CircCCNB1 silencing acting as a miR-106b-5p sponge inhibited GPM6A expression to promote HCC progression by enhancing DYNC1I1 expression and activating the AKT/ERK signaling pathway

Background: Circular RNAs (circRNAs), which generally act as microRNA (miRNA) sponges to competitively regulate the downstream target genes of miRNA, play an essential role in cancer biology. However, few studies have been reported on the role of circRNA based competitive endogenous RNA (ceRNA) network in hepatocellular carcinoma (HCC). Herein, we aimed to screen and establish the circRNA/miRNA/mRNA networks related to the prognosis and progression of HCC and further explore the underlying mechanisms of tumorigenesis.Methods: GEO datasets {"type":"entrez-geo","attrs":{"text":"GSE97332","term_id":"97332"}}GSE97332, {"type":"entrez-geo","attrs":{"text":"GSE108724","term_id":"108724"}}GSE108724, and {"type":"entrez-geo","attrs":{"text":"GSE101728","term_id":"101728"}}GSE101728 were utilized to screen the differentially expressed circRNAs (DE-circRNAs), DE-miRNAs, and DEmRNAs between HCC and matched para-carcinoma tissues. After six RNA-RNA predictions and five intersections between DE-RNAs and predicted RNAs, the survival-related RNAs were screened by the ENCORI analysis tool. The ceRNA networks were constructed using Cytoscape software, based on two models of up-regulated circRNA/down-regulated miRNA/up-regulated mRNA and down-regulated circRNA/up-regulated miRNA/down-regulated mRNA. The qRT-PCR assay was utilized for detecting the RNA expression levels in HCC cells and tissues. The apoptosis, Edu, wound healing, and transwell assays were performed to evaluate the effect of miR-106b-5p productions on the proliferation, invasion, and metastasis of HCC cells. In addition, the clone formation, cell cycle, and nude mice xenograft tumor assays were used to investigate the influence of hsa_circ_0001495 (circCCNB1) silencing and overexpression on the proliferation of HCC cells in vitro and in vivo. Furthermore, the mechanism of downstream gene DYNC1I1 and AKT/ERK signaling pathway via the circCCNB1/miR-106b-5p/GPM6A network in regulating the cell cycle was also explored.Results: Twenty DE-circRNAs with a genomic length less than 2000bp, 11 survival-related DE-miRNAs, and 61 survival-related DE-mRNAs were screened out and used to construct five HCC related ceRNA networks. Then, the circCCNB1/miR-106b-5p/GPM6A network was randomly selected for subsequent experimental verification and mechanism exploration at in vitro and in vivo levels. The expression of circCCNB1 and GPM6A were significantly down-regulated in HCC cells and cancer tissues, while miR-106b-5p expression was up-regulated. After transfections, miR-106b-5p mimics notably enhanced the proliferation, invasion, and metastasis of HCC cells, while the opposite was seen with miR-105b-5p inhibitor. In addition, circCCNB1 silencing promoted the clone formation ability, the cell cycle G1-S transition, and the growth of xenograft tumors of HCC cells via GPM6A downregulation. Subsequently, under-expression of GPM6A increased DYNC1I1 expression and activated the phosphorylation of the AKT/ERK pathway to regulate the HCC cell cycle.Conclusions: We demonstrated that circCCNB1 silencing promoted cell proliferation and metastasis of HCC cells by weakening sponging of oncogenic miR-106b-5p to induce GPM6A underexpression. DYNC1I1 gene expression was up-regulated and further led to activation of the AKT/ERK signaling pathway.     

LncRNA ZNF667-AS1靶向miR-31-5p对食管癌细胞增殖、迁移和凋亡的机制研究

目的 探究长链非编码RNA(lncRNA)ZNF667-AS1通过靶向miR-31-5p对食管癌细胞增殖和迁移的影响及潜在的机制.方法 采用实时荧光定量PCR(qPCR)技术检测ZNF667-AS1在食管癌细胞Eca109、EC1、TE1和正常食管上皮细胞Het-1A的表达水平,并选择表达差异最大的细胞株进行后续实验....     

Efficient incorporation and template-dependent polymerase inhibition are major determinants for the broad-spectrum antiviral activity of remdesivir

Remdesivir (RDV) is a direct-acting antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2. RDV exhibits broad-spectrum antiviral activity against positive-sense RNA viruses, for example, severe acute respiratory syndrome coronavirus and hepatitis C virus, and nonsegmented negative-sense RNA viruses, for example, Nipah virus, whereas segmented negative-sense RNA viruses such as influenza virus or Crimean-Congo hemorrhagic fever virus are not sensitive to the drug. The reasons for this apparent efficacy pattern are unknown. Here, we expressed and purified representative RNA-dependent RNA polymerases and studied three biochemical parameters that have been associated with the inhibitory effects of RDV-triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV-TP, (ii) the effect of the incorporated RDV-monophosphate (MP) on primer extension, and (iii) the effect of RDV-MP in the template during incorporation of the complementary UTP. We found a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Inhibition in primer extension reactions was heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP was seen with all polymerases. Molecular modeling suggests a steric conflict between the 1′-cyano group of the inhibitor and residues of the structurally conserved RNA-dependent RNA polymerase motif F. We conclude that future efforts in the development of nucleotide analogs with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1′-modification.     

Screening differential circular RNA expression profiles reveals the regulatory role of circMARS in anti‐tuberculosis drug‐induced liver injury

Tuberculosis (TB) treatment is plagued by liver damage, which often leads to treatment interruptions. Circular RNAs (circRNAs) are a special class of non‐coding RNAs abundant in body fluids with important biological functions. However, the role of circRNA in anti‐tuberculosis drug‐induced liver injury (ADLI) is unclear. We explored ADLI‐specific circRNAs in TB patients using circRNA microarrays and verified circMARS in a cohort of 300 individuals. In addition to the value assessment of circMARS in patients using a receiver operating characteristic (ROC) curve, cell experiments were also performed under the guidance of bioinformatics analyses. In particular, we found that circMARS acts as a miRNA sponge by binding to miRNAs. Compared with the blank group, the expressions of circMARS, KMT2C gene, and EGFR protein in the ADLI group were increased, while miR‐6808‐5p, miR‐6874‐3p, and miR‐3157‐5p were decreased. Furthermore, when si‐circMARS was used in the ADLI groups, circMARS demotion manifested the opposite results. Subsequently, a self‐controlled cohort of 35 participants was used to verify the circMARS–miR‐6808‐5p/‐6874‐3p/‐3157‐5p–KMT2C–EGFR function axis. Therefore, circMARS may participate in the compensatory repair mechanism of ADLI through the function axis, and may be a potential biomarker for ADLI diagnosis in TB patients.     

Next-generation sequencing: A new avenue to understand viral RNA–protein interactions

The genomes of RNA viruses present an astonishing source of both sequence and structural diversity. From intracellular viral RNA-host interfaces to interactions between the RNA genome and structural proteins in virus particles themselves, almost the entire viral lifecycle is accompanied by a myriad of RNA–protein interactions that are required to fulfill their replicative potential. It is therefore important to characterize such rich and dynamic collections of viral RNA–protein interactions to understand virus evolution and their adaptation to their hosts and environment. Recent advances in next-generation sequencing technologies have allowed the characterization of viral RNA–protein interactions, including both transient and conserved interactions, where molecular and structural approaches have fallen short. In this review, we will provide a methodological overview of the high-throughput techniques used to study viral RNA–protein interactions, their biochemical mechanisms, and how they evolved from classical methods as well as one another. We will discuss how different techniques have fueled virus research to characterize how viral RNA and proteins interact, both locally and on a global scale. Finally, we will present examples on how these techniques influence the studies of clinically important pathogens such as HIV-1 and SARS-CoV-2.     

Downregulation of lncRNA APCDD1L-AS1 due to DNA hypermethylation and loss of VHL protein expression promotes the progression of clear cell renal cell carcinoma

Background: The current studies only indicated that long non-coding RNA (lncRNA) APCDD1L-AS1, as a novel lncRNA, may play a role in oral squamous cell carcinoma and lung cancer. However, its potential role in clear cell renal cell carcinoma (ccRCC) and its possible mechanism of action remain vague.Methods: TCGA-KIRC and GEO data and qRT-PCR and pyrosequencing results of clinical specimens were used to identify the expression level and DNA methylation status of APCDD1L-AS1. The effects of APCDD1L-AS1 overexpression on ccRCC growth and metastasis were determined by function experiments. Western blot and Tandem mass tags (TMT) were utilized to explore the relationship between APCDD1L-AS1 and VHL expression and its downstream underlying mechanisms.Results: The expression of APCDD1L-AS1 was downregulated in ccRCC. Decreased APCDD1L-AS1 expression was related to higher tumor stage and histological grade and shorter RFS (Relapse-free survival). Besides, APCDD1L-AS1 overexpression restrained the growth and metastasis of ccRCC cells in vitro and in vivo. Moreover, reduced APCDD1L-AS1 expression could be caused by DNA hypermethylation and loss of von Hippel Lindau (VHL) protein expression. Furthermore, the dysregulation of histones expression caused by APCDD1L-AS1 overexpression may be one of the important mechanisms to suppress the progression of ccRCC.Conclusion: APCDD1L-AS1 was able to inhibit the progression of ccRCC, and its decreased expression could be caused by DNA hypermethylation and loss of VHL protein expression. Therefore, APCDD1L-AS1 may serve as a new therapeutic target in the treatment of ccRCC.     

Fragrep： An Efficient Search Tool for Fragmented Patterns in Genomic Sequences

Many classes of non-coding RNAs （ncRNAs; including Y RNAs, vault RNAs, RNase P RNAs, and MRP RNAs, as well as a novel class recently discovered in Dictyostelium discoideum） can be characterized by a pattern of short but well-conserved sequence elements that are separated by poorly conserved regions of sometimes highly variable lengths. Local alignment algorithms such as BLAST are therefore ill-suited for the discovery of new homologs of such ncRNAs in genomic sequences. The Fragrep tool instead implements an efficient algorithm for detecting the pattern fragments that occur in a given order. For each pattern fragment, the mismatch tolerance and bounds on the length of the intervening sequences can be specified separately. Furthermore, matches can be ranked by a statistically well-motivated scoring scheme.