Advertisement

LINC01232 promotes lung squamous cell carcinoma progression through modulating miR-181a-5p/SMAD2 axis

Open AccessPublished:December 17, 2022DOI:https://doi.org/10.1016/j.amjms.2022.12.014

      Abstract

      Background

      LINC01232 has been implicated in the progression of multiple malignancies. Yet, the function of LINC01232 in the carcinogenesis of lung squamous cell carcinoma (LUSC) remains unclear. This study aims to examine the role LINC01232 plays in LUSC progression.

      Methods

      mRNA and protein levels were assessed using qRT-PCR and western blot, respectively. Cell proliferation was assessed by CCK-8 and colony formation assays. Cell migration and invasion were evaluated by transwell assay. The interactions between LINC01232, miR-181a-5p, and SMAD2 were assessed using luciferase reporter, RNA pull-down, and RNA immunoprecipitation (RIP) assays. The subcellular distribution of LINC01232 was examined by cytosolic/nuclear fractionation assay

      Results

      LINC01232 was upregulated in both LUSC tissues and cell lines. Knockdown of LINC01232 impaired cell proliferation, migration and invasion capability in H1229 and A549 cells, a phenotype that could be reversed by miR-181a-5p silencing. In addition, LINC01232 silencing reduced levels of N-cadherin, Vimentin, and Snail in H1229 and A549 cells, but increased the level of E-cadherin, which can be abrogated by miR-181a-5p inhibitors.

      Conclusions

      In summary, our study demonstrates that LINC01232 expression increases in LUSC tissues and cell lines and promotes LUSC progression by modulating the miR-181a-5p/SMAD2 signaling, providing new potential drug targets for LUSC treatment.

      Key Indexing Terms

      Introduction

      Lung cancer is a common malignancy in respiratory system, causing highest mortality and morbidity worldwide.
      • Siegel R.L.
      • Miller K.D.
      • Jemal A.
      Cancer Statistics, 2017.
      ,
      • Goldstraw P.
      • Ball D.
      • Jett J.R.
      • et al.
      Non-small-cell lung cancer.
      Non-small-cell lung cancer (NSCLC), including lung squamous cell carcinoma (LUSC), neuroendocrine carcinoma, large cell carcinoma, and lung adenocarcinoma, is the main subtype of lung cancer, accounting for more than 80% of lung cancer cases.
      • Bray F.
      • Ferlay J.
      • Soerjomataram I.
      • et al.
      Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
      • Chansky K.
      • Sculier J.P.
      • Crowley J.J.
      • et al.
      The international association for the study of lung cancer staging project: prognostic factors and pathologic TNM stage in surgically managed non-small cell lung cancer.
      • Osmani L.
      • Askin F.
      • Gabrielson E.
      • et al.
      Current WHO guidelines and the critical role of immunohistochemical markers in the subclassification of non-small cell lung carcinoma (NSCLC): moving from targeted therapy to immunotherapy.
      LUSC, the dominant subtype of NSCLC, is notorious for the great propensity for recurrence and metastasis.
      • Tsai J.H.
      • Donaher J.L.
      • Murphy D.A.
      • et al.
      Spatiotemporal regulation of epithelial-mesenchymal transition is essential for squamous cell carcinoma metastasis.
      Although advances have been made in lung cancer treatment, the prognosis of patients is still poor, with the 5‑year survival rate being merely 4∼17%.
      • Hirsch F.R.
      • Scagliotti G.V.
      • Mulshine J.L.
      • et al.
      Lung cancer: current therapies and new targeted treatments.
      Therefore, there is an urgent need to explore the potential molecular mechanisms of LUSC progress.
      Long noncoding RNAs (lncRNAs) are a class of non-coding RNAs that are greater than 200 nt in length.
      • Jarroux J.
      • Morillon A.
      • Pinskaya M.
      History, discovery, and classification of lncRNAs.
      ,
      • Renganathan A.
      • Felley-Bosco E.
      Long noncoding RNAs in cancer and therapeutic potential.
      Growing evidence indicates that lncRNAs regulate a range of biological and cellular processes, including cell proliferation,
      • Wei G.H.
      • Wang X.
      lncRNA MEG3 inhibit proliferation and metastasis of gastric cancer via p53 signaling pathway.
      metastasis,
      • Luan X.
      • Wang Y.
      LncRNA XLOC_006390 facilitates cervical cancer tumorigenesis and metastasis as a ceRNA against miR-331-3p and miR-338-3p.
      and epithelial-mesenchymal transition (EMT).
      • Lu W.
      • Zhang H.
      • Niu Y.
      • et al.
      Long non-coding RNA linc00673 regulated non-small cell lung cancer proliferation, migration, invasion and epithelial mesenchymal transition by sponging miR-150-5p.
      As such, dysregulation of lncRNAs has been reported to be tightly associated with the progression of multiple malignancies,
      • Xue X.
      • Yang Y.A.
      • Zhang A.
      • et al.
      LncRNA HOTAIR enhances ER signaling and confers tamoxifen resistance in breast cancer.
      • You Z.
      • Liu C.
      • Wang C.
      • et al.
      LncRNA CCAT1 promotes prostate cancer cell proliferation by interacting with DDX5 and MIR-28-5P.
      • Huang Y.
      • Zhang J.
      • Hou L.
      • et al.
      LncRNA AK023391 promotes tumorigenesis and invasion of gastric cancer through activation of the PI3K/Akt signaling pathway.
      • Li B.
      • Mao R.
      • Liu C.
      • et al.
      LncRNA FAL1 promotes cell proliferation and migration by acting as a CeRNA of miR-1236 in hepatocellular carcinoma cells.
      including lung cancer. For instance, lncRNA-H19 promotes lung cancer cell proliferation and metastasis by inhibiting miR-200a expression
      • Zhao Y.
      • Feng C.
      • Li Y.
      • et al.
      LncRNA H19 promotes lung cancer proliferation and metastasis by inhibiting miR-200a function.
      ; lncRNA-DANCR increases lung cancer cell growth and colony formation by sequestering miR-216a
      • Zhen Q.
      • Gao L.N.
      • Wang R.F.
      • et al.
      LncRNA DANCR promotes lung cancer by sequestering miR-216a.
      ; LncRNA-SNHG4 facilitates lung cancer progression by sponging the anti-tumor miR-98-5p.
      • Tang Y.
      • Wu L.
      • Zhao M.
      • et al.
      LncRNA SNHG4 promotes the proliferation, migration, invasiveness, and epithelial-mesenchymal transition of lung cancer cells by regulating miR-98-5p.
      LINC01232 is a newly discovered long noncoding RNA. Recently, abnormal expression of LINC01232 has been observed in several malignancies, including oral carcinoma,
      • Chen M.
      • Xu X.
      • Ma H.
      Identification of oncogenic long noncoding RNAs CASC9 and LINC00152 in oral carcinoma through genome-wide comprehensive analysis.
      esophageal squamous cell carcinoma,
      • Zhao M.
      • Cui H.
      • Zhao B.
      • et al.
      Long intergenic noncoding RNA LINC01232 contributes to esophageal squamous cell carcinoma progression by sequestering microRNA6543p and consequently promoting hepatomaderived growth factor expression.
      and pancreatic cancer.
      • Meng L.D.
      • Shi G.D.
      • Ge W.L.
      • et al.
      Linc01232 promotes the metastasis of pancreatic cancer by suppressing the ubiquitin-mediated degradation of HNRNPA2B1 and activating the A-Raf-induced MAPK/ERK signaling pathway.
      However, the expression pattern and function of LINC01232 in LUSC progress remain enigmatic.
      MicroRNAs (miRNAs) are a group of small non-coding RNAs (∼22 nucleotides) that can inhibit gene expression by post-transcriptional regulation.
      • Lewis B.P.
      • Burge C.B.
      • Bartel D.P.
      Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.
      ,
      • Jiang C.
      • Hu X.
      • Alattar M.
      • et al.
      miRNA expression profiles associated with diagnosis and prognosis in lung cancer.
      During malignant tumor development, miRNAs can act as either tumor suppressor genes or oncogenes. Growing evidence supports that miRNAs play a crucial role in lung cancer progression.
      • Li H.
      • Yang T.
      • Shang D.
      • et al.
      miR-1254 promotes lung cancer cell proliferation by targeting SFRP1.
      • Li J.
      • Jin H.
      • Yu H.
      • et al.
      miRNA1284 inhibits cell growth and induces apoptosis of lung cancer cells.
      • Li Z.
      • Wang X.
      • Li W.
      • et al.
      miRNA-124 modulates lung carcinoma cell migration and invasion.
      As a newly discovered miRNA, miR-181a-5p plays a pivotal role in cell growth, metastasis, and apoptosis through regulating the expression of specific target genes,
      • Han P.
      • Li J.W.
      • Zhang B.M.
      • et al.
      The lncRNA CRNDE promotes colorectal cancer cell proliferation and chemoresistance via miR-181a-5p-mediated regulation of Wnt/beta-catenin signaling.
      • Yu J.
      • Jiang L.
      • Gao Y.
      • et al.
      LncRNA CCAT1 negatively regulates miR-181a-5p to promote endometrial carcinoma cell proliferation and migration.

      Wu L., Song W.Y., Xie Y., et al. miR-181a-5p suppresses invasion and migration of HTR-8/SVneo cells by directly targeting IGF2BP2. Cell Death Dis. Jan 16 2018;9(2):16. https://doi.org/10.1038/s41419-017-0045-0

      • Qi M.
      • He L.
      • Ma X.
      • et al.
      MiR-181a-5p is involved in the cardiomyocytes apoptosis induced by hypoxia-reoxygenation through regulating SIRT1.
      which potentiates it as a tumor biomarker for cancer diagnosis.
      • Xue W.X.
      • Zhang M.Y.
      • Rui L.
      • et al.
      Serum miR-1228-3p and miR-181a-5p as noninvasive biomarkers for non-small cell lung cancer diagnosis and prognosis.
      • Lin Z.
      • Chen Y.
      • Lin Y.
      • et al.
      Potential miRNA biomarkers for the diagnosis and prognosis of esophageal cancer detected by a novel absolute quantitative RT-qPCR method.
      • Zhang H.
      • Zhu M.
      • Shan X.
      • et al.
      A panel of seven-miRNA signature in plasma as potential biomarker for colorectal cancer diagnosis.
      However, the exact molecular mechanism of miR-181a-5p in LUSC progression remains unknown.
      Small mothers against decapentaplegic 2 (SMAD2) functions as a signaling mediator of the TGF-β superfamily in a ligand-specific manner, which regulates an array of biological activities.
      • Yang J.
      • Jiang W.
      The role of SMAD2/3 in human embryonic stem cells.
      • Eppert K.
      • Scherer S.W.
      • Ozcelik H.
      • et al.
      MADR2 maps to 18q21 and encodes a TGFbeta-regulated MAD-related protein that is functionally mutated in colorectal carcinoma.
      • Riggins G.J.
      • Thiagalingam S.
      • Rozenblum E.
      • et al.
      Mad-related genes in the human.
      It has been reported that SMAD2 expression is regulated by miRNAs, typically leading to cancer progression.
      • Zhang C.
      • Wang B.
      • Wu L.
      MicroRNA409 may function as a tumor suppressor in endometrial carcinoma cells by targeting Smad2.
      ,
      • Wang F.
      • Wu D.
      • Chen J.
      • et al.
      Long non-coding RNA HOXA-AS2 promotes the migration, invasion and stemness of bladder cancer via regulating miR-125b/Smad2 axis.
      Yet, in LUSC, whether SMAD2 expression is regulated by miRNAs and how this affects LUSC progression remain elusive.
      Therefore, we set out to explore the role of LINC01232 in LUSC carcinogenesis and molecularly dissect the mechanism of LINC01232 in LUSC development with an aim to provide a potential drug target for LUSC diagenesis and treatment.

      Methods

      Ethical statement

      This study was authorized by the Ethics Committee of Coast Guard Hospital (approval number: [KY-E-2021-01-17]). Informed consent was obtained from all patients involved.

      Clinical samples

      Forty pairs of LUSC and adjacent normal tissues were collected from LUSC patients admitted at Coast Guard Hospital, Jiaxing, who did not receive any chemotherapy or radiation therapy prior to surgery. All tissue specimens were stored directly in liquid nitrogen (Delun, Shanghai, China).

      Cell culture and transfection

      Three LUSC cell lines (A549, CALU3, and H1229) and a normal human bronchial epithelial cell (16HBE) were obtained from ATCC Company (ATCC, USA). These cells were cultured in an RPMI-1640 medium (Gibco, USA) supplemented with 20% fetal bovine serum (FBS) (Gibco, USA), 100 μg/mL streptomycin, and 100 U/mL penicillin at 37 °C in a 5% CO2 incubator. sh-NC, sh-LINC01232, mimics control, miR-181a-5p mimics, inhibitor control, and miR-181a-5p inhibitors were purchased from Genechem Company (Shanghai, China). Cells were seeded in 6-well plates and transfected with the designated construct with Lipofectamine 3000 reagent (Thermo Fisher Scientific, USA). After 48 h, the transfected cells were harvested to perform subsequent experiments.

      Quantitative real-time PCR

      Total RNA from LUSC tissues and cell lines was isolated with Trizol reagent (Invitrogen, USA). Reverse transcription was performed using a Reverse Transcription kit (Thermo Fisher Scientific, USA). Subsequently, q-PCR was performed on a Thermal Cycler Dice Real-Time System (Takara, Japan) with SYBR green PCR master mix (Takara, Japan). Experiments were performed in triplicate and relative expression levels were measured with the 2−ΔΔCt method. The primers used for qRT-PCR were listed in Table 2.

      Western blot

      The protocol of protein extraction was reported previously.
      • Wei X.M.
      • Wumaier G.
      • Zhu N.
      • et al.
      Protein tyrosine phosphatase L1 represses endothelial-mesenchymal transition by inhibiting IL-1beta/NF-kappaB/Snail signaling.
      Briefly, proteins were isolated from LUSC cell lines using RIPA lysis buffer (Sigma-Aldrich, USA) supplemented with an inhibitor cocktail (PIC, Roche, USA). The proteins were separated with a 12% SDS-PAGE gel and then transferred to a PVDF membrane (Millipore, USA). Further, the membrane was blocked with 5% BSA at 37 ˚C for 2 h. After three washes with PBS, the membrane was incubated with primary antibodies against Snail (1:1000, CST, USA), N-cadherin (1:1000, CST, USA), E-cadherin (1:2000, Abcam, USA), Vimentin (1:2000, Abcam, USA), and GAPDH (1:2000, Proteintech, USA) at 4 ˚C for 24 h. Next, the membrane was washed with PBS three times and then treated with secondary antibodies (1:10000, Jackson, USA) at 37 ˚C for 2 h. Finally, the blot was developed with ECL reagents (Amersham, UK), and the protein bands were evaluated using ImageJ software (National Institutes of Health, USA).

      CCK‑8 assay

      Cell proliferation capacity was measured with CCK-8 assay. In short, cells were transfected with indicated constructs for 72 h. Then, the cells were harvested, suspended, and seeded into a 96‑well plate at a density of 1 × 104 cells/well. Subsequently, the cells were cultured for 0 h, 24 h, 48 h, and 72 h., Further, 10 ul of CCK-8 Regent (Beyotime, Shanghai, China) was applied to each well and treated the cells at 37 ˚C for 2 h. Finally, absorbance at 450 nm was read by a microplate reader (BioRad Laboratories, USA).

      Colony formation assay

      For colony formation assay, cells were transfected with specified constructs for 6 h and placed into a 6-well plate (1 × 103 cells/well) to culture for 14 days at 37 ˚C with 5% CO2. Colonies were fixed with 4% Triformol (Sigma Aldrich, USA) and stained with 0.1% crystal violet (Solarbio, China) for 30 min at room temperature. The number of cell colonies was counted manually under a stereomicroscope.

      Transwell assay

      Following transfection with indicated constructs for 72 h, cells were seeded at a density of 1 × 104 cells/chamber into an uncoated (for migration assay) or a Matrigel-coated (for invasion assay) top chamber (BD Biosciences, USA). Then, an RPMI-1640 medium containing 20% FBS was added to a low chamber. After incubation for 24 h, the cells were fixed with 4% Triformol (Sigma Aldrich, USA) for 15 min and stained with 1.5% crystal violet (Solarbio, Beijing, China) at 37 ˚C for 30 min. The migrating and invading cells were observed under an inverted microscope (Leica, Germany).

      Subcellular location of LINC01232

      A Cytoplasmic Nuclear RNA Purification Kit (Norgen Biotek Corp, Canada) was used to separate the nuclear and cytosolic fractions of LUSC cells according to the supplier's protocol. RNAs were extracted from both fractions. Then, qRT-PCR was used to determine the subcellular position of LINC01232.

      Luciferase reporter assay

      Luciferase reporter vectors, LINC01232 wild-type (LINC01232-WT) or mutant (LINC01232-Mut) were subcloned into pGL3-basic plasmid (Promega, USA). Then, the constructs were co-transfected with miR-181a-5p mimics into LUSC cell lines using the Lipofectamine 3000 regents (Thermo Fisher Scientific, USA). The transfected cells were harvested 72 h after transfection, and a Luciferase Reporter Assay System was used to calculate the luciferase activity (Promega, USA). Each experiment was performed in triplicate.

      RNA immunoprecipitation (RIP) assay

      RNA immunoprecipitation assay was used to confirm the relationship between LINC01232 and miR‑654‑3p in LUSC cells. Briefly, cells were lysed with RIP lysis buffer. Anti-Ago2 antibody (1:2000, Abcam, USA) or normal IgG antibody (1:2000, Abcam, USA) was conjugated to magnetic beads and incubated with the cell lysate for 24 h at 4 ˚C. The magnetic beads were then harvested and treated with proteinase K. Finally, the enrichment of LINC01232 and miR-181a-5p in immunoprecipitated RNAs was assessed by qRT-PCR assay.

      RNA pull-down assay

      RNA pull-down assay was used to investigate the interaction between LINC01232, miR-181a-5p, and SMAD2 in LUSC cells. Briefly, for miR-181a-5p pulled down LINC01232 and SMAD2, the cells were transfected with miR-181a-5p probe by Lipofectamine 3000 (Thermo Fisher Scientific, USA) according to the supplier's protocol. After 48 h, the cells were harvested and lysed. Then, the cell lysates were incubated with magnetic beads (Life Technologies, USA) for 2 h. After three washes with PBS, mRNA levels of LINC01232 and SMAD2 were measured using qRT-PCR.

      Statistical analysis

      All data were presented as means ± SD. Student's t-test was used to compare data from two groups, and one-way analysis of variance (ANOVA) was employed to compare data from multiple groups. All statistical analyses were performed with Prism 7.0 software and SPSS version 19.0 software. P < 0.05 was considered different.

      Results

      LINC01232 is upregulated in LUSC tissues and cell lines

      First, we analyzed LINC01232 expression in LUSC in the GEPIA database (http://gepia.cancer‑pku.cn/). We found that LINC01232 expression was significantly upregulated in LUSC (Fig. 1A). To confirm this result, we detected LINC01232 expression in 40 pairs of LUSC tissues and adjacent normal tissues by qRT-PCR. Consistently, LINC01232 expression was elevated in LUSC tissues compared to adjacent normal tissues (Fig. 1B). Next, we examined LINC01232 expression in LUSC cell lines (A549, CALU3, and H1229) and a normal human bronchial epithelial cell line (16HBE) using qRT-PCR. In line with the above results, LINC01232 was upregulated in A549, CALU3, and H1229 cells compared with 16HBE cells (Fig. 1C). In addition, LINC01232 expression was higher in LUSC with stage III and IV tumors than in LUSC with stage I and II tumors (Fig. 1D). Moreover, LINC01232 expression was increased in LUSC with lymph node metastasis compared with LUSC without lymph node metastasis (Fig. 1E). As shown in Table 1, the expression level of LINC01232 was closely related to tumor stage, differentiation, and lymph node metastasis. These findings suggest that LINC01232 may play a promoting role in LUSC progression.
      Fig 1
      Fig. 1LINC01232 is upregulated in LUSC tissues and cell lines. A. LINC01232 levels were upregulated in LUSC compared with normal specimens in TCGA database. B. LINC01232 expression levels in 40 pairs of LUSC tissues were increased compared with adjacent normal tissue specimens detected by qRT-PCR. C. LINC01232 expression in LUSC cell lines (A549, CALU3, and H1229) was elevated compared with the human bronchial epithelial cell (16HBE) assessed by qRT-PCR. D. LINC01232 expression levels in LUSC with different tumor stages quantified by qRT-PCR. E. LINC01232 expression levels were increased in LUSC with lymph node metastasis evaluated by qRT-PCR. All experiments were performed in triplicate. *P < 0.05, **P < 0.01. Abbreviations: LUSC, lung squamous cell carcinoma.
      Table 1LINC01232 expression levels are closely related to tumor stage, differentiation, and lymph node metastasis.
      VariableLINC01232 expressionP value
      Low (20)High (20)
      Age
       <608100.525
       ≥601210
      Sex
       Male11130.519
       Female97
      Tumor stage
       I+II1230.003
      Statistical significance.
       III+IV817
      Differentiation
       Well1290.342
       Moderate-poor811
      Lymph node metastasis
       Negative1120.002
      Statistical significance.
       Positive918
      Smoking status
       Yes8110.342
       No129
      low asterisk Statistical significance.
      Table 2Primers used for qPCR.
      GeneForward 5’-3’Reverse 5’-3’
      LINC012325’-AAAACCTTGAAATCCCTTAATACCA-3’5’-CCTTACCCGTGGAATTCACATATA-3’
      MiR-181a-5p5’-GCCGAACATTCAACGCTGTCG-3’5’-GTGCAGGGTCCGAGGT-3’
      SMAD25’-ACTAACTTCCCAGCAGGAAT-3’5’-GTTGGTCACTTGTTTCTCCA-3’
      U65’-CTCGCTTCGGCAGCACA-3’5’-AACGCTTCACGAATTTGCGT-3’
      GAPDH5’-GCATCCTGGGCTACACTG-3’5’-ACTTCAGGAGCATCTGAAATAGGT-3’

      LINC01232 silencing represses proliferation, migration and invasion of LUSC cells

      To determine the function of LINC01232 in LUSC progression, we knocked down LINC01232 expression in H1229 and A549 cells in which LINC01232 is most abundantly expressed by transfecting them with sh-LINC01232 (sh-LINC01232#1 and sh-LINC01232# 2) plasmids (Fig. 2A). CCK-8 and colony formation analysis showed that LINC01232 silencing inhibited cell viability (Fig. 2B) and reduced cell colony numbers (Fig. 2C). Furthermore, the transwell assay exhibited that LINC01232 silencing could repress the cell migration and invasion capacity (Fig. 2D and E). The results suggest that LINC01232 might act as an oncogene in LUSC progression.
      Fig 2
      Fig. 2LINC01232 silencing represses proliferation, migration and invasion of LUSC cells. A. The efficiency of LINC01232 knockdown was significantly increased in H1229 and A549 cells confirmed by qRT-PCR. B. The inhibited cell viability of H1229 and A549 cells transfected with sh-LINC01232 (sh-LINC01232#1 and sh-LINC01232#2) determined by CCK-8 assay. C. The reduced colony number of H1229 and A549 cells transfected with sh-LINC01232 (sh-LINC01232#1 and sh-LINC01232#2) assessed with colony formation assay. D. The cell migration of H1229 and A549 cells was inhibited by transfecting with sh-LINC01232 (sh-LINC01232#1 and sh-LINC01232#2) investigated with transwell assay. E. The cell invasion of H1229 and A549 cells was suppressed by transfecting with sh-LINC01232 (sh-LINC01232#1 and sh-LINC01232#2) examined with transwell assay. All experiments were performed in triplicate. **P < 0.01.

      LINC01232 facilitates SMAD2 expression via sponging miR-181a-5p

      Next, we detected the distribution of LINC01232 in H1229 and A549 cells using the cell cytoplasmic/nuclear fractionation assay. Our data revealed that LINC01232 was primarily located in the cytoplasm in H1229 and A549 cells (Fig. 3A). Next, we used the online bioinformatics algorithms, StarBase 3.0 database, to predict potential downstream target(s) for LINC01232. The prediction revealed that miR-181a-5p could be a downstream target for LINC01232 (Fig. 3B). Subsequently, we validated the relationship between LINC01232 and miR-181a-5p with luciferase reporter and RIP assays. We observed that the luciferase activity of LINC01232‑WT was significantly decreased in H1229 and A549 cells overexpressing miR-181a-5p (Fig. 3C). Moreover, the RIP assay showed that LINC01232 and miR-181a-5p were enriched in the immunoprecipitated complex of AGO2 (Fig. 3D). Further, we sought to identify the downstream target gene of miR-181a-5p. Using the StarBase 3.0 database, we predicted that SMAD2 could be the target gene of miR-181a-5p (Fig. 3E). Then, we performed luciferase reporter and RNA pull-down assays to validate the interaction between them. The luciferase activity of SMAD2-WT was significantly reduced in H1229 and A549 cells overexpressing miR-181a-5p (Fig. 3F). The RNA pull-down assay showed that the miR-181a-5p probe could enrich more LINC01232 and SMAD2 than the NC probe (Fig. 3G). Furthermore, using qRT-PCR, we found that LINC01232 silencing increased miR-181a-5p levels (Fig. 3H), while decreasing SMAD2 levels in H1229 and A549 cells (Fig. 3I). Together, these results demonstrate that LINC01232 can modulate SMAD2 expression by sponging miR-181a-5p.
      Fig 3
      Fig. 3LINC01232 facilitates SMAD2 expression via sponging miR-181a-5p. A. The subcellular location of LINC01232 in H1229 and A549 cells were confirmed that LINC01232 was located mainly in the cytoplasm determined using cell cytosolic/nuclear fractionation assay. B. The complementary binding sites of miR-181a-5p and LINC01232 predicted by the online bioinformatics algorithm, StarBase 3.0 database. C. The relationship between LINC01232 and miR-181a-5p examined by luciferase reporter assay in H1229 and A549 cells. D. The relationship between LINC01232 and miR-181a-5p confirmed by RNA immunoprecipitation (RIP) assay, the result shown that the abundant enrichment of LINC01232 and miR-181a-5p was observed in Ago2‑containing immunoprecipitation complexes. E. The complementary sites of miR-181a-5p and 3’‑untranslated region of SMAD2 predicted by the StarBase 3.0 database. F. The relationship between SMAD2 and miR-181a-5p confirmed by luciferase reporter assay. The results displayed that the luciferase activity of SMAD2‑WT was significantly decreased in response to miR-181a-5p overexpressed in H1229 and A549 cells. G. The relationship between SMAD2 and miR-181a-5p examined by RNA pull-down assay. Results also exhibited that miR-181a-5p probe could enrich more LINC01232 and SMAD2 than NC probe. H. The increased level of miR-181a-5p in H1229 and A549 cells transfected with sh-LINC01232 (sh-LINC01232#1 and sh-LINC01232#2) detected by qRT-PCR. I. The decreased level of SMAD2 in H1229 and A549 cells transfected with sh-LINC01232 (sh-LINC01232#1 and sh-LINC01232#2) assessed with qRT-PCR. All experiments were performed in triplicate. **P < 0.01. Abbreviations: RIP, RNA immunoprecipitation; SMAD2, small mothers against decapentaplegic 2; wt, wild type; mut, mutant.

      LINC01232 promotes proliferation, migration and invasion of LUSC cells through miR-181a-5p/SMAD2 signaling

      To prove that LINC01232 affects the progression of LUSC cells through modulating the miR-181a-5p/SMAD2 signaling pathway, we conducted a series of rescue experiments. As shown in Fig. 4A and B, LINC01232 silencing reduced both mRNA and protein levels of SMAD2 in H1229 and A549 cells, an inhibitory effect that could be abrogated bymiR-181a-5p inhibitors. Moreover, the impaired cell viability caused by LINC01232 knockdown was rescued by miR-181a-5p inhibitors, which is reflected by the restoration of cell colony numbers (Fig. 4C and D). Additionally, the suppression of the cell migration and invasion capability in H1229 and A549 cells conferred by LINC01232 knockdown was eliminated by co-transfection with miR-181a-5p inhibitors (Fig. 4E and F). Furthermore, in H1229 and A549 cells, LINC01232 silencing reduced the protein levels of N-cadherin, Vimentin, and Snail, while increasing the level of E-cadherin, which were abrogated by co-transfection with miR-181a-5p inhibitors (Fig. 4G). Collectively, these findings strongly suggest that LINC01232 promotes proliferation, migration and invasion of LUSC cells through the miR-181a-5p/SMAD2 signaling.
      Fig 4
      Fig. 4LINC01232 promotes proliferation, migration and invasion of LUSC cells through miR-181a-5p/SMAD2 signaling. A. The presence of miR-181a-5p inhibitor rescues the decreased mRNA level of SMAD2 caused by LINC01232 knockdown in H1229 and A549 cells. B. Inhibition of miR-181a-5p restores the decreased protein level of SMAD2 in H1229 and A549 cells. (C, D). CCK-8 and colony formation assays indicting that inhibition of miR-181a-5p reverses the impaired cell proliferation in H1229 and A549 cells transfected with LINC01232 silencing constructs. (E, F). Transwell assay revealing that the presence of miR-181a-5p inhibitor abrogates the inhibition of cell migration and invasion capability caused by LINC01232 knockdown. G. Protein levels of E-cadherin, N-cadherin, Vimentin, and Snail in H1229 and A549 cells transfected with indicated constructs (sh-NC, sh-LINC01232#1, sh-LINC01232#1, and miR-181a-5p inhibitor) assessed by western blot. All experiments were performed in triplicate. **P < 0.01. Abbreviations: SMAD2, small mothers against decapentaplegic 2.

      Discussion

      LUSC is the main subtype of lung cancer, accounting for 30% of NSCLC cases.
      • Conti L.
      • Gatt S.
      Squamous-cell carcinoma of the lung.
      Although the overall survival rate of patients with lung cancer has been significantly improved by molecularly targeted therapies, the prognosis is still poor.
      • Filipits M.
      New developments in the treatment of squamous cell lung cancer.
      It is, therefore, imperative to explore new LUSC treatments. LncRNAs constitute a large portion of the mammalian transcriptome, involved in an array of cellular processes and mediating the pathogenesis of diverse diseases. It has been reported recently that dysregulation of lncRNAs is involved in LUSC progression.
      • Li R.
      • Yang Y.E.
      • Jin J.
      • et al.
      Identification of lncRNA biomarkers in lung squamous cell carcinoma using comprehensive analysis of lncRNA mediated ceRNA network.
      • Zhang H.Y.
      • Yang W.
      • Zheng F.S.
      • et al.
      Long non-coding RNA SNHG1 regulates zinc finger E-box binding homeobox 1 expression by interacting with TAp63 and promotes cell metastasis and invasion in Lung squamous cell carcinoma.
      • Xu Y.
      • Li J.
      • Wang P.
      • et al.
      LncRNA HULC promotes lung squamous cell carcinoma by regulating PTPRO via NF-kappaB.
      LINC01232 is a newly discovered lncRNA and is found to be involved in the pathogenesis of various cancers.
      • Chen M.
      • Xu X.
      • Ma H.
      Identification of oncogenic long noncoding RNAs CASC9 and LINC00152 in oral carcinoma through genome-wide comprehensive analysis.
      • Zhao M.
      • Cui H.
      • Zhao B.
      • et al.
      Long intergenic noncoding RNA LINC01232 contributes to esophageal squamous cell carcinoma progression by sequestering microRNA6543p and consequently promoting hepatomaderived growth factor expression.
      • Meng L.D.
      • Shi G.D.
      • Ge W.L.
      • et al.
      Linc01232 promotes the metastasis of pancreatic cancer by suppressing the ubiquitin-mediated degradation of HNRNPA2B1 and activating the A-Raf-induced MAPK/ERK signaling pathway.
      However, the function of LINC01232 in LUSC progression needs to be further elucidated. In our study, we found that LINC01232 expression in both LUSC tissues and cell lines was increased, with higher expression levels in LUSC tissues with advanced tumor stage and lymph node metastasis. To investigate the role of LINC01232, we silenced LINC01232 in H1229 and A549 cells by transfecting them with the -sh-LINC01232 plasmid. We found that LINC01232 silencing inhibited the proliferation, migration and invasion of H1229 and A549 cells, suggesting that LINC01232 could function as an oncogene for LUSC development.
      LncRNAs act as competitive endogenous RNAs to sponge miRNAs, thereby regulating the expression of mRNAs.
      • Tay Y.
      • Rinn J.
      • Pandolfi P.P.
      The multilayered complexity of ceRNA crosstalk and competition.
      ,
      • Huarte M.
      The emerging role of lncRNAs in cancer.
      Recent studies suggest that the lncRNA-miRNA-mRNA axis plays a vital role in lung cancer progression.
      • Wang X.
      • Yin H.
      • Zhang L.
      • et al.
      The construction and analysis of the aberrant lncRNA-miRNA-mRNA network in non-small cell lung cancer.
      ,
      • Zhao J.
      • Cheng W.
      • He X.
      • et al.
      Construction of a specific SVM classifier and identification of molecular markers for lung adenocarcinoma based on lncRNA-miRNA-mRNA network.
      In our study, we found that LINC01232 was mainly distributed in the cytoplasm of H1229 and A549 cells. By screening the StarBase 3.0 database, we predicted that miR-181a-5p could be a downstream target of LINC01232. Using luciferase reporter and RNA immunoprecipitation (RIP) assays, we validated that LINC01232 can sponge the miR-181a-5p. Similarly, prediction by the StarBase 3.0 database revealed that miR-181a-5p could bind to the 3’-untranslated region of SMAD2, suggesting SMAD2 is the target gene of miR-181a-5p. Subsequently, using luciferase reporter and RNA pull-down assays, we confirmed that miR-181a-5p can directly target the 3’‑untranslated region of SMAD2, thereby hindering the expression of SMAD2. Further, we observed that LINC01232 silencing inhibited the proliferation, migration and invasion of H1229 and A549 cells, an effect that could be abrogated by the miR-181a-5p inhibitor. In addition, LINC01232 silencing reduced protein levels of N-cadherin, Vimentin, and Snail in H1229 and A549 cells, while protein level of E-cadherin was elevated, which can be abrogated by co-transfection with miR-181a-5p inhibitors.

      Conclusions

      In conclusion, our study first finds an increase in LINC01232 in LUSC tissues and cell lines. We demonstrate that LINC01232 can facilitate LUSC progression by modulating the miR-181a-5p/SMAD2 signaling, providing a new potential drug target for LUSC treatment.

      Declaration of Competing Interest

      The authors declared no competing interests in this work.

      Funding

      No funding.

      CRediT authorship contribution statement

      Dongliang Zhang: Conceptualization, Formal analysis. Minglei Hua: Data curation, Writing – original draft. Nan Zhang: Conceptualization, Data curation, Formal analysis, Writing – original draft.

      Acknowledgments

      We thank the reviewers for their constructive comments.

      References

        • Siegel R.L.
        • Miller K.D.
        • Jemal A.
        Cancer Statistics, 2017.
        CA Cancer J Clin. 2017; 67: 7-30https://doi.org/10.3322/caac.21387
        • Goldstraw P.
        • Ball D.
        • Jett J.R.
        • et al.
        Non-small-cell lung cancer.
        Lancet. 2011; 378: 1727-1740https://doi.org/10.1016/S0140-6736(10)62101-0
        • Bray F.
        • Ferlay J.
        • Soerjomataram I.
        • et al.
        Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
        CA Cancer J Clin. 2018; 68: 394-424https://doi.org/10.3322/caac.21492
        • Chansky K.
        • Sculier J.P.
        • Crowley J.J.
        • et al.
        The international association for the study of lung cancer staging project: prognostic factors and pathologic TNM stage in surgically managed non-small cell lung cancer.
        J Thorac Oncol. 2009; 4: 792-801https://doi.org/10.1097/JTO.0b013e3181a7716e
        • Osmani L.
        • Askin F.
        • Gabrielson E.
        • et al.
        Current WHO guidelines and the critical role of immunohistochemical markers in the subclassification of non-small cell lung carcinoma (NSCLC): moving from targeted therapy to immunotherapy.
        Semin Cancer Biol. 2018; 52: 103-109https://doi.org/10.1016/j.semcancer.2017.11.019
        • Tsai J.H.
        • Donaher J.L.
        • Murphy D.A.
        • et al.
        Spatiotemporal regulation of epithelial-mesenchymal transition is essential for squamous cell carcinoma metastasis.
        Cancer Cell. 2012; 22: 725-736https://doi.org/10.1016/j.ccr.2012.09.022
        • Hirsch F.R.
        • Scagliotti G.V.
        • Mulshine J.L.
        • et al.
        Lung cancer: current therapies and new targeted treatments.
        Lancet. 2017; 389: 299-311https://doi.org/10.1016/S0140-6736(16)30958-8
        • Jarroux J.
        • Morillon A.
        • Pinskaya M.
        History, discovery, and classification of lncRNAs.
        Adv Exp Med Biol. 2017; 1008: 1-46https://doi.org/10.1007/978-981-10-5203-3_1
        • Renganathan A.
        • Felley-Bosco E.
        Long noncoding RNAs in cancer and therapeutic potential.
        Adv Exp Med Biol. 2017; 1008: 199-222https://doi.org/10.1007/978-981-10-5203-3_7
        • Wei G.H.
        • Wang X.
        lncRNA MEG3 inhibit proliferation and metastasis of gastric cancer via p53 signaling pathway.
        Eur Rev Med Pharmacol Sci. 2017; 21: 3850-3856
        • Luan X.
        • Wang Y.
        LncRNA XLOC_006390 facilitates cervical cancer tumorigenesis and metastasis as a ceRNA against miR-331-3p and miR-338-3p.
        J Gynecol Oncol. 2018; 29: e95https://doi.org/10.3802/jgo.2018.29.e95
        • Lu W.
        • Zhang H.
        • Niu Y.
        • et al.
        Long non-coding RNA linc00673 regulated non-small cell lung cancer proliferation, migration, invasion and epithelial mesenchymal transition by sponging miR-150-5p.
        Mol Cancer. 2017; 16: 118https://doi.org/10.1186/s12943-017-0685-9
        • Xue X.
        • Yang Y.A.
        • Zhang A.
        • et al.
        LncRNA HOTAIR enhances ER signaling and confers tamoxifen resistance in breast cancer.
        Oncogene. 2016; 35: 2746-2755https://doi.org/10.1038/onc.2015.340
        • You Z.
        • Liu C.
        • Wang C.
        • et al.
        LncRNA CCAT1 promotes prostate cancer cell proliferation by interacting with DDX5 and MIR-28-5P.
        Mol Cancer Ther. 2019; 18: 2469-2479https://doi.org/10.1158/1535-7163.MCT-19-0095
        • Huang Y.
        • Zhang J.
        • Hou L.
        • et al.
        LncRNA AK023391 promotes tumorigenesis and invasion of gastric cancer through activation of the PI3K/Akt signaling pathway.
        J Exp Clin Cancer Res. 2017; 36: 194https://doi.org/10.1186/s13046-017-0666-2
        • Li B.
        • Mao R.
        • Liu C.
        • et al.
        LncRNA FAL1 promotes cell proliferation and migration by acting as a CeRNA of miR-1236 in hepatocellular carcinoma cells.
        Life Sci. 2018; 197: 122-129https://doi.org/10.1016/j.lfs.2018.02.006
        • Zhao Y.
        • Feng C.
        • Li Y.
        • et al.
        LncRNA H19 promotes lung cancer proliferation and metastasis by inhibiting miR-200a function.
        Mol Cell Biochem. 2019; 460: 1-8https://doi.org/10.1007/s11010-019-03564-1
        • Zhen Q.
        • Gao L.N.
        • Wang R.F.
        • et al.
        LncRNA DANCR promotes lung cancer by sequestering miR-216a.
        Cancer Control. 2018; 251073274818769849https://doi.org/10.1177/1073274818769849
        • Tang Y.
        • Wu L.
        • Zhao M.
        • et al.
        LncRNA SNHG4 promotes the proliferation, migration, invasiveness, and epithelial-mesenchymal transition of lung cancer cells by regulating miR-98-5p.
        Biochem Cell Biol. 2019; 97: 767-776https://doi.org/10.1139/bcb-2019-0065
        • Chen M.
        • Xu X.
        • Ma H.
        Identification of oncogenic long noncoding RNAs CASC9 and LINC00152 in oral carcinoma through genome-wide comprehensive analysis.
        Anticancer Drugs. 2019; 30: 356-362https://doi.org/10.1097/CAD.0000000000000725
        • Zhao M.
        • Cui H.
        • Zhao B.
        • et al.
        Long intergenic noncoding RNA LINC01232 contributes to esophageal squamous cell carcinoma progression by sequestering microRNA6543p and consequently promoting hepatomaderived growth factor expression.
        Int J Mol Med. 2020; 46: 2007-2018https://doi.org/10.3892/ijmm.2020.4750
        • Meng L.D.
        • Shi G.D.
        • Ge W.L.
        • et al.
        Linc01232 promotes the metastasis of pancreatic cancer by suppressing the ubiquitin-mediated degradation of HNRNPA2B1 and activating the A-Raf-induced MAPK/ERK signaling pathway.
        Cancer Lett. 2020; 494: 107-120https://doi.org/10.1016/j.canlet.2020.08.001
        • Lewis B.P.
        • Burge C.B.
        • Bartel D.P.
        Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.
        Cell. 2005; 120: 15-20https://doi.org/10.1016/j.cell.2004.12.035
        • Jiang C.
        • Hu X.
        • Alattar M.
        • et al.
        miRNA expression profiles associated with diagnosis and prognosis in lung cancer.
        Expert Rev Anticancer Ther. 2014; 14: 453-461https://doi.org/10.1586/14737140.2013.870037
        • Li H.
        • Yang T.
        • Shang D.
        • et al.
        miR-1254 promotes lung cancer cell proliferation by targeting SFRP1.
        Biomed Pharmacother. 2017; 92: 913-918https://doi.org/10.1016/j.biopha.2017.05.116
        • Li J.
        • Jin H.
        • Yu H.
        • et al.
        miRNA1284 inhibits cell growth and induces apoptosis of lung cancer cells.
        Mol Med Rep. 2017; 16: 3049-3054https://doi.org/10.3892/mmr.2017.6949
        • Li Z.
        • Wang X.
        • Li W.
        • et al.
        miRNA-124 modulates lung carcinoma cell migration and invasion.
        Int J Clin Pharmacol Ther. 2016; 54: 603-612https://doi.org/10.5414/CP202551
        • Han P.
        • Li J.W.
        • Zhang B.M.
        • et al.
        The lncRNA CRNDE promotes colorectal cancer cell proliferation and chemoresistance via miR-181a-5p-mediated regulation of Wnt/beta-catenin signaling.
        Mol Cancer. 2017; 16: 9https://doi.org/10.1186/s12943-017-0583-1
        • Yu J.
        • Jiang L.
        • Gao Y.
        • et al.
        LncRNA CCAT1 negatively regulates miR-181a-5p to promote endometrial carcinoma cell proliferation and migration.
        Exp Ther Med. 2019; 17: 4259-4266https://doi.org/10.3892/etm.2019.7422
      1. Wu L., Song W.Y., Xie Y., et al. miR-181a-5p suppresses invasion and migration of HTR-8/SVneo cells by directly targeting IGF2BP2. Cell Death Dis. Jan 16 2018;9(2):16. https://doi.org/10.1038/s41419-017-0045-0

        • Qi M.
        • He L.
        • Ma X.
        • et al.
        MiR-181a-5p is involved in the cardiomyocytes apoptosis induced by hypoxia-reoxygenation through regulating SIRT1.
        Biosci Biotechnol Biochem. 2020; 84: 1353-1361https://doi.org/10.1080/09168451.2020.1750943
        • Xue W.X.
        • Zhang M.Y.
        • Rui L.
        • et al.
        Serum miR-1228-3p and miR-181a-5p as noninvasive biomarkers for non-small cell lung cancer diagnosis and prognosis.
        Biomed Res Int. 2020; 20209601876https://doi.org/10.1155/2020/9601876
        • Lin Z.
        • Chen Y.
        • Lin Y.
        • et al.
        Potential miRNA biomarkers for the diagnosis and prognosis of esophageal cancer detected by a novel absolute quantitative RT-qPCR method.
        Sci Rep. 2020; 10: 20065https://doi.org/10.1038/s41598-020-77119-6
        • Zhang H.
        • Zhu M.
        • Shan X.
        • et al.
        A panel of seven-miRNA signature in plasma as potential biomarker for colorectal cancer diagnosis.
        Gene. 2019; 687: 246-254https://doi.org/10.1016/j.gene.2018.11.055
        • Yang J.
        • Jiang W.
        The role of SMAD2/3 in human embryonic stem cells.
        Front Cell Dev Biol. 2020; 8: 653https://doi.org/10.3389/fcell.2020.00653
        • Eppert K.
        • Scherer S.W.
        • Ozcelik H.
        • et al.
        MADR2 maps to 18q21 and encodes a TGFbeta-regulated MAD-related protein that is functionally mutated in colorectal carcinoma.
        Cell. 1996; 86: 543-552https://doi.org/10.1016/s0092-8674(00)80128-2
        • Riggins G.J.
        • Thiagalingam S.
        • Rozenblum E.
        • et al.
        Mad-related genes in the human.
        Nat Genet. 1996; 13: 347-349https://doi.org/10.1038/ng0796-347
        • Zhang C.
        • Wang B.
        • Wu L.
        MicroRNA409 may function as a tumor suppressor in endometrial carcinoma cells by targeting Smad2.
        Mol Med Rep. 2019; 19: 622-628https://doi.org/10.3892/mmr.2018.9642
        • Wang F.
        • Wu D.
        • Chen J.
        • et al.
        Long non-coding RNA HOXA-AS2 promotes the migration, invasion and stemness of bladder cancer via regulating miR-125b/Smad2 axis.
        Exp Cell Res. 2019; 375: 1-10https://doi.org/10.1016/j.yexcr.2018.11.005
        • Wei X.M.
        • Wumaier G.
        • Zhu N.
        • et al.
        Protein tyrosine phosphatase L1 represses endothelial-mesenchymal transition by inhibiting IL-1beta/NF-kappaB/Snail signaling.
        Acta Pharmacol Sin. 2020; 41: 1102-1110https://doi.org/10.1038/s41401-020-0374-x
        • Conti L.
        • Gatt S.
        Squamous-cell carcinoma of the lung.
        N Engl J Med. 2018; 379: e17https://doi.org/10.1056/NEJMicm1802514
        • Filipits M.
        New developments in the treatment of squamous cell lung cancer.
        Curr Opin Oncol. 2014; 26: 152-158https://doi.org/10.1097/CCO.0000000000000049
        • Li R.
        • Yang Y.E.
        • Jin J.
        • et al.
        Identification of lncRNA biomarkers in lung squamous cell carcinoma using comprehensive analysis of lncRNA mediated ceRNA network.
        Artif Cells Nanomed Biotechnol. 2019; 47: 3246-3258https://doi.org/10.1080/21691401.2019.1647225
        • Zhang H.Y.
        • Yang W.
        • Zheng F.S.
        • et al.
        Long non-coding RNA SNHG1 regulates zinc finger E-box binding homeobox 1 expression by interacting with TAp63 and promotes cell metastasis and invasion in Lung squamous cell carcinoma.
        Biomed Pharmacother. 2017; 90: 650-658https://doi.org/10.1016/j.biopha.2017.03.104
        • Xu Y.
        • Li J.
        • Wang P.
        • et al.
        LncRNA HULC promotes lung squamous cell carcinoma by regulating PTPRO via NF-kappaB.
        J Cell Biochem. 2019; 120: 19415-19421https://doi.org/10.1002/jcb.29119
        • Tay Y.
        • Rinn J.
        • Pandolfi P.P.
        The multilayered complexity of ceRNA crosstalk and competition.
        Nature. 2014; 505: 344-352https://doi.org/10.1038/nature12986
        • Huarte M.
        The emerging role of lncRNAs in cancer.
        Nat Med. 2015; 21: 1253-1261https://doi.org/10.1038/nm.3981
        • Wang X.
        • Yin H.
        • Zhang L.
        • et al.
        The construction and analysis of the aberrant lncRNA-miRNA-mRNA network in non-small cell lung cancer.
        J Thorac Dis. 2019; 11: 1772-1778https://doi.org/10.21037/jtd.2019.05.69
        • Zhao J.
        • Cheng W.
        • He X.
        • et al.
        Construction of a specific SVM classifier and identification of molecular markers for lung adenocarcinoma based on lncRNA-miRNA-mRNA network.
        Onco Targets Ther. 2018; 11: 3129-3140https://doi.org/10.2147/OTT.S151121