Epigenetic Silencing of TET1 Mediated Hydroxymethylation of Base Excision Repair Pathway During Lung Carcinogenesis

Backgroud: The methylcytosine dioxygenase Ten-eleven translocation 1 (TET1) is an important regulator for the balance of DNA methylation and hydroxymethylation through various pathways. Increasing evidence has suggested that TET1 probably involved in DNA methylation and demethylation dysregulation during chemical carcinogenesis. However, the role and mechanism of TET1 during lung cancer remains unclear. Methods: The gene and protein expression were detected by qRT-PCR, Western blot and immunohistochemistry during lung carcinogenesis. Methylation and hydroxymethylation status were evaluated by MeDIP-qPCR and hMeDIP-qPCR. The effect and mechanism of TET1 on lung cancer were identied both in vitro and in vivo models. Results: In this study, we found that TET1 expression was signicantly down-regulated and the methylation level was signicantly up-regulated in 3-MCA-induced cell malignant transformation model, rat chemical carcinogenesis model, and human lung cancer tissues. Demethylation experiment further conrmed that DNA methylation negatively regulated TET1 gene expression. TET1 overexpression inhibited cell proliferation, migration and invasion in vitro and in vivo, while knockdown of TET1 resulted in an opposite phenotype. DNA hydroxymethylation level in the promoter region of base excision repair (BER) pathway key genes XRCC1, OGG1, APEX1 signicantly decreased and the degree of methylation gradually increased in malignant transformed cells. After differential expression of TET1, the level of hydroxymethylation, methylation and expression of these genes also changed signicantly. Furthermore, TET1 binds to the promoter of XRCC1, OGG1, and APEX1 to maintain them hydroxymethylated. Blockade of BER pathway key gene alone or in combination signicantly diminished the effect of TET1. Conclusions: Our study demonstrated for the rst time that TET1 gene expression is regulated by DNA methylation and TET1-mediated hydroxymethylation regulates BER pathway to inhibit the proliferation, migration and invasion during the 3-MCA-induced lung carcinogenesis. These results

for early diagnosis and prevention of cancer, screening of susceptible populations and assessment of carcinogenic risk of environmental exposure [6][7][8].
DNA methylation is mainly 5-methylcytosine (5-mC), which strictly regulates gene expression in a dynamic equilibrium by DNMT1, DNMT3a, and DNMT3b in higher eukaryotic cells [9]. Recent studies have found Ten-eleven translocation (TET) family can catalyze the conversion of 5-mC to 5hydroxymethylcytosine (5-hmC), then thus regulated the balance of DNA methylation [10]. TET protein family consists of three members, TET1, TET2 and TET3, which belong to ketoglutarate and Fe 2+ dependent dioxygenase and can produce catalytic oxidation. TET1 and 5-hmC levels are signi cantly correlated with the occurrence, development and prognosis of human solid tumors [11]. However, the regulatory mechanism of TET1 gene and 5-hmC during lung cancer is still unknown. TET1 protein could oxidize 5-hydroxymethyl cytosine to 5-carboxyl cytosine, and that glycosidase TDG could speci cally recognize and remove 5-carboxyl cytosine from DNA, and then initiate base excision repair (BER) pathway to complete DNA demethylation [10]. OGG1, APEX1 and XRCC1 genes are mainly responsible for the process of removing damaged bases, producing single strand breaks and repairing and closing after base excision [12,13]. Reduced expression or functional de ciency of any of these genes may affect the e ciency of repairing DNA damage, lead to damage and increase the susceptibility of tumors. TET1-mediated demethylation requires the involvement of BER pathway in embryonic stem cells [14]. Inhibitor treatment of the key genes APEX1 and PARP1 of the BER pathway, or knockdown of TDG expression can block the demethylation process [15]. However, how TET1 regulates the coordination of BER pathway and participates in demethylation process during lung carcinogenesis remains unclear. In this study, we explored the epigenetic regulation, function and mechanism of TET1 gene and BER pathway during lung carcinogenesis.

Cell culture
The human bronchial epithelial cell line HBE and lung cancer cell lines A549, SPC-A-1, LTEP-a-2, NCI-A proper amount of cells or tissue samples were added to the RIPA protein lysate for lysis, and the protein was extracted after centrifugation at low temperature. Protein concentration was measured according to the instructions of BCA protein concentration test kit. After the preparation of SDS-PAGE gel, about 80 µg for each sample were taken to be electrophoretic and transferred to the PVDF membrane. The PVDF membrane was blocked with 5% skimmed milk powder and then incubated with primary antibodies at 4 ℃ overnight. The antibodies used in this study were anti-TET1 (1:1000; Sant Cruz, USA), anti-XRCC1 (1:800; Boster, China), anti-OGG1 (1:1000; Boster, China), anti-APEX1 (1:1000; Boster, China), anti-betaactin (1:1000; Beyotime, China). The membrane was washed with TBST buffer for 3 times, then incubated secondary antibody at room temperature, and nally detected by ECL chemiluminescent substrate (Thermo, USA).

Immunohistochemistry
Immunohistochemistry was performed on serial sections and used to detect the expression of TET1 protein expression using protocols previously described [19]. Thick sections with 4-µm thickness were incubated with anti-TET1 antibody. The immunohistochemical analyses were evaluated by two individuals who were blinded to the sample information. Nucleoplasm immunostaining was considered evidence of expression. Expression of the proteins was scored according to the staining intensity (0, negative; 1, weak; 2, moderate; and 3, strong) and the proportion of positive cells (0, negative; 1, positive in < 25%; 2, positive in 25-49%; 3, positive in 50-74%; 4, positive in ≥ 75% of cells). If the multiplication of the two scores were more than 6, the expression of TET1 was considered to be high expression.

Demethylation experiment
The P45 3-MCA treated HBE cells were inoculated into six-well plates at the concentration of 4 × 10 5 cells per well. Demethylation agent 5-aza-2'-deoxycytidine (5-aza-dC) (Sigma) with the concentration of 10 µM was used to continuously treat the cells in the six-well plates for 3 days. The cells were then collected and used to extract the DNA, RNA and protein. MeDIP-qPCR was used to detect the methylation level of TET1.
QRT-PCR and Western blot were used to detect the mRNA and protein expression of TET1. The analyses were repeated at least three times.

Cell transfection experiment
For overexpression, the cDNA of the human TET1 gene was cloned into the mammalian expression vector pCMV6-AC-GFP. For gene knockdown, TET1 shRNAs and a negative control shRNA were designed and synthesized, and then subcloned into shRNA-GFP vector. Cell transfection was performed as previously described [22]. Brie y, cells were seeded in plats and cultured for 24 hours. Then, the mixed system of ViaFect™ transfection reagent (Promega, USA), the target plasmid, Opti-MEM medium and complete medium were prepared and replaced the medium in the culture dishes. After 48 h of transfection, the uorescence of the cells was observed with an inverted uorescence microscope. Cells were obtained for the extraction of RNA and protein. The stably cells were screened by G418. Stable cells with TET1 overexpression or knockdown were con rmed by RT-PCR and Western blot.
Cell proliferation assay Cells were evenly seeded in 96-well plates and transfected after culture for 24 hours. The mixed system of CCK-8 solution and RPMI-1640 complete medium were prepared following the instructions of the CCK-8 kit (Dojindo, Japan). Then, the proliferation of cells was detected respectively, after transfected for 24 h, 48 h, 72 h and 96 h (named as D1, D2, D3 and D4). Finally, OD value was quanti ed by measuring the absorbance at 450 nm. All assays were performed at least three times in triplicate.

Clone formation experiment
Approximately 1000 cells transfected for 48 hours were seeded in six-well plates, and appropriate concentration of G418 was used to treat the cells for 14-21 days. When clones with a stable plasmid integrated into genomic DNA were formatted, 4% paraformaldehyde was used to x the cells and crystal violet staining solution (Sigma) was used to stain the clones. Clones contained more than 50 cells were considered to be positive clones. All assays were performed at least three times in triplicate.

Soft agar assay
Soft agar assay was performed as previously described [22]. Brie y, the lower layer agar and the upper layer agar were prepared in a 24-well plate. Cells were mixed with the upper layer agar, and incubate in a 37 °C cell incubator for 2-3 weeks. Clones that contained more than 50 cells were considered as positive clones and counted by phase-contrast microscopy. All assays were performed at least triplicate.

Scratch healing experiment
Cells were seeded in six-well plates, and transfected after cultured for 24 h. Toothpicks were used to scratch in the six-well plates after transfected for 24 h. After scratching for 0 h and 48 h, the same point of the scratched cells were took photos to observe the migration. Finally, the distance of cell migration was analyzed statistically. All assays were carried out at least three times in triplicate.

Transwell assay
Transwell assay was performed as previously described [22]. Brie y, approximately 2 × 10 4 cells that were transfected for 24 hours were seeded in the Transwell chamber (Corning, USA). Transwell chamber with or without Matrigel gel was used for the detection of cell invasion and cell migration, respectively. After culturing for another 24 h, the cells were xed by 4% paraformaldehyde at room temperature and stained by crystal violet staining solution for 30 min. The inverted microscope was used to take photos of the stained cells. Finally, ve different elds were randomly selected for analysis. All assays were performed at least three times in triplicate.

Nude mouse tumorigenesis experiment
All experimental procedures were approved by the Animal Ethics Committee of the Third Military Medical University. Nude mice with six-week-old were randomly divided into the experimental group and the control group with four animals in each group. Lung cancer cells with and without TET1 gene overexpression were harvested, and single-cell suspensions of 1 × 10 7 in 200 µL of phosphate-buffered saline were injected into the right abdominal wall of the nude mice and assigned to the experimental group and the control group, respectively. From the rst day of injection, the major diameter (D) and the minor diameter (d) of tumor volume were measured every 5 days with a vernier caliper. The formula (D × d 2 ) /2 was used to calculate the volume of the tumor. All mice were killed after one month. The tumors were surgically dissected, and were then embedded in para n for HE staining and immunohistochemistry analysis.

Methylated DNA immunopreciption (MeDIP) and hydroxymethylated DNA immunopreciption (hMeDIP)
According to the instructions of the EpiQuik™ methylated DNA immunoprecipitation kit (EpiGentek, USA) and EpiQuik™ hydroxymethylated DNA immunoprecipitation kit (EpiGentek, USA), MeDIP and hMeDIP were performed to detect the level of methylation and hydroxymethylation in the promoter of genes. Brie y, 1 µg of sonicated DNA was diluted and incubated with 150 µl of antibody buffer at room temperature for 90 minutes in strip wells. For MeDIP, normal mouse IgG as the negative control and anti-5-mC for samples were added in the antibody buffer. For hMeDIP, non-Immune IgG was added to the negative control well, anti-5-hmC antibody was added to the sample wells and the positive control wells. Next, the wells were washed once with TE buffer. DNA release buffer containing proteinase K was added to the samples followed by reverse buffer. At last, Filter column was used to elute puri ed DNA. DNA was analyzed by qRT-PCR, and primer sequences were provided in Supplementary Table S3. All analyses were performed at least triplicate and repeated three times.

Statistical analysis
Statistical analysis was performed using SPSS 19.0 software (SPSS, Inc., Chicago, IL). Measurement data were expressed as the mean value ± standard deviation (SD) and the signi cances among groups were analyzed using Student's t test (normal distribution) or F test. P values less than 0.05 were considered statistically signi cant.

TET1 expression was down-regulated during 3-MCA induced lung carcinogenesis and human tumor samples
Firstly, in order to detect the expression of TET1 gene during the process of chemical-induced lung carcinogenesis, TET1 expression of the 3-MCA-induced malignant transformation in HBE cell was detected by qRT-PCR (Fig. 1A) and Western blot (Fig. 1B). With the increase of 3-MCA treatment time, the mRNA and protein expression of TET1 gene gradually decreased signi cantly (P < 0.01). Then, qRT-PCR and Western blot was used to detect TET1 gene expression in the lung tissues of the 3-MCA-exposed rats in vivo. It was found that the mRNA (Fig. 1C) and protein (Fig. 1D) expression of TET1 gene was signi cantly down-regulated in precancerous tissues and cancer tissues compared to normal lung tissues in rats (P < 0.01). It further proved that 3-MCA exposure may result in down-regulation of TET1 gene. Next, the human lung cancer and adjacent normal tissue samples from lung cancer patients were used for immunostaining experiments. As shown in Fig. 1E and 1F, the expression level of TET1 gene in cancer tissues was signi cantly lower than that in adjacent normal lung tissues. These results suggested that TET1 gene may play an important role during the process of lung cancer.

DNA methylation regulated the expression of TET1 during lung carcinogenesis
In order to explore the epigenetic regulatory mechanism of TET1 gene, we rstly detected the methylation level of TET1 in 3-MCA-induced malignant transformation cells through MeDIP-qPCR. As shown in Fig. 2A, the methylation of TET1 in 3-MCA-exposed HBE cells was signi cantly up-regulation compared with the DMSO exposed HBE cells (P < 0.01). To demonstrate whether TET1 expression is regulated by DNA methylation, 3-MCA-exposed HBE cells were used for demethylation experiment. As shown in

Overexpression of TET1 inhibits cell proliferation, migration and invasion
To explore the role of TET1 gene during 3-MCA-induced lung cancer, the TET1 overexpression vector was constructed. We rstly examined TET1 mRNA expression in the lung cancer cell lines and normal HBE cell line by qRT-PCR. As shown in Supplementary Fig. 1, the TET1 transcript level was down-regulated in lung cancer cell lines, especially in SPC-A-1, but was readily expressed in the normal lung cell line HBE. Then, we chose SPC-A-1 with TET1 low expression as cell model for TET1 overexpression ( Fig. 3A and Supplementary Fig. 2). CCK-8 assay found that after over-expressing TET1, the cell growth rate was signi cantly inhibited (P < 0.01) (Fig. 3B). Next, soft agar experiments and colony formation experiments were used to further verify the effect of over-expressing TET1 on cell growth. The results showed that the colony forming ability of the cells was signi cantly lower than that of the control group after overexpressing TET1 (P < 0.01) (Fig. 3C and 3D). Then, scratch healing experiments were used to detect the effect of over-expressing TET1 on cell migration ability. As shown in Fig. 3E, compared with the control group, the migration distance of cells was signi cantly reduced after overexpression of TET1 (P < 0.01). At the same time, transwell was used to detect the migration and invasion ability of cells after overexpressing TET1. The results showed that the number of cells crossing the membrane was signi cantly lower in the over-expressed TET1 group than that in the control group (P < 0.01) (Fig. 3F). The results above suggest that overexpression of TET1 can inhibit cell growth, migration and invasion.

Knockdown of TET1 promotes cell proliferation, migration and invasion
To further demonstrate that TET1 can inhibit cell growth, migration and invasion, TET1 knockdown expression vector was constructed. TET1 overexpression stably transfected cells were used to knock down TET1 expression. The e ciency of knocking down TET1 expression was shown in Fig. 4A and Supplementary Fig. 3. After knocking down TET1 expression, TET1 expression was signi cantly lower than the control group (P < 0.01). CCK-8 assay, soft agar experiments and colony formation experiments detected the cell growth after knocking down TET1 expression, and found that after knocking down TET1 expression, cell growth was signi cantly promoted (P < 0.01) (Fig. 4B-4D). Next, scratch healing experiment found that the migration distance of cells with TET1 knockdown was signi cantly higher than that of the control group (P < 0.01) (Fig. 4E). What's more, transwell results showed that the migration and invasion of knock down TET1 expressing cells were signi cantly up-regulated (P < 0.01) (Fig. 4F). These results further suggested that TET1 expression can inhibit cell growth, migration and invasion of lung cancer cells.

Overexpression of TET1 inhibits cell proliferation in vivo
To verify the effect of TET1 on the growth of lung cancer cells in vivo, TET1 overexpression stably transfected cells were used in nude mice tumor formation experiments. After 30 days, obviously tumor mass was formed subcutaneously in nude mice. As shown in Figs. 5A and 5B, the volume of tumor mass formed in the over-expressed TET1 group was signi cantly lower than that of the control group (P < 0.01). What's more, the weight of the tumor mass in over-expressed TET1 group was signi cantly downregulated compared with the control group (Fig. 5C) (P < 0.01). These results suggested that the growth of over-expressed TET1 cells in vivo was signi cantly inhibited. In order to further demonstrate the effect of TET1 on lung cancer cells in vivo, TUNEL and Ki67 immunostainingin embedded sections of the nude mice tumors were performed. It can be found from the immunohistochemical results shown in Fig. 5D and 5E that the apoptosis was higher in the over-expressed TET1 group than that in the control group, and that the expression of Ki67 in the over-expressed TET1 group was lower than that in the control group. It reveals that over-expression of TET1 could promote the apoptosis and inhibit the proliferation of the lung cancer cells in vivo.

TET1 plays a tumor suppressing role through BER signaling pathway in lung cancer
To explore the mechanism of TET1 during the 3-MCA-induced lung cancer cells, some key genes of BER signaling pathway were detected by qRT-PCR and Western blot. As shown in Fig. 6A and 6C, the mRNA and protein expression of TET1 in the 3-MCA-exposed group were signi cantly lower than that in the control group (P < 0.01), meanwhile the mRNA and protein expression of OGG1, XRCC1, and APEX1 were signi cantly lower compared with the control group (P < 0.01). When TET1 was over-expressed, the mRNA and protein expression of OGG1, XRCC1, and APEX1 were signi cantly up-regulated ( Fig. 6B and 6D) (P < 0.01). When the expression of TET1 was knocked down, the mRNA and protein expression of OGG1, XRCC1, and APEX1 were signi cantly down-regulated ( Fig. 6B and 6D) (P < 0.01). These results suggested that TET1 function as a tumor suppressor gene through the BER signaling pathway. In order to further explore the mechanism how TET1 regulates BER pathway, MeDIP-qPCR and hMeDIP-qPCR were performed. The methylation of the promoter region of OGG1, XRCC1, and APEX1 were signi cantly downregulated (Fig. 6E), and the hydroxymethylation were signi cantly up-regulated (Fig. 6G), when the expression of TET1 was over-expressed (P < 0.01). In addition, after knocking down TET1 expression, the methylation of OGG1, XRCC1, and APEX1 were signi cantly up-regulated (Fig. 6F), and the hydroxymethylation were signi cantly down-regulated (Fig. 6H). These results revealed that epigenetic modi cation played an important role during TET1-mediated BER pathway regulation in lung cancer.
Knocking down of OGG1, XRCC1, and APEX1 can promote the cell growth, migration and invasion of lung cancer To demonstrate that whether the regulation of TET1 on the cell growth, invasion, and migration of lung cancer depends on the BER signaling pathway, the expression of OGG1, XRCC1, and APEX1 were knocked down while TET1 was overexpression in lung cancer cells. Firstly, soft agar assay and cloning formation experiment were performed to detect the cell growth. As shown in Fig. 7A and 7B, over-expressed TET1 resulted in the signi cantly down-regulation of cell growth compared with the control (P < 0.01). Compared to the TET1 over-expression group, the cell growth of TET1 and siOGG1, TET1 and siXRCC1, TET1 and siAPEX1, and TET1 and siBER (the combination of siOGG1, siXRCC1 and siAPEX1) group were signi cantly up-regulated (P < 0.01). What's more, the growth of TET1 and siBER group was signi cantly up-regulated compared with TET1 and siOGG1, TET1 and siXRCC1, and TET1 and siAPEX1, respectively.
Next, scratch healing and transwell experiments were performed to detect the migration and invasion. Knocking down of OGG1, XRCC1, and APEX1 could promote the migration and invasion of lung cancer cells, and simultaneously knocking down of OGG1, XRCC1, and APEX1 is particularly effective (Fig. 7C  and 7D). These results suggested that BER signaling pathway is required for the anti-tumorigenesis function of TET1 in lung cancer. Taken together, the above data demonstrated that TET1 inhibits cell growth, migration and invasion in lung cancer by repressing the BER pathway, via demethylation of the promoters of the BER key regulator OGG1, XRCC1, and APEX1.

Discussion
Accumulating evidence has discovered that TET1 played important roles in the occurrence and development of neoplastic diseases [23,24]. It has reported that overexpression of TET1 could signi cantly inhibit cell growth, migration and invasion of colon cancer and cervical cancer [23,25,26]. In addition, the recent study also found that TET1 involved in p53 mediated lung cancer cellular aging as an oncogene [27]. However, there are other studies showing that TET1 was down-regulated in lung cancer and related to high expression of immune markers and high in ltration of immune cells [28]. Therefore, the role and speci c mechanism of TET1 during the process of lung cancer is not yet clear. In this study, we highlighted the importance of TET1 and BER pathway in lung cancer.
Recent research has found that K-ras promotes DNA methylation and malignant transformation by inhibiting TET1 expression [29]. Recovery of TET1 expression can induce re-expression of tumor suppressor genes and inhibit malignant transformation of cells. These results suggest that TET1mediated DNA demethylation is a crucial link in K-ras-mediated malignant transformation [29]. Our results indicate that the expression of TET1 is signi cantly down-regulated in lung cancer cell lines, 3-MCA-induced malignant transformed cells, animal exposure models, and clinical samples. It suggested that TET1 abnormal expression may be the core link and key factor as early biological event during chemical-induced lung carcinogenesis.
The oxidation of 5-mC by TET proteins to 5-hmC inhibits the binding of DNA methyl-binding proteins, and 5-hmC cannot be recognized by DNMT1, resulting in the loss of methylation markers through subsequent DNA replication cycles and the formation of new unmethylated cytosine derivatives in subchains [30,31].
Studies have shown that DNMTs inhibitor can enhance the osteogenic differentiation potential of adipose-derived mesenchymal stem cells by promoting the expression of TET1 [32]. These results suggest that DNMT proteins compete functionally with TET1 proteins to bind DNA strands, and there may be competition or inhibition between them. In our study, we proved that TET1 is highly methylated through DNMTs, resulting in down-regulation of TET1 expression, thereby promoting the process of 3-MCA-induced malignant transformation in HBE cells. However, whether there is corsstalk between DNMTs and TET1 during tumorigenesis needs further study.
It has been reported that TET1 can increase the level of hydroxymethylation and decrease the level of methylation by binding the promoter region of cell growth and apoptosis-related genes, and promote the level of gene expression, thus participating in the regulation of the growth and metastasis of cancer cells and stem cells [28,[33][34][35]. Microarray cluster analysis of human embryonic cancer cell lines showed that TET1 was mainly enriched in promoter regions of genes involved in cell proliferation, exercise, angiogenesis and DNA damage repair by mediating 5-hmC [36]. Increasing evidence suggested that TET1 is a tumor suppressor in many types of cancer [23,26,[37][38][39]. These reports can further con rm our results. However, there are several reports indicating that TET1 may function as an oncogene by mediating hypomethylation upon targeting by partner proteins in different cancers [27,40]. Recent study showed that TET1 protein can interact with speci c partners and play a biological role through its catalytic and noncatalytic domains [41]. We speculated that the possible explanation for the distinct roles of TET1 is different upstream regulation factors in speci c status and different interacting partners in speci c cell type.
BER pathway is an indispensable DNA repair pathway that involves maintaining genome stability and thus preventing a series of human disease. BER plays a critical role in transcriptional regulation during epigenetic reprogramming [13]. DNA demethylation can passively occur through DNA replication, or it can actively occur with DNA replication-independent [42][43][44]. Activation of DNA methylation may involve TETs mediated oxidation of the methyl group, or activation-induced deaminase mediated deamination of methylated or nearby bases, and the modi ed nucleotide is replaced by the BER pathway [45]. Our study suggested that TET1 inhibits cell growth, migration and invasion by mediating DNA hydroxymethylation to activate BER pathway during the process of chemically induced lung cancer. Previous studies have shown that genetic alteration and protein posttranslational modi cations have emerged as important contributors in regulating the BER pathway through controlling cellular BER protein levels, protein-protein interactions, and enzymatic activities [12,46]. Our study focused on the level of transcriptional regulation to explore the regulatory mechanism of BER pathway during the process of lung cancer. It further enriches the role and importance of BER pathway regulation in a series of diseases including tumorigenesis. It has reported that TET1 plays a biological role in regulating BER pathway through protein-protein interaction [14]. Our results suggested that TET1 plays an anti-tumor role through transcriptional regulation of key molecules in BER pathway. This further enriched and supplemented the regulation mode of TET1 in different physiological and pathological states.

Conclusion
We clari ed for the rst time that TET1 down-regulated associated with DNA methylation and acted as a novel tumor suppressor that inhibited cell growth and metastasis through BER pathway in lung cancer.
TET1 regulated the level of hydroxymethylation and methylation in the promoter region of key gene in BER pathway, which participates in the whole process of lung cancer induced by environmental chemicals. Our study is not only of great signi cance to elucidate the pathogenesis of chemical carcinogenesis and lung cancer, but also provides a scienti c basis for searching for early biomarkers of environmental chemical carcinogenesis and prevention strategies.

Consent for publication
All authors read the nal approval manuscript and agreed for publication.    Knockdown of TET1 gene promoted proliferation, migration and invasion of lung cancer cells. A. Knockdown e ciency of TET1 mRNA and protein level was determined by qRT-PCR (left panel) and Western blot (right panel). B. CCK-8 assay was performed for growth curve of lung cancer cells transfected with control and shTET1 vectors. C. Soft agar assay was used to detect the proliferation of lung cancer cells after the expression of TET1 gene was knocked down. D. Clone formation assay was performed for detecting the proliferation of lung cancer cells after knockdown of TET1 gene expression. E. Relative mobility of lung cancer cells was detected by scratch healing experiments after knocking down the expression of TET1 gene. F. Transwell assay was used to detect the migration and invasion of lung cancer cells after knocking down the expression of TET1 gene. The data were expressed as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01.   of key genes in BER pathway was detected by MeDIP-qPCR after TET1 overexpression (E) and knockdown (F). G and H. the level of hydroxymethylation of the key gene of BER pathway was detected by hMeDIP-qPCR after overexpression (G) and knockdown (H) of TET1. ** P < 0.01.

Figure 7
The effect of BER pathway intervention on the function of TET1 gene in lung cancer. A and B. Soft agar colony formation experiment (A) and Clone formation experiment (B) of lung cancer cells. OGG1, XRCC1, APEX1 was knocked down alone or in combination to treat the cells while over-expressing TET1 gene, the colony formation ability of cell growth was signi cantly up-regulated. C and D. The scratch healing experiment (C) and Transwell experiment (D) was performed to detected the migration and invasion, after cells was treated with BER pathway invention alone or in combination while overexpression of TET1 gene. It was found that the ability of migration and invasion was signi cantly increased. ** P < 0.01.