MicroRNA-29b attenuates non-small cell lung cancer metastasis by targeting matrix metalloproteinase 2 and PTEN
- Hongyan Wang†1,
- Xiaoying Guan†1,
- Yongsheng Tu2,
- Shaoqiu Zheng1,
- Jie Long1,
- Shuhua Li1,
- Cuiling Qi1,
- Xiaobin Xie1,
- Huiqiu Zhang1 and
- Yajie Zhang1Email author
© Wang et al. 2015
Received: 17 March 2015
Accepted: 8 May 2015
Published: 11 June 2015
Our pilot study using miRNA PCR array found that miRNA-29b (miR-29b) is differentially expressed in primary cultured CD133-positive A549 cells compared with CD133-negative A549 cells.
Ten human non-small cell lung cancer (NSCLC) cell lines and samples from thirty patients with NSCLC were analyzed for the expression of miR-29b by quantitative RT-PCR. Bioinformatics analysis combined with tumor metastasis PCR array showed the potential target genes for miR-29b. miR-29b lentivirus and inhibitors were transfected into NSCLC cells to investigate its role on regulating cell proliferation which was measured by CCK-8 assay in vitro and nude mice xenograft tumor assay in vivo. Cell motility ability was evaluated by transwell assay. The target genes of miR-29b were determined by luciferase assay, quantitative RT-PCR and western blot.
Bioinformatics analysis combined with tumor metastasis PCR array showed that matrix metalloproteinase 2 (MMP2) and PTEN could be important target genes of miR-29b. The expression of miR-29b was down regulated in NSCLC tissues compared to the normal tissues. Clinicopathological analysis demonstrated that miR-29b had significant negative correlation with lymphatic metastasis. The gain-of-function studies revealed that ectopic expression of miR-29b decreased cell proliferation, migration and invasion abilities of NSCLC cells. In contrasts, loss-of-function studies showed that inhibition of miR-29b promoted cell proliferation, migration and invasion of NSCLC cells in vitro. Nude mice xenograft tumor assay confirmed that miR-29b inhibited lung cancer growth in vivo. High-invasion (A549-H) and low-invasion (A549-L) NSCLC cell sublines from A549 cells were created by using the repeated transwell assay aimed to confirm the effect of miR-29b on migration and invasion of NSCLC. Furthermore, the dual-luciferase reporter assay demonstrated that miR-29b inhibited the expression of the luciferase gene containing the 3’-UTRs of MMP2 and PTEN mRNA. Western blotting and quantitative RT-PCR indicated that miR-29b down-regulated the expression of MMP2 at the protein and mRNA levels.
Taken together, our results demonstrate that miR-29b serves as a tumor metastasis suppressor, which suppresses NSCLC cell metastasis by directly inhibiting MMP2 expression. The results show that miR-29b may be a novel therapeutic candidate target to slow NSCLC metastasis.
Lung cancer is characterized by a low survival and high relapse rate after surgery [1, 2]. NSCLC, the most frequently occurring category of lung cancer, accounts for approximately 80 % of all cases . Tumor invasion and metastasis are the main factors responsible for NSCLC treatment failure [4, 5].
Emerging evidence has revealed that microRNAs (miRNAs) play key roles in various biological processes, including metastasis, proliferation, apoptosis, stress resistance, tumorigenesis, and cell differentiation [6, 7]. Compared with normal tissues, different tumors have distinct miRNA expression characteristics. It has been determined that abnormal miRNA expression is a critical carcinogenesis signal. In tumors, miRNAs perform functions similar to those of oncogenes and tumor suppressors. miRNA expression patterns are more finely regulated than those of proteins . Given these findings, miRNA may have great potential as a biomarker in early tumor diagnosis and treatment targets . Many studies have attempted to identify the metastasis-related miRNAs in metastatic tumors using miRNA microarrays analysis. For example, several miRNAs have been identified to be involved in development of NSCLC metastasis, including let-7 , miR-200 , miR-125b  and miR-10b . Although miRNAs have been the subject of extensive research in recent years, the molecular regulatory mechanisms of miRNAs and their effects on cancer are not well understood.
In the present study, to screen the metastasis-related miRNAs of NSCLC, CD133-positive and CD133-negative subpopulation from human lung adnocarcinoma A549 cells were isolated through immunomagnetic bead separation method. CD133 has been considered a specific stem cell marker and NSCLC prognosis marker [14, 15]. CD133-positive cells have greater potential for proliferation, metastasis, and chemo-radioresistance [16–18]. Through bioinformatics analysis and miRNA PCR array and tumor metastasis PCR array, PTEN, ETV4, COL4A2 and MMP2 were logically been speculated as miR-29b target genes. Our results showed that miR-29b inhibited growth and metastasis of NSCLC cells in vitro and in vivo. Additionally, dual-luciferase reporter assay and western blot results further elucidated that the miR-29b inhibited the expression of the luciferase gene containing the 3’-UTRs of MMP2 and PTEN mRNA. While miR-29b down-regulated the expression of MMP2 at the protein and mRNA levels. The results showed that miR-29b maybe a novel therapeutic candidate target or strategy for seeking to control NSCLC metastasis.
Tissue samples, cell culture and animals
Information about tissue specimens, NSCLC cell lines and animals is given in the Additional file 1.
Microarray screening of differentially expressed genes between CD133-positive/negative NSCLC cells
Detailed information about isolation of CD133-positive/negative A549 cells and microarray screening of differentially expressed genes is provided in the Additional file 1.
Using “miRNA” as the index word for predicting target genes in the TargetScan (www.targetscan.org), PicTar (www.pictar.org), and miRanda (www.microrna.org) databases, target genes were identified from overlapping results from the three databases. Subsequently, we extracted the overlap of these results with that of the tumor metastasis PCR array.
Quantitative RT-PCR was performed using kits for U6 and mature miR-29b (ABI, Foster City, CA, USA), according to the manufacturer’s instructions. SYBR green real-time RT-PCR was performed to detect MMP2 and PTEN. Detailed information is provided in the Additional file 1.
All miRNA duplexes (Additional file 2: Table S1) were purchased from Genepharma (Shanghai, P.R. China). H460 cells were transfected with inhibitor or inhibitor NC at a final concentration of 100 nmol/L using Lipofectamine RNAiMAX instructions (Invitrogen). A549 subline stably expressing miR-29b (A549-miR-29b) and its control line (A549-NC) were established as described in the Additional file 1.
The cells were lysed with radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China). The antibodies used for western blotting are described in the Additional file 1.
We purchased psiCHECK-2 plasmids from Promega (Madison, WI, USA). Human genome DNA was used as the template for the MMP2 3’ untranslated region (UTR) and PTEN 3’ UTR PCR. XhoI and NotI restriction sites were introduced at the 5’ ends of both the forward and reverse primers (Additional file 2: Table S1). Following double digestion, the linear psiCHECK-2 fragment was connected with the 3’ UTRs using T4 DNA ligase; positive clones were selected for sequencing validation after transformation. The sequencing-validated, target recombinant plasmid was designated psiCHECK-2-Wt-MMP2/PTEN-3’ UTR, and was used as a template for constructing psiCHECK-2-Mut-MMP2/PTEN-3’ UTR using antisense PCR and a site-specific mutagenesis kit (Toyobo, Osaka, Japan).
Luciferase reporter activity assay
Plasmid (0.5 μg) and 50 nmol/L miR-29b mimic/mimic NC were cotransfected using Lipofectamine LTX reagent (Invitrogen). Three replicates and three parallel lines were used each time. Following 48-h transfection, luciferase activity was measured using a GloMax 20/20 Luminometer (Promega) according to the dual luciferase reporting system instructions. Relative luciferase activity was compared using the ratio of Renilla reniformis and firefly luciferase activity (Rn/Ff).
Cell proliferation assay
To measure the effect of miRNA and inhibitor on cellular proliferation rates, cells were incubated in 10 % CCK-8 (DOJINDO) diluted in normal culture media at 37 °C until visual color conversion appears. Proliferation rates were determined at 24, 48, 72, 96, 120 h post-transfection, and quantification was done on a microplate reader set according to the manufacturer’s protocol.
In vitro assays of migration and invasion
The 24-well Boyden chamber with 8-μm pore size polycarbonate membrane (Corning, NY) was used to analyze the migration and invasion of tumor cells. Details are in the Additional file 1.
In vivo studies
H460 subline stably knockdown miR-29b (H460-LV-miR-29b inhibitor) and its control line (H460-LV-CON), were established as described in Additional file 1. Analysis for tumorigenicity was performed as described in Additional file 1.
All data were analyzed using SPSS 13.0 (SPSS Inc, Chicago, IL, USA); A paired t test was used to investigate the difference in the expression level of miR-29b between normal and cancerous tissues. A 2-sample t test was used to analyse the clinicopathologic characteristics of miR-29b expression in the tissues of patients with NSCLC. Quantitative RT-PCR, CCK-8 assay, migration and invasion assay, and luciferase reporter assay were tested using 1-way analysis of variance for factorial design. P value < 0.05 was considered statistically significant.
Screening and identifying the metastasis-related miRNAs and target genes of NSCLC
miR-29b is down-regulated in NSCLC tissues
Clinicopathologic characteristics of miR-29b expression in NSCLC patients
−2.044 ± 1.311
= < 60
−1.807 ± 0.989
−1.883 ± 0.722
−1.957 ± 1.508
−2.182 ± 0.858
−1.877 ± 1.182
Well + Moderate
−1.931 ± 1.344
−1.886 ± 0.401
−1.122 ± 0.638
−1.881 ± 0.453
−2.671 ± 1.398
−1.505 ± 0.799
−2.389 ± 1.302
miR-29b suppresses cell proliferation, migration and invasion in A549 cells
miR-29b deficiency alters the metastasis ability of H460 cells
Effect of miR-29b on cell migration and invasion ability in A549-L and A549-H cells
MMP2 as a target gene of miR-29b in NSCLC
miR-29b affected PTEN expression by binding directly with the PTEN 3’ UTR
In the present study, we provided evidence that miR-29b expression in high-metastatic CD133-positive A549 lines was down-regulated when compared to miR-29b expression in paired low-metastatic CD133-negtive A549 cell lines, miR-29b was confirmed directly targeted 3’-UTR of PTEN and MMP2 mRNAs and down-regulated MMP2 protein expression to suppress lung cancer metastasis in vitro and in vivo.
As a MMP superfamily member, MMP2 specifically degrades type IV collagen, a major component of the extracellular matrix and basal lamina, and is a major factor in tumor invasion and angiogenesis . It has been demonstrated that high MMP2 expression is an independent prognostic factor in NSCLC and is closely related to clinical stage, pathological grade, lymphatic metastasis, and prognosis . The regulatory mechanisms of a miRNA could differ among different microenvironments, miR-29b is upregulated in metastatic breast cancer tissues and indolent lymphocytic leukemia, functioning as an oncogene [21, 22]. However, miR-29b is down-regulated in lung carcinoma tissues . In our study, low-level expression of miR-29b in NSCLC tissues was significantly associated with lymphatic metastasis. We performed gain-of-function in A459 cells and loss-of-function in H460 cells of miR-29b. Our data demonstrated that miR-29b inhibited in vitro cell proliferation, invasion and migration and in vivo suppressed NSCLC growth in a nude mice xenograft model. Furthermore, the dual-luciferase reporter assay demonstrated that miR-29b inhibited the expression of luciferase gene containing the 3’-UTR of PTEN and MMP2. Western blotting indicated that miR-29b down-regulated the endogenous protein expression of MMP2. Based on these results, It’s concluded that miR-29b was related to metastasis in NSCLC.
PTEN regulates tumor cell growth, cell cycle, apoptosis, and metastasis by regulating multiple signal transduction pathways negatively [24, 25]. All three databases used (TargetScan, PicTar, miRanda) identified the two PTEN 3’ UTR miR-29b binding sites. Both sites were conserved among different species and fully complementary to the miR-29b seed sequence, corresponding to the basic rules for predicting miRNA target genes . Our present results showed that miR-29b bound directly to the two PTEN 3’ UTR binding sites and PTEN was a miR-29b target gene. Through miR-29b overexpression or knockdown analysis, the fact was determined that miR-29b variations were not accompanied with the alteration of PTEN expression. As multiple miRNAs could regulate the same target gene , we speculate that other miRNAs could also bind directly to the PTEN 3’ UTR and regulating PTEN expression. Several research reported that PTEN function as a target gene of miR-21 , miR-214 , miR-494 , miR-26a , miR-144  and miR-153 . of these, miR-21, miR-214, and miR-494 are upregulated in NSCLC. Another reason that might explain our contrasting findings was that miR-29b directly inhibits CDC42 and p85α to activate p53 expression . P53 activates PTEN transcription, binding directly to the PTEN promoter and activating PTEN expression . PTEN gene was not only indirectly regulated by miR-29b-p53-PTEN positively, but also directly regulated by miR-29b negatively. The inhibition of Sp1 by miR-29b resulted in the upregulation of PTEN in tongue squamous cell carcinoma .
In summary, our studies demonstrated that down-regulated miR-29b expression was found to be associated with increased MMP2 expression in CD133-positive NSCLC cells through microarrays and bioinformatics analysis. miR-29b played a strong inhibitory role in tumor metastasis. We provided important evidence that miR-29b could suppress NSCLC cells proliferation, migration and invasion by targeting the 3’-UTR of MMP2 and PTEN mRNA to down-regulate MMP2 protein expression. Our findings provided novel evidence for the involvement of miR-29b in NSCLC metastasis, and suggested that miR-29b could be a potential new target for treatment of NSCLC metastasis.
The patient consent of Written informed consent was obtained from the patient for the publication of this report and any accompanying images.
This work was funded by the National Nature Science Foundation of China (No. 81401391), the Doctoral Fund of Ministry of Education of China (No.20134423110001), Science and Technology Program of Guangzhou (No.2014Y2-00171), Guangzhou Municipal Education Department Innovation team grant (No.13C06), Medical Scientific Research Foundation of Guangdong Province (No.A2014278, No.A2013247), and Guangzhou City-belonged Universities Scientific Research Program (No.2012C135).
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