Nc‐RNA‐mediated low expression of AZIN1 correlated with unfavorable prognosis in kidney renal clear cell carcinoma

Abstract Objective Kidney renal clear cell carcinoma (KIRC, ccRCC) is the most common type of renal cancer with high recurrence and mortality. It has long been recognized that Antizyme inhibitor 1 (AZIN1) serves as a pro‐oncogenic molecule in multiple cancers. However, the clinicopathological features of AZIN1 in KIRC remain unexplored. Materials and Methods The Cancer Genome Atlas (TCGA, TIMER, and GEPIA) were employed for pan‐cancer expression and survival analysis of AZIN1, indicating the unique anti‐tumor role of AZIN1 in KIRC. The expression and clinical characteristics of AZIN1 in KIRC were further proven via Human Protein Atlas and TCGA. single‐sample GSEA was employed to investigate the immune infiltration of AZIN1. Then the downstream pathways were illustrated via the LinkedOmics, Metascape, and Cytoscape databases. The possible upper regulating noncoding RNAs (ncRNAs) were analyzed from five programs‐TargetScan, StarBase, miRanda, PITA, and miRmap. Results AZIN1 is downregulated in KIRC patients. Lower levels of AZIN1 were linked with unfavorable outcomes in KIRC patients. The AZIN1 expression was positively related to immune cell infiltration in KIRC. We also elucidated a possible upstream regulatory ncRNA of AZIN1 in KIRC namely STK4‐AS1/AC068338.2‐miR‐106b‐5p‐AZIN1 axis as well as the downstream signaling pathways. Conclusion This study illustrated the unique anti‐tumor role of AZIN1 in KIRC and provided potential value for guiding immunotherapy and targeted therapy.


| INTRODUCTION
Antizyme inhibitor 1 (AZIN1) serves as a crucial mediator in polyamine metabolism.Antizyme inhibitor (AZI) increases the ornithine decarboxylase (ODC) activity, accelerating intracellular polyamine biosynthesis. 1Polyamines, which include spermidine, putrescine, and spermine, are crucial amino acid metabolites participating fundamental intracellular processes. 2Polyamine metabolism is frequently dysregulated in many neoplastic states.Elevated polyamines can play a major role in carcinogenesis via participating in cancer-driving signaling pathways-PI3K-AKT, RAS, and WNT pathways etc. 3 However, recent studies indicate that polyamines have also been reported in regulating the antitumor response. 4,5Given that AZIN1 participates in polyamine metabolism, AZIN1 has been mostly recognized as a pro-oncogenic molecule in cancers. 6idney renal clear cell carcinoma (KIRC), which is featured as metabolic reprogramming, accounts for approximately 70%-80% of all subtypes of renal cancers. 7As KIRC is insensitive to tranditional radiotherapy and chemotherapy, surgical treatment, targeted therapy, and immunotherapy have brought clinical benefits for KIRC patients.Yet KIRC patients are still at high risk of stepping into relapse or metastasis stages after the initial nephrectomy, which is associated with high mortality. 8,9Limited options left KIRC patients with poor prognosis.Therefore, finding new biomarkers not only can provide a better understanding of the molecular mechanisms but also is the key to implementing individualized treatment and predicting the prognosis of KIRC.
In the current study, we first showed that AZIN1 is downregulated in KIRC patients and high AZIN1 is associated with favorable clinical outcomes in KIRC.Next, the upperstream noncoding RNAs (ncRNA)-associated regulation of AZIN1 was explored in KIRC.Moreover, the relationships of AZIN1 expression with biomarkers of immune cells as well as immune cell infiltration in KIRC were illustrated.In conclusion, this novel antitumor role of AZIN1 in KIRC was identified in this study.

| TIMER data mining and GEPIA database analysis
The mRNA expression data of 32 cancer types were obtained from TIMER1.0 (https:// cistr ome.shiny apps.io/ timer/ ) based on The Cancer Genome Atlas (TCGA).The differential expression analysis of AZIN1 was then performed on the normalized data.The correlation of AZIN1 expression with immune cell infiltration level in KIRC was also analyzed by TIMER. 10 GEPIA (http:// gepia.cance r-pku.cn/ ) was employed for gene expression between cancer and normal tissues, 11 as well as for the prognostic analysis of candidate AZIN1 in KIRC.Univariate analysis of AZIN1 in clinical KIRC samples was performed via GEPIA.

| Immunohistochemistry analysis
The immunohistochemical analysis of AZIN1 levels in KIRC and normal tissues was directly obtained from Human Protein Atlas (HPA, https:// www.prote inatl as.org/ ).

| Patient samples
We used the KIRC samples from Huadong Hospital Affiliated to Fudan University in June 2024.The study protocols were approved by the ethical committee of Huadong Hospital (approval number: 2024K178).The patients provided written informed consent.This clinical investigation was conducted according to the principles of the Declaration of Helsinki.

| Cell culture
Non-cancer kidney cell line (HA1E), KMRC1, and OSRC2 were cultured in DMEM (Dulbecco's Modified Eagle Medium) and UMRC3 were mainteined in minimal essential medium.All cell lines were supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine and 100 IU/mL penicillin/streptomycin.All cells were cultured at 37°C in a humidified atmosphere with 5% CO 2 .

| RNA extraction and real-time polymerase chain reaction
Total RNA was isolated from cells using an RNA isolation kit (Beyotime, Shanghai, China) following the manufacturer's instructions.Then the extracted RNA was reverse-transcribed to complementary DNA using the PrimeScript RT reagent kit (Takara Bio, Kusatsu, Japan).Quantitative real-time PCR was applied using a TB Green Premix Ex Taq II kit (Takara Bio).Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.Each experiment was performed in triplicate.Differences in gene expression level, expressed as fold changes, were calculated using the 2−ΔΔCt method.The forward and reverse primers for GAPDH were 5′-TGACTTCAACAGCGACACCCA and 5′-CACCCTGTTGCTGTAGCCAAA, respectively.

| Immune infiltration analysis by single-sample GSEA
The GSVA R package was employed for single-sample GSEA (ssGSEA).Relative tumor infiltration levels of 24 immune cell types were quantified by interrogating expression levels of genes in published signature gene lists. 12Infiltration levels were compared between AZIN1 high-and low-groups.

| Analysis of gene cluster identification and associated pathways of AZIN1
The LinkedOmics database was introduced to obtain the different-expressed genes (DEG) of AZIN1.Then Using the Metascape database and Cytoscape, and protein cluster analysis of DEGs was conducted using a molecular complex detection plug-in to explore key signaling pathways in biological networks and the protein-protein interaction network.
2.8 | Candidate miRNA and lncRNA screening Upstream MicroRNAs (miRNAs) of AZIN1 were predicted by five target gene prediction bioinformatic programs-TargetScan, StarBase, miRanda, PITA, and miRmap.The candidate miRNAs of AZIN1 which appeared in all five platforms were able to be included in further analyses.Besides, StarBase (http:// starb ase.sysu.edu.cn/ ) 13 was applied for expression correlation of miRNA-AZIN1, as well as prediction of upperstream lncRNAs of miR-106b-5p and its expressions in KIRC and normal control.

| Statistical analysis
We employed R software (version 4.0.0) with appropriate packages and GraphPad Prism v8.0 software.The comparison of two groups was performed by a two-tailed student's t-test.The Kaplan-Meier method and log-rank test were used for survival analysis in patients.The optimum cut-off value for eight lncRNAs expression was generated using the survfit function via the R package "survminer."All other cut-off values were showed as medians.p-value <0.05 was considered as statistically significant.

| AZIN1 is downregulated in KIRC patients and high AZIN1 is associated with favorable clinical outcomes in KIRC
The pan-cancer analysis of AZIN1's expression (TPM standardized data) was shown in Figure 1A using TIMER 1.0.AZIN1 mRNA levels were markedly downregulated in five types of cancer, including GBM, THCA, KICH, KIRC, and KIRP.However, the mRNA expressions of AZIN1 were significantly higher in BRCA, CHOL, COAD, ESCA, HNSC, LIHC, LUAD, LUSC, and STAD when compared with normal tissues.High expression of AZIN1 was also observed in SKCM metastatic patients.For those cancers lacking normal tissues in the TIMER dataset, we employed the GEPIA database to illustrate the AZIN1 gene expression.These data showed a significant decrease in AZIN1 expression in DLBC, LGG, SKCM, and TCGT.(Figure 1B).
These data showed that the AZIN1 expression levels seem to vary depending on the cancer type, indicating its diverse roles in cancer.Interestingly, AZIN1 was lower in all types of kidney cancer, as seen in KICH, KIRC, and KIRP.
Next, the prognostic value of AZIN1 was validated in those cancers using GEPIA.Patients from each cancer type were then divided into high-and low-expression groups based on the median AZIN1 expression.The results revealed that low AZIN1 expression was associated with an unfavorable overall survival (OS) in KIRC.However, AZIN1's downregulation was associated with better OS in KIRP (Figure 1C).No statistical significance of AZIN1 expression was observed in other cancer types.
For disease-free survival (DFS), lower expression of AZIN1 in BLCA and KIRP indicated longer DFS, but KIRC patients with lower AZIN1 expression indicated poorer clinical outcomes (Figure 1D), which is consistent with AZIN1's prognostic role of OS in KIRC.Given the above, decreased expression of AZIN1 was identified in KIRC patients, which was correlated with unfavorable prognosis by a combination of OS and DFS.Taken together, AZIN1 seems to play an anti-tumor role and serves as a favorable prognostic factor in KIRC.

| AZIN1 is downregulated in KIRC samples
We further confirmed the protein expression of AZIN1 in KIRC patients and normal tissues are illustrated using HPA.The immunohistochemical analysis showed that compared with three normal tissues, AZIN1 protein expressions were low in three KIRC tissues (Figure 2A).Low levels of AZIN1 protein expression were negatively correlated with advanced clinicopathological features in KIRC (Figure 2B).Based on the TCGA database, patients with stage III/IV RCC exhibited lower levels of AZIN1 expression than patients with I/II disease.T stage and histologic grade showed consistent AZIN1 expression in KIRC.In terms of OS, KIRC survivors showed higher levels of AZIN1 than patients who died of KIRC.We then tested the AZIN1 mRNA expression via qPCR in HA1E and three KIRC cell lines (KMRC1, OSRC2, and UMRC3) in Figure 2C.The mRNA levels of AZIN1 in clinical specimens from the tumor samples and paired normal tissues of seven patients with KIRC are shown in Figure 2D.Furthermore, univariate Cox regression analysis showed that AZIN1, TNM stage, serum calcium, and hemoglobin levels were significantly associated with OS in KIRC patients (p < 0.05; Table 1).Besides, T and M stage, as well as AZIN1 levels were independent prognostic factors for OS in KIRC patients sourced from the TCGA database in the multivariate Cox regression analysis (p < 0.05; Table 1).
Taken together, we further proved that low expression of AZIN1 in KIRC was linked with unfavorable clinical outcomes, indicating the antitumor character of AZIN1 in KIRC.We then investigated the further regulatory mechanism of AZIN1 in KIRC.

| AZIN1 downstream signaling pathway in KIRC
To investigate the possible downstream signaling pathways of AZIN1, the linkedomics database was used to identify associated genes of AZIN1 in KIRC (Figure 3A).The heatmap for positively correlated genes was showed in Figure 3B while negatively correlated genes were illustrated in Figure 3C.Then the enrichment analysis of positively enriched genes was performed using Metascape.Protein post-translational modifications (ER to Golgi transport, ubiquitin-dependent protein catabolic process) were highly enriched (Figure 3D and Table 2), which is consistent with the primary role of AZIN1 in metabolism.Moreover, the results also highlighted that AZIN1 influences the adaptive immune system and neutrophil degranulation.The top 200 positively correlated genes were then depicted with shared functional networks in the same color using Cytoscape as illustrated in Figure 3E-G, including the adaptive immune system and ER-to-Golgi transport system, etc.Interestingly, AZIN1 seems to negatively participate in certain metabolic and immune pathways, such as peptidyl-lysine modification and antigen-processing events (Figure S1 and Table S1), indicating AZIN1 might regulate the metabolic and immune signaling in a more sophisticated manner.

| Correlation analysis between mRNA expression of AZIN1 and immune infiltration
Polyamines have been knowed to be associated with immune system regulation and altered polyamine metabolism may lead to immune suppression or anti-tumor immune response depending on the cancer type.The prognosis of KIRC is closely related to immune infiltration and activation of immune cells. 14AZIN1 is essential for polyamine homeostasis.AZIs have been considered to function as positive regulators of polyamine levels.The correlation between AZIN1 expression and markers of immune cells was then further validated.With the ssGSEA algorithm, we analyzed the tumor immune microenvironment of high and low AZIN1 groups in the TCGA cohort.(Figure 4A).Most immune cells were highly infiltrated in the low-AZIN1 group, such as Treg cells, NK CD56 bright cells, and cytotoxic cells, whereas macrophages, mast cells, DC cells, neutrophils, Tcm, Tem, Tgd, T helper cells, Th1 cells, and Th2 cells were highly enriched in the high-AZIN1 group.The correlation between AZIN1 levels and immune cell infiltration levels was also analyzed.As illustrated in Figure 4B, AZIN1 expression was positively associated with all analyzed immune cells, including macrophage, neutrophil, dendritic cells, as well as B cells, CD8 + T cells, and CD4 + T cells in KIRC.The significant change in infiltration level of CD8 T cells and neutrophils under various copy numbers of AZIN1 in KIRC was observed (Figure 4C).As provided in Table 3, AZIN1 was significantly positively correlated with M2 macrophage's biomarkers (CD163, VSIG4, and MS4A4A), CD4 + T cell's biomarkers, monocyte's biomarkers (CD86 and CSF1R), M1 macrophage's biomarkers (NOS2 and PTGS2), neutrophil's biomarkers (ITGAM and CCR7), and dendritic cells' biomarkers (HLA-DRA, HLA-DPA1, HLA-DPB1, NRP1, and CD1C).It showed that KIRC samples displayed a significant increase of monocytes, T A B L E 2 Functional enrichment analysis of positively correlated genes.

| Analysis and prediction of miRNAs of AZIN1
It is well recognized that non-coding RNAs (ncRNAs), with no capability in encoding proteins, are highly involved in regulating gene expression.miRNAs are small ncRNAs that generally repress target gene expression. 15ysregulated lncRNAs are also reported to be involved in carcinogenesis in KIRC. 16Here, we intended to identify these lncRNA-miRNA regulatory pathways of AZIN1 involved in KIRC patients.First, we analyzed upperstream miRNAs which could potentially bind to AZIN1 via online prediction databases including TargetScan, miRmap, PITA, miRanda and StarBase, and finally found 117 overlapping miRNAs (Figure 5A and Table S2).Among the 117 miRNAs, we found that six miRNAs were negatively correlated with AZIN1 expression from the website (http:// starb ase.sysu.edu.cn/ ).The six miRNA levels in KIRC patients were demonstrated in TCGA samples and only miR-106b-5p is highly expression in KIRC samples which may lead to the lower expression of AZIN1 in KIRC (Figure S2A and Figure 5B,C).In 517 KIRC samples, miR-106b-5 was negatively correlated with AZIN1 expression.The area under the curve was calculated for the diagnostic value of miR-106b-5p as indicated in Figure S2B.Moreover, KIRC patients with higher miR-106b-5p expression possessed poorer OS, progression-free survival and disease-specific survival (Figure 5D-F).According to these results, miR-106b-5p might serve as the most potential miRNA that regulates AZIN1 expression in KIRC.

| Analysis and prediction of upstream lncRNAs of miR-106b-5p
Next, the upstream lncRNAs of miR-106b-5p were predicted using the starBase database.From all the lncRNAs, only eight lncRNAs are downregulated in KIRC samples compared with normal tissues (Figure 6A-H and Table S3).The prognostic analysis of these lncRNAs in KIRC were then assessed.As illustrated in Figure 6I-P, only overexpressed STK4-AS1 and AC068338.2indicated a better prognosis.Besides, both of them were independent prognostic factors for OS among KIRC patients (Tables S3 and S4).Given that lncRNA negatively correlated with miRNA in cancer, STK4-AS1, and AC068338.2were screened as candidate upstream ncRNAs which negatively regulate miR-106b-5p expression in KIRC, suggesting that STK4-AS1 and AC068338.2as an independent risk factor and preclinical marker in KIRC and may be associated with KIRC pathogenesis (Tables S4  and S5).Taken above, the graphical representation of STK4-AS1/AC068338.2-miR-106b-5p-AZIN1 axis in KIRC is illustrated in Figure 7.

| DISCUSSION
Here, our study first reported the anti-cancer role of AZIN1 in KIRC.We identified that AZIN1 played a critical role in KIRC among all types of cancers.AZIN1 is downregulated in KIRC and high expression of AZIN1 was associated with favorable outcomes in KIRC patients.Further, AZIN1 performance in immunocytes infiltrated was demonstrated.Finally, we elucidated an upstream regulatory mechanism of AZIN1 in KIRC namely STK4-AS1/AC068338.2-miR-106b-5p-AZIN1 axis, as well as the downstream signaling pathways.To our knowledge, this is the first time to illuminate the anti-oncogenic function of AZIN1 in KIRC.
AZIN1 has been reported to play an essential part in polyamine metabolism.In polyamine biosynthesis, the ratelimiting process is that Ornithine is converted to putrescene via ODC.Ornithine decarboxylase antizymes (OAZs) usually target ODC for proteasomal degradation.However, AZINs proved to be biochemically similar to ODC, binding to OAZs with higher affinity instead.This rescues ODC activity leading to increased polyamine synthesis.It is worth noting that, between the two isoforms of AZINs, AZIN1 showed increased binding with OAZs than AZIN2. 6aken together, higher expression of AZIN1 is correlated with increased polyamine levels via maintaining the ODC activation.
Polyamines and their metabolites are required for cell proliferation, including the fundamental protein and nucleic acid synthesis as well as cell-to-cell communications. 17Besides, crosstalks between polyamine metabolism and oncogenic pathways, including RAS-MAPK, and PI3K-mTOR pathway, have been reported in multiple cancers.Thus, elevated polyamines are usually recognized as biomarkers of cancer.Given the above, AZIN1 acts as a pro-oncogenic role in most cancers by increasing polyamine levels. 18nterestingly, AZIN1 seems to play quite the opposite role in KIRC in this study.Recent data has provided greater insight into the anti-tumor role of polyamines.
KIRC is an aggressive malignancy characterized by metabolic reprogramming. 74][25] It's worth noting that AZIN1 A-to-I editing, triggered by renal inflammation, not only enhances polyamine biosynthesis but also engages glycolysis and nicotinamide biosynthesis, promoting recovery phenotype. 26Yet, limited knowledge of AZIN1's role in glycolysis nicotinamide biosynthesis in KIRC still requires further investigation.It is worth noting that KIRC cells have also been shown to take up excessive levels of polyamines from the tumor microenvironment in hypoxia states. 27Dysregulated metabolic and oncogenic signaling pathways also endow tumor cells significantly increasing polyamine synthesis and accumulation even more. 28hese metabolic changes make KIRC cells particularly vulnerable to polyamine toxicity than other tumor cells. 29urthermore, another research work uncovered that CA9 knockdown led to the accumulation of putrescine which was toxic to KIRC cells, inhibiting KIRC cell growth eventually. 30Collectively, these reaffirmed that increased polyamines resulted in the opposite of the Warburg effect and inhibition of tumor cells, which might be the reason for the anti-tumor role of AZIN1 in KIRC.
As for miRNA, miR-106b-5p functions as a prooncogenic role to mediate KIRC cell migration and invasion by targeting PDCD4. 31In another study, it has also been shown that downregulation of miR-106b-5p made KIRC/CRC-derived cells more vulnerable to anti-tumor drugs used in the clinic. 32These studies indicated miR-106b-5p exerts pro-oncogenic function in KIRC not only through AZIN1 as a downstream target but also other genes, indicating the pivotal role of miR-106b-5p in KIRC, calling for a more comprehensive study.
As for the immune system, polyamines have been widely involved in the activation, proliferation, and differentiation of multiple immune cells. 28,33For instance, Tregs and M2-typed macrophages can facilitate the immunosuppressive microenvironment, contributing to tumor progression. 346][37] Emerging evidence indicates that the activation of specific metabolic pathways plays a crucial role in regulating angiogenesis and inflammatory responses. 38,39][42][43][44] Immune dysfunction has been closely linked to cancer progression and prognosis in KIRC. 45AZIN1 can modulate immune cell infiltration and regulate immunoflogosis.Features of the tumor microenvironment heavily affect disease biology and may affect responses to systemic therapy.Accumulating data continue to highlight activation of specific metabolic pathway have a pivot role in regulating angiogenesis and inflammatory signatures in KIRC. 38,46A recent study proved that tumorinfiltrating Tregs inhibited anti-tumor activity in KIRC patients. 47In our study, Tregs were highly associated with low expression of AZIN1 in KIRC (Figure 4A).The results also indicated that low levels of AZIN1 predict dismal prognosis in KIRC (Figure 1A,B), which might be associated with Tregs via dampening the immune system in KIRC discussed above.Our studies indicated that the possible mechanism of the anti-tumor effect of increased AZIN1 and polyamine levels in KIRC might be the result of the production of toxic metabolites as well as their influence on cancer immunity.However, there are still several limitations to this study.First, while our research included comprehensive data analysis, the lack of wet lab experiments conducting functional assays to investigate the effects of AZIN1 modulation on KIRC cell behavior, and validating the proposed regulatory mechanisms involving ncRNAs limits the confirmation and validation of our findings.We just barely scratched the surface of our understanding of the underlying mechanisms of AZIN1 in KIRC.To what extent AZIN1 levels affect polyamines and immune infiltration in KIRC is unclear.Second, lack of basic experiments and animal studies calls for future analysis for validation of expression and prognosis of AZIN1 in KIRC.Further investigations are needed to understand the precise molecular mechanisms which made AZIN1 unique in KIRC, as different from other cancers.Third, instead of ncRNAs, more regulatory mechanisms of AZIN1 in KIRC are worth exploring.Given that multiple biomarkers hold promise for RCC staging and drug efficacy evaluation, the identification of AZIN1 as a potential biomarker presents a new-emerging target of diagnostic utility and prognostic value, including early detection, risk stratification, and treatment optimization in KIRC patients.

| CONCLUSION
In conclusion, our study provided that AZIN1 is downregulated in KIRC and high levels of AZIN1 were correlated with favorable outcomes among KIRC patients.This study may serve as a starting point for future investigation of AZIN1 in KIRC, providing potential value for guiding immunotherapy and targeted therapy.

F I G U R E 1
The expression and survival analysis of Antizyme inhibitor 1 (AZIN1) between multiple cancers.(A) AZIN1 expression in tumor and normal tissues in 34 types of cancers via the TIMER database.(B) AZIN1 expression between tumor and normal tissues in eight types of cancers via GEPIA database.(C) The overall survival (OS) plot of statistical significance of AZIN1 in Kidney renal clear cell carcinoma and KIRP.(D)The DFS plot of statistical significance of AZIN1 in BLCA, KIRC, and KIRP.*p < 0.05, **p < 0.01, and ***p < 0.001.

F I G U R E 2
Antizyme inhibitor 1 (AZIN1) expression in clinical samples in kidney renal clear cell carcinoma (KIRC).(A) Immunohistochemistry analysis in tumor tissues and corresponding normal tissues is shown by HPA.Scale bars: 100 μM (B) AZIN1 expression between different stages and associated with clinical outcome in KIRC patients using TCGA database.(C) Levels of AZIN1 mRNA expression in a non-cancer kidney cell line (HA1E) and three KIRC cell lines (KMRC1, OSRC2, and UMRC3).(D) Real-time PCR analysis of AZIN1 gene expression in the tumor samples and paired normal tissues of seven KIRC patients.*p < 0.05, and **p < 0.01.

F
I G U R E 3 Antizyme inhibitor 1 (AZIN1) downstream signaling pathways.(A) Associated genes of AZIN1 are displayed via linkedomics.(B) Positively correlated genes of AZIN1 are shown in the heatmap.(C) Negatively correlated genes of AZIN1 are shown in the heatmap.(D) Metascape database was used to analyze the GO enrichment results of differentially expressed genes screened based on AZIN1 expression.(E) Cytoscape was used to show the correlated genes of AZIN1.(F) Functional networks of AZIN1 were illustrated in modes.(G) Functional networks of AZIN1 were described.| 7of 14 ZENG et al.

F I G U R E 4
The relationship of immune cell infiltration with Antizyme inhibitor 1 (AZIN1) level in kidney renal clear cell carcinoma (KIRC) (A) Enrichment analysis of immune cells between high and low AZIN1 expression.(B) The correlation of AZIN1 expression with B cell, CD8 + T cell, CD4 + T cell, macrophage, neutrophil, or dendritic cell infiltration level in KIRC.(C) The infiltration level of various immune cells under different copy numbers of AZIN1 in KIRC.ns, not significant.*p < 0.05, **p < 0.01, and ***p < 0.001.

F I G U R E 6
The expression level and OS plots of eight lncRNAs associated with miR-106b-5p.(A-H) The expression of eight lncRNAs in kidney renal clear cell carcinoma (KIRC) and control normal samples were determined by the starBase database.(I-P) The association between the expression of eight lncRNAs and overall survival was assessed by the Kaplan-Meier plot.
Correlation analysis between AZIN1 and various gene markers of immune cells in the TCGA cohort.