[Clinical Study of the Impact of ACE I/D Gene Variation on the Clinical Parameters of Patients with Polycystic Ovary Syndrome].

OBJECTIVE: To investigate the relationship between angiotensin Ⅰ-converting enzyme (ACE) insertion/deletion (I/D) gene polymorphism and the genetic risks for polycystic ovary syndrome (PCOS) and to evaluate the impact of ACE I/D genotypes on clinical, hormonal, metabolic and oxidative stress parameters in patients with PCOS. METHODS: This was a retrospective case-control study involving a total of 1 020 PCOS patients and 825 female controls who visited the outpatient clinic of the Department of Reproductive Endocrinology, West China Second Hospital of Sichuan University between 2006 and 2019. The ages of the subjects ranged between 17 and 44. The ACE I/D genotypes were determined by polymerase chain reaction (PCR) and gel electrophoresis. 667 PCOS patients and 527 controls were selected for an analysis of their genotypes and the hormonal, metabolic and oxidative stress parameters. RESULTS: The genotype distributions of the ACE I/D single nucleotide polymorphism was in Hardy-Weinberg equilibrium in both the PCOS group and the control group (all P>0.05), which was representative of the population. There were no statistically significant differences in genotype and allele frequencies between the PCOS and the control groups ( P>0.05). After adjusting for both age and body mass index (BMI), there was no statistically significant difference in clinical characteristics among all genotypes in either the PCOS group or the control group. In the PCOS group, compared with the II genotype subgroup, the ID genotype subgroup had lower luteinizing hormone (LH)/follicle-stimulating hormone (FSH) ratio, while the DD genotype subgroup had higher homeostatic model assessment of insulin resistance (HOMA-IR) and malondialdehyde (MDA) levels. Compared with the ID genotype subgroup, the DD genotype subgroup had lower serum sex hormone binding globulin (SHBG) level, but higher total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels ( P<0.05). In the control group, II genotype subgroup had a higher level of total oxidant status (TOS) than that of the DD genotype subgroup. CONCLUSION: ACE I/D genetic polymorphism is not associated with risks for PCOS. The I/D variation of ACE gene may be related to insulin resistance, dyslipidaemia, hyperandrogenemia and oxidative stress in PCOS patients.

[The Effect of Methylation Level of microRNA Promoter on the Expression of microRNAs and on the Proliferation, Migration and Invasion of Lung Cancer Cells].

OBJECTIVE: To study the effect of methylation level of microRNA promoter on the expression of microRNAs (miRNA34a, miRNA34b, miRNA148a, miRNA203a) and on the proliferation, migration and invasion of lung cancer A549 cells. METHODS: The proliferation of A549 cells treated with different concentrations of demethylated drug 5-aza-2'-deoxycytidine (5-Aza-CdR) was measured by CCK8 assay and calculated the inhibitory rate in 24 h, 48 h and 72 h, respectively. After 72 h of treatment with 20 µmol/L 5-Aza-CdR, methylation-specific PCR (MSP) was used to detect the methylation level of A549 cells in miRNAs gene promoter regions, and real-time quantitative PCR (real-time PCR) was used to test the expression of miRNAs. The migration abilities of A549 cells treated with 20 µmol/L 5-Aza-CdR in 24 h and 48 h were performed with wound healing assay, while the invasion abilities in 48 h were evaluated by Transwell assay, respectively. RESULTS: The proliferation inhibition rate of A549 cells gradually increased with the treatment concentration of 5-Aza-CdR increased and the treatment time prolonged. Compared with the control group, the methylated band of the experimental group was weaker and the unmethylated band was stronger, and the miRNAs gene promoter regions methylation level of the experimental group was lower than that of the control group. The expression level of miRNAs was significantly increased in the experimental group (P<0.05) . The migration and invasion of the experimental group of A549 cells were inhibited compared with the control group (P<0.05) . CONCLUSION: 5-Aza-CdR can reverse methylation levels of miRNAs promoter regions and upregulate the expression level of miRNA34a, miRNA34b, miRNA148a, miRNA203a, resulting in significantly inhibiting the proliferation, migration and invasion of lung cancer cells.

[The Study of Methylated Regulation MicroRNA in Pancreatic Carcinoma Cell and Its Effect on Cell Proliferation,Migration and Invasion].

OBJECTIVE: To study the epigenetic regulation of pancreatic carcinoma related microRNA (miR34a,miR34b,miR148a and miR203a) expression by gene promoter methylation,and its effect on the proliferation,migration and invasion of pancreatic carcinoma cells. METHODS: The pancreatic carcinoma cells were divided into two groups:control group and treatment group.Control group was treated with 0 µmol/L DNA methyltransferase inhibitor 5-Aza-CdR and treatment group was treated with 60 µmol/L 5-Aza-CdR. The methylation status of microRNA gene promoter regions was detected by MSP (methylation-specific PCR). The microRNAs' expression levels were evaluated by real-time PCR. The CCK-8 assay,wound healing assay and Transwell assay were employed to study the proliferation,migration and invasion of pancreatic carcinoma cells,respectively. RESULTS: The results of MSP showed that the methylated band of the treated group was weaker than that of the untreated group and the unmethylated band of the treated group was stronger than that of the untreated group. Real-time PCR results showed that the relative expression levels of microRNAs in the treatment group were higher than those in the control group ( P<0.05). The CCK-8 assay showed that inhibition rate of the treatment group showed dose-dependent effect with the increase of drug concentration. Wound healing assay showed that the wound healing rate of Treatment group was lower than that of untreated group ( P<0.01). The results of transwell assay showed that the number of migrated cells in the treated group was less than that in the untreated group ( P<0.01). CONCLUSION: Decreased methylation levels in microRNA promoter region caused by 5-Aza-CdR treatment increased the expression of miR34a,miR34b ,miR148a and miR203a,leading to inhibition of the proliferation,migration and invasion of pancreatic carcinoma cells.

[Study on the Effect of Overexpression of miR-18a on Cellular Proliferation and Migration by Targeting ATM in Human Colorectal Cancer Cells].

OBJECTIVES: To study the regulation to colon cancer cellular biological properties through miR-18a targeting ataxia-telangiectasia mutated gene (ATM). METHODS: A target of miR-18a was predicted by using bioinformatics tools. The miR-18a mimics and inhibitors were designed and synthesized. The expression of endogenous miR-18a in colon cancer cell line HCT116 was up-regulated or down-regulated by transfection. The effect of overexpression of miR-18a on cellular proliferation, invasion and migration via regulation of ATM gene expression was confirmed in vitro by using qRT-PCR, Western blot, MTT assay, clone forming assay and Transwell method, respectively. RESULTS: ATM was identified as a potential target gene of miR-18a in the bioinformatics analysis. In addition, through transient transfection leading to the overexpression of miR-18a in HCT116 cell, the expression level of ATM was decreased. Down-regulation of HCT116 cell proliferation activity while significantly reducing HCT116 cell clone forming ability, lateral migration ability and longitudinal invasion ability were observed after transfected with miR-18a mimics. All of the changes were related to the overexpression of miR-18a. CONCLUSIONS: miR-18a inhibited the proliferation and migration of colon cance cell HCT116 through negative regulation of ATM expression.

[MiRNA224 Regulation Role on RKIP Gene Expression and Methylation of miRNA224 Gene Promoter Region in Esophageal Squamous Cell Carcinoma].

OBJECTIVES: To study the expression levels of tumor suppressor gene RIKP and miRNA224 in esophageal squamous cell carcinoma (ESCC) tissues. To determine whether miRNA224 targets RKIP and the methylation status of miRNA224 gene promoter region in esophageal carcinoma. METHODS: The expression levels of RKIP and miRNA224 in ESCC and normal tissue were detected by using immunohistochemistry and real-time qPCR, respectively. Luciferase assay was used to determine the targeting of miRNA224 to RKIP. The methylation status of miRNA224 promoter region was studied by bisulfite sequencing PCR (BSP). RESULTS: In 40 cases of ESCC, RKIP expression was significantly lower than that of normal tissue; miRNA224 expression was higher in ESCC than in paracancerous tissue. Luciferase assay showed that miRNA224 targets RKIP 3'UTR thus inhibit its expression. The miRNA224 gene promoter region was hypomethylated in ESCC. CONCLUSIONS: Compared with normal tissue, in ESCC, RKIP was downregulated, while miRNA224 was upregulated, and the promoter region of miRNA224 gene was hypomethylated. RKIP is the target of miRNA224, which may be closely related to esophageal squamous cell carcinoma.

Longnon-coding RNA BLACAT2 promotes gastric cancer progression via the miR-193b-5p/METTL3 pathway.

Gastric cancer is one of the leading prevalent and malignant cancers worldwide, especially in east Asia. However, the in-depth molecular mechanism underlying gastric cancer progression remains uncertain. Recently, studies have identified that long non-coding RNA (lncRNA) could play critical roles in the tumorigenesis of multiple types of cancer. Studies on long non-coding RNA BLACAT2 have proven that it participates in bladder cancer and colorectal cancer regulation and was identified as highly expressed using the cBioPortal for Cancer Genomics in gastric cancer. However, the precise function of lncRNA-BLACAT2 in the carcinogenesis and progression of gastric cancer remains largely unexplored. Our study discovered that lncRNA-BLACAT2 was significantly upregulated in gastric cancer. Different studies have illustrated that BLACAT2 promoted gastric cancer progression through regulating proliferation, migration, invasion, and apoptosis in terms of biological function. Furthermore, BLACAT2 was verified to perform its function through interaction with miR-193b-5p using a luciferase reporter assay. On the other hand, MiR-193b-5p specific inhibitor treatment reversed the inhibitory effect of BLACAT2 on cell biological functions. Additional studies also discovered that Methyltransferase Like 3 (METTL3) was the downstream target of miR-193b-5p. Subsequently, restoration of METTL3 eliminated the suppressive effect of proliferation or the promotive effect of apoptosis caused by BLACAT2 knockdown. To sum up, these experimental results demonstrated that BLACAT2 acted as an oncogene in gastric cancer progression through the regulation of the miR-193b-5p/METTL3 pathway, hence providing new insights regarding the pathogenesis of gastric cancer.

Decreased expression of chromodomain helicase DNA-binding protein 9 is a novel independent prognostic biomarker for colorectal cancer.

Previous studies suggested that chromodomain helicase DNA-binding proteins (CHDs), including CHD 1-8, were associated with several human diseases and cancers including lymphoma, liver cancer, colorectal cancer, stomach cancer, etc. To date, little research on CHD 9 in human cancers has been reported. In this study, we assessed the prognostic value of CHD 9 in patients with colorectal cancer (CRC). We screened for CHD 9 expression using immunohistochemical analysis in 87 surgical CRC specimens and found that the expression was upregulated in 81.5% of the cases, while 7.4% were decreased; in the remaining 11.1% of the cases, levels were not altered. Kaplan-Meier analysis showed that patients with high CHD 9 expression had better prognosis than those with low CHD 9 expression (54.5 vs 32.1%, P=0.034). Subsequently, Cox multi-factor survival regression analysis revealed that expression of CHD 9 protein was an independent predictor for CRC, with a hazard ratio of 0.503 (P=0.028). In addition, we found that CHD 9 expression was positively correlated with MSH2 (rs=0.232, P=0.036). We speculated that CHD9 might be a putative tumor suppressor gene, and could inhibit the development of CRC by participating in DNA repair processes. Our findings suggest that CHD 9 could be a novel prognostic biomarker and a therapeutic target for CRC. Further studies are needed to detect the effect of CHD 9 on cellular function and the expression of mismatch repair genes.

Low vitamin D status is associated with advanced liver fibrosis in patients with nonalcoholic fatty liver disease.

Several studies explored the association between vitamin D status and nonalcoholic fatty liver disease with contradictory results. We aimed to investigate the association between vitamin D status, inflammatory cytokines and liver fibrosis in nonalcoholic fatty liver disease patients. Two hundred nineteen nonalcoholic fatty liver disease patients and 166 age- and gender- matched healthy controls were recruited for this study. Serum 25(OH)D was measured by radioimmunoassay. Serum interleukin-8 and transforming growth factor-ß1 were measured using ELISA. Serum 25(OH)D was only marginally decreased in nonalcoholic fatty liver disease patients. Interestingly, serum 25(OH)D was markedly reduced in nonalcoholic fatty liver disease patients with advanced liver fibrosis compared to nonalcoholic fatty liver disease patients with indeterminate liver fibrosis and no advanced fibrosis. Logistic regression analysis showed that there was an inverse association between serum 25(OH)D and severity of liver fibrosis in nonalcoholic fatty liver disease patients. Further analysis showed that serum interleukin-8 was elevated in nonalcoholic fatty liver disease patients, the highest interleukin-8 in patients with advanced fibrosis. An inverse correlation between serum 25(OH)D and interleukin-8 was observed in nonalcoholic fatty liver disease patients with and without liver fibrosis. Although serum transforming growth factor-ß1 was slightly elevated in nonalcoholic fatty liver disease patients, serum transforming growth factor-ß1 was reduced in nonalcoholic fatty liver disease patients with advanced fibrosis. Unexpectedly, a positive correlation between serum 25(OH)D and transforming growth factor-ß1 was observed in nonalcoholic fatty liver disease patients with advanced fibrosis. In conclusion, low vitamin D status is associated with advanced liver fibrosis in nonalcoholic fatty liver disease patients. Interleukin-8 may be an important mediator for hepatic fibrosis in nonalcoholic fatty liver disease patients with low vitamin D status.

Decreased expression of chromodomain helicase DNA-binding protein 9 is a novel independent prognostic biomarker for colorectal cancer

Previous studies suggested that chromodomain helicase DNA-binding proteins (CHDs), including CHD 1-8, were associated with several human diseases and cancers including lymphoma, liver cancer, colorectal cancer, stomach cancer, etc. To date, little research on CHD 9 in human cancers has been reported. In this study, we assessed the prognostic value of CHD 9 in patients with colorectal cancer (CRC). We screened for CHD 9 expression using immunohistochemical analysis in 87 surgical CRC specimens and found that the expression was upregulated in 81.5% of the cases, while 7.4% were decreased; in the remaining 11.1% of the cases, levels were not altered. Kaplan-Meier analysis showed that patients with high CHD 9 expression had better prognosis than those with low CHD 9 expression (54.5 vs 32.1%, P=0.034). Subsequently, Cox multi-factor survival regression analysis revealed that expression of CHD 9 protein was an independent predictor for CRC, with a hazard ratio of 0.503 (P=0.028). In addition, we found that CHD 9 expression was positively correlated with MSH2 (rs=0.232, P=0.036). We speculated that CHD9 might be a putative tumor suppressor gene, and could inhibit the development of CRC by participating in DNA repair processes. Our findings suggest that CHD 9 could be a novel prognostic biomarker and a therapeutic target for CRC. Further studies are needed to detect the effect of CHD 9 on cellular function and the expression of mismatch repair genes.

CXCR4 antagonist AMD3100 modulates claudin expression and intestinal barrier function in experimental colitis.

Ulcerative colitis is a gastrointestinal disorder characterized by local inflammation and impaired epithelial barrier. Previous studies demonstrated that CXC chemokine receptor 4 (CXCR4) antagonists could reduce colonic inflammation and mucosal damage in dextran sulfate sodium (DSS)-induced colitis. Whether CXCR4 antagonist has action on intestinal barrier and the possible mechanism, is largely undefined. In the present study, the experimental colitis was induced by administration of 5% DSS for 7 days, and CXCR4 antagonist AMD3100 was administered intraperitoneally once daily during the study period. For in vitro study, HT-29/B6 colonic cells were treated with cytokines or AMD3100 for 24 h until assay. DSS-induced colitis was characterized by morphologic changes in mice. In AMD3100-treated mice, epithelial destruction, inflammatory infiltration, and submucosal edema were markedly reduced, and the disease activity index was also significantly decreased. Increased intestinal permeability in DSS-induced colitis was also significantly reduced by AMD3100. The expressions of colonic claudin-1, claudin-3, claudin-5, claudin-7 and claudin-8 were markedly decreased after DSS administration, whereas colonic claudin-2 expression was significantly decreased. Treatment with AMD3100 prevented all these changes. However, AMD3100 had no influence on claudin-3, claudin-5, claudin-7 and claudin-8 expression in HT-29/B6 cells. Cytokines as TNF-α, IL-6, and IFN-γ increased apoptosis and monolayer permeability, inhibited the wound-healing and the claudin-3, claudin-7 and claudin-8 expression in HT-29/B6 cells. We suggest that AMD3100 acted on colonic claudin expression and intestinal barrier function, at least partly, in a cytokine-dependent pathway.