Stage- and smoking-associated microRNA expression in lung adenocarcinoma

Introduction

In recent decades, 5-year survival rates of patients with lung cancer have increased in the United States (1). This improvement is attributable to advances in early diagnosis, the development of combination therapies for lung cancer including targeted therapies, and reduced tobacco use. In patients with non-small cell lung cancer (NSCLC), the 5-year survival rate is significantly higher for patients with early-stage (stage I) disease than for patients with disease in the more advanced stages (stage II or higher). Unfortunately, only a small percentage of NSCLC cases (15.7%) are diagnosed at stage I (1). To prolong patient survival, continued advances in early detection and targeted therapy are critical; these advances will be made through improved understanding of the molecular networks that drive lung cancer, specifically the genomic and epigenomic changes that occur during tumor initiation and progression. In recent years, studies of microRNA (miRNA) expression in NSCLC have improved our understanding of these molecular networks, leading to exploration of miRNA markers for early detection and assessment of treatment response (2-5).

miRNAs are small noncoding RNAs that regulate gene transcription and protein expression (6-8). During the last decade, convincing evidence has shown that miRNAs are involved in the expression and deregulation of a wide spectrum of genes in multiple malignancies, including lung adenocarcinoma (3,9-13). Published miRNA profiling studies have identified many altered miRNAs in lung adenocarcinoma, including miRNAs associated with specific tumor types or with prognosis (4,5). These miRNAs have been shown to be involved in numerous pathways associated with lung cancer development, including cell proliferation, replicative immortality, apoptosis, genomic instability, and immune evasion (14,15). In addition, researchers have evaluated the potential uses of miRNAs as biomarkers for the detection of lung cancer in a variety of specimen types (4,5).

Although it is clear that miRNAs are involved in lung cancer, our understanding of how miRNA alterations may be associated with tumor stage and/or smoking status is limited. Identifying such changes, especially those associated with the early stage of lung cancer, may enhance our understanding of the epigenetic events that give rise to lung cancer. Such an understanding may lead to the development of new tests for early detection, prognosis, and assessment of treatment response in lung cancer patients as well as new targets for lung cancer prevention and treatment. The purpose of this miRNA profiling study was to compare genome-wide miRNA expression using microarrays from patient-derived lung adenocarcinomas and paired nonneoplastic lung tissue specimens and to determine the effects of disease stage and smoking status on miRNA expression. We present this article in accordance with the TRIPOD reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-636/rc).

Methods

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Institutional Review Boards of The University of Texas MD Anderson Cancer Center (No. LAB07-0640), Fox Chase Cancer Center (No. 07-825), and Houston Methodist Hospital (No. Pro000016860). Informed consent was waived in this retrospective study.

Patients and specimens

This study used 58 primary lung adenocarcinoma tissue samples with paired nonneoplastic lung tissue samples that were collected from 1991 to 2006. Of these paired specimens, 50 pairs were collected from the tumor bank at Fox Chase Cancer Center (Philadelphia, PA, USA) and 8 pairs from the tumor bank at the Houston Methodist Hospital (Houston, TX, USA). The tissue specimens had been snap-frozen in liquid nitrogen immediately after resection and stored at −80 °C. The pathologic diagnosis and classification were based on the 2004 World Health Organization Classification of Lung Tumors (16). Hematoxylin and eosin-stained tissue sections were reviewed by pathologists (M.H., Y.G.) to confirm the pathologic diagnosis and specimen adequacy. Tumor specimens chosen for analysis consisted of more than 70% viable tumor cells without extensive necrosis.

Patient data (demographics, smoking status) were obtained from the databases of the tumor banks at Fox Chase Cancer Center and the Houston Methodist Hospital. Selected demographic and clinical characteristics of the 58 patients are summarized in Table 1. None of the patients had received neoadjuvant therapy. Twenty-five (43%) had no history of tobacco smoking, and 33 (57%) had a history of smoking. The proportion of specimens from patients with stage I disease (52%) was similar to that of patients with stage II or higher disease (48%).

RNA isolation and miRNA profiling

Total RNA was isolated with a mirVana miRNA Isolation Kit (Cat #1560, Ambion, Foster City, CA, USA). Total RNA quality was assessed according to the instruction of Agilent’s 2100 Bioanalyzer. Only RNA with a 260/230 ratio above 1.8 was used in the study. miRNA expression profiling analysis was conducted using the Human miRNA V2 Oligo Microarray Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. The microarrays assessed expression of 706 unique human miRNAs. Most of the miRNAs were represented by 16 replicate spots within the arrays. Feature Extraction software (version 9.5.3.1; Agilent Technologies) was used to create data tables from the arrays.

The quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to confirm the altered expression in selected miRNAs (Figure S1, miRNA-139-5p expression, signal of normal/tumor pairs after RNU6B normalization).

Identification of altered miRNAs

To identify miRNAs with significantly upregulated or downregulated expression in lung adenocarcinoma, we used the following combined cut-offs: (I) raw P value ≤0.05; (II) fold change (FC) of at least 50% upregulation or downregulation compared with the mean expression level in the paired nonneoplastic lung tissue; and (III) local false discovery rate (FDR) ≤0.05. All miRNAs determined to be altered in lung adenocarcinoma met all 3 criteria. The miRNAs are ranked according to FC in the tables.

Using the above criteria, we first selected the miRNAs with altered expression across the overall cohort of 58 cases. Then, we identified miRNAs with altered expression in 4 subgroups: stage I adenocarcinomas, stage II or higher adenocarcinomas, smokers, and nonsmokers to identify shared altered miRNAs between stage and smoking status groups. The selected miRNAs with altered expression in each subgroup were then compared with the miRNAs with altered expression in the entire cohort.

Statistical analysis

Patient age at resection was compared between smokers and nonsmokers by the Wilcoxon rank sum test with continuity correction. Patient sex, patient ethnicity, and tumor stage were compared between smokers and nonsmokers using the Pearson Chi-squared test with the Yates continuity correction. P values (two-sided test) less than 0.05 were considered significant.

miRNA expression levels were background-subtracted, quantile-normalized between arrays, and log₂ transformed (17,18). The average of the log₂-transformed expression levels of the replicate spots for each miRNA was computed before statistical analysis. The log differences between miRNA expression levels in tumor and paired nonneoplastic lung samples were compared between patient groups by their smoking status (smoker vs. nonsmoker) and disease stage (stage I vs. stage II and higher) using one-way analysis of variance (ANOVA) followed by pairwise comparisons on a miRNA-by-miRNA basis. The model included fixed effects of smoking status, disease stage, and the interaction between smoking status and disease stage. We assessed the local FDRs for all terms of the model using the Bum method (19). All analyses were performed using R version 4.0.3 software (https://www.r-project.org/) with Bioconductor pages (https://www.bioconductor.org/).

The miRTarBase (http://miRtarbase.mbc.nctu.edu.tw), TarBase v.8 (https://dianalab.e-ce.uth.gr/tarbasev8), and miRWalk (http://mirwalk.umm.uni-heidelberg.de/) tools were used to gather the confirmed targets of the miR-200 family. Pathway analysis was performed using the Reactome database, and we chose pathways that were of interest to us. The ReactomePA package, under R version 4.2.1, was used to generate pathway visualization.

The miR-200 family in The Cancer Genome Atlas (TCGA) was searched for early stage of lung adenocarcinoma in nonsmokers. The miRNA expression data were obtained from UCSC Xena (https://xenabrowser.net/datapages/), underwent batch effect normalization, and were log₂ transformed.

Results

miRNAs with overall altered expression in lung adenocarcinoma tissue compared to paired nonneoplastic lung tissue

A total of 64 of the 706 miRNAs examined were identified as having overall altered expression in lung adenocarcinoma compared to paired nonneoplastic lung tissue: 25 were upregulated in tumor tissues and 39 were downregulated (Table 2). The most highly upregulated miRNA was miR-21 (FC, 3.6), and the most highly downregulated miRNA was miR-451 (FC, −7.8). Of the 64 miRNAs with overall altered expression in lung adenocarcinoma tissue, 38 were identified in lung adenocarcinomas of all stages (Table 2). Five of the altered miRNAs were altered only in stage I adenocarcinomas, and 18 were altered only in stage II or higher tumors. Three miRNAs (miR-205, miR-923, and miR-34b) showed overall altered expression in lung adenocarcinomas, but these alterations were not associated with a specific stage (Table 2).

Of the 64 miRNAs identified as having overall altered expression in lung adenocarcinoma, 41 were observed in adenocarcinomas of both smokers and nonsmokers (Table 3), 20 in nonsmokers, and 1 in smokers only. Two miRNAs (miR-630 and miR-205) showed overall altered expression but no altered expression in either smokers or nonsmokers.

Overall, 36 out of the 64 miRNAs (56.3%) identified as having overall altered expression in lung adenocarcinoma were altered in all stages of adenocarcinoma and in both smokers and nonsmokers.

miRNA expression alterations associated with disease stage

In the 30 stage I adenocarcinoma samples, 58 miRNAs with altered expression were identified. Of these, 38 were also identified in stage II or higher adenocarcinomas (Table 2). The remaining 20 miRNAs whose expression was altered in stage I adenocarcinoma are listed in Table 4. Of these, 14 miRNAs were upregulated and 6 downregulated. The most highly upregulated miRNA was miR-200b (FC, 2.5). Five of these 20 miRNAs also showed overall altered expression (Table 4). The remaining 15 miRNAs with altered expression were identified only in stage I adenocarcinomas (Table 4). Upregulation of 4 miR-200 family members (miR-200b, miR-141, miR-429, miR-200a) was observed in this group.

In the 28 stage II or higher adenocarcinoma samples, 84 miRNAs with altered expression were identified. With exclusion of 38 altered miRNAs that were also identified in the stage I adenocarcinomas, 46 altered miRNAs were associated with stage II or higher disease (Table 5). Of these 46 miRNAs, 19 were upregulated and 27 downregulated. Nineteen of the 46 miRNAs had overall altered expression in lung adenocarcinomas compared to nonneoplastic lung tissue (Table 5), predominantly owing to their expression in stage II or higher adenocarcinomas. The remaining 27 miRNAs were identified exclusively in stage II or higher adenocarcinomas (Table 5) without overall altered expression. None of altered miR-200 family were observed in this group.

miRNA expression alterations associated with smoking status

In the 25 lung adenocarcinomas from nonsmokers, 38 miRNAs with altered expression were observed, 23 of which were upregulated and 15 downregulated. Of these, 20 miRNAs had both altered expression in lung adenocarcinoma samples from nonsmokers and overall altered expression (Table 6). Eighteen miRNAs had altered expression in nonsmokers but did not have overall altered expression (Table 6). Among the upregulated miRNAs in nonsmokers were 3 miR-200 family members (miR-200b, miR-141, miR-200a).

In the 33 lung adenocarcinoma samples from smokers, most miRNAs with altered expression also had overall altered expression in both smokers and nonsmokers (Table 3). Only 6 miRNAs were altered in smokers alone. Of these, miR-31 also showed overall altered expression. The remaining 5 miRNAs with altered expression were identified in smokers but not overall; of these, 4 were upregulated and 1 downregulated (Table 7).

miRNA expression alterations associated with smoking status in early-stage lung adenocarcinoma

In stage I adenocarcinoma specimens from nonsmokers, 14 miRNAs with altered expression were identified, including 8 with overall altered expression (Table 8) and 6 that were altered only in nonsmokers’ stage I tumors. Five upregulated miR-200 family members, miR-200b (FC, 4.1), miR-200a (FC, 3.6), miR-141 (FC, 3.5), miR-429 (FC, 2.9) and miR-200c (FC, 2.5), were observed in this group (Figure S2).

Fifteen miRNAs had altered expression in stage I adenocarcinoma specimens from smokers, including 5 with overall altered expression and 10 altered only in these samples (Table 9; Figure S3). Of these, miR-7, miR-193b, miR-9-3p, and miR-572 were also observed in stage II and higher adenocarcinoma (Figure S4).

Selected pathways and genes targeted by miR-200 family

In stage I adenocarcinoma specimens from nonsmokers, 5 miRNAs of the miR-200 family, miR-200b, miR-200a, miR-141, miR-429, and miR-200c, exhibited upregulation. Using the miRTarBase, TarBase v.8, and miRWalk tools, we selected the top miR-200 family targeted pathways and genes known to be associated with carcinogenesis. The selection criteria were based on the adjusted P value that provided the most significant relevant pathways and genes (Table 10). We observed an overlap of the pathways and genes associated with the miR-200 family (miR-200a, miR-200b, miR-200c).

TCGA data were searched to compare miRNA-200 family expression levels in 448 lung adenocarcinoma and 45 nonneoplastic samples. This analysis revealed that members of the miR-200 family were significantly differentially expressed. However, comparison of the 143 nonsmokers and 305 smokers in the TCGA cohort showed that only miR-141-3p was significantly differentially expressed. Among 71 nonsmokers and 169 smokers with early-stage lung adenocarcinoma, no significant differences in miR-200 family expression were observed.

Discussion

In this miRNA profiling study of lung adenocarcinoma, we found that expression of several miRNAs in lung adenocarcinomas depends on stage and/or smoking status, suggesting that these miRNA alterations may play important roles in carcinogenesis.

We identified 64 miRNAs with overall altered expression in the overall study cohort, 36 of which did not exhibit significant differences in expression by either disease stage or smoking status. We found additional altered miRNAs on disease stage-based analysis comparing stage I with stage II or higher lung adenocarcinoma. These altered miRNAs associated with disease stage did not show altered expression in the full cohort. For example, in 20 miRNAs with altered expression identified in stage I lung adenocarcinoma, 15 did not show overall altered expression (Table 4). Similarly, in the smoking status analysis, we found 38 miRNAs with altered expression in nonsmokers; 18 of these did not show overall altered expression (Table 6).

A wide spectrum of miRNA expression alterations and their clinical implications in lung adenocarcinoma have been reported in profiling studies (4,5,14,20,21). The diversity of these miRNA alterations is attributable to the following factors: the heterogeneity of lung adenocarcinoma, different detection assays used for miRNA profiling, the selection criteria, and the variable cut-off criteria used for miRNA selection. Recently, some consensus has been reached on the predominant miRNA expression alterations in lung adenocarcinoma based on analysis of the pooled published data. In a meta-analysis of 14 pooled studies, Guan et al. (20) demonstrated that 54 miRNAs (26 upregulated and 28 downregulated) were most consistently reported to have altered expression in lung adenocarcinoma. Of these, upregulation of miR-21 and miR-210 and downregulation of miR-30a and miR-126 were the most consistently identified alterations (20). Our findings similarly identified overall altered expression of miR-21 and miR-210 as the first and the third most upregulated miRNAs and miR-126 and miR-30a as the second and the third most downregulated miRNAs (20). Overall, 76% (37/49) of the miRNAs we identified as having altered expression matched those identified by the meta-analysis, after exclusion of the miRNAs that were not included in the assay used in our study. Four altered miRNAs were identified only in our stage and/or smoking status analysis. These included upregulation of miR-200a in nonsmokers with stage I disease, miR-193b alterations in smokers, and miR-708 in stage II or higher disease. Among downregulated miRNAs, miR-30d were only identified in stage II or higher tumors. We also found a discrepancy regarding miR-203 expression, which was upregulated in the meta-analysis but downregulated in our analysis. Interestingly, downregulated miR-203 was only observed in nonsmokers in our study. In short, the miRNA markers identified in our miRNA profiling study were substantially consistent with those in the meta-analysis data, and some miRNAs showed specific association with disease stage and/or smoking status.

We also compared our data with the 14 miRNAs whose expression was reported as altered in lung adenocarcinoma by a TCGA study of 230 paired samples. We matched 10 miRNAs: upregulated miR-21, miR-210, miR-183, miR-200b, miR-31, and miR-205 and downregulated miR-30a, miR-486-5p, miR-143, and miR-145 (22). After exclusion of the miRNAs that were not included in our profiling assay, 91% (10/11) of the miRNAs matched between our data and the TCGA results. In summary, our selection of miRNA markers in this miRNA profiling study yielded substantial consistency with those in the published data, specifically the miRNAs that showed the most significant upregulation or downregulation.

Among the miRNAs that we found to have overall altered expression, the most upregulated was miR-21 and the most downregulated was miR-451. miR-21 has been reported to play roles in suppressing expression of the tumor suppressor gene PTEN and activating the ALK and MEK/ERK signaling pathways in lung cancer (15). Seike et al., in a study of 28 never-smokers, reported that aberrant expression of miR-21 was associated with EGFR gene mutations (23). In our study, we observed upregulated expression of miR-21 in all adenocarcinoma stages and in both smokers and nonsmokers, indicating its constant activity in lung adenocarcinoma carcinogenesis and progression regardless of smoking status. miR-451 has been reported to dysregulate c-Myc-Survivin/Rad51 signaling (24). As noted above, downregulated expression of miR-451 has been found in NSCLC with lymph node metastasis (25), and upregulated expression of miR-451 was identified in radiosensitive lung cancers (26). These findings provide further evidence supporting a possible role for miR-451 as a regulator of tumor suppression in lung cancer.

A unique finding of our study is that expression of several miRNAs was associated with lung adenocarcinoma stage, especially the early stage. To our knowledge, very little is known about stage-associated miRNA expression in lung adenocarcinoma. Of the 20 miRNAs that we identified as having expression alterations specifically associated with stage I adenocarcinoma, 4 of the upregulated miRNAs were from the miR-200 family: miR-200b, miR-141, miR-200a, and miR-429 (Table 4; Figure 1). Another miR-200 family member, miR-200c, had both overall upregulation and upregulation in stage I carcinomas of nonsmokers (Table 8). No altered expression of the miR-200 family members was observed in stage II or higher tumor tissues (Table 5). In addition to stage I disease, the upregulation of miR-200 family miRNAs appeared predominantly in nonsmokers. The upregulation of miR-141, miR-200b, and miR-200a was observed in nonsmokers but not in smokers (Tables 6,7). These findings are consistent with the published miRNA profiling data in 28 never-smokers by Seike et al. showing that 2 miR-200 family members (miR-141 and miR-200b) were upregulated in lung adenocarcinoma of nonsmokers (23). Interestingly, if only stage I adenocarcinoma from nonsmokers were analyzed, all 5 miRNAs from the miR-200 family (miR-200b, miR-200a, miR141, miR-429, and miR-200c) were upregulated (Table 8). The significant upregulation of the miR-200 family in stage I lung adenocarcinoma from nonsmokers diminished in more advanced-stage tumors, suggesting that these miRNAs have roles during carcinogenesis and early cancer development predominantly in nonsmokers.

Figure 1 Upregulated expression of miR-200 family members in stage I lung adenocarcinoma in nonsmokers. (A) miR-200a; (B) miR-200b; (C) miR-429. Box plot shows the IQR (25^th–75^th percentile), with the black horizontal lines in each box representing the median expression levels; whiskers indicate the minimum and maximum values within 1.5 × the IQR, and outliers are displayed as individual points. P values were determined using one-way ANOVA. ANOVA, analysis of variance; IQR, interquartile range.

Several reported mechanisms for the miR-200 family support its possible roles in lung adenocarcinoma initiation and progression. The miR-200 family is known to regulate multiple genes in NSCLC cell lines, including genes involved in the PD-L1/ZEB1 axis, pathways of immune response, metastasis signaling, cell-cell communication, proliferation, and DNA repair (27,28). The expression level of miR-200 family members has been reported to be inversely associated with poor outcomes in patients with NSCLC. For instance, lower miR-200c expression was associated with poor tumor differentiation, lymph node metastases (29), and recurrence of localized stage I disease (30). The miR-200 family was also reported to play an important role in regulation of the epithelial-mesenchymal transition process (31,32). Inhibition of miR-200 can reduce E-cadherin expression, promoting epithelial-mesenchymal transition, indicating that the miR-200 family are maintainers of the epithelial phenotype of cancer cells (33). Recently, expression of miR-200 family, especially miR-200a and miR-200b, was reported to be inversely correlated with PD-L1 levels in NSCLC, indicating their roles in regulating PD-L1 expression in lung cancer (34). Our data showing pathways and genes targeted by the miR-200 family also suggest that miR-200a, miR-200b, and miR-200c targeted not only the above-mentioned important pathways but also some known genes that play critical roles in carcinogenesis, such as TP53, EGFR, MYC, MET, and KRAS.

Despite our findings of aberrant miR-200 family expression in early-stage lung adenocarcinoma in nonsmokers, our TCGA data search showed aberrant expression of only miR-141-3p in nonsmokers. The discrepancy could be caused by multiple factors, such as the heterogeneity of lung adenocarcinoma, different clinical stages, different testing assays with variable selection criteria and cut-offs, or the relatively small numbers of never-smokers in both cohorts. In addition, patients’ ethnicity, especially for Asian populations, may be a factor underlying the discrepancy. In Seike and colleagues’ miRNA profiling study of 28 never-smokers, 75% (21/28) of whom had stage I adenocarcinoma, 2 miR-200 family members (miR-141 and miR-200b) were reported to be upregulated. Notably, the cancer subtypes in that study were diverse, with 22 cases of adenocarcinoma, and 6 carcinoma cases of other carcinoma types (4 squamous cell carcinoma, 1 adenosquamous cell carcinoma, and 1 unclassified carcinoma) were included in that study (23). Such complex miRNA profiles were also seen in our study for other miRNAs. We compared our miRNA markers with those in a pooled analysis by Zhong et al., and found 5 altered miRNAs identified in our sub-group analysis matched those reported by Zhong et al. Specifically, miR-125a and miR-30d were associated with stage II or higher adenocarcinoma, miRNA-708 with stage II or higher adenocarcinoma in nonsmokers, miR-224 with stage I adenocarcinoma in nonsmokers and miR19b with stage I adenocarcinoma in smokers. These 5 miRNAs were not identified in our whole cohort analysis (4). Further study with more early-stage nonsmoker cases may be required to clarify the issue.

In stage I lung adenocarcinoma samples, we identified only 6 miRNAs with downregulated expression. Among these, miR-134 was significantly downregulated, predominantly in nonsmokers. Published studies demonstrate that miR-134 might act as a cancer suppressor via pathways regulating cancer cell proliferation, apoptosis, invasion, and metastasis (35). In lung cancer, however, the role of altered expression of miR-134 has been controversial, with some studies finding downregulation and others upregulation. Our findings suggest that stage and smoking status may be factors in this discrepancy. In our study, the overall expression of miR-134 was downregulated, but it was upregulated in stage II or higher lung adenocarcinomas. In stage II or higher tumors, miR-134 was more strongly upregulated in nonsmokers than in smokers.

In stage II or higher lung adenocarcinoma, we identified 46 miRNAs with altered expression. Of these, 19 had overall altered expression. One of the miRNAs that had overall altered expression, miR-135a-3p, was upregulated in stage II or higher adenocarcinomas. Upregulation of miR-135 family members has been reported to play an important role in apoptosis resistance to an EGFR inhibitor (36). In addition, our observation of downregulation of miR-29c in stage II or higher cancers is consistent with the observations that miR-29c is a tumor suppressor that inhibits cancer cell proliferation and metastasis and induces apoptosis (37).

In lung adenocarcinoma samples from nonsmokers, we identified 38 miRNAs with altered expression. Of these, 18 showed altered expression only in nonsmokers, without overall altered expression. Notably, these miRNAs also tended toward association with disease stage. miR-513c and miR-513b were more strongly upregulated in nonsmokers than in smokers, predominantly in stage II or higher tumors (Figure 2). miR-513 has been reported to negatively regulate PD-L1, thereby affecting the immune response to tumors (38). Also, in nonsmokers, miR-198, which was reported to be associated with prognosis of lung adenocarcinoma by inhibiting cell growth and enhancing apoptosis (39), was upregulated, predominantly in stage II or higher carcinomas. miR-34c-5p, a member of the miR-34 family that was significantly downregulated in nonsmokers and more prominently in stage II or higher tumors, has been reported to be a tumor suppressor but also to protect lung cancer cells from paclitaxel-induced apoptosis (40,41).

Figure 2 Upregulated miRNA expression in stage II or higher lung adenocarcinomas in nonsmokers. (A) miR-513c; (B) miR-513b; (C) miR-198. Box plot shows the IQR (25^th–75^th percentile), with the black horizontal lines in each box representing the median expression levels; whiskers indicate the minimum and maximum values within 1.5 × the IQR, and outliers are displayed as individual points. P values were determined using one-way ANOVA. ANOVA, analysis of variance; IQR, interquartile range; miRNA, microRNA.

Most of the miRNAs with significantly altered expression in smokers also had overall altered expression in nonsmokers. In the whole cohort analysis, we identified 41 miRNAs with altered expression in both smokers and nonsmokers, 12 of which were upregulated and 29 down-regulated (Table 3). This finding suggests common pathways of carcinogenesis in smokers and nonsmokers. In the patients with a smoking history, we identified 6 miRNAs with altered expression exclusively in smokers (Table 7). Of these, both miR-31 and miR-31-3p were upregulated. miR-31 has been reported to be an important miRNA in both adenocarcinoma and squamous carcinoma of the lung and a driver of lung carcinoma through promoting mutant KRAS-mediated oncogenesis (20,42,43). Our finding of aberrant miR-31 expression in lung adenocarcinoma in smokers provided new evidence of the unique role of this miRNA in carcinogenesis of lung adenocarcinoma. In the stage I lung adenocarcinoma samples from smokers, we identified 15 altered miRNAs, including 10 altered in smokers only; the most upregulated was miR-106b and the most downregulated miR-195 (Table 9). miR-106b-5p has been reported to be associated with recurrent tumors in stage I lung adenocarcinoma (44). Low miR-195-3p expression has been reported to predict poor overall survival and disease-specific survival (45). Upregulated miR-7, miR-193b, and miR-9-3p and downregulated miR-572 were also observed in adenocarcinoma of all stages from smokers (Tables 7,9). miR-7 has been reported as an important modulator of EGFR-mediated oncogenesis in lung adenocarcinoma through the RAS/ERK/MYC pathway (46). Altered expression of miR-193b has been shown to be more frequent in squamous carcinoma, and miR-9-3p was reported to promote cell proliferation and suppress ferroptosis in TP53 wild-type lung adenocarcinoma cells (47,48).

Thus, our miRNA profiling study in lung adenocarcinoma identified several miRNA expression alterations that are associated with specific disease stages, especially the early stage of lung adenocarcinoma. These miRNAs may play important roles in lung cancer carcinogenesis. Because the generalizability of our results is limited by our relatively small sample size, further study is required to confirm our findings.

Conclusions

Our findings on differences in miRNA expression in lung adenocarcinomas of different stages may enable a better understanding of carcinogenesis in the lung, especially the sequence and timing of miRNA alterations as cancers progress. Our findings of alterations in the miR-200 family that are associated with early-stage lung adenocarcinoma in nonsmokers suggests a unique role of the miR-200 family in early lung carcinogenesis and progression in this population. These findings may help to develop markers for early lung cancer detection and lung cancer prognosis in nonsmokers.

Acknowledgments

We thank Amy Ninetto, Scientific Editor, Research Medical Library, MD Anderson Cancer Center, for editing the manuscript.

Footnote

Reporting Checklist: The authors have completed the TRIPOD reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-636/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-636/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-636/prf

Funding: This work was supported by an intramural grant to M.G. from MD Anderson Cancer Center and by MD Anderson’s Cancer Center Support Grant (NIH/NCI grant P30CA016672). This work was also supported by the Charles B. Barker Endowed Chair (held by G.A.C.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-636/coif). C.W. is a current employee of Pfizer, Inc. C.I. is a current employee of Caris Life Sciences. G.A.C. is the scientific founder of Ithax Pharmaceuticals, Inc. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Institutional Review Boards of The University of Texas MD Anderson Cancer Center (No. LAB07-0640), Fox Chase Cancer Center (No. 07-825), and Houston Methodist Hospital (No. Pro000016860). Informed consent was waived in this retrospective study.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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