Decreased PANK1 expression in kidney renal clear cell carcinoma: impact on cell apoptosis, invasion, migration, and epithelial-mesenchymal transition

Abstract

Objective

To investigate pantothenate kinases 1 (PANK1) expression in kidney renal clear cell carcinoma (KIRC) tissues, analyze its correlation with clinicopathological features and prognosis, and explore its impact on invasion, migration, and apoptosis in KIRC cells.

Methods

GEPIA (gene expression profiling interactive analysis), UALCAN and LinkedOmics, were employed to analyze PANK1 expression in KIRC tissues and its correlation with clinical characteristics. Comparative analyses were performed between KIRC (Caki-1 and 786-O) and noncancerous renal cells (HK-2 and RPTEC). Transfection with PANK1 activation particles was conducted, followed by Wound healing, Transwell assay, Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and Western blotting.

Results

PANK1 was down-regulated in KIRC tissues and cells compared to normal tissues and noncancerous cells. Correlation analyses linked PANK1 expression with clinicopathological features in KIRC, with high PANK1 expression associated with a favorable outcome. High PANK1 expression correlated positively with E-cadherin (CDH1), tight junction protein 1 (TJP1), Fas cell surface death receptor (FAS), caspase-8 (CASP8), and CASP9, while showing a negative correlation with vimentin (VIM), snail family transcriptional repressor 1 (SNAIL1), twist family BHLH transcription factor 1 (TWIST1), and TWIST2. PANK1 overexpression increased CDH1, TJP1, FAS, CASP8, and CASP9 while downregulating SNAIL1, VIM, TWIST1, and TWIST2, inhibiting invasion and migration, and promoting apoptosis in KIRC cells.

Conclusion

PANK1 down-regulation in KIRC tissues correlated with clinicopathological features and prognosis. Its overexpression modulated epithelial-mesenchymal transition (EMT)-related gene, inhibited invasion, promoted apoptosis in KIRC cells, highlighting its role in disease progression and therapeutic potential.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12672-024-01251-2.

Introduction

Renal cell carcinoma (RCC), ranking 13th in the prevalence of all tumors globally, is among the most common malignant tumors in the urinary system, with increasing incidence and mortality rates [1, 2]. Kidney clear cell carcinoma (KIRC) is most common histology of RCC, followed by papillary RCC and chromophobe RCC [3], constituting approximately 80% of all RCC cases [4]. Unfortunately, around 30% of patients are diagnosed with metastatic RCC, contributing to the challenges in effective treatment [5]. Due to alterations in metabolic pathways contributing to its development, KIRC is classified as a cell metabolism disease, where cancer cells undergo various metabolic changes that facilitate their uncontrolled growth and proliferation [6]. Emerging evidence suggests that the activation of specific metabolic pathway have a role in regulating angiogenesis and inflammatory signatures [7, 8]. The characteristics and conditions within the tumor microenvironment, such as the presence of various cell types, signaling molecules, blood vessels, and extracellular matrix components, play a crucial role in influencing the biological behavior of the disease [9]. These features can affect tumor growth, metastasis, and the immune system’s ability to recognize and attack cancer cells [10]. Moreover, the tumor microenvironment can significantly impact the effectiveness of systemic therapies, including chemotherapy, immunotherapy, and targeted treatments, by either enhancing or hindering their therapeutic effects [11]. Despite the utilization of molecular tumor-targeted drugs, patients with advanced or recurrent KIRC still face limited disease-free and overall survival due to drug side effects and individual variations in drug sensitivity [12]. Understanding the mechanisms underlying tumorigenicity and tumor progression in KIRC remains incomplete [13], and the identification of reliable biomarkers for prognosis and treatment outcomes is an ongoing research focus [14].

Pantothenate kinases (PANKs), as crucial enzymes catalyzing coenzyme A (CoA) biosynthesis, exist in three types (Type I, II, and III) [15]. Among them, human PANK genes (PANK1-3) encode four characterized Type II PANK isoforms [16]. Previous studies have implicated PANKs in tumorigenic processes, with specific isoforms linked to various cancers [17, 18]. PANK1 have been implicated in various diseases, showcasing their significance in physiological processes. Research by Audam TN et al. demonstrated that PANK1 depletion exacerbated post-overload cardiac fibrosis, ventricular remodeling, and heart failure [19]. Additionally, PANK1 knockout in obese, high-glucose mice (ob/ob) was shown to suppress gluconeogenesis in the liver, leading to reduced hyperglycemia and hyperinsulinemia without affecting insulin signal transduction [20]. Notably, PANK1 is highly expressed in the liver, and its knockout results in decreased CoA levels, causing hypoglycemia and steatosis [16]. Studies by Yang L et al. uncovered a link between P53/PANK1/miR-107 and metabolic reprogramming-induced insulin resistance due to a high-fat diet [21]. PANK1, located on chromosome 10q23.31, has been frequently deleted in prostate cancer [22]. Additionally, miR-107, a microRNA located within the intron 5 of PANK1, has been recognized as a promising prognostic biomarker in cancer [23]. Notably, miR-107 functions as a potential tumor suppressor, inhibiting proliferation and invasion in KIRC and delaying tumor growth in nude mice [24].

Given these findings, we hypothesize that PANK1 may play a significant role in KIRC. In this study, we aimed to analyze the expression of PANK1 in KIRC and investigate its impact on the biological characteristics of KIRC cells through in vitro experiments, contributing to the understanding of potential therapeutic targets in KIRC.

Materials and methods

Bioinformatics

The expression of PANK1 in KIRC tissues and normal tissues was assessed using bioinformatics tools, including UALCAN [25], GEPIA (gene expression profiling interactive analysis) [26], LinkedOmics [27], and the Human Protein Atlas (https://www.proteinatlas.org/). These analyses aimed to investigate the correlation between PANK1 expression and clinicopathological features as well as the prognosis of KIRC patients. GEPIA database was also employed for the correlation analysis between PANK1 expression and genes related to epithelial-mesenchymal transition (EMT) and apoptosis in KIRC.

Cell culture

KIRC cell lines, Caki-1 (HTB-47) and 786-O (CRL-1932), and noncancerous human renal proximal tubular cell lines, HK-2 (CRL-2190) and RPTEC (CRL-4031), were procured from the American Type Culture Collection (ATCC, USA). Caki-1 and 786-O cells were cultured in McCoy’s 5a medium (30-2007, ATCC) and RPMI-1640 Medium, (30–200, ATCC), respectively, with the addition of fetal bovine serum (FBS, 30-2020, ATCC) to a final concentration of 10%. RPTEC cells were maintained in DMEM: F12 medium (30-2006, ATCC) supplemented with an hTERT RPTEC growth kit (ACS-4007, ATCC). HK-2 cells were cultured in keratinocyte serum-free medium (K-SFM, 17005-042, ATCC). The cells were incubated in a 37 ℃ humidified atmosphere with 5% CO₂ and 95% air.

Cell grouping and transfection

KIRC cell lines (Caki-1 and 786-O) were categorized into the Mock group, Control activation particles group, and PANK1 activation particles group. Cell transfection was conducted in plasmid transfection medium (sc-108062, Santa Cruz Biotechnology, Inc., USA) using Ultracruz^® transfection reagent (sc-395739, Santa Cruz Biotechnology, Inc., USA). PANK1 activation particles (sc-408890-LAC) and control activation particles (sx-437282) were supplied by Santa Cruz Biotechnology, Inc. (USA).

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)

Cells in each group were digested and rinsed in phosphate-buffered saline (PBS) three times to remove the supernatant through centrifugation. TRIzol™ reagent (15596018, Thermo Fisher, China) was added to extract total RNA from cells, and the purity and concentration of RNA were determined. Reverse transcription was carried out using the SuperScript^™ II reverse transcription enzyme (18064022, Thermo Fisher, China). Subsequently, qRT-PCR was performed using the SYBR GreenERTM qPCR SuperMix Universal (11762500, Thermo Fisher, China), with GAPDH as the endogenous reference. Primer sequence was shown in Table 1.

Table 1.

Primer sequences of quantitative reverse-transcription polymerase chain reaction (qRT-PCR)

Genes	Forward Primer	Reverse Primer
PANK1	5′-TGGAACGCTGGTTAAATTGGT-3′	5′-CCCAGTTTTCCCATAAGCAGTAT-3′
CDH1	5′-CGAGAGCTACACGTTCACGG-3′	5′-GGGTGTCGAGGGAAAAATAGG-3′
TJP1	5′-CAACATACAGTGACGCTTCACA-3′	5′-CACTATTGACGTTTCCCCACTC-3′
SNAIL1	5′-TCGGAAGCCTAACTACAGCGA-3′	5′-AGATGAGCATTGGCAGCGAG-3′
VIM	5′-GACGCCATCAACACCGAGTT-3′	5′-CTTTGTCGTTGGTTAGCTGGT-3′
TWIST1	5′-GTCCGCAGTCTTACGAGGAG-3′	5′-GCTTGAGGGTCTGAATCTTGCT-3′
TWIST2	5′-CATGTCCGCCTCCCACTA-3′	5′-CATGTCCGCCTCCCACTA-3′
FAS	5′-TCTGGTTCTTACGTCTGTTGC-3′	5′-CTGTGCAGTCCCTAGCTTTCC-3′
CASP8	5′-GTTGTGTGGGGTAATGACAATCT-3′	5′-TCAAAGGTCGTGGTCAAAGCC-3′
CASP9	5′-CTTCGTTTCTGCGAACTAACAGG-3′	5′-GCACCACTGGGGTAAGGTTT-3′
GAPDH	5′-GGAGCGAGATCCCTCCAAAAT-3′	5′-GGCTGTTGTCATACTTCTCATGG-3′

Western blotting

Cells were washed with PBS three times, and proteins were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (89900, Thermo Fisher Scientific Inc., Shanghai, China). The protein concentration was determined using the Pierce^™ Bradford Protein Detection Kit (23200, Thermo Fisher Scientific Inc., Shanghai, China). Protein samples (40 µg per well) were loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 89888, Thermo Fisher Scientific Inc., Shanghai, China) for isolation, followed by transfer onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in Tris-buffered saline Tween (TBST) supplemented with 5% non-fat milk. Anti-PANK1 antibody (ab69354, Abcam, USA) at 1/500 dilution and anti-beta actin (β-actin) antibody-loading control (ab8226, Abcam, USA) at 1/1000 dilution were used for membrane inoculation at 4 °C overnight. To prevent non-specific bands, after the transfer process, the membrane was cut based on the size of the target fragment, selecting only the area of interest for subsequent experiments. This step was crucial to ensure the specificity of the results. Consequently, original full-length membrane images are not available. After rinsing with 1 × TBST three times, the membrane was immersed in horseradish peroxidase-labeled enhanced chemiluminescent substrate (Immobilon Western HRP Substrate kit) and exposed to a chemiluminescent apparatus. Images were saved for analysis. The experiment was repeated three times.

Wound healing

A marker pen was used to draw a straight line on the back of a 6-well plate, dividing the wells. In each well, 5 × 10⁵ cells were inoculated. The next day, a pipette tip was used to draw lines perpendicular to the marked line, and three washes with PBS were performed to remove detached cells. Subsequently, cells were cultured in serum-free medium at 37 ℃ and 5% CO₂ and photographed at 0 h and 48 h. This experiment was repeated three times.

Transwell invasion assay

Matrigel and serum-free RPMI 1640 medium were mixed in a 1:7 ratio and added to the upper Transwell chambers (40 µL per well), followed by incubation at 37 ℃ for 4 to 5 h for coagulation. Cells were collected, and a cell suspension (200 µL, 5 × 10⁵ cells/mL) was added to the upper chamber, with RPMI 1640 medium supplemented with 20% FBS (600 µL) in the lower chamber. After 48 h of incubation, chambers were removed, rinsed in serum-free PBS twice, and cells in the upper chambers were scrubbed with a cotton swab. Cells in the lower chambers were fixed in methanol, stained with crystal violet, and mounted in neutral balsam for observation under an inverted microscope. Five different visions were selected for observation. This experiment was repeated three times.

Annexin V-fluorescein isothiocyanate/propidium iodide (annexin V-FITC/PI) staining

Cells in each group were digested in 0.05% trypsin, harvested, and resuspended in 200 µL PBS buffer. Annexin-V FITC reagent (5 µL) was added, followed by light-free incubation at room temperature for 15 min. Subsequently, 10 µL PI solution (50 µg/mL) was added, and 200 µL of buffer was added to the mixture. Fluorescent signals were detected using the FACS420 flow cytometer (Becton Dickinson, USA). The apoptotic rate was expressed as a percentage. This experiment was repeated three times.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.0. Experimental data were expressed as mean ± standard deviation (SD). Comparison of data among multiple groups was conducted using One-way analysis of variance (One-way ANOVA), followed by Tukey’s post hoc test for pairwise comparison. A significance level of P < 0.05 indicated statistical significance.

Results

PANK1 down-regulation observed in KIRC Tissues and cell lines

Comprehensive analyses confirmed significant down-regulation of PANK1 in KIRC tissues, supported by GEPIA (P < 0.001) and UALCAN (P = 1.62E-12) databases (Fig. 1A, B). To gain further insights into its expression pattern, we conducted additional experiments. Comparative analysis demonstrated a notable down-regulation of PANK1 in KIRC cell lines (Caki-1 and 786-O) in comparison to noncancerous human renal proximal tubular cell lines HK-2 and RPTEC (all P < 0.05, Fig. 1C–E). Thus, PANK1 expression is significantly reduced in KIRC tissues and cell lines compared to noncancerous controls.

Fig. 1.

PANK1 Down-Regulation Observed in KIRC Tissues and Cell Lines. Comprehensive analyses, including GEPIA (A) and UALCAN (B) databases, confirm significant down-regulation of PANK1 in KIRC tissues. C–E Quantitative reverse-transcription polymerase chain reaction (qRT-PCR, C) and western blotting (D–E) were employed to determine PANK1 expression in noncancerous human renal proximal tubular cell lines (HK-2 and RPTEC) and KIRC cell lines (Caki-1 and 786-O). *P < 0.05 vs. HK-2 cells, #P < 0.05 vs. RPTEC cells

Correlation of PANK1 expression with clinical features and in KIRC

LinkedOmics analysis (Supplementary Fig. 1A) revealed a significant association between PANK1 and key clinical features in KIRC, including pathologic stage (P = 6.51E-07), T stage (P = 1.00E-06), M stage (P = 4.46E-04) and N stage (P = 6.32E-04). Additionally, the GEPIA database illustrated a gradual decrease in PANK1 expression with increasing tumor stage (P = 2.22E-07, Supplementary Fig. 1B). UALCAN data further supported the correlation, associating PANK1 down-regulation with tumor stage, grade, and nodal metastasis (all P < 0.05, Supplementary Fig. 1C). These findings indicate that PANK1 down-regulation is significantly associated with advanced clinical features in KIRC.

Correlation of PANK1 expression with prognosis in KIRC

GEPIA survival analysis (Supplementary Fig. 2A-B) revealed that PANK1 expression correlated with disease-free survival (DFS, P = 2.4E-06, HR = 0.41) and overall survival (OS, P = 3.5E-11, HR = 0.35). The Cox Regression Test results from LinkedOmics database indicated a significant correlation between past PANK1 expression and overall survival in the context of KIRC (P = 1.69E-13, HR = 0.42, Supplementary Fig. 2C). Similar results were also obtained in the UALCAN database (Supplementary Fig. 2D). The Human Protein Atlas highlighted the favorable impact of elevated PANK1 expression in KIRC. Among patients with high PANK1 expression (n = 212), the 5-year survival rate was significantly higher compared to those with low expression (n = 316) (82% vs. 51%, P = 1.2E-11). Notably, this survival advantage was particularly pronounced in advanced disease stages, with statistically significant differences observed in Stage III (P = 0.005) and Stage IV (P = 0.006) patients (Table 2). Therefore, higher PANK1 expression is associated with improved prognosis and survival in KIRC patients.

Table 2.

The Human Protein Atlas demonstrated that high expression of PANK1 was favorable in KIRC

Stage	Total no.	Dead (no.)	Alive (no.)	5-year survival rate		P
				High expression %	Low expression %
Stage I	263	44	219	85	70	0.050
Stage II	57	13	44	90	72	0.076
Stage III	123	49	74	71	40	0.005
Stage IV	82	66	16	37	16	0.006

Effects of PANK1 on the Invasion and Migration of KIRC cells via mediating EMT

Subsequently, KIRC cell lines (Caki-1 and 786-O) were transfected with PANK1 activation particles, resulting in a significant up-regulation of PANK1 gene and protein expression (all P < 0.05, Fig. 2A–C). The subsequent investigation revealed that PANK1 overexpression effectively restrained the invasion and migration abilities of Caki-1 and 786-O cells (all P < 0.05, Fig. 2D–F). The GEPIA database analysis demonstrated that in KIRC, PANK1 expression correlated with various EMT-related genes, including E-cadherin (CDH1), tight junction protein 1 (TJP1), vimentin (VIM), snail family transcriptional repressor 1 (SNAIL1), twist family BHLH transcription factor 1 (TWIST1), and TWIST2 (Fig. 3A). Consequently, PANK1 exhibited a positive correlation with epithelial markers CDH1 (r = 0.18, P = 5.5E-05) and TJP1 (r = 0.28, P = 5.1E-11), while demonstrating a negative correlation with the expression of VIM (r = − 0.094, P = 0.032), SNAIL1 (r = − 0.13, P = 0.003), TWIST1 (r = − 0.12, P = 0.006), and TWIST2 (r = − 0.098, P = 0.025). Initially, the expression of EMT-related genes, including CDH1, TJP1, SNAIL1, VIM, TWIST1, and TWIST2, was assessed in the transfected cells (Fig. 3B). Consequently, PANK1 overexpression up-regulated CDH1 and TJP1 gene expression, while down-regulating the gene expression of SNAIL1, VIM, TWIST1, and TWIST2 in Caki-1 and 786-O cells (all P < 0.05). In summary, PANK1 overexpression suppresses KIRC cell invasion and migration by modulating EMT-related gene expression.

Fig. 2.

Effects of PANK1 on the Invasion and Migration of KIRC Cells. A–C Quantitative reverse-transcription polymerase chain reaction (qRT-PCR, A) and western blotting (B–C) were utilized to assess the expression of PANK1 in KIRC cell lines (Caki-1 and 786-O) transfected with PANK1 activation particles and control activation particles. D Wound healing and Transwell assays were conducted to evaluate the invasion and migration of Caki-1 and 786-O cells. E Comparison of wound closure (%) among groups. F Comparison of the number of invasive cells among groups. * P < 0.05 vs. the Mock group, # P < 0.05 vs. the cells transfected with Control activation particles

Fig. 3.

Effects of PANK1 on Epithelial-Mesenchymal Transition (EMT) in KIRC. A Correlation of PANK1 expression with the expression of EMT-related genes in KIRC via the GEPIA database; B Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to detect the expression of EMT-related genes in Caki-1 and 786-O cells transfected with PANK1 activation particles and control activation particles. * P < 0.05 vs. the Mock group; # P < 0.05 vs. the Control activation particles

PANK1 overexpression enhanced KIRC Cell apoptosis

Correlation analysis in the GEPIA database revealed a positive association between PANK1 expression and pro-apoptotic genes, including Fas cell surface death receptor (FAS) (r = 0.31, P = 8.2E-13), caspase-8 (CASP8) (r = 0.31, P = 9E-13), and CASP9 (r = 0.093, P = 0.033) (Fig. 4A). Further Annexin V-FITC/PI double staining demonstrated that PANK1 activation particles enhanced apoptosis in Caki-1 and 786-O cells (all P < 0.05, Fig. 4B–C). Additionally, PANK1 overexpression facilitated the gene expression of FAS, CASP8, and CASP9 in KIRC Caki-1 and 786-O cells (all P < 0.05, Fig. 4D–E). Thus, PANK1 overexpression promotes apoptosis in KIRC cells through up-regulation of pro-apoptotic genes.

Fig. 4.

PANK1 Overexpression Enhanced KIRC Cell Apoptosis. A GEPIA analysis revealed the correlation of PANK1 expression in KIRC with the pro-apoptotic genes, including FAS, CASP8, and CASP9. B–C Annexin V-FITC/PI double staining was applied to detect cell apoptosis. D–E Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was applied to detect the expression of FAS, CASP8, and CASP9 in KIRC cells (Caki-1 and 786-O). * P < 0.05 vs. the Mock group, # P < 0.05 vs. the Control activation particles

Discussion

RCC, recognized for its significant level of immune infiltration [28, 29], has an increased risk of development associated with conditions such as obesity, diabetes, and various metabolic disorders [30]. KIRC, the most common and metastatic urological cancer, is essentially a metabolic disease characterized by a reprogramming of energetic metabolism [31, 32]. Specifically, in KIRC, there is a significant alteration in metabolic flux through glycolysis, which becomes partitioned [33, 34]. Additionally, mitochondrial bioenergetics and oxidative phosphorylation (OxPhos) are impaired, alongside disruptions in lipid metabolism [35, 36]. In this scenario it has been shown that PANK1 is an important regulator of cell metabolism to be affected with the expression of p53 [37], thus regulating many biological characteristics of renal cancer cells [38]. Analysis utilizing GEPIA, UALCAN, and The Human Protein Atlas unveiled distinct PANK1 expression patterns in KIRC patients, positioning it as a potential prognostic indicator. Remarkably, the diminished expression of PANK1 in KIRC tissues exhibited positive correlations with advanced tumor stage and grade, further decreasing in cases with nodal metastasis. These collective findings strongly suggest that PANK1 functions as a tumor suppressor gene in KIRC. Consistently, PANK1 emerges as a promising prognostic marker for KIRC [39], showcasing associations with clinical outcomes, metabolism, and immune modulation based on a thorough analysis of TCGA data [40]. This could be attributed to the upregulation of the PANK1 protein, catalyzing the rate-limiting step of coenzyme A biosynthesis, by the tumor suppressor protein p53, often regarded as a “guardian of the genome” [21, 41–43].

To further elucidate the role of PANK1 in KIRC, in vitro experiments were conducted using KIRC cells (Caki-1 and 786-O) and renal proximal tubular cell lines (HK-2 and RPTEC). Consistent with clinical observations, PANK1 exhibited down-regulation in KIRC cells. Given the involvement of miR-107, a microRNA located within the intron of the PANK1 gene, in inhibiting the invasion and migration of KIRC [21, 24]. PANK1 overexpression was undertaken, revealing a significant constraint on the invasion and migration of KIRC cells.

EMT is a pivotal biological process essential for embryogenic development, chronic inflammation, tissue remodeling, tumor metastasis, and various fibrotic diseases, characterized by the conversion of epithelial cells to mesenchymal-like cells with reduced expression of epithelial markers (CDH1 and TJP1) and increased expression of mesenchymal markers (SNAIL1, VIM, TWIST1, and TWIST2) [44]. This process is particularly critical when epithelium-derived malignant tumor cells acquire enhanced abilities for migration and invasion. [45]. Our bioinformatic analysis revealed a compelling association between PANK1 expression and key EMT markers. Specifically, PANK1 expression exhibited a positive correlation with epithelial markers CDH1 and TJP1, while demonstrating a negative correlation with mesenchymal markers SNAIL1, VIM, TWIST1, and TWIST2. Furthermore, the experimental overexpression of PANK1 in KIRC cells yielded consistent results. PANK1 overexpression resulted in an increase in CDH1 and TJP1 expression, the epithelial markers, and a concurrent decrease in SNAIL1, VIM, TWIST1, and TWIST2, the mesenchymal markers. This dual impact of PANK1 on EMT markers strongly suggests that PANK1 plays a regulatory role in EMT processes. Given the established link between EMT and the acquisition of migration and invasion capabilities in malignant cells, our findings suggest that PANK1 may exert its inhibitory effect on KIRC cell invasion and migration through the modulation of EMT. By promoting an epithelial phenotype and suppressing mesenchymal features, PANK1 may contribute to the impairment of the invasive and migratory potential of KIRC cells.

Apoptosis, a critical process in maintaining cellular homeostasis, was explored in the context of PANK1 expression. Previous reports have shown that p53 could induce cell apoptosis [46], which played an important role in regulating energy homeostasis through the transcriptional control of PANK1, independent of its canonical functions in apoptosis [43]. Generally, cell apoptosis is fulfilled via the endogenous or exogenous pathways, while the latter is triggered by the ligation between FAS and FASL, which could further activate CASP8 and then CASP9 [47]. GEPIA analysis demonstrated a positive correlation between PANK1 and apoptosis-related genes (FAS, CASP8, and CASP9). Following transfection with PANK1 activation particles, Caki-1 and 786-O cells exhibited up-regulated expression of FAS, CASP8, and CASP9, accompanied by an increased apoptotic rate. These results suggest that PANK1 may function in KIRC cells through pathways associated with p53-mediated apoptosis or the exogenous apoptotic pathway.

However, several limitations need careful consideration in interpreting these findings. Firstly, the downstream pathways through which PANK1 influences KIRC development and progression remain to be fully elucidated. Understanding these molecular mechanisms could provide deeper insights into the specific role of PANK1 in the context of KIRC. Secondly, while our in vitro experiments provide valuable insights, validating the antagonizing effect of PANK1 on KIRC in in vivo models is crucial to establishing the clinical relevance of these observations. In vivo studies would offer a more comprehensive understanding of PANK1’s impact within the complex tumor microenvironment and its potential as a therapeutic target.

In conclusion, the comprehensive analysis revealed a significant down-regulation of PANK1 in both KIRC tissues and cell lines compared to noncancerous controls. Moreover, the correlation of PANK1 expression with clinical features in KIRC, such as pathologic stage and tumor stage, suggests its involvement in disease progression. Notably, higher PANK1 expression correlates with better disease-free and overall survival rates, particularly in advanced stages of the disease. Mechanistically, PANK1 overexpression suppresses KIRC cell invasion and migration by modulating EMT-related gene expression, while also promoting apoptosis through the up-regulation of pro-apoptotic genes. These findings underscore the potential clinical significance of PANK1 as a diagnostic marker, prognostic indicator, and therapeutic target in the management of KIRC.

Author contributions

Xiang Liu, Wei-Li Zhang and Song Gao conceived and designed the study. Ye-Min Qin, Wei-Li Zhang, and Peng Li performed the experiments and collected the data. Song Gao and Xiao-Yun Xiang analyzed and interpreted the data. Xiao-Yun Xiang and Peng Li drafted the manuscript. All authors critically reviewed and approved the final version of the manuscript.

Funding

No applicable.

Data availability

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

Declarations

Ethics approval and consent to participate

All experimental procedures were approved by the Ethics Committee of Lishui People’ s Hospital.

Consent for publication

All authors consent to the publication of this manuscript.

Competing interests

The authors declare no competing interests.