KIAA0319 influences cilia length, cell migration and mechanical cell–substrate interaction

Following its association with dyslexia in multiple genetic studies, the KIAA0319 gene has been extensively investigated in different animal models but its function in neurodevelopment remains poorly understood. We developed the first human cellular knockout model for KIAA0319 in RPE1 retinal pigment epithelia cells via CRISPR-Cas9n to investigate its role in processes suggested but not confirmed in previous studies, including cilia formation and cell migration. We observed in the KIAA0319 knockout increased cilia length and accelerated cell migration. Using Elastic Resonator Interference Stress Microscopy (ERISM), we detected an increase in cellular force for the knockout cells that was restored by a rescue experiment. Combining ERISM and immunostaining we show that RPE1 cells exert highly dynamic, piconewton vertical pushing forces through actin-rich protrusions that are surrounded by vinculin-rich pulling sites. This protein arrangement and force pattern has previously been associated to podosomes in other cells. KIAA0319 depletion reduces the fraction of cells forming these actin-rich protrusions. Our results suggest an involvement of KIAA0319 in cilia biology and cell–substrate force regulation.

MiR-497-5p down-regulates CDCA4 to restrains lung squamous cell carcinoma progression

Background So far, few have concerned miR-497-5p in lung squamous cell carcinoma (LUSC). Methods MiR-497-5p expression in LUSC was measured by qRT-PCR. Its impacts on tumor-related cell behaviors were investigated by CCK8 assay, scratch healing assay, flow cytometry and Transwell invasion methods. In addition, interaction between miR-497-5p and CDCA4 in LUSC was also elucidated through rescue experiment, western blot, dual-luciferase, and bioinformatics analysis. Results Low level of miR-497-5p was confirmed in LUSC tissue and cells. Overexpressed miR-497-5p markedly inhibited cancer progression. miR-497-5p restrained CDCA4 expression. Rescue assay showed that overexpressing miR-497-5p eliminated effect of overexpressed CDCA4. Conclusion By targeting CDCA4, miR-497-5p restrained development of LUSC.

miR-30a-5p inhibits osteogenesis and promotes periodontitis by targeting Runx2

Background Periodontitis is the most extensive chronic inflammatory bone resorption disease. MiRNAs offer a potential way for potential therapy. Indeed, miR-30a-5p had an increasing expression in periodontitis gingivae, but whether it promotes osteogenesis and inhibits inflammation remains unknown. Methods Periodontitis model was exhibited by wire ligation and verified by micro-CT and HE staining; qPCR was used to detect the expression of miR-30a-5p; miR-30a-5p inhibitors and mimics were transfected into MC3T3-E1 cell line by lipofectamine 3000; The dual luciferase reporter gene experiment and RIP experiment were used to detect the relationship between miR-30a-5p and Runx2; Rescue experiment was used to verify the relationship between miR-30a-5p and Runx2. Results Periodontitis model was exhibited successfully and miR-30a-5p was overexpressed at the bone and gingival tissues of this model. miR-30a-5p inhibitors not only promoted the osteogenesis but also relieved inflammation. Runx2 is a target of miR-30a-5p by dual luciferase reporter gene experiment and RIP experiment. Rescue experiments revealed that miR-30a-5p inhibitors would promote osteogenesis and relieve inflammation by targeting Runx2 in inflammation of MC3T3-E1 cell line. Conclusions That all suggested that miR-30a-5p-mediated-Runx2 provided a novel understanding of mechanism of periodontitis.

MicroRNA-17-5p Downregulation Inhibits Autophagy and Myocardial Remodeling after Myocardial Infarction by Targeting STAT3

Objective: Myocardial infarction (MI) is acknowledged as a major cause of heart disease related death in the world. This study aims to explore the function and mechanism of miR-17-5p-induced STAT3 in myocardial remodeling after MI.Methods: MI mice were infected by lentivirus antago-miR-17-5p vector. The expression of miR-17-5p in myocardial tissues was detected. The levels of STAT3 and specific markers of autophagy LC3-II/LC3-I, Beclin-1 and p62 were measured. Myocardial remodeling and scar size were assessed and cell apoptosis was determined. The binding of miR-17-5p and STAT3 was analyzed. The functions of miR-17-5p and STAT3 in autophagy and myocardial remodeling after MI were confirmed by functional rescue experiment after injecting STAT3 inhibitor S31-20.Results: Decreased fractional shortening, increased left ventricular systolic diameter and ventricular filling velocity were detected in MI mice. High expression of miR-17-5p was found in myocardial tissues after MI. The inhibition of miR-17-5p expression led to reduction of fibrosis of myocardial tissues, scarring, cardiomyocyte apoptosis, and expressions of LC3-II/LC3-I and Beclin-1, and increased p62 expression after MI. miR-17-5p targeted STAT3 and negatively regulated its expression. Inhibition of STAT3 level reversed the improving effect of antago-miR-17-5p on MI.Conclusion: Inhibition of miR-17-5p can inhibit myocardial autophagy through targeting STAT3, and then inhibit myocardial remodeling, thereby protecting myocardium after MI.

Synonymous Mutation Generator: a web tool for designing RNAi-resistant sequences

RNA interference (RNAi) is a useful technique for knocking down a protein of interest, allowing for the study of the function of a gene product. However, RNAi techniques are prone to off-target effects, such as non-specific knockdown of genes besides the protein of interest. An important control and companion to RNAi knockdown experiments is the rescue experiment, wherein gene function is restored by expression of an RNAi-resistant construct of the protein of interest. Generating an RNAi-resistant construct of the protein of interest involves generating silent mutations within the coding sequence of the protein so that the resulting amino acid product is the same, but the protein mRNA is no longer a target for the RNAi. Here, Synonymous Mutation Generator, a Python-based web tool that takes an input DNA coding sequence and outputs a synonymous DNA coding sequence that is RNAi-resistant, is described. This web tool should be a useful resource for researchers cloning RNAi-resistant constructs. Synonymous Mutation Generator is easy to use and can be found at jong2.pythonanywhere.com, and the source code is available on GitHub.

miR-17-5p promotes the invasion and migration of colorectal cancer by regulating HSPB2

Background: MicroRNA (miRNA) can affect tumor progression by regulating cell proliferation, apoptosis and metastasis. After miRNA microarray chip analysis of colorectal cancer (CRC) tissues and adjacent normal tissues, a significant upregulation of miR-17-5p expression was found in CRC tissues. However, the underlying mechanism of miR-17-5p in CRC is still unclear.Methods: The levels of miR-17-5p in 47 paired CRC and adjacent normal tissue samples were determined by quantitative real-time PCR (qRT-PCR). CCK-8, colony formation, flow cytometry and transwell assays were used to explore the biological effects of miR-17-5p on CRC cells. In addition, the transcriptome sequencing and miRNA target prediction software were employed to identify targets of miR-17-5p. Luciferase reporter detection was used to demonstrate the direct binding of target genes by miR-17-5p. The rescue experiment was conducted to investigate the biological function of target genes and regulatory mechanism of miR-17-5p on target genes.Results: The expression of miR-17-5p was significantly higher in CRC tissues than in adjacent normal tissues. In CRC group, the expression of miR-17-5p in cancer tissues with lymph node metastasis was higher compared with those without lymph node metastasis. Overexpression of miR-17-5p inhibited CRC cell apoptosis, as well as promoting proliferation, migration and invasion. We hypothesized that HSPB2 might be a target gene of miR-17-5p and validated for the first time that miR-17-5p binds directly to the 3’-UTR of HSPB2. In the rescue experiment, the tumor suppressive effect of HSPB2 was detected and miR-17-5p could promote cell proliferation, migration and invasion by targeting HSPB2.Conclusion: MiR-17-5p promotes invasion and migration by inhibiting HSPB2 in CRC, thereby implicating its potential as a novel diagnostic biomarker and therapeutic target for CRC.

On the lifetime of bioinformatics web services

Web services are used through all disciplines in life sciences and the online landscape is growing by hundreds of novel servers annually. However, availability varies, and maintenance practices are largely inconsistent. We screened the availability of 2396 web tools published during the past 10 years. All servers were accessed over 133 days and 318 668 index files were stored in a local database. The number of accessible tools almost linearly increases in time with highest availability for 2019 and 2020 (∼90%) and lowest for tools published in 2010 (∼50%). In a 133-day test frame, 31% of tools were always working, 48.4% occasionally and 20.6% never. Consecutive downtimes were typically below 5 days with a median of 1 day, and unevenly distributed over the weekdays. A rescue experiment on 47 tools that were published from 2019 onwards but never accessible showed that 51.1% of the tools could be restored in due time. We found a positive association between the number of citations and the probability of a web server being reachable. We then determined common challenges and formulated categorical recommendations for researchers planning to develop web-based resources. As implication of our study, we propose to develop a repository for automatic API testing and sustainability indexing.

miR-371b-5p promotes cell proliferation, migration and invasion in non-small cell lung cancer via SCAI

Objective: Multiple gene targets have been reported for treatment of non-small cell lung cancer (NSCLC), however, the accompanying genetic tolerance was reported increasingly. Therefore, it is important to find new biomarkers or therapeutic targets in treatment of NSCLC. Methods: The expression levels of miR-371b-5p were detected by qRT-PCR in NSCLC tissues and cell lines. To evaluate the effect of miR-371b-5p on NSCLC progression, we first transfected the miR-371b-5p inhibitor for construction of the miR-371b-5p down-regulated cell model. Then the cell proliferation, migration, invasion and cell apoptosis were detected. In addition, the expression levels of adhesion factors were detected. The target gene of miR-371b-5p was identified by bioinformatics analysis, and rescue experiment was conducted to validate the effect of miR-371b-5p on proliferation, migration and invasion of NSCLC. Results: Our findings revealed that the miR-371b-5p was overexpressed in NSCLC and could markedly promote the cell proliferation, migration and invasion. Expression levels of both intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were significantly down-regulated when treated by miR-371b-5p inhibitor. Moreover, dual-luciferase reporter assay showed that the miR-371b-5p targeted SCAI in regulation of cell proliferation, migration and invasion, and the expression of miR-371b-5p was negatively associated with SCAI in NSCLC tissues and cell lines. Rescue experiment revealed that the miR-371b-5p could rescue the effect of SCAI on cell proliferation, migration and invasion. Conclusion: Our results suggest that the miR-371b-5p and SCAI may serve as novel prognostic biomarkers and therapeutic targets for NSCLC.

LncRNA NRON Inhibits Osteosarcoma Cell Proliferation, Invasion by Regulating MVB12B

Aims: Long noncoding RNA have been proved as important regulator in various diseases. NRON was a newly identified tumor-related lncRNA, and previous studies have reported its function in hepatocellular carcinoma and heart failure. However, the function and mechanism of lncRNA NRON in osteosarcoma still unknown. Methods: Cell proliferation, invasion, migration and apoptosis were detected via CCK-8, transwell assay and Western. Bioinformatics analysis was used to predict the potential target of NRON. Rescue experiment was performed to identify the relationship between NRON and MVB12B. Results: The expression of lncRNA NRON was significantly downregulate in osteosarcoma tissues and cell lines. Knockdown of NRON promoted cell proliferation, invasion and EMT. Overexpression of NRON inhibited cell proliferation, invasion and EMT. Bioinformatics analysis predicted that MVB12B was the direct target. The expression of MVB12B was significantly upregulated in osteosarcoma tissues and cell lines. Rescue experiment further confirmed the relationship between NRON and MVB12B. Overexpression of MVB12B completely reversed the function of NRON. Conclusion: Taken together, our results comprehensively analyzed the function of NRON in osteosarcoma and provided possible mechanism that NRON inhibited osteosarcoma development by regulating MVB12B. Thus, our study may offer a potential therapeutic target for treating osteosarcoma.