Dissertation > Medicine, health > Oncology > Gastrointestinal Cancer > Liver tumors

MiR-221Enhances Anti-HCV Effect of Interferon and All-trans Retinoic Acid Protects Hepatocellular Carcinoma Cells against Serum Starvation Induced Cell Death

Author XuGang
Tutor RenHao
School Second Military Medical University
Course Microbiology
Keywords HCV miR-221 SOCS IFN-α HCC retinoic acid COL8A2
CLC R735.7
Type Master's thesis
Year 2013
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Hepatitis C virus (HCV) is a blood-transmitted human virus and it is a global healthproblem with estimated of170to200million infected individuals worldwide. Seventypercent of acute hepatitis C is rapidly established as chronic infections, which is a leadingcause of liver cirrhosis and hepatocellular carcinoma (HCC). Combination of a pegylatedinterferon (IFN) with ribavirin is the current therapy for HCV infection, and it is stillfraught with limited effciency, resistance problems, poor tolerability, and high cost.Hepatocellular carcinoma is the fifth global pandemic tumor, more than50new patientsarise each year around the world. HCV infection is one of the important factors leading toHCC, but the mechanisms are still not entirely clear. Multiple HCV proteins including coreprotein, non-structural protein5A (NS5A) have been reported to be associated with HCC.At present, the treatment of HCC is based on operation resection, supplemented bychemotherapy or drug therapy.MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs that interact with the3’-untranslated region (UTR) of target mRNAs, resulting in the inhibition of translation or thepromotion of mRNA degradation. MiRNAs play an important role in proliferation,development and differentiation of many cell types and are also involved in thedevelopment of diseases including cancer. The recent research data suggest that miRNAsmight be used as a new molecular target in the diagnosis, therapy and prognosis of tumors.Meantime, Because of the limitation of the current liver cancer therapy, induction ofdifferentiation is a new and effective method for liver cancer differentiation therapy, anddifferentiation inducer has therefore received more and more attention. All-trans retinoicacid (atRA) is by far the most important class of differentiation-inducing agents, has beensuccessfully used in the treatment of acute promyelocytic leukemia (APL). In this study,we focused on the mechanism of miR-221regulating HCV replication and atRA protectingthe liver cancer cell survival.Part1: Mir-221Enhances the Anti-HCV Function of IFN by Targeting toSOCS1/SOCS3With the development of molecular biology technology, RNA interference and microarrayhave been used in studying of HCV life cycle. For instance, four receptors (CD81, SR-BI,Claudin-1and Occludin) correlated with HCV entry were identified, and PI4kinaseregulating HCV replication and the lipoproteins involving in virus assembly and release were also reported. Recent studies suggest that miRNAs play an important role in HCVinfection. Such as miR-122promoting HCV replication by binding to the5,-UTR,miR-193b regulation the sensitivity of HCV for Sorafenib.MiR-221is widely expressed in a large variety of cells, encoded by genes on chromosomeX, functioned as oncogenic factor by downregulation of p16, p27, p57, DDIT4and PTENin HCC. We first found elevated miR-221in both sera of HCV patients and supernatant ofHCVcc (HCV cell culture-derived virus) infected cells. And then, Huh-7.5.1cells werefirst transfected with miR-221mimics and inhibitor, and infected with HCVcc, FQ-PCRand immunofluorescence were used to analysis the replication and infectivity of HCV. Theresults showed that overexpression of miR-221or knock down of miR-221has no effect inHCV replication. Adding IFN to Huh-7.5.1cells culture rescued miR-221inhibition ofHCV infectivity, indicating that miR-221depends on IFN to inhibit HCV infection.The IFN level of Huh-7cell did not change after transfection with miR-221mimics andinhibitor suggested that signal pathway downstream to IFN might be affected. Therefore,we tested the JAK1, STAT1and STAT3downstream to IFN-α/JAK/STAT pathway, andconfirmed that miR-221could upregulate their phosphorylation. We then conducted ananalysis of miR-221’s target via internet database Targetscan and PicTar.Although miR-221did not target on JAK/STAT directly,3’-UTR of SOCS (suppressors ofcytokine signaling)1and3were found to bind with miR-221. To confirm the prediction, aluciferase reporter with wt/mutant3’-UTR of SOCS1and SOCS3was constructed, and theresults of luciferase assay showed SOCS1and SOCS3were the targets of miR-221.Small interfering RNA of SOCS (SiSOCS1and SiSOCS3) were used to knock down theexpression of SOCS1and SOCS3. The data showed that when SOCS1and SOCS3wereknocked down, the signaling of JAK/STAT is enhanced, phosphorylation level of JAK1,STAT1and STAT3increased compared with control, replication of HCV decreased andthe effect of interferon anti-HCV enhanced. The above results showed that SOCS familyis the main negative regulator of JAK/STAT pathway, miR-221promote the JAK/STATpathway by downregulating its repressive modulators SOCS1and SOCS3. Therefore,miR-221could rescue IFN/JAK/STAT signaling, and then, repress HCV replication moreefficiently by targeting SOCS1and SOCS3.The virus life cycle in host cell is a complicated process with the countering force of hostresistance and virus escape fighting in competition. MiR-221plays dual roles in thereplication and infection of HCV, on the one hand keeping the stability of HCV genomeand promote its replication, and on the other hand, targeting SOCS1/SOCS3and functions as an IFN enhancer in anti-HCV infection. Because of the complication of host-virusinteraction, the role of miR-221in the replication and infection of HCV needs furtherstudying.Part2: All-trans retinoic acid protects HCC cells against serum-depletion inducedcell death by upregulating COL8A2Retinoic acid (RA), a vitamin A derivative, is the oxidation product of retinene, whichcomes from the oxidation of vitamin A. RA regulates cell proliferation, differentiation, andmaturation, and is an important in the organism’s normal growth and physiological activity.RA mainly functions through binding to retinoid-x receptor to form a dimer, which bindsto target DNA and regulate the expression of downstream genes. AtRA (all-trans retinoicacid) is the most important differentiation inducer found to date. AtRA is used in thetreatment of HCC because following three characteristics: induce apoptosis, suppressproliferation and induce differentiation of liver cancer cells.AtRA has been reported with contrary function when used for treatment for HCC,normally, atRA possess antitumoral effects, inducing apoptosis and differentiation, but itcould also prolong the survival of HCC cells under serum-free conditions. In this study,HepG2, Hep3B and Huh7cells were cultured under serum, serum-free conditionwith/without atRA, and the survival and apoptosis were observed. In HepG2cells,apoptosis started at36hours after serum depletion, half the cells died at48hours, andfinally all cells died at96hours. Yet with the protection of10μM atRA, apoptosis isdelayed to60hours after serum depletion,30%of the cells still survived at120hours, andthe cells did not die out until168hours. Similar results were observed in Hep3B andHuh-7cell lines, which indicated that atRA protects HCC cells in serum depleted culture,and this protection is dose dependent.In order to study the mechanism, Flow-cytometry assay was carried out to detect the cellcycle and cell apoptosis of test and control cells. The data showed that no change in cellcycle and in cell apoptosis treat with or without atRA. Further studying showed that atRAtreated cells showed enhanced cell adhesion ability. Since cell adhesion is influenced byextracellular-matrix-related molecular, Illumina Human HT-12v4microarray was used tosort out380genes with differential expression (207upregulated and173downregulated) inatRA treated compared with untreated cells. The genes with most significant upregulationwere CYP26A1, HINT3, miR-1282and CYP26B1. Function analysis of the differentiallyexpressed genes showed that extracellular matrix genes mainly got upregulated, and COL8A was distinguished among them, and further confirmed with FQ-PCR and westernblotting. These results indicated that COL8A2might be involved in the protection ofhepatocellular carcinoma cells against serum-starvation induced by atRA. To prove this,we knocked down the expression of COL8A2with siRNA, and as predicted, protectionform atRA as well as the enhancement in cell adhesion during serum depletion eliminated.Moreover, without serum, COL8A2also boost HCC cell migration and invasion. The cellmigration and invasion increased up to31.6%and77.8%with overexpression of COL8A2,and decreased up to30.2%and59.6%with COL8A2knocking down. The above datashowed that atRA protect hepatocellular carcinoma cells against serum-starvation byupregulating collagen8A2.As a novel tumor therapy method, the study of induction and differentiation therapy is stillin the preliminary stage and with many unknown field. Efficient differentiation inducer isthe key problem. AtRA is the most efficient differentiation inducer even if with side effects.Our study enriches the understanding of the role of atRA in antitumor therapy, andprovides basic research data for its clinical applicationSummary:1. Mir-221represses HCV replication by targeting socs, the negative regulator ofJAK/STAT pathway to enhance IFN anti-HCV function.2. AtRA protects HCC cells against serum starvation induced cell death by up-regulatingcollagen8A2. The molecule of ECM is involved in enhancement of cell adhesion byatRA.3. Our reseach focuses on the role of miRNA in HCV infection and the different functionof atRA in HCC theapy, these data

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