequentially dehydrated, treated with 3% hydrogen peroxide, and rinsed with double-distilled water for antigen retrieval. Finally, 1% BSA was applied dropwise for 20 min. On the following day, the sections were removed from the refrigerator and washed with PBS. The appropriate amount of DAB was added to enhance color development, followed by the application of about 100 μl of goat anti-rabbit IgG polymer for enzyme labeling for 20 min. The nuclei were rinsed with PBS, and hematoxylin was added dropwise. The differentiation and return-to-blue solutions were then applied dropwise, followed by quick washes. The sections were dehydrated through a gradient of ethanol (70%, 80%, 90%, 95%, and 100%), immersed in xylene three times (I, II, and III), naturally air-dried, and sealed with drops of neutral resin before being observed and photographed under a microscope. Cell culture Renal cancer cell lines and normal renal cells were obtained from Wuhan Punosai Life Sciences Company. HK2 and ACHN were cultured in DMEM medium supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin. The 769-P cell line was cultured in 1640 medium with 10% fetal bovine serum (Gibco, USA) and 1% penicillin, while Caki-1 cells were cultured in McCoy’s 5A medium containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin at 37 °C in a 5% CO[2] atmosphere. Three shRNA sequences targeting PDLIM7 (sh1: GCACCTGAAGAAATCAAGCCA; sh2: GCTCAGAACAAGATCCGGGCC; sh3: GATGAGGAGCACCTGAAGAAA, BioScien, Shanghai, China). These sequences were packaged into lentiviral particles using 293 T cells following standard viral packaging protocols. The resulting lentiviral supernatant was used to infect target cells. 48 h post-infection, stable transfectants were selected with puromycin at a concentration of 2 μg/mL to establish cell lines with sustained PDLIM7 knockdown. Western blot Tissues and cells were lysed with a protease inhibitor lysate containing 1% protease inhibitor, and the lysate was incubated on ice for 30 min. The mixture was then centrifuged at 4 °C at 12,000 rpm for 15 min to collect the supernatant. Protein concentration was determined using the BCA method. SDS-PAGE was performed to separate the proteins, which were then transferred to a PVDF membrane. The membrane was blocked with 6% skimmed milk for 2 h, followed by the addition of primary rabbit anti-human antibodies: PDLIM7 (1:1000, finetest, China), E-cadherin (1:2000, immunoway, China), N-cadherin (1:2000, immunoway, China), and GAPDH (1:5000, immunoway, China). The membrane was incubated at 4 °C overnight, then washed three times with TBST (10 min each wash). Subsequently, a secondary antibody was applied and incubated for 1 h, followed by another TBST wash for 10 min. The membrane was incubated with the secondary antibody for 1 h, followed by three washes with TBST (10 min each). A chemiluminescence imaging system was used for data visualization and analysis, with GAPDH serving as an internal reference to normalize the target proteins. Wound healing assay To investigate the effect of PDLIM7 on the lateral migration ability of renal clear cell carcinoma 769-P and Caki-1 cells, PDLIM7-knockdown cancer cells were digested and resuspended in either 1640 or McCoy’s 5A medium, and subsequently inoculated into 6-well plates. When the cell confluence reached 70–80%, a straight line scratch was made using a 10 μl pipette tip. Floating debris cells were gently washed away with PBS, and the culture was continued in serum-free medium. All photographs were captured using an inverted phase contrast microscope (Carl Zeiss, Germany) at 100 × magnification after 24 h of incubation. Plate cloning experiment Trypsin was used to digest the cells from each experimental group in the logarithmic growth phase. The cells were then resuspended to create a cell suspension, counted on a counting plate, and inoculated into 6-well culture plates, with each experimental group containing 800–1000 cells per well and three replicate wells. The inoculated cells were placed in an incubator at 37 °C for 14 days, during which the medium was changed every 3–4 days, and the cell status was monitored. Cell colony formation was observed under an inverted microscope, and the cells were washed three times with PBS. An appropriate amount of 4% paraformaldehyde was added to each well to fix the cells for 30 min, followed by three washes with PBS. Five hundred microliters of crystal violet staining solution was added to each well to stain the cells for 30 min. Distilled water (ddH2O) was used to wash the cells several times, and the cells were allowed to dry naturally before being photographed and counted for colonies. Transwell assay for cell migration capacity Successfully knocked-out 769-P or Caki-1 cells were collected, resuspended in serum-free medium, and counted. Then, 600 μl of complete 1640 or McCoy’s 5A medium containing 10% fetal bovine serum was added to the lower chamber, and 200 μl of an equal number of cells was inoculated into the upper chamber. The cells were then incubated in the incubator for 24 h. Cells were gently removed from the upper chamber using a sterile cotton swab. Four percent paraformaldehyde was used to fix the cells on the membrane surface, followed by staining with crystal violet for 10 min. Images were captured under the microscope and subsequently counted. KEGG/GO enrichment Genes strongly associated with PDLIM7 were utilized for KEGG and GO enrichment analysis (KEGG: Kyoto Encyclopedia of Genes and Genomes). The org.Hs.eg.db package was used for gene name transformation, while the R package clusterProfiler was employed for KEGG and Gene Ontology (GO) enrichment analysis, which included biological processes (BP), cellular components (CC), and molecular functions (MF). A threshold value of P < 0.05 was set for all results. Drug sensitivity analysis Drug sensitivity data were obtained from the Cellminer database ([44]https://discover.nci.nih.gov/cellminer/). Drugs linked with PDLIM7 sensitivity were analyzed using Pearson correlation. Correlations greater than 0.3 with a p-value of less than 0.05 were deemed significant and visualized using the R package ggplot2. Results PDLIM7 expression is elevated in renal clear cell carcinoma tissue samples and cells and correlates with poor prognosis To investigate the expression of PDLIM7 in ccRCC cells and tissues, we initially downloaded RNA-seq data of ccRCC from public databases and analyzed the expression of PDLIM7. The results indicated that PDLIM7 expression was significantly higher in ccRCC tissues compared to normal renal tissues (Fig. [45]1a–c). Immunohistochemical staining further revealed that PDLIM7 expression was significantly lower in normal kidney tissues than in ccRCC tissues, with PDLIM7 predominantly localized in the cytoplasm (Fig. [46]1d). We subsequently examined the expression of PDLIM7 in HK-2, a normal renal cell line, as well as in three renal carcinoma cell lines (ACHN, 769-P, and Caki-1) using Western blot analysis. The results demonstrated that PDLIM7 expression was significantly higher in renal carcinoma cell lines compared to normal renal cells (Fig. [47]1e). Collectively, these results suggest that PDLIM7 may be associated with the development of renal clear cell carcinoma. Fig. 1. [48]Fig. 1 [49]Open in a new tab PDLIM7 expression is increased in renal clear cell carcinoma tissue samples and cells and correlates with poor prognosis. (A–C) The public database shows elevated expression of PDLIM7 in ccRCC. (D) Immunohistochemical staining shows elevated expression of PDLIM7 in ccRCC. (E) PDLIM7 expression in ccRCC cells was significantly higher than that in normal cells HK2. Scale bar:10 µm and 5 µm (F,G) Effect of PDLIM7 on OS and PFS in ccRCC patients. Subsequently, we analyzed the impact of PDLIM7 expression on overall survival and progression-free survival of ccRCC patients using a database. We found that the high-expression group of PDLIM7 had significantly lower overall and progression-free survival rates compared to the low-expression group (Fig. [50]1f–g). PDLIM7 expression is elevated in renal clear cell carcinoma tissue samples and cells and correlates with poor prognosis In this study, 521 ccRCC patients with PDLIM7 expression were categorized into two groups based on high and low PDLIM7 expression levels, and their correlation with clinical characteristics was analyzed. The results indicated a strong correlation high PDLIM7 expression and patient age, as shown in Table [51]1. Table 1. Association of PDLIM7 expression with clinicopathologic features of ccRCC patients. Low group(n =) High group(n =) P Gender Male (n = 337) 220 117  = 0.792 Female (n = 184) 118 66 Laterality Left(n = 242) 162 80  = 0.273 Right(n = 278) 176 102 Bilateral(n = 1) 0 1 Age, years  < 60 (n = 259) 154 105  = 0.010*  ≥ 60(n = 262) 184 78 Tumor Stage T1-T2 (n = 335) 224 111  = 0.202 T3-T4 (n = 186) 114 72 Lymph Node Stage N0 (n = 236) 153 83  = 0.116 N1 (n = 15) 6 9 NX (n = 270) 179 91 Metastasis Stage M0(n = 442) 289 153  = 0.565 M1(n = 79) 49 30 ACJJ Stage I–II (n = 318) 215 103 0.102 III–IV (n = 203) 123 80 [52]Open in a new tab PDLIM7 promotes the value-added, migration, and invasion of ccRCC To investigate the effect of PDLIM7 on ccRCC development, we first examined the effects of sh1-PDLIM7, sh2-PDLIM7, and sh3-PDLIM7 transfection on PDLIM7 expression in ccRCC cells using Western blot analysis. The results indicated that sh1-PDLIM7 differed significantly from the control group, with the highest knockdown efficiency (Fig. [53]2a). Subsequently, we selected 769-p and Caki-1 cells with stable low expression of sh1-PDLIM7 for further functional experiments. Fig. 2. [54]Fig. 2 [55]Open in a new tab PDLIM7 promotes cell proliferation, migration, and invasion in ccRCC. (A) Validation of western blot assay for transfection with sh-PDLIM7 in 769-p and Caki-1 cells. (B) Cloning assay for ccRCC cell viability. (C) Detection of cellular wound healing capacity by scratch assay. Scale bar:500 µm (D) Transwell assay for cell migration and invasion. Scale bar:10 µm (E) Western blot for EMT-related protein expression. The value-adding ability of the cells was assessed using a cloning assay. The results demonstrated that cell viability was significantly reduced in the sh1-PDLIM7 group compared to the control group (Fig. [56]2b), suggesting that PDLIM7 knockdown significantly inhibited the value-adding ability of ccRCC cells. To investigate the effect of PDLIM7 on ccRCC metastasis, we assessed the wound healing ability of 769-p and Caki-1 cells using a scratch assay. The results indicated that wound healing was significantly inhibited in the sh-PDLIM7 group compared to the control group (Fig. [57]2c). The invasive abilities of 769-p and Caki-1 cells were assessed using a Transwell assay. The results indicated that the sh1-PDLIM7 group significantly reduced cell invasion (Fig. [58]2d). EMT plays a crucial role in the invasion and metastasis of various cancers and is a significant factor contributing to poor cancer prognosis. We assessed EMT-related proteins via western blot and found that E-cadherin expression was significantly higher in the NC group compared to the sh1-PDLIM7 and sh3-PDLIM7 groups, while N-cadherin expression was significantly lower (Fig. [59]2e). KEGG/GO functional enrichment and drug sensitivity KEGG pathway enrichment analysis of PDLIM7-related genes revealed that motor protein signaling pathways were the most enriched^[60]14 (Fig. [61]3a). Additionally, GO analysis indicated significant enrichment in biological processes, including regulation of ATP-dependent activity and muscle cell apoptosis (Fig. [62]3a). Myotomes and adhesion spots were significantly enriched in cellular components (Fig. [63]3a), while actin binding, myosin heavy chain binding, α-actinin binding, and actinin binding were significantly enriched in molecular functions (Fig. [64]3a). Fig. 3. [65]Fig. 3 [66]Open in a new tab KEGG/GO functional enrichment and drug sensitivity. (A) Functional enrichment of PDLIM7 and related genes KEGG/GO. (B) PDLIM7 and drug correlation. Drug sensitivity analysis results demonstrated the expression level of PDLIM7 in the NCI-60 human cancer cell line and its correlation with several chemotherapeutic agents. PDLIM7 correlated with the sensitivity to multiple tumor chemotherapeutic drugs (Fig. [67]3b), including Everolimus (cor = 0.36, p = 0.004), Deforolimus (cor = 0.37, p = 0.003), XAV-939 (cor = 0.39, p = 0.002), H-89 (cor = 0.32, p = 0.012), Lenvatinib (cor = 0.37, p = 0.004), JNJ-38877605 (cor = 0.40, p = 0.002), and Staurosporine (cor = 0.31, p = 0.016). Statistical analysis All experiments were performed in triplicate, and results are expressed as mean ± standard deviation. A T-test was conducted to analyze significance between two groups, while ANOVA was used for multi-group comparisons. Additionally, a chi-square test was applied for count data analysis. All experimental data were analyzed and plotted using GraphPad Prism (version 9.0) statistical software, with p < 0.05 considered statistically significant. Discussion Extensive systemic metastasis can occur in patients with advanced clear cell renal cell carcinoma (ccRCC), contributing significantly to its poor prognosis. In healthy organisms, mRNAs play a crucial role in cell growth, development, and various pathophysiological functions. Aberrant mRNA expression, leading to dysregulation of corresponding proteins, is commonly observed in various diseases, including cancer^[68]15,[69]16. Notably, mRNA-encoded proteins can function as both oncogenes and tumor suppressor genes, presenting a significant challenge for cancer treatment. A comprehensive understanding of this dual role is essential for advancing cancer development and treatment^[70]17–[71]19. The PDLIM7 protein consists of both PDZ and LIM structural domains, forming a specialized structure that facilitates binding to other protein molecules and performing various biological functions^[72]20. Numerous studies indicate that its primary roles include participation in cell differentiation, organ development, and neural signaling, with aberrant expression of PDLIM7 potentially linked to various diseases, including cancer^[73]21–[74]23. For instance, Yang et al. reported that PDLIM7 expression was significantly elevated in thyroid cancer, where its high levels correlated strongly with clinicopathologic features and promoted invasion and metastasis^[75]24. Additionally, another study identified the fusion gene PDLIM7 in cranial soft tissue myoepithelial carcinoma, although its specific biological role remains unclear^[76]25. These findings collectively suggest that PDLIM7 may regulate the invasion and metastasis of ccRCC. Although the specific factors contributing to the development of clear cell renal cell carcinoma (ccRCC) remain unidentified, studies indicate that obesity, genetic mutations, age, and other factors significantly influence its pathogenesis^[77]26,[78]27. This study demonstrates the potential application of PDLIM7 in the clinical treatment of ccRCC. Analysis of multiple bioinformatics databases revealed that PDLIM7 expression in the tissues and cells of ccRCC patients was significantly elevated compared to normal tissues. Additionally, patients with higher PDLIM7 expression exhibited poorer prognoses, including overall survival (OS) and progression-free survival (PFS). Several studies have demonstrated that mRNA expression levels correlate significantly with the clinicopathologic features and prognosis of patients^[79]28–[80]31. To further explore the relationship between PDLIM7 and clinical features, this study collected pathological information from clinical specimens in bioinformatics databases and analyzed the association between PDLIM7 expression and clinicopathological features. The results indicated that high PDLIM7 expression was significantly correlated with the age of ccRCC patients, suggesting that advanced age may increase the risk of developing ccRCC. These findings suggest that PDLIM7 may serve as a prognostic marker for ccRCC and could play a role in the risk stratification of these patients. EMT endows cancer cells with the capability to metastasize to distant sites. Its most prominent characteristics include the enhancement of invasive potential, motility, and resistance to apoptotic stimuli, thereby facilitating tumor dissemination and metastasis. During EMT, a hallmark molecular change is the downregulation of epithelial markers such as E-cadherin, coupled with an upregulation of mesenchymal markers like N-cadherin, which collectively contribute to the acquisition of a more motile and invasive phenotype in cancer cells^[81]32,[82]33. For example, a study reported that ADAM12 expression was significantly elevated in clear cell renal cell carcinoma (ccRCC). Following ADAM12 knockout, there was a notable decrease in E-cadherin expression accompanied by a substantial increase in N-cadherin protein levels^[83]23. Furthermore, another study found that deletion/knockout of LTB4R also resulted in a significant decrease in E-cadherin expression and an increase in N-cadherin protein expression in ccRCC^[84]34. Consistent with previous studies, our research demonstrates that knockout of PDLIM7 leads to a significant reduction in the epithelial marker E-cadherin expression, accompanied by a marked increase in the mesenchymal marker N-cadherin protein levels. Moreover, the proliferation, migration, and invasion capacities of clear cell renal cell carcinoma (ccRCC) cells are markedly suppressed. These findings suggest that PDLIM7 may regulate ccRCC invasion and metastasis through modulating the epithelial-mesenchymal transition (EMT) process. Consequently, early intervention targeting EMT could represent a viable strategy to impede the progression of ccRCC. Further in vitro experiments demonstrated that PDLIM7 was significantly upregulated in ccRCC tissues and cell lines. Knockdown of PDLIM7 notably inhibited ccRCC cell proliferation, migration, and invasion, suggesting that PDLIM7 is involved in regulating invasion and metastasis in ccRCC. To further explore its specific mechanisms, this study identified relevant genes and performed enrichment analysis, showing that PDLIM7 primarily functions in myosin differentiation and regulation. It may also regulate signaling pathways such as GMP-PKG and cAMP; however, further experiments are needed to confirm this. The poor outcomes in ccRCC are primarily due to the high rate of postoperative recurrence and the absence of effective surveillance markers, leading to treatment resistance in patients with advanced ccRCC^[85]2,[86]35,[87]36. This study analyzed the drugs sensitive to PDLIM7, revealing that Everolimus, Deforolimus, XAV-939, H-89, Lenvatinib, JNJ-38877605, and Staurosporine were significantly effective against cells with high PDLIM7 expression. Everolimus and Lenvatinib have been approved for clinical application in the treatment of ccRCC^[88]37. As the most prevalent cancer subtype arising from VHL gene deletions, ccRCC results in the upregulation of hypoxia-inducible factor (HIF) target gene expression, which further induces alterations in cellular metabolism and signaling pathways. These changes promote angiogenesis within the tumor microenvironment and lead to dysregulation of lipid metabolism. Such mechanisms elucidate the therapeutic efficacy of mTOR pathway inhibitors like Everolimus and VEGF inhibitors such as Lenvatinib in ccRCC management^[89]38,[90]39. These insights provide a valuable foundation for the development of targeted therapeutic strategies and offer theoretical support for the design of novel pharmacological interventions. The above studies suggest that PDLIM7 regulates invasion and metastasis of ccRCC and plays a pro-carcinogenic role in ccRCC, and that PDLIM7 can act as an inhibitor of ccRCC and may be a potential therapeutic target for ccRCC. In conclusion, we examined the relationship between PDLIM7 expression in ccRCC and clinical features, and investigated its potential function and mechanisms of action in ccRCC proliferation and invasion. The results indicate that upregulation of PDLIM7 significantly contributes to ccRCC cell growth and metastasis. Furthermore, the PDZ structural domain has considerable potential for developing related drugs due to its well-defined binding sites. Notably, PDLIM7 may serve as a novel prognostic marker or an effective therapeutic target for ccRCC. Supplementary Information [91]Supplementary Information.^ (134.9KB, pdf) Author contributions Chong Shen and Jianying Wang conceived and designed the study. Jian Zhuo participates in the statistics and analysis of the data. All authors completed experiments. Chong Shen analyzed the data and wrote the manuscript. Funding This work was supported by the Hebei Province 2024 annual medical science research topic. SUBSIDIZE NUMBER:20240024. Data availability The datasets used and analysed during the current study available from the corresponding author on reasonable request. Declarations Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Contributor Information Chong Shen, Email: Shenchong66@163.com. Jianying Wang, Email: byzswjy@163.com. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-025-11495-9. References