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.

Stepwise response of MeJA-induced genes and pathways in leaves of C. roseus.

Catharanthus roseus is a perennial herb known for the production of important terpenoid indole alkaloids (TIAs) in addition to a variety of phenolic compounds. The goal of the present work was to detect the prolonged effects of MeJA (6 uM) treatment across time (up to 24 days) in order to detect the stepwise response of MeJA-induced genes and pathways in leaves of C. rouses. Prolonged exposure of plants to MeJA (6 uM) treatment for different time points (6, 12 and 24 days) indicated that genes in the indole alkaloid biosynthesis pathway and upstream pathways were triggered earlier (e.g., 6 days) than those in the anthocyanin biosynthesis pathway and its upstream pathways (e.g., 12 days). Three enzymes, e.g., T16H, OMT, and D4H, in the six-step vindoline biosynthesis and two enzymes, e.g., TDC and STR, acting consecutively in the conversion of tryptophan to strictosidine, were activated after 6 days of MeJA treatment. Two other key enzymes, e.g., TRP and CYP72A1, acting concurrently upstream of the TIA biosynthesis pathway were upregulated after 6 days. The genes encoding TDC and STR might concurrently act as a master switch of the TIA pathway towards the production of the indole alkaloids. On the other hand, we speculate that the gene encoding PAL enzyme also acts as the master switch of phenylpropanoid biosynthesis and the downstream flavonoid biosynthesis and anthocyanin biosynthesis pathways towards the production of several phenolic compounds. PAL and the downstream enzymes were activated 12 days after treatment. Cluster analysis confirmed the concordant activities of the flower- and silique-specific bHLH25 transcription factor and the key enzyme in the TIA biosynthesis pathway, e.g., STR. Due to the stepwise response of the two sets of pathways, we speculate that enzymes activated earlier likely make TIA biosynthesis pathway a more favourable target in C. roseus than anthocyanin biosynthesis pathway.

Multifunctional activities of ERF109 as affected by salt stress in Arabidopsis.

Transcriptomic analysis was conducted in leaves of Arabidopsis T-DNA insertion ERF109-knocked out (KO) mutant or plants overexpressing (OE) the gene to detect its role in driving expression of programmed cell death- (PCD-) or growth-related genes under high salt (200 mM NaCl) stress. The analysis yielded ~22-24 million reads, of which 90% mapped to the Arabidopsis reference nuclear genome. Hierarchical cluster analysis of gene expression and principal component analysis (PCA) successfully separated transcriptomes of the two stress time points. Analysis indicated the occurrence of 65 clusters of gene expression with transcripts of four clusters differed at the genotype (e.g., WT (wild type), KO ERF109 or OE ERF109 ) level. Regulated transcripts involved DIAP1-like gene encoding a death-associated inhibitor of reactive oxygen species (ROS). Other ERF109-regulated transcripts belong to gene families encoding ROS scavenging enzymes and a large number of genes participating in three consecutive pathways, e.g., phenylalanine, tyrosine and tryptophan biosynthesis, tryptophan metabolism and plant hormone signal transduction. We investigated the possibility that ERF109 acts as a "master switch" mediator of a cascade of consecutive events across these three pathways initially by driving expression of ASA1 and YUC2 genes and possibly driving GST, IGPS and LAX2 genes. Action of downstream auxin-regulator, auxin-responsive as well as auxin carrier genes promotes plant cell growth under adverse conditions.