Cross talk between oxidative stress and hypoxia via thioredoxin and HIF-2α drives metastasis of hepatocellular carcinoma.

Oxidative stress and hypoxia are two opposite microenvironments involved in HCC metastasis. Thioredoxin (TXN) and hypoxia-inducible factor 2α (HIF-2α) are typical proteins involved in these two different microenvironments, respectively. How these two factors interact to influence the fate on tumor cells remains unknown. Hypoxia facilitated HCC cells withstood oxidative stress and eventually promoted HCC cells metastasis, in which TXN and HIF-2α were mostly involved. Upregulation of TXN/HIF-2α correlated with poor HCC prognosis and promoted HCC metastasis both in vitro and in vivo. Epithelial-mesenchymal transition (EMT) process was involved in TXN/HIF-2α-enhanced invasiveness of HCC cells. Additionally, the stability and activity of HIF-2α were precisely regulated by TXN via SUMOylation and acetylation, which contributed to HCC metastasis. Our data revealed that the redox protein TXN and HIF-2α are both associated with HCC metastasis, and the fine regulation of TXN on HIF-2α contributes essentially during the process of metastasis. Our study provides new insight into the interaction mechanism between hypoxia and oxidative stress and implies potential therapeutic benefits by targeting both TXN and HIF-2α in the treatment of HCC metastasis.

[Determination of human papillomavirus in oral leukoplakia,oral lichen planus and oral squamous cell carcinoma].

OBJECTIVE: To investigate the possibility for human papillomavirus (HPV) infection to be a predictable signal for the carcinogenesis of oral mucosa by comparing the prevalences of HPV in each stage of oral mucosal carcinogenesis and to compare the sensitivity differences of the two methods in detecting HPV infection in oral cavity. METHODS: The hybrid capture (HC-II) was used to detect infection of HPV in 255 samples taken from 12 cases of healthy oral mucosa, 211 cases of patients with pathological diagnosis and 32 cases of patients with clinical diagnosis. The diagnosed cases included 8 cases of benign lesions of the oral mucosa, precancerous lesions [74 cases of oral leukoplakia (OLK) with hyperplasia and 42 cases of OLK with oral epithelial dysplasia (OED)], 91 cases of precancerous condition [oral lichen planus (OLP)] and 28 cases of oral squamous cell carcinoma (OSCC). And in situ hybridization (ISH) was used to detect infection of HPV in 33 cases of OSCC and 76 cases of OLK, including 30 cases of hyperplasia, 15 cases of mild OED, 15 cases of moderate OED and 16 cases of severe OED. RESULTS: The prevalence of HPV in OLP samples was higher (12.12%, 8/66) than that of OLK (2.59%, 3/116) (χ(2)=4.666, P=0.031) and OSCC(7.14%, 2/28, χ(2)=0.513, P=0.474). The prevalence of HPV in OSCC (7.14%, 2/28) was higher than that of OLK (2.59%, 3/116), and no significant difference was found. There was only one case of smoke spot and statistical analysis was not carried out. ISH was used to detect type 16/18 and type 31/33 HPV DNA in 109 cases of oral mucosal lesions in paraffin sections and only one case of OSCC was HPV positive. Thirty-seven cases were detected by HC-II and ISH methods at the same time. The same negative results by the two methods were found in 94.6% samples (35/37). In the other two samples, one was OSCC with early infiltration and the other was OLK with hyperplasia, The HC-II results were positive while the ISH results were negative. The patients with OLP and HPV testing results were followed up and the average follow-up period was (36.2 ± 10.5) months. It was found that three of them had a malignant transformation, and the malignant transformation rate of HPV positive patients was 12.50% (1/8), which was higher than that of HPV negative patients (3.45%, 2/58), and the difference was not statistically significant, P=0.249. CONCLUSION: HC-II assay was more sensitive in detecting HPV infection of oral mucosal lesions than ISH. The results of this study showed that there was insufficient evidence for taking HPV infection as a predictor of OLK carcinogenesis. Patients suffering from OLP were in a precancerous condition. The prevalence of HPV in OLP patients of this study was higher than that in OLK and OSCC patients, suggesting that for some reason, OLP patients were susceptible to HPV. HPV testing can be considered as routine in patients with OLP, and HC-II assay was recommended. And patients with OLP and HPV positive should be followed up regularly.

[Downregulation of chromobox protein homolog 7 expression in multiple human cancer tissues].

OBJECTIVE: To investigate the relationship between chromobox protein homolog 7 (cbx7) expression and the occurrence and development of colorectal carcinoma (CRC), gastric carcinoma (GC) and hepatocarcinoma (HCC) tissues. METHODS: The samples of neoplastic tissues and the corresponding cutting-edge normal tissues from 22 cases of CRC, 20 cases of GC, 30 cases of HCC were surgically collected. Level of cbx7 mRNA was detected with a fluorescent quantitative RT-PCR assay, and the correlationship among expression of cbx7 mRNA, the patients' clinicopathologic features and the surviving time after surgery was analyzed. RESULTS: The relative copy number of cbx7 mRNA in carcinomas and the normal tissues was 0.010 ± 0.015 vs 0.053 ± 0.042 for CRCs, 0.197 ± 0.195 vs 1.891 ± 1.254 for GCs, and 0.008 ± 0.008 vs 0.030 ± 0.021 for HCCs, respectively. Compared with the corresponding normal tissues, cbx7 expression was significantly downregulated in CRCs, GCs, and HCCs (t = -7.351, -5.417 and -6.680, respectively, P < 0.01). The expression of cbx7 mRNA in CRCs had significant differences not only between two age groups (the relative copy number of cbx7 mRNA in age > 55 group was 0.007 ± 0.015, but 0.017 ± 0.012 in age ≤ 55 group, t = -2.586, P = 0.022); but also between vascular embolus-positive and negative groups (the level of cbx7 mRNA in positive and negative group was 0.022 ± 0.021 vs 0.006 ± 0.011, t = -3.175, P = 0.010). The area under the receiver operating characteristics (ROC) curve is 0.769 (P = 0.033). when the Cut-off value of the relative copy number of cbx7 mRNA was 0.002 in CRCs. The values less-than 0.002 were defined as low expression. The CRC patients with low expression of cbx7 had a shorter overall survival time; whose 5 years survival rate was only 30.8% (4/13); while the rate was 77.8% (7/9) in high expression of cbx7 group. The difference had statistical significance (χ(2) = 4.329, P = 0.037). The similar differences could not be found among GC and HCC patients. CONCLUSION: Downregulation of cbx7 expression was very common among multiple carcinomas cases, and the downregulation influenced the prognosis of CRC patients.

Impact of GFRA1 gene reactivation by DNA demethylation on prognosis of patients with metastatic colon cancer.

BACKGROUND: The expression of the membrane receptor protein GFRA1 is frequently upregulated in many cancers, which can promote cancer development by activating the classic RET-RAS-ERK and RET-RAS-PI3K-AKT pathways. Several therapeutic anti-GFRA1 antibody-drug conjugates are under development. Demethylation (or hypomethylation) of GFRA1 CpG islands (dmGFRA1) is associated with increased gene expression and metastasis risk of gastric cancer. However, it is unknown whether dmGFRA1 affects the metastasis of other cancers, including colon cancer (CC). AIM: To study whether dmGFRA1 is a driver for CC metastasis and GFRA1 is a potential therapeutic target. METHODS: CC and paired surgical margin tissue samples from 144 inpatients and normal colon mucosal biopsies from 21 noncancer patients were included in this study. The methylation status of GFRA1 islands was determined by MethyLight and denaturing high-performance liquid chromatography and bisulfite-sequencing. Kaplan-Meier analysis was used to explore the effect of dmGFRA1 on the survival of CC patients. Impacts of GFRA1 on CC cell proliferation and migration were evaluated by a battery of biological assays in vitro and in vivo. The phosphorylation of AKT and ERK proteins was examined by Western blot analysis. RESULTS: The proportion of dmGFRA1 in CC, surgical margin, and normal colon tissues by MethyLight was 68.4%, 73.4%, and 35.9% (median; nonparametric test, P = 0.001 and < 0.001), respectively. Using the median value of dmGFRA1 peak area proportion as the cutoff, the proportion of dmGFRA1-high samples was much higher in poorly differentiated CC samples than in moderately or well-differentiated samples (92.3%% vs 55.8%, Chi-square test, P = 0.002) and significantly higher in CC samples with distant metastasis than in samples without (77.8% vs 46.0%, P = 0.021). The overall survival of patients with dmGFRA1-low CC was significantly longer than that of patients with dmGFRA1-high CC (adjusted hazard ratio = 0.49, 95% confidence interval: 0.24-0.98), especially for 89 CC patients with metastatic CC (adjusted hazard ratio = 0.41, 95% confidence interval: 0.18-0.91). These data were confirmed by the mining results from TCGA datasets. Furthermore, GFRA1 overexpression significantly promoted the proliferation/invasion of RKO and HCT116 cells and the growth of RKO cells in nude mice but did not affect their migration. GFRA1 overexpression markedly increased the phosphorylation levels of AKT and ERK proteins, two key molecules in two classic GFRA1 downstream pathways. CONCLUSION: GFRA1 expression is frequently reactivated by DNA demethylation in CC tissues and is significantly associated with a poor prognosis in patients with CC, especially those with metastatic CC. GFRA1 can promote the proliferation/growth of CC cells, probably by the activation of AKT and ERK pathways. GFRA1 might be a therapeutic target for CC patients, especially those with metastatic potential.

Promoter methylation of p16 associated with Helicobacter pylori infection in precancerous gastric lesions: a population-based study.

To investigate the relationship between p16 methylation and Helicobacter pylori infection in precancerous gastric lesions, a population-based study was conducted in Linqu County, a high-risk area of gastric cancer in China. Methylation status of p16 was evaluated by methylation-specific polymerase chain reaction in 920 subjects with precancerous gastric lesions. H. pylori status was determined by 13C-urea breath test and the density of H. pylori in biopsy specimens used for detecting methylation status was assessed by the modified Giemsa stain. The frequency of p16 methylation was significantly higher in subjects with H. pylori positive than those with H. pylori negative in each category of gastric lesion (p<0.001, respectively). Compared with H. pylori negative, the odds ratios (ORs) of p16 methylation were markedly elevated in subjects with H. pylori positive for superficial gastritis (OR, 9.45; 95% confidence interval [CI]: 2.94-30.41), chronic atrophic gastritis (OR, 15.92; 95%CI: 7.60-33.36), intestinal metaplasia (OR, 4.46; 95%CI: 2.44-8.13), indefinite dysplasia (OR, 3.67; 95%CI: 1.90-7.10), and dysplasia (OR, 2.48; 95%CI: 1.02-5.99). Moreover, the frequencies of p16 methylation increased steadily with the severity of H. pylori density in gastric mucosa. Compared with H. pylori negative, the OR of p16 methylation was 1.02-16.13 times higher in subjects with mild H. pylori infection, and 2.69-38.73 times higher in those with moderate/severe infection, respectively. Our findings indicate that p16 methylation was significantly associated with H. pylori infection in precancerous gastric lesions, suggesting that H. pylori infection could potently induce methylation of p16 CpG island.

[Effects of methylation status of CpG islands on results of luciferase reporter assay].

OBJECTIVE: To investigate the effects of methylation status of CpG islands of endogenous E-cadherin (CDH1) gene on the promoter activity of corresponding genes in reporter assays. METHODS: The methylation statuses of CpG island of CDH1 in 8 different cell lines were detected by methylation-specific PCR. CDH1 protein was analyzed by Western blotting. Two sets of pGL3 reporter vectors with different genotypes/haplotypes of the CDH1 promoter were constructed [ pGL3-A(-73)/-C(-73) pGL3-H1/-H4] and used to transfect these cell lines. The differences between these promoter reporter vectors were analyzed by t-test. RESULTS: (1) CDH1 CpG island was unmethylated in AGS, MCF7, MKN74, and PC-3 cell lines, expressed in MCF7, MKN74, and PC-3, but not in AGS. Expression of CDH1 was silenced by methylation in HeLa, BGC823, A549, and RKO cell lines. (2) In the four CDH1-unmethylated MCF7, MKN74, PC-3, and AGS cell lines, the promoter activities of pGL3-C(-73), (as 0.78 +/- 0.10, 0.17 +/- 0.01, 0.11 +/- 0.01, 1.19 +/- 0.18) were significantly higher than those of pGL3-A(-73) (as 0.30 +/- 0.08, 0.07 +/- 0.01, 0.07 +/- 0.01, 0.39 +/- 0.04) (t values are -6.298, - 12.349, -8.128, -7.388, and P <. 0.1). However, in the four CDH1-methylated HeLa, BGC823, A549, and RKO cell lines, the promoter activity of pGL3-C(-73) (as 0.09 +/- 0.02, 0.13 +/- 0.02, 0.05 +/- 0.01, 0.01 +/- 0.00) was significantly lower than that of pGL3-A(-73) (as 0.16 +/- 0.01, 0.25 +/- 0.01, 0.11 +/- 0.03, 0.03 +/- 0.00) (t valued at 5.958, 11.189, 3.661, 13.866, and P<0.05). (3) In the unmethylated MKN74 and methylated RKO cell lines, the promoter activities of pGL3-H1/-H4 were obviously and contrarily different (as 1.57 +/- 0.23/0.94 +/- 0.06 and 0.38 +/- 0.02/0.50 +/- 0.04, t values were 4.577 and -4.915, P values were 0.010 and 0.003). CONCLUSION: The methylation status of CpG island of the target gene in the tested cell lines affects the promoter activity in Reporter Assay significantly. The most active one may be the most suppressive one.

[Increase of saliva nitrate and nitrite level in patients with oral candidiasis].

OBJECTIVE: To observe the alterations of saliva nitrate and nitrite level in patients with oral candidiasis. METHODS: Parotid saliva and whole saliva were collected from 33 patients and 34 healthy volunteers. Concentrations of nitrate and nitrite in saliva were determined by high-performance liquid chromatography. Follow-up observation was performed on 10 patients after treatment. The data were statistically analyzed with independent-samples t test or paired-samples t test at alpha = 0.05. RESULTS: There was significant increase of the concentrations and secretion rate of parotid saliva nitrate in patient group as compared with controls: (49.70 +/- 0.50) vs (21.51 +/- 0.60) mg/L (t = 2.692, P = 0.009) and (27.71 +/- 0.50) vs (12.55 +/- 0.60) microg/min (t = 2.554, P = 0.013), respectively. Significantly increased concentrations and secretion rate of nitrate and nitrite [nitrate: (6.46 +/- 0.94) vs (1.11 +/- 0.70) mg/L (t = 3.792, P = 0.000); nitrite: (8.48 +/- 0.58) vs (3.39 +/- 0.53) mg/L (t = 2.888, P = 0.005); nitrate secretion rate: (10.57 +/- 0.91) vs (2.10 +/- 0.74) microg/min (t = 3.464, P= 0.001); nitrite secretion rate: (13.91 +/- 0.55) vs (6.42 +/- 0.58) microg/min (t = 2.397, P = 0.020)] were revealed in whole saliva of patients group. Significantly decreased nitrate and nitrite levels were also observed in patients after treatment, especially the changes of parotid saliva nitrate secretion rate [(37.50 +/- 0.50) vs (14.34 +/- 0.64) microg/min (t = 3.142, P = 0.012)], whole saliva nitrate [(14.29 +/- 1.01) vs (2.59 +/- 1.03) mg/L (t = 3.475, P = 0.007)] and whole saliva nitrate secretion rate [(25.97 +/- 0.93) vs (4.12 +/- 1.00) microg/min (t = 3.922, P = 0.003)]. CONCLUSION: The present study revealed the significant increase of salivary nitrate and nitrite level in patients with oral candidiasis is considered to be associated with the host defense reaction.

Telomere Length of Circulating Cell-Free DNA and Gastric Cancer in a Chinese Population at High-Risk.

Background: Telomeres have long been found to be involved in cancer development, while little was known about the dynamic changes of telomere length in carcinogenesis process. Methods: The present study longitudinally investigated telomere alterations of cell-free DNA (cfDNA) in 86 gastric cancer (GC) subjects recruited through a 16-year prospective cohort with 2-4 serums collected before each GC-diagnosis from baseline and three follow-up time-points (a total of 276 samples). As the control, 86 individual-matched cancer-free subjects were enrolled with 276 serums from the matched calendar year. Results: In the 73 pairs of baseline serums from GC and control subjects, shortened telomeres showed increased subsequent GC risk [odds ratio (OR) = 9.17, 95% CI: 2.72-31.25 for 1 unit shortening]. In each baseline gastric lesion category, higher risks of GC progression were also found with shortened cfDNA telomeres; ORs per 1 unit shortening were 6.99 (95% CI: 1.63-30.30) for mild gastric lesions, 6.06 (95% CI: 1.89-19.61) for intestinal metaplasia and 15.63 (95% CI: 1.91-125.00) for dysplasia. With all measurements from baseline and follow-up time-points, shortened telomeres also showed significant association with GC risk (OR = 7.37, 95% CI: 2.06-26.32 for 1 unit shortening). In temporal trend analysis, shortened telomeres were found in GC subjects compared to corresponding controls more than 3 years ahead of GC-diagnosis (most P < 0.05), while no significant difference was found between two groups within 3 years approaching to GC-diagnosis. Conclusion: Our findings suggest that telomere shortening may be associated with gastric carcinogenesis, which supports further etiological study and potential biomarker for risk stratification.

[The correlative analysis of indefinite dysplasia and dysplasia, and its p16 methylation in human gastric mucosa with Helicobacter pylori infection].

OBJECTIVE: To compare the CpG island methylation status of p16 in subjects with gastric indefinite dysplasia or dysplasia, and investigate its association with exposure factors. METHODS: Methylation status of p16 was determined by methylation specific PCR method and denaturing high-performance liquid chromatography analysis in 223 indefinite dysplasia and 130 dysplasia from Linqu County in Shandong Province, an area with high GC mortality rates. Multivariable analysis was used to analyze the relationship between p16 methylation and age, gender, cigarette smoking, alcohol drinking, and Helicobacter pylori infection. RESULTS: The methylation frequency of p16 in indefinite dysplasia was not significantly different from that of dysplasia (28.3% vs 24.6%, P=0.46). No significant association was found between p16 methylation and age, gender, cigarette smoking, and alcohol drinking. In dysplasia, the association between p16 methylation and Helicobacter pylori infection was close to the statistical significance by univariate analysis that the relative risk of p16 methylation of the infected subjects(n=93) is higher than that of the uninfected subjects (n=37)(OR=2.62, 95% CI: 0.92 to 7.43, P=0.07).But by multivariate analysis, there was no association between them(P=0.11). CONCLUSION: The frequency of p16 methylation in gastric mucosa of indefinite dysplasia was similar to that of dysplasia. p16 methylation status was not associated with age, but might be significantly associated with Helicobacter pylori infection.

[Analysis of demethylation-related HPV16 reactivation by DHPLC-primer extension assay].

OBJECTIVE: To establish a sensitive assay to detect methylation status of the critical 7862nt CpG site related to transcription of HPV16 E6 and E7 genes. METHODS: Genomic DNA of two HPV16-infected cell line SiHa and CaSki was extracted and modified by sodium bisulfite to convert the unmethylated Cs to Us (Ts in PCR products). The target sequence of HPV16 including the 7862nt CpG site was pre-amplified by PCR. Then, the methylation status of the 7862nt site was differentiated in a primer extension reaction with an HPV16-specific primer, and separated by DHPLC at 80 degrees C. RESULTS: The primers without extension and with extension, whether matched to CpG or TpG, could be separated by DHPLC completely. The peak for ddTTP-extension products corresponding to the demethylated CpG site was observed at retention time 6.7 min in both cell lines. However, the peak for ddCTP-extension products representing the methylated CpG site could be detected at retention time 6.3 min in CaSki cell line only, which integrated with 499 methylated and one demethylated HPV16 copies. CONCLUSION: The established DHPLC-primer extension assay can be used to detect methylated and demethylated HPV16 copies simultaneously with a sensitivity up to 1/500 (0.2%).

[Quantification of methylation of SNCG CpG islands in human tissue samples by the combined COBRA-DHPLC assay].

OBJECTIVE: To setup a quantitative assay for detection of methylation of SNCG CpG island in human tissue samples. METHODS: Methylation status of the 16 tested CpG sites within the CpG island was analyzed by bisulfite-clone-sequencing for 2 gastric carcinoma cell lines, 2 normal gastric mucosa samples, and 2 pairs of primary gastric carcinomas and their corresponding non-neoplastic tissues, respectively. RESULTS: The methylation of -88 and other four CpG sites was well correlated with the methylation of the overall CpG island. Thus, a combined bisulfite-restriction assay (COBRA) was developed based on the enzyme AciI, which digested the only one GCGG sequence in the PCR products of the methylated CpG island, but not the GTGG in the demethylated one. The digested fragments (144 bp and 85 bp) and undigested fragment (229 bp) could be completely separated by denaturing high performance liquid chromatography (DHPLC). According to the peak areas of these fragments, the proportion of the methylated copies of the SNCG CpG island was calculated easily. The result of the COBRA-DHPLC assay was reproducible and consistent with that of clone-sequencing. CONCLUSION: A COBRA-DHPLC assay is setup successfully for quantification of methylation of the SNCG CpG island.

[Methylation specific binding activity of zinc finger protein Kaiso].

OBJECTIVE: To compare binding activity of different zinc finger domain of human Kaiso with methylated CpG. METHODS: pGEX constructs with different human Kaiso domain were generated and then corresponding fusion proteins were induced and purified. Electrophoretic mobility shift assays were applied to evaluate the binding activity of fusion proteins with methylated CpG. RESULTS: The purified GST-KaisoZF fusion protein (without the POZ protein binding domain) could bind with methylated CpG probe specifically, but not for three or two zinc fingers without flanking domains. CONCLUSION: Human zinc finger protein Kaiso could bind with methylated CpG specifically, only in the assistance of the neighboring flank sequence of the zinc finger domain.

[Aberrant methylation of APC and Bikunin CpG islands in sporadic breast carcinomas].

OBJECTIVE: To investigate relationship between methylation status of the APC and Bikunin CpG islands and clinicopathological characteristics of breast carcinomas. METHODS: The methylation status of APC and Bikunin CpG islands in 152 sporadic breast carcinoma samples were analyzed by methylation specific PCR. RESULTS: 40.8% of breast carcinomas examined showed methylated signals for the APC. The methylation frequency of APC was significantly correlated to the tumor size (chi(2) = 4.041; P = 0.044), but not to patients' age, pathologic type of tumor, clinical stage, histological grade, lymph node metastasis and the status of estrogen or progestogen receptor. In addition, 24.6% of carcinoma samples examined revealed strong methylated signals for Bikunin. No significant correlation was found between the aberrant methylation of Bikunin and the clinicopathological characteristics of sporadic breast carcinomas. CONCLUSION: The aberrant methylations of APC and Bikunin are frequent events during breast carcinogenesis. APC methylation might play a role in the progression of breast cancer.

[Effect of selenium-enriched garlic on chronic gastritis of the glandular stomach of Mongolian gerbils induced by H. pylori].

OBJECTIVE: To detect the therapeutic effect of selenium-enriched garlic (SeG) on chronic gastritis. METHODS: Chronic gastritis was induced of the glandular stomach of male Mongolian Gerbils via gastric instillation of H. pylori TN2 strain once every 4 days for 5 consecutive times followed by random classification into six groups. Fresh SeG suspension was administrated daily at dosages of 4.70, 1.5, 0.47, 0.15 g.kg(-1).d(-1) for four weeks. The gerbils in the positive control group were treated with omeprazole, clarithromycin, and amoxicillin for one week. The gerbils were killed for pathological examination four weeks after SeG-treatment. RESULTS: Chronic gastritis (CAG), low-grade dysplasia or gastric intraepithelial neoplasia (DYS/GIN) were observed among 77% and 38.5% of the 13 H. pylori-treated animals in the negative control group, respectively; whereas 40% and 26.7% in the positive control group (n = 15), respectively. The incidences of CAG and DYS/GIN in the SeG groups (n = 21 - 27) were reduced dose-dependently, 16.7% - 38.7% and 11.1% - 14.3% for CAG and DYS/GIN, respectively. CONCLUSION: SeG administration inhibits the development and progression of CAG induced by H. pylori remarkably.

[Relationship between myocyte proliferation and cardiac function in adult rats with heart failure].

OBJECTIVE: To confirm whether there is myocytes proliferation in the adult rat with heart failure or not, and to investigate the relationship between myocyte proliferation and heart function. METHODS: Descending anterior branch of left coronary artery was ligated in 20 adult male SD rats so as to establish an heart failure models. Eight rats were used as controls. Hemodynamic parameters, blood pressure (BP), left ventricle end systolic pressure (LVESP), left ventricle end diastolic pressure (LVEDP), +LVdp/dt(max), and -LVdp/dt(max), were measured 30 days after the coronary occlusion. Based on the results of heart function examination, the heart infarct rats were divided into 2 subgroups: cardiac functional compensation subgroup (8 rats), and cardiac functional decompensation subgroup (6 rats). Then the rats were killed and their hearts were taken out and stained with propidium iodide (PI) and antibody to alpha-sarcomeric actin. Immunohistochemistry was used to detect the proliferation cell nuclear antigen (PCNA). Confocal microscopy was used to observe the mitotic image. Light microscopy was used to observe the PCNA positive rate in the myocardium. RESULTS: (1) Mitotic images of myocytes could be identified by confocal microscopy in the left ventricle of all rats. (2) PCNA expression was detected in the nuclei of both infarct and normal hearts. The PCNA positive rate of the cardiac functional compensation subgroup was 7.2% +/- 1.4%, significantly higher than that of the control group (2.2% +/- 0.8%, P = 0.648). However, the PCNA positive rate of the cardiac functional decompensation subgroup was 3.0% +/- 1.3%, not significantly different from that of the control group (P = 0.648). (3) The correlation coefficient between PCNA-positivity of cardiomyocytes and +LVdp/dt(max) in the infarct rats were 0.80 (P < 0.01) and the correlation coefficient between PCNA-positivity of cardiomyocytes and -LVdp/dt(max) was -0.76 (P = 0.01). CONCLUSION: (1) There is myocyte proliferation in the adult rat heart. (2) Myocyte proliferation is positively correlated with heart systolic function, and negatively correlated with heart diastolic function in chronic heart failure.

Genome-wide DNA methylation profiles altered by Helicobacter pylori in gastric mucosa and blood leukocyte DNA.

PURPOSE: To investigate Helicobacter pylori (H.pylori) associated genome-wide aberrant methylation patterns in gastric mucosa and blood leukocyte DNA, a population-based study was conducted in Linqu County. RESULTS: A total of 3000 and 386 CpGs were differentially methylated after successful H.pylori eradication in gastric mucosa and blood leukocyte DNA respectively, and 17 were the same alteration trend in the both tissues. The differentially methylated CpGs were located more frequently in promoters or CpG islands for gastric mucosa and gene body or open sea for blood leukocyte DNA. In eradicated gastric mucosa, the hypermethylated CpGs were enriched across inflammatory pathways, while the hypomethylated CpGs in tube morphogenesis, development and so on. The final validation found lower SPI1, PRIC285 and S1PR4 methylation levels in H.pylori positive subjects by case-control comparison, and increased methylation levels in H.pylori eradicated gastric mucosa by self-comparison. The Cancer Genome Atlas (TCGA) database analysis suggested that the up-regulation of the three genes by hypomethylation might be associated with gastric carcinogenesis. EXPERIMENTAL DESIGN: Infinium HumanMethylation 450K BeadChip was used to compare methylation profiles prior to and after eradication treatment. The methylation levels of identified candidate differentially methylated genes before and after H.pylori eradication were further validated by two stages (Stage I: self-comparison of 16 subjects before and after anti-H.pylori treatment; Stage II: case-control comparison of 25 H.pylori positive and 25 negative subjects and self-comparison of 50 anti-H.pylori treated subjects). CONCLUSIONS: Novel H.pylori associated aberrant methylated genes were identified across the whole genome both in gastric mucosa and blood leukocyte DNA.

Expression of ECRG4, a novel esophageal cancer-related gene, downregulated by CpG island hypermethylation in human esophageal squamous cell carcinoma.

AIM: To study the mechanisms responsible for inactivation of a novel esophageal cancer related gene 4 (ECRG4) in esophageal squamous cell carcinoma (ESCC). METHODS: A pair of primers was designed to amplify a 220 bp fragment, which contains 16 CpG sites in the core promoter region of the ECRG 4 gene. PCR products of bisulfite-modified CpG islands were analyzed by denaturing high-performance liquid chromatography (DHPLC), which were confirmed by DNA sequencing. The methylation status of ECRG 4 promoter in 20 cases of esophageal cancer and the adjacent normal tissues, 5 human tumor cell lines (esophageal cancer cell line-NEC, EC109, EC9706; gastric cancer cell line- GLC; human embryo kidney cell line-Hek293) and 2 normal esophagus tissues were detected. The expression level of the ECRG 4 gene in these samples was examined by RT-PCR. RESULTS: The expression level of ECRG 4 gene was varied. Of 20 esophageal cancer tissues, nine were unexpressed, six were lowly expressed and five were highly expressed compared with the adjacent tissues and the 2 normal esophageal epithelia. In addition, 4 out of the 5 human cell lines were also unexpressed. A high frequency of methylation was revealed in 12 (8 unexpressed and 4 lowly expressed) of the 15 (80 %) downregulated cancer tissues and 3 of the 4 unexpressed cell lines. No methylation peak was observed in the two highly expressed normal esophageal epithelia and the methylation frequency was low (3/20) among the 20 cases in the highly expressed adjacent tissues. The methylation status of the samples was consistent with the result of DNA sequencing. CONCLUSION: These results indicate that the inactivation of ECRG 4 gene by hypermethylation is a frequent molecular event in ESCC and may be involved in the carcinogenesis of this cancer.

Rapid screening mitochondrial DNA mutation by using denaturing high-performance liquid chromatography.

AIM: To optimize conditions of DHPLC and analyze the effectiveness of various DNA polymerases on DHPLC resolution, and evaluate the sensitivity of DHPLC in the mutation screening of mitochondrial DNA (mtDNA). METHODS: Two fragments of 16s gene of mitochondrial DNA (one of them F2 is a mutant fragment) and an A3243G mutated fragment were used to analyze the UV detection limit and determine the minimum percentage of mutant PCR products for DHPLC and evaluate effects of DNA polymerases on resolution of DHPLC. Under the optimal conditions, we analyzed the mtDNA mutations from muscle tissues of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and screened blindly for variances in D-loop region of mtDNA from human gastric tumor specimen. RESULTS: Ten A3243G variants were detected in 12 cases of MELAS, no alterations were detected in controls and these results were consistent with the results obtained by analysis of RFLP with ApaI. We also identified 26 D-loop variances in 46 cases of human gastric cancer tissues and 38 alterations in 13 gastric cancer cell lines. The mutation of mtDNA at 80 ng PCR products containing a minimum of 5% mutant sequences could be detected by using DHPLC with UV detector. Moreover, Ampli-Taq Gold polymerase was equally as good as the proofreading DNA polymerase (e.g., Pfu) in eliminating the false positive produced by Taq DNA polymerases. CONCLUSION: DHPLC is a powerful, rapid and sensitive mutation screening method for mtDNA. Proofreading DNA polymerase is more suitable for DHPLC analysis than Taq polymerase.

Silencing-specific methylation and single nucleotide polymorphism of hMLH1 promoter in gastric carcinomas.

AIM: To investigate CpG methylation and single nucleotide polymorphism (SNP) of a specific promoter region of hMLH1 in primary gastric carcinoma. METHODS: Primary gastric carcinomas (n=80), their corresponding normal mucosal samples, and gastric mucosal biopsies from normal/gastritis control patients (n=54) were used. Hypermethylation at -253 nt and -251 nt in relation with the translational start site and SNP of a silencing specific region (-339 nt-46 nt) in the hMLH1 promoter were analyzed by Bst UI-combined bisulfite assay (COBRA), denaturing high performance liquid chromatogram (DHPLC), and sequencing. RESULTS: (A) The specific methylation at -253 nt and -251 nt was observed in 2 of 60 primary gastric carcinomas, but neither in all of the corresponding mucosa nor in normal/gastritis samples, by Bst UI-COBRA and DHPLC. (B) The hMLH1 promoter was methylated homogeneously in the xenograft of the primary gastric carcinoma with the methylated and unmethylated hMLH1. (C) The pattern of SNP at -93 nt of the hMLH1 promoter in 54 Chinese patients with gastric carcinoma was the same as that in the control patients: 51 % was A/G heteroalleles, 34 % and 15 % were A/A and G/G homoalleles, respectively. CONCLUSION: Biallelic inactivation of hMLH1 by epigenetic silencing existed in human primary gastric carcinoma homogeneously. Hypermethylation of hMLH1 may play a role in the early stage of development of a few gastric carcinomas. The SNP at -93 nt is not related to the susceptibility of gastric carcinomas.

Methylation of GATA-4 and GATA-5 and development of sporadic gastric carcinomas.

AIM: To understand the implication of GATA-4 and GATA-5 methylation in gastric carcinogenesis. METHODS: Methylation status of GATA-4 and GATA-5 CpG islands in human gastric mucosa samples, including normal gastric biopsies from 45 outpatients, gastric dysplasia [low-grade gastric intraepithelial neoplasia (GIN), n = 30; indefinite, n = 77], and 80 paired sporadic gastric carcinomas (SGC) as well as the adjacent non-neoplastic gastric tissues was analyzed by methylation specific polymerase chain reaction (MSP) and confirmed by denatured high performance liquid chromatography (DHPLC). Immunohistochemical staining was used to detect protein expression. The correlation between GATA-4 and GATA-5 methylation and clinicopathological characteristics of patients including Helicobacter pylori (H. pylori) infection was analyzed. RESULTS: GATA-4 and GATA-5 methylation was frequently observed in SGCs (53.8% and 61.3%, respectively) and their corresponding normal tissues (41.3% and 46.3%) by MSP. The result of MSP was consistent with that of DHPLC. Loss of both GATA-4 and GATA-5 proteins was associated with their methylation in SGCs (P = 0.01). Moreover, a high frequency of GATA-4 and GATA-5 methylation was found in both gastric low-grade GIN (57.1% and 69.0%) and indefinite for dysplasia (42.9% and 46.7%), respectively. However, GATA-4 and GATA-5 methylation was detected only in 4/32 (12.5%) and 3/39 (7.7%) of normal gastric biopsies. GATA-4 methylation in both normal gastric mucosa and low-grade GIN was also significantly associated with H. pylori infection (P = 0.023 and 0.027, two-sides). CONCLUSION: Epigenetic inactivation of GATA-4 (and GATA-5) by methylation of CpG islands is an early frequent event during gastric carcinogenesis and is significantly correlated with H. pylori infection.