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The N-ethylmaleimide-sensitive fusion protein attachment protein receptor YKT6 is a key protein that controls the release of exosomes, was reported to play important roles in multiple cancers. However, the role of YKT6 in hepatocellular carcinoma (HCC) is still unknown. Here we first used bioinformatics tools to analyze the YKT6 mRNA expression in HCC. In addition, we retrospectively collected 330 cancer tissue specimens from HCC patients and 180 para-cancerous tissue specimens, and detected YKT6 expression using immunohistochemical staining. Then the relationship between YKT6 expression and the clinical characteristics of HCC was analyzed, Kaplan–Meier analysis and Cox regression model were also performed to evaluate the impact of YKT6 expression on prognosis of HCC. Protein-protein interaction network of YKT6, and the gene enrichment analysis (GSEA) database were used to predict possible signal pathways regulated by YKT6 in HCC. The high expression rate of YKT6 in HCC (72.40%, 239/330) was higher than that in adjacent tissues (17.80%, 32/180, p <.001), and high expression of YKT6 was correlated with tumor size (p = 0.002), Edmondson Grade (p <.001), metastasis (p <.001), microvascular invasion (p =.005), AFP level (p =.002). Kaplan–Meier survival analysis showed that HCC patients with high YKT6 expression level had poorer prognosis. Meanwhile, multivariate Cox regression analysis showed that Edmondson grade (p =.009), metastasis (p =.049), YKT6 expression (p =.037) are independent risk factors for poor prognosis of HCC. Conclusions: Our results suggested that the upregulated expression of YKT6 is closely related to the progression HCC, which may be used as a potential biomarker for poor prognosis in HCC. [ABSTRACT FROM AUTHOR]

Circular RNAs are a class of RNAs with a covalently closed configuration, and several members of them have been reported to be capable of regulating various biological processes and predicting the outcome of disease. Among them, circular RNA circ-ITCH has been identified to be aberrantly expressed and associated with disease progression in diverse cancers. However, the correlation of circ-ITCH expression with clinicopathological features, as well as the prognosis of cancers, remains inconclusive. Therefore, a meta-analysis was performed to investigate the clinical significance of circ-ITCH in cancers by systematically summarizing all eligible literatures. Up to August 31, 2020, relevant articles were searched in PubMed, Web of Science, Cochrane library, Embase, CNKI, and Wanfang databases. Pooled hazard ratios (HRs) and odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were calculated. A total of 1604 patients from 14 studies were included in this meta-analysis. The results showed that cancer patients with low circ-ITCH expression were more susceptible to develop lymph node metastasis (OR = 2.25 , 95% CI: 1.67-3.02, p ≤ 0.01), larger tumor size (OR = 3.01 , 95% CI: 2.01-4.52, p ≤ 0.01), advanced TNM stage (OR = 2.82 , 95% CI: 1.92-4.14, p ≤ 0.01), and poor overall survival (OS) (HR = 2.45 , 95% CI: 2.07–2.90, p ≤ 0.01 , univariate analysis; HR = 2.69 , 95% CI: 1.82-3.96, p ≤ 0.01 , multivariate analysis). Thus, low circ-ITCH expression was significantly associated with aggressive clinicopathological features and unfavorable outcome in various cancers. Therefore, circ-ITCH may serve as a molecular therapy target and a prognostic marker in human cancers. [ABSTRACT FROM AUTHOR]

Background: Plasmid construction of small fragments of interest (such as insertion of small fragment marker genes, expression of shRNA, siRNA, etc) is the basis of many biomolecular experiments. Here, we describe a method to clone short DNA into vectors by polymerase chain reaction (PCR), named one‐step PCR cloning. Our method uses PCR to amplify the entire circular plasmid. The PCR was performed by the primers containing the gene of short DNA with overlapping sequences between 10–15 bp. The PCR products were then transformed into E. coli and cyclized by homologous recombination in vivo. Methods: The pEGFP‐N1‐HA plasmid was constructed by one‐step PCR and transformation. Cells were transfected with pEGFP‐N1‐HA and pEGFP‐N1 plasmid using TurboFect transfection reagent. Protein expression was detected by western blotting and the HA‐GFP fusion protein was detected by confocal microscopy. Results: The pEGFP‐N1‐HA plasmid was successfully constructed and HA expression in cells. Conclusions: Free from the limitations of restriction enzyme sites and omitting the ligation process, our method offers a flexible and economical option of plasmid construction. Key points: Significant findings of the study: A method to clone short DNA into plasmids was found. What this study adds: Our study provides a flexible and economical option to clone short DNA into plasmids. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the expression of tribbles homologue 3 (TRB3) and its regulation on endoplasmic reticulum stress (ERS)-induced photoreceptor apoptosis after retinal detachment (RD) using a rat model. Methods: RD animal model was created in Wistar rats by subretinal injection of 1% sodium hyaluronate. At various time points after RD, expression of TRB3 was detected by quantitative real-time PCR and Western blotting. TRB3 protein distribution in retina was evaluated by immunohistochemistry. RNA interference was used to inhibit TRB3 expression and subretinal injection of lentivirus TRB3 shRNA (LV-TRB3-sh) was performed. The rats were then randomly divided into four groups: normal control group, RD group, vehicle + RD group and LV-TRB3-sh + RD group. The mRNA and protein level of TRB3 as well as Caspase-12 were detected. TdT-mediated fluorescein-16-dUTP nick-end labeling (TUNEL) assay was used to detect the apoptosis of retinal cells. Retinal outer nuclear layer (ONL) thickness was measured to assess retina damage in each group. Results: TRB3 expression and TRB3-positive cell count were significantly increased after RD and peaked at day 3 after RD. The ratio of TUNEL-positive photoreceptors and expression of ERS-induced apoptosis marker Caspase-12 in LV-TRB3-sh + RD group were significantly reduced. The ONL thickness in LV-TRB3-sh + RD group was thicker than that both in RD group and vehicle + RD group. Conclusion: TRB3 expression is up-regulated in retinas after RD and knockdown of TRB3 protects photoreceptors against ERS-induced apoptosis. TRB3 may be a crucial molecule in photoreceptor apoptosis induced by ERS after RD. [ABSTRACT FROM AUTHOR]

Abstract: Skin barrier dysfunction is involved in the pathogenesis of atopic dermatitis (AD). The impaired barrier function is related to disturbed keratinocyte differentiation through improper Ca2+-regulated pathway in AD. Transient receptor potential canonical (subtype) 6 (TRPC6) is a Ca2+-permeable nonselective cation channel expressed in keratinocytes. TRPC6 contributes to the process of keratinocyte differentiation by regulating Ca2+ influx. Furthermore, TRPC6 alone is considered to be sufficient for nearly full physiological response of keratinocyte differentiation. Until now, the correlation between TRPC6 and AD has not been investigated. We hypothesize that dysfunction of TRPC6 may trigger the process of AD, and that restoring or enhancing the activity of TRPC6 may serve as a new modality in treating AD. [Copyright &y& Elsevier]