Identification Of The MicroRNA Targeting MTA1 Gene In Non-small Cell Lung Cancer And The Underlying Mechanism

BackgroundLung cancer is the leading cause of cancer mortality worldwide with 80-85% of lung cancers of cases due to non-small cell lung cancer (NSCLC). Approximately 56% of lung cancers are diagnosed at a distant stage, whilst only 15% of patients are diagnosed at a local stage. The overall 5-year survival rate for NSCLC is as low as 17.1%. Metastatic disease seriously impacts treatment outcomes and overall survival rate in NSCLC patients. Further insight into the molecular mechanisms underpinning metastasis is significantly important in disease outcomes and may facilitate the identification of new molecular targets for the treatment of the disease.Metastasis-associated gene 1 (MTA1) was discovered by screening a cDNA library from rat metastatic breast tumors which identified the gene to be one of the metastasis related genes that is overexpressed in numerous carcinomas, including NSCLC.colorectal carcinoma, hepatocellular carcinoma and breast cancer. In the past decades, MTA1 has been confirmed to be a critical component of nucleosome remodeling and the histone deacetylase (NuRD) complex. It is a key regulator of the DNA damage response, is involved in epithelial-mesenchymal transition (EMT) and inflammation in both a transcription dependent and -independent manner. However, the mechanism of MTA1 deregulation remains to be fully elucidated in order to achieve MTA1 targeted therapies.MicroRNAs (miRNAs) are a category of small non-coding RNA molecules with 18-23 nucleotides. MiRNAs affect gene expression through interaction between their seed region and the 3’or 5’untranslated region (UTR) of the target mRNA, which leads to mRNA cleavage, translational repression or activation and the formation of heterochromatin. Numerous studies have shown that different miRNAs in cancer might function either as activators or suppressors of tumors via regulation of different targets. For instance, the miR-200 family was shown to abrogate the ability of cancer cells to undergo EMT. Through modulation of the PTEN signaling pathway, upregulation of miR-21 in NSCLC might promote the invasion and migration ability of cancer cells. However, the relationship between MTA1 signaling and miRNA remains largely undetermined.Here, we set out to characterize the regulation of MTA1 expression by tumor suppressive miRNAs using bioinformatics analysis. Based on two commonly used algorithms, Targetscan and microrna.org, complete complementary sequences to three miRNAs (miR-199a/b-5p, miR-125a-3p) were found in the 3’-untranslated region (3’UTR) of MTA1. Each of the 3 candidate miRNAs have been reported to be anti-oncogenically involved in carcinogenesis, invasion and metastasis.In this study, we performed luciferase reporter assays to try to find that miR-125a-3p was a regulator of MTA1 protein expression through interaction with its 3’UTR. Next, in-vitro assays were used to demonstrated that miR-125a-3p induced suppression of the motility and proliferation of NSCLC cells that was completely or partially mediated by silencing of MTA1 expression respectively. Furthermore,we attempted to prove that MTA1 protein expression was inversely correlated with miR-125a-3p expression in NSCLC tissues.Materials and methodsTissue sampleEight pairs of primary NSCLC tissues and their corresponding normal lung tissue samples were obtained from 3 squamous cell lung carcinoma (SCC) patients (one highly differentiated and two moderately differentiated) and 5 lung adenocarcinoma patients (one highly differentiated, two moderately differentiated and two poorly differentiated). Consent forms were signed by all patients. The procedures were approved by the Clinical Research Ethics Committee of Nanfang Hospital. None of the patients had been treated with radiotherapy or chemotherapy prior to surgery. All samples were immediately placed in liquid nitrogen and stored at-80℃. Both normal and tumor tissues were verified by histological analysis.Cell cultureHEK-293T, SPC-A-1 and 95D cell lines were purchased from Shanghai Cell Bank of Chinese Academy of Science (Shanghai, China) and cultured in DMEM (Gibco,Shanghai, China) supplemented with 10% fetal bovine serum (FBS; Gibco, USA). Cells were incubated in a humidified atmosphere with 5% CO2 at 37℃.miRNA mimics and siRNAsmiRNA mimics, small interfering RNA (siRNA) against MTA1 (si-MTA1)and their negative controls were synthesized by GenePharma (Shanghai, China). The sequences were as follows:miR-199a-5p mimic: 5’-CCCAGUGUUCAGACUACCUGUUC-3’; miR-199b-5p mimic: 5’-CCCAGUGUUUAGACUAUCUGUUC-3’; miR-125a-3p mimic: 5’-ACAGGUGAGGUUCUUGGGAGCC-3’;scramble mimic: 5’-UUCUCCGAACGUGUCACGUTT-3’(negative control for miRNA mimics); si-MTA1:5’-CCAGCAUCAUUGAGUACUATT-3’; and scramble siRNA 5’-GCGACGAUCUGCCUAAGAUdTdT-3’(negative control for si-MTA1).TransfectionCells were seeded in six-well plates and cultured for 18-24 h. Then, cells were transfected with miRNA mimics or siRNAs (100μM total) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Co-transfection of miR-125a-3p mimics and si-MTA1 in SPC-A-1 and 95D cells were performed according to the similar procedures. At 48 h post transfection, the cells were harvested for further analysis.Western blot analysisProteins were extracted from whole cell lysates and separated by SDS-polyacrylamide gel electrophoresis, then transferred to PVDF membranes (Merck Millpore, Milford, MA). The following primary antibodies were used:mouse anti-MTA1 (1:1500; Abcam, Cambridge, USA) and mouse anti-β-actin (1:5000; Sigma-Aldrich, St. Louis, Missouri, USA). Membranes were incubated with the horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz, CA). LuminataTM Chemiluminescent Detection Substrates (Merck Millpore, Darmstadt, Germany) was used to visualize the immunoreactive bands and the density of each band was analyzed using Image J software.RNA preparation and quantitative real-time PCR (qRT-PCR) analysis Total RNAs were isolated from cells or tissues using Trizol reagent (Invitrogen). The TaqMan Reverse Transcription Kit (Takara, Dalian, China) was used to obtain cDNA for mRNA detection, whereas TaqMan MicroRNA Reverse Transcription Kit (Takara) was applied to reverse transcribe RNA for miRNA detection. For miRNA and mRNA, real-time PCR was performed using miRscript SYBR Green PCR Kit and SYBR Green PCR Kit (Takara) respectively, on a Roche LightCycler 480 platform (Roche, Switzerland). The PCR steps included enzyme activation for 10 min at 95℃ followed by 40 cycles of 95℃ for 15 s and 60 s annealing at 60℃, according to manufacturer’s protocol. U6 and GAPDH were used as internal controls for miRNA and mRNA analysis respectively. The primers for miRNAs and mRNAs are listed in Supplementary Table S2. Data were expressed as fold changes relative to GAPDH or U6 and calculated basing on the following formula:RQ= 2-AACt.Luciferase reporter assayThe pmirGLO vectors containing wild-type or mutant putative miR-125a-3p binding site in human MTA13’UTR were synthesized by GenePharma (Shanghai, China). HEK293T or SPC-A-1 cells were seeded into 24 well plates the day before transfection and then co-transfected with 50 ng of wild-type or mutant-type luciferase vector and 20μM miR-125a-3p mimics or negative control. After 48 h, luciferase activity was assayed by using the Dual-luciferase Reporter Assay System (Promega, Madison, WI, USA).Cell proliferation assayCells were seeded in 96-well plates at a density of 3×103 cells/well and culture overnight. After transfection, cells were cultured for 1-5 days. On the indicated days, 20μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent (MTT; 5 mg/ml; Sigma, St Louis, USA) was added to each well and incubated for 4 h. Then, the formazan was dissolved in 150 μl of dimethyl sulfoxide (DMSO) after the removal of the medium. Finally, the absorbance at 490 nm for each sample was measured using a microplate reader (ELx800; BioTek, Winooski, USA).The 5-ethynyl-2’-deoxyuridine (EdU) assay was performed using a Cell-LightTM EdU imaging detecting kit (RiboBio, Guangzhou, China) according to the manufacturer’s recommendations. Forty eight hours after transfection, NSCLC cells (500 cells/well) were cultured in 6-well plates with DMEM containing 10% FBS. After 10-14 days, the cells were fixed in 4% methyl alcohol and stained with 0.1% crystal violet, and the number of colonies (≥50 cells/colony) was counted.Invasion and migration assaysFor the wound-healing assay, cells were seeded in 6-well plates (5×105 cells/well) and incubated for 24 h, and then transfected with miRNA mimics, negative control siRNA, si-MTA1 or scrambled control siRNA. After 24 h culture in complete culture medium, a pipette tip was used to create a wound. The cells were then cultured in serum free medium. The ability of wound healing was measured under a microscope (Olympus, Tokyo, Japan) at 0 and 48 h.The invasion assay was performed in 24-well transwell plates. Briefly, cells (5 x 104 cells/well) were resuspended in serum free DMEM and grown in the upper chambers with Matrigel-coated membrane (BD Bioscience, San Jose, USA), and 500 μl of DMEM containing 10% FBS was added into the bottom of the chambers. Cells were allowed to migrate through the 8 μm polyethylene terephthalate membrane for 24 h. Cells passed through the membrane were fixed in 4% formaldehyde and stained with 0.1% crystal violet. Statistical analysis The data were expressed as the mean ±SD of three independent experiments. All statistical analyses were conducted using SPSS 14.0 statistics software package (SPSS Inc., Chicago, USA). A P value of<0.05 was considered to be statistically significant.ResultsMTA1 is a direct target of miR-125a-3pMTA1 was demonstrated to be an oncogene that plays a pivotal role in NSCLC. To gain further insight into which miRNAs regulate the expression of MTA1 from 3 candidate miRNAs, qRT-PCR and Western blotting was performed to confirm if restoration of miR-199a-5p, miR-199b-5p or miR-125a-3p resulted in down regulation of MTA1 in SPC-A-1 and 95D cells. It was found that none of the 3 miRNAs had any effect on MTA1 mRNA expression, whereas at the protein expression level, MTA1 was significantly repressed in miR-125a-3p transfected cells compared to mock, miR-control, miR-199a-5p, or miR-199b-5p transfected cells. These data suggest that miR-125a-3p down-regulated MTA1 expression at the post-transcriptional level.One putative miR-125a-3p-binding site within the 3’UTR of MTA1 was predicted by Targetscan and the microrna.org database. To validate the direct interaction between MTA1 and miR-125a-3p, we performed luciferase reporter assays in HEK293T and SPC-A-1 cells by co-transfection with MTA13’UTR reporter plasmid or its mutant and miR-125a-3p mimics or negative control. The luciferase reported activity analysis showed that ectopic expression of miR-125a-3p significantly suppressed the luciferase activity of the wild-type vector but not the mutant reporter. These results were reproducible both in HEK293T and SPC-A-1 cells and suggest that miR-125a-3p binds directly to specific sites in the 3’UTR of MTA1 mRNA.Knockdown of MTA1 by siRNA elicits similar reponses as re-expression of miR-125a-3pTo determine the functional relationship between miR-125a-3p and MTA1 in the proliferation, migration and invasion of NSCLC cell lines, SPC-A-1 and 95D cells were transfected with plasmids expressing si-MTA1 which resulted in greatly decreased MTA1 mRNA and protein levels. A similar reduction in MTA1 protein but not mRNA expression was also observed in cells overexpressing miR-125a-3p. This was in good agreement with the above results obtained.Knockdown of MTA1 by siRNA led to decreased cell viability and colony formation rate, which mimicked the inhibitory effects of miR-125a-3p on cell proliferation and further enhanced the effect of miR-125a-3p on suppressing proliferation. These results were also replicated using the Edu cell proliferation assay.As observed in the wound-healing and transwell plate assays with or without matrigel, ectopic expression of miR-125a-3p and knockdown of MTA1 had almost exactly the same inhibitory effect on cell migration and invasion. Notably, the migration and invasion ability of SPC-A-1 and 95D cells co-transfected with miR-125a-3p mimics and si-MTAl was almost entirely similar to that of cells overexpressing miR-125a-3p or with decreased MTA1 expression. These observations suggest that down-regulation of miR-125a-3p may stimulate proliferation, migration and invasion by promoting the expression of MTA1.Inverse relationship between the expression of MTA1 protein and miR-125a-3p in NSCLC tissues.As the direct and negative regulation of MTA1 by miR-125a-3p was confirmed in vitro, we speculated that an inverse relationship between miR-125a-3p and MTA1 protein expression may exist in NSCLC clinical samples. Therefore, the expression level of miR-125a-3p and MTA1 protein in 8 pairs of NSCLC and matched adjacent normal tissues was examined by qRT-PCR and western blot analysis. Spearman’s correlation analysis revealed a significant negative correlation between the expression of miR-125a-3p and MTA1 in NSCLC tissues (r=-0.762, P=0.028), indicating that miR-125a-3p down-regulation was significantly associated with higher MTA1 protein levels.Conclusion:1. miR-125a-3p could bind directly to specific sites in the 3’UTR of MTAl mRNA and in turn, decreasing the protein level of MTA1 at a posttranscriptional level.2. Down-regulation of miR-125a-3p may stimulate proliferation, migration and invasion by promoting the expression of MTA1.3. A significant negative correlation was revealed between the expression of miR-125a-3p and MTA1 in NSCLC tissues.