Huadong Zhao

Reduced expression of N-Myc downstream-regulated gene 2 in human thyroid cancer

BackgroundNDRG2 (N-Myc downstream-regulated gene 2) was initially cloned in our laboratory. Previous results have shown that NDRG2 expressed differentially in normal and cancer tissues. Specifically, NDRG2 mRNA was down-regulated or undetectable in several human cancers, and over-expression of NDRG2 inhibited the proliferation of cancer cells. NDRG2 also exerts important functions in cell differentiation and tumor suppression. However, it remains unclear whether NDRG2 participates in carcinogenesis of the thyroid.MethodsIn this study, we investigated the expression profile of human NDRG2 in thyroid adenomas and carcinomas, by examining tissues from individuals with thyroid adenomas (n = 40) and carcinomas (n = 35), along with corresponding normal tissues. Immunohistochemistry, quantitative RT-PCR and western blot methods were utilized to determine both the protein and mRNA expression status of Ndrg2 and c-Myc.ResultsThe immunostaining analysis revealed a decrease of Ndrg2 expression in thyroid carcinomas. When comparing adenomas or carcinomas with adjacent normal tissue from the same individual, the mRNA expression level of NDRG2 was significantly decreased in thyroid carcinoma tissues, while there was little difference in adenoma tissues. This differential expression was confirmed at the protein level by western blotting. However, there were no significant correlations of NDRG2 expression with gender, age, different histotypes of thyroid cancers or distant metastases.ConclusionOur data indicates that NDRG2 may participate in thyroid carcinogenesis. This finding provides novel insight into the important role of NDRG2 in the development of thyroid carcinomas. Future studies are needed to address whether the down-regulation of NDRG2 is a cause or a consequence of the progression from a normal thyroid to a carcinoma.

N-MYC downstream-regulated gene 2, a novel estrogen-targeted gene is involved in the regulation of NA+/K+-ATPase

Na+/K+-ATPase, a plasma membrane protein abundantly expressed in epithelial tissues, has been identified and linked to numerous biological events, including ion transport and reabsorption. In Na+/K+-ATPase, the β-subunit plays a fundamental role in the structural integrity and functional maturation of holoenzyme. Estrogens are important circulating hormones that can regulate Na+/K+-ATPase abundance and activity; however, the specific molecules participating in this process are largely unknown. Here, we characterize that N-myc downstream-regulated gene 2 (NDRG2) is an estrogen up-regulated gene. 17β-Estradiol binds with estrogen receptor β but not estrogen receptor α to up-regulate NDRG2 expression via transcriptional activation. We also find that NDRG2 interacts with the β1-subunit of Na+/K+-ATPase and stabilizes the β1-subunit by inhibiting its ubiquitination and degradation. NDRG2-induced prolongation of the β1-subunit protein half-life is accompanied by a similar increase in Na+/K+-ATPase-mediated Na+ transport and Na+ current in epithelial cells. In addition, NDRG2 silencing largely attenuates the accumulation of β1-subunit regulated by 17β-estradiol. Our results demonstrate that estrogen/NDRG2/Na+/K+-ATPase β1 pathway is important in promoting Na+/K+-ATPase activity and suggest this novel pathway might have substantial roles in ion transport, fluid balance, and homeostasis.

Secretory miRNAs as novel cancer biomarkers.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that degrade or block target mRNAs at the post-transcriptional level. Many studies have shown that miRNA dysregulation is involved in cancer initiation, invasion, metastasis, and so forth. Notably, recent studies have revealed secretory miRNA levels in blood and other body fluids to correlate significantly with cancer progression, therapeutic response and patient survival. Thus, secretory miRNAs have demonstrated great potential as powerful and non-invasive cancer biomarkers. Herein, we summarize the current progress of secretory miRNAs in different cancer types and analyze the potential mechanisms of miRNA secretion. Then, we discuss the different approaches to miRNA detection in body fluids and the advantages of secretory miRNAs as biomarkers for early cancer diagnosis and the prediction of therapeutic efficacy. Finally, we list the current progress of secretory miRNAs as cancer biomarkers in clinical trials. Although several issues remain to be clarified, such as the mechanisms of miRNA secretion, it is only a matter of time before miRNAs are widely utilized as cancer biomarkers.

Prognostic value of HMGB1 overexpression in resectable gastric adenocarcinomas

IntroductionHMGB1(High mobility group box 1), originally described as a nuclear protein, is now regarded as a multifunctional protein with a paradoxical dual effect in tumors. In the present study, HMGB1 overexpression and its correlation with the clinicopathologic characteristics and recurrence-free survival were evaluated in gastric adenocarcinomas.Methods76 gastric adenocarcinomas surgically removed entered the study. The immunohistochemical staining was used to assess HMGB1 expression through tissue microarray procedure. The clinicopathologic characteristics of all patients were recorded, and the regular follow-up was made for all patients.ResultsAlmost all the gastric adenocarcinomas showed HMGB1 positive staining mainly in the nucleus, and the overexpression of HMGB1 was found in cancerous tissues with higher strong reactivity rate, compared with non-cancerous tissues (total expression score ≥ 9, 42.0% vs. 9.0%, P < 0.001). Survival analysis revealed that tumor stage negatively correlated with cancer-free survival (P = 0.022). Furthermore, HMGB1 overexpression positively associated with cancer-free survival of resectable gastric adenocarcinomas (P = 0.023).ConclusionsThe overexpression of HMGB1 protein indicates that HMGB1 may play a role in the tumorigenesis of gastric adenocarcinomas. And the overexpression of HMGB1 may be a marker of good prognosis of gastric adenocarcinoma given curative resection combined with adjuvant chemotherapy.

Expression of NDRG2 in esophageal squamous cell carcinoma

N‐Myc downstream‐regulated gene 2 (NDRG2), a new member of the N‐Myc downstream‐regulated gene family, has been found to be a differentially expressed gene involved in a variety of cancers. The present study aimed to investigate the expression of NDRG2 in esophageal squamous cell carcinoma (ESCC). Immunohistochemistry was performed in 154 samples from patients with ESCC to detect the expression level of NDRG2 and C‐MYC. Results indicated that the expression level of NDRG2 in the cancer samples was significantly lower than that in normal tissues; the trend of C‐MYC was the reverse. The Wilcoxon–Mann–Whitney test showed significant difference in the expression of NDRG2 in patients with different T stage, TNM stage, and differentiation degree of cancers (P = 0.036, 0.031, 0.001, respectively). Patients in stages I and II were followed up for 5 consecutive years and Kaplan–Meier survival analysis demonstrated that the survival time of ESCC patients with high expression of NDRG2 was longer than those with low expression during the 5‐year follow‐up period (P = 0.0018). Cox regression analysis indicated that low expression of NDRG2, cancer stage of pT1, and distant organ metastasis (pM1) were the independent poor prognostic factors of ESCC (P = 0.004, 0.019, 0.0013, respectively). Furthermore, up‐regulation of NDRG2 was introduced to ESCC cell lines (EC9706 and EC109) by plasmid transfection. In vivo and in vitro studies indicated that overexpression of NDRG2 markedly reduced proliferation and promoted the apoptosis of EC9706 and EC109 cells. In summary, our results demonstrated that NDRG2 played an important role in the proliferation of ESCC cells and the expression of NDRG2 in ESCC was closely related with the prognosis.

Clinicopathological significance of SOX4 expression in primary gallbladder carcinoma

AimSOX4, as a member of the SRY-related HMG-box (SOX) transcription factor family, has been demonstrated to be involved in tumorigenesis of many human malignancies; however, its role in primary gallbladder carcinoma (PGC) is still largely unknown. The aim of this study was to investigate SOX4 expression in PGC and its prognostic significance.MethodsFrom 1997 to 2006, 136 patients underwent resection for PGC. The median follow-up was 12.8 months. Immunostainings for SOX4 were performed on these archival tissues. The correlation of SOX4 expression with clinicopathological features including survival was analyzed.ResultsSOX4 was expressed in 75.0% (102/136) of PGC but not in the normal epithelium of the gallbladder. In addition, the over-expression of SOX4 was significantly associated with low histologic grade (P = 0.02), low pathologic T stage (P = 0.02), and early clinical stage (P = 0.03). The levels of SOX4 immunostainings in PGC tissues with positive nodal metastasis were also significantly lower than those without (P = 0.01). Moreover, Kaplan-Meier curves showed that SOX4 over-expression was significantly related to better overall (P = 0.008) and disease-free survival (P = 0.01). Furthermore, multivariate analyses showed that SOX4 expression was an independent risk factor for both overall (P = 0.03, hazard ratio, 3.682) and disease-free survival (P = 0.04, hazard ratio, 2.215).ConclusionOur data indicate for the first time that the over-expression of SOX4 in PGC was significantly correlated with favorable clinicopathologic features and was an independent prognostic factor for better overall and disease-free survival in patients. Therefore, SOX4 might be an auxiliary parameter for predicting malignant behavior for PGC.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1534825818694957.

Expression and biological significance of c-FLIP in human hepatocellular carcinomas

Backgroundc-FLIP can be considered as a tumor-progression factor in regard to its anti-apoptotic functions. In the present study, we intended to investigate the expression of c-FLIP in human HCC tissues, and its relation with drug-induced cell apoptosis through the specific inhibition of c-FLIP expression by siRNA in 7721 cells.Methodsc-FLIP expression was quantified immunohistochemically in HCC tissues(eighty-six cases), and corresponding noncancerous tissues (fifty-seven cases). Patients with HCC were followed up for cancer recurrence. Then, the c-FLIP gene was silenced with specific siRNA in 7721 HCC cells. c-FLIP expression was detected by RT-PCR, Western Blot and immunocytochemical staining. The cellular viability and cell apoptosis were assayed in vitro with cells treated with doxorubicin.ResultsPositive immunostaining was detected for c-FLIP in 83.72% (72/86) human HCC tissues, 14.81% (4/27) hepatic cirrhosis, 11.11% (2/18) hepatic hemangioma tissues, and absent in normal hepatic tissues. The overexpression(more than 50%) of c-FLIP in HCC adversely affected the recurrence-free survival. Through c-FLIP gene silencing with siRNA, the expressions of c-FLIP mRNA and protein were remarkably down-regulated in 7721 HCC cells. And doxorubicin showed apparent inhibition on cell proliferations, and induced more apoptosis.ConclusionThese results indicate that c-FLIP is frequently expressed in human HCCs, and its overexpression implied a lesser probability of recurrence-free survival. The specific silencing of c-FLIP gene can apparently up-regulate drug-induced HCC cell apoptosis, and may have therapeutic potential for the treatment of human HCC.

NDRG2 suppresses the proliferation of clear cell renal cell carcinoma cell A-498

BackgroundRecently, the anti-tumor activity of N-myc downstream-regulated gene 2 (NDRG2) was shown decreased expression in clear cell renal cell carcinoma (CCRCC), but the role of the down-expression of NDRG2 has not been described.MethodsThe NDRG2 recombinant adenovirus plasmid was constructed. The proliferation rate and NDRG2 expression of cell infected with recombinant plasmid were mesured by MTT, Flow cytometry analysis and western blot.ResultsThe CCRCC cell A-498 re-expressed NDRG2 when infected by NDRG2 recombinant adenovirus and significantly decreased the proliferation rate. Fluorescence activated cell sorter analysis showed that 25.00% of cells expressed NDRG2 were in S-phase compared to 40.67% of control cells, whereas 62.08% of cells expressed NDRG2 were in G1-phase compared to 54.39% of control cells (P < 0.05). In addition, there were much more apoptotic cells in NDRG2-expressing cells than in the controls (P < 0.05). Moreover, upregulation of NDRG2 protein was associated with a reduction in cyclin D1, cyclin E, whereas cyclinD2, cyclinD3 and cdk2 were not affected examined by western blot. Furthermore, we found that p53 could upregulate NDRG2 expression in A-498 cell.ConclusionsWe found that NDRG2 can inhibit the proliferation of the renal carcinoma cells and induce arrest at G1 phase. p53 can up-regulate the expression of NDRG2. Our results showed that NDRG2 may function as a tumor suppressor in CCRCC.

Genetic polymorphisms in circadian negative feedback regulation genes predict overall survival and response to chemotherapy in gastric cancer patients

Circadian negative feedback loop (CNFL) genes play important roles in cancer development and progression. To evaluate the effects of single nucleotide polymorphisms (SNPs) in CNFL genes on the survival of GC patients, 13 functional SNPs from 5 CNFL genes were genotyped in a cohort of 1030 resected GC patients (704 in the training set, 326 in the validation set) to explore the association of SNPs with overall survival (OS). Among the 13 SNPs, three SNPs (rs1056560 in CRY1, rs3027178 in PER1 and rs228729 in PER3) were significantly associated with OS of GC in the training set, and verified in the validation set and pooled analysis. Furthermore, a dose-dependent cumulative effect of these SNPs on GC survival was observed, and survival tree analysis showed higher order interactions between these SNPs. In addition, protective effect conferred by adjuvant chemotherapy (ACT) on GC was observed in patients with variant alleles (TG/GG) of rs1056560, but not in those with homozygous wild (TT) genotype. Functional assay suggested rs1056560 genotypes significantly affect CRY1 expression in cancer cells. Our study presents that SNPs in the CNFL genes may be associated with GC prognosis, and provides the guidance in selecting potential GC patients most likely responsive to ACT.

MSP58 Knockdown Inhibits the Proliferation of Esophageal Squamous Cell Carcinoma in Vitro and in Vivo

Esophageal carcinoma (EC) is one of the most aggressive cancers with a poor prognosis. Understanding the molecular mechanisms underlying esophageal cancer progression is a high priority for improved EC diagnosis and prognosis. Recently, MSP58 was shown to behave as an oncogene in colorectal carcinomas and gliomas. However, little is known about its function in esophageal carcinomas. We therefore examined the effects of MSP58 knockdown on the growth of esophageal squamous cell carcinoma (ESCC) cells in vitro and in vivo in order to gain a better understanding of its potential as a tumor therapeutic target. We employed lentiviral-mediated small hairpin RNA (shRNA) to knock down the expression of MSP58 in the ESCC cell lines Eca-109 and EC9706 and demonstrated inhibition of ESCC cell proliferation and colony formation in vitro. Furthermore, flow cytometry and western blot analyses revealed that MSP58 depletion induced cell cycle arrest by regulating the expression of P21, CDK4 and cyclin D1. Notably, the downregulation of MSP58 significantly inhibited the growth of ESCC xenografts in nude mice. Our results suggest that MSP58 may play an important role in ESCC progression.