RNA sequencing (RNA-seq), somatic mutations, and related clinical data were downloaded from TCGA (https://portal.gdc.cancer.gov/), containing 11,069 samples from 33 types of cancer (25). RNA sequence data from 539 patients with KIRC and 72 normal tissues were also downloaded from TCGA database. RNA-seq data and patient clinical information (Workflow Type: HTSeq-FPKM) were acquired using UCSC Xena (https://xena.ucsc.edu/), an online tool for the exploration of gene expression and clinical and phenotypic data. UALCAN (http://ualcan.path.uab.edu) (26) and the Human Protein Atlas (HPA) (https://www.Proteinatlas.org/) (27) were used to analyze GMFB protein expression patterns and immunohistochemical results in normal kidney tissue and KIRC samples.

Survival and clinical phenotype data were extracted for each sample downloaded from TCGA database. Overall survival (OS) and disease-free survival (DFS) were evaluated to determine the relationship between Gmfb expression and patient prognosis. The Kaplan–Meier curve and Cox regression analyses were performed to assess the association between Gmfb expression and OS in KIRC patients. Subsequently, multivariate analysis was employed to estimate whether Gmfb expression was an independent prognostic factor for survival in patients with KIRC.

Gene expression profiling interactive analysis (GEPIA) (http://gepia2.cancer-pku.cn/#index) was used to confirm the correlation between Gmfb expression and clinicopathological information in multiple cancer types (28).

The top 2,000 upregulated and downregulated differentially expressed genes (DEGs) between the high and low Gmfb expression groups, defined by Spearman’s rho above 0.30, were used for enrichment analyses. Pathways within the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) terms were assessed for enrichment using the KEGG Orthology-Based Annotation System (KOBAS) platform (http://kobas.cbi.pku.edu.cn/) (29–32) and Metascape (https://metascape.org/gp/index.html#/main/step1) (33, 34).

CIBERSORTx (http://cibersort.stanford.edu/) is a deconvolution algorithm that predicts the cell type components of intricate tissues based on normalized gene expression profiles; its results are usually in agreement with true estimates for many types of cancer (35). As so, CIBERSORTx was used to evaluate the relative proportions of 22 infiltrating immune cell subtypes in KIRC and LIHC (35). The percentages of the 22 immune cell subtypes derived from CIBERSORTx were regarded as a correction at p < 0.05 (36). The relationship between the expression of 22 subtypes of tumor-infiltrating immune cells (TIICs) and the survival of KIRC and LIHC patients was conducted using GEPIA2021 (http://gepia2021.cancer-pku.cns/) (37). The results of the survival analysis were considered statistically significant at p < 0.05.

Protein–protein interaction (PPI) networks were constructed using the Search Tool for the Retrieval of Interacting Genes (STRING; http://string-db.org/) database (38) (version 11.5) and Metascape (http://metascape.org). Hub genes were identified using the CytoHubba plugin (39) in the Cytoscape software (40) (version 3.7.7). The maximal clique centrality (MCC) algorithm was employed to identify hub genes.

The human embryonic kidney cell line 293T purchased from American Type Culture Collection (ATCC) and Caki-2 purchased from the Fenghui Institute of Biotechnology were used in this study. The HEK293T and Caki-2 cells were separately maintained in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose and DMEM/nutrient mixture F12, respectively, supplemented with 10% fetal bovine serum (FBS) (Cat. No. 10091148, Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (Cat. No. 60162ES76, Yeasen, Shanghai, China) at 37°C and 5% CO₂.

The three pairs of small interfering RNA (siRNA) sequences of human Gmfb are shown in Supplementary Table 1A . A scrambled sequence was used as a siRNA control. The pcDNA-GMFB plasmid and pcDNA3.1 were kindly provided by Wan Sun. Caki-2 cells were transfected using Lipofectamine 2000 (Cat. No. T101, Vazyme, Nanjing, China), according to the manufacturer’s instructions. After transfection for 12, 24, and 48 h, cells were collected for subsequent experiments.

Caki-2 cells were lysed in radioimmunoprecipitation assay buffer (Cat. No. P0013B, Beyotime, Jiangsu, China) with protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA) to extract whole-cell proteins. Protein concentration was quantified using a bicinchoninic acid (BCA) kit (Cat. No. 20201ES76, Yeasen, Shanghai, China). A sample (30 μg) of each protein was then separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA), and blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. After incubation overnight at 4°C with primary antibodies against GMFB (1:1,000; Cat. No. 10690-1-AP, ProteinTech, Rosemont, IL, USA), GMFG (1:1,000; Cat. No. D121824, Sangon Biotech, Shanghai, China), p21 (1:1,000; Cat. No. 10355-1-AP, ProteinTech, Rosemont, IL, USA), p27 (1:1,000; Cat. No. #3686, CST, Danvers, MA, USA), and actin (1:10,000; Cat. No. 20536-1-AP, ProteinTech, Rosemont, IL, USA), membranes were incubated with a secondary antibody for 2 h. The secondary antibody used was horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:10,000; Cat. No. SA00001-2, ProteinTech, Rosemont, IL, USA). The proteins were visualized using enhanced chemiluminescence (ECL) (Cat. No. E412-02, Vazyme, Jiangsu, China). Tanon 5200S (Tanon Science & Technology, Shanghai, China) was used to evaluate the density of protein bands, and relative protein levels were quantified using ImageJ software (https://imagej.nih.gov/ij/).

Total RNA was extracted with TRIzol reagent (Cat. No. R401-01, Vazyme) and reverse transcribed into cDNA using the Revert Aid Reverse Transcription System (Takara, Dalian, China) according to the manufacturer’s protocol. PrimeScript RT polymerase (Cat. No. RR036A, Takara, Dalian, China) and SYBR Green Master Mix (Cat. No. FP205-02, Tiangen Biotech, Beijing, China) were used for conducting qRT-PCR on a LightCycler 96 Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Data were analyzed using the 2^−ΔΔCt method (24). Oligos were synthesized by Sangon Biotech. The oligo sequences are shown in Supplementary Table 1B .

Immunofluorescence analysis was performed to detect the colocalization of GMFB and Arp2/3 complex. 293T and Caki-2 cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 in phosphate-buffered saline (PBS) for 30 min, blocked with 3% BSA for 1 h, and incubated with primary antibodies against GMFB (1:200 Cat. No. 10690-1-AP, 60062-1-Ig, ProteinTech, Rosemont, IL, USA), Arp2 (1:50; Cat. No. D260865, Sangon Biotech, Shanghai, China), and Arp3 (1:50; Cat. No. sc-48344, Santa Cruz, Dallas, TX, USA) overnight with immunofluorescence buffer at 4°C. Cells were then incubated with secondary antibodies (Invitrogen, Waltham, MA, USA), goat anti-mouse IgG(H+L) cross-adsorbed secondary antibody, Alexa Fluor 488 (Cat. No. A-11001); goat anti-mouse IgG(H+L) cross-adsorbed secondary antibody, Alexa Fluor 555 (Cat. No. A-21422); goat anti-rabbit IgG(H+L) cross-adsorbed secondary antibody, Alexa Fluor 488 (Cat. No. A-11008); and goat anti-rabbit IgG(H+L) cross-adsorbed secondary antibody, Alexa Fluor 555 (Cat. No. A-21428) for 1 h at room temperature. After three washes for 10 min each with 1× PBS, cell sections were counterstained and mounted with DAPI Fluoromount-G (Cat. No. D9542, Sigma-Aldrich, St. Louis, MO, USA) and examined under a confocal microscope (Leica, Wetzlar, Germany).

Caki-2 cells were transfected with siRNA and pcDNA-GMFB using Lipo2000 (Cat. No. T101-01, Vazyme, Jiangsu, China). At 12, 24, and 48 h post-transfection, cell viability was tested using a Cell Counting Kit-8 (Cat. No. C0037, Beyotime, Jiangsu, China), according to the manufacturer’s instructions. This experiment was repeated four times.

Caki-2 cells were transfected with an empty vector or Gmfb overexpression (OE) plasmid. JC-1 staining (Cat. No. C2003S, Beyotime, Jiangsu, China) was performed to determine mitochondrial membrane potential (MMP), according to the manufacturer’s instructions. Cell visualization was performed using fluorescence microscopy (Leica, Wetzlar, Germany).

Caki-2 cells were seeded in six-well plates and grown to 80%–90% confluence. Confluent cell monolayers were scratched using a sterile 200-µl pipette tip and rinsed with PBS to remove scratched cells according to our previous work (24). Wound closure was observed after 24 h. The wound closure rate was calculated as follows: wound closure (%) = (the area of initial wound − the area of the final wound)/area of initial wound × 100.

The migration and invasion abilities of cells were assessed using transwell migration (without Matrigel) and invasion (with Matrigel) assays in 24 wells according to our previous work (24). Cell suspensions in an FBS-free medium were added to the upper transwell chambers (24-well, 8-μm pores; BD, Franklin Lakes, NJ, USA), whereas 600 µl of the medium was supplemented with 10% FBS and added to the lower transwell chambers. The cell count after positive staining with crystal violet indicated migration or invasion.

Detailed information on the clinical and gene expression data of the downloaded 539 samples is presented in Table 1 . The median age of participants was 61 years. Among the 539 patients, 353 were male (65.5%) and 186 were female (34.5%). The 269 samples of participants under the age of 60 accounted for 49.9% of all samples. The topographic distribution included 51.6% T1 (n = 278), 13.2% T2 (n = 71), 33.2% T3 (n = 179), and 2% T4 (n = 11) cases. Only 16 patients (6.2%) showed lymph node involvement, while 78 cases (15.4%) had distant metastases. Most patients (467, 87.8%) were white.

The contribution of Gmfb to pan-cancer OS and DFS in the GEPIA2 database was compared using the Mantel–Cox test. The criterion for significant differences was a false discovery rate (FDR) <0.05, also known as p-value adjustment. OS analysis showed that Gmfb expression was negatively correlated with the hazard ratio (HR) of KIRC but positively correlated with the HR of LIHC in pan-cancers ( Figure 1A ). This was consistent with a previous report (24), in which Gmfb expression was negatively correlated with the HR of KIRC but positively correlated with the HR of adrenocortical carcinoma (ACC) ( Figure 1A ). Based on the correlation of Gmfb with OS and DFS in pan-cancer analysis, GMFB may be a promising novel prognostic factor for KIRC.

Gmfb expression levels were analyzed in pan-cancer tissues using TCGA dataset. Analysis of unpaired samples of 63 types of cancer tissues showed that Gmfb expression differed in 15 types of cancer tissues ( Figure 1B ), and it was downregulated in KIRC (p < 0.001) ( Figure 1B ). In the paired KIRC samples, the expression of Gmfb was also downregulated (p < 0.001) ( Figure 1C ). Protein expression analysis indicated that GMFB was weakly expressed in KIRC (p < 0.001) ( Figure 1E ). To verify GMFB expression at the protein level, we used the immunohistochemical results of KIRC provided by the HPA database ( Figure 1F ), which were consistent with those obtained from TCGA database. Gmfb was generally downregulated in renal cancer tissues.

In vitro, we confirmed that the Gmfb mRNA ( Figure 1D ) and protein ( Figure 1G ) levels were significantly decreased in Caki-2 cells when compared with HEK293T cells, which was consistent with the results of immunostaining of GMFB in HEK293T and Caki-2 cells ( Figures 1H–I ). We also detected the colocalization of GMFB and Arp2/3, which were known interaction partners of GMFB ( Figures 1H,I ), supporting that GMFB was an intracellular protein.

The association between Gmfb expression and clinical features of KIRC patients is summarized in Table 2 . T stage (p < 0.001), M stage (p < 0.004), primary therapy outcome (p = 0.016), pathologic stage (p < 0.001), gender (p = 0.002), histological grade (p < 0.001), and OS events (p < 0.001) were all significantly associated with Gmfb expression.

The differential expression of Gmfb was examined according to age, gender, pathologic stage, T classification, N classification, M classification, and histological grade (G) of KIRC patients ( Figure 2 ). Expression of Gmfb was significantly downregulated in men compared to women (p = 0.004) ( Figure 2A ); it was also significantly associated with pathologic stage (stage IV compared to stage I, p = 0.002; stage III compared to stage I, p < 0.001) ( Figure 2B ), T stage (T3 compared to T1, p < 0.001) ( Figure 2C ), M stage (M1 compared to M0, p = 0.007) ( Figure 2D ), and histological grade (G4 compared to G2, p < 0.001; G3 compared to G2, p = 0.003) ( Figure 2E ). There was no significant relationship between Gmfb expression and age ( Figure 2F ) or N classification ( Figure 2G ).

OS analysis showed that Gmfb was a low-risk gene in kidney tissue (p = 0.00018) ( Figure 3A ). The Kaplan–Meier DFS analysis also demonstrated that patients with high Gmfb expression levels survived longer than patients with low Gmfb expression (p = 0.00013) ( Figure 3B ). Cox OS analysis demonstrated that a significant prognostic difference existed between the high and low Gmfb expression groups in both univariate (HR 0.541, 95% CI, 0.397–0.736, p < 0.001) and multivariate (HR 0.578, 95% CI, 0.374–0.892; p = 0.013) analyses ( Table 3 ). A receiver operating characteristic (ROC) curve was used to evaluate the prediction accuracy of intensity. The Gmfb score, in terms of the ROC curve, was 0.905 ( Figure 3C ), indicating that Gmfb was a very sensitive biomarker for KIRC.

The Sankey diagram further demonstrated the association between age, gender, Gmfb expression, and clinical status ( Figure 3D ). Each column represents a characteristic (gender; T, N, and M stages; and expression of Gmfb; different colors represent the various states of these characteristics, and lines represent the distribution of the same sample in other characteristic variables). As shown in the diagram, most female and male patients progressed to stage I. Most patients with stage I disease had high Gmfb expression and a high survival rate. Conversely, patients with more severe diseases tended to have lower Gmfb expression and lower survival rates. A nomogram of OS was constructed to integrate Gmfb expression and some prognostic factors, including T, N, and M classifications and age ( Figure 3E ). A higher nomogram score represented worse predictive probability. The calibration curve assessed the performance of the nomogram for Gmfb, and the C-index for OS was 0.755 ( Figure 3F ). In conclusion, this nomogram is a good model for predicting the survival of patients with KIRC.

To clarify the association between Gmfb expression and cell viability in KIRC, the effects of Gmfb OE and knockdown (KD) on the viability of Caki-2 cells were examined. First, we designed three siRNA sequences for Gmfb, and KD efficiency was determined in HEK293T by Western blotting. As shown in Figure 4A , the siRNA1 with the highest KD efficiency in HEK 293T cells was used for the subsequent experiment ( Figure 4A ). Then, Gmfb KD and OE in Caki-2 cells were confirmed by Western blotting ( Figures 4B, C ) and qRT-PCR ( Figures 4D, E ). In parallel, the expression of GMFG protein, a homolog of GMFB, remained unchanged after transfection with siGmfb or Gmfb OE plasmid, indicating that Gmfb KD specificity was reasonable ( Figures 4F, G ). After transfection with siRNA and OE plasmids at 12, 24, and 48 h, a significant increase in viability after Gmfb KD was obtained at 24 h (p < 0.05) and 48 h (p < 0.01) ( Figure 4H ), while a significant decrease in viability after Gmfb OE was observed at 24 h (p < 0.01) and 48 h (p < 0.001) ( Figure 4I ). These results indicated that GMFB expression level was negatively correlated with tumor cell viability. Moreover, we investigate the effect of altered Gmfb expression on the Caki-2 cell cycle. At 48 h post-transfection of siGmfb in Caki-2 cells, the expression levels of p21 (CDKN1A) and p27 (CDKN1B) significantly decreased when compared with those in the scramble group ( Figures 4J, K ). The downregulation of p21 and p27 was required for the cellular transition from quiescence to the proliferative state (41). Gmfb OE in Caki-2 cells led to a lower number of migrated cells than in the empty vector group ( Figures 4L, M ). Invasion assays showed that Gmfb OE inhibited Caki-2 cell invasion ( Figures 4L, N ) and significantly reduced wound closure ( Figures 4O, P ). Detection of MMP using the JC-1 assay revealed a shift from red to green fluorescence in Gmfb OE Caki-2 cells ( Figure 4Q ), indicating that a high level of GMFB led to MMP loss. Taken together, we proposed that Gmfb was a suppressor gene in KIRC.

To elucidate the molecular mechanism of GMFB in KIRC, Gmfb co-expressed DEGs were used for functional enrichment analysis. The 3493 DEGs identified based on |log2 fold-change| > 1 (adjusted p < 0.05) corresponded to 912 upregulated genes and 2,581 downregulated genes. Detailed information on the DEGs is presented in Supplementary Table 2 . DEG expression is shown in the volcano plot ( Figure 5A ), and the heatmap exhibits a corresponding hierarchical clustering analysis ( Figure 5B ). The details of the top 10 DEGs with upregulation and downregulation are listed in Table 4 and Supplementary Table 3 , respectively. Most of these DEGs have not been reported in previous KIRC studies, except for ALDH6A1 and NCR3LG1. Six DEGs with unknown biological functions and three non-coding RNAs were not identified, including AC003072.1, AC073896.4, AC108673.3, AP001505.1, AC012510.1, AC084036.1, PWAR5, SCARNA10, and SNORA53. Among these, the mRNA levels of seven upregulated and four downregulated DEGs with known functions were further validated by qRT-PCR. Due to the very high cycle threshold values for ZNF196 and SELENOP (38 to 39), we did not redesign the primer for the validation experiment. Consistent with the results of the meta-analysis, INAFM1, HCFC1R1, and METTL26 were downregulated, and ATP6V0D2, TBC1D14, OXCT1, ALDH6A1, FAM160A1, and NCR3LG1 were upregulated in Gmfb OE Caki-2 cells ( Figures 5C, D ). The details of the nine validated DEGs are listed in Table 4 , and the remaining 11 DEGs that were not validated are provided in Supplementary Table 3 .

The KEGG pathway and GO enrichment analyses using KOBAS are shown in Figure 6 . The neuroactive ligand–receptor interaction ranked 1 in the top 10 DEGs enriched KEGG pathways, followed by collecting duct acid secretion, linoleic acid metabolism, alpha-linolenic acid metabolism, and mineral absorption ( Supplementary Figure 1A ). GO enrichment analysis showed that cornification, extracellular region, and serine-type endopeptidase activity were the most enriched terms in biological process (BP), cellular compartment (CC), and molecular function (MF) categories, respectively ( Supplementary Figure 1B and Supplementary Table 4 ). The top ten enriched KEGG pathways, GO terms, and the heatmap of associated DEGs are shown in Figures 6A–D (upregulated DEGs) and Figures 6E–H (downregulated DEGs). Upregulated DEGs were most enriched in collecting duct acid secretion (KEGG pathway), cellular protein metabolic process (BP category of GO), an integral component of the plasma membrane (CC of GO), and serine-type endopeptidase activity (MF of GO) ( Figures 6A–D ). Downregulated DEGs were most enriched in mineral absorption (KEGG pathway), cornification (BP of GO), extracellular region (CC of GO), and antigen-binding (MF of GO) ( Figures 6E–H ). There was more overlap in the KEGG pathway and GO analyses between total DEGs and downregulated DEGs than between upregulated DEGs, indicating that the downregulated DEGs may play a more critical and dominant role in KIRC. To this end, we selected five genes involved in the mineral absorption pathway (MT2A, 1G, 1F, TF, and S100G) for qRT-PCR validation of the RNA-seq results. The qRT-PCR revealed that MT2A, MT1G, and MT1F were downregulated, whereas S100G and TF did not show a significant change in Gmfb OE Caki-2 cells ( Figures 6I, J ).

To clarify the links between terms, PPI network and MCODE component analyses of the top 2,000 DEGs ( Supplementary Figure 1C ), upregulated ( Figure 7A ) and downregulated ( Figure 7B ), were performed using Metascape. Supplementary Table 5 provides detailed data. The PPI network showed that the predominant GO annotations of MCODE1 were cornified envelopes and keratinization in the total DEGs. Among the DEGs associated with upregulated Gmfb, GO annotations of MCODE1 were mainly linked with nucleosome assembly, nucleosome organization, and chromatin assembly. Among the DEGs related to downregulated Gmfb, GO annotations of MCODE1 were specifically associated with G-protein-coupled receptor (GPCR) downstream signaling, GPCR signaling, and GPCR ligand binding ( Supplementary Table 5 ).

Upregulated DEGs were projected separately into the human PPI network in the STRING database, and the hub genes were filtered using the MCC algorithm ( Figure 8 ). The details of the top ten hub genes are shown in Supplementary Table 6 . The top six upregulated hub genes (HIST1H2BB, HIST1H1A, HIST1H4F, HIST1H3J, HIST2H2AB, and HIST1H1B) ( Figure 8B ) belong to the histone cluster family and are mainly involved in nucleosome assembly, nucleosome organization, and chromatin assembly. The top ten downregulated hub genes included SPRR1B, SPRR1A, IVL, SPRR3, SPRR2A, TGM1, SPRR2D, SPRR2E, PI3, and CASP14 ( Figure 8D ). Except for CASP14 and TGM1, these genes were mainly associated with keratinization, and none has been reported in KIRC. These results suggest that GMFB may play a vital role in modulating nucleosome structure, cellular senescence, mitotic prophase, and keratinization in KIRC.

Based on the importance of TME, the relationship between immunity and Gmfb expression was further explored. Tumor RNA-seq data (retrieved from TCGA) of 33 different tumor patients and matched normal tissue samples from the Genomic Data Commons (GDC) website were downloaded to analyze the relationship between Gmfb expression and 22 types of TIICs. The heatmap of KIRC and LIHC analysis indicated Gmfb mRNA expression significantly correlated with TIICs ( Figure 9A ). The analysis by CIBERSORTx showed that the Gmfb mRNA expression positively correlated with the infiltration of T CD4+ memory resting cells (p = 0.00007, R = 0.172), monocytes (p = 5.68E−06, R = 0.196), mast resting cells (p = 0.047, R = 0.086), mast activated cells (p = 0.0017, R = 0.136), macrophage M2 cells (p = 1.81E−09, R = 0.257), macrophage M1 T cells (p = 0.0028, R = 0.13), eosinophil (p = 7.74E−11, R = 0.278), and B naïve cells (p = 9.95E−08, R = 0.229) but negatively correlated with the infiltration of regulatory T cells (Tregs) (p = 1.09E−31, R = −0.479), T follicular helper cells (TFH) (p = 1.43E−15, R = −0.337), T CD8+ cells (p = 4.97E−14, R = −0.319), natural killer activated cells (p = 7.42E−13, R = −0.305), B plasma cells (p = 0.0058, R = −0.12), and B memory cells (p = 0.00001, R = −0.185) ( Figure 9A ). However, our previous work showed that higher expression levels of Gmfb predicted poor prognostic outcomes in LIHC (26). To explore the possible mechanism underlying the differential effects of Gmfb on LIHC and KIRC, the correlation between Gmfb expression and TIIC in LIHC was analyzed. As shown in Supplementary Figure 2 , the infiltration of B memory cells, mast activated cells, and neutrophils was significantly increased in LIHC ( Supplementary Figures 2A–B ) and was uniformly correlated with worse OS ( Supplementary Figures 2C–E ). None of these three immune cell types affected the OS of KIRC patients ( Supplementary Figure 3 ). Moreover, the levels of B memory cells, mast activated cells, and neutrophils did not correlate significantly with Gmfb expression in the LIHC group ( Figure 9A ).

The association between TIICs and KIRC tumor tissues was examined. The relationship between the 22 TIICs and KIRC prognosis was investigated using GEPIA2021. The Kaplan–Meier survival curves (p < 0.05) are shown in Figures 9B–F , and further results are summarized in Supplementary Figure 3 . The fractions of macrophages M2 cells (p = 0.05) and resting mast cells (p = 0.000403) were associated with improved survival ( Figures 9B, C ), but high levels of TFH cells (p = 0.05), Tregs (p = 0.00138), and B plasma cells (p = 0.00844) were associated with worse outcomes ( Figures 9D–F ).

Taken together, the higher expression of Gmfb in KIRC predicted a better prognosis, partially due to the optimal TME.