Inactivation of SMARCA2 by promoter hypermethylation drives lung cancer development

Accepted Manuscript Inactivation of SMARCA2 by promoter hypermethylation drives lung cancer development

Jixiang Wu, Keshuai He, Yajun Zhang, Jixiang Song, Zhan Shi, Weiwei Chen, Yongfeng Shao PII: DOI: Reference:

S0378-1119(18)31177-6 https://doi.org/10.1016/j.gene.2018.11.032 GENE 43377

To appear in:

Gene

Received date: Revised date: Accepted date:

29 September 2018 8 November 2018 13 November 2018

Please cite this article as: Jixiang Wu, Keshuai He, Yajun Zhang, Jixiang Song, Zhan Shi, Weiwei Chen, Yongfeng Shao , Inactivation of SMARCA2 by promoter hypermethylation drives lung cancer development. Gene (2018), https://doi.org/10.1016/j.gene.2018.11.032

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ACCEPTED MANUSCRIPT Inactivation of SMARCA2 by promoter hypermethylation drives lung cancer development

Jixiang Wua, b, Keshuai Hea, Yajun Zhangb, Jixiang Songb, Zhan Shib, Weiwei Chenc, Yongfeng Shaoa* Department of Cardio-Vascular Surgery, The First Affiliated Hospital of Nanjing

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a

Medical University, Nanjing 211166, China

Department of Cardiothoracic Surgery, The Third People’s Hospital of Yancheng

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City, Yancheng 224000, China

Department of Radiotherapy, The Third People’s Hospital of Yancheng City,

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c

Yancheng 224000, China

Corresponding to: Yongfeng Shao, Department of Cardio-Vascular Surgery, The

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*

First Affiliated Hospital of Nanjing Medical University, Nanjing 211166, China.

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E-mail address: [email protected].

ACCEPTED MANUSCRIPT Abstract The SWI/SNF complex is a multimeric chromatin remodeling complex that has vital roles in regulating gene expression and cancer development. However, to date few studies have deeply explored the mechanism of SMARCA2 inactivation. We applied multi-omics analysis to explore the mechanism of SMARCA2 inactivation in The

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Cancer Genome Atlas (TCGA) database and performed the dCas9-DNMT3a system to evaluate the role of promoter methylation in SMARCA2 transcriptional regulation.

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We also assessed the tumor suppressing roles of SMARCA2 in lung cancer development by in vitro experiments. SMARCA2 promoter hypermethylation was

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significantly associated with decreased expression of SMARCA2. This result was further confirmed in the results of our own tissues. In addition, we observed that the

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mRNA level decreased by about 3 folds while the CpG island of promoter is nearly 30% hypermethylated by dCas9-DNMT3a system in H1299 cells. We identified SMARCA2

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as a tumor suppressor gene whose expression was downregulated in lung cancers. Its inactivation was significantly associated with the poor survival of lung cancer patients

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[hazard ratio, HR=0.35 (0.27-0.45)]. Besides, we found that SMARCA2 was a tumor

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suppressor and can significantly inhibit lung cancer cell vitality. We found that promoter hypermethylation contribute to the inactivation of SMARCA2. We also verified its oncogenetic roles of BRM inactivation in lung adenocarcinoma, which

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may provide a potential target for the clinical treatment.

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Key words: SMARCA2, hypermethylation, dCas9-DNMT3a and lung cancer

ACCEPTED MANUSCRIPT 1. Introduction Lung cancer is the leading cause of cancer death in the world, with almost 1.6 million deaths per year (Siegel et al., 2016). In recent decades, our understanding of the molecular mechanisms in lung cancer development has been revolutionized by the identification of genetic abnormalities, such as epidermal growth factor receptor

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(EGFR) mutations, MET amplifications and so on (Janku et al., 2011). Similarly, it has become increasingly obvious that epigenetic alternations play equally important

attracted much attention (Wilson and Roberts, 2011).

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roles in tumorigenesis, and among them, chromatin remodeling factors have recently

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The SWI/SNF complex is a multimeric chromatin remodeling complex that has vital roles in regulating gene expression and cancer development such as

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differentiation, metabolism, DNA repair and cell cycle control (Peterson and Workman, 2000; Klochendler-Yeivin et al., 2002; Simone, 2006; Reisman et al.,

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2009). The key ATP catalytic subunit of the SWI/SNF complex is SMARCA2 ( also known as BRM) or SMARCA4 (also known as BRG1), which have now emerged as

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bona fide tumor suppressor genes (Reisman et al., 2003; Fukuoka et al., 2004; Glaros

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et al., 2008; Medina et al., 2008; Reisman et al., 2009). Evidence suggested that loss of SMARCA4 expression through mutations is associated with cancer development including NSCLC (Reisman et al., 2003;

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Fukuoka et al., 2004; Medina et al., 2004; Medina et al., 2008; Weissman and Knudsen, 2009; Wilson and Roberts, 2011). The SMARCA2 gene, encodes part of the

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large ATP-dependent chromatin remodeling complex SNF/SWI, which is required for transcriptional activation of genes normally repressed by chromatin. Interestingly, SMARCA2 is rarely mutated in various cancers (Bourachot et al., 2003; Yamamichi et al., 2005; Glaros et al., 2007), but usually epigenetically silenced in about 15-40% of many primary solid tumors (Decristofaro et al., 2001; Glaros et al., 2007; Yamamichi et al., 2007; Shen et al., 2008), including in 17-30% of NSCLC (Reisman et al., 2003; Glaros et al., 2007). However, to date few studies have deeply explored the mechanism of SMARCA2 inactivation in a multi-omics database, and lack of investigating the function of SMARCA2 in lung adenocarcinoma.

ACCEPTED MANUSCRIPT In this study, we found that copy number variations and promoter hypermethylation contribute to the inactivation of SMARCA2. We also verified its oncogenetic roles of BRM inactivation in lung adenocarcinoma, which may provide a potential target for

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the clinical treatment.

ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1 Cell culture Involved cell lines in this study were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM (Gibco, USA) containing 10% fetal bovine serum (Gibco), 100 units/ml penicillin, 100 μg/ml

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streptomycin, and 5.5 M MD-glucose (normal glucose) and incubated at 37°C in 5%

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CO2 and saturated humidity.

2.2 Plasmid construction

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A synthetic plasmid coding Cas9 was obtained from Addgene corporation (http://www.addgene.org). The constructs of Fuw-dCas9-DNMT3a plasmid (Addgene

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plasmid#84475) was reported previously (Liu et al., 2016). The sequence of SMARCA2-expression plasmid was amplified according to the full-length of

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SMARCA2 exons (based on the SMARCA2 sequence in NCBI website) and then sub-cloned into a pCDNA3.1 vector (Invitrogen, Shanghai, China). All constructs

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were sequenced before transfection.

2.3 Construction of single guide RNAs (sgRNAs) As described previously (Morita et al., 2016), the demethylation effect of

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Fuw-dCas9-DNMT3a system can maintain more than 90% within 200bp whereas not extend to about 1 kb away from the target sequence. According to the position of the

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interesting CpG island, we designed sgRNAs for CRISPR/dCas9 system as listed in Table s1. The efficiency verification experiment was conducted in vitro after sgRNAs were constructed and sequenced.

2.4 RNA extraction The harvested cells were dissolved in 1ml Trizol® reagent (Invitrogen, MA, USA) and then total mRNA was extracted following the manufacturer's protocol.

2.5 Reverse transcription and quantitative real-time PCR (RT-qPCR)

ACCEPTED MANUSCRIPT The total mRNA was reverse transcribed to complementary DNA (cDNA) using the High Capacity RNA-to-cDNA Kit (Takara) after the concentration and purity of RNA were determined with the Beckman DU-640 Spectrophotometer (Beckman Coulter, Inc., Brea, CA, USA). Real-time PCR was performed with the SYBR Premix Ex Taq™ (Takara, Japan) on a QuantStudio™ 7 Flex Real-Time PCR System. The

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cDNA-special primers were presented in Table S1. The cycle threshold (Ct) is defined as the number of cycles required for the fluorescent signal to cross the

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threshold in real-time PCR. 2 −ΔΔCt formula was used to calculate the relative expression of gene mRNA, where ΔΔCt = Ct (SMARCA2) − Ct (GAPDH) (Livak and

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Schmittgen, 2001). Therefore, 2−ΔΔCt value shows the mRNA expressions relative to the endogenous control gene (GAPDH). All reactions were performed in triplicate and

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comparison was based on Student’s t test.

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the result was presented as mean ± standard deviation (SD). Further grouped

2.6 Western blot analysis

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The cells were washed three times with PBS, and total protein was isolated using

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protein lysis buffer. After centrifugation at 12,000 х g for 15 min at 4°C, cell debris was removed, and the supernatant (cell lysate) was used for Western blotting. Protein concentrations were measured using the BCA assay (Beyotime, Shanghai, China).

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Equal amounts of proteins were separated on 10% SDS-PAGE gels and then transferred onto PVDF membranes (Millipore, MA, USA). The membranes were

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blocked in blocking buffer (Tris-buffered saline, pH 7.6, 5% skim milk and 0.05% Tween) at room temperature for 1.5 h. Then, the membranes were incubated at 4°C overnight with a primary antibody diluted in blocking buffer followed by incubation with the corresponding secondary anti-IgG horseradish peroxidase conjugate (Santa Cruz Biotechnology, CA, USA) for 1.5 h. Antibody binding was visualized with ECL solution (Pierce Biotechnology, Inc., Rockford, IL, USA). The expression of SMARCA2 was assessed by immunoblotting using an antibody purchased from Abcam (ab15597, Abcam, Cambridge, MA, USA) and normalized to that of GAPDH.

ACCEPTED MANUSCRIPT 2.7 Cell migration and invasion assay Migration and invasion assays were performed using transwell migration chambers (Corning, Cat# 354578), respectively. The control and transfected cells were seeded at a density of 4×104 cells/well. A volume of 100 μl of cells was added to the upper chamber, while 600 μl of DMEM containing 10% FBS was added to the lower

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chamber and incubated at 37°C in 5% CO2. The cells attached to the upper surface of the membrane were removed with a cotton swab, and the cells on the underside were

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fixed, stained with Giemsa (Dingguobio, Shanghai, China) for 3-5 min, and counted (nine random fields) by two independent investigators. The results were normalized to

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the controls.

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2.8 CCK8 assay

Cell-counting assay kit (CCK8) was purchased from Dojindo (Japan). Briefly, at 2 h

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before each indicated time point, 10 μl of CCK-8 solution was added to each well on a plate containing 100 μl of DMEM. Then, the absorbance at 450 nm was recorded

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using a microplate absorbance reader. Each count was determined as an average of

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three replicates, and each data point was the average of at least three experiments. All data were normalized to the control group.

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2.9 Statistical analysis

The multiomics data of The Cancer Genome Atlas (TCGA) were downloaded from

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cBioPortal for Cancer Genomics in Jun 2017 (http://www.cbioportal.org/tutorial.jsp). SMARCA2 gene expression data from TCGA data were analyzed for differential expression in tumor tissues versus adjacent normal tissues. Furthermore, the association between SMARCA2 expression in all tumor tissue samples and survival was analyzed by Cox regression. Kaplan–Meier analysis was used to fit the survival curves and was tested by log-rank test. For experimental data, continuous values were described with mean ± SE and tested by ANOVA analyses. A value of P<0.05 was accepted as statistically significant. All statistical analyses were performed using Graphpad Prism 6.01.

ACCEPTED MANUSCRIPT 3. Results 3.1 Molecular mechanism underlying the inactivation of SMARCA2 We first sought to elucidate the transcriptional regulation underlying low SMARCA2 gene expression. Somatic mutations of lung adenocarcinoma patient samples, including amplification, deep deletion, truncating mutations and missense mutations,

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frequently occurring in a set of SWI/SNF complex genes(Witkowski et al., 2014). While there was only one sample bears truncation mutation which might induce loss

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of function of SMARCA2 (Figure 1A). Thus, we speculated that the inactivation of SMARCA2 is not primarily due to somatic mutations. Combined analysis of copy

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number alterations and gene expression data revealed that the mRNA expression of SMARCA2 gene was lower in deletions compared with that in diploid, gain and

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amplification SMARCA2 genes in 512 lung adenocarcinoma tissues (Figure 1B). We also found that in all types of copy number alterations, sallow deletion and diploid

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account for the highest proportion (50.00% and 34.38%, respectively) as shown in Figure 1C. Further studies suggested that the SMARCA2 promoter hypermethylation

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was significantly associated with decreased expression of SMARCA2 (Figure 1D),

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implicating DNA hypermethylation as a novel mechanism for SMARCA2 inhibition (Pcrude=2.0010-4; Padjust=1.5910-2). Together, these finding suggested that copy number deletion and promoter hypermethylation play important roles in the

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inactivation of SMARCA2 in lung adenocarcinoma.

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3.2 Hypermethylation of the promoter inactivates expression of SMARCA2 We detected the methylation levels of SMARCA2 promoter in 15 pairs of normal tissues of lung cancer and adjacent tissues. As shown in Figure 2A, SMARCA2 is hypermethylated in tumors compared with normal tissues. Further, we identified that hypermethylation of the CpG island at SMARCA2 promoter is significantly correlated with its expression (P=0.026, Figure 2B). To verify the activation mechanism of SMARCA2 in adenocarcinoma, we induced targeted demethylation in SMARCA2 promoter in adenocarcinoma cells with a specific CRISPR/dCas9 system. Figure 2C provides an illustration of the CRISPR/dCas9 system that H1299 cell co-transfected

ACCEPTED MANUSCRIPT with Fuw-dCas9-DNMT3a and sgRNA is regarded as treatment group while only delivery of Fuw-dCas9-DNMT3a was regarded as control group. The targeted methylation was measured by Bisulfite Genomic Sequence (BSP) while the transcriptional level of SMARCA2 was determined by RT-qPCR. As expected, we observed that the mRNA level decreased by about 3 folds while the CpG island of

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promoter is nearly 30% hypermethylated (Figure 2D-E), indicating targeted demethylation in SMARCA2 promoter could directly lead to the aberrant expression of

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SMARCA2 in adenocarcinoma.

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3.3 SMARCA2 is absent in lung adenocarcinoma and low SMARCA2 expression is correlated with poorer overall survival

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To understand the involvement of SMARCA2 in lung adenocarcinoma, we analyzed the mRNA level of SMARCA2 in the TCGA transcriptome data from 57 pairs of lung

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adenocarcinoma patients. Tumor tissues showed lower levels of SAMRCA2 (Figure 3A-B) compared to the adjacent tissues. Besides, Kaplan–Meier curve demonstrated

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that lower expression was significantly correlated with reduced patient survival

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(Figure 3C). We also detected SAMRCA2 in paired of lung cancer tissues by western blotting, and observed a significantly lower levels of SMARCA2 in lung cancer samples compared with adjacent tissues (Figure 3D and 3E). Next, we examined the

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levels of SAMRCA2 mRNA and protein in a panel of adenocarcinoma cell lines, including H1299, A549 and H1703. We observed significantly higher levels of

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SMARCA2 in H1299 compared to the others, so we chose H1299 for the next experiments (Figure 3F and 3G).

3.4 SMARCA2 induces abnormal cell viability in lung adenocarcinoma cells To investigate the tumor suppressing roles of SMARCA2 in lung cancer cells, we overexpressed SMARCA2 in H1299 cells. The cells were divided into two groups, OE group (LV-BRM lentivirus) and NC group (CON235 lentivirus). Control lentivirus and SAMRCA2 overexpressing lentivirus were transfected into H1299 cell lines. Western blot analysis was used to detect the expression levels of SMARCA2 (Figure 4A and

ACCEPTED MANUSCRIPT 4B). The results of the CCK8 experiment showed that overexpressing SMARCA2 inhibited cell growth of H1299 cells (Figure 4E). Furthermore, overexpression of SMARCA2 led to significantly decrease in the ability of colony formation in soft agar (Figure 4F). Finally, SMARCA2-overexpression H1299 cells showed a significant reduction in their ability to migration (Figure 4G). SMARCA2 knocked down in

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H1299 cells showed increased number of colonies and the ability to migration

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(Figure 4C, 4F, 4H-4J).

ACCEPTED MANUSCRIPT 4. Discussion To our knowledge, this is the first study using CRISPR/dCas9 system to precisely modify the CpG island of SMARCA2 promoter, and verified the causal correlation between promoter hypermethylation and inactivation of SMARCA2 in lung cancer. Besides, we confirmed the tumor suppressing role of SMARCA2 in vitro in lung

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adenocarcinoma. SWI/SNF has been linked to cancer since the discovery that BAF47 is inactivated

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due to a combination of mutations and deletions in human rhabdoid tumors and the subsequent discovery that BAF47 induces malignant tumors in mice in a relatively

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short period of time(DeCristofaro et al., 1999; Betz et al., 2002). In contrast, the knockout of either SAMRCA4 or SAMRCA2 has not yielded robust malignant

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phenotypes (Hoffman et al., 2014; Orvis et al., 2014). Considering the heterogeneity of SAMRCA2 expression in composite tumors, SAMRCA2 may be a potential

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“second hit” candidate in cancers and may play an important role in tumor progression (Orvis et al., 2014). Thus, further studies focusing on SAMRCA2 are

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required to broaden our understanding of its involvement in tumor formation and

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progression. In this study, tumor tissues showed lower levels of SAMRCA2 compared to the adjacent tissues. Besides, Kaplan–Meier curve demonstrated that lower expression was significantly correlated with reduced patient survival. Furthermore,

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we also found that SAMRCA2 overexpression can inhibit the proliferation and migration of lung cancer cells in vitro. Studies have investigated the effect of

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SMARCA2 on cancer evolution through various ways. For example, its functional linkage to proteins such as Rb and p53 may partly explain how SMARCA2 silencing results in the loss of cellular growth control (Reisman et al., 2002; Xu et al., 2007). Also, the aberration of DNA repair proteins such as GADD45, BRCA1 AND p53, functionally linked to SMARCA2, is known to potentiate cancer development (Bochar et al., 2000; Wang et al., 2007). Loss of SMARCA2 expression has been linked to the progression of lung adenocarcinoma with the features of epithelium-mesenchymal transition (EMT), a major step towards more aggressive disease. Together, all these evidence indicated that inactivation of SAMRCA2 plays

ACCEPTED MANUSCRIPT oncogenic roles in lung cancers. In previous studies, the association between the SAMRCA2 promoter variants and cancers were observed and has potential therapeutic implications (Wong et al., 2014; Segedi et al., 2016). Thus further studies will be required to clarify the role of epigenetic SAMRCA2 silencing as an oncogenic driver in the pathogenesis of lung

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cancers. To illustrate this mechanism underlying the inactivation of SAMRCA2, we firstly resorted to the multiomics database of the TCGA, and found that copy number

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deletion and promoter hypermethylation are tightly correlated with the decreased expression of BRM in lung cancer. The correlation of promoter methylation and

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SAMRCA2 expression were confirmed in our own tumor tissues. In addition, we used CRISPR/dCas9 system to precisely modify the methylation levels of CpG island in

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SAMRCA2 promoter, and verified the casual correlation between promoter hypermethylation and inactivation of SAMRCA2. Our current data raise the possibility

approach in these malignancies.

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of reversing this epigenetic dysregulation as a novel therapeutic and/or preventive

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Taken together, our study confirms that the tumor suppressor gene SAMRCA2

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inhibits lung cancer cell vitality in vitro. In addition, we used a combination of multi-omics analysis and CRISPR/dCas9 system to explore the epigenetic regulation of SAMRCA2 in lung cancer. These results indicated that hypermethylation of

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SAMRCA2 promoter plays oncogenic roles in lung cancer development.

ACCEPTED MANUSCRIPT Conflict of interest disclosure statement The authors have declared that no conflict of interest exists.

Grant Support This study was supported by the Natural Science Foundation of Jiangsu Province

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(BK20151590) and The “333” Project of Jiangsu Province (BRA2014336). The funders had no role in the study design, data collection and analysis, decision to

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publish, or preparation of the manuscript.

ACCEPTED MANUSCRIPT Legends to Figures Figure 1. Molecular mechanisms underlying SMARCA2 inactivation. (A) Schematic diagram showing the positions of individual somatic alterations in the SMARCA2 gene identified in lung adenocarcinoma. Missense mutations as putative driver (black green) are displayed. (B) Copy number alterations of SMARCA2 in lung

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adenocarcinoma samples from TCGA data and it shows the association between mRNA levels and copy number variation of SMARCA2. Data are expressed as the

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means ± SD. (C) Pie chart showing the proportion of different status of copy number variations. (D) The expression of SMARCA2 negatively correlates with SMARCA2

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methylation in lung adenocarcinoma patients (P < 0.0001). The Spearman test was used with linear regression. The expression and methylation levels of SMARCA2 were

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downloaded from https://github.com/cBioPortal. The values are calculated by correlation analysis between the SMARCA2 mRNA expression level and methylation

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using linear regression. The band around the regression line indicates the 95%

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confidence interval of regression coefficients in multiple linear regression.

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Figure 2. The correlation of promoter methylation and SMARCA2 expression in lung adenocarcinoma tissues.

(A) The methylation levels of BRM promoter is detected from 15 pairs of lung

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adenocarcinoma tissues (Tumor) and adjacent tissues (Adjacent). N=15 pairs of tissues, ** P < 0.01. (B) The image shows the correlation between methylation levels

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of CpG island and SMARCA2 expression. (C) The image shows the detailed information of CRISPR/dCas9 system. (D) The methylation level of the CpG island in SMARCA2 promoter was analyzed by BSP in treated H1299 cell lines (E) The transcriptional level of SMARCA2 was measured by RT-qPCR. N=4/group, data are expressed as the means ± SEM, ** P < 0.01.

Figure 3. SMARCA2 expression levels are lower in lung adenocarcinoma tissues. (A) The tendency is calculated from 57 pairs of lung adenocarcinoma tissues (Tumor) and adjacent tissues (Adjacent). (B) Box plot showing lower levels of SMARCA2

ACCEPTED MANUSCRIPT mRNA in tumor tissues than in adjacent tissues. Data are expressed as the mean ± SEM. ** P < 0.01, Ntumor=488, Nadjacent=57 (C) Kaplan-Meier curve of the survival of patients with high and low levels of SMARCA2 mRNA (P < 0.05). The definitions of high and low expression are classified according to the median. The Kaplan-Meier curve indicates cancer-specific survival. (D and E) SMARCA2 protein expression in

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tumor and adjacent tissues in adenocarcinoma patient samples. Data are expressed as pairs of tissues. ** P < 0.01, Npairs=9. (F and G) The mRNA (F) and protein (G)

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expression of SMARCA2 in different lung cancer cell lines.

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Figure 4. SMARCA2 is critical for lung cancer cell proliferation and migration. (A and B) mRNA and protein expression of SMARCA2 in control and overexpressed

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cells. (C and D) mRNA and protein expression of SMARCA2 in control and knocked down cells. (E) CCK8 assays were used to determine the viability of knocking down

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SAMRCA2 in H1299 cells. (F) Colony formation assays were performed to determine the proliferation of knocking down SAMRCA2 in H1299 cells. (G) Transwell assays

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were performed to determine the metastasis of knocking down SMARCA2 in H1299

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cells. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01. (H) CCK8 assays were used to determine the viability of SAMRCA2 overexpressed A549 cells. (I) Colony formation assays were performed to determine the proliferation of

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SAMRCA2 overexpressed A549 cells. (J) Transwell assays were performed to determine the metastasis of SAMRCA2 overexpressed A549 cells. Data are expressed

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as the mean ± SEM. ** P < 0.01.

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ACCEPTED MANUSCRIPT Roberts, H., Knox, J., Marquez, S., Wong, R., Darling, G., Waldron, J., Goldstein, D., Leighl, N., Shepherd, F.A., Tsao, M., Der, S., Reisman, D. and Liu, G., 2014. Two BRM promoter insertion polymorphisms increase the risk of early-stage upper aerodigestive tract cancers. Cancer Med 3, 426-33. Xu, Y., Zhang, J. and Chen, X., 2007. The activity of p53 is differentially regulated by Brm- and Brg1-containing SWI/SNF chromatin remodeling complexes. J Biol Chem 282, 37429-35. Yamamichi, N., Inada, K., Ichinose, M., Yamamichi-Nishina, M., Mizutani, T., Watanabe, H., Shiogama, K., Fujishiro, M., Okazaki, T., Yahagi, N., Haraguchi, T., Fujita, S., Tsutsumi, Y., Omata, M. and features and differentiation state. Cancer Res 67, 10727-35.

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Iba, H., 2007. Frequent loss of Brm expression in gastric cancer correlates with histologic Yamamichi, N., Yamamichi-Nishina, M., Mizutani, T., Watanabe, H., Minoguchi, S., Kobayashi, N.,

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Kimura, S., Ito, T., Yahagi, N., Ichinose, M., Omata, M. and Iba, H., 2005. The Brm gene suppressed at the post-transcriptional level in various human cell lines is inducible by

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transient HDAC inhibitor treatment, which exhibits antioncogenic potential. Oncogene 24,

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5471-81.

ACCEPTED MANUSCRIPT Abbreviations

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NSCLC , Non -small cell lung cancer; TCGA, The Cancer Genome Atlas; sgRNA, single guide RNA; RT-qPCR, quantitative real-time PCR; SD, standard deviation; BSP, Bisulfite Genomic Sequence; CI, Confidence interval; HR, hazard ratio.

ACCEPTED MANUSCRIPT Highlights 1. We found that copy number variations and promoter hypermethylation contribute to the inactivation of SMARCA2. 2. We used CRISPR/dCas9 system to precisely modify the CpG island of SAMRCA2 promoter.

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3. We verified its oncogenetic role of SAMRCA2 inactivation in lung

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adenocarcinoma, which may provide a potential target for the clinical treatment.

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