Long noncoding RNA SNHG12 suppresses esophageal squamous cell carcinoma progression through competing endogenous RNA networks.

PURPOSE: Esophageal squamous cell cancer (ESCC) has high rates of recurrence and mortality. Small nucleolar RNA host gene 12 (SNHG12) is known to promote the progression of several cancers. Therefore, we aimed to investigate the expression and role of SNHG12 in ESCC. METHODS: The expression and clinical value of SNHG12 in esophageal cancer were explored using data from The Cancer Genome Atlas (TCGA) and the online server GEPIA. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to verify the expression levels of SNHG12 in ESCC tissues and cell lines. Furthermore, loss-of-function assays were performed to examine the effect of SNHG12 on ESCC cells in vitro and in vivo. The potential competing endogenous RNA networks of SNHG12 in ESCC were explored. RESULTS: SNHG12 was downregulated in human ESCA tissues compared to control tissues. The expression of SNHG12 was strongly associated with T stage, N stage, and TNM stage. Low SNHG12 expression in esophageal tumor tissues was significantly correlated with poor prognosis. Furthermore, knockdown of SNHG12 not only promoted proliferation, colony formation, migration, and invasion and inhibited apoptosis in ESCC cells in vitro, but also increased tumor growth in vivo. Additionally, this proves that the SNHG12/miRNA-195-5p/BCL9 network might be involved in ESCC. CONCLUSION: This is the first study to reveal that SNHG12 is downregulated in ESCC tissues and could be used as a prognostic tool. SNHG12 suppressed tumor progression in ESCC cells, serving as a potential biomarker. The SNHG12/miRNA-195-5p/BCL9 network is proposed to be the mechanism leading to ESCC progression.

Molecular Characterization of Goose Phosphoenolpyruvate Carboxylase Kinase 1 (Pepck) Gene and Its Potential Role in Hepatic Steatosis Induced by Overfeeding

Over-accumulation of triglycerides (TGs) in goose hepatocytes leads to the formation of fatty acid liver. Phosphoenolpyruvate carboxylase kinase 1 (PEPCK) is regarded as the rate-limiting enzyme for gluconeogenesis, and there is evidence that PEPCK is involved in regulating hepatic glucolipid metabolism. Hence, we proposed that PEPCK may have a role in goose hepatic steatosis. To test our hypothesis, the present study was conducted to firstly determine the sequence characteristics of goose PEPCK and then to explore its role in overfeeding-induced fatty liver. Our results showed that goose PEPCK encodes a 622-amino-acids protein that contains highly conserved oxaloacetate-binding domain, kinase-1 and kinase-2 motifs. PEPCK had higher mRNA levels in goose liver, and overfeeding markedly increased its expression in livers of both Sichuan White and Landes geese (p 0.05). Besides, expression of PEPCK was positively correlated with hepatic TG levels as well as plasma glucose and insulin concentrations. Additionally, in cultured goose primary hepatocyte, treatment with either oleic acid (0.8, 1.2 or 1.6 mM) or linoleic acid (0.125 or 0.25 mM) significantly (p 0.05) enhanced the expression of PEPCK. Taken together, these data suggested a role for PEPCK in the occurrence of overfeeding-induced goose hepatic steatosis.(AU)

Molecular Characterization of Goose Phosphoenolpyruvate Carboxylase Kinase 1 (Pepck) Gene and Its Potential Role in Hepatic Steatosis Induced by Overfeeding

Over-accumulation of triglycerides (TGs) in goose hepatocytes leads to the formation of fatty acid liver. Phosphoenolpyruvate carboxylase kinase 1 (PEPCK) is regarded as the rate-limiting enzyme for gluconeogenesis, and there is evidence that PEPCK is involved in regulating hepatic glucolipid metabolism. Hence, we proposed that PEPCK may have a role in goose hepatic steatosis. To test our hypothesis, the present study was conducted to firstly determine the sequence characteristics of goose PEPCK and then to explore its role in overfeeding-induced fatty liver. Our results showed that goose PEPCK encodes a 622-amino-acids protein that contains highly conserved oxaloacetate-binding domain, kinase-1 and kinase-2 motifs. PEPCK had higher mRNA levels in goose liver, and overfeeding markedly increased its expression in livers of both Sichuan White and Landes geese (p 0.05). Besides, expression of PEPCK was positively correlated with hepatic TG levels as well as plasma glucose and insulin concentrations. Additionally, in cultured goose primary hepatocyte, treatment with either oleic acid (0.8, 1.2 or 1.6 mM) or linoleic acid (0.125 or 0.25 mM) significantly (p 0.05) enhanced the expression of PEPCK. Taken together, these data suggested a role for PEPCK in the occurrence of overfeeding-induced goose hepatic steatosis.

Genetic polymorphisms and haplotype of hormone-related genes are associated with the risk of breast cancer in Chinese women.

Sex hormones play important roles in breast cancer (BC) development. This study investigated associations between BC risk and hormone-related gene variants in Chinese women. In a cohort of 336 patients with histopathologically confirmed BC and 390 age-matched controls, we genotyped seven single nucleotide polymorphisms (SNPs) in five hormone-related genes: estrogen receptor-α (ESR1), aromatase (CYP19), catechol-O-methyl transferase (COMT), sex hormone-binding globulin (SHBG), and glutathione S-transferase (GSTP1). Among these seven SNPs, the SNPs in GSTP1 rs1695 [A/G; odds ratio (OR): 1.68; 95% confidence interval (CI): 1.23-2.30] and ESR1 rs2046210 (C/T; OR: 1.39; 95%CI = 1.02-1.91) were associated with an increased risk among heterozygote carriers. Homozygotes of minor alleles of CYP19 rs10046 (CC) were associated with a reduced risk of BC with OR: 0.61 (95%CI = 0.39-0.95). In addition, a stratified analysis by menopausal status indicated that the association of the CYP19 polymorphisms (rs10046 and rs700519) with BC risk was mainly evident in premenopausal women, and the association of CYP19 rs700519 with BC risk was significant in women less than 50 years old. Haplotype analysis identified 15 common haplotypes (>1%). The haplotype TGGGGTC was significantly associated with BC risk compared with the reference haplotype CGAGGTC (OR > 1000, P < 0.0001). Our data demonstrate that these ESR1, GSTP1, and CYP19 polymorphisms are associated with risk of BC, and the risk haplotype TGGGGTC could help to identify populations with high susceptibility to BC in Chinese women.

Effect of progranulin (PGRN) on the proliferation and senescence of cervical cancer cells.

We investigated the effect of progranulin (PGRN) expression on the proliferation and senescence of cervical cancer cells. PGRN small interfering RNA (siRNA) was introduced into the SiHa and HeLa cell lines of human cervical carcinoma using liposome-mediated transfection. The expression levels of PGRN in each cell line after transfection of PGRN siRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). Senescence in the cell lines was detected using the ß-galactosidase-staining test, and proliferation was detected by clone formation. The RT-PCR assay showed that the expression of PGRN in all of the cell lines transfected with PGRN siRNA markedly decreased. In the clone-forming test, compared with the control group, the colony-forming ability in all cell lines decreased significantly after transfection with PGRN siRNA. The ß-galactosidase-staining experiments showed that the phenomenon of cell aging in the PGRN interference group was more obvious than in the control group. After the cervical cancer cells had been transfected with PGRN siRNA, cell senescence was accelerated and clone-forming ability was markedly reduced. This suggests that PGRN can promote the proliferation of the cervical cancer cell line; proliferation of cervical cancer cells is achieved by inhibiting their senescence.

Relationship of the APOA5/A4/C3/A1 gene cluster and APOB gene polymorphisms with dyslipidemia.

We determined the alleles of ten single nucleotide poly-morphisms (SNPs) in the APOA5/A4/C3/A1 gene cluster and in APOB in Han Chinese from Xinjiang Shihezi, China using MALDI-TOF mass spectrometry, and explored the correlation between these SNPs and dyslipidemia through a case-control study design with 250 pa-tients and 250 normal controls. All SNPs except for APOA5 rs2072560 conformed to Hardy-Weinberg equilibrium (all P > 0.05). APOA5 rs651821, APOA4 rs5104, APOC3 rs734104, and APOC3 rs5128 geno-type and allele frequencies were significantly different between groups (all P < 0.01). For rs651821, the risks of dyslipidemia for the CC or CC+CT genotypes were 9.917 or 1.859 times that of TT, and the risk of the C vs T allele was 2.027. For rs5104, the AG, GG, or AG+GG risks were 1.797, 1.861, and 1.809 times AA, and the G vs A risk was 1.427. For rs734104, the CT, CC, or CC+CT risks were 1.851, 2.570, and 1.958 times TT, and the C vs T risk was 1.610. For rs5128, the GC or CC+GC risks were 1.738 or 1.749 times GG, and the C vs G risk was 1.477. Compared with the wild-type haplotype TATG, the risks of dyslipidemia with CGCC, TGCC, or CATG haplotypes (odds ratios = 2.434, 1.503, and 2.740, respectively) were significantly higher. Our results suggested that these four SNPs were significantly associated with dyslipidemia in Xinjiang Shihezi Han Chinese, and might serve as risk factors for dyslipidemia. Individuals carrying the CGCC, TGCC, or CATG haplotypes were prone to dyslipidemia.

Study of the methylation patterns of the EGFR gene promoter in non-small cell lung cancer.

We investigated the methylation state of the epidermal growth factor receptor (EGFR) gene promoter in non-small cell lung cancer (NSCLC) and analyzed its effect on tumor biology. We enrolled 120 patients with NSCLC who had been confirmed by pathologic diagnosis and had been operated on. The methylation states of the EGFR gene promoter were detected and analyzed and a prognosis was given. NSCLC cell lines and nude mice were used to study the treatment reactivity of gefitinib (an EGFR inhibitor) with or without 5-aza-2'-deoxycytidine (5-aza-CdR) intervention. EGFR expression was high when the methylation degree was lower in patients with adenocarcinoma and poor pathological differentiation of tumor than in patients with squamous cell carcinoma and good pathological differentiation. NSCLC cells with low expression of EGFR and high methylation in the promoter region were insensitive to EGFR-targeted therapy. However, apoptosis and proliferation inhibition of cancer cells were even more pronounced when 5-aza-CdR was used to inhibit methylation. An in vivo study confirmed that methylation adjuvant therapy can improve the sensitivity of cancer to EGFR-targeted therapy. Application of a demethylating agent could be an important supplement for improving EGFR inhibition in the treatment of NSCLC, especially in those who are insensitive to the use of an EGFR inhibitor alone.

Effect of age on the immune system and pathology of mice with chronic graft-versus-host disease lupus nephritis.

We investigated the effect of age on the expression of immune molecules [ANA, C4, double stranded DNA (dsDNA), CD16/32, CD19, CD3, and CD64], urine protein, and pathology in mice with chronic graft-versus-host disease (cGVHD) lupus nephritis (LN), and their relationship with reactivity index score. Mouse models of cGVHD LN were established, and mice were randomly divided into four aged-based groups of nine mice each. Serum levels of ANA, C4, and dsDNA were determined, the urine protein levels were assessed, and expression levels of CD16/32, CD19, CD3, and CD64 were measured. Expression levels of CD16/32+CD19(T1), CD16/CD32+CD3(T2), and CD64+CD3 or CD19(T3) were defined in the thymus, in bone marrow they were defined as CD16/32+CD19(B1), CD16/32+CD3(B2), CD64+CD3 or CD19(B3), and in spleen they were defined as CD16/32+CD19(P1), CD16/32+CD3(P2), CD64+CD3 or CD19(P3), respectively. There were significant differences in the levels of dsDNA and urine protein among the four groups (P < 0.05), which were negatively correlated with age. B1, B2, S1, and S2 were significantly different among the four groups (P < 0.05), with a positive correlation with age for B1 and B2. There was no correlation of expression of ANA, C4, dsDNA, T1-T3, B1-B3, S2-S3 with reactivity index score; S1 was the exception (r = -0.440, P = 0.011). Age influenced levels of dsDNA and urine protein in the mouse cGVHD model of LN. S1 was associated with reactivity index score and might also affect pathological changes.

Analysis and clinical significance of microRNA-499 expression levels in serum of patients with acute myocardial infarction.

The aim of this study was to investigate the expression level of microRNA-499 and its clinical significance in serum of patients with acute myocardial infarction (AMI). We recruited 59 patients with AMI and 60 healthy individuals undergoing physical examination in our hospital during the same period as controls. Peripheral blood was drawn in the morning on the same day of microRNA extraction. The expression level of microRNA-499 was analyzed by real-time fluorescent quantitative polymerase chain reaction (qPCR). The sensitivity and specificity of the clinical diagnosis of AMI were analyzed by a receiver operating characteristic (ROC) curve. Fluorescent qPCR analysis showed that the expression of microRNA-499 in serum of patients with AMI was significantly higher than in controls (P < 0.05). MicroRNA-499 was detected in blood serum 3 h post-AMI, reaching a peak after 12 h and declining after 15 h. The area under the ROC curve (AUC) for the gold standard cardiac troponin I (cTnI) was 0.971 [95% confidence interval (CI): 0.951-1.000], and for the microRNA-499, AUC = 0.915 (95%CI: 0.826-1.000). When the microRNA-499 levels in patient and control (> 1.5) sera were compared, the sensitivity of microRNA-499 in judging AMI was found to be 86.37% and the specificity was 93.47%. Our results demonstrated that the expression levels of microRNA-499 in serum of patients with AMI were abnormal. Its high sensitivity and specificity for the diagnosis of AMI suggest that it would be useful as an auxiliary index for clinical diagnosis of AMI.

Germline mutations of DICER1 in Chinese women with BRCA1/BRCA2-negative familial breast cancer.

Germline mutations in identified breast cancer susceptibility genes account for less than 20% of Chinese familial breast cancers. Dicer is an essential component of the microRNA-producing machinery; germline mutations of DICER1 have been confirmed in familial pleuropulmonary blastoma, ovarian sex cord-stromal tumors, and other cancers. Low expression of DICER1 is frequently detected in breast cancer. However, whether germline mutations of DICER1 occur in familial breast cancers remain unknown. Sixty-five breast cancer probands from BRCA1/BRCA2-negative Chinese breast cancer families were screened for germline mutations in DICER1. In addition, 100 unrelated healthy females were enrolled as controls. A polymerase chain reaction sequencing assay was used to screen for mutations in coding regions and at the exon-intron boundaries of DICER1. All variants in introns were evaluated using the NNSplice software to determine the potential splicing effect. A total of 12 germline variants were found, including 11 variants in introns and 1 variant in the 3'-non-coding region. Four variants (IVS8-205 C>T, IVS11+131 delGAAA, IVS16+42 delTA, and IVS19+160 T>C) were novel. Three variants (IVS11+105 C>T, IVS16+42 delTA, and 6095 T>A) may affect splice sites. None of the observed variants appeared to be disease-related, suggesting that germline mutations in DICER1 are rare or absent in familial breast cancer patients.

Genetic relationship between lodging and lodging components in barley (Hordeum vulgare) based on unconditional and conditional quantitative trait locus analyses.

Lodging (LD) is a major constraint limiting the yield and forage quality of barley. Detailed analyses of LD component (LDC) traits were conducted using 246 F2 plants generated from a cross between cultivars ZQ320 and 1277. Genetic relationships between LD and LDC were evaluated by unconditional and conditional quantitative trait locus (QTL) mapping with 117 simple sequence repeat markers. Ultimately, 53 unconditional QTL related to LD were identified on seven barley chromosomes. Up to 15 QTL accounted for over 10% of the phenotypic variation, and up to 20 QTL for culm strength were detected. Six QTL with pleiotropic effects showing significant negative correlations with LD were found between markers Bmag353 and GBM1482 on chromosome 4H. These alleles and alleles of QTL for wall thickness, culm strength, plant height, and plant weight originated from ZQ320. Conditional mapping identified 96 additional QTL for LD. Conditional QTL analysis demonstrated that plant height, plant height center of gravity, and length of the sixth internode had the greatest contribution to LD, whereas culm strength and length of the fourth internode, and culm strength of the second internode were the key factors for LD-resistant. Therefore, lodging resistance in barley can be improved based on selection of alleles affecting culm strength, wall thickness, plant height, and plant weight. The conditional QTL mapping method can be used to evaluate possible genetic relationships between LD and LDC while efficiently and precisely determining counteracting QTL, which will help in understanding the genetic basis of LD in barley.

miR-34a targets the inhibin beta B gene, promoting granulosa cell apoptosis in the porcine ovary.

During ovarian follicular growth and development, only a few follicles actually ovulate. Recently, it was found that follicular atresia is triggered by granulosa cell apoptosis, but the molecular mechanism of follicular atresia was not understood. Using flow cytometry, we found that miR-34a promotes granulosa cell apoptosis in pig ovarian follicles. In addition, inhibin beta B was found to be a miR-34a target gene, based on luciferase reporter assays, quantitative RT-PCR and Western blotting. Taken together, our data indicate that miR-34a plays an important role in granulosa cell apoptosis by targeting the INHBB gene in the porcine ovary.

TLR4 gene expression in pig populations and its association with resistance to Escherichia coli F18.

TLR4 is the main recognition receptor of bacterial lipopolysaccharides, which play an important role in innate and adaptive immunity. We used real-time PCR to analyze the tissue expression profile and differential expression of TLR4 in 4 pig populations (Escherichia coli F18-resistant Sutai, E. coli F18-sensitive Sutai, Large White, Meishan), in order to determine the role that the TLR4 gene plays in resistance to E. coli F18. We found that TLR4 expressed consistently in the 4 populations, with relatively high levels in immune tissues and the highest level in the lung. Generally, the expression of TLR4 in E. coli F18-sensitive individuals was the highest, followed by that in E. coli F18-resistant, Large White and Meishan. In the spleen, lung, kidney, lymph nodes, and thymus gland, TLR4 expression is significantly higher in the E. coli F18-sensitive than in the other 3 populations; there were no significant differences among E. coli F18-resistant Sutai, Large White, and Meishan. In addition, Gene Ontology and pathway analysis showed that TLR4 takes part in the inflammatory response. We found that porcine TLR4 has consistent tissue specificity in each breed, and downregulation of expression of the TLR4 gene is related to resistance to E. coli F18 in weaning piglets.

Evaluation of a sexuality education program for young adolescents in Jamaica.

Despite their increasing numbers, few of the sexuality education and pregnancy prevention programs in developing countries have been evaluated. This study, conducted in 1995-1997, assesses the impact of a school-based sexuality education program, the Grade 7 Project, on 945 Jamaican seventh graders (aged 11-14) and their initiation of sexual activity and use of contraception at first intercourse, as well as the knowledge and attitudes that influence their behaviors. Using a quasi-experimental design, the study measured the effects of the Grade 7 Project when the nine-month intervention was completed (short term) and one year after that (long term). Multivariate logistic regression analysis indicated that the project had no effect on initiation of sexual activity, but it had a positive short-term impact on use of contraception at first intercourse (P = .08); adolescents in the intervention group were more than twice as likely to use contraception. The project also had a positive short-term influence on several aspects of the adolescents' knowledge of and attitudes about sexuality and pregnancy. The modest impact of the Grade 7 Project is encouraging, as school-based sexuality education programs of limited duration rarely have a long-term impact. Moreover, competing socioeconomic and cultural forces in Jamaica encourage early sexuality and parenthood among adolescents. The use of more participatory teaching methods and smaller class sizes might strengthen the Grade 7 Project and enhance its impact.