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. Author manuscript; available in PMC: 2021 Mar 1.
Published in final edited form as: Hepatology. 2019 Nov 3;71(3):780–793. doi: 10.1002/hep.30893

Hepatitis C virus mediated inhibition of miR-181c activates ATM signaling and promotes hepatocyte growth

Tapas Patra 1, Keith Meyer 1, Ratna B Ray 2, Ranjit Ray 1,3,*
PMCID: PMC7008082  NIHMSID: NIHMS1045939  PMID: 31400158

Abstract

Hepatitis C virus (HCV) infection promotes hepatocyte growth and progress to hepatocellular carcinoma (HCC). We previously observed that HCV infection of hepatocytes transcriptionally downregulates miR-181c expression through C/EBP-β. Here, we examined the role of miR-181c in the regulation of cell cycle progression in context to HCV infection. In silico analysis suggested ataxia-telangiectasia mutated (ATM) protein, a protein kinase, is a direct target of miR-181c. ATM is a central mediator of response for cellular DNA double-strand break. Our results demonstrated that ATM expression is higher in HCV infected hepatocytes and chronic HCV infected liver biopsy specimens. We have shown a direct interaction of miR-181c with the 3’ UTR of ATM, and the presence of ATM in miR-181c associated RISC complex. Exogenous expression of miR-181c inhibited ATM expression and activation of its downstream molecules, Chk2 and Akt. On the other hand, introduction of anti-miR-181c restored ATM and phosphorylated Akt. Further, introduction of miR-181c significantly inhibited phospho-CDK2 and Cyclin-A expression arresting cell cycle progression, while overexpression of miR-181c promoted apoptosis of HCV infected hepatocytes and can be inhibited by overexpression of ATM from a clone lacking miR-181c binding sites. In addition, miR-181c significantly regressed tumor growth in xenograft human HCC mouse model.

Conclusions:

Together, our results suggested that HCV infection suppresses miR-181c in hepatocytes resulting in ATM activation and apoptosis inhibition for promotion of cell cycle progression. The results provided a novel mechanistic insight in understanding the role of miR-181c in HCV associated hepatocyte growth promotion, and may have potential for therapeutic intervention.

Keywords: HCV, hepatocyte, miR-181c, ATM, cell cycle progression, apoptosis

Introduction

HCV is a hepatotropic, single stranded, positive sense RNA virus that belongs to the hepacivirus genus of the Flaviviridae family (1). In about 30% of HCV infected patients, the virus is spontaneously cleared during the acute phase of infection, while the remaining 70% of chronically infected patients eventually progress to serious liver disease, including steatosis, cirrhosis and hepatocellular carcinoma (HCC) (2). Several HCV proteins exhibit oncogenic potential. For example, HCV core protein promotes metabolic disorder, immortalizes primary human hepatocytes, and enhances reactive oxygen species (ROS) formation and cell growth (3). Transgenic mice with liver specific core protein expression display HCC. HCV NS5A protein is also involved with a variety of host signal transduction pathways, such as anti-apoptosis, ROS production, immune evasion and cell proliferation (4). HCV-associated metabolic disorders, reactive oxidative stress and inflammation result in fibrogenesis, cirrhosis, and create genomic instability or pro-oncogenic microenvironment. Therefore, long time interaction of HCV proteins with host potentiates HCC (5).

HCV infection induces elevated levels of reactive oxygen species, resulting in oxidative stress and causing severe DNA damage in hepatocytes (6). The DNA damage response pathway is coordinated with DNA repair, cell cycle progression and proliferation (7). During DNA repair, several processes occur, including mismatch repair, homologous recombination, non-homologous end joining, and chromosome segregation, defects in which genomic instability may occur and eventually lead to HCC (8). On the other hand, impairment of the DNA damage response pathway leads to a consequence of cell cycle arrest and apoptosis (9). The foremost sensor of DNA damage is the MRN complex, consisting of three proteins- Mre11, Rad50 and Nbs1. The active complex subsequently recruits ATM, an enzyme involved in repairing DNA damage, to promote cell survival (10, 11). ATM was initially recognized in individuals with ataxia telangiectasia; a neurodegenerative autosomal recessive disorder, exhibiting motor neuropathy, immunodeficiency and cancer predisposition (12). Several exogenous and endogenous factors regulate the kinase activity of ATM, including microRNAs (miRNAs) (13, 14).

microRNAS (miRNAs) are endogenous non-coding short RNA molecules that have been highly conserved during evolution. miRNAs generally bind to the 3’ untranslated region of mRNAs and regulate many cellular processes, including metabolism, proliferation, differentiation and apoptosis (15, 16). HCV infection modulates the expression of several miRNAs (17). We reported previously that HCV infection of hepatocytes transcriptionally downregulates miR-181c expression by modulating CCAAT/enhancer binding protein β (C/EBP-β). HCV mediated miR-181c suppression enhances homeobox A1 expression (18). In the present study, we examined whether miR-181c has a regulatory role upon hepatocyte growth regulation. Our results suggested that HCV mediated miR-181c suppression modulates ATM signaling pathway and treatment with this miRNA regresses HCC tumor growth in mouse model.

Materials and Methods

PATIENT SAMPLES

Chronically HCV-infected liver biopsy specimens from eight adult patients and non-HBV/HCV liver biopsy specimens from seven patients were kindly provided by Adrian M Di Bisceglie, Saint Louis University from the repository and used in our study. The liver specimens were collected from all subjects with their written informed consent, and the human studies protocol was approved by the Saint Louis University Internal Review (number 10592).

HEPATOCYTES, HCV INFECTION AND TRANSFECTION OF miR-181c

Huh7.5 cells, and Huh7.5 cells harboring HCV full-length replicon (Rep2a) were grown in Dulbecco’s modified Eagle’s medium (Hyclone) containing 10% FBS, 100 U of penicillin/ml, and 100 mg of streptomycin/ml. Multiple passages of immortalized human hepatocytes (IHH) generated a transformed and tumorigenic phenotype, named as THH (19). HCV genotype 2a (clone JFH1) was grown in Huh7.5 cells. Culture medium containing HCV was filtered through a 0.45μm pore size cellulose acetate membrane (Nalgene) to remove cell debris. HCV RNA in cell culture supernatant was quantified by real-time PCR in an ABI Prism 7000 real-time Thermo cycler (Department of Pathology, Saint Louis University) using HCV analyte-specific reagents (ASRs, Abbott Molecular). Huh7.5 and THH cells were infected with HCV genotype 2a and incubated for 72 h. Control-miR, mimic hsa-miR-181c-5p, anti-miR-181c-5p, miR-19a-5p, or miR-10b-5p (Ambion, Life Technology) was transfected into hepatocytes using Lipofectamine RNAimax (Invitrogen, Life Technology) following manufacturer’s instruction and incubated for 48 h. The full-length 3’-UTR of ATM cloned in pRL-CMV plasmid (kindly provided by Hailiang Hu, Duke University), transfected into hepatocytes along with control or miRNAs using Lipofectamine-3000 (Invitrogen Life Technology).

RNA ISOLATION AND QUANTITATIVE REVERSE TRANSCRIPTASE PCR (qPCR)

Total RNA was isolated from cells using TRIzol reagent (Invitrogen). cDNA was synthesized using Superscript III reverse transcriptase kit (Invitrogen) with a random hexamer following manufacturer’s protocol. Real-time PCR was performed for quantitation of gene expression using TaqMan and SYBR-green PCR master-mix and detected by RT-PCR (Applied Biosystems). For detection, miR-181c (assay identification number 000482), ATM (Forward primer: 5’-TGGATCCAGCTATTTGGTTTGA-3’ and Reverse primer: 5’-CCAAGTATGTAA CCAACAATAGAAGAAGTAG-3’), Bcl2 (Forward primer: 5’-GGGAGGATTGTGGCCTTCT TT-3’ and Reverse primer: 5’-GCCTTTGTGGAACTGTACGGC-3’), and Bax (Forward primer: 5’-AACATGGAGCTGCAGAGGAT-3’ and Reverse primer: 5’-GGAGGAAGTCCAATGTC CAG-3’) were used. U6 (assay identification number 001973) and GAPDH (Forward primer: 5’-CATGTTCGTCATGGGTGTGAACCA-3’ and Reverse primer: 5’-AGTGATGGCATGGACT GTGGTCAT −3’) was used as endogenous control. The relative gene expression was analyzed by using the 2ΔΔCT formula (ΔΔCT = ΔCT of the sample – ΔCT of the untreated control).

AGO2 RNA CO-IMMUNOPRECIPITATION

miR-181c transfected Rep2a-Rluc cells were lysed with lysis buffer (150 mM KCl, 25 mM Tris-HCl, 5 mM EDTA, 1% Triton X-100, 5 mM dithiothreitol, protease inhibitor mixture, and 100 U/ml RNase-OUT). Cell lysates were clarified, incubated with an anti-Ago2 monoclonal antibody (Mab 11A9, Sigma) or isotype control IgG2a at 4°C overnight, and mixed with protein G-Sepharose (GE Healthcare) for 2 h. The beads were washed four times, and RNA was isolated by using the RNeasy mini-kit (Qiagen).

LUCIFERASE REPORTER ASSAY

Huh7.5 cells were co-transfected with the ATM 3’-UTR luciferase reporter plasmid (provided by Hailiang Hu, Duke University) and control-miR or two different doses of miR-181c mimic (50 nM and 100 nM), and miR-19a mimic (100 nM) or miR-10b mimic (100 nM). The relative Renilla luciferase activity was measured following manufacturer’s protocol (Glomax Luminometer, Promega).

WESTERN BLOT ANALYSIS

Cell lysates were prepared by using 2× SDS sample buffer and proteins were resolved by SDS-PAGE, transferred onto a nitro-cellulose membrane, and blocked with 3% non-fat dry milk. The membrane was incubated at 4°C overnight with specific primary antibody, followed by a secondary antibody conjugated with horseradish peroxidase. The protein bands were detected by chemiluminescence (Amersham). The blot was reprobed with β-Actin HRP conjugated antibody (Sigma) to compare protein load in each lane. Densitometry analysis was done using Image J software. Commercially available antibodies to ATM, phospho-ATM (Ser1981), cyclin A, and CDK2 were procured (Santa Cruz Biotechnology); Chk2, phospho-Chk2 (Thr68), Akt, phospho-Akt (Ser473), caspase 3, and cleaved-PARP were procured (CST); and phospho-CDK2 (Thr39) was procured (Abcam) for western blot analyses.

CELL CYCLE PHASE DISTRIBUTION

Huh7.5 cells were transfected with miR-181c mimic or control miR. After 48 h of transfection, cells were fixed with 70% cold ethanol and permeabilized using 0.5% Triton X 100. Cells were stained with propidium iodide (Invitrogen) for 40 min at room temperature. FACS Caliber flow cytometer (BD Bioscience) FL2 channel was used to capture 104 events per sample, and cell cycle phase distribution was analyzed using FlowJo 7.5 software.

APOPTOSIS ASSAY

Huh7.5 cells were transfected with miR-181c or control-miR. Cells were trypsinized 48 h after transfection and suspended in Annexin binding buffer. The apoptotic effect of the hepatocytes was measured with Annexin V fluorescein isothiocynate (FITC) apoptosis detection kit (Invitrogen). FACS Caliber flow cytometer (BD Bioscience) FL1 channel was used to capture 104 events per sample, and cellular apoptosis was measured using FlowJo 7.5 software.

ATM RESCUE EXPERIMENT

Huh7.5 cells and THH were co-transfected with miR-181c mimic (50 nM) and the full-length ATM clone devoid of 3’-UTR (provided by Roger Williams, Cambridge University, UK) in two different doses. After 48 h of co-transfection, cell lysates were prepared for western blot analysis using specific antibodies.

HCC XENOGRAFT MODEL

Six-week-old male athymic nude mice were purchased from Charles River Laboratories. All animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of Saint Louis University and NIH guidelines. A total of ~106 THH were mixed with 40% Matrigel for subcutaneous injections in a volume of 100 μl in mouse flank. Once, the tumor volume reached 50 mm3, 10 μg of miR-181c or control-miR associated with siPort transfection reagent was delivered intra-tumoral (five times) at three day intervals. Tumor growth was examined weekly until the end of each experiment. Tumor size was measured with a slide caliper and the volume calculated using the formula (L × W2) × 0.5, where L is length and W is width. Mice were sacrificed 28 days after introduction of microRNA treatment. The tumors were collected for total RNA preparation and qRT-PCR analysis.

STATISTICAL ANALYSIS

All the analyses were performed from three independent experiments. GraphPad Prism 7 was used to analyze the experimental data. The results are presented as means ± standard deviations. Unpaired two-tailed Student’s t-tests and Pearson’s correlation analysis were used to compare the mean values between two groups. Statistical significance was considered as p <0.05.

Results

ATM IS A DIRECT TARGET OF miR-181c

In silico analysis using TargetScan, miRDB, and miRWalk algorithms predicted with high scores that there are two putative binding sites of miR-181c in the 3’-UTR of ATM (Fig. 1, panel A). Our previous study suggested that HCV infection suppresses miR-181c expression in hepatocytes by transcriptional downregulation (18). This result led us to examine whether there is a relationship between miR-181c and ATM expression. To examine potential modulatory effects of miR181c upon ATM expression, the full length 3’-UTR of ATM cloned at the downstream of a Renilla luciferase reporter in pRL-CMV plasmid was co-transfected with miR-181c into Huh7.5 cells. miR-10b and miR-19a were used as negative controls since they do not bind at the 3’-UTR of ATM. Luciferase activity was measured 48 h post-transfection. We observed miR-181c reduced luciferase activity in a concentration dependent manner (Fig. 1, panel B). On the other hand, miR-10b or miR-19a as a control did not show any effect.

FIG. 1.

FIG. 1.

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miR-181c targets ATM regulation. In silico analysis of the 3’-UTR of ATM reveals two putative miR-181c binding sites 1 and 2 (panel A). Huh7.5 cells were co-transfected with the 3’-UTR of ATM cloned into pRL-CMV luciferase vector and different concentrations of miR-181c mimic (50 and 100 nM). Luciferase activity was measured in luminometer after 48 h of transfection. Control-miR (100 nM) was used as a control, and miR-10b or miR-19a were used as unrelated negative controls (panel B). Huh7.5 and HCV full-length replicon cell (Rep2a) lysates were immune-precipitated with Ago2 specific monoclonal antibody or an unrelated IgG2a isotype control and RNA was isolated. Relative expression level of ATM mRNA from Ago2 immuno-precipitates were analyzed by qRT-PCR (panel C). Huh7.5 and THH cells were transfected with control-miR, miR-181c mimic, or anti-miR-181c, and total RNA or cell lysates were prepared after 48 h of transfection. The ATM transcript was analyzed by qPCR from Huh7.5 cells or THH, and normalized with the GAPDH expression level (panel D). Phospho-ATM (Ser1981) and total ATM protein expression status were analyzed by western blot (panel E). Expression level of actin in each lane is shown as a loading control for comparison. Densitometric analyses of the western blot after normalization with actin are shown below. The results were presented as mean with standard deviation from at least three independent experiments. ‘*’, p value of <0.05 was considered statistically significant.

To further examine the in vivo association of miR-181c and ATM, we immune-precipitated miR-181c-Ago2 RISC complex with Ago2 antibody or isotype control IgG from Huh7.5 and Huh7.5 cells harboring full length HCV genome (Rep2a) in the presence of miR-181c. The presence of miR-181c-bound ATM mRNA was significantly higher in Ago2 immuno-precipitate as compared to the isotype control (Fig. 1, panel C), suggesting an association between these two molecules. We also observed that overexpression of miR-181c mimic causes suppression of ATM mRNA expression, and treatment with anti-miR181c restored ATM mRNA in Huh7.5 cells and THH (Fig. 1, panel D). The results indicated that miR181c induce ATM mRNA degradation. Next, we observed overexpression of miR-181c mimic inhibiting total and phosphorylated ATM expression (Ser1981) in Huh7.5 cells or THH. On the other hand, introduction of anti-miR-181c restored total and phosphorylated ATM expression (Fig. 2, panel E). Thus, our results indicated that ATM is a direct target of miR-181c.

FIG. 2.

FIG. 2.

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HCV infection induces ATM expression. Huh7.5 cells and THH were mock-treated or infected with HCV genotype 2a. Cell lysates or total RNA was prepared after three days of viral infection. The status of phospho-ATM (Ser1981) and total ATM protein expression analyzed by western blot are shown (panel A). Expression level of actin in each lane is shown as a loading control for comparison. Densitometric analyses of the western blot after normalization with actin are shown on the right side. The transcript levels of ATM were measured by qPCR from HCV infected Huh7.5 cells and THH cells or mock-treated cells, and normalized with the expression level of GAPDH (panel B). A comparison of ATM transcript in HCV infected (n=8) and unrelated control (HCV or HBV uninfected) (n=7) liver biopsy specimens are shown (panel C). Pearson’s correlation plot of miR-181c and ATM transcript analyzed by qPCR in the same liver biopsy specimens (r=−0.4353, p=0.1048) are shown (panel D). Huh7.5 and THH cells were infected with HCV genotype 2a. Cells were transfected with miR-181c mimic after 24 h of infection and cell lysates were prepared after 48 h of transfection. Phospho-ATM (Ser1981) and total ATM expression were analyzed by western blot (panel E). Expression level of actin in each lane was shown as a loading control for comparison. The results were presented as mean with standard deviation from at least three independent experiments. Densitometric analyses of the western blot after normalization with actin are shown in below. ‘*’, p value of <0.05 was considered statistically significant.

HCV INFECTION ENHANCES ATM EXPRESSION

ATM is critically involved in repairing DNA damage to enhance cell survival (20). To investigate the role of ATM in HCV infected hepatocytes, we determined the status of ATM expression. ATM expression was significantly higher (~2 fold) for phosphorylated (Ser1981) ATM and total ATM in HCV genotype 2a infected Huh7.5 and THH cells, as compared to mock infected cells (Fig. 2, panel A). We also observed that the expressed mRNA level of ATM was higher in HCV infected Huh7.5 and THH as compared to mock-treated control cells (Fig. 2, panel B), suggesting HCV infection enhances ATM expression at the transcriptional level. We further investigated ATM expression in liver biopsy samples from patients infected with HCV. Our results demonstrated that the level of ATM transcripts were significantly higher in chronically HCV infected patients (Fig. 2, panel C). These results suggest that HCV infection of hepatocytes leads to activation of ATM which acts as a DNA damage response element. Next, we examined the potential link between miR-181c and ATM expression in liver biopsy from HCV infected patients or unrelated control liver specimens (Fig. 2, panel D). Our results indicated that ATM mRNA was inversely correlated to miR-181c expression in HCV infected samples. Further, we observed that miR-181c mimic reduces the expression of total and phosphorylated ATM expression (Ser1981) in HCV infected Huh7.5 or THH cells (Fig. 2, panel E). Together, our results suggested that the overexpression of miR-181c directly suppresses ATM expression and HCV induced ATM expression combats DNA damage by suppressing miR-181c.

miR-181c REGULATES DOWNSTREAM SIGNALING MOLECULES OF ATM

ATM phosphorylates several downstream molecules, such as Chk2 and Akt (21, 22). Western blot analysis indicated that overexpression of miR-181c reduces the level of phosphorylated Chk2 in Huh7.5 cells and THH (Fig. 3, panel A). We next examined the status of Akt activation. Overexpression of miR-181c downregulated phosphorylated Akt (Ser473), but not total Akt expression in both Huh7.5 cells and THH (Fig. 3, panel B). Further, introduction of anti-miR-181c restored phosphorylated Akt expression. Taken together, the results suggested that HCV mediated miR-181c suppression induces phospho-ATM, which phosphorylates Chk2 and Akt.

FIG. 3.

FIG. 3.

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miR-181c alters downstream signaling molecules of ATM. Huh7.5 cells or THH were transfected with miR-181c mimic or control-miR. Cell lysates were prepared after 48 h of transfection. Phospho-Chk2 (Thr68) and total Chk2 expression were analyzed by western blot using specific antibodies (panel A). Cells were separately transfected with control-miR, miR-181c mimic or anti-miR-181c and cell lysates were prepared after 48 h of transfection. Phospho-Akt (Ser473) and total Akt expression were analyzed by western blot (panel B). Expression level of actin in each lane is shown as a loading control for comparison. Densitometric analyses after normalization with actin are shown below. The results are presented as mean with standard deviation from at least three independent experiments. ‘*’, p value of <0.05 was considered statistically significant.

ATTENUATION OF CELL CYCLE PROGRESSION BY miR-181c

Cyclin-dependent kinase 2 (CDK2) is one of the key cell cycle regulators. The phosphorylated form of CDK2, by binding to its partner Cyclin E or Cyclin A, plays an important role in cell cycle progression, specifically acting at the G1-S-G2 phase (23). Akt regulates cell cycle progression from the S to G2 phase by direct phosphorylation at Thr39 site of CDK2 (24). In addition, Akt activates histone deacetylase 3 (HDAC3) mediated CDK2-Cyclin A complex stability (25, 26). Western blot analysis was performed to examine expression of Cyclin A and phosphorylated CDK2 (Thr39) when miR-181c mimic suppresses Akt activation. The exogenous expression of miR-181c downregulated both Cyclin A and phosphorylated CDK2 expression as compared to the control in both Huh7.5 cells and THH, while introduction of anti-miR-181c restored Cyclin A and phosphorylated CDK2 expression, although total CDK2 expression did not change (Fig. 4, panel A).

FIG. 4.

FIG. 4.

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miR-181c inhibits cell cycle progression. Huh7.5 cells and THH were transfected with control-miR, miR-181c mimic, or anti-miR-181c, and cell lysates were prepared after 48 h of transfection. The levels of Cyclin A, phospho-CDK2 (Thr39) and total CDK2 expression were analyzed by western blot (panel A). Expression level of actin in each lane is shown as a loading control for comparison. Densitometric analyses of the western blot after normalization with actin are shown below. Cell cycle progression was analyzed by FACS in Huh7.5 cells treated with miR-181c mimic or control-miR after propidium iodide staining (panel B). Quantitative representation of different phases of cell cycle was shown below. The results were presented as mean with standard deviation from at least three independent experiments. ‘*’, p value of <0.05 was considered statistically significant.

To understand whether miR-181c mediated suppression of phosphorylated CDK2 and Cyclin A expression affects cell cycle phase distribution, flow cytometry analysis was performed with Huh7.5 cells transfected with miR-181c mimic or control miR. We observed a higher number of cells accumulated in G2 phase following the over expression of miR-181c mimic in a phase distribution analysis when compared with other phases of cell cycle (Fig. 4, panel B). The miR-181c mediated downregulation cell cycle progression correlates with our observation for a decrease in phosphorylation of Akt and Chk2 expression in hepatocytes. Thus, our results indicated that overexpression of miR-181c in hepatocytes causes’ cell cycle arrest in the G2 phase.

miR-181c OVEREXPRESSION INDUCES APOPTOSIS

We next examined whether miR-181c mediated downregulation cell cycle progression is associated with apoptotic cell death. For this, we performed a flow cytometry analysis of Huh7.5 cells transfected with miR-181c mimic after Annexin V staining. Accumulation of Annexin V indicated miR-181c expression increased apoptosis in hepatocytes (Fig. 5, panel A). We also observed induction of caspase-3 cleavage (~17 kD fragment) in miR-181c overexpressed Huh7.5 cells and THH (Fig. 5, panel B). PARP cleavage is another characteristic hallmark of apoptosis. Overexpression of miR-181c displayed a higher accumulation of the cleaved PARP fragment (~ 89 kD) when compared to the control Huh7.5 cells or THH (Fig. 5, panel C). Thus, our results suggested that overexpression of miR-181c leads to apoptosis in hepatocytes.

FIG. 5.

FIG. 5.

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miR-181c induces apoptosis. Apoptosis was analyzed by FACS in Huh7.5 cells treated with miR-181c mimic or control-miR after Annexin V staining. Quantitative representation of apoptosis is shown (panel A). Huh7.5 cells or THH were transfected with miR-181c mimic and cell lysates were prepared after 48 h of transfection. The level of cleaved Caspase 3 expression was analyzed by western blot (panel B). The level of cleaved PARP accumulation was analyzed by western blot (panel C). The inhibition of miR-181c mediated ATM expression, Chk2 expression and PARP cleavage were determined from rescue experiment after co-transfection of miR-181c and ATM from a clone lacking miR-181c binding sites (panel D). Expression level of actin in each lane is shown as a loading control for comparison. Densitometric analyses of the western blot after normalization with actin are shown on the right. The results are presented as mean with standard deviation from at least three independent experiments. ‘*’, p value of <0.05 was considered statistically significant.

To rescue the inhibitory effects of miR181c upon ATM expression, a full-length ATM clone devoid of 3’-UTR was co-transfected with miR-181c into Huh7.5 cells and THH. Western blot analyses were performed for p-ATM (S1981), total ATM, p-Chk2 (T68), total Chk2, and cleaved PARP after 48 h of transfection. We observed that the inhibitory effect of miR-181c is restored by ATM overexpression form total and phosphorylated ATM, phosphorylated Chk2, and decrease in cleaved PARP accumulation (Fig. 5, panel D). Downregulation of pro-apoptotic gene Bax mRNA further indicated that overexpressed ATM can rescue miR-181c induced apoptosis (data not shown).

INTRODUCTION OF miR-181c REGRESSES XENOGRAFT TUMOR GROWTH

We further examined whether miR-181c mimic treatment regresses tumor growth in a xenograft mouse model. For this, we subcutaneously implanted transformed hepatocytes into the flank of nude mice. When tumors were palpable (~80 mm3), we randomly divided the mice in two groups. Intra-tumoral injection of miR-181c mimic or control-miR was administrated every 3rd day until the end of the experiment. miR-181c treated mice displayed significant inhibition of tumor growth as compared to control-miR treated mice (Fig. 6, panel A). Representative tumors from control and experimental groups are shown (Fig. 6, panel B). Analysis of tumor RNA in miR-181c treated or untreated control mice suggested reduced ATM expression as expected (Fig. 6, panel C). Further, the tumors treated with miR-181c mimic showed downregulation of anti-apoptotic Bcl2 mRNA and upregulation of pro-apoptotic Bax mRNA expression, when compared with untreated control tumors (Fig. 6, panel D). The results suggested that miR-181c mimic causes apoptosis in xenograft tumors.

FIG. 6.

FIG. 6.

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miR-181c impairs hepatocyte growth. THH were subcutaneously implanted into the flank of nude mice (n=5 per group). Once the tumor volume reached 50 mm3, miR-181c or control-miR was delivered intra-tumorally at 3-day interval until the end of experiment. Mice were sacrificed after 28 days of miRNA treatment. Tumor volume is presented and indicated the time of 1st miR-181c administration (panel A). Representative photographs of tumor from control or miR-181c introduced group are shown (panel B). Total RNA was prepared from the tumor for qRT-PCR. U6 and GPDH were used as the internal controls for relative mRNA and normalization in quantitative analyses. Comparative histogram analysis of miR-181c and ATM transcripts in control-miR and miR-181c mimic treated xenograft tumors are shown (panel C). The transcript levels of Bcl2 and Bax were also analyzed by qPCR from untreated control or miR-181c mimic treated xenograft tumors and normalized with GAPDH expression level (panel D). ‘*’, p value of <0.05 was considered statistically significant.

Discussion

HCV infection exerts a profound effect on the expression of cellular miRNAs. Altered expression of miRNAs is involved in HCV associated pathogenesis by controlling signaling pathways for proliferation and apoptosis. Suppression of miR-181c in the liver upon HCV infection results in ATM regulation at the transcriptional level. ATM plays a hierarchical regulatory role in promoting repair of the double-strand break induced DNA damage response. DNA double-strand breaks can originate from a variety of sources including ionizing radiation, alkylating agents, reactive oxygen species (ROS) production. Our results suggested that ATM is a direct target for miR-181c related to suppression and hepatocyte growth promotion and can be rescued by overexpression of ATM from a clone lacking miR-181c binding sites. Machida et al. (27) suggested that HCV infection or expression of viral core or NS3 protein induces DNA double-strand breaks. Our current study suggests that DNA damage is accompanied by ATM kinase activation in hepatocytes. Phosphorylation of Ser1981 stabilizes ATM at the damaged DNA sites and recruits downstream effector proteins, like Chk2 and Akt (21, 22). Our results indicated that exogenous expression miR-181c suppresses ATM activation, and inhibits the expressions of phosphorylated Chk2 and Akt. Ariumi et al. (28) demonstrated that ATM preferentially binds to NS3–4A, and ATM partially co-localizes with NS3–4A in the perinuclear region. This result indicated that HCV might hijack ATM for its replication, resulting in enhancement of mutation frequency in cellular genes, including proto-oncogenes, and leading to development of hepatocellular carcinoma. A recent report suggests that ATM enhances DNA damage signaling response and may provide novel insights of epigenetic regulation during tumorigenesis (29).

Several reports suggest the role of miRNAs in targeting genes involved in inflammation and fibrosis in chronic HCV infection (15). Lower miR-29 expression in the liver of HCV infected patients may help in viral RNA replication and inhibition of miR-29 is linked with HSC activation and collagen synthesis (30). Reduced expression of miR-449a and miR-107 was also observed in chronic HCV patients, but not in alcoholic and non-alcoholic liver disease by genome wide miRNA analyses (31, 32). miR-449a regulates the expression of YKL40 by targeting the NOTCH signaling pathway following HCV infection (31), and YKL40 is known to promote extracellular matrix synthesis. Downregulation of miR-107 and miR-449a modulates the expression of CCL2 by targeting components of the interleukin-6 receptor (IL-6R) complex in patients with HCV related liver disease. miR-449a and miR-107 target IL-6R and JAK1, inhibiting IL-6 signaling and impairing STAT3 activation in human hepatocytes (32). We previously demonstrated that HCV transcriptionally downregulates miR-181c expression (16). Suppression of miR-181c may be a prognostic indicator in rapidly proliferating cells, like glioblastoma, osteosarcoma, and ovarian carcinoma (33–35). Multiple molecules act as targets for miR-181c, including Six2 in metanephric mesenchymal cells, FoxO1 in diabetic endothelial cells, TGF-β in glioblastoma cells, PRKCD in ovarian and Erk in prostate cancer cells (35–39). Here, we focused on the functional importance of miR-181c downregulation in HCV infected hepatocytes. We have observed that HCV associated downregulation of miR-181c targets the 3’-UTR leading to enhanced ATM expression. Further, our results suggested that the functional activation of ATM inversely correlates with miR-181c expression in HCV infected patient liver samples.

Nuclear serine/threonine kinase protein, Chk2, regulates cell cycle in a way to prevent cells from dividing in an uncontrolled manner. Prevention of Chk2 activation seems to impose cell cycle arrest in the G2 phase (40). Akt activation overcomes DNA damage induced G2 phase for cell cycle arrest and apoptosis (24, 41). Our previous study suggested that HCV activates Akt signaling in hepatocytes (42). Here, we observed that introduction of miR-181c mimic results in the downregulation of Akt and associates with regulatory proteins for attenuation of cell cycle progression by arresting at the G2 phase. We also observed that introduction of the miR-181c mimic leads to apoptosis as noted by caspase 3 activation and PARP cleavage, and can be inhibited by overexpression of ATM from a clone lacking miR-181c binding sites. Akt is one of the multiple substrates activated by ATM and play a role on cell cycle regulatory molecules (43), and apoptosis by activating the anti-apoptotic Bcl-2 protein (44).

Our study suggests HCV mediated suppression of miR-181c and regulation of Akt signaling by modulating ATM expression. Enhanced ATM results in activation of downstream effector molecules, like Chk2 and Akt. These activities modulate cell cycle progression, inactivate apoptosis, and promote hepatocyte growth leading to disease progression (Fig. 7). Other miRNAs, including miR-155, miR-146a-5p, miR-135a-5p, miR-373, were shown to either promote or inhibit HCV associated hepatocarcinogenesis (45–48). Our study highlighted intra-tumoral introduction of synthetic miR-181c inhibits tumor progression in a HCC xenograft tumor model. Recent introduction of direct-acting antivirals (DAAs) with the combinations of virus replication inhibitors may achieve a decrease in virus titer to a sustained virological response (SVR) in HCV infected patients (49). However, treatment of advanced liver disease will be difficult with DAAs alone and alternate therapies, including the use of RNAi and miRNAs may be a useful for therapeutic intervention (50). The anti-proliferative effect of miR-181c underlines the potential for this microRNA in combination with other therapeutic modalities to prevent HCV associated liver diseases progression.

FIG. 7.

FIG. 7.

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Schematic presentation of HCV mediated miR-181c regulation and consequences upon ATM signaling on hepatocyte growth promotion.

Acknowledgements:

We thank Adrian M. Di Bisceglie for providing archived liver samples, Hailiang Hu for full-length 3’-UTR of ATM clone, and Roger Williams for providing full-length ATM clone devoid of 3’-UTR. We appreciate the help from Subhayan Sur in animal experiments.

Funding: This work was supported by research grants DK113645 (R.R.) and DK081817 (R.B.R) from the National Institutes of Health, and from Presidential and Liver Center Research Funds of Saint Louis University.

REFERENCES

  • 1.Pfaender S, Brown RJ, Pietschmann T, Steinmann E. Natural reservoirs for homologs of hepatitis C virus. Emerg Microbes Infect 2014;3:e21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Kanwal F, Hoang T, Kramer JR, Asch SM, Goetz MB, Zeringue A, Richardson P, et al. Increasing prevalence of HCC and cirrhosis in patients with chronic hepatitis C virus infection. Gastroenterology 2011;140:1182–1188 e1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Kwon YC, Ray RB, Ray R. Hepatitis C virus infection: establishment of chronicity and liver disease progression. EXCLI J 2014;13:977–996. [PMC free article] [PubMed] [Google Scholar]
  • 4.Gimenez-Barcons M, Wang C, Chen M, Sanchez-Tapias JM, Saiz JC, Gale M Jr. The oncogenic potential of hepatitis C virus NS5A sequence variants is associated with PKR regulation. J Interferon Cytokine Res 2005;25:152–164. [DOI] [PubMed] [Google Scholar]
  • 5.Ray RB, Ray R. Hepatitis C virus manipulates humans as its favorite host for long term relationship. Hepatology 2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Ivanov AV, Bartosch B, Smirnova OA, Isaguliants MG, Kochetkov SN. HCV and oxidative stress in the liver. Viruses 2013;5:439–469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Bartek J, Bartkova J, Lukas J. DNA damage signalling guards against activated oncogenes and tumour progression. Oncogene 2007;26:7773–7779. [DOI] [PubMed] [Google Scholar]
  • 8.Yang SF, Chang CW, Wei RJ, Shiue YL, Wang SN, Yeh YT. Involvement of DNA damage response pathways in hepatocellular carcinoma. Biomed Res Int 2014;2014:153867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Jackson SP, Bartek J. The DNA-damage response in human biology and disease. Nature 2009;461:1071–1078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Shiloh Y ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 2003;3:155–168. [DOI] [PubMed] [Google Scholar]
  • 11.Uziel T, Lerenthal Y, Moyal L, Andegeko Y, Mittelman L, Shiloh Y. Requirement of the MRN complex for ATM activation by DNA damage. EMBO J 2003;22:5612–5621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Lavin MF. Ataxia-telangiectasia: from a rare disorder to a paradigm for cell signalling and cancer. Nat Rev Mol Cell Biol 2008;9:759–769. [DOI] [PubMed] [Google Scholar]
  • 13.Lung RW, Hau PM, Yu KH, Yip KY, Tong JH, Chak WP, Chan AW, et al. EBV-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma. J Pathol 2018;244:394–407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Hu H, Du L, Nagabayashi G, Seeger RC, Gatti RA. ATM is down-regulated by N-Myc-regulated microRNA-421. Proc Natl Acad Sci U S A 2010;107:1506–1511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009;136:215–233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281–297. [DOI] [PubMed] [Google Scholar]
  • 17.Shrivastava S, Steele R, Ray R, Ray RB. MicroRNAs: Role in Hepatitis C Virus pathogenesis. Genes Dis 2015;2:35–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Mukherjee A, Shrivastava S, Bhanja Chowdhury J, Ray R, Ray RB. Transcriptional suppression of miR-181c by hepatitis C virus enhances homeobox A1 expression. J Virol 2014;88:7929–7940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Kwon YC, Bose SK, Steele R, Meyer K, Di Bisceglie AM, Ray RB, Ray R. Promotion of Cancer Stem-Like Cell Properties in Hepatitis C Virus-Infected Hepatocytes. J Virol 2015;89:11549–11556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Huen MS, Chen J. The DNA damage response pathways: at the crossroad of protein modifications. Cell Res 2008;18:8–16. [DOI] [PubMed] [Google Scholar]
  • 21.Matsuoka S, Rotman G, Ogawa A, Shiloh Y, Tamai K, Elledge SJ. Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro. Proc Natl Acad Sci U S A 2000;97:10389–10394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Viniegra JG, Martinez N, Modirassari P, Hernandez Losa J, Parada Cobo C, Sanchez-Arevalo Lobo VJ, Aceves Luquero CI, et al. Full activation of PKB/Akt in response to insulin or ionizing radiation is mediated through ATM. J Biol Chem 2005;280:4029–4036. [DOI] [PubMed] [Google Scholar]
  • 23.Rosenblatt J, Gu Y, Morgan DO. Human cyclin-dependent kinase 2 is activated during the S and G2 phases of the cell cycle and associates with cyclin A. Proc Natl Acad Sci U S A 1992;89:2824–2828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Maddika S, Ande SR, Wiechec E, Hansen LL, Wesselborg S, Los M. Akt-mediated phosphorylation of CDK2 regulates its dual role in cell cycle progression and apoptosis. J Cell Sci 2008;121:979–988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Lv WW, Wei HM, Wang DL, Ni JQ, Sun FL. Depletion of histone deacetylase 3 antagonizes PI3K-mediated overgrowth of Drosophila organs through the acetylation of histone H4 at lysine 16. J Cell Sci 2012;125:5369–5378. [DOI] [PubMed] [Google Scholar]
  • 26.Vidal-Laliena M, Gallastegui E, Mateo F, Martinez-Balbas M, Pujol MJ, Bachs O. Histone deacetylase 3 regulates cyclin A stability. J Biol Chem 2013;288:21096–21104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Machida K, Cheng KT, Sung VM, Lee KJ, Levine AM, Lai MM. Hepatitis C virus infection activates the immunologic (type II) isoform of nitric oxide synthase and thereby enhances DNA damage and mutations of cellular genes. J Virol 2004;78:8835–8843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Ariumi Y, Kuroki M, Dansako H, Abe K, Ikeda M, Wakita T, Kato N. The DNA damage sensors ataxia-telangiectasia mutated kinase and checkpoint kinase 2 are required for hepatitis C virus RNA replication. J Virol 2008;82:9639–9646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Filipponi D, Emelyanov A, Muller J, Molina C, Nichols J, Bulavin DV. DNA Damage Signaling-Induced Cancer Cell Reprogramming as a Driver of Tumor Relapse. Mol Cell 2019;74:651–663 e658. [DOI] [PubMed] [Google Scholar]
  • 30.Bandyopadhyay S, Friedman RC, Marquez RT, Keck K, Kong B, Icardi MS, Brown KE, et al. Hepatitis C virus infection and hepatic stellate cell activation downregulate miR-29: miR-29 overexpression reduces hepatitis C viral abundance in culture. J Infect Dis 2011;203:1753–1762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Sarma NJ, Tiriveedhi V, Subramanian V, Shenoy S, Crippin JS, Chapman WC, Mohanakumar T. Hepatitis C virus mediated changes in miRNA-449a modulates inflammatory biomarker YKL40 through components of the NOTCH signaling pathway. PLoS One 2012;7:e50826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Sarma NJ, Tiriveedhi V, Crippin JS, Chapman WC, Mohanakumar T. Hepatitis C virus-induced changes in microRNA 107 (miRNA-107) and miRNA-449a modulate CCL2 by targeting the interleukin-6 receptor complex in hepatitis. J Virol 2014;88:3733–3743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Ruan J, Lou S, Dai Q, Mao D, Ji J, Sun X. Tumor suppressor miR-181c attenuates proliferation, invasion, and self-renewal abilities in glioblastoma. Neuroreport 2015;26:66–73. [DOI] [PubMed] [Google Scholar]
  • 34.Mori F, Sacconi A, Canu V, Ganci F, Novello M, Anelli V, Covello R, et al. miR-181c associates with tumor relapse of high grade osteosarcoma. Oncotarget 2015;6:13946–13961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Yao L, Wang L, Li F, Gao X, Wei X, Liu Z. MiR181c inhibits ovarian cancer metastasis and progression by targeting PRKCD expression. Int J Clin Exp Med 2015;8:15198–15205. [PMC free article] [PubMed] [Google Scholar]
  • 36.Lv X, Mao Z, Lyu Z, Zhang P, Zhan A, Wang J, Yang H, et al. miR181c promotes apoptosis and suppresses proliferation of metanephric mesenchyme cells by targeting Six2 in vitro. Cell Biochem Funct 2014;32:571–579. [DOI] [PubMed] [Google Scholar]
  • 37.Yang G, Wu Y, Ye S. MiR-181c restrains nitration stress of endothelial cells in diabetic db/db mice through inhibiting the expression of FoxO1. Biochem Biophys Res Commun 2017;486:29–35. [DOI] [PubMed] [Google Scholar]
  • 38.He X, Liu Z, Peng Y, Yu C. MicroRNA-181c inhibits glioblastoma cell invasion, migration and mesenchymal transition by targeting TGF-beta pathway. Biochem Biophys Res Commun 2016;469:1041–1048. [DOI] [PubMed] [Google Scholar]
  • 39.Su Z, Zhang M, Xu M, Li X, Tan J, Xu Y, Pan X, et al. MicroRNA181c inhibits prostate cancer cell growth and invasion by targeting multiple ERK signaling pathway components. Prostate 2018;78:343–352. [DOI] [PubMed] [Google Scholar]
  • 40.Zannini L, Delia D, Buscemi G. CHK2 kinase in the DNA damage response and beyond. J Mol Cell Biol 2014;6:442–457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Kandel ES, Skeen J, Majewski N, Di Cristofano A, Pandolfi PP, Feliciano CS, Gartel A, et al. Activation of Akt/protein kinase B overcomes a G(2)/m cell cycle checkpoint induced by DNA damage. Mol Cell Biol 2002;22:7831–7841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Bose SK, Shrivastava S, Meyer K, Ray RB, Ray R. Hepatitis C virus activates the mTOR/S6K1 signaling pathway in inhibiting IRS-1 function for insulin resistance. J Virol 2012;86:6315–6322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Liu P, Begley M, Michowski W, Inuzuka H, Ginzberg M, Gao D, Tsou P, et al. Cell-cycle-regulated activation of Akt kinase by phosphorylation at its carboxyl terminus. Nature 2014;508:541–545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Pugazhenthi S, Nesterova A, Sable C, Heidenreich KA, Boxer LM, Heasley LE, Reusch JE. Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein. J Biol Chem 2000;275:10761–10766. [DOI] [PubMed] [Google Scholar]
  • 45.Zhang Y, Wei W, Cheng N, Wang K, Li B, Jiang X, Sun S. Hepatitis C virus-induced up-regulation of microRNA-155 promotes hepatocarcinogenesis by activating Wnt signaling. Hepatology 2012;56:1631–1640. [DOI] [PubMed] [Google Scholar]
  • 46.Bandiera S, Pernot S, El Saghire H, Durand SC, Thumann C, Crouchet E, Ye T, et al. Hepatitis C Virus-Induced Upregulation of MicroRNA miR-146a-5p in Hepatocytes Promotes Viral Infection and Deregulates Metabolic Pathways Associated with Liver Disease Pathogenesis. J Virol 2016;90:6387–6400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Van Renne N, Roca Suarez AA, Duong FHT, Gondeau C, Calabrese D, Fontaine N, Ababsa A, et al. miR-135a-5p-mediated downregulation of protein tyrosine phosphatase receptor delta is a candidate driver of HCV-associated hepatocarcinogenesis. Gut 2018;67:953–962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Sur S, Sasaki R, Devhare P, Steele R, Ray R, Ray RB. Association between MicroRNA-373 and Long Noncoding RNA NORAD in Hepatitis C Virus-Infected Hepatocytes Impairs Wee1 Expression for Growth Promotion. J Virol 2018;92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Au JS, Pockros PJ. Novel therapeutic approaches for hepatitis C. Clin Pharmacol Ther 2014;95:78–88. [DOI] [PubMed] [Google Scholar]
  • 50.Fan R, Xiao C, Wan X, Cha W, Miao Y, Zhou Y, Qin C, et al. Small molecules with big roles in microRNA chemical biology and microRNA-targeted therapeutics. RNA Biol 2019;16:707–718. [DOI] [PMC free article] [PubMed] [Google Scholar]

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