MiroRNA-188 Acts as Tumor Suppressor in Non-Small-Cell Lung

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MiroRNA-188 acts as tumor suppressor in nonsmall-cell lung cancer by targeting MAP3K3 Lili Zhao, Xin Ni, Linlin Zhao, Yao Zhang, Dan Jin, Wei Yin, Dandan Wang, and Wei Zhang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00071 • Publication Date (Web): 12 Mar 2018 Downloaded from http://pubs.acs.org on March 13, 2018

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Molecular Pharmaceutics

MiroRNA-188 acts as tumor suppressor in non-small-cell lung cancer by targeting MAP3K3 Lili Zhao 1, Xin Ni 1, Linlin Zhao1, Yao Zhang1, Dan Jin2, Wei Yin3, Dandan Wang1, Wei Zhang1*

1

Department of Respiratory Medicine, Affiliated Hongqi Hospital of Mudanjiang

Medical University, Mudanjiang 157011, Heilongjiang, P.R. China 2

Department of Ultrasound, Mudanjiang Women and Children’s Hospital, Mudanjiang

157000, Heilongjiang, P.R. China 3

Department of Bone Surgery, Mudanjiang Forestry Hospital, Mudanjiang 157000,

Heilongjiang, P.R. China

*Corresponding author: Wei Zhang, Department of Respiratory Medicine, Affiliated Hongqi Hospital of Mudanjiang Medical University, Mudanjiang 157011, Heilongjiang, P.R. China E-mail: [email protected]

Running Head: functional implication of miR-188 in NSCLC

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ABSTRACT Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer. MicroRNAs have been increasingly implicated in NSCLC and may serve as novel therapeutic targets to combat cancer. Here we investigated the functional implication of miR-188 in NSCLC. We first analyzed miR-188 expression in both NSCLC clinical samples and cancer cell lines. Next we investigated its role in A549 and H2126 cells with cell proliferation, migration and apoptosis assays. To extend the in vitro study, we employed both xenograft model and LSL-K-ras G12D lung cancer model to examine the role of miR-188 in tumorigenesis. Last we tested MAP3K3 as miR188 target in NSCLC model. MiR-188 expression was significantly downregulated at the NSCLC tumor sites and lung cancer cells. In vitro transfection of miR-188 reduced cell proliferation and migration potential and promoted cell apoptosis. In xenograft model, miR-188 inhibited tumor growth derived from cancer cells. Intranasal miR-188 administration reduced tumor formation in NSCLC animal model. MAP3K3 was validated as direct target of miR188. Knocking down MAP3K3 in mice also inhibited tumorigenesis in LSL-K-ras G12D model. Our results demonstrate that miR-188 and its downstream target MAP3K3 could be a potential therapeutic target for NSCLC.

KEYWORDS: MiR-188, NSCLC, K-ras G12D, MAP3K3

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INTRODUCTION Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer. The types of NSCLC include adenocarcinoma, large-cell carcinoma, squamous cell carcinoma, and more poorly differentiated variants. Patients with NSCLC are often diagnosed at advanced stages of the disease. Conventional chemotherapy only prolongs marginally survival of such individuals. Therefore, the mechanistic study of the pathogenesis of NSCLC followed by the identification of specific genetic or molecular events that are associated with specific subtypes of NSCLC will provide more rational and promising targeting treatments to improve the clinical outcome. Activating mutations in K-RAS (10–30%) and loss of function point mutations in p53 (50–70%) are among the most common mutations in NSCLC.(1-3) And a subset of NSCLC was shown to carry activating mutations in epidermal growth factor receptor gene (EGFR) which are markedly responsive to EGFR inhibitor gefitinib.(4) Besides these proteincoding oncogenes, microRNAs (miRNAs) have also been increasingly implicated in NSCLC and may serve as novel therapeutic targets to combat cancer.(5, 6) MiRNAs are sequence specific regulators for gene expression and were first discovered in worm in 1990s.(7) They usually contain 21~25 nucleotides and fine-tune the expression of thousands mRNAs, with each mRNA regulated by multiple miRNAs. Most miRNAs bind to the complementary sites in 3’-untranslated regions (UTRs) of target mRNAs to suppress gene expression by either directing mRNAs degradation or protein translation repression.(8) During the last 20 years, miRNAs have been proposed as key regulators in various biological processes including cell differentiation, cell growth and apoptosis.(9, 10) Dysregulation of miRNAs in cells has been reported in a variety of human cancers.(11, 12) Based on the latest bioinformatics prediction studies, the link of miRNA targets and a wide range of diseases including cancers have been

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predicted (13-16). The alteration of miRNA expression can act as either oncogenic factors or tumor suppressors to regulate the tumorigenesis.(17) In experimental validation studies, many miRNAs such as let-7,(18, 19) miR-34,(20) miR-21 (21) and miR-31(22) have being reported as potential therapeutic targets in various NSCLC tumor models. MiR-188 is located on the X chromosome in human and was first discovered in 2003.(23) It has been reported to control synaptic transmission and dendritic plasticity by targeting neuropilin-2.(24) It has also been found in regulating age-associated switch between osteoblast and adipocyte differentiation.(25) In cancer research, miR-188 has been reported as tumor suppressor in various cancer models including hepatocellular carcinoma, oral squamous cell carcinoma and prostate cancer.(26-28) MiR-188-5p down-regulation is an independent prognostic factor for poor overall and recurrencefree survival in prostate cancer.(26) In another study, authors have identified unique miRNA molecular profiles in the diagnosis and prognosis of lung cancer.(29) MiR-188 was found differentially expressed in adenocarcinoma tissues compared with noncancerous lung tissues. Therefore, the past studies on miR-188 all suggested its potential predictive role and therapeutic potential in cancer therapy. However, little is known about the molecular function of miR-188 in lung cancer model. In this study, to establish the clinical association of miR-188 with NSCLC, we examined miR-188 expression in 52 NSCLC tissue samples in comparison with the matched non-tumor lung tissues from the same donors. We also confirmed the downregulation of miR-188 in six cancer cell lines. In order to understand the role of miR188 in tumorigenesis, we investigated the effects of over-expressing miR-188 in cell proliferation, migration and cell death in A549 and H2126 cells. For in vivo study, we used both xenograft and orthotropic mouse lung cancer model to test miR-188 as tumor

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suppressor in lung cancer. Last but not least, we examined MAP3K3 as a predictive miR-188 target in our in vitro and in vivo lung cancer models. Further on, we tried to validate MAP3K3 as downstream target of miR-188 for suppressing tumor growth with knocking-down approach.

EXPERIMENTAL SECTION Clinical samples NSCLC tissue samples were acquired from hospital under an Institutional Review Board of Affiliated Hongqi Hospital of Mudanjiang Medical University approved protocol. Patients included in the study were given written informed consents. Pathological examination validated the presence of at least 80% tumor tissue in cancerous samples, while cancerous tissue was not found at all in the corresponding benign regions. Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA,USA) method according to the manufacturer’s protocol. Cell culture Lung cancer lines (A549, H2126, SHP-77, H522, H1299 and H2085) and normal lines (BEAS-2B and NuLi-1) were obtained from the American Type Culture Collection (Manassas, VA, USA). The NSCLC cell lines were cultured in either Dulbecco’s modified Eagle’s medium or RPMI-1640 medium.BEAS-2B cells were cultured in LHC-9 medium and NuLI-1 cells were cultured in DMEM. All media were added with 10% fetal bovine serum (HyClone Laboratories, Logan, UT, USA), and an antibiotic cocktail of 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco, Grand Island, NY, USA). cDNA synthesis and qRT–PCR

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The isolated RNA was first reverse transcribed with the TaqMan miRNA reverse transcription kit (Applied Biosystems, Waltham, MA, USA) according to the instruction of the manufacturer. The quantitative PCR was performed using TaqMan Advanced miRNA Assay (Applied Biosystem, Waltham, MA, USA) in an ABI PRISM 7900 realtime PCR machine. Primers for hsa-miR-188 (#A25576) were also acquired from Applied Biosystems. MiRNA expression level was normalized against RNU43. All experiments were performed in triplicate and the differential expression levels were determined with 2- ∆∆CT method. Cell transfection MiR-188 mimic and non-targeting miRNA mimic were acquired from Ruibo (Guangzhou, China). At 48 hours post seeding, cells were transfected with 50 nM miRNAs by the Lipofectamine 2000 kit (Life Technologies, NY, USA) following the manufacturer’s protocol. At 48 h after transfection, cells were used for experiments including cell migration, cell apoptosis and proliferation assays and Western blotting. Cell viability assay Cells transfected with miR-188 or control miRNA mimics were harvested and plated in 96-well plate at 2,000 cells per well and incubated at 37°C. Cell viability was analyzed with the Cell Counting Kit-8. Trans-well cell migration assay Cells transfected with miR-188 or control miRNA mimics were cultured at 5×105/well in the upper chamber of a trans-well plate (Corning, NY, USA). At 24 h post seeding, cells migrated to the bottom chamber were fixed and stained with Toluidine Blue O dye according to manufacturer’s instruction. Flow cytometry

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To evaluate apoptosis status, cells expressing miR-188 or control mimics were stained with FITC-Annexin V staining Detection kit (Biosea Biotechnology, BJ, China) on FACSCalibur. Western blotting Total protein from tissues or cells was extracted in RIPA buffer. The extracted protein was measured using a BCA protein quantification kit (Thermo Scientific, Rockford, IL, USA). 40 µg of protein were subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5 % non-fat milk and incubated with primary antibody at 4oC for 16 h. After washing step, the membrane was incubated with a secondary antibody conjugated with HRP for 1 h at room temperature. Chemilluminance signals were detected using an ECL plus kit (Thermo Fisher Scientific). Caspase3 (#9662), Actin (#3700) and MAP3K3 (#5727) antibodies were from Cell Signaling (MA, USA). Lentiviral vectors construction and viral particles production Lentivirus production was performed as described previously (30). Pri-miR-188 sequence or non-targeting scramble sequence were sub-cloned into pLenti vector (Invitrogen, CA, USA) to build lenti-mir-188 or lenti-control vectors. Lenti-MAP3K3 shRNA or lenti-scramble shRNA were purchased from Dharmacon. Xenograft study NOD/SCID mice were obtained from Jackson Laboratories (ME, USA). 5×106 cells transfected with miRNAs were injected into either flank of NOD/SCID mice. Measurement of tumor volumes was taken weekly after grafting. The detailed protocol was described previously (18). All animal experiments were approved by the Institutional Animal Care and Use Committee of Affiliated Hongqi Hospital of Mudanjiang Medical University.

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In vivo lung cancer model of Lox-Stop-Lox K-ras G12D (LSL-K-ras G12D) The heterozygote mouse strain was acquired from the NCI-Frederick Mouse Repository. To induce NSCLC formation, 6-weeks old LSL-K-ras G12D mice were anesthetized and intranasally inoculated with 5×106 PFU pre-packaged adenovirus expressing cre (Ad-cre). Ad-cre in 65 µl instillations as described previously.(20) A group of mice was sacrificed 7 weeks after infection to establish the tumor burden base line. The remaining mice were received lentiviral particles as described in the main text at 3×106 TU per animal in the presence of 100 g/ml polybrene resuspended in 1×MEM. Lung tissues were harvested for histological examination to study tumor burden following O.C.T. frozen section procedure. Lung tumor areas were quantified with ImageJ software. The overall tumor burden was calculated as a ratio of total tumor area to total lung area by taking the average of every sixth slide section throughout the tissue. Statistical analysis All experiments performed in this study have been repeated at least 3 times. Statistical analysis was performed with SPSS software. Results were presented as mean ± standard deviation (SD). Data was compared with Student’s t test. Differences were considered significant at p < 0.05.

RESULTS MiR-188 is downregulated in NSCLC To assess the association of miR-188 expression in human NSCLC, we performed qRTPCR on 52 human cancer tissue samples. As baseline control, miR-188 expression was measured in corresponding adjunct non-tumor tissue from the same sample. In these samples, miR-188 level was significantly lower in the majority of tumor regions as

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compared with matched non-tumor lung tissues (Figure 1A). The expressional difference is regardless of the patient’s gender, age and pathological stage (data not shown). To further confirm this association, we also characterised the miR-188 expression in six NSCLC lines (A549, H2126, SHP-77, H522, H1299 and H2085) obtained from ATCC. BEAS-2B and NuLi-1 are derived from normal lung tissues. We used NuLi-1 as baseline control. Intriguingly, miR-188 was found significantly lower in all these six cancer cell lines with the reduction range from around 20% to 80% (Figure 1B). Therefore, our tissue and cell expression data strongly suggested the downregulation of miR-188 may play a protective role in NSCLC pathogenesis. MiR-188 expression suppresses the proliferation and migration of NSCLC cells To investigate the role or miR-188 in NSCLC, we used A549 and H2126 as in vitro model to perform the mechanistic study. We first transfected A549 and H2126 with miR-188 mimic and control mimic. We started to measure the cell proliferation at 24 h, 48 h and 72 h post transfection. MiR-188 mimic significantly down-regulated the cell proliferation in both A549 and H2126 from 48 h to 72 h post transfection. Next, we proceeded to study the cell migration ability in transwell assay. We found cells transfected with miR-188 migrated in slower rate than control cells in transwell system (Figure 2B). Since both proliferation and migration represent key aspects of cancer progression, these results suggested miR-188 may play a negative role in NSCLC tumorigenesis. MiR-188 expression promotes cell apoptosis in NSCLC lines Next we examine the effect of miR-188 in apoptotic pathway of NSCLC lines. We transfected A549 and H2126 with miR-188 or control mimic and performed flow cytometry to measure cell apoptosis 48 h post transfection. As shown (Figure 3A), the percentages of apoptotic cells in control groups were below 10%. MiR-188 over-

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expression significantly induced the apoptotic cell population with nearly two folds in both lines. We then harvested the cells to analyse the underlying apoptotic signalling by examining Caspase 3 cleavage, a key indicator of apoptotic activation in cells. In line with our observation in FACS assay, we found cleaved Caspase 3 was induced in A549 and H2126 expressing miR-188 mimic as compared with control groups (Figure 3B). Taken together, miR-188 could promote cell apoptosis by activating conventional apoptosis pathway. MiR-188 reduces tumor growth in lung cancer xenograft models. So far our in vitro studies suggested miR-188 as tumor suppressor in NSCLC. To evaluate this hypothesis more directly, we proceeded to analyse the tumor growth in xenograft models derived from A549 and H2126 cells. To induce tumor growth, cells transfected with either miR-188 or negative control miRNA were injected subcutaneously into immunodeficient NOD/SCID mice. To control the host variability, each animal received miR-188 transfected cells in one flank and control cells in the opposite flank. We measured the tumor volumes weekly till week 8 after cells injection. As shown (Figure 4A), the site injected with A579 expressing control miRNA exhibited robust tumor growth. Interestingly, miR-188 inhibited tumor growth of the A579 xenograft. Throughout the course, tumors that developed from cells expressing miR-188 were markedly smaller than their corresponding control tumors. And the tumors at the sites receiving miR-188-transfected A579 cells progressed in a much slower pace than the control group. Similarly, miR-188 robustly interfered with tumorigenesis in the H2126 xenograft (Figure 4B). At week 8 post injection, the average tumor volume from control group was more than twice larger than miR-188 xenograft group. Therefore, our in vivo study also supported the hypothesis that miR-188 acts as a tumor suppressor. MiR-188 suppresses tumor growth in KRAS mutation induced lung cancer model.

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Since miR-188 suppressed lung cancer cells growth in vitro and in vivo, we proceeded to determine if miR-188 could directly inhibit tumor growth in relevant NSCLC animal model. Various mouse genetic models of lung cancer have relied on the induction by oncogenic K-ras mutants.(31) Here we employed LSL-K-ras G12D mouse model. A loxP flanked stop codon has been inserted before the mutant KRAS (G12D) which has been engineered into the endogenous locus. Cre-mediated excision of the stop element can lead to the physiologically relevant level of KRAS (G12D) that in turn triggers tumor formation in the animal. For lung tumor development, viral system can be used to deliver Ad-cre via intranasal delivery. In this study we developed a protocol to study the tumor suppression effect of miR-188 in KRAS (G12D) NSCLC model as illustrated in Figure 5A. We firstly induced tumor formation in the lung via Ad-cre. At week 7 after K-ras G12D induction, a group of mice were sacrificed to set the tumor score baseline. The remaining mice were divided randomly into two groups and received with either exogenous miR-188 or control miRNA via lentiviral infection to their tumor sites. At week 14, all animals were sacrificed to examine their tumor burden in the lung tissues. In control group, tumors continued to progress from about 4% of average tumor burden in the baseline to about 17%. In contrast, tumors exposed to lenti-miR-188 progressed in a much slower rate reaching only to around 9% of gross tumor burden in average, suggesting that the tumor growth was inhibited by miR-188 (Figure 5B). MAP3K3 is a target of miR-188 and may be implicated lung tumorigenesis. To dissect the molecular mechanism by which miR-188 confers tumor suppression on lung cancer model, we predicted miR-188 potential targeting genes in Targetscan database (32). MAPK pathways act downstream of many growth-factor receptors and their activations play important parts in various cancer progression.(33) Many signalling components in MAPK pathways could be molecular targets for cancer treatments.(34)

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MAP3K3 is among the most potential miR-188 targets in the prediction, with a cumulative weighted context score of -0.25 (Figure 6A). To confirm this, we examined the mRNA expression level of MAP3K3 in the cells transfected with miR-188. As shown (Figure 6A), MAP3K3 expression was markedly downregulated in both A549 and H2126 cells expressing miR-188 mimic. Next, we examined the protein level of MAP3K3 in the tumor tissues harvested from LSL-K-ras G12D mice. The tumor samples from treatment group infected with lentivirus expressing miR-188 showed markedly reduced amount of MAP3K3 as compared to those from the control group infected with lentiviral control vector. These results have suggested MAP3K3 is a direct target of miR-188 in cells and in vivo. Then, we sought to ask whether miR188/MAP3K3 axis played a role in suppressing tumor growth in our model. Similar to the study described in Figure 5A, we induced lung tumors in LSL-K-ras G12D mice with intranasal delivery of Ad-cre. At week 7 post Ad-cre delivery when the lung tumors were established, we tested the therapeutic effect of knocking-down MAP3K3 with lentiviral infection of MAP3K3 shRNA through intranasal pathway. Lentiviral vector expressing scramble shRNA was used in the control group. Intriguingly, 7 weeks after lentiviral delivery, average tumor size from the treatment group became significantly smaller than that in the control group. But the inhibitory effect of knocking down MAP3K3 in tumor progression was less than that of miR-188 delivery, suggesting other unidentified pathways regulated by miR-188 for tumor suppression. Taken together, MAP3K3 is one of the downstream targets of miR188 for inhibiting tumor progression.

DISCUSSION

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Molecular Pharmaceutics

In this study, we have investigated the function of miR-188 in NSCLC tumorigenesis. We first demonstrated altered miR-188 expression was correlated to the tumorigenesis of lung cancer by confirming miR-188 expression was suppressed in clinical lung cancer samples and six cancer lines. Cell culture studies showed the expression miR188 in two lung cancer lines potently inhibited their proliferation and migration and enhanced the cell death via Caspase 3 activation. To extend the in vitro studies, we also investigated the functional consequence of miR-188 expression in xenografts and LSLK-ras G12D mice, a lung cancer animal model. We observed substantial tumor suppression by miR-188 in these two in vivo models. To elucidate the mechanistic function of miR-188 as tumor suppressor, we identified MAP3K3 as target gene regulated by miR-188. Knocking-down MAP3K3 directly was shown to reduce tumor growth in LSL-K-ras G12D mice, suggesting MAP3K3 pathway is negatively regulated by miR-188 lung tumorigenesis. MiRNA expression signature for lung cancers was characterized by previous studies (5, 6, 29) to explore novel prognostic biomarker or therapeutic targets for lung cancers. MiR-188 was reported downregulated in adenocarcinoma tissues in a microarray profiling study.(29) Our study was first time to establish such correlation in clinical NSCLC tissues. Interestingly, miR-188 down-downregulation was also described in human hepatocellular carcinoma,(28) acute myeloid leukaemia,(35) prostate cancer (26) and oral squamous cell carcinoma.(27) These similar observations strongly suggest miRNA-188 play a key role in suppressing tumorigenesis across different cancer types. It will be promising to test this hypothesis in other types of cancers. It is plausible that miRNA-188 or its downstream targets participate in a highly conserved mechanism in regulating oncogenesis. The next interesting question will be whether inhibiting miRNA-188 expression promotes tumor formation. Therefore,

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knocking-down or even knocking-out experiments with CRISPR/Cas9 system to suppress miRNA-188 expression in human cell lines will provide more robust results in testing this hypothesis.(36) Although miR-188 expression potently suppressed NSCLC progression in two animal models, it did not completely stop tumor growth, suggesting other miRNA regulatory circuits are also relevant to lung cancer.(6) Research interests in exploring miRNAs as therapeutic targets for lung cancer have grown exponentially in recent years since the landmark papers that firstly revealed the over-expression of let-7 reduces tumor growth in lung cancer mouse model.(19) Many other miRNA targets have been characterized in lung cancer model. For instance, miR-181a has shown to inhibit NSCLC proliferation by targeting CDK1.(37) MiR-29b can attenuate NSCLC metastasis by targeting MMP2 and PTEN.(38) And miRNA-142-3p can act as a potential tumor suppressor by targeting HMGB1 directly in NSCLC model.(39) However, there are lack of studies investigating the upstream events that lead to the altered expression of these miRNAs implicated in NSCLC pathogenesis. To understand the causes of the functional dysregulation of microRNA in cancer will provide more opportunities for the discovery and development of future miRNA based therapies. Recent studies have also reported a combinatorial approach targeting multiple components of tumor pathways that will likely reduce the occurrence of acquired resistance and enhance the efficacies (40, 41). MiR-34 and let-7 both function as tumor suppressors by targeting different pathways in NSCLC.(40) Combining let-7 and miR34 into a single therapeutic displayed superior effects than the use of single agent in NSCLC mouse models.(40) One interesting direction in future will be to explore synergistic effects by targeting miR-188 with other miRNA candidates simultaneously for lung cancer treatment.

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In our study, we discovered MAP3K3 as direct target of miR-188 and showed the reduction of MAP3K3 inhibits KRAS mutation driven tumorigenesis. MAP3K3 belongs to a family of serine/threonine kinases that encode important functions in a wide range of biological processes. It acts at the upstream of SEK and MEK and activates NFĸB activation to confer cell resistance to apoptosis.(33) Over-expression of MAP3K3 has been reported to be linked to ovarian cancer and associated with poor survival rate.(42) Another study has showed amplification of MAP3K3 gene promotes tumor initiation and survival of breast cancer cells (43). In addition, MAP3K3 expression is proposed as a predictor of poor disease prognosis in esophageal squamous cell carcinoma.(44) Our study also supports MAP3K3 acts as oncogenic factor in human cancer. To further establish the link of between MAP3K3 and NSCLC, it will be worthwhile to evaluate the expression of MAP3K3 in NSCLC patients or the clinical samples from different pathological stage. During the preparation of our paper, a very recent study has also reported MAP3K3 as a direct target of miR-188 in Lineage-negative bone marrow cells. The downregulation of MAP3K3 by miR-188 leads to cell senescence and decreased cell proliferation and migration.(45) Therefore, it is intriguing to examine whether senescence associated pathway could contribute to the tumor regression observed in our study models. Another interesting study showed miR-188 can induce cell cycle arrest by targeting multiple cyclin/CDK complexes.(46) This could be another underlying mechanism that accounts for miR-188 mediated lung tumor suppression. Since knocking-down MAP3K3 shows less inhibitory effect on tumor growth that expressing miR-188, it is likely miR-188 could target multiple components at the downstream to deliver anti-tumor effects in NSCLC. Therefore, it will also be interesting to assess the change of cyclin/CDK complexes in NSCLC model.

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In conclusion, our study strongly supports that miR-188 act as a tumor suppressor miRNA in the lung and the beneficial effects of expressing miR-188 in two different tumor models also make clinical relevant predictions to using miR-188 based therapeutic agents in NSCLC.

Competing interests The authors declare that they have no competing interests.

Funding This work was supported by Health and Family Planning Commission of Heilongjiang Province (2017-340; 2013-199; 2010-283); Planning from Mudanjiang Medical University (2011-28).

Acknowledgments None.

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FIGURE LEGENDS Figure 1. The clinical significance of miR-188 levels in Non-Small Cell Lung Cancer (NSCLC). (A) miR-188 is downregulated in 52 NSCLC tissues as compared with the matched non-tumor sites. *** p