Up-regulating methylation of CAP1 promoter can promote apoptosis and inhibit migration and invasion of lung adenocarcinoma cells

Abstract

Background

Lung adenocarcinoma (LUAD) is the most common histological subtype of lung cancer, which poses a significant threat to human health. Adenylate cyclase-associated protein 1 (CAP1) is an important protein closely linked to cancer initiation and progression.

Method

Target Gene fragments were amplified by PCR, and the products of 2 fragments were ligated to construct pdCas9-Dnmt3a-BSD plasmid. Stable cell lines with methylation of CAP1 promoter up-regulated were then established through transfection and screening. Cell proliferation was assessed using colony formation and proliferation assays, while apoptosis was assessed by flow cytometry. Wound healing, transwell migration, and invasion assays were conducted to evaluate cell migration and invasion. Western blot and PCR assays were used to study the expression of molecules involved in apoptosis, migration, and invasion.

Result

CAP1 protein expression was higher in early-stage LUAD tissues than in adjacent normal tissues, and elevated in A549, H1299, and PC9 cells as compared to Beas-2B control cells. In addition, CAP1 promoter was abnormally hypo-methylated in LUAD cells and tissues. Up-regulating CAP1 promoter methylation through the CRISPR-dCas9-Dnmt3a system which reduced CAP1 expression can induce apoptosis via the Bax/Bcl-2/Caspase-3 pathway, and inhibited migration and invasion. We also found that the methylation of the CAP1 promoter was regulated by Dnmt3a, Tet1, and/or Tet2.

Conclusion

Up-regulating CAP1 promoter methylation promotes apoptosis and inhibits migration and invasion of LUAD cells. This suggests that methylating the CAP1 promoter could be a potential therapeutic approach for early-stage LUAD.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40001-025-03135-9.

Introduction

Lung cancer (LC) is the first leading cause of cancer morbidity and mortality worldwide, [1] with a 5 year survival rate of less than 20% [2]. Lung adenocarcinoma (LUAD) is the most common histological subtype of LC, which accounts for about 40% of all LC [3]. The etiology of LC is not fully understood, and its occurrence and development are believed to be closely related to genetic and environmental factors. Abnormal DNA methylation modification often occurs in the early stage of tumor formation, which is regarded as one of the important causes and a marker of human tumor [4]. Studies have shown that environmental factors can alter the DNA methylation status of cells, and confirmed the close relationship between cancer and the environment at the molecular level [5–7]. Therefore, the study of DNA methylation in some specific genes has important clinical value for the early diagnosis, treatment and prognosis of LC.

CAP (adenylate cyclase associated protein) was originally discovered in budding yeast S. cerevisiae and is thought to regulate the actin cytoskeleton and mediate Ras regulation of adenylate cyclase [8–10]. It was later found that CAP promotes actin filament turnover in yeast by facilitating all key rate-limiting steps in the cofilin-driven actin dynamics [11]. In mammals, there are two subtypes of CAP: CAP1 and CAP2 [12, 13]. CAP1 is the universally expressed isoform believed to fulfill the protein functions in most tissue or cell types with CAP2 in a complementary role. Earlier studies showed that CAP1 has the dual functions of signal transduction and maintenance of actin cytoskeleton [10, 14]. With the development of research, more functions of CAP1 have been found. Zhou et al. found that CAP1 can promote matrix adhesion, [15] and recently reported that cAMP/CAP1/Rap1 pathway is a new signaling pathway involved in matrix adhesion in colon cancer cells [16]. In addition, CAP1 was found to promote tumor cell migration [17, 18]. CAP1 was also reported to be associated with breast cancer cell proliferation in vitro and metastasis in breast cancer patients [19]. It has been found that CAP1 is overexpressed in pancreatic cancer, which is related to the invasion of pancreatic cancer [20]. Liu et al. found that CAP1 was also overexpressed in hepatocellular carcinoma and associated with metastasis of human hepatocellular carcinoma [21]. Kolegova et al. found higher expression of CAP1 in cancer tissues compared to normal tissues, and considered that CAP1/Cofilin series and intracellular proteolytic system play an important role in the process of tumor transformation and lymphatic metastasis in NSCLC [22]. These studies show that CAP1 protein is highly expressed in some malignant tumors, and the high expression of CAP1 protein is positively correlated with cancer progression. However, these studies have mostly focused on protein level of CAP1, and it is unclear whether the overexpression of CAP1 protein is related to epigenetics in cancers.

The clustered regularly interspaced short palindromic repeats (CRISPR)/catalytically inactivated Cas9 (dCas9) methylation editing system is a technology that uses RNA-guided Cas nuclease to make specific DNA modification of targeted genes [23]. DNA methyltransferase (Dnmt) can regulate the methylation of genes. The CRISPR-dCas9-Dnmt3a system fuses dCas9 with Dnmt3a enzyme and guides it to the target gene via sgRNA, up-regulating methylation levels of the target gene. In this study, we found abnormal hypo-methylation of CAP1 promoter in LUAD cell lines and tissues. Using the CRISPR-dCas9-Dnmt3a system, we investigated the effects of up-regulating methylation of CAP1 promoter on biological characteristics of LUAD cells. In addition, we explored the factors regulating methylation levels of CAP1 gene in LUAD cells.

Materials and methods

Clinical specimens

Six pairs of LUAD tumor tissues and corresponding adjacent normal lung tissues were collected from patients who underwent pulmonary surgery at the Ningbo No.2 Hospital (Ningbo, China) from January to August 2019. Patient demographics and clinicopathological information are shown in supplemental Table S1. The pathology of all the LUAD patients was confirmed by the pathologists of the hospitals; the pathological staging was determined according to the American Joint Committee on Cancer TNM staging system (8th edition). The study design was evaluated and approved by the Ethics Committee of the Shanghai Tenth People's Hospital. All experiments were performed following the regulations of the Ethics Committee of the Shanghai Tenth People’s Hospital (SHDSYY-2022-4308). Written informed consent was obtained from all the patients or their relatives.

Cell lines and cell culture

Human LUAD cell lines H1299, A549, PC9 and human normal lung epithelial cells Beas-2B as well as human embryonic kidney cells 293 T were from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Beas-2B and 293 T cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco; Thermo Fisher Scientific, USA), H1299, A549 and PC9 cells in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, USA). All media were supplemented with 10% fetal bovine serum (FBS, Gibco; Thermo Fisher Scientific, USA) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, USA) and the cells were cultured at 37 °C in a humidified incubator with 5% CO₂.

Plasmid design and construction

The UBC-dCas9-Dnmt3a domain (red frame segment) (Fig. S1A) of Fuw-dCas9-Dnmt3a (Addgene plasmid: 84476) and the non-EF1-Cas9 domain (red frame segment) (Fig. S1B) of lentiCas9-Blast (Addgene plasmid: 52962) were amplified by using polymerase chain reaction (PCR), respectively. These two amplification products were connected with DNA ligase to build a new plasmid named pdCas9-Dnmt3a-BSD. Five pairs of single guide RNA (sgRNA) primers targeting CAP1 promoter were designed and labeled as sg-CAP1 − 1, − 2, − 3, − 4, − 5, respectively. A pair of out-of-order sgRNA were designed and labeled as sg-control. These 6 pairs of primers were inserted into the BsmBI position of plasmid lentiGuide-Puro (Addgene plasmid: 52963), respectively. All selected products were sequenced before transfection. Primers information was listed in supplemental Table S2. Plasmid Fuw-dCas9-Dnmt3a, lentiCas9-Blast and lentiGuide-Puro were all from Addgene.

Stable cell line generation

Our new plasmid pdCas9-Dnmt3a-BSD and the package vector plasmid (pMD2.G and psPAX2) were transfected into 293 T cells with PEI transfection reagent. Next, the 293 T cells were cultured for 48 h (h), the supernatant culture medium was collected, and the cell debris was filtered out. These packaged viruses containing dCas9-Dnmt3a structure were respectively transfected into H1299 and A549 cells using polybrene reagent. Stably expressing dcas9-Dnmt3a cells were selected with blasticidin (10 µg/ml) for about 10 days. And then the sgRNA plasmids were packaged into virus and transfected into stably expressing dcas9-Dnmt3a cell strain using polybrene reagent. After about 10 days of treatment with puro (10 µg/ml), the untransfected cells sgRNA plasmid died out and the surviving cells were successfully transfected with sgRNA plasmid. These cells were then divided into individual cell for amplification, and the amplified cells were detected for methylation of CAP1 promoter by Mass-array sequencing. Thus, we obtained the stable cell lines that could up-regulate the methylation level of CAP1 promoter.

Immunohistochemistry

Fresh tissue specimens were fixed in 4% paraformaldehyde, and then were dehydrated in increasing concentrations of isopropyl alcohol, followed by clearing of alcohol by xylene. The specimens were subsequently embedded in paraffin wax. The paraffin-embedded tissue was sectioned into 4 μm slides, and each slide was stained with hematoxylin and eosin standard staining (H&E). For immunohistochemistry, tissue sections were deparaffinized and incubated in citrate buffer at 95 °C for 40 min for antigen retrieval and then incubated overnight at 4 °C with the primary antibodies including anti-CAP1 (1:100 dilution). After three times washing, tissue sections were incubated with biotinylated anti-mouse IgG (1:200 dilution) for 1 h at room temperature and then washed three times, after which streptavidin–horseradish peroxidase conjugates were added and the slides incubated for 45 min. After three washes with PBS, DAB solution was added and the slides were counterstained with haematoxylin. Negative controls were treated in the same way except without adding the primary antibodies. Immunohistochemistry staining was assessed by independent pathologists. The staining extent score was on a scale of 0–3, corresponding to the percentage of immunoreactive tumor cells (0–10%, 11–25%, 26–75% and 76–100%, respectively) and the staining intensity (negative, score = 0; weak, score = 1; strong, score = 2; very strong, score = 3). A score ranging from 0–3 was calculated by multiplying the staining extent score with the intensity score, resulting in a low (0–1) level or a high (2–3) level value for each specimen.

Polymerase chain reaction (PCR)

A 20ul PCR reaction system was prepared with a cDNA template, primers, 2 × Taq PCR Mix (TIANGEN, China) and deionized water. Then the PCR reaction conditions were set: step 1: 94 °C for 3 min; step 2: 94 °C for 30 s, 55 °C for 30 s, 72 °C for 3 min, 30 cycles in total; step 3: 72 °C for 5 min. The PCR products were detected by 1.5% agarose gel electrophoresis. Primer sequences are listed in supplemental Table S2.

RNA extraction and quantitative real-time polymerase chain reaction (qPCR)

The total RNA was extracted from cells or frozen human tissues using RNA Easy Fast tissue/cell Kit (TIANGEN, China) according to the instructions. The concentration and purity of RNA samples were measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). The reverse transcription of RNA was performed using cDNA kit (Vazyme Biotech, China). The qPCR was performed using SYBR Green PCR kits (Vazyme Biotech, China) on the ABI Prism 7500 sequence detection system (Applied Biosystems, USA) and the CT values were determined. All primer sequences are listed in supplemental Table S2. The relative expression of mRNA were calculated using the 2-ΔΔCt method; GAPDH was used as internal standards.

Methylation specific PCR (MSP)

The extracted genomic DNA needs to be transformed by bisulfite before MSP. The DNA disulfite conversion was performed according to the instructions of the DNA Bisulfite Conversion Kit (TIANGEN, China). MSP was performed according to the instructions of the methylation-specific PCR kit (TIANGEN, China). The templates were the product of DNA disulfide conversion. Primer sequences are listed in supplemental Table S2.

Western blotting analysis

Proteins were extracted by lysing cells on ice with RIPA buffer (Beyotime, China) containing protease inhibitors. The protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, USA). Protein lysates (50 µg/lane) were separated using 10% sodium dodecyl sulfate–polyacrylamide gels electrophoresis and transferred onto polyvinylidene fluoride membranes (Merck, USA). The membranes were subsequently blocked for using 5% skim milk for 1 h and incubated with primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with respective secondary antibodies (mouse or rabbit) at room temperature for 1 h. After washing thrice with PBST, the signals were detected on a Tanon (Shanghai, China) chemiluminescence image analysis system. All antibodies used against the proteins are listed in supplemental Table S3.

Cell proliferation assay

Cell proliferation assay was performed with Cell Counting Kit-8 (Corning, USA) abiding by the manufacturer’s protocols. Briefly, 1000 cells were seeded onto 96-well plates (Corning, USA) and allowed to grow for 24 h, 48 h, 72 h and 96 h (or no 96 h). And then, 10 µl of CCK8 solution (Yeasen, China) and 100 µl of serum-free medium were added to each well and incubated at 37 °C in dark for 2 h. The optical density values were determined at 450 nm by using a multifunctional microplate detector (Molecular Devices, USA).

Wound healing assay

Cells (wild type and transfected H1299 or A549) were seeded in triplicate onto 6-well plates (Corning, USA). After the cells were attached to the surface and reached about 90% confluence, the monolayers were scratched using a 200 μL pipette; cell debris was removed by washing thrice with 1 × PBS. Subsequently, cells were provided with medium supplemented with 1% FBS. Images of cell migration were captured at the same locations at 0 h and 24 h after injury, and the wound area was estimated using the ImageJ software (NIH, USA).

Colony formation assay

Cells were seeded at a density of 500 cells/well onto 6-well plates (Corning, USA) and cultured in complete media for approximately 12 days; cells were replenished with fresh media every three days. And then, the colonies were fixed using formaldehyde and then stained with 0.1% crystal violet (Vicmed, China). These colonies were subsequently photographed and counted.

Flow cytometry assay

Cell apoptosis was measured by flow cytometry assay using the Annexin V-EGFP/PI Apoptosis Detection Kit (KeyGEN BioTECH, China) according to the manufacturer’s instructions. Briefly, cells were cultured into 6-well plates at a density of 1 × 10⁴/well, and 5 μM/ml of apoptosis promoter cisplatin (Beyotime BioTECH, China) was added to each well. After incubation for 24 h, cells were washed twice with cold 1 × PBS and suspended in Annexin V binding buffer. The cells were then stained for 15 min using fluorescein isothiocyanate and propidium iodide at room temperature in the dark. Finally, a BD FACS Calibur (Beckman Coulter, CA, USA) was used to detect the apoptosis rate.

Trans-well invasion and migration assays

The invasion and migration ability of the cells were assayed using trans-well chambers (8 μm pore size, Corning, USA) pre-covered or uncovered with Matrigel (BD Biosciences, USA), respectively. Specifically, transfected cells (5 × 10⁴) were inoculated in the upper chamber and a culture medium supplemented with 10% FBS was provided in the lower chamber. After 24 h of incubation, the invading and migrating cells were fixed, stained with 0.1% crystal violet (Vicmed, China), and photographed and counted using an inverted microscope.

Mass-array methylation sequencing

The methylation detection of cytosine phosphate-guanosine (CpG) islands was performed by Mass-array methylation sequencing provided by the Oebiotech technology Co., LTD (Shanghai, China). The procedure of Mass-array methylation detection was summarized as follows: CpG islands of CAP1 were divided into two parts to design and synthesize primers, respectively. DNA was extracted from cells or tissues and tested for its concentration and purity. The DNA samples to be detected were treated with NaHSO₃ and amplified by PCR. The PCR products were detected by 1.5% agarose gel electrophoresis. The qualified PCR products will be continued for subsequent experiments: SAP reaction, T cleavage transcription/RNase A digestion reaction and resin purification. The purified product will be detected by a MassARRAY NanodispenserRS1000 apparatus. Finally, the test data was obtained with EpiTYPER™ software. Primer sequences are listed in supplemental Table S2.

Statistical analysis

The data were analyzed by SPSS software (Version 24.0, SPSS, Inc., Chicago, IL, USA) and GraphPad Prism software (Version 8.3, GraphPad Prism Software Inc., San Diego, CA), and a P value < 0.05 was considered significant. All continuous data are presented as the means ± standard deviation (SD).

Results

CAP1 gene is highly expressed at nucleic acid level and its promoter is abnormally hypo-methylated in LUAD cells

We detected the expression of CAP1 gene at the nucleic acid level in LUAD cells H1299, A549 and PC9, and normal lung epithelial cells Beas-2B. Total RNA of H1299, A549, PC9 and Beas-2B were extracted respectively, and reverse transcribed into cDNA. These reverse transcription products were used as templates for PCR and qPCR. The results of PCR showed that the expressions of CAP1 in A549, H1299 and PC-9 were higher than that in Beas-2B (Fig. 1A). The qPCR could more accurately detect the expression of CAP1 in these four cells. The result of qPCR showed that the expression of CAP1 in A549, H1299 and PC-9 was higher than that in Beas-2B (P < 0.05) (Fig. 1B).

Fig. 1.

CAP1 is highly expressed and hypo-methylated in LUAD cells. A Comparing the nucleic acid expression of CAP1 in A549, H1299, PC-9 and Beas-2B via agarose gel electrophoresis. B Comparing the nucleic acid relative expression of CAP1 in A549, H1299, PC-9 and Beas-2B by qPCR. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). C The blue areas indicated by the arrow are the two CpG islands of the CAP1 promoter. CpG island-1 runs from the 101 st to the 538th base (relative to the sequences initiation site). CpG island-2 runs from the 582nd to the 905th base (relative to the sequences initiation site). D Using MSP to detect the methylation of CpG island-1 in H1299, A549, PC9 and Beas-2B cells. M: methylated; U: un-methylated. E Lollipop chart shows MassARRAY methylation sequencing of CpG island-1 in Beas-2B and H1299 cells. Note 1: Each dot represents the position of the corresponding CpG site, and the color of the dot represents the methylation level of CpG. The methylation level goes from 0 to 1. Methylation level = methylated/(methylated + un-methylated) 100%. Gray indicates that it cannot be detected. F Methylation levels of individual CpGs of CpG island-1 in Beas-2B and H1299 cells. Shown is the mean percentage ± SD of two cells. G Lollipop chart shows MassARRAY methylation sequencing of CpG island-2 in Beas-2B and H1299 cells. The Note is the same as Note 1. H Methylation levels of individual CpGs of CpG island-2 of CAP1 in Beas-2B and H1299 cells. Shown is the mean percentage ± SD of two cells

Gene expression at the nucleic acid level is considered to be regulated by the promoter, and the methylation status of CpG islands in the promoter is an important factor regulating gene expression. Using http://switchgeargenomics.com/ products, we determined the region of CAP1 promoter, which contained 1011 bases. According to the CpG island criteria: CpG island span > 300 bp, GC content > 50%, and CpG frequency > 0.6, we confirmed that there are two CpG islands in CAP1 promoter (Fig. 1C). Table S4 shows the sequences of CAP1 promoter and CpG islands sequences marked by underline. The CAP1 promoter has two CpG islands, which are named CpG island-1 and CpG island-2, respectively. We firstly used MSP to detect the methylation status of CpG island-1 of CAP1 promoter in H1299, A549, PC9 and Beas-2B cell. The result showed that the methylation degree of CpG island-1 was lower in H1299 and A549 cells compared to Beas-2B control cells (Fig. 1D).

In order to further understand the methylation status of individual CpGs of CpG islands in CAP1 promoter, we extracted DNA from H1299 and Beas-2B cells, and performed methylation sequencing of promoters respectively using MassARRAY methylation sequencing technology. There are 45 CpGs could be detected, including 33 CpGs in CpG Island-1 and 12 CpGs in CpG Island-2. The sequencing results showed that the methylation at CpG sites 5, 11, 12, 14, 15, 19 and 22 (relative to the initiation site of CpG island-1) of CpG island-1 in H1299 cells were lower than that in Beas-2B cells (P < 0.05) (Fig. 1E, F). The methylation at CpG site 12 (relative to the initiation site of CpG island-2) of CpG island-2 in H1299 cells was lower than that in Beas-2B cells (P < 0.05) (Fig. 1G, H).

These results indicate that the methylation degree of CAP1 promoter is lower in several LUAD cell lines than in Beas-2B control cells. This hypo-methylation of CAP1 promoter should be abnormal and may be closely related to its high expression at nucleic acid level in LUAD cell lines.

CAP1 protein is highly expressed and its promoter is abnormally hypo-methylated in early stage LUAD tissues

We also detected the expression of CAP1 protein in cancerous tissues and para-carcinoma tissues of patients with early stage LUAD by immunohistochemistry. The results showed that the expression of CAP1 protein was strong or medium positive in cancerous tissues and weakly positive in para-carcinoma tissues (Figs. 2A, S1C). There was significant difference in the expression of CAP1 protein between cancerous tissues and para-carcinoma tissues (P < 0.05) (Table S5).

Fig. 2.

CAP1 is highly expressed and hypo-methylated in early stage LUAD tissues. A Immunohistochemistry staining of CAP1 in human early stage LUAD tissues. Scale bar = 200 μm for 5 × and 25 μm for 40 ×. B Using MSP to detect the methylation of CpG island-1 in early stage LUAD tissues. M: methylated; U: un-methylated. C The gray values of B by ImageJ (*P < 0.05). D Lollipop chart shows MassARRAY methylation sequencing of CpG island-1 in early stage LUAD tissues. The Note is the same as Note 1. E Methylation levels of individual CpGs of CpG island-1 in early stage LUAD tissues. Shown is the mean percentage ± SD of two tissues. F Lollipop chart shows MassARRAY methylation sequencing of CpG island-2 in early stage LUAD tissues. The Note is the same as Note 1. G Methylation levels of individual CpGs of CpG island-2 in early stage LUAD tissues. Shown is the mean percentage ± SD of two tissues

We extracted genomic DNA from cancerous and para-carcinoma tissues of three patients, and detected the methylation levels of CpG island-1 of CAP1 by using MSP. The result showed that the methylation level of CpG island-1 in cancerous tissues was significantly lower than that in para-carcinoma tissues (P < 0.05) (Fig. 2B, C).

We also examined the global methylation status of CpG islands in CAP1 promoter in cancerous and para-carcinoma tissues of early stage LUAD patients. We extracted genomic DNA from cancerous and para-carcinoma tissues of the above three patients. The genomic DNA was used to detect the methylation level of individual CpGs of CpG islands in CAP1 promoter by using MassARRAY methylation sequencing. The results showed that the methylation at CpG sites 1, 14, 15, 18, 19, 22, 24, 25 and 31 (relative to the CpG island-1 initiation site) of CpG island-1 in cancerous tissues were lower than that in para-carcinoma tissues (P < 0.05) (Fig. 2D, E). There was no significant difference in the methylation of CpG island-2 between cancerous and para-cancer tissues (Fig. 2F, G).

These results indicate that the methylation degree of CAP1 promoter in cancerous tissues is lower than in para-carcinoma tissues in early stage LUAD. This hypo-methylation of CAP1 promoter should be abnormal and may be closely related to its high expression at protein level in early stage LUAD.

Construct stable transfected LUAD cell strains up-regulating methylation of CAP1 promoter

Since the promoter of CAP1 is abnormally hypo-methylated in LUAD cell lines and human LUAD tissues, we will investigate the effects of up-regulating methylation of CAP1 promoter on biological characteristics of LUAD cells. Liu et al. used lentiviral plasmid CRISPR-dCas9-Dnmt3a named Fuw-dCas9-Dnmt3a to target specific CpGs of target gene and successfully increased the methylation levels of these CpGs. [24] We will also target the CAP1 promoter with this CRISPR-dCas9-dnmt3a methylation editing system. Figure 3A and B show a pattern of targeting the CAP1 promoter and reducing its expression using CRISPR-dCas9-Dnmt3a system. Since Liu’s CRISPR-dCas9-Dnmt3a system [25] lacks anti-blasticidin sequences for constructing stably transfected LUAD cell strains, we need to construct a new lentiviral plasmid containing dCas9-Dnmt3a and anti-blasticidin sequences. The BSD sequences of lentiviral plasmid lentiCas9-Blast was resistant to blasticidin. We constructed new lentiviral plasmids by linking part of the lentivirus plasmid Fuw-dCas9-Dnmt3a (red frame segment) (Fig. S1A) with part of the lentivirus plasmid lentiCas9-Blast (red frame segment) (Fig. S1B) through seamless cloning experiments. Fig. S1D shows a simple structure of the new lentiviral plasmids containing dCas9-Dnmt3a and BSD sequences. We first screened seven potential dCas9 expression plasmids from these new plasmids by PCR. The seven plasmids, as well as the plasmid Fuw-dCas9-Dnmt3a and blank plasmid were transfected into 293 T cells, respectively. The plasmid Fuw-dCas9-Dnmt3a was the positive control, and the blank plasmid was the negative control. Total proteins of 293 T cells transfected with these plasmids were extracted for western blotting analysis, respectively. As shown in Fig. S1E, plasmid 4 expressed dCas9 protein. This plasmid was then sequenced, and the results showed that its sequences contained dCas9-Dnmt3a and BSD sequences. We named this plasmid pdCas9-Dnmt3a-BSD. This plasmid was used to construct H1299 cell strain that could stably express dCas9, and the cell strain was named H1299-dCas9 cell strain. Subsequently, we respectively transferred five sg-CAP1 plasmids into H1299-dCas9 cell strain, and screened out H1299-dCas9 cell strain that could up-regulate the methylation level of CAP1 promoter. The sg-control plasmid was also transferred into H1299-dCas9 cell strain as a control cell strain, which was named H1299-dCas9-sg-control cell strain. Fig. S1F showed that the nucleic acid expression of CAP1 was decreased in H1299-dCas9 cells transfected with plasmid sg-CAP1 − 4 or − 5. Fig. S1G showed that the protein expression of CAP1 was also decreased in H1299-dCas9 cells transfected with plasmid sg-CAP1 − 4 or − 5. In order to obtain stable experimental results, monoclonal cell screening was performed in H1299-dCas9 cell strain transfected with plasmid sg-CAP1 − 4 or − 5. We amplified these monoclonal cells and selected a monoclonal cell strain with reduced CAP1 expression. This cell strain was named H1299-dCas9-sg-CAP1 cell strain and was used in subsequent experiments. By the same method, we obtained A549-dCas9-sg-CAP1 cell strain and A549-dCas9-sg-control cell strain.

Fig. 3.

Construct stable transfected LUAD cell strains up-regulating methylation of CAP1 promoter. A Upper panel: schematic representation of a catalytic inactive mutant Cas9 (dCas9) fused with Dnmt3a for de novo methylation of specific sequences. Lower panel: a dCas9-Dnmt3a construct and a guide RNA construct with puro cassettes. B Schematic representation of targeting the CAP1 promoter by dCas9-Dnmt3a with sgRNA to up-regulate CAP1 gene methylation and inhibit expression of CAP1 protein. C, D The expression of CAP1 protein in H1299-dCas9-sg-CAP1 cell strain and H1299-dCas9-sg-control cell strain. (*P < 0.05, **P < 0.01). E, F The expression of CAP1 protein in A549-dCas9-sg-CAP1 cell strain and A549-dCas9-sg-control cell strain. (*P < 0.05, **P < 0.01). G Lollipop chart shows MassARRAY methylation sequencing of CpG island-1 in H1299-dCas9-sg-CAP1 cell strain and H1299-dCas9-sg-control cell strain. The Note is the same as Note 1. H Methylation levels of individual CpGs of CpG island-1 in H1299-dCas9-sg-CAP1 cell strain and H1299-dCas9-sg-control cell strain. Shown is the mean percentage ± SD of two cells. I Lollipop chart shows MassARRAY methylation sequencing of CpG island-2 in H1299-dCas9-sg-CAP1 cell strain and H1299-dCas9-sg-control cell strain. The Note is the same as Note 1. J Methylation levels of individual CpGs of CpG island-2 in H1299-dCas9-sg-CAP1 cell strain and H1299-dCas9-sg-control cell strain. Shown is the mean percentage ± SD of two cells

Next, we examined the expression of CAP1 protein in H1299-dCas9-sg-CAP1 cell strain. The results showed that the expression of CAP1 protein in H1299-dCas9-sg-CAP1 cell strain was significantly lower than that in H1299-dCas9-sg-control cell strain (P < 0.05) (Fig. 3C, D). Similarly, the expression of CAP1 protein in A549-dCas9-sg-CAP1 cell strain was significantly lower than that in A549-dCas9-sg-control cell strain (P < 0.05) (Fig. 3E, F). Subsequently, we examined methylation levels of CpG sites of CpG islands of CAP1 promoter in H1299-dCas9-sg-CAP1 cell strain and control cell strain by MassARRAY methylation sequencing. The result showed that the methylation degree at CpG sites 1, 18, 19, and 22 (relative to the CpG island-1 initiation site) of the CpG island-1 in CAP1 promoter was significantly up-regulated in H1299-dCas9-sg-CAP1 cell strain compared to the control cell strain (Fig. 3G, H). The methylation degree of CpG sites of CpG island-2 in CAP1 promoter was not significantly up-regulated in H1299-dCas9-sg-CAP1 cell strain compared to the control cell strain (Figs. 3I, J).

In this section, we have constructed H1299-dCas9-sg-CAP1 and A549-dCas9-sg-CAP1 cell strains to up-regulate methylation degree of CAP1 promoter. Both H1299-dCas9-sg-CAP1 and A549-dCas9-sg-CAP1 cell strains can reduce the expression of CAP1 protein.

Effects of up-regulating methylation of CAP1 promoter on biological characteristics of LUAD cells

Cell proliferation

Cell proliferation assay and colony formation assay were used to examine the effect of up-regulating methylation of CAP1 promoter on proliferation capacity of LUAD cells. The result of cell proliferation assay showed that the proliferation curve of the H1299-dCas9-sg-CAP1 cell strain was decreased compared with H1299-dCas9-sg-control cell strain (Fig. 4A). The proliferation curve of the H1299-dCas9-sg-CAP1 cell strain was also decreased compared with A549-dCas9-sg-control cell strain (Fig. 4B). The results of colony formation assay showed that the numbers of clones of the H1299-dCas9-sg-CAP1 cell strain and A549-dCas9-sg-CAP1 cell strain were significantly reduced compared with their respective control cell strain (Figs. 4C–E). These results indicate that up-regulating methylation of CAP1 promoter can inhibit the proliferation of LUAD cells.

Fig. 4.

Up-regulating methylation of CAP1 promoter inhibited proliferation and promoted apoptosis of LUAD cells. A Growth curves for 96 h were measured by CCK-8 in H1299-dCas9-sg-CAP1 cell strain and H1299-dCas9-sg-control cell strain. (*P < 0.05, **P < 0.01). B Growth curves for 72 h were measured by CCK-8 in A549-dCas9-sg-CAP1 cell strain and A549-dCas9-sg-control cell strain. (*P < 0.05, **P < 0.01). C Colony formation assays were performed to detect cell apoptosis in H1299-dCas9-sg-CAP1 cell strain and H1299-dCas9-sg-control cell strain. D Colony formation assays were performed to detect cell apoptosis in A549-dCas9-sg-CAP1 cell strain and A549-dCas9-sg-control cell strain. E Relative colony number in C (***P < 0.001) and D (**P < 0.01). F Flow cytometry assays were performed to detect cell apoptosis in H1299-dCas9-sg-CAP1 cell strain and H1299-dCas9-sg-control cell strain. G Flow cytometry assays were performed to detect cell apoptosis in A549-dCas9-sg-CAP1 cell strain and A549-dCas9-sg-control cell strain. H Apoptosis rate in F (*P < 0.05) and G (*P < 0.05)

Cell apoptosis

Flow cytometry assay was used to detect the effect of up-regulating methylation of CAP1 promoter on apoptosis of LUAD cells. As illustrated in the Fig. 4F and G, the early apoptotic rate is in the Q3 area, while the late apoptosis rate is in the Q2 area. The total apoptosis rate is the early apoptotic rate plus the late apoptosis rate. As is shown, the total apoptosis rate of H1299-dCas9-sg-CAP1 cell strain was higher than that of H1299-dCas9-sg-control cell strain (P < 0.05) (Fig. 4F, H). The total apoptosis rate of A549-dCas9-sg-CAP1 cell strain was higher than that of A549-dCas9-sg-control cell strain (P < 0.05) (Fig. 4G, H). These results indicate that up-regulating methylation of CAP1 promoter can promote apoptosis of LUAD cells.

Cell migration on horizontal direction

Wound healing assay was used to detect the effect of up-regulating methylation of CAP1 promoter on horizontal migration ability of LUAD cells. The results showed that the migration distances of H1299-dCas9-sg-CAP1 cell strain and A549-dCas9-sg-CAP1 cell strain were significantly reduced compared with their respective control cell strain (Fig. 5A–C). The results indicate that up-regulating methylation of CAP1 promoter can significantly attenuate the horizontal migration ability of H1299 cells and A549 cells.

Fig. 5.

Up-regulating methylation of CAP1 promoter inhibited migration and invasion ability of LUAD cells. A Wound healing assays were performed to detect cell migration ability in H1299-dCas9-sg-CAP1 cell strain and H1299-dCas9-sg-control cell strain. B Wound healing assays were performed to detect cell migration ability in A549-dCas9-sg-CAP1 cell strain and A549-dCas9-sg-control cell strain. C Cell migration area in A (**P < 0.01) and B (***P < 0.001). Cell migration rate = (0 h wound area–24 h wound area)/0 h wound area﹡100%. D, E Transwell migration assays were performed to detect cell migration ability in vertical direction (Upper panel). Transwell invasion assays were performed to detect cell invasion ability (Lower panel). F Cell migration number in H1299-dCas9-sg-CAP1 cell strain and H1299-dCas9-sg-control cell strain (**P < 0.01), and in A549-dCas9-sg-CAP1 cell strain and A549-dCas9-sg-control cell strain (**P < 0.01). G Cell invasion number in H1299-dCas9-sg-CAP1 cell strain and H1299-dCas9-sg-control cell strain (**P < 0.01), and in A549-dCas9-sg-CAP1 cell strain and A549-dCas9-sg-control cell strain (**P < 0.01)

Cell migration on vertical direction and invasion

Transwell migration and invasion assays were used to detect the effect of up-regulating methylation of CAP1 promoter on the migration and invasion ability of H1299 and A549 cells in the vertical direction. The results showed that the migration numbers of H1299-dCas9-sg-CAP1 cell strain and A549-dCas9-sg-CAP1 cell strain were significantly reduced compared with their respective control cell strain (P < 0.05) (Figs. 5D–F). The invasion numbers of H1299-dCas9-sg-CAP1 cell strain and A549-dCas9-sg-CAP1 cell strain were significantly reduced compared with their respective control cell strain (P < 0.05) (Figs. 5D, E, G).

These results indicated that up-regulating methylation of CAP1 promoter can attenuate the proliferation, migration and invasion ability of LUAD cells, and promote the apoptosis of LUAD cells.

Mechanism of CAP1 methylation affecting apoptosis of LUAD cells

The western blot assay was used to study the mechanism of CAP1 methylation affecting apoptosis of LUAD cells. Bax, Bcl-2, Cleaved-Caspase-3, and Caspase-3 are several critical proteins in the apoptotic pathway. The Bax is a promoting apoptotic protein and Bcl-2 is an anti-apoptotic protein. The ratio of Bax to Bcl-2 is a key factor in the regulation of apoptosis [25]. Caspase-3 is the most dominant terminal cleaved enzyme during apoptosis, Cleaved-Caspase-3 is its activated form [25]. The ratio of Cleaved-Caspase-3 to Caspase-3 is an important indicator of cell apoptosis. The western blotting analysis showed that the expression of CAP1 protein in H1299-dCas9-sg-CAP1 cell strain was decreased (Fig. 6A, B), and the ratios of Bax to Bcl-2 (Fig. 6A, C) and Cleaved-Caspase 3 to Caspase-3 (Fig. 6A, D) were increased compared with H1299-dCas9-sg-control cell strain. Similarly, the expression of CAP1 protein in A549-dCas9-sg-CAP1 cell strain was decreased (Fig. 6E, F), and the ratios of Bax to Bcl-2 (Fig. 6E, G) and Cleaved-Caspase 3 to Caspase-3 (Fig. 6E, H) were increased compared with A549-dCas9-sg-control cell strain.

Fig. 6.

Mechanisms of CAP1 methylation on apoptosis, migration and invasion. A The expression of CAP1, Bax, BCL-2, Cleaved-Caspase-3 and Caspase-3 in H1299-dCas9-sg-CAP1 cell strain and H1299-dCas9-sg-control cell strain. B Relative expression of CAP1 (**P < 0.01). C The ratio of Bax/Bcl-2 (**P < 0.01). D The ratio of Cleaved-Caspase-3/Caspase-3 (*P < 0.05). E The expression of CAP1, Bax, BCL-2, Cleaved-Caspase-3 and Caspase-3 in A549-dCas9-sg-CAP1 cell strain and A549-dCas9-sg-control cell strain. F Relative expression of CAP1 (***P < 0.01). G The ratio of Bax/Bcl-2(**P < 0.01). H The ratio of Cleaved-Caspase-3/Caspase-3 (*P < 0.05). I–L The expression of CAP1, Cofilin and total Actin in H1299-dCas9-sg-CAP1 cell strain and H1299-dCas9-sg-control cell strain. I Relative expression of CAP1 (*P < 0.05). J Relative expression of Cofilin (**P < 0.01). K Relative expression of total Actin (*P < 0.05). M–P The expression of CAP1, Cofilin and total Actin in A549-dCas9-sg-CAP1 cell strain and A549-dCas9-sg-control cell strain. I Relative expression of CAP1 (**P < 0.01). J Relative expression of Cofilin (*P < 0.05). K Relative expression of total Actin (*P < 0.05)

These results indicate that up-regulating methylation of CAP1 promoter can reduce the expression of CAP1 protein, inhibit the proliferation and promote the apoptosis of LUAD cells through Bax/Bcl-2/Caspase-3 signaling pathway.

Mechanism of CAP1 methylation affecting migration and invasion of LUAD cells

The western blot assay was used to investigate the mechanism of CAP1 methylation affecting migration and invasion of LUAD cells. The results showed that the expressions of CAP1, Cofilin and Actin in H1299-dCas9-sg-CAP1 cell strain were decreased compared with H1299-dCas9-sg-control cell strain (Fig. 6I–L). Similarly, we found that the expressions of CAP1, Cofilin and Actin in A549-dCas9-sg-CAP1 cell strain were decreased compared with A549-dCas9-sg-control cell strain (Fig. 6M–P).

These results suggest that up-regulation of CAP1 promoter methylation can reduce the expression of CAP1 and lead to decreased expression of Cofilin and Actin, thereby attenuating the migration and invasion ability of LUAD cells.

Regulatory factors of CAP1 methylation

Currently, it has been found that Dnmts can up-regulate methylation level of genes, while ten-eleven translocations (Tets) can down-regulate methylation level of genes. Dnmts and Tets can work together to maintain the dynamic balance of gene methylation. Through the Cancer Genome Atlas (TCGA) database, we found that the expressions of Dnmt3a (Fig. 7A, B) and Tet1 (Figs. 7C, D) were higher (P < 0.05) in tumor tissues than in normal tissues, and there was no statistically significant difference in Tet2 expression between tumor tissues and normal tissues (P > 0.05) (Figs. S1H, S1I). Decitabine is a strong Dnmts inhibitor that can down-regulate the methylation of genome. When H1299 cells were treated with different concentrations of Decitabine, the nucleic acid expression of CAP1 was up-regulated, and there was a dose–response relationship between the expression level and drug dose (Figs. 7E, F). This indicates that Dnmts could regulate the expression of CAP1. Moreover, we found that Dnmt3a can up-regulate the methylation level of the CAP1 promoter in H1299-dCas9-sg-CAP1 cell strain. These indicate that Dnmt3a can up-regulate the methylation level of CAP1 promoter.

Fig. 7.

Mechanisms regulating CAP1 methylation. A, B Difference box diagram (P = 3.901e-20) and pairwise difference analysis plot (***P < 0.001) show the expression of Dnmt3a in LUAD using TCGA database, respectively. C, D Difference box diagram (P = 1.163e-14) and pairwise difference analysis plot (***P < 0.001) show the expression of Tet1 in LUAD using TCGA database, respectively. E Nucleic acid expression of CAP1 treated with different concentrations of Descitabine by agarose gel electrophoresis in H1299 cells. F Relative expression of CAP1 treated with different concentrations of Descitabine by qPCR in H1299 cells (*P < 0.05, **P < 0.01, ***P < 0.001). G The inhibition rate of different concentrations of Bobcat339 on H1299-dCas9-sg-CAP1 cell strain and H1299-dCas9-sg-control cell strain. H, I The expression of CAP1 protein in H1299 cells and H1299 cells treated with Bobcat339 (60 μM) (**P < 0.01). J Growth curves for 96 h were measured by CCK-8 in H1299-dCas9-sg-CAP1 cell strain, H1299-dCas9-sg-CAP1 cell strain treated with Bobcat339 (60 μM), H1299-dCas9-sg-control cell strain and H1299-dCas9-sg-control cell strain treated with Bobcat339 (60 μM). (*P < 0.05, **P < 0.01)

Bobcat339 (MCE, China) is a potent and selective inhibitor of Tet enzymes that inhibit Tet1 and Tet2. Cytotoxicity assay was performed to test the relationship between drug concentration of Bobcat339 and inhibition rate in LUAD cells. The half maximum inhibitory concentration (IC50) is the concentration corresponding to 50% apoptosis of a tumor cell induced by a drug, and can be used to measure the ability of this drug to induce apoptosis. Figure 7G shows that Bobcat339 has an IC50 of about 63 μM in H1299-dCas9-sg-CAP1 cell strain, which is lower than that in H1299-dCas9-sg-control cell strain with an IC50 of about 72 μM. This suggests that the apoptosis of H1299 cells with up-regulated methylation of CAP1 promoter is more easily induced by Bobcat339. Then, we extracted total protein from H1299 cells with Bobcat339 intervention and performed western blot assay. The result shows that the expression of CAP1 protein was decreased in H1299 cells treated with Bobcat339 (Figs. 7H, I). This indicates that the inhibitors of Tet1 and Tet2 can reduce CAP1 expression by up-regulating the methylation level of the CAP1 promoter. In other words, Tet1 and/or Tet2 can increase CAP1 expression by down-regulating the methylation level of the CAP1 promoter. Subsequently, Bobcat339 was used for cell proliferation assay. As shown in Fig. 7J, the proliferation curves of H1299-dCas9-sg-control cell strain treated by Bobcat339, H1299-dCas9-sg-CAP1 cell strain treated by Bobcat339 and H1299-dCas9-sg-CAP1 cell strain were all decreased compared with H1299-dCas9-sg-control cell strain. Moreover, the proliferation curve of H1299-dCas9-sg-control cell strain treated with Bobcat339 was similar to that of H1299-dCas9-sg-CAP1 cell strain. These results suggest that Bobcat339 may inhibit the proliferation of H1299 cells partly by up-regulating methylation level of CAP1.

Discussion

Previous studies have found that CAP1 protein is an important functional protein and its expression in NSCLC tissues is higher than that in normal tissues [22, 26]. In this study, we found that the expression of CAP1 was higher in cancerous tissues when compared with para-carcinoma tissues in early stage LUAD, and higher in LUAD cell lines A549, H1299, and PC9 than in normal lung epithelial cell line Beas-2B. Studies have found that the DNA methylation of some genes has altered in the early stage of cancer, [27–29] and this change can alter its own expression [30, 31]. The methylation status of a gene’s promoter region controls the “on/off” of this gene. CpG islands are regions rich in cytosine-guanine dinucleotides (CG), which are usually present in the promoter of genes and involved in the regulation of gene expression [32]. In this study, we found that there are two CpG islands in the core region of the promoter of CAP1 gene. Further studies showed that some CpGs of CpG island-1 in CAP1 promoter occurred with lower methylation in H1299 cells than in Beas-2b cells. Meanwhile, some CpGs of CpG island-1 in CAP1 promoter also occurred lower methylated in cancerous tissues than in para-carcinoma tissues in early stage LUAD. Furthermore, the CpGs that undergo methylation changes in LUAD tissues partially overlap with those that undergo methylation changes in LUAD cell lines. These results suggest that lower methylation of these CpGs may be associated with higher expression of CAP1 in LUAD. This phenomenon of CAP1 gene has not been reported before.

The CRISPR-dCas9 methylation editing system is a technology that uses RNA-guided Cas nuclease to make specific DNA modification of targeted genes. [23]. In the CRISPR-dCas9 platform, dCas9 is mainly fused with other effector proteins for gene regulatory activation, [33] which provides an efficient tool for targeted gene modification such as DNA methylation, gene expression modulation, and epigenetic regulation [24, 34, 35]. Liu et al. reported that fusion of Tet1 or Dnmt3a with a dCas9 enables targeted DNA methylation editing [24]. They targeted the dCas9-Tet1 or -Dnmt3a fusion proteins to methylated or unmethylated promoter sequences, respectively, resulting in the activation or silencing of endogenous reporter. Owing to the hypo-methylation of CAP1 promoter in LUAD cells, we explored the effects of up-regulating methylation of CAP1 promoter on biological characteristics of LUAD cells. In our study, we used the CRISPR-dCas9-Dnmt3a system to target CAP1 promoter and constructed H1299 and A549 cell strains steadily up-regulating methylation of CAP1 promoter. According to methylation sequencing results, four CpGs of CpG island-1 in CAP1 promoter were significantly up-regulated in H1299-dCas9-sg-CAP1 cell strain compared with H1299-dCas9-sg-control cell strain. These four CpGs also occurred methylation changes in LUAD tissues. Therefore, we speculated that these four CpGs of CAP1 promoter were prone to methylation changes. Based on the results of western blot assays, we found that the expression of CAP1 protein decreased significantly in H1299-dCas9-sg-CAP1 cell strain. This result suggests that methylation changes in these four CpGs of CAP1 promoter have significant effects on their own expression.

Cell apoptosis is an autonomic and orderly process of cell death, which is controlled by multiple genes to maintain the stability of internal environment [36]. Currently, it has been found that there are three pathways involved in the occurrence of apoptosis, and mitochondria-mediated apoptosis pathway is a very important mechanism [37]. Previous studies have found that CAP1 promoted apoptosis through Caspase [38]. In this study, we found that up-regulating methylation of CAP1 promoter can increase apoptosis of LUAD cells. Further research showed that up-regulating methylation of CAP1 promoter can reduce its expression, increase the ratio of Bax/Bcl-2 and the ratio of Cleaved-Caspase-3/Caspase-3 in LUAD cells. The Bcl-2 protein family is the main component regulating mitochondrial permeability. There are many members of this family, including both pro-apoptotic and anti-apoptotic proteins, which can regulate apoptosis by forming homologous (Bax/Bax, Bcl-2/Bcl-2) or heterologous (Bax/Bcl-2) dimers [39]. Bax is a pro-apoptotic protein in the Bcl-2 family, which can destroy the permeability of mitochondrial outer membrane, migrate from cell fluid to mitochondria and nuclear membrane, and promote the release of apoptotic factors [40]. Bcl-2 is an anti-apoptotic protein that can inhibit apoptosis by maintaining the integrity of the mitochondrial membrane [41]. Under normal conditions, Caspase-3 in the cytoplasm is not active, but exists in the form of Pro-Caspase-3 [42]. When cells receive apoptotic stimulation, Pro-Caspase-3 is cleaved to Cleaved-Caspase-3 and activated, which induces cell apoptosis [43]. Previous study found that CAP1 promoted apoptosis through Caspase. Our results suggest that up-regulating methylation of CAP1 promoter can reduce its expression, and promote apoptosis of LUAD cells through Bax/Bcl-2/Caspase-3 signaling pathway.

The invasion and migration of tumor cells are important reasons for the poor prognosis of malignant tumors. Previous studies have found that CAP1 is related to the metastasis and invasion of tumor cells. [17–19, 21] In this study, we found that up-regulating methylation of CAP1 promoter can inhibit the migration and invasion ability of LUAD cells. Further research showed that up-regulating methylation of CAP1 promoter can reduce the expression of itself, and decrease the expression of Actin and Cofilin in LUAD cells. In recent years, Cofilin has been found to be closely related to tumor cell invasion and metastasis [11]. Cofilin is a binding protein of Actin, which has the ability to promote cell migration and movement [44]. It has been reported that Cofilin can promote cytoskeletal recombination and participate in the invasion and metastasis of malignant tumors by altering the conformation of Actin [45]. When Cofilin binds to Actin, it can change the cell migration ability by changing the conformation of Actin, causing it to separate and depolymerize, and changing the cytoskeletal conformation [46]. Studies have shown that CAP1 can coordinate with Cofilin to regulate the integration and depolymerization of G-Actin and F-Actin, with dual functions of signal transduction and maintenance of actin cytoskeleton [10, 14].

Therefore, our results suggest that up-regulating the methylation of CAP1 promoter can reduce the expression of CAP1, Cofilin and Actin proteins, thereby weakening the invasion and migration of LUAD cells.

In addition, we investigated the mechanisms that regulated methylation level of CAP1 promoter in LUAD cells. It has been found that Dnmts and Tets can regulate the methylation or demethylation of genes, respectively [47]. In this study, we found that Dnmt3a and Tet1 were highly expressed in tumor tissues and closely related to the occurrence of cancer by using the TCGA database. Dnmt3a, known as de novo methylase, can methylate unmethylated DNA strands [48]. Further studies showed that Dnmt3a could up-regulate the methylation level of the CAP1 promoter. Tet-mediated active DNA demethylation pathway is the only clear active DNA demethylation pathway at present [49]. In this study, we also found that the inhibitors of Tet1 and Tet2 can reduce CAP1 expression by up-regulating the methylation level of the CAP1 promoter. This in turn suggests that Tet1 and/or Tet2 can increase CAP1 expression by down-regulating the methylation level of the CAP1 promoter. Currently, Dnmts and Tets inhibitors have poor specificity in the treatment of cancer, and the potential cancer-promoting and side effects limit their clinical application. In this study, we found that Bobcat339 may inhibit the proliferation of H1299 cells partly by up-regulating methylation level of CAP1. Therefore, our study may provide a new idea for the treatment of LUAD by up-regulating methylation of CAP1 promoter.

Conclusion

In this study, we found that the relatively high expression of CAP1 protein is associated with hypo-methylation of its promoter in LUAD cell lines and tissues. Up-regulating methylation of CAP1 promoter can promote apoptosis and inhibit migration and invasion of LUAD cells. These results suggest that up-regulating methylation of CAP1 promoter may be a potential treatment for patients with early stage LUAD.

Abbreviations

CAP

Lung adenocarcinoma: LUAD; Adenylate cyclase associated protein

LC

Lung cancer

CRISPR

Clustered regularly interspaced short palindromic repeats

dCas9

Catalytically inactivated Cas9

Dnmt

DNA methyltransferase

PCR

Polymerase chain reaction

qPCR

Quantitative real-time polymerase chain reaction

MSP

Methylation specific PCR

H

Hours

CpG

Cytosine phosphate-guanosine

SD

Standard deviation

TCGA

The cancer genome atlas

IC50

Half maximum inhibitory concentration

Tet

Ten-eleven translocations

Author contributions

G.S.L., Y.L.G., Y.E.M., H.X.L., and C.H.W. performed the research and analyzed results. G.S.L., Y.L.G., S.S.X., and J.S.Z. wrote the paper. K.W. provided help for Dnmt3a, Tet1 and Tet2 expression analysis from TCGA database. J.Y.Z. provided LUAD tumor tissues. M.T., S.S.X., Y.E.M., and J.S.Z. provided critical comments. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No.82272673) and National clinical key specialty construction project of China(Z155080000004).

Availability of data and materials

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The study design was evaluated and approved by the Ethics Committee of the Shanghai Tenth People’s Hospital.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.