TIMM17A overexpression in lung adenocarcinoma and its association with prognosis

Lung adenocarcinoma (LUAD), a leading cause of cancer-related mortality worldwide, demands a deeper understanding of its molecular mechanisms and the identification of reliable biomarkers for better diagnosis and targeted therapy. Leveraging data from the Cancer Genome Atlas (TCGA), the Clinical Proteomic Tumor Analysis Consortium (CPTAC), and the Human Protein Atlas (HPA), we investigated the mRNA and protein expression profiles of TIMM17A and assessed its prognostic significance through Kaplan–Meier survival curves and Cox regression analysis. Through Gene Set Enrichment Analysis, we explored the regulatory mechanisms of TIMM17A in LUAD progression and demonstrated its role in modulating the proliferative capacity of A549 cells, a type of LUAD cell, via in vitro experiments. Our results indicate that TIMM17A is significantly upregulated in LUAD tissues, correlating with clinical staging, lymph node metastasis, overall survival, and progression-free survival, thereby establishing it as a critical independent prognostic factor. The construction of a nomogram model further enhances our ability to predict patient outcomes. Knockdown of TIMM17A inhibited the growth of LUAD cells. The potential of TIMM17A as a biomarker and therapeutic target for LUAD presents a promising pathway for improving patient diagnosis and treatment strategies.


Survival rate and ROC analysis
We used the 'survival' and 'survminer' packages for Kaplan-Meier survival curve analysis and survival ROC analysis within the R software framework (ver.4.1.3).Through these analytical procedures, we were able to derive an AUC value that served as a reliable metric for evaluating the accuracy of our predictions.The ROC curves were generated using analytical tools available through the xiantao academic website (https:// www.xiant ao.love/), facilitating precise and effective visualization of our diagnostic predictions 19 .

HPA database and CPTAC database
In the present inquiry, an approach was employed, whereby immunohistochemical chips derived from the Human Protein Atlas (HPA) database 20 (located at https:// www.prote inatl as.org/) were utilized in conjunction with protein expression data obtained from the Clinical Proteomic Tumor Analysis Consortium (CPTAC), which was sourced from the University of Alabama Cancer Database (UALCAN) (found at https:// ualcan.path.uab.edu/ index.html 21 .

Nomogram model
To predict the 1-year, 3-year, and 5-year overall survival (OS) rates for patients with lung adenocarcinoma (LUAD), nomogram models were carefully constructed using the "rms" package in R statistical software (version  4.1.3).These models, designed to consider various factors affecting survival rates, were then evaluated for their reliability and accuracy through the plotting of calibration curves.This step allowed for a detailed assessment of the model's predictions against observed outcomes, ensuring the models' accuracy and reliability 22 .

Gene set enrichment analysis (GSEA)
To thoroughly investigate the multifaceted and complex functions of TIMM17A within intricate signaling pathways, computational tool GSEA software was employed, utilizing the TCGA LUAD dataset.In order to ensure the robustness and significance of the study findings, 1000 sequencing tests were conducted.Through these numerous tests, statistically significant pathways with a remarkable False Discovery Rate (FDR) q value of less than 0.05 were pinpointed and extensively analyzed 23 .

Univariate and multivariate Cox regression analysis
To thoroughly investigate the independent factors influencing the overall survival (OS) rate in patients with lung adenocarcinoma, we applied standard univariate and multivariate Cox regression analyses.This approach was implemented to meticulously scrutinize the impacts of pivotal variables, namely age, gender, T, N, M, and TIMM17A expression levels, on the prognosis of LUAD, with utmost scrupulousness 19 .

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Complementary DNA (cDNA) was synthesized using 20 μl of HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing), following the manufacturer's instructions.qRT-PCR was then performed as per the kit's guidelines to ensure accuracy and reproducibility 24 .In order to evaluate the expression levels of the target genes, specific primer sequences were utilized: GAPDH Forward: TGC ACC ACA ACT GCT TAG C; GAPDH Reverse: GGC ATG GAC TGT GGT CAT GAG; TIMM17A Forward: GCT CCA CAG TTA GGA GGT AGC;

Patient samples
85 pairs of fresh tumor and paracancerous tissue samples from patients with LUAD were obtained from YiZheng People's Hospital.None of the patients had undergone radiotherapy, chemotherapy, or immunotherapy prior to sample collection.This study received approval from the hospital's ethics committee, and written informed consent was provided by all participating patients (No. 2019-27).

Cell culture and transfection
A549 cells were purchased from Pricella (Wuhan, China).The cells were cultured in Ham's F-12k medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.The culture was maintained at 37 °C in a incubator with 5% CO 2 .After passaging cells and culturing them in a six-well plate for 24 h, transfection was performed when the cell density reached 60-70%.Transfection of TIMM17A siRNA (Ruibo, Guangzhou) was carried out using Lipofectamine 3000 transfection reagent (Invitrogen, USA).Cells were collected 48 h post-transfection to extract RNA for assessing transfection efficiency, all experiments performed in triplicate 25 .

Cloning experiment and edu assay
Cells were collected 48 h post-transfection, resuspended after centrifugation, and counted using a cell counting plate.Cell density was adjusted, and 500 cells were seeded per well in a six-well plate.Each well was supplemented with 2 ml of complete medium.The medium was changed every 3-5 days, and the cells were cultured for approximately 2 weeks.After removing the six-well plate, the culture medium was aspirated, and cells were fixed with 4% paraformaldehyde at room temperature for 30 min.After fixation, crystal violet staining was performed for 5 min, followed by washing with PBS for 2-3 times.The plates were air-dried, observed, and photographed.Edu experiments were conducted following the instructions of the Edu-488 Cell Proliferation Assay Kit (Bi Yun Tian, Shanghai) 26 , with all experiments performed in triplicate.

Statistical analysis
In the conduct of this empirical inquiry, we employed an assortment of statistical techniques to dissect information in various contexts.For data that resided within a dyadic framework, we harnessed the analytical power of the independent Wilcoxon test, thereby securing a statistically significant reckoning.In the event that data accrued from a tripartite or quadrilateral schema, we opted to implement the Kruskal-Wallis test, which furnished a statistically significant determination.We conducted univariate survival analysis using Kaplan-Meier survival analysis and the log-rank test to assess statistical significance.Additionally, for multivariate survival analysis, we employed the Cox regression model, considering variables with a p value below the critical threshold of 0.05 as statistically significant.

Ethics statement
This study was approved by the Institutional Research Ethics Committees of the YiZheng People's Hospital.We confirmed that all methods were carried out in accordance with relevant regulations and written informed consent was obtained from patients.

Result TIMM17A is abnormally up-regulated in LUAD
To investigate the expression levels of TIMM17A, we analyzed transcriptome data from 33 types of cancer in the TCGA database.Among these, the tumor data for LGG, LAML, MESO, OV, TGCT, UVM, UCS, ACC, and DLBC lack corresponding normal control groups.In the remaining 24 types of tumors, TIMM17A expression demonstrated varying patterns.It was downregulated in only four tumor types: GBM, THCA, KICH, and KIRC.Aberrant expression of TIMM17A was observed in 12 different cancers, including LIHC, CESC, LUAD, COAD, BRCA, ESCA, STAD, UCEC, HNCS, LUSC, BLCA, and CHOL.However, no expression differences of TIMM17A were observed in SARC, KIRP, PRAD, SKCM, THYM, READ, PAAD, and PCPG.(Fig. 1A,B).Furthermore, in LUAD tissues, abnormal upregulation of TIMM17A protein expression was validated using the CPTAC database (Fig. 1C).Subsequently, paired analysis of TCGA LUAD dataset consistently showed aberrant upregulation of TIMM17A expression in LUAD tissues (Fig. 1D).To validate these findings, we collected 85 pairs of LUAD tissues and their corresponding normal lung tissues.qRT-PCR confirmed the abnormal upregulation of TIMM17A expression in LUAD tissues (Fig. 1E).Additionally, a thorough examination of the HPA database corroborated these observations, demonstrating a similarly elevated expression of TIMM17A protein in LUAD (Fig. 2A).The aforementioned results highlight the significant upregulation of TIMM17A expression in LUAD.

TIMM17A is associated with poor prognosis in LUAD.
Given the aberrant expression of TIMM17A in LUAD, we conducted a thorough investigation into its relationship with LUAD prognosis.Initially, the TIMM17A gene demonstrated notable effectiveness in LUAD detection, evidenced by an area under the curve (AUC) of 0.899 (Fig. 3A).Subsequent analysis using time-dependent receiver operating characteristic (ROC) curves revealed TIMM17A's significant diagnostic potential for advanced LUAD, with an AUC of 0.725 at 10 years (Fig. 3B).Additionally, there was a positive correlation between increased TIMM17A expression and reduced OS and PFI in LUAD patients (Fig. 3C,D).While there were no significant statistical differences in DFI and DSS associated with TIMM17A expression (Fig. 3E,F www.nature.com/scientificreports/prognosis with higher TIMM17A levels was observed.These findings suggest that TIMM17A could be a viable prognostic biomarker for LUAD.

Relationship between TIMM17A expression and clinical features in patients with LUAD
Our investigation explored the TCGA LUAD dataset to examine the complex relationship between TIMM17A expression and the clinical features of LUAD patients.While there was no marked difference in TIMM17A expression across different tumor stages (Fig. 4A) and N-stage (Fig. 4B), a notable increase in TIMM17A expression was observed across LUAD Stage I-IV.Significantly, TIMM17A levels were higher in patients with lymph node metastasis compared to those without.In terms of T-stage, TIMM17A expression notably escalated with advancing disease stage, a trend that was statistically significant (Fig. 4C).Contrarily, TIMM17A expression was strikingly higher in patients with M1 stage compared to M0 stage (Fig. 4D).Additionally, in Stage III LUAD samples, TIMM17A expression significantly exceeded that in normal, Stage I, and Stage II groups, as confirmed by the CPTAC database (Fig. 4E).Furthermore, in our analysis of tumor grade, TIMM17A protein expression substantially increased with advancing grade (Fig. 4F).In summation, the intricacies of the intricately woven relationship between TIMM17A and the malignancy and prognosis of LUAD, render TIMM17A a potential harbinger of LUAD prognosis.

Univariate and multivariate analysis of TIMM17A in LUAD
In order to thoroughly investigate the multifaceted and complex nature of LUAD prognosis, our team engaged in a rigorous and comprehensive analysis utilizing the one-way Cox method.We meticulously examined a range of potentially impactful factors, including gender, age, T-stage, N-stage, M-stage, and TIMM17A.The results of this exhaustive analysis revealed a number of key findings, most notably the fact that T-stage, N-stage, M-stage, and TIMM17A all played a significant role in the prognosis of LUAD (Fig. 5A).However, we did not stop there.In order to more fully verify the independence of these factors, and to gain a more nuanced understanding of their individual contributions to the overall picture, we conducted a multifactorial Cox analysis.Our efforts bore fruit, as this analysis revealed TIMM17A to be one of the truly independent prognostic factors for LUAD, as evinced by the striking and revealing Fig. 5B.The implications of this groundbreaking finding cannot be overstated.www.nature.com/scientificreports/Indeed, it is abundantly clear that the role of TIMM17A in the prognosis of LUAD is of paramount importance, and that it has significant implications for the development of effective treatments and prognostic strategies moving forward.In light of these exciting and novel findings, we believe that TIMM17A should be considered as a leading candidate for further exploration as a potential marker for the treatment and prognosis of LUAD.

GSEA of TIMM17A in LUAD
In our endeavor to decipher the complex role of TIMM17A in regulating LUAD staging, we employed GSEA.This meticulous analysis revealed a significant association between TIMM17A expression and various biological processes, including Butanoate_metabolism, Cell_cycle, DNA_replication, RNA_degradation, TGF_beta signal-ing_pathway, and WNT_signaling_pathway, among others (Fig. 6A, Table 1).This finding is a pivotal contribution to our understanding of how TIMM17A influences the diverse biological pathways integral to LUAD staging.The complexity of these interactions is immense, yet our use of GSEA has enabled us to make substantial strides in elucidating this intricate network.

Nomogram model
After utilizing TIMM17A in the Nomogram model construction, we proceeded to examine its effectiveness in prognosticating LUAD patients, as illustrated in Fig. 7A.Notably, the results of our calibration efforts revealed a remarkable degree of predictive accuracy (Fig. 7B-D).It is thus safe to assert that the model is a highly reliable tool for forecasting the prognosis of patients suffering from LUAD.

The prognostic and in vitro cell experiment of TIMM17A in LUAD
In our final analysis, we conducted a logical examination of the pathological characteristics and expression of TIMM17A in 85 cases of LUAD patients, revealing a correlation between TIMM17A and LUAD T stage, N stage, and overall stage (Table 2).Kaplan-Meier survival curves indicated a significant association between high TIMM17A expression and adverse prognosis (Fig. 8A).Univariate Cox regression demonstrated that T stage, N stage, and TIMM17A may serve as adverse prognostic factors for LUAD.Further multivariate Cox analysis identified TIMM17A as an independent prognostic factor for LUAD, particularly associated with T stage (Fig. 8B).To delve deeper into the functional significance of TIMM17A, we performed TIMM17A gene knockdown experiments in A549 cells (Fig. 8C).Clone formation assays and Edu cell proliferation experiments following TIMM17A knockdown demonstrated a significant suppression of A549 cell proliferation (Figs.8D,E).In summary, the inhibition of A549 cell proliferation upon TIMM17A knockdown suggests its crucial role in the development of LUAD.

Discussion
LUAD, a malignancy originating from glandular epithelium, stands as one of the most prevalent cancers globally 27 .In spite of notable progress in contemporary treatment modalities, LUAD remains a major cause of mortality, underscoring the imperative need for continued research into innovative therapeutic targets and prognostic biomarkers 28 .The pursuit of molecular markers that can accurately reflect the complex nature of LUAD, coupled with the elucidation of their intricate associations with the clinical characteristics of affected patients, has emerged as a critical area of focus in the ongoing battle against this formidable disease 29,30 .TIMM17A has been increasingly recognized as a key player in tumorigenesis, with its aberrant expression linked to the progression and prognosis of various cancers.The role of TIMM17A in breast cancer has been extensively studied.Overexpression of TIMM17A in breast cancer consistently correlates with accelerated disease progression, decreased OS, and reduced distant metastasis survival, suggesting its potential as a therapeutic www.nature.com/scientificreports/target 12 .In invasive and triple-negative breast cancer (TNBC), TIMM17A has been identified as a key target gene of miR-24-2 and miR-133b, with its low expression associated with improved survival, indicating diagnostic and therapeutic potential 31,32 .High expression of TIMM17A mRNA in breast cancer is linked to adverse pathological parameters and poor clinical outcomes, positioning TIMM17A as an independent predictor of OS and a potential prognostic marker 13 .In fibrolamellar hepatocellular carcinoma (FLC), including primary and metastatic tumors, TIMM17A is frequently overexpressed on the 1q chromosome, suggesting its potential role as an oncogene in this rare subtype of liver cancer 33 .However, research on TIMM17A in LUAD is relatively limited.Therefore, we embarked on an in-depth investigation into the expression of the TIMM17A gene in LUAD and its potential significance in the prognosis and clinical features of the disease.Through the analysis of TIMM17A mRNA expression in the TCGA LUAD dataset and combining protein expression data from CPTAC and HPA databases, we concluded that TIMM17A is aberrantly upregulated in LUAD.To validate this finding, we conducted qRT-PCR verification in 85 LUAD patients, and the results were consistent with the database expression, strengthening our observations.Regarding the relationship between TIMM17A and the prognosis of LUAD patients, we employed Kaplan-Meier survival curves and Cox regression analysis, revealing a correlation between high TIMM17A expression and adverse outcomes.Furthermore, in the TCGA LUAD dataset, TIMM17A showed associations with pathological features of LUAD, and logical analysis of TIMM17A expression and pathological features in 85 LUAD patients indicated correlations with T staging, N staging, and Stage grading.These results further underscored the potential role of TIMM17A in LUAD.To comprehensively understand the impact of TIMM17A on LUAD, we conducted GSEA analysis, revealing TIMM17A enrichment in various critical biological processes and pathways, such as Butanoate_metabolism, Cell_cycle, DNA_replication, RNA_degradation, TGF_beta_signaling_pathway, and WNT_signaling_pathway.These findings suggest that TIMM17A may participate in influencing the progression of LUAD through these pathways.In addition, considering the multifactorial Cox analysis showing the correlation between TIMM17A and T staging in 85 LUAD patients, we performed TIMM17A mRNA knockdown experiments to validate this association.The results demonstrated that the knockdown of TIMM17A inhibited the proliferation ability of A549 cells, further confirming the crucial role of TIMM17A in the development of LUAD.
While our study presents significant insights, it is important to acknowledge certain limitations.First, the reliance on bioinformatics analysis and cellular experiments in this research underlines the need for additional validation through clinical samples 30 .Second, the specific molecular mechanisms underpinning TIMM17A's role remain unexplored, warranting further experimental investigation for a more comprehensive understanding.Lastly, although this study highlights TIMM17A's potential relevance in LUAD, the practicality of its application in clinical settings necessitates more extensive prospective research for definitive validation 34 .

Conclusions
Overall, our bioinformatics analysis substantiates the role of TIMM17A as a promising diagnostic biomarker and therapeutic target in LUAD.The experimental findings underscore TIMM17A's critical involvement in LUAD pathogenesis, especially its influence on cellular proliferation.Furthermore, this study offers an initial insight into the role of TIMM17A in LUAD, laying a solid foundation for future research endeavors to expand upon.

Figure 2 .Figure 3 .
Figure 2. TIMM17A protein expression in LUAD.TIMM17A protein expression is abnormally elevated in LUAD according to the HPA database.

Figure 4 .
Figure 4. Relationship between TIMM17A expression and clinical features in patients with LUAD.(A) TIMM17A expression is not significantly different between different stages of LUAD patients.(B) No significant difference in TIMM17A expression was observed between N stages.(C) TIMM17A expression significantly increases with T stage.(D) In patients with M1 stage LUAD, TIMM17A expression is significantly higher than in those with M0 stage.(E) TIMM17A expression is substantially higher in Stage III LUAD samples compared to those in normal, Stage I, and Stage II groups, as confirmed by the CPTAC database.(F) The expression of TIMM17A protein increases significantly with Grade stage.

Figure 5 .
Figure 5. Univariate and multivariate analysis of TIMM17A in LUAD.(A) The results of the one-way Cox method show that T-stage, N-stage, M-stage, and TIMM17A are all significant prognostic factors.(B) The multifactorial Cox analysis further highlights the independent prognostic significance of TIMM17A.

Figure 6 .
Figure 6.GSEA analysis of TIMM17A in LUAD.The GSEA analysis shows that TIMM17A is associated with several signaling pathways, including Butanoate metabolism, Cell cycle, DNA replication, RNA degradation, TGF beta signaling pathway, and WNT signaling pathway.

Figure 7 .
Figure 7. Nomogram model.(A) After incorporating TIMM17A into the Nomogram model, the effectiveness of the model in prognosticating LUAD patients was examined.(B-D) Calibration curves for the Nomogram model at 1.3.5 years.

Figure 8 .
Figure 8. Prognostic and In vitro Cell Experiment of TIMM17A in LUAD.(A) Kaplan-Meier survival curve analysis of TIMM17A in a cohort of 85 LUAD cases.(B) Univariate Cox and multivariate Cox analyses of TIMM17A in the 85 LUAD cases.(C) qRT-PCR analysis showing the knockout efficiency of TIMM17A in A549 cells.(D) Clone formation experiment following TIMM17A knockdown in A549 cells.(E) Edu cell proliferation experiment after TIMM17A knockdown in A549 cells; scale bar: 100 μm.

Table 1 .
Gene set enrichment analysis results.

Table 2 .
Correlation between TIMM17A expression in LUAD tissues and clinicopathological features of LUAD patients.