Chongchang Zhou

Detection of 14-3-3 sigma (σ) promoter methylation as a noninvasive biomarker using blood samples for breast cancer diagnosis

As a tumor suppressor gene, 14-3-3 σ has been reported to be frequently methylated in breast cancer. However, the clinical effect of 14-3-3 σ promoter methylation remains to be verified. This study was performed to assess the clinicopathological significance and diagnostic value of 14-3-3 σ promoter methylation in breast cancer. 14-3-3 σ promoter methylation was found to be notably higher in breast cancer than in benign lesions and normal breast tissue samples. We did not observe that 14-3-3 σ promoter methylation was linked to the age status, tumor grade, clinic stage, lymph node status, histological subtype, ER status, PR status, HER2 status, or overall survival of patients with breast cancer. The combined sensitivity, specificity, AUC (area under the curve), positive likelihood ratios (PLR), negative likelihood ratios (NLR), diagnostic odds ratio (DOR), and post-test probability values (if the pretest probability was 30%) of 14-3-3 σ promoter methylation in blood samples of breast cancer patients vs. healthy subjects were 0.69, 0.99, 0.86, 95, 0.31, 302, and 98%, respectively. Our findings suggest that 14-3-3 σ promoter methylation may be associated with the carcinogenesis of breast cancer and that the use of 14-3-3 σ promoter methylation might represent a useful blood-based biomarker for the clinical diagnosis of breast cancer.

Promoter hypermethylation of miR-34a contributes to the risk, progression, metastasis and poor survival of laryngeal squamous cell carcinoma

MiR-34a is a direct transcriptional target of p53, which induces cell cycle arrest, senescence, and apoptosis. Recently, we and others identified abnormal expression of miR-34a in laryngeal squamous cell carcinoma (LSCC). The aim of our present study was to investigate the contribution of miR-34a promoter methylation to LSCC. Bisulfite pyrosequencing technology was applied to measure DNA methylation levels of six CpG sites in the miR-34a promoter from 104 LSCC tumor tissues and their corresponding adjacent tissues. Our results showed that the methylation levels of the miR-34a promoter were significantly higher in cancer tissues compared with the adjacent tissues (adjusted P=5.05E-10). A breakdown analysis for cigarette smoking behavior indicated a significantly elevated tendency of miR-34a methylation level in LSCC patients with smoking behavior but not in LSCC patients without smoking behavior (Smoking: Tumor vs Normal, adjusted P=3.12E-9; Non-smoking: Tumor vs Normal, adjusted P=0.073). In addition, miR-34a promoter methylation frequency remarkably increased in the advanced stage patients (adjusted P=0.003) and advanced T classified tumors (adjusted P=0.015). Moreover, significant association of miR-34a promoter hypermethylation with LSCC lymph metastasis was observed (adjusted P=0.002). Meanwhile, Kaplan-Meier survival curves results showed that high methylation of miR-34a promoter were associated with poor overall survival (log-rank test, P=0.023). Our study revealed that miR-34a promoter hypermethylation was a risk factor for LSCC, played a critical role in the disease progression and metastasis, and could serve as a poor prognostic factor for LSCC.

DNA methylation of CMTM3 , SSTR2 , and MDFI genes in colorectal cancer

Colorectal cancer (CRC) is increasingly common worldwide, including in China. Therefore, there is an increasing need to detect CRC at an early stage and to discover and evaluate diagnostic and prognostic biomarkers. DNA methylation of genes in CRC is a potential epigenetic biomarker for the early detection of CRC. This study was performed to analyze the methylation frequency of six candidate genes, CMTM3, SSTR2, MDFI, NDRG4, TGFB2, and BCL2L11, in fresh-frozen CRC tissues and adjacent normal colorectal tissues, from 42 patients with CRC. DNA isolation, bisulphite modification, and pyrosequencing were performed. The sensitivity, specificity, and the area under the receiver operator characteristic (ROC) curve (AUC) were evaluated to determine whether these genes showed any associations with tumor grade, stage, or diagnostic features. Among the tested genes, three genes, CMTM3, SSTR2, and MDFI were significantly methylated in CRC tissues when compared with adjacent normal colorectal tissues. The ROC analysis showed that a multigene model, including CMTM3, SSTR2, and MDFI, had a sensitivity of 81% and a specificity of 91% with an AUC value of 0.92. The findings of this study have shown that DNA methylation of the genes, CMTM3, SSTR2, and MDFI should be studied further with a view to determining their potential role as biomarkers for CRC.

Long non-coding RNA AC026166.2-001 inhibits cell proliferation and migration in laryngeal squamous cell carcinoma by regulating the miR-24-3p/p27 axis

Long non-coding RNA (lncRNA) AC026166.2-001 was found to be down-regulated in laryngeal squamous cell carcinoma (LSCC) tissues and metastatic neck lymph nodes. Decreased AC026166.2-001 was associated with poorer prognosis and may act as a novel biomarker for LSCC patients. In this study, AC026166.2–001 was overexpressed by a lentivirus vector and down-regulated by a small interfering RNA (siRNA). The results of real-time cell analysis (RTCA) and a plate colony formation assay showed that AC026166.2–001 inhibited LSCC cell proliferation and the clone-forming capacity. Cell cycle distribution and related protein changes were measured by flow cytometry. AC026166.2–001 arrested the cell cycle at the G1 phase and induced apoptosis. In addition, AC026166.2–001 decreased cell migration as measured by wound healing assays and transwell migration assays. Moreover, luciferase reporter assay and Western blotting results suggested that AC026166.2–001 acts as a sponge of miR-24-3p and regulates the expression of p27 and cyclin D1. The in vivo results showed that AC026166.2–001 significantly suppressed the growth of LSCC xenografts and promoted apoptosis. We validated the molecular mechanisms underlying AC026166.2–001 in LSCC. This is the first report of AC026166.2–001 acting as a tumor suppressor in LSCC by regulating the miR-24-3p/p27 axis.

CDKN2A methylation in esophageal cancer: a meta-analysis

CDKN2A is a tumor suppressor gene and is frequently inactivated in human cancers by hypermethylation of its promoter. However, the role and diagnostic value of CDKN2A methylation in esophageal cancer (EC) remains controversial. Therefore, we performed a meta-analysis, including data from 42 articles (2656 ECs, 612 precancerous lesions, and 2367 controls). A significant increase in the frequency of CDKN2A methylation was identified during EC carcinogenesis: cancer vs. controls, odds ratio (OR) = 12.60 (95 % CI, 8.90–17.85); cancer vs. precancerous lesions, OR = 2.89 (95% CI, 2.20–3.79); and precancerous lesions vs. controls, OR = 7.38, 95% (CI, 4.31–12.66). CDKN2A promoter methylation was associated with EC tumor grade (OR = 1.79; 95% CI, 1.20–2.67) and clinical stage (OR = 2.56; 95% CI, 1.33–4.92). Additionally, the sensitivity, specificity, and area under the summary receiver operating characteristic curve (AUC) for diagnosis of EC based on CDKN2A methylation were 0.52 (95% CI, 0.44–0.59), 0.96 (95% CI, 0.93–0.98), and 0.83 (95% CI, 0.79–0.86), respectively. AUCs for blood and tissue sample subgroups were 0.90 and 0.82, respectively. Our findings indicate that CDKN2A methylation has a vital role in EC tumorigenesis and could be a biomarker for early diagnosis of EC.

Role of CDH13 promoter methylation in the carcinogenesis, progression, and prognosis of colorectal cancer: A systematic meta-analysis under PRISMA guidelines.

Background: H-cadherin (CDH13) is commonly downregulated through promoter methylation in various cancers. However, the role of CDH13 promoter methylation status in patients with colorectal cancer (CRC) remains to be clarified. Methods: Eligible articles were identified from online electronic database based on the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement criteria. The pooled odds ratio (OR) and the corresponding 95% confidence interval (95% CI) were calculated and analyzed. Results: Eventually, a total of nine studies were included in this meta-analysis, including 488 CRC, 298 adjacent, 144 normal, 68 premalignant tissues. The results demonstrated that CDH13 promoter methylation was notably higher in CRC than in normal, adjacent, and premalignant tissues (cancer tissues vs normal tissues: OR = 16.94, P < 0.001; cancer tissues vs adjacent tissues: OR = 20.06, P < 0.001; cancer tissues vs premalignant tissues: OR = 2.23, P = 0.038). CDH13 promoter methylation had a significantly increased risk for poorly differentiated CRC (OR = 4.07, P = 0.001). CDH13 promoter methylation was not associated with sex status, tumor stage, and lymph node status (all P > 0.05). One study with 85 CRC patients reported that CDH13 promoter methylation was correlated with poor prognosis in overall survival (OS). Conclusions: CDH13 promoter methylation may play an important role in the initiation and progression of CRC, and may be correlated with OS of patients with CRC. Additional studies with large sample sizes are needed to further confirm our findings in the future.

SHISA3 Promoter Methylation Is a Potential Diagnostic and Prognostic Biomarker for Laryngeal Squamous Cell Carcinoma

The purpose of this study was to evaluate the contribution of SHISA3 promoter methylation to laryngeal squamous cell carcinoma (LSCC). SHISA3 promoter methylation status and expression were determined using methylation-specific polymerase chain reaction (MSP) and quantitative real-time PCR (qRT-PCR) in 93 paired LSCC and adjacent normal tissues, respectively. Furthermore, the regulatory function of the SHISA3 promoter fragment was analyzed using a luciferase reporter assay. The results reveal that there is a significant increase in SHISA3 methylation in LSCC tissues compared with corresponding nontumor tissues (P = 4.58E − 12). The qRT-PCR results show a significant association between SHISA3 methylation and expression in LSCC (P = 1.67E − 03). In addition, the area under the receiver operating characteristic curve was 0.91. Consequently, a log-rank test and multivariate Cox analysis suggest that SHISA3 promoter hypermethylation is a predictor of poor overall survival for LSCC (log-rank P = 0.024; HR = 2.71; 95% CI = 1.024–7.177; P = 0.047). The results indicate that SHISA3 promoter hypermethylation might increase the risk of LSCC through regulation of gene expression and is a potential diagnostic and prognostic biomarker for LSCC.

Elevated methylation of CMTM3 promoter in the male laryngeal squamous cell carcinoma patients

OBJECTIVE CKLF-like MARVEL transmembrane domain containing 3 (CMTM3), as a tumor suppressor gene, plays an important role in the suppression of cell growth and apoptosis. The goal of our study is to investigate the association between CMTM3 promoter methylation and laryngeal squamous cell carcinoma (LSCC). DESIGN AND METHODS Using the bisulfite pyrosequencing technology, DNA methylation levels of seven CpG sites in CMTM3 promoter are measured in tumor tissues and their adjacent tissues of 76 male LSCC patients. RESULTS Our results reveal a significantly elevated promoter methylation of CMTM3 in tumor tissues compared with their adjacent tissues (P<0.001). A breakdown analysis by age shows that significant association of CMTM3 promoter methylation with cancer risk is specific to the LSCC patients older than 55years (P<0.001) but not in the younger patients (P=0.305). Moreover, the association is only observed in the LSCC patients with smoking behavior (P=0.001). Breakdown analysis also shows that CMTM3 promoter methylation is associated with cancer risk among patients with stage I LSCC (P<0.001). CONCLUSION In conclusion, our study indicates that elevated CMTM3 methylation is a risk factor in male LSCC patients, especially in the patients with age over 55years and with smoking behavior.

Synthesis and Properties of Flexible Polyurethane Using Ferric Catalyst for Hypopharyngeal Tissue Engineering.

Biodegradable polyurethane is an ideal candidate material to fabricate tissue engineered hypopharynx from its good mechanical properties and biodegradability. We thus synthesized a hydrophilic polyurethane via reactions among polyethylene glycol (PEG), e-caprolactone (e-CL) and hexamethylene diisocyanate (HDI), and thrihydroxymethyl propane (TMP). The product possessed a fast degradability due to its good wettability and good mechanical parameters with the elongations at break (137 ± 10%) and tensile strength (4.73 ± 0.46 MPa), which will make it a good matrix material for soft tissue like hypopharynx. Its biological properties were evaluated via in vitro and in vivo tests. The results showed that this hydrophilic polyurethane material can support hypopharyngeal fibroblast growth and owned good degradability and low inflammatory reaction in subcutaneous implantation. It will be proposed as the scaffold for hypopharyngeal tissue engineering research in our future study.

LZTS2 promoter hypermethylation: a potential biomarker for the diagnosis and prognosis of laryngeal squamous cell carcinoma

BackgroundLZTS2 (leucine zipper tumor suppressor 2), a candidate tumor suppressor gene, suppresses cell growth and plays a vital role in the carcinogenesis and development of tumors. No studies to date have described methylation of the LZTS2 promoter in human cancers, including LSCC (laryngeal squamous cell carcinoma). Therefore, the aim of this study was to explore the relationship between LZTS2 promoter methylation and risk of LSCC.MethodsIn our study, LZTS2 promoter methylation levels in LSCC tumor and adjacent normal tissues from 96 patients were measured using quantitative methylation-specific polymerase chain reaction (qMSP) assays.ResultsThe qMSP analyses revealed that LZTS2 promoter methylation levels in the LSCC tumor samples were significantly higher than those in paired adjacent healthy tissue samples. Furthermore, LZTS2 methylation levels were elevated in smokers, advanced T classified, and clinically staged patients, as well as in patients with lymph node metastases. In addition, Kaplan-Meier survival curves results showed that overall survival of LSCC patients with hypomethylated LZTS2 promoters was significantly higher than that in patients with hyper-methylated LZTS2 promoters (log-rank test P = 0.028). Meanwhile, the area under the receiver operating characteristic curve was 0.920. The diagnostic threshold value for LZTS2 methylation was 11.63% (94.7% sensitivity and 80.4% specificity).ConclusionsLZTS2 promoter hypermethylation is associated with risk, progression, and prognosis of LSCC in a cohort of 96 human subjects; LZTS2 promoter hypermethylation is a candidate diagnostic and prognostic biomarker for LSCC.