Downregulation of m6A reader YTHDC2 promotes the proliferation and migration of lung cancer and cigarette smoke-exposed BEAS-2B cells

Background: Lung cancer is one of the most common carcinomas worldwide. Cigarette smoking is considered as the leading cause of lung cancer. The aberrant expression of several YTH family proteins has been reported to be closely associated with several cancers. This study aims to evaluate the expression prole and function of YTHDC2, an RNA m6 A methylation reader protein, by in vitro and animal experiments and bioinformatics analysis. Methods: The expression of YTHDC2 gene and protein was analyzed by the GEPIA online tool and Gene Expression Omnibus (GEO) database, as well as immunohistochemistry of tissue arrays. The YTHDC2 expression in cigarette smoke-exposed cells was examined by RT-qPCR and western blot assays. The TCGA datasets and proteomic analysis of YTHDC2 knockdown cells were used to predict the clinical signicance and biological function of YTHDC2. Functional assays, including wound healing, transwell migration, cell cycle and cell proliferation, were used to determine the biological role of YTHDC2 in lung cancer. The xenograft animal model further validated the tumor suppressor effect of YTHDC2 in vivo. The copy number variation of YTHDC2 was analyzed by TCGA datasets and TaqMan copy number assay. Results: YTHDC2 was signicantly low-expressed in lung cancer and cigarette smoke-exposed cells. Notably, decreased YTHDC2 was found highly associated with smoking history, pathological stage, invasion depth, and lymph node metastasis, as well as poor outcomes. The YTHDC2 related genes and proteins were enriched in several cancer-related pathways, and several tumor suppressor genes were found positively correlated with YTHDC2. The in vivo and in vitro study revealed that overexpression of YTHDC2 inhibited the proliferation and migration ability of lung cancer cells, as well as the tumor growth in nude mice. Besides, a signicant positive correlation between the copy number and expression level of YTHDC2 was found in lung cancer. Conclusions: Our results indicated that the smoking-related down-regulation of YTHDC2 was associated with enhanced proliferation and migration of lung cancer cells and appeared to be regulated by DNA copy number variation. Importantly,

YTHDC2 inhibited the proliferation and migration ability of lung cancer cells, as well as the tumor growth in nude mice. Besides, a signi cant positive correlation between the copy number and expression level of YTHDC2 was found in lung cancer.
Conclusions: Our results indicated that the smoking-related down-regulation of YTHDC2 was associated with enhanced proliferation and migration of lung cancer cells and appeared to be regulated by DNA copy number variation. Importantly, YTHDC2 may serve as a tumor suppressor gene and a favorable prognostic indicator in lung cancer patients.

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However, the manuscript can be downloaded and accessed as a PDF. Figure 1 The YTHDC2 gene and protein were down-regulated in lung cancer patients from the TCGA, GEO and HPA databases, as well as in cigarette smoke-exposed cells. (A) Differential analysis of YTHDC2 mRNA expression in lung cancer tissues based on the GEPIA tool. *p < 0.05 versus normal tissues. Differential analysis of YTHDC2 mRNA expression in lung cancer tissues in GSE32665 (B) and GSE19188 (C). (D) The representative IHC images showed that the YTHDC2 staining was found in the cytoplasm of the cells Page 4/32 in lung cancer or normal lung tissues. High expression of YTHDC2 could be found in adjacent normal tissues, while its expression was decreased in most lung cancer tissues. (E) Differential analysis of YTHDC2 staining positive ratio quantitated by IHC pro ler in lung cancer tissue arrays. (F) The relative mRNA expression level of YTHDC2 in cigarette smoke-exposed cells (S10, S20 and S30) and normal BEAS-2B cells. (G) Western blot analysis and quantitative results of YTHDC2 protein expression in cigarette smoke-exposed cells (S10, S20 and S30) and normal BEAS-2B cells. S10, S20 and S30 represent BEAS-2B cells exposed to cigarette smoke for 10, 20 and 30 passages separately. **p < 0.01 versus normal BEAS-2B cells. LUAD: lung adenocarcinoma; LUSC: Squamous cell carcinoma; IHC:

Figure 1
The YTHDC2 gene and protein were down-regulated in lung cancer patients from the TCGA, GEO and HPA databases, as well as in cigarette smoke-exposed cells. (A) Differential analysis of YTHDC2 mRNA expression in lung cancer tissues based on the GEPIA tool. *p < 0.05 versus normal tissues. Differential analysis of YTHDC2 mRNA expression in lung cancer tissues in GSE32665 (B) and GSE19188 (C). (D) The representative IHC images showed that the YTHDC2 staining was found in the cytoplasm of the cells Page 6/32 in lung cancer or normal lung tissues. High expression of YTHDC2 could be found in adjacent normal tissues, while its expression was decreased in most lung cancer tissues. (E) Differential analysis of YTHDC2 staining positive ratio quantitated by IHC pro ler in lung cancer tissue arrays. (F) The relative mRNA expression level of YTHDC2 in cigarette smoke-exposed cells (S10, S20 and S30) and normal BEAS-2B cells. (G) Western blot analysis and quantitative results of YTHDC2 protein expression in cigarette smoke-exposed cells (S10, S20 and S30) and normal BEAS-2B cells. S10, S20 and S30 represent BEAS-2B cells exposed to cigarette smoke for 10, 20 and 30 passages separately. **p < 0.01 versus normal BEAS-2B cells. LUAD: lung adenocarcinoma; LUSC: Squamous cell carcinoma; IHC: Immunohistochemistry.

Figure 1
The YTHDC2 gene and protein were down-regulated in lung cancer patients from the TCGA, GEO and HPA databases, as well as in cigarette smoke-exposed cells. (A) Differential analysis of YTHDC2 mRNA expression in lung cancer tissues based on the GEPIA tool. *p < 0.05 versus normal tissues. Differential analysis of YTHDC2 mRNA expression in lung cancer tissues in GSE32665 (B) and GSE19188 (C). (D) The representative IHC images showed that the YTHDC2 staining was found in the cytoplasm of the cells in lung cancer or normal lung tissues. High expression of YTHDC2 could be found in adjacent normal tissues, while its expression was decreased in most lung cancer tissues. (E) Differential analysis of YTHDC2 staining positive ratio quantitated by IHC pro ler in lung cancer tissue arrays. (F) The relative mRNA expression level of YTHDC2 in cigarette smoke-exposed cells (S10, S20 and S30) and normal BEAS-2B cells. (G) Western blot analysis and quantitative results of YTHDC2 protein expression in cigarette smoke-exposed cells (S10, S20 and S30) and normal BEAS-2B cells. S10, S20 and S30 represent BEAS-2B cells exposed to cigarette smoke for 10, 20 and 30 passages separately. **p < 0.01 versus normal BEAS-2B cells. LUAD: lung adenocarcinoma; LUSC: Squamous cell carcinoma; IHC: Immunohistochemistry.

Figure 2
Identi cation of TSGs and MRGs in YTHDC2 related genes. Volcano plots showing the genes correlated with YTHDC2 in LUAD (A) and LUSC (B) in the TCGA database by using the LinkedOmics online tool. Scatter plots showing the KEGG enrichment results of the genes associated with LUAD (C) and LUSC (D). Scatter plots showing the biological process enrichment results of the genes associated with LUAD (E) and LUSC (F). Rich Factor is the ratio of the related genes annotated in a pathway to all the genes in this pathway. The larger the Rich factor, the greater the degree of enrichment. The color gradient from red to green represents the p value; the closer to green color, the lower the p value and the higher the signi cance level corresponding to the enrichment. Volcano plots showing the TSGs in YTHDC2 related genes in LUAD (G) and LUSC (H). Volcano plots showing the MRGs in YTHDC2 related genes in LUAD (I) and LUSC (J). The red dots in volcano plots represent the positively related genes (Pearson coe cient > 0.3, p < 0.001), and the green dots represent the negatively related genes (Pearson coe cient < -0.3, p < 0.001), while the black dots represent genes have no signi cant correlation with YTHDC2 (Pearson coe cient between -0.3 and 0.3). The relative mRNA expression level of APC (K), CHD1 (L), DMXL1 (M) in cigarette smoke-exposed cells (S10, S20 and S30) and normal BEAS-2B cells.  Identi cation of TSGs and MRGs in YTHDC2 related genes. Volcano plots showing the genes correlated with YTHDC2 in LUAD (A) and LUSC (B) in the TCGA database by using the LinkedOmics online tool. Scatter plots showing the KEGG enrichment results of the genes associated with LUAD (C) and LUSC (D). Scatter plots showing the biological process enrichment results of the genes associated with LUAD (E) and LUSC (F). Rich Factor is the ratio of the related genes annotated in a pathway to all the genes in this pathway. The larger the Rich factor, the greater the degree of enrichment. The color gradient from red to green represents the p value; the closer to green color, the lower the p value and the higher the signi cance level corresponding to the enrichment. Volcano plots showing the TSGs in YTHDC2 related genes in LUAD (G) and LUSC (H). Volcano plots showing the MRGs in YTHDC2 related genes in LUAD (I) and LUSC (J). The red dots in volcano plots represent the positively related genes (Pearson coe cient > 0.3, p < 0.001), and the green dots represent the negatively related genes (Pearson coe cient < -0.3, p < 0.001), while the black dots represent genes have no signi cant correlation with YTHDC2 (Pearson coe cient between -0.3 and 0.3). The relative mRNA expression level of APC (K), CHD1 (L), DMXL1 (M) in cigarette smoke-exposed cells (S10, S20 and S30) and normal BEAS-2B cells.  Scatter plots showing the biological process enrichment results of the genes associated with LUAD (E) and LUSC (F). Rich Factor is the ratio of the related genes annotated in a pathway to all the genes in this pathway. The larger the Rich factor, the greater the degree of enrichment. The color gradient from red to green represents the p value; the closer to green color, the lower the p value and the higher the signi cance level corresponding to the enrichment. Volcano plots showing the TSGs in YTHDC2 related genes in LUAD (G) and LUSC (H). Volcano plots showing the MRGs in YTHDC2 related genes in LUAD (I) and LUSC (J). The red dots in volcano plots represent the positively related genes (Pearson coe cient > 0.3, p < 0.001), and the green dots represent the negatively related genes (Pearson coe cient < -0.3, p < 0.001), while the black dots represent genes have no signi cant correlation with YTHDC2 (Pearson coe cient between -0.3 and 0.3). The relative mRNA expression level of APC (K), CHD1 (L), DMXL1 (M) in cigarette smoke-exposed cells (S10, S20 and S30) and normal BEAS-2B cells.             Copy number variation in LUAD samples with different smoking histories. Different letters (a, b, c, and d) represent statistically signi cant group differences. (I) The copy number variation of YTHDC2 in BEAS-2B cells and cigarette smoke-exposed cells (grey block), as well as two lung cancer cell lines (black block).
The dotted line (copy number = 2) represents the copy number of the reference gene RNase P. correlation between YTHDC2 mRNA expression and copy numbers in LUAD (C) and LUSC (F). Line represents linear regression of data (LUAD: y=1.065x+9.177, R2 = 0.385; LUSC: y=0.965x+9.318, R2 = 0.198). (G) The oncomine datasets for respective YTHDC2 copy numbers in lung cancer were retried under the parameters with a threshold p value of 0.001 and a minimum 2-fold change. The data in the graphic show signi cant downregulation (blue column) of the YTHDC2 copy numbers in lung cancer versus normal tissue. The intensity of blue represents the respective levels of YTHDC2 copy number. (H) Copy number variation in LUAD samples with different smoking histories. Different letters (a, b, c, and d) represent statistically signi cant group differences. (I) The copy number variation of YTHDC2 in BEAS-2B cells and cigarette smoke-exposed cells (grey block), as well as two lung cancer cell lines (black block).
The dotted line (copy number = 2) represents the copy number of the reference gene RNase P.