CLIC1 knockout inhibits invasion and migration of gastric cancer by upregulating AMOT-p130 expression.

PURPOSE: To explore the regulatory relationship between Chloride intracellular channel 1 (CLIC1) and Angiomotin (AMOT)-p130, and reveal the role of AMOT-p130 in gastric cancer (GC). METHODS: Immunohistochemistry was performed to analyze the expression of CLIC1 and AMOT-p130 in GC tissues and adjacent tissues. The expression of AMOT-p130 upon CLIC1 silencing was analyzed using RT-PCR, western blot, and immunofluorescence in GC cells. Transwell and wound-healing assays were performed to detect migration and invasion in GC cells. The changes in EMT-related proteins were detected using western blot. RESULTS: Our study found that high CLIC1 expression was significantly associated with low AMOT-p130 expression in GC tissues. Silencing CLIC1 expression in MGC-803 cells (MGC-803 CLIC1 KO) and AGS cells (AGS CLIC1 KO) decreased the invasive and migratory abilities of tumor cells, which were induced by the upregulation of AMOT-p130. Subsequently, we demonstrated that AMOT-p130 inhibits the invasive and migratory abilities of GC cells by inhibiting epithelial-mesenchymal transition. CONCLUSIONS: Our study suggests that AMOT-p130 could inhibit epithelial-mesenchymal transition in GC cells. CLIC1 may participate in the metastatic progression of GC by downregulating the expression of AMOT-p130.

The application value of PAX1 and ZNF582 gene methylation in high grade intraepithelial lesion and cervical cancer.

PURPOSE: To investigate the possibility of using the methylation level of PAX1/ZNF582 gene as molecular marker to differentiate the progression of cervical cancer. METHODS: From January 2016 to March 2018, 150 patients, who were admitted to Cervical Disease Diagnosis and Treatment Center of Xuzhu Maternity and Child Care Hospital, were enrolled in this study. Patients were classified into chronic cervicitis (for 19 cases), low-grade squamous intraepithelial lesion (LSIL) (18 cases), high-grade squamous intraepithelial lesion (HSIL) (37 cases) and squamous cell carcinoma (SCC) (31 cases). All patients underwent several tests including Thin-prep cytology test (TCT), HPV DNA detection and detection of methylation level of PAX1/ZNF582 genes. RESULTS: For diagnosis of HSIL, the area under curve (AUC) was 0.878 (95% CI 0.806 ~ 0.950); the threshold for PAX1 was 12.285, the sensitivity and specificity were 91.9% and 72.8%, respectively. The AUC of ZNF582 gene detection was 0.900 (95% CI 0.842 ~ 0.959), the threshold was 11.56, while the sensitivity and specificity were 97.3% and 76.7%, respectively. Among various tests we conducted, PAX gene detection methods showed the highest specificity (97.30%). PAX1/ZNF582 gene detection method demonstrated the highest accuracy. CONCLUSIONS: For patients with high-grade cervical lesion and cervical cancer, the methylation level of PAX1/ZNF582 gene could be applied as a noteworthy biomarker for diagnosis and for cervical cancer classification.

Effect of exosome biomarkers for diagnosis and prognosis of breast cancer patients.

PURPOSE: Exosomes are gradually detected as an indicator for diagnosis and prognosis of breast cancer in clinic and a systematic review was conducted. METHODS: A search for clinical studies published before July 1, 2017 was performed. Methods of exosome purification and identification from all studies were extracted. For diagnosis evaluation, the comparison of exosome biomarkers expression between breast cancer patients and healthy women was obtained; for prognosis prediction, the correlation between exosome biomarkers expression and chemotherapy resistance, overall survival (OS), disease-free survival (DFS), recurrence and metastasis of breast cancer was also extracted. RESULTS: A total of 11 studies with 921 breast cancer patients were included. Ultracentrifugation is the most frequent method to purify exosomes and transmission electron microscopy is commonly used to identify exosomes. Exosome biomarkers (such as HER2, CD47, Del-1, miR-1246 and miR-21) in breast cancer patients are significantly higher than those in healthy controls, exosomal GSTP1 and TRPC5 are related to chemotherapy resistance, exosome-carrying TRPC5, NANOG, NEUROD1, HTR7, KISS1R and HOXC are correlated to PFS, DFS or OS, and some exosomal proteins (HER2, KDR, CD49d, CXCR4 and CD44) as well as miRNAs (miR-340-5p, miR-17-5p, miR-130a-3p, miR-93-5p) are associated with tumor recurrence or distant organ metastasis. CONCLUSIONS: Exosome biomarkers can be used for early diagnosis and prognosis of breast cancer patients in clinic.

MicroRNA-455 suppresses the oncogenic function of HDAC2 in human colorectal cancer.

Colorectal cancer (CRC) is the fourth leading cause of cancer-induced mortality. Histone deacetylase 2 (HDAC2) is involved in prognosis and therapy of CRC. This study aimed to explore novel therapeutic targets for CRC. The alteration of HDAC2 expression in CRC tissues was estimated by qRT-PCR. After lentivirus transfection, HDAC2 knockdown was confirmed by western blot analysis. The effect of HDAC2 knockdown on cell proliferation was then assessed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Screened by TargetScan, microRNA (miR)-455 was predicted to bind to 3'UTR of HDAC2 and the prediction was verified by luciferase assay. Finally, cells were transfected, respectively, with miR-455 mimics or miR-455 negative control (miR-NC) and the expression of HDAC2, cell proliferation and apoptosis of transfected cells were respectively evaluated by western blot analysis, MTT assay and flow cytometry. Results showed that the HDAC2 expression was up-regulated in CRC tissues (P<0.05). HDAC2 knockdown significantly decreased cell viability at day 3 (P<0.05), day 4 (P<0.01), and day 5 (P<0.001) after infection. Then, miR-455 was verified to directly target HDAC2, resulting in a significant difference in luciferase activity (P<0.01). Moreover, miR-455 decreased the expression of HDAC2 (P<0.01). miR-455 remarkably decreased cell viability at day 3 (P<0.05), day 4 (P<0.01), and day 5 (P<0.001) after transfection while inducing cell apoptosis (P<0.001). In conclusion, miR-455 inhibited cell proliferation while inducing cell apoptosis by targeting HDAC2 in CRC cells.

MicroRNA-455 suppresses the oncogenic function of HDAC2 in human colorectal cancer

Colorectal cancer (CRC) is the fourth leading cause of cancer-induced mortality. Histone deacetylase 2 (HDAC2) is involved in prognosis and therapy of CRC. This study aimed to explore novel therapeutic targets for CRC. The alteration of HDAC2 expression in CRC tissues was estimated by qRT-PCR. After lentivirus transfection, HDAC2 knockdown was confirmed by western blot analysis. The effect of HDAC2 knockdown on cell proliferation was then assessed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Screened by TargetScan, microRNA (miR)-455 was predicted to bind to 3′UTR of HDAC2 and the prediction was verified by luciferase assay. Finally, cells were transfected, respectively, with miR-455 mimics or miR-455 negative control (miR-NC) and the expression of HDAC2, cell proliferation and apoptosis of transfected cells were respectively evaluated by western blot analysis, MTT assay and flow cytometry. Results showed that the HDAC2 expression was up-regulated in CRC tissues (P<0.05). HDAC2 knockdown significantly decreased cell viability at day 3 (P<0.05), day 4 (P<0.01), and day 5 (P<0.001) after infection. Then, miR-455 was verified to directly target HDAC2, resulting in a significant difference in luciferase activity (P<0.01). Moreover, miR-455 decreased the expression of HDAC2 (P<0.01). miR-455 remarkably decreased cell viability at day 3 (P<0.05), day 4 (P<0.01), and day 5 (P<0.001) after transfection while inducing cell apoptosis (P<0.001). In conclusion, miR-455 inhibited cell proliferation while inducing cell apoptosis by targeting HDAC2 in CRC cells.

RETRACTION of "Correlation analysis between an IL-6 genetic polymorphism and non-small cell lung cancer prognosis", by Zhao K, Xu J, Tian H published in Genetics and Molecular Research 15 (1): 15017021 (2016) DOI: 10.4238/gmr.15017021.

Zhao Kai, first author wishes to retract the following article: Zhao K, Xu J and Tian H (2016). Correlation analysis between an IL-6 genetic polymorphism and non-small cell lung cancer prognosis. Genet. Mol. Res. 15: gmr.15017021 According to the author, the reasons for the retraction are as follows: - All patients enrolled in the study received surgical resection and lymph node dissection. So the patients' information was incorrect. Then, the corresponding information and data need to be collected again and reanalyzed. - The P value for Figure 1 was calculated by the log-rank test. Since the data need to be reanalyzed, the 95% confidence intervals for the average survival times also need to be recalculated. - The author intends to synthesize new primer sequences for the genotyping of the IL-6 SNPs and restart the detection. The follow-up data need to be collected again and then the survival analysis also needs to be reanalyzed and investigate IL-6 levels in the patients to determine if the levels of IL-6 correlate with the survival effects. The first author contacted GMR to voluntarily report this inaccuracy and to request a retraction. He takes full responsibility for the error and apologizes to the journal, its readers, and the other authors.

Characterization and comparison of transgenic Artemisia annua GYR and wild-type NON-GYR plants in an environmental release trial.

The anti-malarial drug, artemisinin, is quite expensive as a result of its slow content in Artemisia annua. Recent investigations have suggested that genetic engineering of A. annua is a promising approach to improve the yield of artemisinin. In this study, the transgenic A. annua strain GYR, which has high artemisinin content, was evaluated in an environmental release trial. First, GYR plants were compared with the wild-type variety NON-GYR, with regard to phenotypic characters (plant height, crown width, stem diameter, germination rate, leaf dry weight, 1000-seed weight, leave shape). Second, stress resistance in the two varieties (salt, drought, herbicide, and cold resistance) was evaluated under different experimental conditions. Finally, gene flow was estimated. The results indicated that there were significant differences in several agronomic traits (plant height, stem diameter, and leave dry weight) between the transgenic GYR and NON-GYR plants. Salt stress in transgenic and control plants was similar, except under high NaCl concentrations (1.6%, w/w). Leaf water, proline, and MDA content (increased significantly) were significantly different. Transgenic A. annua GYR plants did not grow better than NON-GYR plants with respect to drought and herbicide resistance. The two varieties maintained vitality through the winter. Third, gene flow was studied in an environmental risk trial for transgenic GYR. The maximum gene flow frequency was 2.5%, while the maximum gene flow distance was 24.4 m; gene flow was not detected at 29.2 m at any direction. Our findings may provide an opportunity for risk assessment in future commercialization of transgenic A. annua varieties.

Correlation analysis between an IL-6 genetic polymorphism and non-small cell lung cancer prognosis.

Interleukin-6 (IL-6) is a multifunctional cytokine that is involved in tumor cell proliferation, apoptosis, and differentiation. The purpose of this study was to evaluate the impact of the single nucleotide polymorphism (SNP) -174G/C in IL-6 on the prognosis and pain tolerance of non-small cell lung cancer (NSCLC) patients. DNA was extracted from the peripheral blood of 434 patients with NSCLC, which was diagnosed by cytology or histology. Polymerase chain reaction-restriction fragment length polymorphism was used to detect the IL-6 -174G/C genotypes and their correlation with survival was analyzed. The IL-6 -174G/C genotypes were high IL-6 production type (G carriers - GG or GC genotypes) and low IL-6 production type (CC genotype). The correlation between the IL-6 SNP and pain level/analgesic use was also analyzed. Survival analysis showed that patients carrying the G allele (CG/GG) had a shorter survival time than patients with the CC genotype. The -174G/C SNP is in the promoter region of the IL-6 gene and may be associated with changes in gene transcription and serum cytokine levels. Presence of the IL-6 -174G/C SNP is significantly correlated with morphine equivalent daily dose. Patients with the CC genotype needed a higher opioid dose than patients with the GG or GC genotypes. In conclusion, we found that the IL-6 -174G/C SNP is closely related to survival, analgesic use and pain tolerance in NSCLC patients. However, it is necessary to further validate the results with a larger patient cohort and elucidate the mechanisms of this SNP.

Molecular cloning, tissue expression pattern, and copy number variation of porcine SCUBE3.

The signal peptide CUB EGF-like domain-containing protein 3 (SCUBE3) gene is a member of SCUBE gene family and plays important roles in bone cell biology and the determination of limb bone length. In this study, the full-length transcript of porcine SCUBE3 was cloned using reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The full-length sequence of porcine SCUBE3 cDNA was 4131 base pairs and included 21 exons. The SCUBE3 gene contained a 2895-base pair open reading frame that encoded a peptide of 965 amino acids. Comparison of the deduced amino acid sequences of porcine SCUBE3 with those of human, mouse, zebrafish, and rat showed 96, 95, 73, and 95% identities, respectively. Porcine SCUBE3 mRNA expression levels were highest in the backfat, bone marrow, and cartilage tissues. Copy number variation was detected in porcine SCUBE3 and validated by real-time quantitative polymerase chain reaction. Different copy number variations were present in randomly selected individuals and may, therefore, be a good marker for identifying phenotypic traits. Our findings provide a basis for further investigation of the functions and regulatory mechanisms of SCUBE3 in pigs.

Comparison of extraction methods of total microbial DNA from freshwater.

The demand for molecular analysis of aquatic microbial communities in freshwater has highlighted the need for efficient methods of DNA extraction. The centrifugation method and filtration-membrane method are 2 widely used methods for extracting DNA. The objective of this study was to compare the extraction efficiency of 3 methods, including the centrifugation method, filtration-membrane method, and modified filtration-membrane method, by evaluating the quantity and purity of DNA extracts obtained from water. DNA extraction was analyzed by agarose gel electrophoresis, ultraviolet-spectroscopy, restriction enzyme digestion, and polymerase chain reaction. The results showed that the modified filtration-membrane method was the most efficient for extracting microbial DNA from freshwater with high integrity and purity and is suitable for molecular applications.

Association of the FABP2 Ala54Thr polymorphism with type 2 diabetes, obesity, and metabolic syndrome: a population-based case-control study and a systematic meta-analysis.

Previous studies have reported associations between the functional FABP2 Ala54Thr (rs1799883) polymorphism and type 2 diabetes mellitus (T2DM), obesity, and metabolic syndrome in different populations with conflicting results. We investigated the association between the FABP2 Ala54Thr polymorphism and T2DM (235 cases, 431 controls), obesity (377 cases, 431 controls), and metabolic syndrome (315 cases, 323 controls) by logistic regression analysis in a Chinese study cohort recruited from Yichang, Hubei Province. We then comprehensively reviewed the association of the FABP2 Ala54Thr polymorphism with T2DM, obesity, and metabolic syndrome via meta-analysis. The strength of association was assessed by odds ratios (ORs) with 95% confidence intervals (CIs). The FABP2 Ala54Thr polymorphism was significantly associated with obesity (AT vs AA: OR = 2.633, 95%CI = 1.065-6.663, P = 0.036; TT vs AA: OR = 4.160, 95%CI = 1.609-10.757, P = 0.003) and metabolic syndrome (TT vs AA: OR = 2.273, 95%CI = 1.242-4.156, P = 0.008) by logistic regression with adjustment for covariates. However, no significant association was found between T2DM and the FABP2 Ala54Thr polymorphism. We identified 24 studies on T2DM (4517 cases, 5224 controls), 9 studies on obesity (949 cases, 2002 controls), and 6 studies on metabolic syndrome (2194 cases, 3282 controls) by literature search. The meta-analyses revealed significant associations for metabolic syndrome (T allele: OR = 1.179, 95%CI = 1.015-1.362, P = 0.031) and T2DM (T allele: OR = 1.160, 95%CI = 1.08-1.24, P < 0.001), but no association for obesity (T allele: OR = 1.069, 95%CI = 0.925-1.235, P = 0.367).

Range-wide phylogeography and conservation genetics of a narrowly endemic stream salamander, Pachyhynobius shangchengensis (Caudata, Hynobiidae): implications for conservation.

The Shangcheng stout salamander (Pachyhynobius shangchengensis) is an endangered amphibian endemic to the Dabie Mountains, southeast China, and is currently threatened by habitat loss and illegal poaching. Here we used the mitochondrial DNA control region sequence (768 bp) to conduct a comprehensive investigation of genetic diversity, phylogeographic pattern, and demographic history of the species across its geographic distribution to assist its conservation. We concluded that the levels of genetic variation are relatively low in all four populations. Analysis of molecular variance indicated that the most likely phylogeographic pattern is [JGT] [KHJ] [TM, BYM]. Two distinct clades were identified in the phylogenetic tree of 28 haplotypes, corresponding to the two southern populations (TM, BYM) and two northern populations (JGT, KHJ). Significant population differentiation (FST) was detected among all populations. Among the four populations, historical demographic analyses (e.g., the g parameter, the Tajima D test, and the Fu Fs test) did not reveal definite information on population expansion except for the BYM population, which had undergone a strong population expansion event. Based on the analysis of a Bayesian skyline plot, the total population underwent a significant population fluctuation around 20 kya. This may have been triggered by the end of the last glacial maximum. In conclusion, the existence of three evolutionarily significant units (BMY-TM, KHJ, and JGT) and four management units (BMY, TM, KHJ, and JGT) is supported by our study.

Evidence for inbreeding depression in the tree Robinia pseudoacacia L. (Fabaceae).

The magnitude of inbreeding depression within populations is important for the evolution and maintenance of mixed mating systems. However, data are sparse on the magnitude of inbreeding depression in Robinia pseudoacacia. In this study, we compared differences in the mature seed set per fruit, seed mass, germination success, and seedling growth between self- and cross-pollination treatments and estimated the inbreeding depression at 3 stages: seed maturation, seedling emergence, and seedling growth at 10 and 20 weeks. We found that progenies resulting from cross-pollination treatments showed significantly higher fitness than progenies resulting from self-pollination, causing high levels of inbreeding depression. Inbreeding depression was not uniformly manifested, however, over the 3 stages. Inbreeding depression was the greatest between fertilization and seed maturation stage (δ = 0.5419), and the seedling emergence (0.3654) stage was second. No significant differences in seedling growth were observed between selfed and crossed progenies. The cumulative inbreeding depression (δ) across all 3 stages averaged 0.7452. Inbreeding depression may promote outcrossing in R. pseudoacacia by acting as a post-pollination barrier to selfing. The large difference in the seed set between self- and cross-pollination that we detected indicated that inbreeding depression would probably be a reasonable explanation for the high abortion and low seed set in R. pseudoacacia.