miR-145-5p/CAMSAP2 axis modulates cisplatin sensitivity in non-small cell lung cancer

Abstract

Background

Drug resistance remains a major obstacle to effective lung cancer treatment. Cisplatin, which is a platinum-based chemotherapeutic agent, is widely used as a first-line treatment for non-small cell lung cancer (NSCLC); however, its clinical efficacy is often limited by the development of resistance. MicroRNAs (miRNAs) are key regulators of the epigenetic landscape and have been reported to play critical roles in various cancer-related processes. This study aimed to identify potential miRNAs associated with the DNA damage response (DDR), which is a major mechanism underlying cisplatin sensitivity, and to investigate the role of miRNAs in modulating the cisplatin response.

Methods

Differential expression analysis of DNA damage response-related miRNAs was performed using lung cancer datasets from The Cancer Genome Atlas (TCGA). The role of miR-145-5p in cisplatin responsiveness was evaluated by transfecting lung cancer cell lines with a miR-145-5p mimic. The IC₅₀ of cisplatin was assessed using the MTT assay. Cisplatin-induced apoptosis was investigated through apoptosis assays. In addition, changes in apoptosis markers and associated signaling pathways were analyzed by immunoblotting. The potential target of miR-145-5p was identified by integrating data from multiple miRNA databases. The regulatory relationship between miR-145-5p and its target in relation to cisplatin sensitivity was further validated using luciferase reporter assays, RNA interference, and rescue experiments.

Results

miR-145-5p was identified as a strong candidate, as it was observed to significantly downregulated in lung tumor tissues and associated with poor prognosis. In vitro experiments demonstrated that miR-145-5p expression was positively correlated with cisplatin sensitivity across a panel of NSCLC cell lines and that its overexpression reduced the cisplatin IC₅₀ while increasing apoptosis. By integrating target prediction and validation by a luciferase reporter assay, CAMSAP2 was identified as a potential direct target of miR-145-5p. CAMSAP2 expression was negatively associated with cisplatin responsiveness, and rescue experiments confirmed that miR-145-5p could enhance cisplatin-induced apoptosis by downregulating CAMSAP2.

Conclusions

miR-145-5p directly targets and suppresses CAMSAP2, sensitizing lung cancer cells to cisplatin. These findings highlight the miR-145-5p/CAMSAP2 regulatory axis as a critical modulator of cisplatin sensitivity and a potential therapeutic target for overcoming drug resistance in lung cancer.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12935-026-04209-2.

Background

Lung cancer has emerged as the leading cause of cancer-related incidence and mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 80% of all cases [1]. NSCLC includes several histological subtypes, such as lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and large cell carcinoma, with each subtype exhibiting distinct molecular and clinical characteristics. Despite significant progress in early diagnosis and therapeutic intervention, the five-year overall survival rate for patients with lung cancer remains low, which is primarily due to poor responses to therapy and the emergence of drug resistance [2].

Cisplatin, which is a platinum-based chemotherapeutic agent, is widely used as a standard first-line treatment for NSCLC because of its ability to induce DNA cross-links, thereby leading to apoptosis in rapidly dividing tumor cells. However, its clinical efficacy is often limited by the emergence of acquired resistance, which severely compromises treatment outcomes [3]. Several cellular mechanisms underlie cisplatin resistance, including increased DNA damage repair capability, altered drug uptake and efflux, evasion of apoptosis, and dysregulation of key signaling pathways [4, 5]. In response to cisplatin-induced DNA damage, the DNA damage response (DDR) activates cell cycle checkpoints and initiates either DNA repair or apoptosis. Cancer cells with an efficient DDR can repair damage and continue to proliferate, thus contributing to resistance. In contrast, cells with impaired DDR accumulate irreparable DNA lesions, thereby leading to cell death [6]. Therefore, a more in-depth understanding of the molecular mechanisms driving cisplatin resistance is essential for developing strategies to enhance therapeutic efficacy and improve patient outcomes.

Emerging evidence highlights the critical role of microRNAs (miRNAs) in the development and progression of NSCLC. miRNAs are small noncoding RNAs that regulate gene expression at the posttranscriptional level. Moreover, miRNAs function by binding to complementary sequences within the 3’ untranslated regions (3’-UTRs) of their target mRNAs, thus resulting in mRNA degradation or translational repression [3]. These regulatory molecules have been demonstrated to control various biological processes, including cell proliferation, differentiation, apoptosis, and metastasis [3]. Recent studies have provided insight on the role of miRNAs in modulating the sensitivity of NSCLC cells to cisplatin. For example, miR-192 confers cisplatin resistance by downregulating Bim, which is a key proapoptotic protein of the Bcl-2 family [7]. Conversely, miR-138 enhances cisplatin sensitivity by targeting ERCC1, which is a DNA repair protein involved in nucleotide excision repair, thereby impairing the ability of cells to recover from cisplatin-induced DNA damage [8]. These findings underscore the potential roles of miRNAs in overcoming resistance and improving treatment outcomes in NSCLC patients. However, the current understanding of the involvement of miRNAs in cisplatin responsiveness remains largely unknown, and further studies are needed to elucidate their precise molecular mechanisms and therapeutic applications.

In this study, we aimed to identify the miRNAs that are associated with the DDR pathway, which is a central mechanism underlying cisplatin sensitivity. We further investigated their functional relevance, potential mRNA targets, and the molecular mechanisms by which they influence cisplatin responsiveness. By comprehensive analyses, miR-145-5p was identified as the most potential regulator relevant to lung cancer progression. Previous studies have demonstrated that miR-145-5p is commonly downregulated in NSCLC. Reduced expression of miR-145-5p has been associated with poor prognosis and aggressive tumor behavior in lung cancer patients [9, 10]. Despite accumulating evidence supporting the tumor-suppressive role of miR-145-5p, its mechanistic involvement in cisplatin sensitivity has not been fully elucidated. Our study addresses this gap by defining a novel miR-145-5p–regulated pathway that governs cisplatin response, supported by rigorous functional and rescue experiments, which may support the development of novel therapeutic strategies to overcome cisplatin resistance.

Methods

MiRNA and gene expression datasets and differential expression analysis

The differential expression analysis of miRNA was conducted using R version 4.4.3. miRNA expression data for LUAD and LUSC were retrieved from The Cancer Genome Atlas (TCGA) database using the TCGAbiolinks R package. The clinical characteristics of the patients were shown in Supplementary Table S1 and S2. Expression profiles of tumor and adjacent normal tissues were extracted, and differential expression analysis was performed using the edgeR package. Differentially expressed miRNAs (DEmiRNAs) were identified by comparing tumor samples to normal tissues using edgeR package. Thresholds for significance were set at |log2 fold change (logFC)| > 1 and a false discovery rate (FDR) < 0.05. A volcano plot visualizing the distribution of DEmiRNAs was generated using the ggplot2 package. To further link with cisplatin responsiveness, DEmiRNAs implicated in the DDR regulation were evaluated. The list of miRNAs which significantly enriched in the ‘signal transduction in response to DNA damage’ pathway was retrieved from the miRpathDB v.2.0 database [11] (https://mpd.bioinf.uni-sb.de/overview.html). The overlap among DEmiRNA from LUAD and LUSC datasets and miRNAs related to DDR were demonstrated as a Venn diagram generated by Bioinformatics & Evolutionary Genomics online platform (https://bioinformatics.psb.ugent.be/webtools/Venn/). The list of overlapping miRNAs was shown in Supplementary Table S3.

The expression data of miR-145 and CAMSAP2 mRNA and the corresponding clinical data of lung cancer patients were acquired from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/gds/) [12]: GSE102287 (24 tumor and 24 normal lung tissues), The expression level of miR-145 and CAMSAP2 were compared between normal and tumor lung tissues.

The expression data of CAMSAP2 mRNA in lung cancer patients carried a wild type or mutant CAMSAP2 gene in both LUAD and LUSC subtypes were obtained from The Cancer Genome Atlas (TCGA) database [13] via the cBioPortal platform (https://www.cbioportal.org/) [14]: LUAD dataset (487 wide type and 16 mutant lung tissues) and LUSC dataset (454 wide type and 12 mutant lung tissues). The expression level of CAMSAP2 was compared between CAMSAP2 wide type and mutant lung tissues.

TCGA expression data were obtained from normalized datasets, and GEO datasets were batch-corrected and normalized according to the original data processing pipelines provided by the respective studies. For GEO datasets (GSE102287) that contained matched tumor and adjacent normal samples, paired-sample analyses were performed to minimize inter-individual variability and improve statistical robustness.

Survival analysis and gene set enrichment analysis (GSEA)

The Kaplan Meier analysis was performed using KM-plotter database (https://kmplot.com/analysis/index.php? p=home) with the “best cutoff” auto-selection option set to “by expression value.” The cutoff was therefore determined automatically by the platform [15]. The Pan-cancer miRNA datasets were applied to evaluate the relationship between miR-145 and overall survival (OS) in LUAD and LUSC patients. The Lung cancer mRNA datasets were also applied for the analysis of CAMSAP2 (CAMSAP1L1), SRGAP1, PPP3CA, MDFIC, and SEMAD as related to OS in LUAD patients. The patients were classified into high vs. low expression group based on the best performing expression value of each target as the cutoff. The hazard ratio (HR) with 95% confidence interval was reported and log rank P value was used to determine the statistical significance (p < 0.05).

GSEA was performed using 4.3.2 software. Gene expression data of LUAD datasets from the TCGA database were analyzed with 1000 gene-set permutations using the gene-ranking metric T-test. The analysis utilized the “Hallmarks” gene set collection (h.all.v2022.1.Hs.symbols) from the Molecular Signatures Database (MsigDB) (https://www.gsea-msigdb.org/gsea/msigdb/human/collections.jsp#H) [16, 17]. Patients were divided into high and low expression groups based on the median expression level of each target gene.

Cell culture and reagents

Human NSCLC cells namely H460, H292, A549 and H23, and normal bronchial epithelial BEAS-2B cells were purchased from American Type Culture Collection (ATCC, VA, USA). Cisplatin-resistant A549 cells (A549-CR) were generated and characterized as described [18]. H460, H292 and H23 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium. A549 and BEAS-2B cells were grown in Dulbecco’s Modified Eagle Medium (DMEM). All cell culture media were supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. All the media and supplements were purchased from Gibco (NY, USA). Cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂. LY294002 was obtained from MedChemExpress (NJ, USA).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted by using the GENEzol reagent (Geneaid Biotech, New Taipei City, Taiwan) following the manufacturer’s instructions. RNA samples were reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad Laboratories, CA, USA). The quantification of miR-145-5p and CAMSAP2 expression was performed using SensiFAST SYBR NO-ROX Kit (Bioline, TN, USA). The expression levels were normalized to U6 for miR-145-5p or GAPDH for CAMSAP2 levels. The relative expression levels were analyzed using the 2^−∆∆Ct method. The primers and condition used were listed in Table 1.

Table 1.

List of primer sequences for qRT-PCR analysis

Name	Primer sequences (5’−3’)	Tm (℃)
miR-145-5p	F-5’-GTCCAGTTTTCCCAGGAATCCCT-3’ R-5’-GCTGTCAACGATACGCTACCTA-3’	58.8
CAMSAP2	F-5’-AAAATCGCCTGCAATCTGGC-3’ R-5’-AGAATGAGACTCCCAGCACG-3’	57.8
U6	F-5’-CGCTTCGGCAGCACATATACTA-3’ R-5’-CGCTTCACGAATTTGCGTGTCA − 3’	58.9
GAPDH	F-5’-ACATCGCTCAGACACCAT-3’ R-5’-TGTAGTTGAGGTCAATGAAGGG-3’	61.0

Drug responsiveness data analysis

The drug response information and RNAi screen data were obtained from DepMap PRISM (https://depmap.org/repurposing/) via the DepMap data portal [19]. The AUC (PRISM Repurposing Secondary Screen) of chemotherapies and gene expression data set from 33 NSCLC with LUAD subtype were retrieved. The correlation analysis between AUC and gene expression was performed.

Cell viability

Cells were treated with various concentrations of cisplatin for 24 h. After treatment, cells were subsequently incubated with MTT solution (0.5 mg/mL) for 4 h at 37 °C. Following incubation, the formazan crystals were dissolved in 100 µL of DMSO. The optical density was then measured at 570 nm using a CLARIOstar plus microplate reader (BMG LABTECH, Ortenberg, Germany). The mean optical density of each experimental group was calculated to determine cell viability, expressed as a percentage, with the control group being set at 100%.

Apoptosis assay

Cells were treated with varying concentration of cisplatin for 24 h. Following treatment, cells were incubated with a fluorescent nuclear staining dye Hoechst 33342 (10 µg/mL) for 30 min in the dark. Apoptotic characteristics including DNA fragmentation, chromatin condensation and apoptotic body formation were visualized under fluorescence microscope (Olympus Ix51 Invert, Tokyo, Japan). At least five random field/group were captured. Apoptotic cells were calculated and presented as a percentage to total cell number.

MiRNA, siRNA and plasmid transfections

Cells were transfected with miR-145-5p mimic and negative control mimic (miR-NC) using Lipofectamine RNAiMAX (Invitrogen, MA, USA) as previously described [20]. The miR-145-5p mimic and miR-NC were purchased from GenePharma (Shanghai, China), and the siRNA sequence of CAMSAP2 and its control were purchased from Thermo Fisher Scientific (MA, USA). Briefly, 50 nM of miRNA or siRNA in Opti-MEM medium (Thermo Fisher Scientific, MA, USA) was mixed with Lipofectamine RNAiMAX for 15 min at room temperature, followed by addition to the cells. After 48 h, the transfection efficiency was evaluated by qRT-PCR.

CAMSAP2 and mock plasmids were constructed and co-transfected with miR-145-5p by using Lipofectamine 2000 (Invitrogen, MA, USA) as described [21]. After 48-post transfection, the miR-145-5p and CAMSAP2 expressions were examined by qRT-PCR.

Dual-luciferase reporter assay

miRNA target prediction tools, including TargetScan 8.0 (http://targetscan.org/) [22], miRDB (https://mirdb.org/index.html) [23], miRmap (https://mirmap.ezlab.org/) [24], and microT-CDS (https://dianalab.e-ce.uth.gr/html/dianauniverse/index.php? r=microT_CDS) [25, 26] were utilized to identify putative targets of miR-145-5p. Either pmirGLO-CAMSAP2 wide-type (pGLO-C2-WT) or pmirGLO-CAMSAP2 mutant (pGLO-C2-MUT) (Promega Corporation, WI, USA) was co-transfected with miR-145-5p mimic or negative control mimic using Lipofectamine 2000 (Invitrogen, MA, USA). After 48-post transfection, the luciferase activities were analyzed using the Dual-Luciferase Reporter assay system (Promega Corporation, WI, USA) and Renilla luciferase activity was used for normalization.

Immunoblot analysis

Cells were lysed with TMN lysis buffer (20 mM Tris-HCl, pH 7.5; 1 mM MgCl₂; 150 mM NaCl; 20 mM NaF; 0.5% sodium metavanadate; 1% nonidet-P40; 0.1 mM phenylmethylsulfonyl fluoride; and protease inhibitor cocktail) for 30 min at 4 °C as previously described [27]. Protein content was evaluated by the BCA Protein Assay Reagent Kit (Thermo Scientific, MA, USA). An equal amount of protein of each group was separated by SDS–polyacrylamide gel electrophoresis and transferred to PVDF membranes. Subsequently, non-specific binding on membranes was blocked in 5% skim milk in TBST (Tris-buffer saline with 0.075% Tween-20) solution. Membranes were incubated with specific primary antibody overnight at 4 °C, triple washed with TBST and incubated with secondary antibody for 2 h at room temperature. Protein expression was visualized using an enhanced chemiluminescence system with Immobilon Western chemiluminescent HRP substrate (Merck Millipore, MA, USA). The densitometry analysis was performed with GAPDH served as loading control. Primary antibodies used were anti-CAMSAP2 (Proteintech #17880-1-AP, 1:1000), anti-Poly(ADP-ribose) Polymerase (PARP) (Cell Signaling Technology #9542, 1:1000), anti-actin (Cell Signaling Technology), anti-phosphorylate AKT (Cell Signaling Technology #9271, 1:1000), anti-AKT (Cell Signaling Technology #9272, 1:1000), anti-phosphorylate PI3K (Cell Signaling Technology #4228, 1:1000), anti-PI3K (Cell Signaling Technology #4257, 1:1000), anti-β-catenin (Cell Signaling Technology #8480, 1:1000), anti-phosphorylate GSK3β (Cell Signaling Technology #9336, 1:1000), anti-GSK3β (Cell Signaling Technology #9315, 1:1000), anti-Bcl-2 (Cell Signaling Technology #4223, 1:1000), anti-Bax (Cell Signaling Technology #2772, 1:1000), anti-GAPDH (Santacruz #sc-32233, 1:2000), HRP-conjugated anti-rabbit (Cell Signaling Technology #7074, 1:1000), and HRP-conjugated anti-mouse (Cell Signaling Technology #7076, 1:1000).

Statistical analysis

All data are expressed as the mean ± SEM from at least three independent biological replicates. One-way ANOVA for comparisons among multiple groups was utilized, while unpaired Student’s t-test or the Mann–Whitney U test was used for comparisons between two groups. Pearson’s correlation coefficient was applied for correlation analysis. These analyses were performed using Prism 9 (GraphPad software), with P values < 0.05 considered statistically significant.

Results

Low miR-145-5p expression is correlated with poor prognosis in lung cancer patients

First, to identify miRNAs associated with the DDR and cisplatin sensitivity in NSCLC, DDR-related DEmiRNAs in LUAD and LUSC were analyzed using the TCGA dataset. Differential expression analysis revealed a total of 131 upregulated and 69 downregulated miRNAs in LUAD tumor tissues, as well as 170 upregulated and 52 downregulated miRNAs in LUSC tumor tissues, compared with adjacent normal tissues (|log₂FC| > 1, FDR < 0.05) (Fig. 1A and B). However, no notable differences were observed in the clinical characteristics of the analyzed lung cancer patients (Tables S1 and S2). Given the potential role of downregulated miRNAs in promoting cisplatin resistance by the suppression of DNA repair and antiapoptotic genes, subsequent analyses focused on this subset. To further identify candidate miRNAs involved in DDR regulation, the downregulated DEmiRNAs were compared with a curated list of DDR-related miRNAs. This integrative approach identified five overlapping miRNAs (Fig. 1C, Supplementary Table S3). Among these, miR-145-5p was selected for further investigation because of its previously reported involvement in lung cancer progression and tumor suppressive functions [28].

Fig. 1.

miR-145-5p is downregulated in lung cancer tissues and is associated with poor prognosis. (A-B) Volcano plots illustrating differentially expressed miRNAs (DEmiRNAs) between tumor and normal lung tissues in LUAD (A) and LUSC (B) patients based on TCGA data. The red dots indicate upregulated miRNAs, the blue dots indicate downregulated miRNAs, and the gray dots represent nonsignificant changes. FDR: false discovery rate. (C) Venn diagram showing the overlap between TCGA-derived DEmiRNAs and DDR-related miRNAs. (D-E) Relative expression of miR-145-5p in tumor versus normal tissues in LUAD (D) and in LUSC (E), as analyzed using TCGA data. ****P < 0.001 vs. normal tissues. (F) miR-145-5p expression in paired tumor and adjacent normal tissues obtained from the GEO database (GSE102287). ****P < 0.001 vs. normal tissues. (G-H) Kaplan-Meier survival analysis of lung adenocarcinoma (LUAD) (G) and squamous cell carcinoma (LUSC) (H) patients stratified by high and low miR-145-5p expression levels, as analyzed using the KM-plotter database. HR: hazard ratio. (I) Relative expression of miR-145-5p in normal bronchial epithelial BEAS-2B cells and various NSCLC cell lines, as measured by qRT-PCR. The data are presented as the means ± SEMs (n = 3). *P < 0.05, **P < 0.01 vs. BEAS-2B cells

The expression of miR-145-5p was examined in lung cancer and normal lung tissues using data from both TCGA and GEO databases (GSE102287). The results revealed significant downregulation of miR-145-5p in tumor tissues compared with normal lung tissues in both the LUAD and LUSC cohorts (Fig. 1D and E). Consistently, a marked reduction in miR-145-5p expression was observed in paired tumors compared with adjacent normal tissues (Fig. 1F). Survival analysis further revealed that lower miR-145-5p expression was significantly associated with reduced OS in patients with LUAD, whereas no significant correlation was observed in LUSC (Fig. 1G and H). To validate these findings in vitro, miR-145-5p expression levels were examined in a panel of NSCLC cell lines and compared to those in nonmalignant bronchial epithelial BEAS-2B cells. A consistent decrease in miR-145-5p levels was observed in most of the NSCLC cell lines (Fig. 1I). Collectively, these results suggest that miR-145-5p is downregulated in NSCLC and may serve as a prognostic biomarker, particularly in LUAD.

miR-145-5p expression is positively correlated with cisplatin sensitivity in NSCLC

The above-mentioned findings highlight the clinical relevance of miR-145-5p in lung cancer prognosis. To further investigate the molecular mechanism underlying its role in tumor progression, GSEA was performed. The results indicated that miR-145-5p expression was associated with key cell death-related pathways, particularly regarding apoptosis and the p53 signaling pathway (Fig. 2A). Specifically, high miR-145-5p expression was correlated with increased enrichment scores for apoptosis (enrichment score [ES]: −0.29, normalized enrichment score [NES]: −1.21, p = 0.196) and the p53 pathway (ES: −0.31, NES: −1.36, p = 0.086), suggesting a potential tumor-suppressive role by the regulation of cell death mechanisms.

Fig. 2.

Low expression of miR-145-5p is associated with reduced sensitivity to cisplatin-induced cell death. (A) GSEA of LUAD patients from the TCGA dataset comparing high versus low miR-145-5p expression groups. The enriched pathways included apoptosis and p53 signaling. (B) NSCLC cells were treated with various concentrations of cisplatin (0–250 µM) for 24 h. Cell viability was examined using the MTT assay, and the percentage of viable cells was quantified. The data are presented as the means ± SEMs (n = 3). (C) The half-maximal inhibitory concentration (IC₅₀) of cisplatin for each cell line was calculated using Prism 9 software. (D) Pearson correlation analysis between miR-145-5p expression levels and cisplatin IC₅₀ values across NSCLC cell lines revealed a significant negative correlation

Given that the deregulation of apoptosis is a known contributor to chemotherapy in lung cancer, particularly in response to cisplatin treatment [29], the relevance of miR-145-5p expression and cisplatin sensitivity in NSCLC was evaluated. The half-maximal inhibitory concentration (IC₅₀) of cisplatin was determined in four NSCLC cell lines (A549, H292, H23 and H460) using the MTT assay. The results revealed that A549 and H292 cells exhibited higher IC₅₀ values compared to H23 and H460 cells, thus indicating greater resistance to cisplatin (Fig. 2B and C).

To explore the correlation between miR-145-5p expression and cisplatin sensitivity, the expression levels of miR-145-5p in these cell lines were quantified. Consistent with previous observations (Fig. 1E), A549 and H292 cells exhibited relatively low miR-145-5p expression. Subsequent correlation analysis further confirmed a significant negative association between miR-145-5p expression and IC₅₀ values (Fig. 2D), thereby indicating that NSCLC cells with reduced miR-145-5p expression are less sensitive to cisplatin. These findings suggest that miR-145-5p may enhance cisplatin responsiveness, potentially by promoting apoptotic signaling pathways.

miR-145-5p enhances cisplatin-induced apoptosis in NSCLC cells

To investigate the role of miR-145-5p in modulating cisplatin sensitivity, A549 and H292 cells, which exhibited the lowest endogenous levels of miR-145-5p were transfected with either a miR-145-5p mimic or a negative control mimic (miR-NC) (Fig. 3A). Moreover, qRT-PCR analysis confirmed a significant increase in miR-145-5p expression following transfection with the mimic (Fig. 3B). After transfection, the cells were treated with various concentrations of cisplatin. A cell viability assay revealed that the overexpression of miR-145-5p significantly enhanced cisplatin cytotoxicity, with IC₅₀ values being reduced to 17.79 ± 1.06 µM in A549 cells and 15.09 ± 1.21 µM in H292 cells. In contrast, miR-NC-transfected cells exhibited higher IC₅₀ values of 45.14 ± 5.48 µM in A549 cells and 40.78 ± 4.49 µM in H292 cells (Fig. 3C), thereby indicating a 2–3fold decrease in the IC₅₀ upon miR-145-5p overexpression.

Fig. 3.

miR-145-5p enhances cisplatin-induced apoptosis in NSCLC cells. (A) Sequences of the miR-145-5p mimic and a negative control (miR-NC). (B) A549 and H292 cells were transfected with either the miR-145-5p mimic or the miR-NC. The expression level of miR-145-5p was assessed by qRT-PCR. The data are presented as the means ± SEMs (n = 3). *P < 0.05 vs. miR-NC. (C) Transfected cells were treated with various concentrations of cisplatin (0–250 µM) for 24 h, and cell viability was examined using the MTT assay. IC₅₀ values were calculated using Prism 9 software. The data are shown as the means ± SEMs (n = 3). *P < 0.05 vs. miR-NC. (D) Apoptosis was evaluated by H33342 nuclear staining. The percentage of apoptotic nuclei per field was quantified. The data are presented as the means ± SEMs (n = 3). *P < 0.05 vs. miR-NC. (E) The protein expression levels of PARP and cleaved PARP were analyzed by immunoblotting and normalized to GAPDH expression. The quantitative data are presented as the means ± SEMs (n = 3). *P < 0.05 vs. miR-NC. The charts were plotted from triplicated experiments, in which the original blots were provided in supplementary files

Consistently, H33342 staining revealed a substantial increase in apoptotic nuclear morphology in cells transfected with the miR-145-5p mimic following cisplatin treatment, whereas apoptotic features were less prominent in the miR-NC group (Fig. 3D). Furthermore, Western blot analysis demonstrated enhanced cleavage of PARP in response to cisplatin, with a greater degree of activation being observed in cells overexpressing miR-145-5p compared to controls cells (Fig. 3E). Together, these results suggest that miR-145-5p sensitizes NSCLC cells to cisplatin-induced apoptosis.

miR-145-5p directly targets CAMSAP2

To elucidate the mechanism by which miR-145-5p enhances cisplatin sensitivity, potential target genes were identified using multiple databases, including the TargetScan, miRDB, miRmap and microT-CDS databases (Fig. 4A). Among the five overlapping candidate genes (Supplementary Table S4), CAMSAP2 emerged as the most promising target because of its strong association with lung cancer prognosis (Supplementary Fig. S1 and S2A). Furthermore, CAMSAP2 mRNA contains multiple predicted binding sites for miR-145-5p (Fig. 4B).

Fig. 4.

CAMSAP2 mRNA is a direct target of miR-145-5p. (A) Predicted target genes of miR-145-5p were identified using the TargetScan, miRDB, miRmap, and microT-CDS databases. The Venn diagram shows overlapping genes ranked in the top 25% or with prediction scores greater than 95. (B) Predicted miR-145-5p binding site on the CAMSAP2 3'-UTR, and the sequences used for the luciferase reporter assay are shown. (C) Correlation analysis of miR-145-5p and CAMSAP2 mRNA expression levels in NSCLC tissues obtained from the GSE102287 dataset. (D) A549 and H292 cells were transfected with either a miR-145-5p mimic or a negative control (miR-NC), and CAMSAP2 mRNA levels were assessed by qRT-PCR. The data are presented as the means ± SEMs (n = 3). ***P < 0.005 vs. miR-NC. (E) CAMSAP2 protein levels were analyzed by immunoblotting in transfected cells. The data are presented as the means ± SEMs (n = 3). ***P < 0.005 vs. miR-NC. The charts were plotted from triplicated experiments, in which the original blots were provided in supplementary files. (F) Luciferase activity was measured in A549 and H292 cells cotransfected with either wild-type (pGLO-C2-WT) or mutant (pGLO-C2-MUT) CAMSAP2 3'-UTR luciferase constructs, along with the miR-145-5p mimic or miR-NC. Relative luciferase activity is shown as the fold change compared with that of the miR-NC. The data are presented as the means ± SEMs (n = 3). *P < 0.05, **P < 0.01 vs. miR-NC

Analysis of the GSE102287 dataset revealed that CAMSAP2 expression was significantly lower in NSCLC tissues compared to normal lung tissues (Supplementary Fig. S2A). Additionally, mutation frequency analysis revealed no significant difference in CAMSAP2 mutation rates between lung cancer and normal tissues (Supplementary Fig. S2B), thereby suggesting that the downregulation of CAMSAP2 may occur by posttranscriptional mechanisms rather than due to genetic alterations.

A strong inverse correlation was observed between CAMSAP2 and miR-145-5p expression levels (Fig. 4C). To experimentally validate this relationship, A549 and H292 cells were transfected with either a miR-145-5p mimic or a negative control (miR-NC). The qRT-PCR results demonstrated significant downregulation of CAMSAP2 mRNA in cells transfected with the miR-145-5p mimic compared with those transfected with the miR-NC (Fig. 4D). Immunoblot analysis further confirmed the reduction in CAMSAP2 protein levels following miR-145-5p overexpression (Fig. 4E). In addition, luciferase reporter assays revealed that miR-145-5p significantly decreased the relative luciferase activity of the CAMSAP2 3’-UTR reporter in both A549 and H292 cells (Fig. 4F), thus supporting the conclusion that CAMSAP2 is a direct downstream target of miR-145-5p.

CAMSAP2 negatively regulates cisplatin responsiveness via PI3K/AKT signaling

Pathway enrichment analysis revealed that CAMSAP2 is associated with the PI3K/AKT/mTOR and Wnt/β-catenin signaling pathways in cancer (Supplementary Fig. S2C). The phosphoinositide 3-kinase (PI3K)/AKT signaling pathway is a central regulator of cellular survival and proliferation and is frequently activated in cancer, where it contributes to enhanced resistance to apoptosis and reduced sensitivity to anticancer therapies, while the Wnt/β-catenin signaling pathway is a key regulator of cell fate determination, proliferation, and differentiation, and its aberrant activation in cancer is associated with tumor progression, stemness, and resistance to anticancer therapies. Both of which have been previously implicated in cisplatin responsiveness [7, 30]. The role of CAMSAP2 in cisplatin sensitivity was further investigated using the DepMap data portal (Supplementary Fig. S2D). The analysis revealed that low CAMSAP2 expression was significantly correlated with a low area under the curve (AUC) for cisplatin, thereby indicating increased sensitivity to the drug. Similarly, NSCLC cells with reduced CAMSAP2 expression were more susceptible to cisplatin-induced cell death (Figs. 2B and 5A and B C).

Fig. 5.

CAMSAP2 knockdown enhances cisplatin sensitivity in NSCLC cells. (A) CAMSAP2 mRNA expression levels in NSCLC cell lines and BEAS-2B cells were analyzed by qRT-PCR. The data are presented as the means ± SEMs (n = 3). *P < 0.05 vs. BEAS-2B cells. (B) Correlation analysis of CAMSAP2 expression levels and the IC₅₀ of cisplatin in NSCLC cell lines. (C) A549 and H292 cells were transfected with siCAMSAP2 or control siRNA (siCtrl). The protein levels of CAMSAP2, AKT, phosphorylated AKT (p-AKT), PI3K, and phosphorylated PI3K (p-PI3K) were examined by immunoblotting and normalized to GAPDH expression. The data are presented as the means ± SEMs (n = 3). *P < 0.05, **P < 0.01 vs. siCtrl. The charts were plotted from triplicated experiments, in which the original blots were provided in supplementary files. (D) CAMSAP2-knockdown and control A549 and H292 cells were treated with various concentrations of cisplatin (0–250 µM) for 24 h. Cell viability was assessed using the MTT assay, and the percentage of viable cells was quantified. The data are presented as the means ± SEMs (n = 3). *P < 0.05 vs. siCtrl cells. IC₅₀ values were calculated by Prism 9 software. (E) Apoptotic cell death was assessed by H33342 nuclear staining. The number of cells displaying apoptotic nuclei was quantified as a percentage of the total number of cells per field. The data are presented as the means ± SEMs (n = 3). *P < 0.05 vs. siCtrl cells. (F) The protein expression of PARP and cleaved PARP was analyzed by immunoblotting and normalized to GAPDH expression. The data are presented as the means ± SEMs (n = 3). **P < 0.01 vs. siCtrl cells. The charts were plotted from triplicated experiments, in which the original blots were provided in supplementary files

To validate these findings, A549 and H292 cells (which endogenously express high levels of CAMSAP2) were transfected with CAMSAP2-specific siRNAs. Immunoblotting confirmed the effective knockdown of CAMSAP2 in both cell lines (Fig. 5C). Notably, CAMSAP2 silencing resulted in a marked reduction in the levels of phosphorylated PI3K (p-PI3K) and phosphorylated AKT (p-AKT), whereas the expression of other proteins associated with the Wnt/β-catenin signaling pathway remained unchanged (Fig. 5C and S2E). These results support the functional involvement of CAMSAP2 in cancer-associated pathways, particularly regarding its regulatory role in PI3K/AKT signaling.

Functional assays revealed that CAMSAP2 silencing enhanced cisplatin-induced cytotoxicity. The IC₅₀ of cisplatin in CAMSAP2-knockdown A549 cells was 23.50 ± 4.15 µM, which was significantly lower than that in control siRNA transfected cells (siCtrl; 44.37 ± 4.93 µM). Similarly, in H292 cells, CAMSAP2 knockdown reduced the IC₅₀ to 18.09 ± 2.19 µM compared with the IC₅₀ of 30.01 ± 4.21 µM in siCtrl cells (Fig. 5D). An apoptosis assay revealed a marked increase in the number of apoptotic CAMSAP2 depleted cells upon cisplatin treatment compared to siCtrl cells (Fig. 5E). Furthermore, immunoblotting revealed a significantly greater ratio of cleaved to total PARP in the cisplatin-treated CAMSAP2-knockdown cells, whereas this effect was less prominent in the control cells (Fig. 5F). These findings suggest that CAMSAP2 knockdown enhances cisplatin responsiveness in NSCLC cells.

To further confirm the role of CAMSAP2 in cisplatin sensitivity, CAMSAP2 was overexpressed in A549 and H292 lung cancer cells by transfection with a CAMSAP2 expression plasmid. Western blot analysis confirmed a marked increase in CAMSAP2 protein levels in both cell lines compared with control cells. Concomitantly, phosphorylation levels of PI3K and AKT were significantly elevated, whereas total PI3K and AKT levels remained unchanged, indicating activation of the PI3K/AKT signaling pathway upon CAMSAP2 overexpression (Supplementary Fig. S3A). Next, to verify whether CAMSAP2-mediated cisplatin resistance is dependent on PI3K/AKT signaling, control and CAMSAP2-overexpressing cells were pretreated with LY294002 for 1 h, followed by exposure to cisplatin for 24 h. Cell viability was assessed using the MTT assay, and apoptosis was evaluated by Hoechst 33342 staining. As shown in Supplementary Figure S3B and S3C, CAMSAP2 overexpression significantly attenuated cisplatin-induced cytotoxicity and apoptosis in both A549 and H292 cells. Importantly, pharmacological inhibition of PI3K/AKT signaling by LY294002, which the inhibitory activity was verified by western blot analysis (Supplementary Fig. S3D). It markedly reversed the protective effect conferred by CAMSAP2, restoring cisplatin sensitivity and apoptotic response. Collectively, these findings demonstrate that CAMSAP2 induces cisplatin resistance in lung cancer cells through activation of the PI3K/AKT signaling pathway.

miR-145-5p sensitizes cells to cisplatin-induced apoptosis by targeting CAMSAP2

To confirm that miR-145-5p enhances cisplatin-induced apoptosis by targeting CAMSAP2, A549 and H292 cells were cotransfected with miR-145-5p mimics and a CAMSAP2 overexpression plasmid. The results demonstrated successful overexpression of both miR-145-5p and CAMSAP2 following transfection. Notably, CAMSAP2 expression was significantly reduced by miR-145-5p transfection and was restored by cotransfection with the CAMSAP2 plasmid (Fig. 6A).

Fig. 6.

miR-145-5p sensitizes cells to cisplatin-induced apoptosis by targeting CAMSAP2 mRNA. (A) A549 and H292 cells were cotransfected with either a miR-145-5p mimic or a control miR-NC, along with the CAMSAP2 overexpression plasmid. The expression levels of miR-145-5p and CAMSAP2 were evaluated by qRT-PCR. The data are presented as the means ± SEMs (n = 3). *P < 0.05, **P < 0.01, ***P < 0.005 vs. NC; ##P < 0.01 vs. miR-145-5p. (B) All of the transfectants were treated with or without cisplatin (40 M) for 24 h. Cell viability was examined using the MTT assay, and the results are expressed as a percentage of viable cells. The data are presented as the means ± SEMs (n = 3). *P < 0.05 vs. cisplatin-treated NC group; #P < 0.05 vs. cisplatin-treated miR-145-5p group. (C) Apoptosis cell death was analyzed by H33342 nuclear staining. Apoptotic cells were quantified as a percentage of total cells per field. The data are presented as the means ± SEMs (n = 3). **P < 0.01, ***P < 0.005 vs. cisplatin-treated NC group; ##P < 0.01, ###P < 0.005 vs. cisplatin-treated miR-145-5p group. (D) PARP and cleaved PARP levels were analyzed by immunoblotting and normalized to GAPDH expression. The data are presented as the means ± SEMs (n = 3). *P < 0.05 vs. cisplatin-treated NC group; #P < 0.05 vs. cisplatin-treated miR-145-5p group. The charts were plotted from triplicated experiments, in which the original blots were provided in supplementary files

The cells were subsequently treated with 40 µM cisplatin and analyzed using the MTT assay. The results demonstrated that miR-145-5p significantly reduced cell viability to less than 20% in response to cisplatin, whereas cells transfected with miR-NC exhibited increased viability. In contrast, CAMSAP2 overexpression partially rescued the viability of the miR-145-5p transfected cells, increasing it to approximately 60% (Fig. 6B). These findings indicate that CAMSAP2 overexpression mitigates the sensitizing effect of miR-145-5p on cisplatin-induced cytotoxicity.

Consistently, apoptotic cell death as assessed by nuclear morphology was markedly increased (60–80%) in the miR-145-5p transfected cells following cisplatin treatment (Fig. 6C). However, the overexpression of CAMSAP2 in these cells reduced cisplatin-induced apoptosis to approximately 40–50%. Consistent with these observations, the levels of cleaved PARP were elevated in miR-145-5p-transfected cells treated with cisplatin but were notably decreased upon CAMSAP2 overexpression (Fig. 6D). In addition, miR-145-5p mimic markedly suppressed the phosphorylation of both PI3K and AKT. Importantly, co-transfection with a CAMSAP2 expression plasmid substantially attenuated this inhibitory effect, restoring PI3K and AKT activation (Supplementary Fig. S4). Consistently, miR-145-5p increased the Bax/Bcl-2 ratio, indicating enhanced apoptotic signaling, whereas CAMSAP2 overexpression reduced this ratio and reverse the pro-apoptotic effect of miR-145-5p. These findings indicate that miR-145-5p enhances cisplatin sensitivity by promoting apoptosis through modulation of the CAMSAP2/PI3K/AKT signaling axis.

miR-145-5p restores cisplatin sensitivity in cisplatin-resistant cells by targeting CAMSAP2

To further validate the role of miR-145-5p and CAMSAP2 in cisplatin resistance, A549-CR cells were established. The resistance phenotype was confirmed by MTT assay, which revealed that the IC₅₀ in A549-CR cells was approximately fivefold greater than that in parental A549 cells (Fig. S5A). A549-CR cells were subsequently transfected with either miR-145-5p mimics or siCAMSAP2. The qRT-PCR results confirmed successful overexpression of miR-145-5p following mimic transfection (Supplementary Fig. S5B). In addition, both treatments resulted in significant downregulation of CAMSAP2 expression (Supplementary Fig. S5B and C).

A cell viability assay revealed that miR-145-5p significantly increased the susceptibility of A549-CR cells to cisplatin-induced cytotoxicity, reducing the IC₅₀ by approximately twofold (Fig. S5D). Similarly, CAMSAP2 knockdown by siRNA partially restored cisplatin sensitivity in A549-CR cells (Fig. S5E). These findings suggest that miR-145-5p restores cisplatin sensitivity in resistant NSCLC cells by targeting CAMSAP2.

miR-145-5p sensitizes patient-derived lung cancer cells to cisplatin-induced apoptosis by targeting CAMSAP2

To enhance the translational relevance of our findings, we validated key observations in patient-derived lung cancer cells (ELC12 and ELC16). Rescue experiment was performed using miR-145-5p mimic in combination with CAMSAP2 overexpression (Supplementary Fig. S6A). As shown in Supplementary Figure S6B, miR-145-5p significantly enhanced cisplatin-induced cytotoxicity in both ELC12 and ELC16 cells, as demonstrated by reduced cell viability. In contrast, CAMSAP2 overexpression attenuated cisplatin sensitivity and increased cell viability. Notably, CAMSAP2 co-transfection partially reversed the miR-145-5p–mediated sensitization to cisplatin. Consistently, Hoechst 33342 staining revealed increased nuclear condensation and fragmentation in miR-145-5p–transfected cells following cisplatin treatment, which was markedly diminished upon CAMSAP2 overexpression (Supplementary Fig. S6C). Together, these results indicate that the miR-145-5p–CAMSAP2 axis regulates cisplatin responsiveness in patient-derived lung cancer cells, supporting the clinical relevance of our findings beyond immortalized cell lines.

Discussion

Recent findings have emphasized the critical role of miRNAs in modulating cisplatin responsiveness across various cancer types [7, 31]. Numerous studies have demonstrated that the dysregulation of specific miRNAs significantly contributes to the development of cisplatin resistance [3, 7, 31]. In the present study, we evaluated the regulatory role of miRNAs in cisplatin sensitivity in NSCLC by integrating bioinformatics analyses with in vitro validation. By differential expression profiling of TCGA datasets, we identified miR-145-5p as a key candidate due to its involvement in DNA damage response pathways and its potential clinical relevance. Notably, miR-145-5p was significantly downregulated in tumor tissues compared with normal tissues and was associated with poor OS, particularly among patients with LUAD. Functional assays further validated the role of miR-145-5p in modulating cisplatin sensitivity, as its overexpression led to a significant reduction in the IC₅₀ values and a marked induction of apoptosis in NSCLC cells. These findings highlight the potential of miR-145-5p as a therapeutic sensitizer. Mechanistically, CAMSAP2 was identified as a direct downstream target of miR-145-5p, thus providing important insights into the proapoptotic effects mediated by this miRNA (Fig. 7). The observed inverse correlation between CAMSAP2 expression and cisplatin sensitivity, together with the results of the rescue experiments suggest that CAMSAP2 suppression is a critical mediator of miR-145-5p-induced chemosensitization.

Fig. 7.

Schematic representation of the molecular mechanism by which miR-145-5p contributes to cisplatin resistance in lung cancer. The downregulation of miR-145-5p in lung cancer cells leads to increased CAMSAP2 expression, which suppresses cisplatin-induced apoptosis by the PI3K/AKT pathway and promotes the development of cisplatin resistance

miRNAs are increasingly becoming recognized as being pivotal regulators of cancer biology, orchestrating key processes such as proliferation, apoptosis, metastasis, and therapeutic responses. In the context of cancer, miRNAs may function as oncogenes or tumor suppressors, depending on the biological context and the nature of their target genes [31]. For example, miR-21 is among the most extensively characterized oncogenic miRNAs in NSCLC and has been observed to promote tumor progression by targeting tumor suppressor phosphatase and tensin homolog (PTEN) [32]. Conversely, tumor-suppressive miRNAs such as the let-7 family, miR-34, and miR-200 are frequently downregulated in NSCLC, thereby leading to the inhibition of tumor growth and metastasis, both of which are associated with advanced disease and poor prognosis [33, 34].

Accumulating evidence further suggests that aberrant miRNA expression plays a critical role in lung cancer progression and may serve as a valuable prognostic and predictive biomarker [7, 35]. In addition to their prognostic implications, miRNAs are also extensively involved in the regulation of drug resistance in NSCLC. Several miRNAs have been implicated in the multifaceted mechanisms underlying cisplatin resistance, particularly by the regulation of apoptosis and DNA repair. For example, miR-10b has been observed to confer cisplatin resistance by directly targeting the p53 protein [36], whereas miR-139-5p enhances cisplatin sensitivity by suppressing cell proliferation and promoting apoptosis through targeting of the homeobox protein HOXB2 [37]. Based on these findings, our study further expands upon this perspective by identifying DDR-related DEmiRNAs and establishing the prognostic and functional significance of miR-145-5p in modulating cisplatin sensitivity in NSCLC.

miR-145-5p is widely recognized as a tumor-suppressive miRNA that regulates critical processes involved in cancer progression [28]. Dysregulation of miR-145-5p has been reported in various malignancies, including breast, colorectal, bladder, and notably lung cancers, where it is frequently downregulated, leading to the upregulation of downstream oncogenic targets. For example, in breast cancer, miR-145-5p suppresses tumor progression by inhibiting SOX2 [38]. In colorectal cancer, it inhibits cell proliferation, metastasis, and epithelial–mesenchymal transition (EMT), a dynamic biological process in which epithelial cells acquire mesenchymal traits that enhance migratory capacity, invasiveness, and therapeutic resistance by targeting CDCA3 [39]. Similarly, in bladder cancer, miR-145-5p reduces cell proliferation and migration by targeting TAGLN2 [40].

In lung cancer, miR-145-5p has been shown to inhibit cell proliferation, migration, invasion, and EMT by targeting multiple oncogenic pathways [9, 10, 41]. Beyond its role in tumor progression, emerging evidence indicates that miR-145-5p is also involved in the regulation of drug resistance [7, 28, 42]. Notably, miR-145-5p has been reported to enhance gefitinib sensitivity in lung cancer by suppressing NRAS and MEST expression [43]. In another study of NSCLC, downregulation of miR-145-5p promoted EMT and drug resistance through upregulation of Sp1, particularly in pemetrexed-resistant cells [44]. Moreover, miR-145-5p has been implicated in the modulation of cisplatin resistance via interactions with drug-responsive non-coding RNAs, including circular RNA CircPVT1 and the long non-coding RNA LINC00852 [45, 46]. Several downstream targets of miR-145-5p, such as ABCC1, KLF4, and CDK6, have been reported to mediate cisplatin sensitivity [7, 45, 46]. Despite these advances, the role of miR-145-5p in regulating cisplatin sensitivity in lung cancer remains incompletely characterized, and the underlying molecular mechanisms are not fully understood. Consistent with previous reports, our study demonstrated a significant depletion of miR-145-5p in lung tumor tissues and revealed a positive correlation between miR-145-5p expression and OS in patients, emphasizing its potential clinical relevance.

Importantly, we identified calmodulin-regulated spectrin-associated protein family member 2 (CAMSAP2) as a novel direct target of miR-145-5p that is involved in modulating cisplatin responsiveness. CAMSAP2 is a noncentrosomal minus-end binding protein that is known to regulate microtubule dynamics and modulate cell motility [47]. Although microtubule-associated proteins have been implicated as being prognostic biomarkers in lung cancer [48, 49], CAMSAP2 has recently emerged as being a distinct tumor promoter. For example, CAMSAP2 has been identified as a downstream effector of the circSOD2/miR-2355-5p regulatory axis, contributing to enhanced cell proliferation and migration [50]; moreover, it has been demonstrated to promote cancer metastasis through activation of the RASAL2/ERK signaling pathway [20]. These findings strongly support our observations that CAMSAP2 is overexpressed in NSCLC and plays a critical role in tumor progression. In the present study, we further explored the role of CAMSAP2 in cisplatin resistance. Using a combination of target prediction databases and luciferase reporter assays, we confirmed that miR-145-5p directly binds to the 3’ UTR of CAMSAP2 mRNA, thus leading to translational repression and mRNA degradation. Functionally, CAMSAP2 modulates the downstream PI3K/AKT signaling pathway, which is a well-established pathway that promotes cancer cell survival and drug resistance [7].

In the present study, we uncover a previously unrecognized mechanism by which miR-145-5p enhances cisplatin sensitivity through direct targeting of CAMSAP2 and subsequent modulation of the PI3K/AKT pathway. Importantly, we demonstrate that this pathway plays a causal role in mediating cisplatin-induced cytotoxicity. Using a series of systematic and precisely designed experiments, including gain- and loss-of-function analyses, direct target validation, pathway interrogation, and rescue experiments, we provide compelling evidence that miR-145-5p regulates cisplatin sensitivity through this newly identified axis.

This work advances the field by moving beyond correlative observations and isolated targets to establish a mechanistically integrated miR-145-5p/CAMSAP2/PI3K/AKT regulatory axis. Our findings not only deepen the understanding of miRNA-mediated chemoresistance mechanisms but also highlight miR-145-5p and its downstream pathway as potential therapeutic targets and predictive biomarkers for improving cisplatin efficacy in lung cancer.

Taken together, these findings suggest that the downregulation of miR-145-5p in NSCLC leads to CAMSAP2 upregulation, which suppresses cisplatin-induced apoptosis and contributes to drug resistance (Fig. 7). This study not only corroborates the tumor-suppressive role of miR-145-5p but also reveals a novel regulatory axis influencing cisplatin responsiveness. Despite the compelling evidence provided by this study, several limitations should be acknowledged. First, although our in vitro findings are robust, in vivo validation by animal models or patient-derived xenografts would strengthen the clinical relevance of the miR-145-5p/CAMSAP2 axis. Due to limitations in time and resources, such in vivo experiments were beyond the scope of the present study. Nevertheless, to enhance translational relevance, we validated our key observations using patient-derived lung cancer cells (Supplementary Fig. S6), which represent an intermediate and clinically meaningful model that more closely reflects tumor heterogeneity and drug response in patients. Second, while our data demonstrate that CAMSAP2 activates PI3K/AKT signaling and promotes apoptosis evasion, the partial rescue of miR-145-5p–mediated cisplatin sensitivity by CAMSAP2 overexpression suggests that additional downstream targets and signaling pathways are likely involved. miR-145-5p is known to regulate multiple genes implicated in cell survival, apoptosis, and chemoresistance [45, 51]; therefore, its tumor-suppressive effects are unlikely to be mediated exclusively through CAMSAP2. Accordingly, although the CAMSAP2/PI3K/AKT axis represents a major mechanism contributing to cisplatin resistance, a more comprehensive exploration of alternative pathways and interacting molecular networks may uncover additional mechanisms underlying miR-145-5p–regulated drug responsiveness. In particular, further investigation into how CAMSAP2 interfaces with other components of the DDR response network may reveal novel therapeutic vulnerabilities. Finally, validation of miR-145-5p and CAMSAP2 expression in clinical samples in relation to cisplatin response are needed to further support their potential application as predictive biomarker for cisplatin resistance.

Conclusions

In conclusion, our study identified the miR-145-5p/CAMSAP2 axis as a critical modulator of cisplatin sensitivity in NSCLC. These findings highlight miR-145-5p as a promising dual-function biomarker for prognosis and chemosensitivity and suggest that the therapeutic restoration of miR-145-5p levels could be a novel strategy to overcome cisplatin resistance and improve treatment outcomes in lung cancer.

Abbreviations

A549-CR

Cisplatin-resistant A549 cells

CAMSAP2

Calmodulin-regulated spectrin-associated protein family member 2

DDR

DNA damage response

DEmiRNAs

Differentially expressed miRNAs

GEO

Gene Expression Omnibus

GSEA

Gene Set Enrichment Analysis

LUAD

Lung adenocarcinoma

LUSC

Lung squamous cell carcinoma

miRNAs

microRNAs

NSCLC

Non-small cell lung cancer

siRNAs

Small interfering RNAs

TCGA

The Cancer Genome Atlas

Author contributions

V.P. and B.G. performed study concept and design; N.S., W.L.T., W.A., S.L., C.S., P.K., S.P. and I.I., P.C. and C.V. performed development of methodology and review the paper; N.S. and V.P. wrote and revised the paper; N.S., W.L.T. and V.P. provided acquisition, analysis and interpretation of data, and statistical analysis; V.P. and B.G. provided technical and material support. All authors read and approved the final paper.

Funding

This work was funded by Thailand Science research and Innovation Fund Chulalongkorn University (HEA_FF_68_011_3300_003 for in vitro experiments), the Faculty of Pharmaceutical Sciences, Chulalongkorn University (Phar2564-RG001 for clinical analysis) and the Second Century Fund, Chulalongkorn University (C2F to NS).

Data availability

All data are included in this article and supplementary file.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.