Results Of Real-time Quantitative Polymerase Chain Reaction Research Articles

An Antenna-Enriched Chemosensory Protein Plays Important Roles in the Perception of Host Plant Volatiles in Bactrocera dorsalis (Diptera: Tephritidae).

Olfaction plays indispensable roles in insect behavior such as host location, foraging, oviposition, and avoiding predators. Chemosensory proteins (CSPs) can discriminate the hydrophobic odorants and transfer them to the odorant receptors. Presently, CSPs have been identified in many insect species. However, their presence and functions remain unknown in Bactrocera dorsalis, a destructive and invasive insect pest in the fruit and vegetable industry. Here, we annotated eight CSP genes in the genome of B. dorsalis. The results of quantitative real-time polymerase chain reaction (RT-qPCR) showed that BdorCSP3 was highly expressed in the antennae. Molecular docking and in vitro binding assays showed that BdorCSP3 had a good binding ability to host volatiles methyl eugenol (ME, male-specific attractant) and β-caryophyllene (potential female attractant). Subsequently, CRISPR/Cas9 was used to generate BdorCSP3-/- mutants. Electroantennograms (EAGs) and behavioral assays revealed that male mutants significantly reduced the preference for ME, while female mutants lost their oviposition preference to β-caryophyllene. Our data indicated that BdorCSP3 played important roles in the perception of ME and β-caryophyllene. The results not only expanded our knowledge of the olfaction perception mechanism of insect CSPs but also provided a potential molecular target for the control of B. dorsalis.

FlgL mutation reduces pathogenicity of Aeromonas hydrophila by negatively regulating swimming ability, biofilm forming ability, adherence and virulence gene expression

Aeromonas hydrophila is a serious human and animal co-pathogenic bacterium. Flagellum, a key virulence factor, is vital for bacterium tissue colonization and invasion. flgL is a crucial gene involved in the composition of flagellum. However, the impact of flgL on virulence is not yet clear. In this study, we constructed a stable mutant strain (△flgL-AH) using homologous recombination. The results of the attack experiments indicated a significant decrease in the virulence of △flgL-AH. The biological properties analysis revealed a significant decline in swimming ability and biofilm formation capacity in △flgL-AH and the transmission electron microscope results showed that the ∆flgL-AH strain did not have a flagellar structure. Moreover, a significant decrease in the adhesion capacity of ∆flgL-AH was found using absolute fluorescence quantitative polymerase chain reaction (PCR). The quantitative real-time PCR results showed that the expression of omp and the eight flagellum-related genes were down-regulated. In summary, flgL mutation leads to a reduction in pathogenicity possibly via decreasing the swimming ability, biofilm formation capacity and adhesion capacity, these changes might result from the down expression of omp and flagellar-related genes.

Baicalin Promotes Apoptosis of Human Medullary Breast Cancer via the ERK/p38 MAPK Pathway

Objectives: The present study aims to examine the effects of baicalin on the human medullary breast carcinoma (MBC) cell line Bcap-37 and to determine whether baicalin regulates Bcap-37 cell apoptosis through the ERK/p38 mitogen-activated protein kinase (MAPK) signaling pathway. Materials and Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Transwell assay were utilized to measure the effects of baicalin on the proliferation and migration ability of Bcap-37 cells, respectively. Flow cytometer analysis was implied to detect the effects of baicalin on Bcap-37 cell apoptosis. Real-time quantitative polymerase chain reaction (RT-PCR) was conducted to observe the influence of baicalin on mRNA expressions of apoptosis-related genes. Western blot was executed to further explore the action of baicalin on apoptosis-related proteins. PD98059 (a specific inhibitor of ERK) and SB203580 (a specific inhibitor of p38 MAPK) were used to further clarify the intrinsic mechanism of baicalin regulating apoptosis in Bcap-37 cells. Results: Compared with the control group, baicalin significantly inhibits the proliferation activity of Bcap-37 cells in a concentration- and time-dependent manner, with a p-value < 0.05. The transwell assay indicated that the migration viability of cells decreased further, followed by the increased concentration of baicalin, and the p-value had a statistical difference. Besides, flow cytometry was conducted to assess the effects of baicalin on the early and late apoptosis rates of Bcap-37 cells, and results showed that baicalin highly promoted the apoptosis level both at the early and late stages with a statistical difference in a concentration-dependent manner ( p < 0.05). Results of RT-PCR presented that, compared with the control group, baicalin significantly activated the mRNA expression of Bax, p38, and p-ERK1 and abolished the mRNA expression of Bcl-2 in every dosing group in a concentration-dependent way ( p < 0.05). Western blot exhibited that, compared with the control group, baicalin promoted the protein expression of caspase-3, caspase-9, Bax, p38, P-ERK, and p53 while playing an opposite function to Bcl-2 in each dosing group ( p values have statistical differences). At last, PD98059 and SB203580 were applied to explore the potential mechanism of baicalin in apoptosis promotion. And results revealed that, compared with the group treated with baicalin alone, protein expression of Bax, p38, p-ERK, caspase-3, and caspase-9 was downregulated obviously in the group treated with both baicalin and PD98059 or SB203580 ( p < 0.05). The p53 expression inhibition showed a difference only when compared to the group adding PD98059. Compared with the blank group, a statistical difference was only observed in Bcl-2 expression in the group treated with baicalin in combination with PD98059 or SB203580 ( p < 0.05). Interestingly, a p53 expression difference was only displayed between the blank group and the group treated with baicalin and SB203580 ( p < 0.05). Conclusion: Our study observed that baicalin inhibits the proliferation and migration ability of Bcap-37 cells. In addition, baicalin induces cell apoptosis via the ERK/p38 MAPK signal pathway.

Oleanolic acid attenuates hydrogen peroxide-induced apoptosis of IPEC-J2 cells through suppressing c-Jun and MAPK pathway.

Oleanolic acid (OA) is a natural triterpenoid with therapeutic potential for a multitude of diseases. However, the precise mechanism by which OA influences stress-induced apoptosis of intestinal epithelial cells remains elusive. Therefore, the effect of OA on intestinal diseases under stressful conditions and its possible mechanisms have been investigated. In a hydrogen peroxide (H2 O2 )-induced oxidative stress model, OA attenuated H2 O2 -induced apoptosis in a concentration-dependent manner. To investigate the underlying mechanisms, the gene expression profile of OA on IPEC-J2 cells was analyzed using an RNA sequencing system. Results from gene ontologyand Kyoto encyclopedia of genes and genomesanalysis confirmed that OA may mitigate the cytotoxic effects of H2 O2 by downregulating gene expression through the MAPK signaling pathway. Furthermore, Quantitative real-time polymerase chain reactionresults validated the differentially expressed genes data. Western blot analysis further demonstrated that OA effectively suppressed the expression level of c-Jun protein induced by H2 O2 in IPEC-J2 cells. Collectively, our results indicate that OA pretreatment significantly attenuated H2 O2 -induced apoptosis in intestinal epithelial cells through suppressing c-Jun and MAPK pathway.

Identification of the potential biomarkers associated with circadian rhythms in heart failure.

Heart failure (HF) is a syndrome with multiple clinical symptoms resulting from damage to the heart's structure and/or function with various pathogenic factors, which has developed as one of the most severe threats to human health. Approximately 13% of genes and about 8% of proteins contained in the heart are rhythmic, which could lead to HF if disrupted. Herein, we aimed to identify the circadian rhythms-related hub genes as potential biomarkers contributing to the identification and treatment of HF. Expression data of ischemic and dilated cardiomyopathy samples with or without HF were collected from the GEO database. First, genes with differential expression in HF and healthy samples were identified, named as differentially expressed genes (DEGs), which were then intersected with circadian rhythms-related genes to identify circadian rhythms-related DEGs. A protein-protein interaction (PPI) network was established to screen hub genes. The performance of the hub genes to identify HF among healthy controls was assessed by referring to the receiver operating characteristic (ROC) curve. Additionally, quantitative real-time polymerase chain reaction (RT-PCR) was run to further validate the hub genes depending on clinical human peripheral blood samples. A total of 10,163 DEGs were determined, composed of 4,615 up-regulated genes and 5,548 down-regulated genes in HF patients in comparison to healthy controls. By overlapping the circadian rhythms-related genes in the Circadian Gene DataBase (CGDB), 723 circadian rhythms-related DEGs were obtained, mainly enriched in regulating lipid metabolic process, circadian rhythm and AMPK signaling pathway. Eight hub genes were screened out through the PPI network. The ROC curve indicated the high accuracy of five hub genes with AUC>0.7, which also showed high accuracy validated by the external validation dataset. Furthermore, according to the results of quantitative RT-PCR, the HF group showed significantly increased relative mRNA expression of CRY2 and BHLHE41 while the decreased ARNTL and NPAS2 in comparison to controls, indicating the four hub genes as potential biomarkers of HF. Our study validated that ARNTL, CRY2, BHLHE41 and NPAS2 could serve as potential biomarkers of circadian rhythm in HF. These results may provide a reference for employing novel markers or targets for the diagnosis and treatment of HF.

The effect of Annexin A5 overexpression on invasiveness and expression of the genes involved in epithelial-mesenchymal transition of HCT 116 cell line.

Epithelial-to-mesenchymal transition (EMT) plays a critical role in colorectal cancer (CRC) metastasis. In the present study, we evaluated the effects of annexin A5 (ANXA5) overexpression on invasiveness as well as the expression of genes involved in EMT of HCT 116 cell line. PCMV6-AC-IRES-GFP plasmid harboring ANXA5 cDNA was constructed. HCT 116 cell line was transfected with recombinant plasmids using Lipofectamine 3000. Fluorescent microscopy was used to determine the efficiency of plasmid transfection. Cell viability was determined using the MTT assay. HCT 116 cell migration was evaluated using wound healing assay and transwell migration assay. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure the expression of genes involved in EMT. The results of RT-qPCR showed overexpression of ANXA5 compared to the control group. ANXA5 overexpression had no significant effects on cell viability but significantly decreased the rate of wound closure in the wound healing assay as well as the number of migrated cells in transwell assay. Furthermore, ANXA5 overexpression decreased the expression of N-cadherin, Snail, Slug, MMP-2, and MMP-9 while the expression of E-cadherin increased following ANXA5 overexpression. However, VEGF expression did not significantly change after ANXA5 overexpression. Results of the present study suggest that ANXA5 overexpression might have inhibitory effects on the metastasis of CRC through modulating the expression of EMT- related genes.

Screening of co-pathogenic genes of non-alcoholic fatty liver disease and hepatocellular carcinoma.

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a risk factor for hepatocellular carcinoma (HCC). However, its carcinogenic mechanism is still unclear, looking for both diseases’ transcriptome levels, the same changes as we are looking for NAFLD may provide a potential mechanism of action of HCC. Thus, our study aimed to discover the coexisting pathogenic genes of NAFLD and HCC.MethodsWe performed a variance analysis with public data for both diseases. At the same time, weighted gene correlation network analysis (WGCNA) was used to find highly correlated gene modules in both diseases. The darkturquoise gene module was found to be highly correlated with both diseases. Based on the diagnosis related module genes and the differential genes of the two diseases, we constructed diagnostic and prognostic models by logistic regression, univariate Cox regression, and LASSO regression. Public datasets verified the results. Meanwhile, we built a competing endogenous RNA (ceRNA) network based on the model genes and explored the related pathways and immune correlation involved in the two diseases by using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and gene set enrichment analyses. Immunohistochemistry was used to verify the different expression of ABCC5 and TUBG1 among the normal liver, NAFLD, and HCC tissues. Sodium palmitate/sodium oleate was used to establish high-fat cell models, and Real Time Quantitative Polymerase Chain Reaction (RT-qPCR) was used to verify the messenger RNA (mRNA) expression of ABCC5 in lipidization cells.ResultsA total of 26 upregulated genes and 87 downregulated genes were found using limma package identification analysis. According to WGCNA, the darkturquoise gene module was highly correlated with the prognosis of both diseases. The coexisting genes acquired by the two groups were only three central genes, that is, ABCC5, DHODH and TUBG1. The results indicated that the diagnostic and prognostic models constructed by ABCC5 and TUBG1 genes had high accuracy in both diseases. The results of immunohistochemistry showed that ABCC5 and TUBG1 were significantly overexpressed in NAFLD and HCC tissues compared with normal liver tissues. The Oil Red O staining and triglyceride identified the successful construction of HepG2 and LO2 high-fat models using PA/OA. The results of RT-qPCR showed that the lipidization of LO2 and HepG2 increased the mRNA expression of ABCC5.ConclusionsThe gene model constructed by ABCC5 and TUBG1 has high sensibility and veracity in the diagnosis of NAFLD as well as the diagnosis and prognosis of HCC. ABCC5 and TUBG1 may play an important role in the development of NAFLD to HCC. In addition, lipidization could upregulate the mRNA expression of ABCC5 in HCC.

Identification of Transcriptional Pattern Related to Immune Cell Infiltration With Gene Co-Expression Network in Papillary Thyroid Cancer.

BackgroundA growing body of evidence suggests that immune cell infiltration in cancer is closely related to clinical outcomes. However, there is still a lack of research on papillary thyroid cancer (PTC).MethodsBased on single-sample gene set enrichment analysis (SSGSEA) algorithm and weighted gene co-expression network analysis (WGCNA) tool, the infiltration level of immune cell and key modules and genes associated with the level of immune cell infiltration were identified using PTC gene expression data from The Cancer Genome Atlas (TCGA) database. In addition, the co-expression network and protein-protein interactions network analysis were used to identify the hub genes. Moreover, the immunological and clinical characteristics of these hub genes were verified in TCGA and GSE35570 datasets and quantitative real-time polymerase chain reaction (PCR). Finally, receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic value of hub genes.ResultsActivated B cell, activated dendritic cell, CD56bright natural killer cell, CD56dim natural killer cell, Eosinophil, Gamma delta T cell, Immature dendritic cell, Macrophage, Mast cell, Monocyte, Natural killer cell, Neutrophil and Type 17 T helper cell were significantly changed between PTC and adjacent normal groups. WGCNA results showed that the black model had the highest correlation with the infiltration level of activated dendritic cells. We found 14 hub genes whose expression correlated to the infiltration level of activated dendritic cells in both TCGA and GSE35570 datasets. After validation in the TCGA dataset, the expression level of only 5 genes (C1QA, HCK, HLA-DRA, ITGB2 and TYROBP) in 14 hub genes were differentially expressed between PTC and adjacent normal groups. Meanwhile, the expression levels of these 5 hub genes were successfully validated in GSE35570 dataset. Quantitative real-time PCR results showed the expression of these 4 hub genes (except C1QA) was consistent with the results in TCGA and GSE35570 dataset. Finally, these 4 hub genes had diagnostic value to distinguish PTC and adjacent normal controls.ConclusionsHCK, HLA-DRA, ITGB2 and TYROBP may be key diagnostic biomarkers and immunotherapy targets in PTC.

Effects of grape seed proanthocyanidins on the expression of inflammatory mediators in gingival epithelial cells.

This study aims to determine the effect of grape seed proanthocyanidin (GSP) pretreatment on lipopolysaccharide (LPS)-induced inflammation of human gingival epithelial cells (HGECs). HGECs were cultivated with different concentrations of GSPs (0, 1, 5, 10, 20, 40, 60, 80, 100 μg·mL-1) for 6, 12, 24, and 48 h. CCK-8 was used to detect the proliferation activity of HGECs. HGECs were treated with different concentrations of GSPs (0, 10, 20, and 40 μg·mL-1) for 24 h and then cultured with 1.0 μg·mL-1 LPS. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 and anti-inflammatory cytokines IL-4, IL-10, and transforming growth factor-β (TGF-β). Quantitative real-time polymerase chain reaction (QRT-PCR) was used to detect the mRNA expression levels of TNF-α, IL-1β, IL-6, IL-4, IL-10, and TGF-β. When the GSP concentration was 0-40 μg·mL-1, the cell proliferation had no significant difference. When the action time reached 24 h, the cell proliferation was the highest. The results of ELISA and QRT-PCR showed that 10, 20, and 40 μg·mL-1 GSPS decreased the expression levels of TNF-α, IL-1β, and IL-6 (P<0.05) and increased the expression levels of IL-4, IL-10, and TGF-β compared with 0 μg·mL-1 GSPS (P<0.05). GSPS (0-40 μg·mL-1) has no significant effect on the proliferation activity of HGECs. Pretreatment with GSPS can inhibit the expression of pro-inflammatory factors and enhance the expression of anti-inflammatory factors. Hence, GSPS has a certain preventive effect on the resistance of HGECs to the stimulation of endotoxin.

Effects and Mechanisms of Bone Marrow Mesenchymal Stem Cell Transplantation for Treatment of Ischemic Stroke in Hypertensive Rats.

Background and ObjectivesStroke is the most common cause of human death and functional disability, resulting in more than 5 million deaths worldwide each year. Bone marrow mesenchymal stem cells (BMSCs) are a kind of stem cell that are able to self-renew and differentiate into many types of tissues. Therefore, BMSCs have the potential to replace damaged neurons and promote the reconstruction of nerve conduction pathways and connective tissue. However, it remains unknown whether transplanted BMSCs promote angiogenesis or improve the tissue microenvironment directly or indirectly through paracrine interactions. This study aimed to determine the therapeutic effect of BMSCs on ischemic stroke with hypertension in a rodent model and to explore the possible mechanisms underlying any benefits.Methods and ResultsMiddle cerebral artery occlusion was used to establish the experimental stroke model. The area of cerebral infarction, expression of vascular endothelial growth factor (VEGF) and glial cell line-derived neurotrophic factor (GDNF), and increment of astrocyte were measured by TTC staining, western blot, real-time quantitative polymerase chain reaction (RT-qPCR) and immunocytochemistry. The results showed a smaller area of cerebral infarction and improved neurological function scores in animals treated with BMSCs compared to controls. The results of RT-qPCR and western blot assays showed higher expression of VEGF and GDNF in BMSC-treated animals compared with controls. Our study also showed that one round of BMSCs transplantation significantly promoted the proliferation of subventricular zone and cortical cells, especially astrocytes, on the ischemic side following cerebral ischemia.ConclusionsAbove findings support that BMSCs have therapeutic effects for ischemic stroke complicated with hypertension, which may occur via up-regulated expression of VEGF and GDNF and reduction of neuronal apoptosis, thereby promoting the recovery of nerve function.

Comprehensive analysis of the functional and prognostic value of E2F transcription factors in human prostate cancer through data mining and experimental validation.

BackgroundA growing body of evidence shows that E2F transcription factors play a significant role in the tumorigenesis of prostate cancer. However, their functional and prognostic value has not been fully illustrated. Therefore, we used bioinformatics methods to further analyze the possible roles of E2F transcription factors in the development and progression of prostate cancer.MethodsWe explored the expression levels of E2F transcription factors using data from The Cancer Genome Atlas (TCGA) and Oncomine database in paired and unpaired samples. The clinical correlation and prognostic value of E2F transcription factors were assessed. Using the R package “pROC”, we judged the diagnostic value of E2F transcription factors. The online website tool cBioPortal was also employed to find possible gene alterations of E2F transcription factors in samples from TCGA. The R package “clusterprofiler” was used to conduct functional analysis. Moreover, we also used the Tumor Immune Estimation Resource to search for the associations between E2F transcription factors and the infiltration levels of 6 kinds of immune cells. Finally, quantitative real-time polymerase chain reaction (PCR) was conducted to validate the expression levels of E2F transcription factors in human paired prostate tissues.ResultsE2F1/2/3/5 messenger RNA (mRNA) expression levels were higher in prostate cancer tissues than in normal tissues, while E2F4 and E2F6 mRNA expression levels were lower (P<0.05). All E2F transcription factors were associated with clinical parameters. Kaplan-Meier analysis revealed that E2F1/4/6/8 were notably associated with the overall survival of patients with prostate cancer (P<0.05). Receiver operating characteristic (ROC) curve results showed that except for E2F7, the other E2F transcription factors had diagnostic value for prostate cancer (P<0.05). We further found close associations between E2F transcription factors and the infiltration levels of immune cells. The results of quantitative real-time PCR were consistent with those from public databases.ConclusionsE2F transcription factor family members are differentially expressed in prostate cancer and are significantly related to the prognosis of patients, suggesting that they may be adopted as biomarkers for prognosis prediction and the treatment of prostate cancer.

CXCL10 and its related key genes as potential biomarkers for psoriasis: Evidence from bioinformatics and real-time quantitative polymerase chain reaction.

Although several studies have attempted to investigate the etiology of and mechanism underlying psoriasis, the precise molecular mechanism remains unclear. Our study aimed to explore the molecular mechanism underlying psoriasis based on bioinformatics.GSE30999, GSE34248, GSE41662, and GSE50790 datasets were obtained from the Gene Expression Omnibus database. The Gene Expression Omnibus profiles were integrated to obtain differentially expressed genes in R software. Then a series of analyses was performed, such as Gene Ontology annotation, Kyoto Encyclopedia of Genes and Genomes pathway analysis, protein-protein interaction network analysis, among others. The key genes were obtained by CytoHubba, and validated by real-time quantitative polymerase chain reaction.A total of 359 differentially expressed genes were identified between 270 paired lesional and non-lesional skin groups. The common enriched pathways were nucleotide-binding and oligomerization domain-like receptor signaling pathway, and cytokine-cytokine receptor interaction. Seven key genes were identified, including CXCL1, ISG15, CXCL10, STAT1, OASL, IFIT1, and IFIT3. These key genes were validated as upregulated in the 4 datasets and M5-induced HaCaT cells.Our study identified 7 key genes, namely CXCL1, ISG15, CXCL10, STAT1, OASL, IFIT1, and IFIT3, and 2 mostly enriched pathways (nucleotide-binding and oligomerization domain-like receptor signaling pathway, and cytokine-cytokine receptor interaction) involved in psoriatic pathogenesis. More importantly, CXCL1, ISG15, STAT1, OASL, IFIT1, IFIT3, and especially CXCL10 may be potential biomarkers. Therefore, our findings may bring a new perspective to the molecular mechanism underlying psoriasis and suggest potential biomarkers.

Assessment of the anti-rheumatoid arthritis activity of Gastrodia elata (tian-ma) and Radix aconitic lateralis preparata (fu-zi) via network pharmacology and untargeted metabolomics analyses.

Gastrodia elata and Radix aconiti lateralis preparrata are respectively named as Tian-Ma and Fu-Zi (TF) in Chinese. We explored the active components against rheumatoid arthritis (RA) from an extensively used couplet of Chinese herbs, Gastrodia elata and Radix aconiti lateralis preparata (TF) via untargeted metabolomics and network pharmacological approaches. Water extracts of TF were mixed at ratios 1:1, 3:2 and 2:3 (w/w). Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was then utilized as metabolomics screening. Human Metabolome (http://www.hmdb.ca/) and Lipidmaps (http://www.lipidmaps.org/) databases were used to annotate detected compounds. Further identification of vital genes and important pathways associated with the anti-RA properties of the TF preparations was done via network pharmacology, and verified by real-time quantitative polymerase chain reaction (RT-qPCR). Four key compounds involved in unsaturated fatty acid biosynthesis and isoflavonoid biosynthesis were identified through metabolomics analyses. Three key components of TF associated with anti-RA activity were linoleic acid, daidzein, and daidzin. Results of RT-qPCR revealed that all 3 tested TF couplets (1:1, 3:2, and 2:3) markedly suppressed the transcription of PTGS2. These results were consistent with our network pharmacological predictions. The anti-RA properties of Tian-Ma and Fu-Zi are associated with the inhibition of arachidonic acid metabolism pathway.

Effect of miR-146a on biological behavior of esophageal squamous cell carcinoma cells and its mechanism

Objective: To investigate the effects and mechanisms of miR-146a on the proliferation, invasion, migration, apoptosis and cell cycle of esophageal squamous cell carcinoma cells. Methods: The expressions of miR-146a in 3 esophageal squamous carcinoma cell lines (Eca109, KYSE140, KYSE150) were detected by real-time quantitative polymerase chain reaction (RT-qPCR). MiR-146a mimics was transfected into Eca109 to up-regulate the expression of miR-146a. Effects of miR-146a on cell proliferation, invasion and migration were evaluated by cell counting kit 8 (CCK-8), Transwell assay and wound-healing assay, respectively. The cell apoptosis and cycle were assessed by flow cytometry (FCM). Finally, relevant bioinformatics techniques were used to predict the target gene of miR-146a. Dual luciferase reporter assay was used to identify the interaction of 3' terminal untranslated region (3' UTR) of miR-146a and its target gene, interleukin-1 receptor-associated kinase (IRAK1). RT-qPCR and western blot were used to detect the mRNA and protein expressions of IRAK1, respectively. Results: The relative expressions of miR-146a in 3 esophageal squamous cell carcinoma cells (Eca109, KYSE140, KYSE150) were 0.36±0.05, 0.16±0.06 and 0.09±0.02, respectively, all of which were significantly lower than 1±0.05 of normal esophageal epithelial cells (HEEC) (P<0.01). The model of esophageal squamous cell carcinoma cells was constructed by transfecting miR-146a mimics into Eca109 cells. The results showed that the ability of absorbance value, the number of transmembrane cells (52±18), the reduced scratch distances at 48 hours and 72 hours after transfection [(25.29±0.77) μm, (30.66±0.91) μm] were significantly lower than those of the negative control group and blank control group (all P<0.01). The early apoptosis rate was (6.13±0.91)%, higher than (2.50±0.68)% of the negative control group (P<0.01) and (1.70±0.20)% of blank control group (P<0.01). The percentage of cells in G(1) phase [(44.74±6.76)%] was decreased while the G(2)/M phase [(41.88±2.88)%] was increased when compared with the negative control group and the blank control group (P<0.05 and P<0.01, respectively). The results of dual luciferase reporter gene assay showed that luciferase activity in the group co-transfected with IRAK1-wild-type and miR-146a mimics was significantly lower than that in the control groups (P<0.01). The results of RT-qPCR and western blot showed that the mRNA and protein expressions of IRAK1 in the co-transfected group were 1.02±0.28 and 1.00±0.05, respectively, both lower than those in the negative control group and the blank control group (P<0.01). Conclusions: The expressions of miR-146a are decreased in the esophageal squamous cell lines, which plays a role as tumor suppressor gene. MiR-146a can inhibit the proliferation, invasion and migration of esophageal squamous cell cells, promote apoptosis, and block the cell cycle at G(2)/M stage. MiR-146a may mediate the malignant biological behavior of esophageal cancer cells through the regulation of IRAK1.

Effects of Lactobacillus fermentum CQPC04 on Lipid Reduction in C57BL/6J Mice.

Probiotics are functional foods that can effectively regulate lipid reduction and maintain body health. In this study, a strain of Lactobacillus fermentum CQPC04 (LF-CQPC04) isolated from traditional naturally fermented vegetables (Sichuan pickles) was studied, and its effects on lipid reduction in mice, as well as its mechanism of action, were observed. The results of this experiment show that LF-CQPC04 can reduce the abnormal weight gain and abnormal visceral index of mice caused by a high-fat diet. LF-CQPC04 can decrease TG (triglycerides), TC (total cholesterol), LDL-c (low-density lipoprotein cholesterol), AST (aspartate transaminase), ALT (alanine aminotransferase), and AKP (alkaline phosphatase) levels and increase HDL-c (high-density lipoprotein cholesterol) levels in the serum of high-fat mice. LF-CQPC04 can also decrease the levels of inflammatory cytokines, such as IL-6 (interleukin-6), IL-1β (interleukin-1 beta), TNF-α (tumor necrosis factor alpha), and IFN-γ (interferon gamma), and increase IL-4 and IL-10 levels in the serum of high-fat mice. The results of RT-qPCR (real-time quantitative polymerase chain reaction) and western blot experiments show that LF-CQPC04 can also down-regulate the expression of PPAR-γ (peroxisome proliferator-activated receptor gamma), C/EBP-α (CCAAT/enhances binding protein alpha) mRNA, and protein in the liver tissue of high-fat mice, while up-regulating the expression of Cu/Zn-SOD (copper/zinc superoxide dismutase), Mn-SOD (manganese superoxide dismutase), CAT (catalase), CYP7A1 (cholesterol 7 alpha hydroxylase), PPAR-α (peroxisome proliferator-activated receptor alpha), CPT1 (carnitine palmitoyl transferase 1), LPL (lipoprotein lipase), and ABCA1 (ATP-binding cassette transporter A1). Moreover, LF-CQPC04 shows stronger effects in regulating lipid reduction in mice than L-carnitine and commercial LB (Lactobacillus delbrueckii subsp. Bulgaricus) bacteria. LF-CQPC04 is beneficial for lipid reduction in animals and has good probiotic potential.

Circulating miR-489 as a potential new biomarker for idiopathic dilated cardiomyopathy.

MicroRNAs (miRNA) are functional RNAs that have emerged as pivotal gene expression regulators in cardiac disease. Although several cardiomyocyte miRNAs have been reported to play roles in heart failure progression among patients with idiopathic dilated cardiomyopathy (DCM), the role of circulating miRNAs has not yet been well-examined. After total RNA extraction from the peripheral blood samples of three control participants and six patients with DCM, miRNA profiling was performed using miRNA arrays. Based on the results of this initial screening, real-time polymerase chain reaction (RT-PCR) was used to perform a quantitative analysis of blood samples from a larger number of matched patients (DCM, n=20; controls, n=5). Finally, the correlations between specific miRNA expression levels and hemodynamic parameters were analyzed. A primary screening of 2,565 miRNAs resulted in the identification of nine miRNA candidates. Quantitative RT-PCR results revealed significantly increased miR-489 expression levels in the DCM group. Moreover, there was a significant positive correlation between miR-489 expression level and left ventricular ejection fraction. Our results suggest that circulating miR-489 could be a potential noninvasive diagnostic biomarker for DCM. Additionally, the quantification of circulating miR-489 may have value as a potential prognostic marker for patients with DCM.

Potential False-Negative Nucleic Acid Testing Results for Severe Acute Respiratory Syndrome Coronavirus 2 from Thermal Inactivation of Samples with Low Viral Loads.

BackgroundCorona Virus Disease-2019 (COVID-19) has spread widely throughout the world since the end of 2019. Nucleic acid testing (NAT) has played an important role in patient diagnosis and management of COVID-19. In some circumstances, thermal inactivation at 56 °C has been recommended to inactivate Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) before NAT. However, this procedure could theoretically disrupt nucleic acid integrity of this single-stranded RNA virus and cause false negatives in real-time polymerase chain reaction (RT-PCR) tests.MethodsWe investigated whether thermal inactivation could affect the results of viral NAT. We examined the effects of thermal inactivation on the quantitative RT-PCR results of SARS-CoV-2 particularly with regard to the rates of false-negative results for specimens carrying low viral loads. We additionally investigated the effects of different specimen types, sample preservation times and a chemical inactivation approach on NAT.ResultsOur work showed increased Ct values in specimens from diagnosed COVID-19 patients in RT-PCR tests after thermal incubation. Moreover, about half of the weak-positive samples (7 of 15 samples, 46.7%) were RT-PCR negative after heat inactivation in at least one parallel testing. The use of guanidinium-based lysis for preservation of these specimens had a smaller impact on RT-PCR results with fewer false negatives (2 of 15 samples, 13.3%) and significantly less increase in Ct values than heat inactivation.ConclusionThermal inactivation adversely affected the efficiency of RT-PCR for SARS-CoV-2 detection. Given the limited applicability associated with chemical inactivators, other approaches to ensure the overall protection of laboratory personnel need consideration.

Expression of HOXA terminal transcript antisense RNA in hepatocellular carcinoma tissues and its effect on proliferation, invasion and migration of hepatocellular carcinoma HepG2 cells

Objective To investigate the expression of HOXA terminal transcript antisense RNA (HOTTIP) in hepatocellular carcinoma (HCC) tissues and to explore its effect on proliferation, invasion and migration in HepG2 cells. Methods A total of 60 cases with HCC tissues undergoing excision surgery and 60 cases of corresponding paracancerous tissues from January 2012 to June 2018 in Dandong First Hospital of Liaoning Province were collected. The expressions of HOTTIP in HCC tissues and paracancerous tissues were detected by using real-time quantitative polymerase chain reaction (RT-qPCR), and the relationship between the expression and clinicopathological features was analyzed. HepG2 cell line with high expression of HOTTIP constructed by cell transfer technique was treated as the experimental group, and the empty plasmid pcDNA3.1-NC was treated as the control group. The effect of HOTTIP on the proliferation of HepG2 cells was detected by using CCK-8 method, and the effect of HOTTIP on invasion and migration of HepG2 cells was detected by using Transwell assay. Results The expression of HOTTIP mRNA in HCC tissues was higher than that in paracancerous tissues, and there was no statistically significant difference (1.9±0.6 vs. 0.9±0.7, t = 6.069, P 0.05). The results of RT-qPCR showed that the expression of HOTTIP mRNA in HepG2 cells was increased after transfection of overexpressed HOTTIP and the differences was statistically significant compared with the control group (63±6 vs. 13±9, t = 9.129, P < 0.01). The results of CCK-8 method showed that the proliferation activity of cells was enhanced after the overexpression of HOTTIP in HepG2 cells (24 h, 36 h, 72 h at 490 nm absorbance was 1.497 ± 0.017 vs. 0.826±0.006, 2.002±0.025 vs. 1.211±0.020, 3.257±0.042 vs. 1.772±0.021), and the differences were statistically significant (t values were 5.321, 7.349, 8.793, all P < 0.01). In Transwell invasion assay, the number of cells transferred into the basement membrane in the experimental group was more than that in the control group (101±9 vs. 41±11), and the difference was statistically significant (t = 6.839, P < 0.01). In Transwell migration assay, the number of cells transferred into the basement membrane in the experimental group was more than that in the control group (112±9 vs. 53±11), and the difference was statistically significant (t = 7.105, P < 0.01). Conclusion The expression of HOTTIP in HCC tissues is up-regulated, and the overexpression of HOTTIP can promote the proliferation, invasion and migration of HCC cells. Key words: Liver neoplasms; HOXA terminal transcript antisense RNA; Cell proliferation; Neoplasm invasiveness; Neoplasm metastasis

Simultaneous silencing of two target genes using virus-induced gene silencing technology in Nicotiana benthamiana.

Virus-induced gene silencing (VIGS) is an effective strategy for rapid gene function analysis. It is well established that the NAC transcription factor and salicylic acid (SA) signal pathway play essential roles in response to biotic stresses. However, simultaneous silencing of two target genes using VIGS in plants has been rarely reported. Therefore, in this report, we performed VIGS to silence simultaneously the SA-binding protein 2 (NbSABP2) and NbNAC1 in Nicotiana benthamiana to investigate the gene silencing efficiency of simultaneous silencing of two genes. We first cloned the full-length NbNAC1 gene, and the characterization of NbNAC1 was also analysed. Overlap extension polymerase chain reaction (PCR) analysis showed that the combination of NbSABP2 and NbNAC1 was successfully amplified. Bacteria liquid PCR confirmed that the combination of NbSABP2 and NbNAC1 was successfully inserted into the tobacco rattle virus vector. The results showed that the leaves from the NbSABP2 and NbNAC1 gene-silenced plants collapsed slightly, with browning at the base of petiole or veina. Quantitative real-time PCR results showed that the expression of NbSABP2 and NbNAC1 were significantly reduced in 12 days post silenced plants after tobacco rattle virus infiltration compared with the control plants. Overall, our results suggest that VIGS can be used to silence simultaneously two target genes.

Expression of Rap1, guanosine triphosphate Rap1, vascular endothelial growth factor and β-catenin in experimental choroidal neovascularization

Objective To observe the expression of Rap1, guanosine triphosphate-Rap1 (GTP-Rap1), vascular endothelial growth factor (VEGF) and β-catenin in experimental choroidal neovascularization (CNV). Methods Forty-two brown Norwegian rats were randomly divided into a blank control group (7 rats) and a model group (35 rats). Both eyes were enrolled. The CNV model was established by holmium ion laser photocoagulation in the model group. At 3, 7, 14, 21, and 28 days after photocoagulation, fluorescein fundus angiography (FFA) and choroidal vascular smear were performed to observe the degree of fluorescein leakage and CNV area in rats; Western blot and real-time quantitative polymerase chain reaction (RT-PCR) were used to detect the expression of Rap1, GTP-Rap1, VEGF, β-catenin and mRNA in CNV. Results The results of FFA examination showed that a large disc-shaped fluorescein leaked in the photo-condensation spot 14 days after photocoagulation. Laser confocal microscopy showed that compared with 7 days after photocoagulation, CNV area increased at 14, 21, 28 days after photocoagulation, and the difference were statistically significant (t=3.725, 5.532, 3.605;P<0.05). Western blot showed that there was no significant difference in the relative expression of Rap1 protein in CNV at different time points after photocoagulation between the two groups (P=0.156). Compared with the blank control group, the relative expression of GTP-Rap1 protein was significantly decreased, the relative expression of VEGF and β-catenin protein were significantly increased in the model group (P=0.000). The results of RT-PCR showed that there was no significant difference in the relative expression of Rap1 mRNA at different time points after photocoagulation between the two groups (P=0.645), but there were significant difference in the relative expression of β-catenin mRNA (P=0.000). At 7, 14, 21 and 28 days after photocoagulation, there were significant difference in the relative expression of GTP-Rap1 and VEGF mRNA between the two groups (P=0.000). Conclusions The expression of GTP-Rap1 in experimental CNV is significantly lower than that in normal rats. Key words: Choroidal neovascularization/treatment; rap1 GTP-binding proteins; Vascular endothelial growth factors; beta Catenin