Glycyrrhizin Attenuates c-Src-Mediated Lipopolysaccharide-Induced Inflammatory Response and Apoptosis in Bronchial Epithelial Cells by Upregulating miR-146b-5p

Background: Bronchial asthma is a common chronic inflammatory disease of the respiratory tract, whose pathogenesis involves a variety of factors. The purpose of this study was to explore the effect of traditional Chinese medicine Glycyrrhizin (Gly) on lipopolysaccharide (LPS)-induced inflammation and apoptosis of bronchial epithelial cells and its action mechanism. Methods: Gly (20 µM) was used to treat bronchial epithelial BEAS-2B cells stimulated with LPS. The expression of SRC and miR-146b-5p in BEAS-2B cells was modified by the respective transfections with pcDNA-SRC, miR-146b-5p mimic and miR-146b-5p inhibitor. STRING and Starbase online databases were used to predict the relationship between Gly, miR-146b-5p and SRC. Luciferase reporter assays were performed to verify the binding of miR-146b-5p to SRC. The viability, inflammatory response and apoptosis of BEAS-2B cells were examined by CCK-8, ELISA and Tunel assays respectively. The expressions of apoptosis-related proteins (Bcl-2, Bax, caspase3 and Cleaved-caspase3), SRC and miR-146b-5p were detected by qRT-PCR or western blotting. Results: Gly inhibited LPS-induced inflammation and apoptosis in BEAS-2B cells. The interaction between Gly and SRC was predicted by STRING. SRC expression was high in BEAS-2B cells stimulated with LPS and could be negatively regulated by Gly. Overexpression of SRC effectively alleviated the inhibitory effect of Gly on LPS-induced damages in BEAS-2B cells. In addition, results of luciferase reporter assays verified SRC as a direct target gene of miR-146b-5p. The expression level of miR-146b-5p was downregulated by LPS stimulation in BEAS-2B cells. Gly decreased the expression of SRC in LPS-stimulated BEAS-2B cells. These results could all be reversed by miR-146b-5p knockdown. Conclusion: Gly decreases the expression of SRC by upregulating the level of miR-146b-5p, thus alleviating the inflammation and apoptosis of bronchial epithelial cells treated with LPS. Our results provide a new theoretical basis for applying Gly to the clinical management of asthma.

Lidocaine and Bupivacaine Downregulate MYB and DANCR lncRNA by Upregulating miR-187-5p in MCF-7 Cells

Background: Breast cancer is the most common malignancy and a leading cause of death among women. The majority of patients require surgery, and retrospective studies have revealed an association between anaesthetic techniques during surgery and clinical outcomes. Local anaesthetics (LAs) influence carcinogenesis by interacting with non-coding RNAs (ncRNAs). However, the detailed mechanisms underlying the association between LAs and ncRNAs remain unclear.Methods: In this study, the effects of two commonly used LAs, lidocaine and bupivacaine, on the malignancy of MCF-7 breast cancer cells were investigated. The expression profiles of the microRNAs (miRNAs) that responded to treatment with LAs were determined through next-generation sequencing.Results: Data from the functional assay revealed that the LAs suppressed the proliferation of MCF-7 cells. The result of next-generation sequencing revealed that 131 miRNAs were upregulated, following treatment with the LAs. Validation using polymerase chain reaction (PCR) identified miR-187-5p as a potential biomarker, and it was selected for further analyses. Prediction with bioinformatics tools and luciferase reporter assays revealed that MYB is a direct target gene of miR-187-5p. Based on the hypothesis that lncRNAs acts as miRNA sponges, the target lncRNA, DANCR, of miR-187-5p was predicted using DIANA-LncBase v2 and validated using luciferase reporter assays. In addition, the reciprocal suppressive effect between DANCR and miR-187-5p was determined.Conclusions: This study suggests that one of the anti-tumour mechanisms of lidocaine and bupivacaine is mediated through the DANCR-miR-187-5p-MYB axis. This may provide a novel molecular mechanism of tumour suppression in breast cancer.

lncRNA SNHG15 Induced by SOX12 Promotes the Tumorigenic Properties and Chemoresistance in Cervical Cancer via the miR-4735-3p/HIF1a Pathway

Cervical cancer (CC) is one of the most common malignancies in females, with high prevalence and mortality globally. Despite advances in diagnosis and therapeutic strategies developed in recent years, CC is still a major health burden worldwide. The molecular mechanisms underlying the development of CC need to be understood. In this study, we aimed to demonstrate the role of lncRNA SNHG15 in CC progression. Using qRT-PCR, we determined that lncRNA SNHG15 is highly expressed in CC tumor tissues and cells. lncRNA SNHG15 knockdown also reduces the tumorigenic properties of CC in vitro, as determined using the MTT, EdU, flow cytometry, and transwell assays. Using bioinformatics analysis, RNA pull-down, ChIP, and luciferase reporter assays, we verified the molecular mechanisms of lncRNA SNHG15 in CC progression and found that lncRNA SNHG15 expression in CC cells is transcriptionally regulated by SOX12; moreover, lncRNA SNHG15 promotes CC progression via the miR-4735-3p/HIF1a axis. This study can provide a potential target for CC diagnosis or therapeutic strategies in the future.

Circular RNA CCDC66 Improves Murine Double Minute 4 (MDM4) Expression through Targeting miR-370 in Colorectal Cancer

Introduction. Colorectal cancer (CRC), a common digestive tract tumor that contains colon and rectal cancer, is one of the three most common cancers globally. circRNAs are involved in the occurrence and development of CRC, but the mechanism of how they participate in this process remains unclear. Methods. We adopted PCR for expression measure, CCK-8 for cell proliferation detection, Transwell for cell migration and invasion detection, and dual-luciferase reporter assays to detect the potential downstream targets of CCDC66 in CRC. Results. This study showed that circRNA CCDC66 was overexpressed in CRC tissues, and after knockdown, it inhibited the proliferation, migration, and invasion of CRC cells (RKO and HCT-116) in vitro. In addition, the dual-luciferase reporter assay showed that there was a binding site between circCCDC66 and miR-370, as well as between miR-370 and murine double minute 4 (MDM4). That is, circCCDC66 upregulated the expression of MDM4 through competitively binding to miR-370. The expression of circCCDC66 in CRC tissues was positively correlated with MDM4 and negatively correlated with miR-370. Conclusion. In summary, our results indicate that circCCDC66 is a key upregulation of CRC. circCCDC66 upregulates MDM4 through competitive binding to miR-370, thereby enhancing the metastatic ability of CRC cells and promoting the development of CRC.

Functional dissection of inherited non-coding variation influencing multiple myeloma risk

Thousands of non-coding variants have been associated with increased risk of human diseases, yet the causal variants and their mechanisms-of-action remain obscure. In an integrative study combining massively parallel reporter assays (MPRA), expression analyses (eQTL, meQTL, PCHiC) and chromatin accessibility analyses in primary cells (caQTL), we investigate 1,039 variants associated with multiple myeloma (MM). We demonstrate that MM susceptibility is mediated by gene-regulatory changes in plasma cells and B-cells, and identify putative causal variants at six risk loci (SMARCD3, WAC, ELL2, CDCA7L, CEP120, and PREX1). Notably, three of these variants co-localize with significant plasma cell caQTLs, signaling the presence of causal activity at these precise genomic positions in an endogenous chromosomal context in vivo. Our results provide a systematic functional dissection of risk loci for a hematologic malignancy.

Staphylococcus aureus activates the Aryl Hydrocarbon Receptor in Human Keratinocytes

Staphylococcus (S.) aureus is an important pathogen causing various infections including - as most frequently isolated bacterium - cutaneous infections. Keratinocytes as the first barrier cells of the skin respond to S. aureus by the release of defense molecules such as cytokines and antimicrobial peptides. Although several pattern recognition receptors expressed in keratinocytes such as Toll-like and NOD-like receptors have been reported to detect the presence of S. aureus, the mechanisms underlying the interplay between S. aureus and keratinocytes are still emerging. Here we report that S. aureus induced gene expression of CYP1A1 and CYP1B1, responsive genes of the aryl hydrocarbon receptor (AhR). AhR activation by S. aureus was further confirmed by AhR gene reporter assays. AhR activation was mediated by factor(s) < 2 kDa secreted by S. aureus. Whole transcriptome analyses and real-time PCR analyses identified IL-24, IL-6 and IL-1beta as cytokines induced in an AhR-dependent manner in S. aureus-treated keratinocytes. AhR inhibition in a 3D organotypic skin equivalent confirmed the crucial role of the AhR in mediating the induction of IL-24, IL-6 and IL-1beta upon stimulation with living S. aureus. Taken together, we further highlight the important role of the AhR in cutaneous innate defense and identified the AhR as a novel receptor mediating the sensing of the important skin pathogen S. aureus in keratinocytes.

Neomorphic DNA-binding enables tumor-specific therapeutic gene expression in fusion-addicted childhood sarcoma

Chimeric fusion transcription factors are oncogenic hallmarks of several devastating cancer types including pediatric sarcomas, such as Ewing sarcoma (EwS) and alveolar rhabdomyosarcoma (ARMS). Despite their exquisite specificity, these driver oncogenes have been considered largely undruggable due to their lack of enzymatic activity. Here, we show in the EwS model that - capitalizing on neomorphic DNA-binding preferences - the addiction to the respective fusion transcription factor EWSR1-FLI1 can be leveraged to express therapeutic genes. We genetically engineered a de novo enhancer-based, synthetic and highly potent expression cassette that can elicit EWSR1-FLI1-dependent expression of a therapeutic payload as evidenced by episomal and CRISPR-edited genomic reporter assays. Combining in silico screens and immunohistochemistry, we identified GPR64 as a highly specific cell surface antigen for targeted transduction strategies in EwS. Functional experiments demonstrated that anti-GPR64-pseudotyped lentivirus harboring our expression cassette can specifically transduce EwS cells to promote the expression of viral thymidine kinase sensitizing EwS for treatment to the otherwise relatively non-toxic (Val)ganciclovir and leading to strong anti-tumorigenic, but no adverse effects in vivo. Further, we prove that similar vector designs can be applied in PAX3-FOXO1-driven ARMS, and to express immunomodulatory cytokines, such as IL-15 and XCL1, in tumor types typically considered to be immunologically cold. Collectively, these results generated in pediatric sarcomas indicate that exploiting, rather than suppressing, the neomorphic functions of chimeric transcription factors may open inroads to innovative and personalized therapies, and that our highly versatile approach may be translatable to other cancers addicted to oncogenic transcription factors with unique DNA-binding properties.

HIV-1 Vpr protein upregulates microRNA-210-5p expression to induce G2 arrest by targeting TGIF2

MicroRNAs (miRNAs) are important molecules that mediate virus-host interactions, mainly by regulating gene expression via gene silencing. Here, we demonstrated that HIV-1 infection upregulated miR-210-5p in HIV-1-inoculated cell lines and in the serum of HIV-1-infected individuals. Luciferase reporter assays and western blotting confirmed that a target protein of miR-210-5p, TGIF2, is regulated by HIV-1 infection. Furthermore, HIV-1 Vpr protein induced miR-210-5p expression. The use of a miR-210-5p inhibitor and TGIF2 overexpression showed that Vpr upregulated miR-210-5p and thereby downregulated TGIF2, which might be one of the mechanisms used by Vpr to induce G2 arrest. Moreover, we identified a transcription factor, NF-κB p50, which upregulated miR-210-5p in response to Vpr protein. In conclusion, we identified a mechanism whereby miR-210-5p, which is induced upon HIV-1 infection, targets TGIF2. This pathway was initiated by Vpr protein activating NF-κB p50, which promoted G2 arrest. These alterations orchestrated by miRNA provide new evidence on how HIV-1 interacts with its host during infection and increase our understanding of the mechanism by which Vpr regulates the cell cycle.

The lncRNA H19/miR-766-3p/S1PR3 Axis Contributes to the Hyperproliferation of Keratinocytes and Skin Inflammation in Psoriasis via the AKT/mTOR Pathway

Background. The pathogenesis of long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) are well studied in psoriasis. However, little is known about how specific lncRNAs and miRNAs affect the mechanism of psoriasis development and which pathways are involved. Objectives. To explore the role of the lncRNA H19/miR-766-3p/S1PR3 axis in psoriasis. Methods. miRNA and lncRNA microarrays were performed using IL-22-induced HaCaT cells and psoriatic lesions, respectively. Fluorescence in situ hybridization and quantitative reverse-transcriptase polymerase chain reaction were used to detect the expression of miR-766-3p and lncRNA H19. Luciferase reporter assays were used to identify miR-766-3p/lncRNA H19 and miR-766-3p/S1PR3 combinations. CCK-8 and ELISA were performed to evaluate the proliferation of keratinocytes and the secretion of pro-inflammatory cytokines. Western blot analysis was used to detect the expression of S1PR3 and its downstream effector proteins. Results. MiR-766-3p was upregulated in both HaCaT cells treated with the psoriasis-related cytokine pool (IL-17A, IL-22, IL-1 alpha, oncostatin M, and TNF-alpha) and tissues. Overexpression of miR-766-3p promoted keratinocyte proliferation and IL-17A and IL-22 secretion. LncRNA H19 and S1PR3 were demonstrably combined with miR-766-3p by luciferase reporter assay. lncRNA H19 repressed proliferation and inflammation, which were reduced by the miR-766-3p. AKT/mTOR pathway effected proliferation and inflammation by the lncRNA H19/miR-766-3p/S1PR3 axis. Conclusions. We established that downregulation of lncRNA H19 promoted the proliferation of keratinocytes and skin inflammation by up-regulating miR-766-3p expression levels and inhibiting activation of S1PR3 through the AKT/mTOR pathway in psoriasis.

HOXA-AS3 Promotes Proliferation and Migration of Hepatocellular Carcinoma Cells via the miR-455-5p/PD-L1 Axis

Hepatocellular carcinoma (HCC) is the most prevalent type of hepatic carcinoma. Long noncoding RNAs (lncRNAs) are considered crucial regulators of gene expression; however, their functions in HCC are not well understood. Thus, the present study is aimed at elucidating the functions of the lncRNA HOXA-AS3 in HCC. The functions of the HOXA-AS3/miR-455-5p/programmed death-ligand 1 (PD-L1) axis were investigated in vitro via qRT-PCR and dual-luciferase reporter assays. The effect of HOXA-AS3 expression on tumor growth and metastasis was assessed using a mouse xenograft model. High HOXA-AS3 expression was observed in the HCC cell lines. Furthermore, overexpression of HOXA-AS3 in HCC cells enhanced proliferation, migration, and invasion, regulated the cell cycle, and retarded apoptosis. We also identified an miR-455-5p binding site in HOXA-AS3. By sponging miR-455-5p, HOXA-AS3 increased the expression of PD-L1. Additionally, both the inhibition of PD-L1 and overexpression of miR-455-5p reversed the effects on cell proliferation and invasion triggered by the overexpression of HOXA-AS3. In conclusion, HOXA-AS3 modulated the functions of HCC cells through the miR-455-5p/PD-L1 axis. Therefore, HOXA-AS3 may be a novel therapeutic target for HCC.