16S full-length gene sequencing analysis of intestinal flora in breast cancer patients in Hainan Province.

Breast cancer has become the number one cancer in the world, and intestinal flora may be closely linked to it. Geographic location also has an important impact on human intestinal flora. We conducted the first study on the intestinal flora of breast cancer patients and non-breast cancer patients in a tropical region - Hainan Province in China. At the same time, Pacbio platform based on third-generation sequencing was used for the first time to conduct 16S full-length sequencing of fecal microorganism DNA. We completed the species diversity analysis and differential species analysis of the intestinal flora between the two groups, inferred their functional genetic composition and performed functional difference analysis. There were statistically significant differences in alpha diversity between the two groups in Hainan Province. By species composition difference analysis, at the phylum level, Bacteroidales (P = 0.006) and Firmicutes (P = 0.002) was differed between the two groups, and at the genus level, 17 breast cancer-related differential species such as Bacteroides were screened. According to the five grouping methods including ER level, PR level, HER2 status, Ki67 index and histological grade of breast cancer patients, 4, 1, 9, 6, 5 differential microbiota were screened out respectively, which were in total 25 (P < 0.05 for all subgroups) . The functional prediction and difference analysis revealed two functional metabolisms with significant differences between the two groups of microbes (P < 0.05). These results suggest that breast cancer is associated with changes in the composition and function of intestinal flora. These microflora and functional differences may become biomarkers or new targets for diagnosis and treatment of breast cancer.

[Application value of high-throughput sequencing for the detection of thalassemia in ethnic Li minority areas].

OBJECTIVE: To assess the value of high-throughput sequencing for the detection of thalassemia-associated variants in ethnic Li minority areas of Hainan, China. METHODS: In Baoting Li and Miao Autonomous County of Hainan Province, 1842 middle school students were randomly selected as the subjects, which included 1249 ethnic Lis, 454 ethnic Hans, and 139 individuals from other ethnic minorities. With DNA extracted from peripheral blood samples, gap-PCR combined with high-throughput sequencing were carried out to detect potential variants of the globin genes. RESULTS: In total 22 α-thalassemia genotypes, 5ß-thalassemia genotypes, and 21 α-composite ß-thalassemia genotypes were detected. The carrier rates for ethnic Li, ethnic Han and other ethnic minorities were 78.14%, 24.01%, and 28.06%, respectively. In addition, 22 fusion genes, 8 variants leading to abnormal hemoglobin, and 10 rare mutations were identified. CONCLUSION: High-throughput sequencing can detect a wide range of genetic variants associated with thalassemia in the ethnic Li minority areas and has played an important role for the identification of fusion genes, variants underlying hemoglobin anomalies and rare mutations.

Report of Two Novel Thalassemia Variants, HBB: c.181delG and HBA1: c.121_126delAAGACC, in Chinese Individuals.

In this study, we report two novel thalassemia variants detected in Chinese individuals using targeted NGS technology. We detected a novel frameshift variant, HBB: c.181delG, in a 32-year-old Chinese individual. This novel variant [a single nucleotide deletion at nucleotide 181 of codon 60 (-G)], was detected by targeted next generation sequencing (NGS), resulting in a stop codon at codon 60 in exon 2 of the HBB gene. The impact of this novel variant was further analyzed by an in vitro model. We also identified a novel in-frame variant, HBA1: c.121_126delAAGACC [codons 40/41 (-AAGACC)], in another Chinese individual in this study. We named these two novel variants, HBB: c.181delG and HBA1: c.121_126delAAGACC according to the Human Genome Variation Society (HGVS), which were detected by the first author. These two novel variants have expanded the mutation spectrum of thalassemia and it would be beneficial for carrier screening, genetic counseling and prenatal diagnosis (PND) of thalassemia.

Assessment of the Link between XRCC1 Arg399Gln Polymorphism and Breast Cancer: a Meta-Analysis in a Single Ethnic Group.

BACKGROUND: Although various individual studies have been conducted to determine the association between X-ray repair cross-complementing group 1 (XRCC1) Arg399Gln polymorphism and breast cancer, the results remain inconclusive. To assess the influence of XRCC1 Arg399Gln polymorphism on the risk of breast cancer, a metaanalysis was performed in a single ethnic group. METHODS: Eligible studies were identified via databases such as PubMed, Springer Link, Ovid, Chinese Wanfang Data Knowledge Service Platform, Chinese National Knowledge Infrastructure, and Chinese Biology Medicine, throughout February 2016. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strengths of the associations. RESULTS: Ten studies documenting a total of 4732 breast cancer cases and 5677 controls were included in this metaanalysis. The results indicated no significant association between XRCC1 Arg399Gln polymorphism and breast cancer risk in both total analysis and subgroup analysis stratified by geographical areas and source of controls. CONCLUSIONS: This meta-analysis provided evidence that XRCC1 Arg399Gln variant might not be risk alleles for breast cancer susceptibility in the Chinese population. Further studies conducted in other ethnic groups are required for definite conclusions.

Association Studies of CYP1A1 Exon7 Polymorphism and -GSTM1 Interaction with Esophageal Cancer Risk: a Meta-Analysis in the Chinese Population.

BACKGROUND: Although many epidemiological studies have investigated the CYP1A1 exon7 polymorphism and -GSTM1 interaction with esophageal cancer (EC), definite conclusions cannot be drawn. This study was conducted to explore this association in the Chinese population using meta-analysis. METHODS: Relevant studies were identified from PubMed, Springer Link, Ovid, Chinese Wanfang Data Knowledge Service Platform, Chinese National Knowledge Infrastructure, and Chinese Biology Medicine databases published through August 2015. The association of CYP1A1 exon7 polymorphisms and EC risk was estimated by odds ratio (ORs) with 95% confidence intervals (CIs). In addition, the interaction between the CYP1A1 exon7 and GSTM1 genotypes was assessed. RESULTS: A total of 13 case-control studies including 1781 EC cases and 1996 controls were included in this metaanalysis. Overall, significantly increased EC risk was associated with the CYP1A1 exon7 polymorphism (G vs. A OR = 1.36, 95% CI = 1.14 - 1.64; GG vs. AA: OR = 1.85, 95% CI = 1.22 - 2.79; GG vs. AG: OR = 1.41, 95% CI = 1.01 - 1.96; GG + AG vs. AA: OR = 1.47, 95% CI = 1.28 - 1.68; GG vs. AA + AG: OR = 1.60, 95% CI = 1.10 - 2.31). In a subgroup analyses stratified by geographic areas, histopathology type and source of controls, the significant risk was found in hospital-based population, in South and North China. Analysis of CYP1A1- GSTM1 interaction did find synergistic interaction between these two genes. CONCLUSIONS: This meta-analysis provides the evidence that CYP1A1 exon7 polymorphism may contribute to the EC development in the Chinese population, and CYP1A1- GSTM1 interaction might elevate the risk.

Glutathione-S-transferases M1/T1 gene polymorphisms and endometriosis: a meta-analysis in Chinese populations.

In view of the controversies surrounding the glutathione-S-transferases (GST) M1/T1-endometriosis association, a meta-analysis of the GSTM1/GSTT1 genetic association studies of endometriosis was performed in Chinese populations. PubMed, Springer Link, OvidSP, and Chinese databases were searched for related studies. A total of nine studies on GSTM1-endometriosis involved 874 cases and 997 controls, and five studies on GSTT1 involved 404 cases and 513 controls were included in this meta-analysis. Overall, the null genotype of GSTM1/GSTT1 was significantly related to endometriosis risk in Chinese populations (GSTM1, OR = 2.21, 95% CI: 1.22-4.01; GSTT1, OR = 2.31, 95% CI: 1.34-3.99). In subgroup analyses stratified by ethnicity and source of controls, the same results were observed in Chinese Han and population-based studies. The sensitivity analysis confirmed the reliability and stability of the meta-analysis. No publication bias was found among studies by Egger's test. In conclusion, our meta-analysis supports that the GSTM1/GSTT1 null genotype might contribute to individual susceptibility to endometriosis in Chinese populations, especially in Chinese Han.

Decreased BIRC5-206 promotes epithelial-mesenchymal transition in nasopharyngeal carcinoma through sponging miR-145-5p.

Metastasis significantly contributes to the poor prognosis of advanced nasopharyngeal carcinoma (NPC). Our prior studies have demonstrated a decrease in BIRC5-206 expression in NPC, which promotes disease progression. However, the role of BIRC5-206 in the invasion and metastasis of NPC has not been fully elucidated. In this study, our objective was to explore the biological function and underlying mechanisms of BIRC5-206 in NPC. Additionally, we established an NPC mouse model of lung invasiveness using C666 cells to assess the impact of BIRC5-206 on NPC metastasis. Our results revealed that silencing BIRC5-206 inhibited apoptosis and enhanced the invasion of NPC cells, whereas its overexpression reversed these effects. Moreover, decreased BIRC5-206 expression significantly increased N-cadherin and Vimentin expression while reducing E-cadherin and occludin levels, both in vivo and in vitro. Additionally, silencing BIRC5-206 markedly augmented the formation of invasive foci in lung tissues. Rescue experiments further confirmed that decreased BIRC5-206 expression facilitates NPC metastasis via modulation of the miR-145-5p/CD40 signaling pathway. In summary, our study suggests that BIRC5-206 may serve as a potential prognostic biomarker and therapeutic target in the diagnosis and treatment of NPC.

Low Expression of BIRC5-206 Promotes Cancer Progression in Nasopharyngeal Carcinoma via Enhancing Expression of Stem Cell Markers.

OBJECTIVE: Survivin is highly expressed in various malignant tumor cells and positively related to poor prognosis and drug resistance. This study aimed to explore the role of non-coding splice variant of Survivin, BIRC5-206 (ENST00000589892.1) in the progression of nasopharyngeal carcinoma (NPC), a malignant tumor that highly occurs in the southern region of China. METHODS: shRNA was used to knockdown BIRC5-206 mRNA level in CNE-2 and HOPNE-1 cells. Then, cell death, migration, invasion and clone formation ability of CNE-2 and HOPNE-1 cells were detected by flow cytometry, scratch-healing experiments, transwell invasion assay and clone formation assay, respectively. CD44+ and CD133+ positive cells were determined via Flow cytometry. Oct4, Nanog and SOX2 protein levels in CNE-2 and HOPNE-1 cells were measured by Western blot. RESULTS: BIRC5-206 decreased significantly in NPC cell lines. Silencing of BIRC5-206 suppressed the apoptosis, facilitated the migration, invasion and proliferation of both HONE1 and CNE-2 cells. In addition, knockdown of BIRC5-206 significantly promoted the expression cancer stem cell marker (CD44 and CD133) and pluripotency markers (Oct4, Sox2 and Nanog). CONCLUSIONS: BIRC5-206 might facilitate NPC tumor progression by inducing the transformation of NPC cells to cancer stem cells.

TTN-AS1 accelerates the growth and migration of nasopharyngeal carcinoma cells via targeting miR-876-5p/NETO2.

Nasopharyngeal carcinoma (NPC) is one of the most predominant cancers occurring in China with high morbidity. Lately, large quantities of long non-coding RNAs (lncRNAs) have been highlighted to regulate the biological activities in multiple tumors, including NPC. Our study centered on whether TTN-AS1 was involved in NPC and how it modulated the progression of NPC. Here, qRT-PCR data uncovered that TTN-AS1 expression was conspicuously high in NPC cells. Based on the results of functional assays, TTN-AS1 silence hampered the proliferative, migratory, and invasive abilities but stimulated the apoptotic capability of NPC cells. After a series of mechanism assays, TTN-AS1 was found to competitively bind with miR-876-5p and recruit UPF1 to enhance NETO2 expression. In addition, TTN-AS1 could be transcriptionally activated by YY1 in NPC cells. It was also found that miR-876-5p overexpression or NETO2 downregulation had inhibitory effects on cell proliferation, migration, and invasion in NPC. Moreover, NETO2 upregulation could restore the suppressive impacts of TTN-AS1 depletion on NPC cell and tumor growth. In conclusion, YY1-activated TTN-AS1 interacted with both miR-876-5p and UPF1 to upregulate NETO2, thus strengthening NPC cell malignant behaviors, which might provide more useful information for people to develop effective NPC treatments.

Molecular dynamic and pharmacological studies on protein-engineered hirudin variants of Hirudinaria manillensis and Hirudo medicinalis.

BACKGROUND AND PURPOSE: Hirudin variants are the most powerful thrombin inhibitors discovered to date, with a lower risk of bleeding than heparin. For anticoagulation, the C-termini of hirudin variants bind to the exocite I of thrombin. Anticoagulant effects of gene-recombinant hirudin are weaker than natural hirudin for the reason of lacking tyrosine O-sulfation at C-terminus. EXPERIMENTAL APPROACH: An integrative pharmacological study was carried out using molecular dynamic, molecular biological and in vivo and in vitro experiments to elucidate the anticoagulant effects of protein-engineered hirudins. KEY RESULTS: Molecular dynamic analysis showed that modifications of the C-termini of hirudin variant 1 of Hirudo medicinalis (HV1) and hirudin variant 2 of Hirudinaria manillensis (HM2) changed the binding energy of the C-termini to human thrombin. The study indicated that Asp61 of HM2 that corresponds to sulfated Tyr63 of HV1 is critical for inhibiting thrombin activities. Further, the anticoagulant effects of HV1 and HM2 were improved when the amino acid residues adjacent to Asp61 were mutated to Asp. These improvements were prolongation of the activated partial thromboplastin time, prothrombin time and thrombin time of human blood, and decreased Ki and IC50 values. In the in vivo experiments, mutations at C-termini of HV1 and HM2 significantly changed partial thromboplastin time, prothrombin and thrombin time CONCLUSION AND IMPLICATIONS: The study indicated that the anticoagulant effects of gene-engineered HM2 are stronger than gene-engineered HV1 and HM2-E60D-I62D has the strongest effects and could be an antithrombotic with better therapeutic effects.

Circ_0000215 Exerts Oncogenic Function in Nasopharyngeal Carcinoma by Targeting miR-512-5p.

Background: Increasing circular RNAs (circRNAs) are reported to participate in cancer progression. Nonetheless, the role of circRNAs in nasopharyngeal carcinoma (NPC) has not been fully clarified. This work is aimed to probe the role of circ_0000215 in NPC. Methods: Circ_0000215 expression in NPC tissues and cell lines was examined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8 (CCK-8) assay, 5-bromo-2'-deoxyuridine (BrdU) assay, scratch healing assay and Transwell experiment were executed to investigate the regulatory function of circ_0000215 on the proliferation, migration and invasion of NPC cells. RNA immunoprecipitation (RIP), pull-down and dual-luciferase reporter experiments were utilized to determine the binding relationship between circ_0000215 and miR-512-5p, and between miR-512-5p and phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) 3'UTR. The effects of circ_0000215 on NPC growth and metastasis in vivo were examined with nude mice model. Western blot was applied to detect the regulatory effects of circ_0000215 and miR-512-5p on PIK3R1 expression. Results: Circ_0000215 was overexpressed in NPC tissues and cell lines. The functional experiments confirmed that knockdown of circ_0000215 impeded the growth and metastasis of NPC cells in vitro and in vivo. Additionally, circ_0000215 could also work as a molecular sponge to repress miR-512-5p expression. PIK3R1 was validated as a target gene of miR-512-5p, and circ_0000215 could increase the expression level of PIK3R1 in NPC cells via suppressing miR-512-5p. Conclusion: Circ_0000215 is overexpressed in NPC and exerts oncogenic effects in NPC through regulating miR-512-5p/PIK3R1 axis.

Polymerase Chain Reaction in the Detection of miR-455-5p and Sphingosine-1 Phosphate Proteins in Cervical Carcinoma with the Help of Gold Nanoparticles-Based.

This study aimed to detect miR-455-5p and S1PR1 proteins using nanoparticle-assisted polymerase chain reaction (nano-PCR) to determine their correlation with cervical carcinoma prognosis. To achieve this study's goals, we selected 48 cervical carcinoma patients between January 2014 to January 2016 and subjected them to the miR-455-5p test by nano-PCR. The collected samples were then divided into two groups based on miR-455-5p levels. We had four HeLa cell groups, one group as the control, and one group overexpressed the miR-455-5p protein. A third group was miR-455-5p silent, and a separate group overexpressed both the miR-455-5p and S1PR1 proteins. Results also proved that the nano-PCR had a higher sensitivity than RT-PCR, and patients with poor prognosis had lesser miR-455-5p levels. Similarly, high levels of miR-455-5 contributed to cancer cell apoptosis and migration inhibition by targeting S1PR1 expression negatively. These two biomarkers are therefore significantly related to the prognosis of cervical carcinoma patients.

Transcriptional Start Site Coverage Analysis in Plasma Cell-Free DNA Reveals Disease Severity and Tissue Specificity of COVID-19 Patients.

Symptoms of coronavirus disease 2019 (COVID-19) range from asymptomatic to severe pneumonia and death. A deep understanding of the variation of biological characteristics in severe COVID-19 patients is crucial for the detection of individuals at high risk of critical condition for the clinical management of the disease. Herein, by profiling the gene expression spectrum deduced from DNA coverage in regions surrounding transcriptional start site in plasma cell-free DNA (cfDNA) of COVID-19 patients, we deciphered the altered biological processes in the severe cases and demonstrated the feasibility of cfDNA in measuring the COVID-19 progression. The up- and downregulated genes in the plasma of severe patient were found to be closely related to the biological processes and functions affected by COVID-19 progression. More importantly, with the analysis of transcriptome data of blood cells and lung cells from control group and cases with severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection, we revealed that the upregulated genes were predominantly involved in the viral and antiviral activity in blood cells, reflecting the intense viral replication and the active reaction of immune system in the severe patients. Pathway analysis of downregulated genes in plasma DNA and lung cells also demonstrated the diminished adenosine triphosphate synthesis function in lung cells, which was evidenced to correlate with the severe COVID-19 symptoms, such as a cytokine storm and acute respiratory distress. Overall, this study revealed tissue involvement, provided insights into the mechanism of COVID-19 progression, and highlighted the utility of cfDNA as a noninvasive biomarker for disease severity inspections.

[Identification of the new allele D18S1364-10 by sequence analysis].

OBJECTIVE: To identify a new allele by analyzing the polymorphism of D18S1364 in Power Plex(TM)16. METHODS: An abnormal band was found in paternity test by short tandem repeat-PCR, and collected by gel extraction. Then the DNA was amplified, cloned and sequenced. RESULTS: A fragment containing eight bases less than the minimal allele in the D18S1364 ladder, was found with the core sequence of (ATCT)6(ATGT)1(ATCT)3. The allele was D18S1364-10. CONCLUSION: The D18S1364-10 allele was found and reported for the first time in China.

A genome-wide scan suggests a susceptibility locus on 5p 13 for nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) occurs with high frequency in Southeast Asian populations. The high prevalence and familial clustering of NPC in these populations suggest that genetic factors may contribute to the increased cancer risk by affecting susceptibility. The aim of the present study was to map chromosomal loci linked to susceptibility genes predisposing for NPC. We carried out a genome-wide scan by multipoint affected-only allele-sharing methods in 15 Chinese NPC families with two to six affected members per family. The families were from the Guangdong province in the south of China, where the highest risk of NPC is documented. These samples were genotyped using 800 microsatellite markers covering all autosomal chromosomes with an average marker distance of 5 cM. Using multipoint linkage analysis, four loci (2q, 5p, 12p, and 18p) showed LOD scores above 1.5. After genotyping additional markers in these four regions, only one locus on 5p13 showed an increased LOD of 2.1. In further haplotype analysis, affected individuals in six families shared three marker haplotypes between D5S674 and D5S418. In conclusion, a region on 5p13 may harbor a susceptibility gene for NPC.

[Identification of a rare allele of FGA-13].

OBJECTIVE: To identify a rare allele in the FGA locus in a Hainan Li ethnic family. METHODS: Short tandem repeat-PCR (STR-PCR) and DNA sequencing were used to detect the allele. RESULTS: A rare allele in the FGA locus was identified. It is FGA-13. CONCLUSION: This finding enriched the gene polymorphism database, and may improve the ability of individual identification.

[Study on the expression of survivin mRNA and protein in nasopharyngeal carcinoma].

OBJECTIVE: To investigate the relationship between survivin mRNA and protein expression and nasopharyngeal carcinoma (NPC). METHODS: Survivin mRNA and protein were detected by in situ hybridization and immunohistochemical S-P staining respectively. RESULTS: Among 64 cases of NPC, 42 cases (65.6%) were positive for survivin mRNA expression, 30 cases (46.9%) had high expression level. In 46 cases (71.9%) of NPC were positive for survivin protein expression, and 38 cases (59.4%) had high expression level. In 22 cases of NPC with detailed clinical information, the positive expression rates of survivin mRNA and protein in stages III+ IV of NPC were 66.7% and 61.1% respectively, which were higher than those in stages I+ II of NPC (50.0% and 50.0% respectively). There was no significant difference between survivin mRNA and protein expression regarding age or gender of NPC patients (all P> 0.05). The positive expression rates of survivin mRNA and protein in chronic nasopharyngitis group were 33.3% and 23.3% respectively, which were lower than those in NPC group (chi (2)= 12.04, P< 0.01 and chi (2)= 19.57, P< 0.01, respectively). In 64 cases of NPC, 36 cases were positive for both survivin mRNA and protein, and the expression of survivin mRNA and protein showed positive correlation (phi = 0.43). CONCLUSION: The expression of survivin gene may play some roles in the pathogenesis of NPC. Detection of survivin mRNA and protein will be helpful for diagnosis, clinical staging and prognosis of NPC.

Survivin gene functions and relationships between expression and prognosis in patients with nasopharyngeal carcinoma.

This study aimed to investigate the relationship between prognosis and protein and mRNA expression of an apoptotic inhibitor gene, survivin, in patients with nasopharyngeal carcinoma. Furthermore, functions of the survivin gene in the CNE2 nasopharyngeal carcinoma cell line were assessed. Immunohistochemistry and in situ hybridization were used in detecting the survivin protein and mRNA in 44 nasopharyngeal carcinoma specimens, and 30 chronic nasopharyngitis samples as controls. Survivin gene expression in CNE2 cell line was suppressed with an shRNA (short hairpin RNA). The positive ratios of expression for survivin protein and mRNA in nasopharyngeal carcinoma were 79.5% and 75.0% respectively, obviously higher than in the control group (p<0.01), and there is very good consistency between the two methods. The mean survival time of patients with higher survivin protein or mRNA expression was shorter than in patients with lower levelsv(p<0.01). Proliferation of the CNE2 cell line was distinctly inhibited by the shRNA . The results indicate that overexpression of the survivin gene plays an important role in onset and development of nasopharyngeal carcinoma, and it may be helpful for prognostic appraisal.