Enqiang Linghu

Methylation of DACT2 accelerates esophageal cancer development by activating Wnt signaling

Esophageal cancer is one of the most common malignancies worldwide. DACT2 is frequently methylated in human lung, hepatic, gastric and thyroid cancers. The methylation status and function of DACT2 remain to be elucidated in human esophageal cancer. Ten esophageal cancer cell lines, 42 cases of dysplasia and 126 cases of primary esophageal cancer samples were analyzed in this study. The expression of DACT2 was detected in YES2 cells, while reduced DACT2 expression levels were found in TE8 and KYSE70 cells, and complete loss of DACT2 expression was found in KYSE30, KYSE140, KYSE150, KYSE410, KYSE450, TE3 and TE7 cells. Loss of expression or reduced expression of DACT2 correlated with promoter region hypermethylation in esophageal cancer cells. Restoration of DACT2 expression was induced by 5-aza-2′-deoxycytidine. In human primary esophageal squamous carcinoma, 69% (87/126) of samples were methylated. Methylation of DACT2 was significantly associated with tumor stage and metastasis (P < 0.01, P < 0.05). DACT2 suppressed colony formation, cell migration and invasion in esophageal cancer cells, and it also suppressed esophageal cancer cell xenograft growth. DACT2 inhibited Wnt signaling in human esophageal cancer cells. In conclusion, DACT2 is frequently methylated in human esophageal cancer and its expression is regulated by promoter region methylation. DACT2 suppresses esophageal cancer growth by inhibiting Wnt signaling.

Silencing NKD2 by promoter region hypermethylation promotes gastric cancer invasion and metastasis by up-regulating SOX18 in human gastric cancer

Naked cuticle homolog2 (NKD2) is located in chromosome 5p15.3, which is frequently loss of heterozygosity in human colorectal and gastric cancers. In order to understand the mechanism of NKD2 in gastric cancer development, 6 gastric cancer cell lines and 196 cases of human primary gastric cancer samples were involved. Methylation specific PCR (MSP), gene expression array, flow cytometry, transwell assay and xenograft mice model were employed in this study. The expression of NKD1 and NKD2 was silenced by promoter region hypermethylation. NKD1 and NKD2 were methylated in 11.7% (23/196) and 53.1% (104/196) in human primary gastric cancer samples. NKD2 methylation is associated with cell differentiation, TNM stage and distant metastasis significantly (all P < 0.05), and the overall survival time is longer in NKD2 unmethylated group compared to NKD2 methylated group (P < 0.05). Restoration of NKD2 expression suppressed cell proliferation, colony formation, cell invasion and migration, induced G2/M phase arrest, and sensitized cancer cells to docetaxel. NKD2 inhibits SOX18 and MMP-2,7,9 expression and suppresses BGC823 cell xenograft growth. In conclusion, NKD2 methylation may serve as a poor prognostic and chemo-sensitive marker in human gastric cancer. NKD2 impedes gastric cancer metastasis by inhibiting SOX18.

Methylation of SLFN11 is a marker of poor prognosis and cisplatin resistance in colorectal cancer

AIM The expression of human SLFN11 was reported to sensitize cancer cells to DNA damaging agents. This study is to explore the epigenetic change and the function of SLFN11 in human colorectal cancer (CRC). MATERIALS & METHODS Six CRC cell lines and 128 primary CRC samples were used. RESULTS SLFN11 was methylated in 55.47% (71/128) of primary CRC. The expression of SLFN11 was regulated by promoter region methylation. Methylation of SLFN11 was significantly associated with age, poor 5-year overall survival and 5-year relapse-free survival (all p < 0.05). SLFN11 suppressed CRC cell growth both in vitro and in vivo and sensitized CRC cells to cisplatin. CONCLUSION SLFN11 is frequently methylated in human CRC, and the expression of SLFN11 is regulated by promoter region methylation. Methylation of SLFN11 reduced the sensitivity of CRC cells to cisplatin.

Methylation of DIRAS1 promotes colorectal cancer progression and may serve as a marker for poor prognosis

BackgroundDIRAS1 is a new member of the Ras gene family. It was described as a potential tumor suppressor in human glioblastomas and esophageal cancer. The role of DIRAS1 in colorectal cancer remains unclear.MethodsTo explore the epigenetic changes and function of DIRAS1 in human colorectal cancer, we studied ten colorectal cancer cell lines and 146 primary colorectal cancer samples and 50 matched adjacent samples using semi-quantitative reverse transcription PCR, immunohistochemistry, methylation-specific PCR and bisulfite sequencing, western blot, flow cytometry, and transwell assays.ResultsDIRAS1 expression was found in DKO and HCT116 cells, while reduced expression was detected in LoVo, SW48, LS180, and SW620 cells, and there was no expression detected in DLD1, HT29, RKO, and SW480 cells. Complete methylation was found in the promoter region of DLD1, HT29, RKO, and SW480 cells. Partial methylation was detected in LoVo, LS180, SW48, and SW620 cells, and unmethylation was found in DKO and HCT116 cells. These results indicate that promoter region methylation correlated with loss of/reduced expression of DIRAS1. Re-expression of DIRAS1 was induced by 5-aza-2′-deoxycytidine, suggesting that the expression of DIRAS1 is regulated by promoter region methylation. DIRAS1 was methylated in 47.3% (69/146) of primary colorectal cancer samples, no methylation was found in non-cancerous colonic tissue samples. Methylation of DIRAS1 was significantly associated with TNM stage (P < 0.05) and short survival time (P = 0.0121). DIRAS1 induced apoptosis and inhibited cell proliferation, migration, and invasion in colorectal cancer. Finally, DIRAS1 suppressed colorectal cancer cell xenograft growth in nude mice.ConclusionsDIRAS1 is frequently methylated in human colorectal cancer and the expression of DIRAS1 is regulated by promoter region methylation. Methylation of DIRAS1 is a marker of poor prognosis in human colorectal cancer.

Methylation of ZNF331 is an independent prognostic marker of colorectal cancer and promotes colorectal cancer growth

BackgroundZNF331 was reported to be a transcriptional repressor. Methylation of the promoter region of ZNF331 has been found frequently in human esophageal and gastric cancers. The function and methylation status of ZNF331 remain to be elucidated in human colorectal cancer (CRC).MethodsSix colorectal cancer cell lines, 146 cases of primary colorectal cancer samples, and 10 cases of noncancerous colorectal mucosa were analyzed in this study using the following techniques: methylation specific PCR (MSP), qRT-PCR, siRNA, flow cytometry, xenograft mice, MTT, colony formation, and transfection assays.ResultsLoss of ZNF331 expression was found in DLD1 and SW48 cells, reduced expression was found in SW480, SW620, and HCT116 cells, and high level expression was detected in DKO cells. Complete methylation of the ZNF331 in the promoter region was found in DLD1 and SW48 cells, partial methylation was found in SW480, SW620, and HCT116 cells, and unmethylation was detected in DKO cells. Loss of/reduced expression of ZNF331 is correlated with promoter region methylation. Restoration of ZNF331 expression was induced by 5-aza-2′-deoxycytidine (DAC) in DLD1 and SW48 cells. These results suggest that ZNF331 expression is regulated by promoter region methylation in CRC cells. ZNF331 was methylated in 67.1% (98/146) of human primary colorectal cancer samples. Methylation of ZNF331 was significantly associated with tumor size, overall survival (OS), and disease-free survival (DFS) (p < 0.01, p < 0.01, p < 0.05). Methylation of ZNF331 was an independent poor prognostic marker for 5-year OS and 5-year DFS (both p < 0.05). ZNF331 suppressed cell proliferation and colony formation in CRC cells and suppressed human CRC cell xenograft growth in mice.ConclusionsZNF331 is frequently methylated in human colorectal cancer, and the expression of ZNF331 is regulated by promoter region methylation. Methylation of ZNF331 is a poor prognostic marker of CRC.

Epigenetic silencing of TMEM176A promotes esophageal squamous cell cancer development

The function of human transmembrane protein 176A (TMEM176A) in cancer remains unclear. To understand the function and mechanism of TMEM176A in human esophageal cancer development, 13 esophageal cancer cell lines and 267 cases of primary esophageal squamous cell cancer (ESCC) samples were analyzed by methylation specific PCR (MSP), flow cytometry, immunohistochemistry and transfection assays. TMEM176A was highly expressed in BIC1 cells and loss of TMEM176A expression was found in TE1, TE3, TE13, KYSE140, KYSE180, KYSE410, KYSE450, KYSE520, Segl, KYSE150, YES2 and COLO680N cells. Complete methylation was detected in TE1, TE3, TE13, KYSE140, KYSE180, KYSE410, KYSE450, KYSE520, Segl, KYSE150, YES2 and COLO680N cells, while unmethylation was detected in BIC1 cells. Restoration of TMEM176A expression was induced by 5-aza-2’-deoxycytidine treatment in methylated cell lines. TMEM176A was methylated in 66.7% (178/267) of primary esophageal cancer samples, and promoter region methylation was significantly associated with tumor differentiation (p<0.001) and loss off/reduced expression of TMEM176A (p<0.05). Methylation of TMEM176A was significantly associated with poor 5-year overall survival (p < 0.05). Cox proportional hazards model analysis suggest that TMEM176A methylation is an independent prognostic factor for poor 5-years OS. TMEM176A inhibited cell invasion and migration, and induced apoptosis in esophageal cancer cells. TMEM176A suppressed esophageal cancer cell growth both in vitro and in vivo. In conclusion, TMEM176A is frequently methylated in human ESCC and the expression of TMEM176A is regulated by promoter region methylation. TMEM176A methylation may serve as a diagnostic and prognostic marker in ESCC. TMEM176A is a potential tumor suppressor in human ESCC.

Correction to: Methylation of ZNF331 is an independent prognostic marker of colorectal cancer and promotes colorectal cancer growth

After publication of the original article [1], it came to the authors’ attention that a reference was omitted from the Background.

Methylation of TMEM176A is an independent prognostic marker and is involved in human colorectal cancer development

ABSTRACT Colorectal cancer (CRC) is the third most common malignancy and the fourth most common cause of cancer related death worldwide. This study was designed to find tumor suppressors involved in CRC development by performing RNA-seq. Eight CRC cell lines and 130 cases of primary CRC samples were used. RNA-seq, methylation-specific PCR (MSP), flow cytometry, transwell assays, and a xenograft mouse model were used. Reduction of TMEM176A expression was confirmed in human CRC cells by RNA-seq. TMEM176A was expressed in LS180 and SW620 cells, loss of TMEM176A expression was observed in LOVO, HCT116, RKO, and DLD1 cells, and reduced TMEM176A expression was found in HT29 and SW480 cells. Unmethylation of the TMEM176A promoter was found in LS180 and SW620 cells, whereas complete methylation was found in LOVO, HCT116, RKO, and DLD1 cells, and partial methylation was found in HT29 and SW480 cells. Promoter region methylation correlated with loss of/reduced expression of TMEM176A. Re-expression of TMEM176A was induced by 5-aza-2′-deoxycytidine. TMEM176A was methylated in 50.77% of primary colorectal cancers. Methylation of TMEM176A was associated with tumor metastasis (P<0.05) and was an independent prognostic factor for 5-year overall survival (OS) according to Cox proportional hazards model analysis (P<0.05). TMEM176A induced apoptosis and inhibited cell migration and invasion in CRC cells. TMEM176A suppressed CRC cell growth both in vitro and in vivo. Our results suggest that expression of TMEM176A is regulated by promoter region methylation. TMEM176A methylation is an independent prognostic marker for 5-year OS in CRC, and may act as a tumor suppressor in CRC.