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. 2024 Apr 2;14(5):125. doi: 10.1007/s13205-024-03975-y

LncRNA MRPL39 inhibits cell proliferation and migration by regulating miR-130/TSC1 axis in non-small cell lung cancer

Qinghao Fan 1,✉, Xianrong Bao 1, Han Zhao 1, Sichen Li 1
PMCID: PMC10987421  PMID: 38577417

Abstract

Currently, the effect of miR-130 on non-small cell lung cancer (NSCLC) remains controversial. In this study, the expression of miR-130 and lncRNA MRPL39 in tumor and non-tumor tissues of NSCLC patients was examined using real-time PCR (RT-PCR) and correlated with the prognosis of NSCLC. The phenotypic effects of miR-130 and MRPL39 on proliferation and migration of NSCLC cell line A549 cells were assessed through CCK-8 and Transwell assays with miR-130 mimic and MRPL39 (mitochondrial ribosomal protein L39) overexpressed plasmid transfection. StarBase/TargetScan analysis and dual-luciferase reporter gene assays were conducted to investigate the relationship between MRPL39, miR-130, and Tuberculosis sclerosis 1 (TSC1). MiR-130 was overexpressed, and MRPL39 was downregulated in NSCLC tissues and cells. Inhibition of miR-130 expression and overexpression of MRPL39 resulted in the inhibition of the viability and migration of A549 cells. MRPL39 is a potential upstream regulatory long non-coding RNA of miR-130, and its expression is negatively regulated by miR-130. TSC1 was identified as a target of miR-130, suppressing the antitumor effects of FGD5-AS1 silencing on GBM cells. After overexpression of MRPL39, the mRNA and protein levels of TSC1 in A549 cells significantly increased. However, after transfection with miR-130 mimic, the up-regulation of mRNA and protein was inhibited, leading to the suppression of cell proliferation and migration.

Keywords: Non-coding RNA, Tumor, Clinical marker, microRNA

Introduction

Lung cancer ranks as the second most prevalent malignant tumor, with mortality rates consistently topping the list among all malignancies (Schabath and Cote 2019; Qu et al. 2020; Li et al. 2023; Carolina and Nuria 2020). Non-small cell lung cancer (NSCLC) constitutes over 80% of all lung cancers and stands as the predominant pathological type (Zhi 2019; Jin et al. 2022a, b; Rodriguez de Dios et al. 2020). Large cell carcinoma, squamous cell carcinoma, and adenocarcinoma represent the three most prevalent subtypes of NSCLC. Specifically, NSCLC is characterized by its aggressive nature, marked by a rapid doubling time, a tendency for early dissemination, and, in many cases, a swift onset of symptoms. Currently, the clinical management of NSCLC primarily revolves around surgical resection, chemotherapy, and radiotherapy, yet the prognosis remains suboptimal (Tsuboi et al. 2007; Chaft et al. 2021). The formidable challenges posed by NSCLC, including its aggressive growth, widespread dissemination, and high recurrence rate, contribute significantly to its poor prognosis (Beltran and Blancafort 2011). Therefore, there is a pressing need to delve into the potential molecular mechanisms underlying the onset and progression of NSCLC and to formulate innovative therapeutic strategies.

MicroRNAs (miRNAs) constitute a class of endogenously expressed non-coding small RNA molecules, typically around 22 nucleotides in length, which regulate gene expression through interactions with target genes (Rennie et al. 2016; Kuru and Akgöl 2022). Their dysregulation plays a role in various facets of cancer cell behavior, influencing processes such as cell growth, apoptosis, proliferation, migration, and invasion (Rupaimoole and Slack 2017). Depending on their specific targets, miRNAs can function as either tumor suppressors, impeding cancer progression, or oncogenes, promoting cancer development. Due to their sensitivity, miRNAs serve as valuable biomarkers for diagnosing and treating various diseases, including lung cancer and Alzheimer's disease (Wang et al. 2022). The abnormal expression of several miRNAs, such as miR-486, miR-30d, miR-499, miR-137, miR-375, miR-182, miR-130, and let7a, is associated with the onset and progression of NSCLC (Ye et al. 2017; Qi et al. 2018; Wang et al. 2018; Chen et al. 2021; Zhu et al. 2021; Dezfuli et al. 2022; Jin et al. 2022a; Zeng et al. 2023). The majority of miRNAs serve as crucial indicators for tumors and are explored as potential therapeutic interventions in NSCLC. The observation of changes in miRNA expression levels can help identify the specific type of lung tumor. MiRNAs play a pivotal role in altering the expression of genes associated with lung tumors, influencing various biological processes such as cell division, proliferation, and apoptosis. Depending on their role, miRNAs can function as either tumor suppressors or oncogenes. Extensive research has been conducted on changes in miRNA expression within the context of NSCLC. Specific miRNAs have been pinpointed as potential biomarkers and promising targets for therapeutic interventions in the treatment of NSCLC. The role of miR-130 in NSCLC remains a subject of debate, with conflicting findings reported in the literature (Ye et al. 2017; Zhang et al. 2018). Both members of the miR-130 family, namely miR-130a and miR-130b, have been associated with various malignancies, including NSCLC. It is crucial to emphasize that the precise roles of miR-130a in NSCLC may vary, and additional investigation is necessary to delve deeper into its molecular mechanisms and clinical implications. Furthermore, it's important to consider miR-130b, a distinct member of the miR-130 family, independently of miR-130a, as it may have different effects in cancer. Therefore, it is imperative to identify the correlation between miR-130 and NSCLC, taking into account the potential divergent roles of different members within the miR-130 family.

In addition to miRNA, researchers are investigating the link between NSCLC and the tuberous sclerosis gene (TSC). TSC is known to be associated with an autosomal dominant syndrome and comprises TSC1 and TSC2, encoding hamartin and tuberin, respectively (Pfirmann et al. 2021). Notably, the inactivation of these genes has been confirmed to be associated with NSCLC (Pfirmann et al. 2021). Notably, the inactivation of these genes has been confirmed to be associated with NSCLC (Fuchs et al. 2014). The hamartin/tuberin protein complex plays a central role in the regulation of the mammalian target of rapamycin (mTOR) signaling pathway (Kotulska et al. 2009), Specifically, TSC1 is a vital component of the pro-survival mTOR signaling pathway, contributing significantly to various cellular processes such as development, cell growth, proliferation, survival, autophagy, and ciliary development through its collaboration with a diverse array of regulatory molecules (Huang and Manning 2008; Li et al. 2017; Mallela and Kumar 2021). Additionally, NSCLC commonly features mutations in the tuberous sclerosis complex subunits 1 and 2, although their implications on antitumor immunity have not been thoroughly investigated. This research is specifically focused on identifying the relationship between TSC and NSCLC, shedding light on the potential significance of these mutations in the context of antitumor immune responses.

Long non-coding RNA (LncRNA) refers to a type of non-coding transcript typically exceeding 200 nucleotides in length (Bridges et al. 2021). It constitutes one of the largest and significantly distinct RNA families that have garnered attention in recent years (Xu et al. 2022). Advancements in research have unveiled the crucial role of LncRNA in transcriptional regulation, including genome rearrangement, chromosome modification, and X chromosome silencing (Cech and Steitz 2014; Dykes and Emanueli 2017). LncRNA plays a pivotal role in various biological processes such as the proliferation, invasion, migration, and apoptosis of different cancer cells (Ma et al. 2013; Zhao et al. 2018; Cao et al. 2019). While lncRNAs were initially considered non-functional "noise" in the genome, distinct from messenger RNAs involved in protein production, the past decade has witnessed a wealth of studies demonstrating their involvement in the onset and progression of numerous diseases, including cancer. LncRNAs play essential roles in a variety of cellular processes. For instance, Yu et al. (2018) showcased that LncRNA MRPL39 can inhibit gastric cancer proliferation and progression by directly targeting miR-130. In addition to their higher oncogenic and tumor-suppressive properties, lncRNAs are gaining prominence as significant molecules in lung cancer. The stability of LncRNAs in the bloodstream makes them particularly promising as potential non-invasive tools for early-stage cancer detection. Consequently, this research also aims to explore and establish the potential link between LncRNAs and NSCLC, with a focus on their role in the context of lung cancer.

The literature review has suggested a potential relationship between lncRNA, miR-130, and TSC1 in NSCLC. Therefore, this research aims to uncover the underlying mechanisms involving lncRNA, miR-130, and TSC1 in NSCLC. The expression levels of miR-130 and lncRNA MRPL39 in both tumor and non-tumor tissues of NSCLC patients were identified using RT-PCR. Additionally, cell viability and total protein estimation were analyzed through CCK-8 and Western blot assays, respectively. These assays are instrumental in drawing conclusions about the intricate relationship between miR-130, lncRNA, and TSC1 in NSCLC. The findings from this research have the potential to contribute to the development of highly sensitive diagnostic methods and targeted drugs for NSCLC.

Materials and methods

Clinical sample collection and cell culture

Forty pairs of frozen tissue samples, including both NSCLC patient tissues and non-tumor tissues, were collected from Jinhua People’s Hospital. These samples were rapidly frozen in liquid nitrogen and subsequently stored at − 80 ℃ for the purpose of RNA extraction. It is important to note that this study was carried out with the informed consent of NSCLC patients and received approval from the Ethics Committee of Jinhua People’s Hospital (Approval No. IRB-2021037-R).

The human lung cancer cell lines (HCC827, NCI-H460, A549 and H1299) and normal lung cell line (BEAS-2B) were obtained from ATCC (Rockville, MD, USA). The cells were cultured in RPMI-1640 supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO, USA) in a humidified incubator containing 5% CO2 at 37 °C.

Cell transfection

miR-130 inhibitor, miR-130 mimic and miR-130 NC were purchased from RiboBio (Guangzhou, China). The pcDNA3.1 vector was obtained from Vazyme (Nanjing, Jiangsu, China). Full-length lncRNA MRPL3 was subcloned into pcDNA3.1 vector to overexpress lncRNA MRPL3. The A549 cells at logarithmic growth stage were inoculated into six-well plates overnight for culture, and cell transfection was performed the next day. Lipofectamine 3000 (Thermo Fisher Scientific, Inc. Waltham, MA, USA) was used to prepare the mixture miR-130 inhibitor, miR-130 mimic, miR-130 NC, pcDNA3.1, pcDNA3.1-lncRNA MRPL3 or pcDNA3.1-lncRNA MRPL3 + miR-130 mimic-lipofectamine 3000 according to the instructions. The mixture was dripped into 6-well plates according to the instructions of the transfection kit for transfection. The cells from each group were collected 48 h after transfection for subsequent operations.

RNA extraction and quantification by RT-PCR

The total RNA was extracted from cultured cells or tissue specimens using TRIzol Reagent (Invitrogen, USA). The cDNA was synthesized with First-Strand cDNA Synthesis Kit (APExBIO, Houston, USA). qPCR was performed on ABI 7500-Fast Real-Time PCR System (Applied Biosystem, Foster City, CA, USA) with SYBR Green qPCR Master Mix (APExBIO, Houston, USA). The RT phase required a 20-min incubation period at 55 °C. Initial denaturation at 95 °C for three minutes was followed by 45 cycles of 45 s at 95 °C and 45 s at 60 °C during the PCR cycling conditions. A total volume of 40 μL was used to construct the first strand cDNA synthesis for the two-step procedure. β-actin was used as an internal reference for mRNA and lncRNA, and U6 were used as an internal reference for miRNAs. All qPCR experiments were performed at ≥ 3 times and relative expression was calculated through the 2−ΔΔCt method.

Cell viability by CCK-8 assay

The of A549 was measured with Cell Counting Kit-8 (CCK-8; Solarbio, Beijing, China). A549 cells was inoculated into 96-well plates at 1 × 104 cells per well incubate for 24 h and the cells were transfected with miR-130 inhibitor, miR-130 mimic, miR-130 NC, pcDNA3.1, pcDNA3.1-lncRNA MRPL3 or pcDNA3.1-lncRNA MRPL3 + miR-130 mimic for 48 h. 10 μL of CCK-8 solution was applied per well and incubation for 2 h. Finally, the absorbance value of each well at 450 nm was measured with an enzyme marker (Thermo Fisher Scientific, Inc. Waltham, MA, USA).

Transwell migration assay

Transwell migration assay was were performed using 24-well transwell chamber to detect cell invasion. 200 μL transfected miR-130 inhibitor, miR-130 mimic, miR-130 NC, pcDNA3.1, pcDNA3.1-lncRNA MRPL3 or pcDNA3.1-lncRNA MRPL3 + miR-130 mimic A549 cells suspended in serum-free RPMI-1640 medium were seeded into the upper chamber, and 600 μL RPMI-1640 medium with 10% FBS were added to the lower chamber. The 24-well transwell chamber was incubated in a humidified incubator containing 5% CO2 at 37 °C for 24 h. After adding 4% paraformaldehyde (Solarbio, Beijing, China) for 15 min, adding crystal violet solution (Sigma-Aldrich, USA) for 15 min staining. Then the invasive cells were photographed and counted under a microscope (Leica, Germany).

Total protein estimation and Western blot analysis

A549 cells or tissues were added with RIPA lysate (Pierce, Rockford, IL, USA) to extract total protein, and the protein concentration was detected with BCA kit (Thermo Fisher Scientific, Inc.). The total protein was separated by 10% SDS-PAGE and transferred to PVDF membrane (Merck Millipore, Billerica, MA, USA). The membrane was sealed with blocking solution 1xTBS, 0.1% Tween-20 with 5% W/v nonfat dry milk (Thermo Fisher Scientific, Inc. Waltham, MA, USA) at room temperature for 1 h and incubated with anti-TSC1 antibody (1:1000 ab217328, Abcam, Cambridge, MA, USA) at 4 °C overnight. After washing with 1xTBS, 0.1% Tween-20, add HRP-conjugated against rabbit (Abcam) and incubate for 1 h at room temperature. After washing, the protein bands were visualized by ECL luminescent solution (Thermo Fisher Scientific, Inc. Waltham, MA, USA) and analyzed with Image J (https://imagej.net/ij/).

Dual‐luciferase reporter assay

The wild-type or mutant sequence of LncRNA MRPL39 or TSC1 was constructed into a pGL3 luciferase vector (Promega Corporation, Madison, WI). A549 cells were co-transfected with miR-130 mimic (miR-130 NC), pGL3-LncRNA MRPL39-Wt, pGL3-LncRNA MRPL39-Mut, pGL3-TSC1-Wt or pGL3-TSC1-Mut using Lipofectamine™ 3000 (Thermo Fisher Scientific, Inc. Waltham, MA, USA). 48 h after transfection, A549 cells were collected and cleaved to detect luciferase activity using the Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA).

Statistical analysis

Data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism software. The student’s t-test or one-way analysis of variance were used to assess the significance between the data. P < 0.05 is considered statistically significant.

Results

miR-130 is up-expressed and LncRNA MRPL39 is down-expressed in NSCLC tissues, which is associated with poor prognosis

To elucidate the direct relationship between MRPL39 and miR-130 in NSCLC A549 cells, we utilized miRcode (http://www.mircode.org) to predict the binding sites of miR-130 and MRPL39 (Fig. 1A). Following a comprehensive analysis, we collected 40 pairs of human lung cancer tissues and non-tumor tissues for the evaluation of miR-130 and MRPL39 expression levels through RT-PCR analysis. The results indicated a significant upregulation of miR-130 expression in NSCLC tissues compared to non-tumor tissues (Fig. 1B), while MRPL39 exhibited a significant downregulation (Fig. 1C). Furthermore, we assessed the expression levels of miR-130 and MRPL39 in human lung (bronchial) epithelial cell (BEAS-2B) and NSCLC cell lines (HCC827, NCI-H460, A549, and H1299). The findings revealed higher miR-130 expression in NSCLC cells compared to BEAS-2B cells (Fig. 1D), with MRPL39 expression being lower in NSCLC cells than in BEAS-2B cells (Fig. 1E). For practical applicability and to elucidate the clinicopathological role of miR-130 and MRPL39 in NSCLC, 40 patients were categorized into the miR-130 high-expression group (n = 22) and miR-130 low-expression group (n = 18) or MRPL39 high-expression group (n = 18) and MRPL39 low-expression group (n = 22) based on the cutoff value, defined as the median of the cohort. Patients with higher miR-130 and lower MRPL39 expression were significantly associated with larger carcinoma size, advanced TNM stage, and lymphatic metastasis. Moreover, Kaplan–Meier analysis demonstrated that patients with higher miR-130 or low MRPL39 expression exhibited a shorter overall and disease-free survival period than those with high miR-130 or low MRPL39 expression (Fig. 1F, G). In conclusion, these results suggest that miR-130 expression is upregulated, and MRPL39 is downregulated in NSCLC, closely associated with the poor prognosis of NSCLC.

Fig. 1.

Fig. 1

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miR-130 is up-expressed and LncRNA MRPL39 is down-expressed in NSCLC tissues, which is associated with poor prognosis. A To predict the upstream target and specific binding sites of miR-130; B, C the RT-PCR analysis of the mRNA expression level of miR-130 (A) and lncRNA MRPL39 (B) in NSCLC tissues and non-tumor tissues (n = 40); D, E the RT-PCR analysis of the mRNA expression level of miR-130 (D) and lncRNA MRPL39 (E) in BEAS-2B, HCC827, NCI-H460, A549 and H1299 cells (n = 3). F MRPL39 expression in the tested cells. G Overall and disease-free survival period

miR-130 inhibits cell proliferation and migration

To explore the potential impact of miR-130 on the tumorigenicity of NSCLC A549 cells, the expression of miR-130 was down-regulated in A549 cells through the transfection of a miR-130 inhibitor. Transfection efficiency was confirmed by reliable RT-PCR analysis, demonstrating a significant decrease in the expression of miR-130 after the miR-130 inhibitor transfection (Fig. 2A). Subsequently, the effects of the miR-130 inhibitor on the growth of A549 cells were investigated. As illustrated in Fig. 2B, the down-regulation of miR-130 substantially accelerated the growth of A549 cells. Furthermore, the impact of the miR-130 inhibitor on the migration of A549 cells was assessed using a Transwell assay. The results indicated that the down-regulation of miR-130 significantly inhibited the migration of A549 cells (Fig. 2C, D).

Fig. 2.

Fig. 2

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miR-130 inhibits cell proliferation and migration. A The mRNA expression of miR-130 in A549 cells was detected by RT-PCR analysis after miR-130 inhibitor transfection; B the cell proliferation was detected by CCK-8 assay in A549 cells after miR-130 inhibitor transfection; C the cell migration was evaluated by Transwell assay in A549 cells after miR-130 inhibitor transfection; D the number of cell migration statistics of C

LncRNA MRPL39 is an upstream regulatory lncRNA of miR-130

We initially established the correlation between MRPL39 and miR-130 in NSCLC tissues. Pearson correlation analysis revealed a negative association between the expression of MRPL39 and that of miR-130 in NSCLC tissues (r = − 0.4956, P = 0.0011, Fig. 3A). Subsequently, we cloned the wild-type MRPL39 sequence containing the potential binding site of miR-130, as well as the mutant MRPL39 sequence, into a luciferase reporter gene system. The results demonstrated that miR-130 suppressed the luciferase activity of wild-type MRPL39 but had no effect on the luciferase activity of the mutant MRPL39 in A549 cells (Fig. 3B).

Fig. 3.

Fig. 3

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LncRNA MRPL39 is an upstream regulatory lncRNA of miR-130. A Negative relationship between the MRPL39 level and miR-130 expression in 40 NSCLC tissues. B The dual luciferase assay was conducted in A549 cells cotransfected with the recombinant reporter plasmid and indicated miR-130/miR-NC mimics. C The mRNA expression of miR-130 in A549 cells was detected by RT-PCR analysis after MRPL39 overexpression

LncRNA MRPL39 inhibits cell proliferation and migration

To elucidate the potential effect of MRPL39 on the tumorigenicity of A549 cells. The expression of MRPL39 in A549 cells was over-regulated by the transfection of MRPL39 pcDNA 3.1 overexpression plasmid. Transfection efficiency was verified by RT-PC analysis showed that the expression of MRPL39 found to increase significantly after MRPL39 pcDNA 3.1 overexpression plasmid transfection (Fig. 4A). Additionally, the effects of MRPL39 overexpression on the growth of A549 cells were investigated. As shown in Fig. 4B, MRPL39 overexpression markedly suppressed the cell growth of A549 cells. Furthermore, the effect of MRPL39 overexpression on the migration of A549 cells was detected by Transwell assay. The results in Fig. 4C indicated that overexpression of MRPL39 inhibited the expression level of miR-130. The results showed that MRPL39 overexpression significantly inhibited the migration of A549 cells (Fig. 4C, D).

Fig. 4.

Fig. 4

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LncRNA MRPL39 inhibits cell proliferation and migration. A The mRNA expression of MRPL39 in A549 cells was detected by RT-PCR analysis after MRPL39 overexpression. B The cell proliferation was detected by CCK-8 assay in A549 cells after MRPL39 overexpression. C The cell migration was evaluated by Transwell assay in A549 cells after MRPL39 overexpression. D The number of cell migration statistics of C

TSC1 is a direct functional target of miR-130

To reveal the mechanism underlying the function of miR-130 in A549, we performed TargetScan analysis to explore the target genes of miR-130. A binding site was predicted between miR-130 and TSC1 by TargetScan (Fig. 5A). Dual-luciferase reporter gene assay showed that miR-130 expression remarkably decreased the luciferase activity of TSC1 wt (Fig. 5B). To study the relationship between TSC1 and miR-130, Pearson correlation analysis showed that there was a negative association between the expression of miR-130 and that of TSC1 in NSCLC tissues (r =  − 0.4093, P = 0.0087, Fig. 5C). We then examined the protein expression of TSC1 in A549 cells after miR-130 inhibitor or MRPL39 overexpression. The results of Western blot showed that the protein levels of TSC1 were significantly increased in miR-130 inhibitor or MRPL39 overexpression transfected A549 cells (Fig. 5D, E).

Fig. 5.

Fig. 5

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TSC1 is a direct functional target of miR-130. A The predicted binding sites between miR-130 and TSC1 mRNA. B Negative relationship between the miR-130 level and TSC1 expression in 40 NSCLC tissues. C The 3′UTR reporter assay was performed in A549 cells overexpressed with miR-130, and pGL3-TSC1-3′-UTR-WT or pGL3-TSC1-3′-UTR-Mutation was co-transfected. Luciferase assays were performed 48 h after transfection, and firefly luciferase activity was normalized to the Renilla luciferase control. **P < 0.01. D The expression of TSC1 protein in A549 cells with miR-130 transfection was assessed by Western blot analysis. E The expression of TSC1 protein in A549 cells with lncRNA MRPL39 overexpression was assessed by Western blot analysis

LncRNA MRPL39 affects the progression of NSCLC by regulating miR-130/TSC1

To study the relationship between MRPL39, miR-130, and TSC1, we co-transfected A549 cells with MRPL39 pcDNA 3.1 overexpression plasmid and miR-130 mimic. The expression of TSC1 was markedly upregulated in MRPL39 overexpression group but significantly decreased in miR-130 mimic group. miR-130 mimic reversed the effect of MRPL39 overexpression on TSC1 expression (Fig. 6A). Additionally, CCK-8 results showed that MRPL39 overexpression markedly suppressed the cell growth of A549 cells, miR-130 mimic accelerated the cell growth, while miR-130 mimic reversed the proliferation of A549 cells induced by MRPL39 overexpression (Fig. 6B). The results of cell migration were consistent with the CCK-8 results (Fig. 6C, D).

Fig. 6.

Fig. 6

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LncRNA MRPL39 affects the progression of NSCLC by regulating miR-130/TSC1. A The mRNA expression of TSC1 in A549 cells was detected by RT-PCR analysis after miR-130 or/and MRPL39 overexpression. B The cell proliferation was detected by CCK-8 assay in A549 cells after miR-130 or/and MRPL39 overexpression. C The cell migration was evaluated by CCK-8 assay in A549 cells after miR-130 or/and MRPL39 overexpression. D The number of cell migration statistics of C. E The expression of TSC1 protein in A549 cells with miR-130 or/and lncRNA MRPL39 overexpression was assessed by Western blot analysis

Discussion

In the present study, we explored the impact of the lncRNA MRPL39-miR-130-TSC1 axis on the proliferation and migration of NSCLC A549 cells, along with the underlying molecular mechanisms. Our current findings indicate that: (i) miR-130 is overexpressed, while lncRNA MRPL39 is underexpressed in NSCLC tissues, and this dysregulation is associated with poor prognosis; (ii) LncRNA MRPL39 acts as an upstream regulatory lncRNA of miR-130, and both miR-130 and lncRNA MRPL39 contribute to the inhibition of cell proliferation and migration; and (iii) TSC1 serves as a direct functional target of miR-130, with lncRNA MRPL39 influencing the progression of NSCLC by regulating the miR-130/TSC1 axis.

MiR-130 has been identified as an oncogene in various human tumors, attributed in part to its proliferative and growth-promoting potential (Duan et al. 2016; Egawa et al. 2016). In the context of NSCLC, the role of miR-130 has been investigated in recent years, yielding conflicting findings. Ye et al. reported down-regulated miR-130 expression in NSCLC tissues and cell lines, correlating with aggressive clinicopathological features and poor prognosis in NSCLC patients (Zhang et al. 2018). In contrast, another study by Zhang et al. found that high expression of miR-130b promoted proliferation and inhibited apoptosis in NSCLC in a xenograft tumor model of nude mice (Zhang et al. 2018). In the current study, we observed an up-regulation of miR-130 in NSCLC tissues and cell lines. Inhibition of miR-130 further suppressed the proliferation and migration of A549 cells. These results suggest that miR-130 may act as an oncogene in NSCLC, supporting its potential as a therapeutic target for the disease.

MRPL39 is a gene that spans 1725 base pairs and is located on chromosome 21. Its role in NSCLC has not been previously investigated. However, in gastric cancer, Yu et al. discovered that MRPL39 is notably down-regulated and exerts inhibitory effects on the proliferation and progression of gastric cancer cells by directly targeting miR-130 (Yu et al. 2018). In our study, we observed significant down-regulation of MRPL39 in both NSCLC patient tissue samples and cell lines. Acting as an upstream regulatory lncRNA of miR-130, MRPL39 was found to inhibit the proliferation and migration of NSCLC A549 cells by directly targeting miR-130, as evidenced by CCK-8 and transwell assays. This suggests a potential tumor-suppressive role for MRPL39 in NSCLC.

Furthermore, TSC1 was selected for the target genes of miR-130b-3p from 4453 potential target genes because TSC1 through mTORC1 affect cell metabolism, inhibiting tumor growth, invasion and migration and migration (Lai et al. 2021). Then we explored the interactions of MRPL39-miR-130-TSC1 in A549 cells. The results indicated that miR-130 could directly bind to MRPL39 and TSC1-3’UTR through their own miRNA binding sites to inhibit MRPL39 and TSC1 expression. Functionally, Overexpression of MRPL39 significantly upregulated TSC1 expression, resulting in A549 cell proliferation and migration, while further overexpression of miR-130 reversed these effects. Taken together, all data confirm that MRPL39 inhibits NSCLC proliferation and migration by regulating the miR-130/TSC1/mTORC1 axis. The limitation of this paper is that we did not further verify that TSC1 inhibits A549 cell proliferation and migration through mTORC1 and further confirmed at animal level. In conclusion, our study demonstrates that miR-130 is upregulated and MRPL39 is downregulated in NSCLC compared with non-tumor tissue and contribute to the high proliferation and migration activity of A549 cells. Moreover, MRPL39 inhibits A549 cell proliferation and migration by regulating the miR-130/TSC1/mTORC1 axis.

In this study, the authors demonstrated that the lncRNA MRPL39 is downregulated, and miR-130 is upregulated in NSCLC clinical specimens. Additionally, they revealed a direct interaction between lncRNA MRPL39 and miR-130, identifying TSC1 as a direct functional target of miR-130. Moreover, the study found that the lncRNA MRPL39-miR-130-TSC1 pathway plays a crucial role in NSCLC cell proliferation and migration, highlighting its potential as valuable therapeutic targets. Looking ahead, this study paves the way for new research involving different miRNAs. However, fine-tuning experimental conditions, considering the nature of the sequences, is essential for optimization. Conducting similar research with other miRNAs in various diseases may offer opportunities for future investigations.

Author contributions

The authors are contributed to the preparation of the manuscript and discussion. All authors read and approved the final manuscript.

Funding

The second batch of major (key) science and technology research projects in Jinhua City in 2021, 2021-3-113.

Declarations

Conflict of interest

Authors declare no conflict of interest.

Ethical approval

This study was conducted with the informed consent of NSCLC patients and approved by the Ethics Committee of Jinhua People’s Hospital (Approved No. IRB-2021037-R).

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