Jianming Hu

Inactivation of miR-34a by aberrant CpG methylation in Kazakh patients with esophageal carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is an aggressive tumor with dismal prognosis and high incidence and mortality in Kazakh population. MiR-34a, a direct p53 target gene, possesses tumor-suppressive properties as they mediate apoptosis, cell cycle arrest, and senescence. The reduced expression of miR-34a by methylation in various cancers has been reported.MethodsTo determine whether aberrant miR-34a methylation occurs in esophageal cancer, the DNA methylation of 23 CpGs sites in the miR-34a promoter was quantitatively analyzed in relation to the translation initiation site by MALDI -TOF mass spectrometry in 59 ESCC tissues and 34 normal tissues from the Kazakh population. Real-time PCR was used to detect the inhibition of miR-34a expression levels and to evaluate their association with methylation.ResultsWe found that miR-34a is more frequently methylated in ESCC (0.133 ± 0.040) than in controls (0.066 ± 0.045, P < 0.01). A nearly two-fold increase in miR-34a expression for the hypomethylated promoter was found in normal esophageal tissues than ESCC with hypermethylation (P <0.0001), pointing to a negative relationship between miR-34a CpG sites methylation and expression(r = −0.594, P = 0.042). The hypermethylation of miR-34a CpG_8.9 was associated with the advanced UICC stage III/IV of the esophageal cancers, and the hypermethylation of CpG_8.9 and CpG_5 of miR-34a was significantly correlated with lymph node metastasis.ConclusionsOur findings suggest that miR-34a is involved in the etiology of ESCC and that hypermethylated miR-34a is a potential biomarker for ESCC diagnosis and prognosis. Moreover, targeting miR-34a methylation by demethylating agents may offer a novel strategy for anticancer therapy of ESCC.

Multiple polymorphisms within the PLCE1 are associated with esophageal cancer via promoting the gene expression in a Chinese Kazakh population.

Although recent genome-wide association studies of esophageal squamous cell carcinoma (ESCC) identified a susceptibility locus in phospholipase C epsilon 1 (PLCE1) in Chinese Han populations, few studies further confirmed these findings in pure Kazakh population in which there are higher incidence and mortality of ESCC. Here, we investigated the potential associations between 19 SNPs of PLCE1 and susceptibility to ESCC in 222 cases and 326 controls from a pure ethnic population of Kazakh. Real-time PCR and immunohistochemistry were performed to detect the PLCE1 expression levels and evaluate their association with PLCE1 polymorphism. We found that only 4 SNPs (rs753724, rs11187842, rs2274223, and rs12263737) with moderate linkage disequilibrium (LD) confer significantly increased risk of ESCC, with the ORs ranging from 1.43 to 2.04, and there was a risk allele dose-dependent increase in ESCC risk (P-trend=0.043). Especially, the risk effects of rs2274223 were more evident in poor differentiation and advanced clinical stages of Kazakh ESCC. Additionally, the significantly lowest PLCE1 mRNA expression was found in the KYSE-150 cell line having no risk alleles compared with other three cell lines having risk alleles, and the normal tissues of both homozygous mutant type of PLCE1 rs12263737 and rs2274223 had a higher PLCE1 staining score than that of homozygous wild type. Our findings suggested that genetic variants in PLCE1 might serve as candidate markers for Kazakh ESCC susceptibility, and these LD variants might influence ESCC risk individually and jointly by promoting the messenger RNA and protein expression of the gene.

Overexpression of PLCE1 in Kazakh esophageal squamous cell carcinoma: implications in cancer metastasis and aggressiveness

Three recent large‐scale genome‐wide association studies (GWAS) in Chinese Han populations have identified an esophageal squamous cell carcinoma (ESCC) susceptibility locus within phospholipase C epsilon 1 (PLCE1) gene, which encodes a phospholipase involved in intracellular signaling. The expressed PLCE1 in ESCC, however, are inconsistent. This study examined PLCE1 expression by immunohistochemistry (IHC) from 110 ethnic Kazakh ESCC patients and 50 from adjacent normal esophageal tissues (NETs). The expressed PLCE1 was localized in cytoplasm, especially in the peripheral layers of cancer cell nests, which was significantly higher in tumors than in NETs (p < 0.001). Increased expression of PLCE1 was correlated with advanced tumor‐node‐metastasis (TNM) stages (p = 0.015) and lymph node metastasis (p = 0.003) in patients with ESCC. Of the 110 patients, we examined 50 paired ESCC tissues and corresponding NETs by quantitative RT‐PCR (polymerase chain reaction) and the mean mRNA level of PLCE1 in ESCC was 1.85‐fold higher compared with those in corresponding NETs (p = 0.0012). Meanwhile, 4 of 5 ESCC cell lines also showed elevated expression of PLCE1 mRNA. Furthermore, elevated expression of PLCE1 mRNA in Kazakh ESCC was associated with its immunoreactivity (ρ = 0.297, p = 0.040), lymph node metastasis (p < 0.001), and advanced TNM stages of ESCC (p = 0.013). To our knowledge, this study demonstrates for the first time that PLCE1 overexpression correlates with lymph node metastasis and advanced TNM stages of Kazakh ESCC, implicating a role of PLCE1 in cancer metastasis and aggressiveness in ethnic Kazakh patients with ESCC. Furthermore, the current findings may warrant investigations into whether inhibiting PLCE1 could be a strategy for targeted anticancer therapy particularly for Kazakh ESCC.

Heterozygote of PLCE1 rs2274223 increases susceptibility to human papillomavirus infection in patients with esophageal carcinoma among the Kazakh populations

The involvement of human papillomavirus (HPV) in the carcinogenesis of esophageal squamous carcinoma remains undetermined. However, three genome‐wide association studies of esophageal cancer have identified a shared susceptibility locus at 10q23 (rs2274223: A5780G) in phospholipase C epsilon 1 (PLCE1). The current study aims to present a comprehensive and novel spectrum about the HPV genotype distribution of esophageal carcinoma in Kazakhs and assess its association with PLCE1 polymorphisms. The HPV genotypes in 183 patients with esophageal cancer and 89 controls selected from the Kazakh population were evaluated using the HPV gene chip. The PLCE1 rs2274223 variant was genotyped in esophageal carcinoma patients by MALDI‐ToF Mass Spectrometry. The presence of seven HPV genotypes in esophageal carcinoma tissues—including HPV 16, 18, 35, 52, 6, 11, 43—was significantly higher at 31.7% than those in controls at 9.0% (P < 0.001). Such presence was strongly associated with increased risk of esophageal carcinoma (OR 4.70; 95% CI 2.13–10.36). Among all HPV genotypes detected, HPV16 was the most common genotype identified (29.0%, OR 4.13; 95% CI 1.87–9.13), which is significantly associated with well‐differentiated esophageal carcinoma (P = 0.037). HPV‐positive patients were generally younger than HPV‐negative patients (70.1% vs. 29.3%, P = 0.013). PLCE1 rs2274223 genotypes AG and AG/GG were significantly associated with HPV‐positive patients with esophageal carcinoma (OR 2.05, 95% CI 1.03–4.08 and OR 1.98, 95% CI 1.02–3.84, respectively). These findings suggest that heterozygote of PLCE1 rs2274223 increases susceptibility to HPV infection in patients with esophageal carcinoma among the Kazakh populations. J. Med. Virol. 86:608–617, 2014.

Analysis of PTEN Methylation Patterns in Soft Tissue Sarcomas by MassARRAY Spectrometry

Soft tissue sarcomas (STSs) are a rare and fascinating group of diseases that can be subdivided into specific reciprocal translocations in STSs (SRTSs) and nonspecific reciprocal translocations in STSs (NRTSs). PTEN mutations are rare in STSs, suggesting that PTEN expression may be lost by alternative mechanisms such as methylation. In order to reveal whether aberrant PTEN methylation occurs in STSs, MassARRAY Spectrometry was carried to detect methylation patterns of PTEN in STSs. We evaluated methylation levels in 41 CpG sites from −2,515 to −2,186 bp (amplicon A) and −1,786 to −1,416 bp (amplicon B) relative to the translation initiation site in 110 different cases (46 cases of SRTSs, 40 cases of NRTSs, and 24 cases of normal controls). In addition, immunohistochemistry (IHC) was used to detect the loss of PTEN to determine whether PTEN alterations were responsible for decreased PTEN expression. Our data showed that expression of PTEN was diminished in 49 (57%) STSs, whereas the remaining cases (43%) were classified as high expression. Our previous results found that only 2 of 86 cases (2.3%) had a PTEN mutation suggesting that PTEN may be mainly downregulated in STSs by methylation, but not by mutation of PTEN itself. We observed that amplicon A was hypermethylated in STSs with low PTEN expression, whereas normal controls had low methylation levels (P<0.0001), which was not present in amplicon B (P>0.05), nor were there significant differences in the methylation levels in PTEN between SRTS and NRTS cases. The majority of individual CpG units within two amplicons was demonstrated to be hypermethylated. These findings indicate that PTEN hypermethylation is a common event in STSs suggesting that the inactivation of PTEN may be due to hypermethylation in the promoter of PTEN. The aberrant methylation of the CpG sites within PTEN promoter may serve as a potential candidate biomarker for STSs.

Targeting oncogenic PLCE1 by miR-145 impairs tumor proliferation and metastasis of esophageal squamous cell carcinoma

Phospholipase C epsilon 1 (PLCE1) is a susceptibility gene in esophageal squamous cell carcinoma (ESCC). Nevertheless, the role of PLCE1 in ESCC tumorigenesis has not been elucidated. In this study, we determined the function of PLCE1 and its regulatory microRNA (miRNA) in ESCC. PLCE1 protein was excessively expressed in ESCC and precancerous lesions compared with that in normal tissues. High PLCE1 expression levels in ESCC were significantly linked with poor overall survival. Knockdown of PLCE1 promoted the apoptosis, cytokine-induced apoptosis, and sensitivity of cancer cells to chemotherapeutic drugs but abrogated the proliferation and EMT phenotype of ESCC in vitro. Notably, miR-145 was newly identified as a potent repressor of PLCE1 expression by directly targeting the 3′UTR of PLCE1. MiR-145 also inhibited cell proliferation, migration, and metastasis, as well as controlled the cytoskeleton dynamics of esophageal cancer. Moreover, miR-145 was expressed at low levels in a large cohort of patients with ESCC and was inversely correlated with PLCE1 protein expression in cancer cells and tissues. These findings demonstrate that PLCE1 functions as tumor promoter in ESCC and can be suppressed by miR-145 through inhibition of PLCE1 translation. Hence, delivery of PLCE1-targeting miR-145 is a potential therapeutic approach for esophageal cancer.

ORAOV1 overexpression in esophageal squamous cell carcinoma and esophageal dysplasia: a possible biomarker of progression and poor prognosis in esophageal carcinoma ☆,☆☆

Oral cancer overexpressed 1 (ORAOV1) has been reported to exhibit high amplification levels in esophageal squamous cell cancer (ESCC) and in premalignant lesions. However, ORAOV1 protein expression levels in ESCC and esophageal squamous intraepithelial neoplasia (ESIN) have not yet been reported. We have explored the relationship of ORAOV1 protein expression with ESCC and ESIN by immunohistochemically analyzing tissue microarrays containing esophageal samples from patients with various clinical features and prognoses. The percentage of ESCC, high-grade ESIN (HGESIN), low-grade ESIN (LGESIN), and nontumoral control patients overexpressing ORAOV1 were 70.63% (101/143), 77.36% (41/53), 48.96% (47/96), and 5.79% (7/121), respectively. ORAOV1 overexpression also appears to be significantly higher in ESCC, HGESIN, and LGESIN than in the controls (all P < .001), and the levels observed for ESCC and HGESIN were also significantly higher than that in LGESIN (both P = .001). These results corresponded to high sensitivity and specificity values in ESCC, HGESIN, and LGESIN tissues. Furthermore, the increased expression of ORAOV1 is significantly associated with lymph node metastasis (P = .001) and an advanced TNM stage (III + IV) (P = .014), and patients with ORAOV1 overexpression experienced shorter overall survival time compared with those with lower ORAOV1 (χ(2) = 11.505, P = .001). This study provides the first evidence of ORAOV1 overexpression in ESCC and ESIN and demonstrates a potential role in tumor progression and metastasis. ORAOV1 overexpression could, therefore, be used as a novel biomarker of poor prognosis in patients with ESCC.

Membrane type 1-matrix metalloproteinase induces epithelial-to-mesenchymal transition in esophageal squamous cell carcinoma: Observations from clinical and in vitro analyses.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is associated with enhanced tumorigenicity in many cancers. A recent study has revealed that MT1-MMP induces epithelial-to-mesenchymal transition (EMT) in prostate and breast cancer cells. However, its role in esophageal squamous cell carcinoma (ESCC) has not been studied. Here, we investigated the role of MT1-MMP in the dissemination of ESCC. Expression of MT1-MMP was detected by immunohistochemistry and tissue microarray in 88 Kazakh ESCC patients. Western blotting was performed to detect endogenous and overexpressed exogenous MT1-MMP in the Eca109 and Eca9706 cell lines, respectively. Transwell assay was used to estimate MT1-MMP-induced invasion and metastasis. EMT-associated proteins were detected by immunohistochemistry and western blotting. The associations between the expression of MT1-MMP and EMT-associated proteins with clinicopathologic parameters were analyzed. Overexpression of MT1-MMP was confirmed in Kazakh ESCC patients. MT1-MMP levels were found to be correlated with the depth of tumor infiltration. MT1-MMP induced EMT in ESCC both in vivo and in vitro, N-cadherin and Vimentin expression was upregulated upon MT1-MMP transfection into cells. However, E-cadherin was found to be downregulated. MT1-MMP-induced EMT led to increase migration and invasion in ESCC cell lines. In conclusion, our results suggest that MT1-MMP promotes ESCC invasion and metastasis.

Prognostic value of PLCE1 expression in upper gastrointestinal cancer: a systematic review and meta-analysis.

BACKGROUND A number of studies have identified a shared susceptibility locus in phospholipase C epsilon 1 (PLCE1) for esophageal squamous cell carcinoma (ESCC) and gastric cardia adenocarcinomas (GCA). However, the results of PLCE1 expression in esophageal and gastric cancer remain inconsistent and controversial. Moreover, the effects on clinicopathological features remain undetermined. This study aimed to provide a precise quantification of the association between PLCE1 expression and the risk of ESCC and GCA through meta-analysis. MATERIALS AND METHODS Eligible studies were identified from PubMed, Wanfang Data, ISI Web of Science, and the Chinese National Knowledge Infrastructure databases. Using RevMan5.2 software, pooled odds ratios (ORs) with 95% confidence intervals (CIs) were employed to assess the association of PLCE1 expression with clinicopathological features relative to ESCC or GCA. RESULTS Seven articles were identified, including 761 esophageal and gastric cancer cases and 457 controls. Overall, we determined that PLCE1 expression was associated with tumor progression in both esophageal cancers (pooled OR=5.93; 95%CI=3.86 to 9.11) and gastric cancers (pooled OR=9.73; 95%CI=6.46 to 14.7). Moreover, invasion depth (pooled OR=3.62; 95%CI=2.30 to 5.70) and lymph node metastasis (pooled OR=4.21; 95%CI=2.69 to 6.59) were linked with PLCE1 expression in gastric cancer. However, no significant associations were determined between PLCE1 overexpression and the histologic grade, invasion depth, and lymph node metastasis in esophageal cancer. CONCLUSIONS Our meta- analysis results indicated that upregulated PLCE1 is significantly associated with an increased risk of tumor progression in ESCC and GCA. Therefore, PLCE1 expression can be appropriately regarded as a promising biomarker for ESCC and GCA patients.

Clinicopathological significance of ALDH1A1 in lung, colorectal, and breast cancers: a meta-analysis

AIM Aldehyde dehydrogenase 1 family member A1 (ALDH1A1) is a putative cancer stem cell marker. This meta-analysis evaluated ALDH1A1 expressions clinicopathological associations with lung cancer (LC), colorectal cancer (CRC) and breast cancer (BC). MATERIALS & METHODS Publications were retrieved from various databases and assessed for relevance and quality. Relationships between ALDH1A1 expression and clinicopathological characteristics were evaluated using Review Manager 5.2 software. RESULTS Thirty-eight studies were included (6057 patients). ALDH1A1 expression was significantly associated with the presence of LC; lymph node metastasis, clinical stage and differentiation in LC and BC; and molecular subtype in BC (p < 0.05). There were no significant association with BC tumor size and CRC. CONCLUSION ALDH1A1 may be a stem cell marker in LC and BC.