miR-3613-5p promotes lung cancer progression by targeting and regulating XPO6.

To explore miR-3613-5p’s role in LC, we detected its expression in 112 LC samples. miR-3613-5p expression was significantly higher in LC tissues than in corresponding normal tissues ( P  < 0.05) (Fig.  1 A). Meanwhile, the expression of miR-3613-5p in cell lines was also detected, and our results showed that miR-3613-5p was significantly upregulated in all LC cell lines compared with the normal LC cell line ( P  < 0.05) (Fig.  1 B). The median overall survival was 51 months (95% CI: 48.21, 53.42) in patients with low miR-3613-5p expression and 39 months (95% CI: 34.48, 43.68) in patients with high miR-3613-5p expression in LC. Survival analysis indicated that miR-3613-5p expression strongly correlated with overall survival in LC patients, with LC patients exhibiting high miR-3613-5p expression showing poorer overall survival ( P  < 0.001) (Fig.  1 C). Fig. 1 miR-3613-5p expression in LC and its link to survival. A - B qRT-PCR was performed to detect miR-3613-5p expression in normal and LC tissues (A), as well as in normal and LC cells (B) C High and low miR-3613-5p expression-based survival curves in 112 LC patients miR-3613-5p expression in LC and its link to survival. A - B qRT-PCR was performed to detect miR-3613-5p expression in normal and LC tissues (A), as well as in normal and LC cells (B) C High and low miR-3613-5p expression-based survival curves in 112 LC patients miR-3613-5p expression in LC tissues was not associated with age or gender, but was significantly correlated with smoking history, TNM stage, tumor size, and lymph node metastasis ( P  < 0.05) (Table  1 ). Moreover, multivariate regression results indicated miR-3613-5p ( P  < 0.05, 95% CI = 1.855–12.204) is an independent LC prognostic factor. (Table  2 ). Table 1 Clinical characteristics of 112 LC patients in two groups (stratified by the mean value of miR-3613-5p expression level) All cases ( n  = 112) Low miR-3613-5p ( n  = 51) High miR-3613-5p ( n  = 61) P Age (years) ≤ 60 50 23 27 0.929 > 60 62 28 34 Gender Male 53 22 31 0.417 Female 59 29 30 smoking history No 29 18 11 0.038 Yes 83 33 50 TNM stage I/II 39 25 14 0.004 III/IV 73 26 47 Tumor size < 3 cm 28 18 10 0.021 ≥ 3 cm 84 33 51 Lymph node metastasis Negative 32 22 10 0.002 Positive 80 29 51 Clinical characteristics of 112 LC patients in two groups (stratified by the mean value of miR-3613-5p expression level) Table 2 Multivariate Cox regression analysis HR 95% CI P miR-3613-5p 4.758 1.855–12.204 0.001 Age 1.198 0.567–2.534 0.636 Gender 1.270 0.61–2.644 0.523 Smoking history 1.286 0.548–3.014 0.563 TNM 1.141 0.244–5.343 0.867 Tumor size 1.066 0.256–4.437 0.93 Lymph node metastasis 1.133 0.175–7.353 0.896 Multivariate Cox regression analysis XPO6 was determined as a predicted target gene of miR-3613-5p via the miRDB database, with the predicted binding sites shown in Fig.  2 A. We further used a dual-luciferase reporter gene assay to verify if XPO6 is miR-3613-5p’s direct target in LC cells. Compared to the NC group, miR-3613-5p mimics significantly inhibited wt-XPO6 luciferase activity ( P  < 0.05), whereas no notable inhibition was observed in the mutant type (Fig.  2 B-C). This indicated that miR-3613-5p directly binds to XPO6. XPO6 was significantly underexpressed in LC cells ( P  < 0.05) (Fig.  2 D). Fig. 2 miR-3613-5p directly targets XPO6. A Predicted binding sites between miR-3613-5p and XPO6. B - C Luciferase activity assays of wild-type XPO6 and mutant XPO6 in H23 ( B ) and H1299 cells ( C ). D The expression of XPO6 in LC cell lines miR-3613-5p directly targets XPO6. A Predicted binding sites between miR-3613-5p and XPO6. B - C Luciferase activity assays of wild-type XPO6 and mutant XPO6 in H23 ( B ) and H1299 cells ( C ). D The expression of XPO6 in LC cell lines To investigate whether overexpression of XPO6 can counteract the stimulatory influence of miR-3613-5p, transfection efficiency was first verified using qRT-PCR. Transfection of miR-3613-5p mimics into H23 and H1299 cells confirmed that miR-3613-5p was markedly overexpressed in cells transfected with miR-3613-5p mimics ( P  < 0.05) (Fig.  3 A). When miR-3613-5p was overexpressed, XPO6 was significantly downregulated ( P  < 0.05) (Fig.  3 B). Co-transfection of miR-3613-5p mimics and XPO6 plasmids demonstrated that overexpression of XPO6 could reverse the miR-3613-5p mimic-induced downregulation of XPO6. This indicated that overexpression of XPO6 reverses the downregulatory impact of miR-3613-5p on XPO6. As shown in Fig.  3 C-D, miR-3613-5p could promote cell growth ( P  < 0.05), but overexpression of XPO6 restored the cell growth rate. Likewise, XPO6 overexpression also suppressed cell migration and invasion ( P  < 0.05), reversing the cancer cell growth-promoting effect induced by miR-3613-5p (Figs.  3 E-F). Fig. 3 Overexpression of XPO6 reverses the stimulatory effect of miR-3613-5p. A miR-3613-5p expression detected via qRT-PCR. B Expression level of XPO6 determined via qRT-PCR. (C-D) Cell proliferation of H23 ( C ) and H1299 ( D ) cells. E - F Migration ( E ) and invasion ( F ) of H23 and H1299 cells Overexpression of XPO6 reverses the stimulatory effect of miR-3613-5p. A miR-3613-5p expression detected via qRT-PCR. B Expression level of XPO6 determined via qRT-PCR. (C-D) Cell proliferation of H23 ( C ) and H1299 ( D ) cells. E - F Migration ( E ) and invasion ( F ) of H23 and H1299 cells

112 pairs of LC and corresponding normal tissues were collected from Chongqing Southwest Aluminium Hospital. The Chongqing Southwest Aluminium Hospital Ethics Committee approved this study, with all participants providing written informed consent before enrollment. Before study inclusion, no patients had undergone any anti-tumor treatment. Tissue processing was conducted strictly in compliance with institutional policies and approved experimental protocols. Human LC cell lines and normal LC lines were acquired from the Type Culture Collection of the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. miR-3613-5p mimics and negative controls were generated by Ribobio Technology Co., Ltd. (Guangzhou, China). Cells were maintained in culture according to the manufacturer’s provided instructions. BEAS-2B and H1650 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin; A549 cells were cultured in DMEM/F12 (1:1) medium; H23 and H1299 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum. All cells were cultured in a humidified incubator at 37 °C with 5% CO₂. For transfection, miRNA mimics (50 nM) or NC were transfected into cells using Invitrogen™ Lipofectamine 2000 (Life Technologies, New York, USA) and incubated for 48 h. To generate pcDNA3.1-XPO6, we inserted the XPO6 CDS into the pcDNA3.1(+) plasmid. Total RNA was isolated from cells or tissues via Trizol reagent (Invitrogen, USA) for miR-3613-5p and XPO6 detection. Reverse transcription of miR-3613-5p was conducted with stem-loop RT primers (RiboBio, China). qRT-PCR products were detected using SYBR Green; U6 served as the internal control for miR-3613-5p (and GAPDH for XPO6) to calculate relative expression levels. The qRT-PCR primer sequences and PCR conditions are detailed as follows: The primers are listed as: U6-forward: 5’-GCTTCGGCAGCACATATACTAAAAT-3’, U6-reverse: 5’-CGCTTCACGAATTTGCGTGTCAT-3’; miR-3613-5p-forward: 5’-CTTGTTTTTTTTTTTTCATGTTGT-3’, miR-3613-5p-reverse: 5’-AGTCTCAGGGTCCGAGGTATTC-3’; GAPDH-forward: 5’-CTGGGCTACACTGAGCACC-3’, GAPDH-reverse: 5’-AAGTGGTCGTTGAGGGCAATG-3’; XPO6-forward: 5’-AACTCCTTTTGGCTCACCATAAA-3’, XPO6-reverse: 5’-TGTCACAGGGGACTGGATCAA-3’. For PCR reaction conditions, they start with initial denaturation at 95 °C for 30 s, followed by 40 amplification cycles (each cycle includes denaturation at 95 °C for 5 s and annealing at 60 °C for 31 s), and finally extension at 65 °C for 5 s. XPO6 was forecast as a potential target gene of miR-3613-5p using the miRDB database ( https://mirdb.org/ ). The 3’ UTR sequences containing wild-type or mutant binding sites of XPO6 for miR-3613-5p were synthesized and cloned into the psiCHECK-2 vector plasmid (Promega, Madison, WI, USA). The wild-type or mutant plasmids, along with miR-3613-5p mimic, were co-transfected into H23 and H1299 cells, after which luciferase activity was assessed. To assess cell proliferation, the CCK-8 assay kit (Dojindo, Tokyo, Japan) was utilized. At 0, 24, 48, and 72 h of culture, CCK-8 was added to each well. After incubation, the absorbance at 450 nm was detected. For the Transwell migration assay, H23 and H1299 cells (5 × 10⁴ cells) were placed in the upper chamber’s uncoated membrane. For the invasion assay, H23 and H1299 cells (1 × 10⁵ cells) were plated in the upper chamber with an extracellular matrix gel-coated membrane (Sigma, E1270). For both assays, upper chambers received cells in serum-free medium, and lower chambers contained 15% serum medium as a chemoattractant. After 24 h of incubation for migration and 48 h for invasion, non-migratory and non-invasive cells on the upper membrane were wiped off using a swab. Lower membrane cells were fixed with methanol, stained with 0.1% crystal violet (Sigma), and counted. All data analyses relied on GraphPad Prism 10.1.2. Group differences were analyzed using the T-test, one-way ANOVA, and two-way ANOVA. A P -value < 0.05 was considered statistically significant. The median expression level of miR-3613-5p was used as the cut-off value, the KM curve was generated using SPSS 27, and the COX analysis was performed using SPSS 27. Tukey’s method was used for statistical correction of multiple comparisons. All experiments included ≥ 3 biological replicates and ≥ 3 technical replicates.

miR-3613-5p exhibits an upregulated expression trend in LC tissues and cells, and directly targets XPO6, while XPO6 shows downregulated expression in LC. Meanwhile, clinical correlation and prognostic analyses indicate that miR-3613-5p can serve as an independent prognostic factor. Notably, the prognostic role of the miR-3613-5p-XPO6 axis in LC is demonstrated for the first time. At present, the clinical translation of the miR-3613-5p/XPO6 axis is still in the early stage. Future studies using in vivo models will be needed to further validate this mechanism.

miRNAs play critical regulatory roles in cancer initiation, development, and metastasis, and they hold promise as reliable biomarkers for assessing cancer prognosis [ 26 ]. In the field of LC research, miRNAs not only serve as potential indicators for LC diagnosis but also provide important references for prognostic evaluation [ 27 ]. Meanwhile, as potential therapeutic targets for LC, miRNAs have driven relevant studies that offer novel insights into optimizing clinical intervention strategies for LC [ 28 ]. It has been reported that several miRNAs, such as miR-106a, miR-146, miR-155, miR-150, miR-17-3p, miR-191, miR-197, miR-192, miR-21, miR-203, miR-205, miR-210, miR-212, and miR-214, exhibit an upregulated expression trend in LC [ 29 ]. Given the significant value of miRNAs in LC research, this study focused on miR-3613-5p to explore in depth its prognostic value in LC and the regulatory mechanism. We found that miR-3613-5p expression was upregulated in LC, suggesting that it may function as an oncogene in LC. Concurrently, we observed that miR-3613-5p expression was significantly associated with smoking history, TNM stage, tumor size, and lymph node metastasis. High miR-3613-5p expression correlated with significantly shortened overall survival among LC patients. Additionally, the results of multivariate Cox regression analysis further confirmed that miR-3613-5p could serve as an independent prognostic factor for LC patients. Existing studies have shown that plasma miR-3613-5p can serve as a non-invasive biomarker. For instance, plasma miR-3613-5p may have greater potential as a biomarker for endometriosis [ 30 ]. Based on this, it is speculated that miR-3613-5p could also serve as a biomarker for LC. Similar associations between miRNAs and the clinicopathological features have also been reported in previous studies. For instance, the expression of miR-21 in LC is significantly correlated with TNM stage, lymph node metastasis, and overall survival [ 31 ]. miR-128-3p shows a strong correlation with smoking history, tumor size, and TNM stage in LC patients [ 32 ]. miR-126 has even been identified as an independent prognostic indicator in NSCLC [ 33 ]. This study exhibits certain similarities to the reported functional patterns of miRNAs in LC. Previous studies have demonstrated that miRNAs can participate in the initiation and advancement of cancer by regulating their target mRNAs [ 34 ]. To further clarify the mechanism underlying the role of miR-3613-5p, we predicted its potential target genes using bioinformatics databases and found that miR-3613-5p might target XPO6. XPO6 exerts important functions in various key biological processes, including transcriptional regulation, cell death, cell adhesion, memory formation, and cell differentiation [ 20 ]. Meanwhile, this study also identified that XPO6 was significantly decreased in LC cells, and this result is consistent with findings from relevant studies in other fields. For example, the expression of XPO6 is also downregulated in senescent human fibroblasts, and its expression level is correlated with cell proliferation ability [ 35 ]. In addition, another study reported that the expression of XPO6 is also significantly decreased in thyroid cancer tissues or cells [ 36 ]. In summary, the finding of low XPO6 expression in LC in this study is consistent with the expression patterns of XPO6 in other diseases or physiological processes reported in the aforementioned studies. miR-3613-5p can significantly enhance the proliferation, migration, and invasion capabilities of LC cells, a functional trait aligning with its previously reported roles in cancer cells. For example, miR-3613-5p is closely associated with cancer cell proliferation [ 37 ]. In gastric cancer research, silencing miR-3613-5p alleviates pathological gastric mucosal lesions, in contrast to its overexpression, which markedly promotes cell proliferation and migration [ 38 ]. In LUAD, miR-3613-5p has also been shown to promote LUAD cell proliferation [ 12 ]. In senescent human fibroblasts, downregulation of XPO6 promotes cell proliferation [ 35 ]. The regulation of gene expression by miRNAs is reversible [ 39 ], and this regulatory relationship is not unidirectional—miRNAs can not only regulate mRNAs, but mRNAs can also exert reverse regulation on miRNAs [ 40 ]. For instance, the 3’-UTR of LMO7 contains a sequence that matches miR-96, and the oncogenic effect of miR-96 in LC is reversed by LMO7 overexpression [ 41 ]. Similarly, miR-223 regulates caprin-1 by targeting its 3’-UTR, thereby influencing cell proliferation and invasion capabilities [ 42 ]. miR-3613-5p directly targets NR5A2 to activate the AKT/MAPK pathway [ 12 ]. By extension, it is speculated that miR-3613-5p in the present study may also directly target XPO6 to activate the AKT/MAPK pathway. The present study is the first to explore the regulatory mechanism between miR-3613-5p and XPO6 in LC. In conclusion, this study confirms that miR-3613-5p is closely associated with smoking history, tumor size, TNM stage, and lymph node metastasis in LC patients, and it can serve as an independent prognostic biomarker for LC patients. miR-3613-5p has shown potential as a disease biomarker, but its clinical application is still in the early stage. Such results suggest that miR-3613-5p may promote LC. Mechanistically, miR-3613-5p targets XPO6, and XPO6 can regulate miR-3613-5p to further influence the progression of LC. Based on these findings, downregulating miR-3613-5p or overexpressing XPO6 is expected to be a potential therapeutic strategy for LC patients, thereby providing a novel perspective for treating LC clinically. However, the present study has the following limitations: it is a single-center study with a limited sample size, and lacks validation by in vivo experiments.

Lung cancer (LC) is not only one of the most widely diagnosed malignant tumors worldwide but also remains the leading contributor to global cancer-related mortality [ 1 ]. LC falls primarily into two categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) [ 2 ]. Among these, NSCLC constitutes a larger proportion of cases and can be further divided into main subtypes: squamous cell carcinoma (SCC), adenocarcinoma (AD), and large cell carcinoma (LCC) [ 3 ]. Statistics show that approximately 1.8 million new LC cases are diagnosed and 1.6 million deaths are attributed to this disease annually worldwide, with its mortality rate exceeding the combined mortality rates of colorectal cancer, breast cancer, and prostate cancer [ 4 , 5 ]. Despite significant advancements in early screening techniques and standardized treatment strategies for LC in recent years, the disease is still at an advanced stage when most patients are diagnosed, with overall prognosis remaining poor [ 6 , 7 ]. Therefore, in-depth exploration of the mechanisms associated with LC prognosis remains one of the core focuses in the field of malignancy research [ 8 ]. A class of small endogenous non-coding RNAs, known as microRNAs (miRNAs), has an approximate length of 19–25 nucleotides (nt) [ 9 ]; their mechanism of action involves incomplete base pairing with complementary sites within target mRNAs’ 3’-UTR, which enables them to negatively regulate target gene expression, ultimately leading to the degradation of target mRNAs [ 10 ]. miRNAs perform key regulatory functions in the emergence and advancement of human malignant tumors, including LC [ 11 ]. In existing studies, miR-3613-5p is found to act as an oncogene in lung adenocarcinoma (LUAD) [ 12 ]; in pancreatic cancer, downregulated miR-3613-5p expression relates to poor tumor prognosis [ 13 ], while in breast cancer [ 14 ] and gastric cancer [ 15 ], miR-3613-5p expression is notably overexpressed. Collectively, miR-3613-5p serves as a biomarker that has dual roles (either oncogenic or tumor-suppressive), and its functional pattern is closely linked to cancer type [ 16 ]. XPO6, a member of the importin-β nuclear transport protein family [ 17 ], has one of its core functions in mediating the migration of miRNAs from the nucleus to the cytoplasm [ 18 ], while it is also closely involved in the regulation of inflammatory responses and immune-related genes [ 19 ]. In recent years, growing research evidence points to the fact that XPO6 is closely associated with cancer progression [ 20 ]; as the main regulator of nuclear export for actin family proteins, XPO6 has been clearly identified as an oncogene in prostate cancer and breast cancer [ 21 ], and another study has confirmed that low expression levels of XPO6 are significantly correlated with decreased survival rates in breast cancer patients [ 22 ]. miRNAs can modulate gene expression by targeting mRNAs, thereby participating in the occurrence and development of tumors [ 23 ]. For instance, the expression of XPO6 mRNA in prostate cancer cells is modulated by miR-1 [ 24 ]. Dysregulated miR-1 and miR-133a are closely associated with the negative regulation of mRNA targets, including XPO6 [ 25 ]. However, the prognostic value of miR-3613-5p in LC and the regulatory relationship between miR-3613-5p and XPO6 remain unknown. We conducted the first investigation of the miR-3613-5p-XPO6 axis in LC. Therefore, this study evaluates the prognostic value of miR-3613-5p in LC, laying a foundation for further in-depth exploration of its regulatory mechanism.