Overview of MicroRNA Biology Mohr, Ashley M.; Mott, Justin L.
Seminars in liver disease,
02/2015, Letnik:
35, Številka:
1
Journal Article
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Abstract
In considering an overview of microRNA biology, it is useful to consider microRNAs as a part of cellular communication. At the simplest level, microRNAs act to decrease the expression of ...messenger RNAs that contain stretches of sequence complementary to the microRNA. This function can be likened to the function of endogenous or synthetic short interfering RNA. However, microRNA function is more complicated and nuanced than this “on–off” model would suggest. Further, many microRNA targets are themselves noncoding RNAs. In this review, the authors discuss the role of microRNAs in shaping the proteome of the cell in a way that is consistent with microRNA involvement in a highly regulated conversation, sensitive to outside influence and internal feedback.
MicroRNAs are a class of small regulatory RNAs that function to modulate protein expression. This control allows for fine‐tuning of the cellular phenotype, including regulation of proliferation, cell ...signaling, and apoptosis; not surprisingly, microRNAs contribute to liver cancer biology. Recent investigations in human liver cancers and tumor‐derived cell lines have demonstrated decreased or increased expression of particular microRNAs in hepatobiliary cancer cells. Based on predicted and validated protein targets as well as functional consequences of altered expression, microRNAs with decreased expression in liver tumor cells may normally aid in limiting neoplastic transformation. Conversely, selected microRNAs that are up‐regulated in liver tumor cells can promote malignant features, contributing to carcinogenesis. In addition, microRNAs themselves are subject to regulated expression, including regulation by tumor suppressor and oncogene pathways. This review will focus on the expression and function of cancer‐related microRNAs, including their intimate involvement in tumor suppressor and oncogene signaling networks relevant to hepatobiliary neoplasia. (HEPATOLOGY 2009.)
It has been established that microRNA expression and function contribute to phenotypic features of malignant cells, including resistance to apoptosis. Although targets and functional roles for a ...number of microRNAs have been described in cholangiocarcinoma, many additional microRNAs dysregulated in this tumor have not been assigned functional roles. In this study, we identify elevated miR‐25 expression in malignant cholangiocarcinoma cell lines as well as patient samples. In cultured cells, treatment with the Smoothened inhibitor, cyclopamine, reduced miR‐25 expression, suggesting Hedgehog signaling stimulates miR‐25 production. Functionally, miR‐25 was shown to protect cells against TNF‐related apoptosis‐inducing ligand (TRAIL)‐induced apoptosis. Correspondingly, antagonism of miR‐25 in culture sensitized cells to apoptotic death. Computational analysis identified the TRAIL Death Receptor‐4 (DR4) as a potential novel miR‐25 target, and this prediction was confirmed by immunoblot, cell staining, and reporter assays. Conclusion: These data implicate elevated miR‐25 levels in the control of tumor cell apoptosis in cholangiocarcinoma. The identification of the novel miR‐25 target DR4 provides a mechanism by which miR‐25 contributes to evasion of TRAIL‐induced cholangiocarcinoma apoptosis. (HEPATOLOGY 2012)
Obesity during pregnancy increases the risk for maternal complications like gestational diabetes, preeclampsia, and maternal inflammation. Maternal obesity also increases the risk of childhood ...obesity, intrauterine growth restriction (IUGR) and diabetes to the offspring. Increased circulating free fatty acids (FFAs) in obesity due to adipose tissue lipolysis induces lipoapoptosis to hepatocytes, cholangiocytes, and pancreatic-β-cells. During the third trimester of human pregnancy, there is an increase in maternal lipolysis and release of FFAs into the circulation. It is currently unknown if increased FFAs during gestation as a result of maternal obesity cause placental cell lipoapoptosis. Increased exposure of FFAs during maternal obesity has been shown to result in placental lipotoxicity. The objective of the present study is to determine saturated FFA-induced trophoblast lipoapoptosis and also to test the protective role of monounsaturated fatty acids against FFA-induced trophoblast lipoapoptosis using in vitro cell culture model. Here, we hypothesize that saturated FFAs induce placental trophoblast lipoapoptosis, which was prevented by monounsaturated fatty acids.
Biochemical and structural markers of apoptosis by characteristic nuclear morphological changes with DAPI staining, and caspase 3/7 activity was assessed. Cleaved PARP and cleaved caspase 3 were examined by western blot analysis.
Treatment of trophoblast cell lines, JEG-3 and JAR cells with palmitate (PA) or stearate (SA) induces trophoblast lipoapoptosis as evidenced by a significant increase in apoptotic nuclear morphological changes and caspase 3/7 activity. We observed that saturated FFAs caused a concentration-dependent increase in placental trophoblast lipoapoptosis. We also observed that monounsaturated fatty acids like palmitoleate and oleate mitigates placental trophoblast lipoapoptosis caused due to PA exposure.
We show that saturated FFAs induce trophoblast lipoapoptosis. Co-treatment of monounsaturated fatty acids like palmitoleate and oleate protects against FFA-induced trophoblast lipoapoptosis.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Isolated hepatocytes undergo lipoapoptosis, a feature of hepatic lipotoxicity, on treatment with saturated free fatty acids (FFA) such as palmitate (PA). However, it is unknown if palmitate is ...directly toxic to hepatocytes or if its toxicity is indirect via the generation of lipid metabolites such as lysophosphatidylcholine (LPC). PA-mediated hepatocyte lipoapoptosis is associated with endoplasmic reticulum (ER) stress, c-Jun NH(2)-terminal kinase (JNK) activation, and a JNK-dependent upregulation of the potent proapoptotic BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Our aim was to determine which of these mechanisms of lipotoxicity are activated by PA-derived LPC. We employed Huh-7 cells and isolated murine and human primary hepatocytes. Intracellular LPC concentrations increase linearly as a function of the exogenous, extracellular PA, stearate, or LPC concentration. Incubation of Huh-7 cells or primary hepatocytes with LPC induced cell death by apoptosis in a concentration-dependent manner. Substituting LPC for PA resulted in caspase-dependent cell death that was accompanied by activating phosphorylation of JNK with c-Jun phosphorylation and an increase in PUMA expression. LPC also induced ER stress as manifest by eIF2α phosphorylation and CAAT/enhancer binding homologous protein (CHOP) induction. LPC cytotoxicity was attenuated by pharmacological inhibition of JNK or glycogen synthase kinase-3 (GSK-3). Similarly, short-hairpin RNA (shRNA)-targeted knockdown of CHOP protected Huh-7 cells against LPC-induced toxicity. The LPC-induced PUMA upregulation was prevented by JNK inhibition or shRNA-targeted knockdown of CHOP. Finally, genetic deficiency of PUMA rendered murine hepatocytes resistant to LPC-induced apoptosis. We concluded that LPC-induced lipoapoptosis is dependent on mechanisms largely indistinguishable from PA. These data suggest that FFA-mediated cytotoxicity is indirect via the generation of the toxic metabolite, LPC.
MicroRNAs regulate pathways contributing to oncogenesis, and thus the mechanisms causing dysregulation of microRNA expression in cancer are of significant interest. Mature mir‐29b levels are ...decreased in malignant cells, and this alteration promotes the malignant phenotype, including apoptosis resistance. However, the mechanism responsible for mir‐29b suppression is unknown. Here, we examined mir‐29 expression from chromosome 7q32 using cholangiocarcinoma cells as a model for mir‐29b downregulation. Using 5′ rapid amplification of cDNA ends, the transcriptional start site was identified for this microRNA locus. Computational analysis revealed the presence of two putative E‐box (Myc‐binding) sites, a Gli‐binding site, and four NF‐κB‐binding sites in the region flanking the transcriptional start site. Promoter activity in cholangiocarcinoma cells was repressed by transfection with c‐Myc, consistent with reports in other cell types. Treatment with the hedgehog inhibitor cyclopamine, which blocks smoothened signaling, increased the activity of the promoter and expression of mature mir‐29b. Mutagenesis analysis and gel shift data are consistent with a direct binding of Gli to the mir‐29 promoter. Finally, activation of NF‐κB signaling, via ligation of Toll‐like receptors, also repressed mir‐29b expression and promoter function. Of note, activation of hedgehog, Toll‐like receptor, and c‐Myc signaling protected cholangiocytes from TRAIL‐induced apoptosis. Thus, in addition to c‐Myc, mir‐29 expression can be suppressed by hedgehog signaling and inflammatory pathways, both commonly activated in the genesis of human malignancies. J. Cell. Biochem. 110: 1155–1164, 2010. Published 2010 Wiley‐Liss, Inc.
CHOP and AP-1 cooperatively mediate PUMA expression during lipoapoptosis Cazanave, Sophie C; Elmi, Nafisa A; Akazawa, Yuko ...
American journal of physiology. Gastrointestinal and liver physiology/American journal of physiology: Gastrointestinal and liver physiology,
07/2010, Letnik:
299, Številka:
1
Journal Article
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Endoplasmic reticulum (ER) stress-mediated apoptosis is a key feature of hepatocyte cytotoxicity by saturated free fatty acids (FFA). This lipoapoptosis is dependent, in part, on the transcriptional ...upregulation of the BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Although the activator protein (AP)-1 complex facilitates PUMA expression by saturated FFA, the transcription factor CAAT/enhancer binding homologous protein (CHOP) is also induced by ER stress and promotes apoptosis. To integrate the role of these two transcription factors in ER stress-induced apoptosis, we examined the relative contribution of CHOP and AP-1 in mediating PUMA induction by saturated FFA. Our results demonstrate that short-hairpin RNA-targeted knockdown of CHOP attenuates palmitate-induced apoptosis in Huh-7 cells. Loss of CHOP induction also reduced the increase in PUMA mRNA and protein levels as well as Bax activation by palmitate. No functional CHOP binding sites were identified in the PUMA promoter sequence. Rather, we observed that CHOP physically interacts with the AP-1 complex protein c-Jun upon palmitate treatment, and a CHOP:phosphorylated c-Jun heteromeric complex binds to the AP-1 consensus binding sequence within the PUMA promoter region. Finally, loss of function studies suggest that both transcription factors are necessary for maximal PUMA induction. Collectively, these data suggest that CHOP and AP-1 cooperatively mediate PUMA induction during hepatocyte lipoapoptosis.
Lipoapoptosis occurring due to an excess of saturated free fatty acids such as palmitate is a key pathogenic event in the initiation of nonalcoholic fatty liver disease. Palmitate loading of cells ...activates the endoplasmic reticulum stress response, including induction of the proapoptotic transcription factor C/EBP homologous protein (CHOP). Furthermore, the loss of microRNAs is implicated in regulating apoptosis under conditions of endoplasmic reticulum (ER) stress. The aim of this study was to identify specific microRNAs regulating CHOP expression during palmitate-induced ER stress. Five microRNAs were repressed under palmitate-induced endoplasmic reticulum stress conditions in hepatocyte cell lines (miR-92b-3p, miR-328-3p, miR-484, miR-574-5p, and miR-615-3p). We identified miR-615-3p as a candidate microRNA which was repressed by palmitate treatment and regulated CHOP protein expression, by RNA sequencing and in silico analyses, respectively. There is a single miR-615-3p binding site in the 3'untranslated region (UTR) of the Chop transcript. We characterized this as a functional binding site using a reporter gene-based assay. Augmentation of miR-615-3p levels, using a precursor molecule, repressed CHOP expression; and under these conditions palmitate- or tunicamycin-induced cell death were significantly reduced. Our results suggest that palmitate-induced apoptosis requires maximal expression of CHOP which is achieved via the downregulation of its repressive microRNA, miR-615-3p. We speculate that enhancement of miR-615-3p levels may be of therapeutic benefit by inhibiting palmitate-induced hepatocyte lipoapoptosis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Nonalcoholic steatohepatitis (NASH) patients have elevated plasma saturated free fatty acid levels. These toxic fatty acids can induce liver cell death and our recent results demonstrated that the ...biliary epithelium may be susceptible to lipotoxicity. Here, we explored the molecular mechanisms of cholangiocyte lipoapoptosis in cell culture and in an animal model of NASH. Treatment of cholangiocytes with palmitate (PA) showed increased caspase 3/7 activity and increased levels of cleaved poly (ADP-ribose) polymerase and cleaved caspase 3, demonstrating cholangiocyte lipoapoptosis. Interestingly, treatment with PA significantly increased the levels of microRNA miR-34a, a pro-apoptotic microRNA known to be elevated in NASH. PA induction of miR-34a was abolished in cholangiocytes transduced with forkhead family of transcription factor class O (FoxO)3 shRNA, demonstrating that FoxO3 activation is upstream of miR-34a and suggesting that FoxO3 is a novel transcriptional regulator of miR-34a. Further, anti-miR-34a protected cholangiocytes from PA-induced lipoapoptosis. Direct and indirect targets of miR-34a, such as SIRT1, receptor tyrosine kinase (MET), Kruppel-like factor 4, fibroblast growth factor receptor (FGFR)1, and FGFR4, were all decreased in PA-treated cholangiocytes. SIRT1 and MET were partially rescued by a miR-34a antagonist. Cholangiocyte apoptosis and miR-34a were dramatically increased in the liver of mice with early histologic features of NASH. Our study provides evidence for the pro-apoptotic role of miR-34a in PA-induced cholangiocyte lipoapoptosis in culture and in the liver.
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