Background Tetracycline (Tet)-regulated expression system has become a widely applied tool to control gene activity. This study aimed to improve the Tet-on system with superior regulatory characteristics. Results By comprehensively comparing factors of transactivators, Tet-responsive elements (TREs), orientations of induced expression cassette, and promoters controlling the transactivator, we developed an optimal Tet-on system with enhanced inducible efficiency and lower leakiness. With the system, we successfully performed effective inducible and reversible expression of microRNA, and presented a more precise and easily reproducible fine-tuning for confirming the target of a miRNA. Finally, the system was applied in CRISPR/Cas9-mediated knockout of nuclear factor of activated T cells-5 ( NFAT5 ), a protective transcription factor in cellular osmoregulation. Conclusions This study established an improved Tet-on system for powerful and stringent gene regulation in functional genetic studies. Electronic supplementary material The online version of this article (10.1186/s40104-019-0354-5) contains supplementary material, which is available to authorized users.

Coronary heart disease (CHD) is one of the leading causes of heart‐associated deaths worldwide. Conventional diagnostic techniques are ineffective and insufficient to diagnose CHD with higher accuracy. To use the circulating microRNAs (miRNAs) as non‐invasive, specific and sensitive biomarkers for diagnosing of CHD, 203 patients with CHD and 144 age‐matched controls (126 high‐risk controls and 18 healthy volunteers) were enrolled in this study. The direct S‐Poly(T)Plus method was used to identify novel miRNAs expression profile of CHD patients and to evaluate their clinical diagnostic value. This method is an RNA extraction‐free and robust quantification method, which simplifies procedures, reduces variations, in particular increases the accuracy. Twelve differentially expressed miRNAs between CHD patients and high‐risk controls were selected, and their performances were evaluated in validation set‐1 with 96 plasma samples. Finally, six (miR‐15b‐5p, miR‐29c‐3p, miR‐199a‐3p, miR‐320e, miR‐361‐5p and miR‐378b) of these 12 miRNAs were verified in validation set‐2 with a sensitivity of 92.8% and a specificity of 89.5%, and the AUC was 0.971 (95% confidence interval, 0.948‐0.993, P < .001) in a large cohort for CHD patients diagnosis. Plasma fractionation indicated that only a small amount of miRNAs were assembled into EVs. Direct S‐Poly(T)Plus method could be used for disease diagnosis and 12 unique miRNAs could be used for diagnosis of CHD.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and usually lethal lung disease and it has been widely accepted that fibroblast proliferation is one of the key characteristics of IPF. Long noncoding RNAs (lncRNAs) play vital roles in the pathogenesis of many diseases. In this study, we investigated the role of lncRNA FENDRR on fibroblast proliferation. Human lung fibroblasts stably overexpressing FENDRR showed a reduced cell proliferation compared to those expressing the control vector. On the other hand, FENDRR silencing increased fibroblast proliferation. FENDRR bound serine-arginine rich splicing factor 9 (SRSF9) and inhibited the phosphorylation of p70 ribosomal S6 kinase 1 (PS6K), a downstream protein of the mammalian target of rapamycin (mTOR) signaling. Silencing SRSF9 reduced fibroblast proliferation. FENDRR reduced β-catenin protein, but not mRNA levels. The reduction of β-catenin protein levels in lung fibroblasts by gene silencing or chemical inhibitor decreased fibroblast proliferation. Adenovirus-mediated FENDRR transfer to the lungs of mice reduced asbestos-induced fibrotic lesions and collagen deposition. RNA sequencing of lung tissues identified 7 cell proliferation-related genes that were up-regulated by asbestos but reversed by FENDRR. In conclusion, FENDRR inhibits fibroblast proliferation and functions as an anti-fibrotic lncRNA.

Background Advances in microRNAs (miRNAs) biomarkers have generated disease markers with potential clinical values. However, none of these published results have been applied in clinic until today. The main reason could be the lack of simple but robust miRNA measurements. Methods We built up a simple but ultrasensitive RT-qPCR protocol, Direct S-Poly(T) Plus assay, for detecting miRNAs without RNA purification. In this study, the method was optimized and compared with other RNA purification-based miRNA assays, and the sensitivity was tested. Using Direct S-Poly(T) Plus method, seven potential miRNA biomarkers of colorectal cancer were validated. Results It is possible to detect approximately 100 miRNAs with minimal plasma inputs (20 μl) and time (~ 140 min) with this approach. The sensitivity of this method was 2.7–343-fold higher than that of the stem-loop method, and comparable with S-Poly(T) plus method. 7 validated miRNA biomarkers of colorectal cancer by Direct S-Poly(T) plus assay could discriminate colorectal cancer stage I from healthy individuals, and promised satisfactory discrimination with the area under receiver operating characteristic (ROC) curve ranging from 0.79 to 0.94 (p value < 0.001). Conclusions This simple and robust protocol may have strong impact on the development of specific miRNAs as biomarkers in clinic.

Iron is an essential nutrient for almost all organisms. However, excess iron generates reactive oxygen species and causes tissue injuries. Iron has been shown to accumulate in alveolar macrophages and is implicated in idiopathic pulmonary fibrosis (IPF). In this study, we examined iron accumulation in lung fibroblasts and the underlying mechanisms. We hypothesize that the downregulation of Solute Carrier Family 40 Member 1 ( SLC40A1) results in iron accumulation in lung fibroblasts. Using a Prussian Blue iron staining, we found that iron accumulated in the fibrotic region of the lungs from IPF patients and mice with lung fibrosis induced by bleomycin and asbestos. Iron was partially co-localized with the fibroblast marker vimentin in IPF lungs. By using publicly available scRNA-seq data, we identified SLC40A1, the only known gene involved in iron export, as a down-regulated gene in alveolar fibroblasts. The downregulation of SLC40A1 was confirmed in the lung fibroblasts isolated from IPF patients and bleomycin-treated mice. The treatment of human lung fibroblasts with transforming growth factor-β1 (TGF-β1), a major cytokine elevated in IPF, reduced SLC40A1 mRNA and protein expression. TGF-β1 downregulated SLC40A1 expression via SMAD3 as determined by chromatin immunoprecipitation and luciferase promoter reporter assays. Knockdown of SLC40A1 using lentiviral shRNAs or TGF-β treatment induced iron accumulation in human lung fibroblasts as determined by live cell ferrous dye staining using a SiRhoNox-1 probe. Knockdown of SLC40A1 enhanced the iron-induced lung fibroblast activation. In summary, we conclude that the downregulation of SLC40A1 caused by TGF-β1 induces iron accumulation in lung fibroblasts, resulting in fibroblast activation. R01 HL157450, R01HL135152 and P20GM103648 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.