MicroRNA-141-regulated KLK10 and TNFSF-15 gene expression in hepatoblastoma cells as a novel mechanism in liver carcinogenesis.

Liver cancer is one of the most pivotal global health problems, leading hepatocellular carcinoma (HCC) with a significant increase in cases worldwide. The role of non-coding-RNA in cancer proliferation and carcinogenesis has attracted much attention in the last decade; however, microRNAs (miRNAs), as non-coding RNA, are considered master mediators in various cancer progressions. Yet the role of miR-141 as a modulator for specific cellular processes in liver cancer cell proliferation is still unclear. This study identified the role of miR-141 and its potential functions in liver carcinogenesis. The level of miR-141 in HepG2 and HuH7 cells was assessed using quantitative real-time PCR (qRT-PCR) and compared with its expression in normal hepatocytes. A new miR-141 construct has been performed in a CMV promoter vector tagged with GFP. Using microarray analysis, we identified the potentially regulated genes by miR-141 in transfected HepG2 cells. The protein profile of the kallikrein-related peptidase 10 (KLK10) and tumor necrosis factor TNFSF-15 was investigated in HepG2 cells transfected with either an inhibitor, antagonist miR-141, or miR-141 overexpression vector using immunoblotting and flow cytometry assay. Finally, ELISA assay has been used to monitor the produced inflammatory cytokines from transfected HepG2 cells. Our findings showed that the expression of miR-141 significantly increased in HepG2 and HuH7 cells compared to the normal hepatocytes. Transfection of HepG2 cells with an inhibitor, antagonist miR-141, showed a significant reduction of HepG2 cell viability, unlike the transfection of miR-141 overexpression vector. The microarray data of HepG2 cells overexpressed miR-141 provided a hundred downregulated genes, including KLK10 and TNFSF-15. Furthermore, the expression profile of KLK10 and TNFSF-15 markedly depleted in HepG2 cells transfected with miR-141 overexpression accompanied by a decreasing level of interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α), indicating the role of miR-141 in HepG2 cell proliferation and programmed cell death. Interestingly, the experimental rats with liver cancer induced by Diethylnitrosamine injection further confirmed the upregulation of miR-141 level, IL-10, and TNF-α and the disturbance in KLK10 and TNFSF-15 gene expression compared with their expression in normal rats. The in-silico online tools, IntaRNA and miRWalk were used to confirm the direct interaction and potential binding sites between miR-141 and identified genes. Thus, the seeding regions of potential targeted sequences was cloned upstream of luciferase reporter gene in pGL3 control vector. Interestingly, the luciferase activities of constructed vectors were significantly decreased in HepG2 cells pre-transfected with miR-141 overexpression vector, while increasing in cells pre-transfected with miR-141 specific inhibitor. In summary, these data suggest the crucial role of miR-141 in liver cancer development via targeting KLK10 and TNFSF-15 and provide miR-141 as an attractive candidate in liver cancer treatment and protection.

Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication.

The antiviral response against influenza A virus (IAV) infection includes the induction of the interferon (IFN) signaling pathway, including activation of the STATs protein family. Subsequently, antiviral myxovirus resistance (MxA) protein and other interferon-stimulated genes control virus replication; however, the molecular interaction of viral-mediated IFN signaling needs more investigation. Host microRNAs (miRNAs) are small non-coding molecules that posttranscriptionally regulate gene expression. Here, we sought to investigate the possible involvement of miR-141 in IAV-mediated IFN signaling. Accordingly, the microarray analysis of A549 cells transfected with precursor miR-141 (pre-miR-141) was used to capture the potentially regulated genes in response to miR-141 overexpression independent of IAV infection. The downregulation of targeted genes by miR-141, in addition to viral gene expression, was investigated by quantitative real-time PCR, western blot analysis, and flow cytometric assay. Our findings showed a significant upregulation of miR-141 in infected A549 cells with different strains of IAV. Notably, IAV replication was firmly interrupted in cells transfected with the miR-141 inhibitor. While its replication significantly increased in cells transfected with pre-miR-141 confirming the crucial role of miRNA-141 in supporting virus replication. Interestingly, the microarray data of miR-141 transduced A549 cells showed many downregulated genes, including MxA, STAT3, IFI27, and LAMP3. The expression profile of MxA and STAT3 was significantly depleted in infected cells transfected with the pre-miR-141, while their expression was restored in infected cells transfected with the miR-141 inhibitor. Unlike interleukin 6 (IL-6), the production of IFN-ß markedly decreased in infected cells that transfected with pre-miR-141, while it significantly elevated in infected cells transfected with miR-141 inhibitor. These data provide evidence for the crucial role of miR-141 in regulating the antiviral gene expression induced by IFN and IL-6 signaling during IAV infection to ensure virus replication.

Legionellapneumophila induces methylomic changes in ten-eleven translocation to ensure bacterial reproduction in human lung epithelial cells.

Introduction. Legionella pneumophila is a Gram-negative flagellated bacteria that can infect human lungs and cause a severe form of pneumonia named Legionnaires' disease.Hypothesis. We hypothesize that L. pneumophila infection induces methylomic changes in methylcytosine dioxygenases, ten-eleven translocation (TET) genes, and controls DNA methylation following infection.Aim. In the current research, we sought to further investigate DNA methylation changes in human lung epithelial cells upon L. pneumophila infection and determine how methylation inhibitor agents disturb L. pneumophila reproduction.Methodology. A549 cell line was used in L. pneumophila infection and inhibitors' treatment, including 5-azacytidine (5-AZA) and (-)-epigallocatechin-3-O-gallate (EGCG).Results. Interestingly, DNA methylation analysis of infected A549 using sodium bisulfite PCR and the methylation-sensitive HpaII enzyme showed potential methylation activity within the promoter regions of ten-eleven translocation (TET) genes located on CpG/397-8 and CpG/385-6 of TET1 and TET3, respectively. Such methylation changes in TET effectors decreased their expression profile following infection, indicated by quantitative real-time PCR (RT-qPCR), immunoblotting and flow cytometry. Furthermore, pre-treatment of A549 cells with 5-AZA or EGCG significantly decreased the bacterial reproduction characterized by the expression of L. pneumophila 16S ribosomal RNA and the c.f.u. ml-1 of bacterial particles. Moreover, both methylation inhibitors showed potent inhibition of methionine synthase (MS) expression, which was further confirmed by the docking analysis of inhibitor ligands and crystal structure of MS protein.Conclusion. These data provide evidence for the methylomic changes in the promoter region of TET1 and TET3 by L. pneumophila infection in the A549 cell line and suggest the anti-bacterial properties of 5-AZA and EGCG, as methylation inhibitors, are due to targeting the epigenetic effector methionine synthase.