Hepatitis C virus mediated changes in miRNA-449a modulates inflammatory biomarker YKL40 through components of the NOTCH signaling pathway - PubMed (original) (raw)

Hepatitis C virus mediated changes in miRNA-449a modulates inflammatory biomarker YKL40 through components of the NOTCH signaling pathway

Nayan J Sarma et al. PLoS One. 2012.

Abstract

Liver disease due to hepatitis C virus (HCV) infection is an important health problem worldwide. HCV induced changes in microRNAs (miRNA) are shown to mediate inflammation leading to liver fibrosis. Gene expression analyses identified dysregulation of miRNA-449a in HCV patients but not in alcoholic and non-alcoholic liver diseases. By sequence analysis of the promoter for YKL40, an inflammatory marker upregulated in patients with chronic liver diseases with fibrosis, adjacent binding sites for nuclear factor of Kappa B/P65 and CCAAT/enhancer-binding protein alpha (CEBPα) were identified. P65 interacted with CEBPα to co-operatively activate YKL40 expression through sequence specific DNA binding. In vitro analysis demonstrated that tumor necrosis factor alpha (TNFα) mediated YKL40 expression is regulated by miRNA-449a and its target NOTCH1 in human hepatocytes.NOTCH1 facilitated nuclear localization of P65 in response to TNFα. Further, HCV patients demonstrated upregulation of NOTCH1 along with downregulation of miRNA-449a. Taken together it is demonstrated that miRNA-449a plays an important role in modulating expression of YKL40 through targeting the components of the NOTCH signaling pathway following HCV infection. Therefore, defining transcriptional regulatory mechanisms which control inflammatory responses and fibrosis will be important towards developing strategies to prevent hepatic fibrosis especially following HCV recurrence in liver transplant recipients.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. miRNA-449a is downregulated in HCV patients and YKL40 is upregulated in patients with hepatic fibrosis.

Total RNA was isolated from liver biopsies obtained from 10 chronic HCV patients, 10 alcoholic hepatitis patients, 10 non-alcoholic steatohepatitis (NASH) patients and 10 normal donor livers (control). Expression of miRNA-449a (1A) and YKL40 (1B) were determined by Q-PCR. The ΔΔCt value was calculated by normalizing the threshold (CT) values with GAPDH expression and miRNA-449a (1A) or YKL40 (1B) expression respectively in controls. The ‘*’ represents p value<0.01 obtained by a two-tailed t test. Error bars represent Standard Deviations (SD) calculated from three independent experiments.

Figure 2. TNFα regulates the expression of YKL40 at the transcriptional level.

A. (Upper panel) Hepatocytes were transfected with an −3000 bp _YKL40_-promoter driven reporter construct with (+) or without (−) TNFα. Firefly luciferase activity was measured 48 hours after transfection and normalized to a Renilla luciferase internal control. The numbers represent fold-change over control (average of three independent experiments); error bars represent SD. The ‘*’ represents p value<0.05 obtained by a two-tailed t test. (Lower panel): hepatocytes from 2A were immunoblotted with anti-YKL40 with (+) or without (−) TNFα. ACTIN was used as the loading control. B. HEPG2 cells were immunostained with anti-YKL40 antibody without (−) or with (+) TNFα. C. Quantification of the YKL40 immunostaining signal in HEPG2 cells (1B). The numbers represent the average fluorescence intensity of YKL40 (n = 100). D. Essential regions in the YKL40 promoter required for TNFα mediated expression. Hepatocytes were transfected with luciferase reporters driven by deletion constructs of YKL40 promoter (−3000 bp, −2000 bp, −1000 bp, −500 bp, filled black bars on left) construct with (+) or without (−) TNFα. Firefly luciferase activity was measured 48 hours after transfection and normalized to a Renilla luciferase internal control. The luciferase activity was normalized to the control empty luciferase vector and the numbers represent fold-change over control (average of three independent experiments); error bars represent SD calculated from three independent experiments.

Figure 3. Transcription factors P65 and CEBPα regulate expression of YKL40.

A. Computational prediction of transcription factors binding to the YKL40 promoter. Computational analysis of −4000 bp upstream of the open reading frame using the Transcription Element Search System (TESS). The bars represent the predicted consensus binding sites in the DNA for transcription factors P65 and CEBPα. B. Mutation of NFKB/P65 binding site inhibits TNFα mediated YKL40 induction. Hepatocytes were transfected with luciferase reporters driven by deletion constructs of YKL40 promoter −578 bp wildtype, −578 bp P65 binding site mutated (*) and −500 bp wildtype (P65 site deleted), filled black bars on left) construct with (+) or without (−) TNFα. Firefly luciferase activity was measured 48 hours after transfection and normalized to a Renilla luciferase internal control. The numbers represent fold-change over the −578 wildtype construct without TNFα treatment (average of three independent experiments); error bars represent SD. The P65 binding site mutation is shown in the lower panel. The ‘*’ represents p value <0.05 obtained by a two-tailed t test. C. CEBPα in an upstream transcription factor to activate YKL40 expression. Hepatocytes were transfected with luciferase reporters driven by deletion constructs of YKL40 promoter (−3000 bp, −2000 bp, −1000 bp, −500 bp, filled black bars on left) along with an empty vector or vector expressing CEBPα and treated with TNFα. Firefly luciferase activity was measured 48 hours after transfection and normalized to a Renilla luciferase internal control. The numbers represent fold-change over the control empty luciferase vector (average of three independent experiments); error bars represent SD.

Figure 4. CEBPα interacts with NFKB/P65 to bind YKL40 promoter and cooperates to activate transcription in hepatocytes.

A. YKL40 promoter showing binding sites for P65 and CEBPα. The black horizontal bars represent regions amplified by the PCR primers. B. Chromatin was immunoprecipitated with anti-P65 or anti-CEBPα or isotype control IgG from hepatocytes. Segments of the YKL40 promoter (indicated in 4A) were amplified by PCR. The first three lanes show immunoprecipitated chromatin (IP) and the fourth lane show input chromatin (Input). ACTIN promoter amplification is shown as the negative control. C. Co-immunoprecipitation of P65 with CEBPα in hepatocytes. Whole cell lysates were subjected to immunoprecipitation with either rabbit IgG or anti-P65. CEBPα in the cell lysates (Input) and immunoprecipitated complexes (IP) was detected by immunoblotting with anti-CEBPα. P65 was detected by immunoblotting with anti-P65. D. HEPG2 cells were treated with (+) or without (−) TNFα and co-immunostained with anti-CEBPα and anti-P65. E. Hepatocytes were transfected with a luciferase construct driven by the −3000 bp YKL40 promoter in addition to the control vector or vector expressing P65 or CEBPα or both. Firefly luciferase activity was measured 48 hours after transfection and normalized to a Renilla luciferase internal control. The numbers represent fold-change over the control empty vector (average of three independent experiments); error bars represent SD. Bottom panel, expression of P65 and CEBPα was verified by immunoblotting with anti-P65 and anti-CEBPα respectively.

Figure 5. Notch1 regulates nuclear retention of NFKB/P65 in response to TNFα.

A. (Right panel) Hepatocytes were treated with (+) or without (−) TNFα and cytoplasmic and nuclear fractions were extracted. (Left panel) hepatocytes were transfected with either non-specific siRNA or siRNA specific for NOTCH1 and treated with TNFα and cytoplasmic and nuclear fractions were extracted. Whole cell lysates, cytoplasmic and nuclear extracts were subjected to immunoblotting with anti-P65. B. HEPG2 cells were treated with (+) or without (−) TNFα and immunostained with anti-P65. The arrows indicate nuclear localization of P65. C. Quantification of the P65 immunostaining signal in HEPG2 cells (5B). The numbers represent the average fluorescence intensity of P65 (n = 100). D. HEPG2 cells were transfected with either non-specific siRNA or siRNA specific for NOTCH1 and treated with TNFα. The arrows indicate nuclear localization of P65. E. Quantification of the P65 immunostaining signal in HEPG2 cells (5D). The numbers represent the average fluorescence intensity of P65 (n = 100). The ‘*’ represents p value<0.05 obtained by a two-tailed t test.

Figure 6. microRNA 449a regulates YKL40 expression by targeting NOTCH1 for silencing.

A & B. Hepatocytes were transfected with an empty vector (−) or vector expressing miRNA-449a (+) and expression of NOTCH1 (6A) and miRNA-449a (6B) were determined by Q-PCR. The ΔΔCT value was calculated by normalizing the threshold (CT) values with GAPDH and expression of NOTCH1 and miRNA-449a respectively in controls. The ‘*’ represents p value<0.05 obtained by a two-tailed t test. C. (Upper panel) hepatocytes were transfected with a luciferase construct driven by the YKL40 promoter in addition to the control vector or vector expressing miRNA-449a or non-specific siRNA or siRNA specific for NOTCH1 or siRNA specific for P65 in the presence of TNFα. Firefly luciferase activity was measured 48 hours after transfection and normalized to a Renilla luciferase internal control. The numbers represent fold-change over the control vector (average of three independent experiments); error bars represent SD. (Lower panel) Downregulation of YKL40, NOTCH1 and P65 is verified by immunoblotting with anti-YKL40 or anti-NOTCH1 or anti-P65 respectively. ACTIN is shown as the loading control. D. Hepatocytes were transfected with a luciferase construct driven by the YKL40 promoter in addition to the control vector or vector expressing miRNA-449a construct with (+) or without (−) TNFα. Firefly luciferase activity was measured 48 hours after transfection and normalized to a Renilla luciferase internal control. The numbers represent fold-change over the control vector without TNFα (average of three independent experiments); error bars represent SD. The ‘*’ represents p value<0.01 obtained by a two-tailed t test.

Figure 7. NOTCH1 expression is upregulated in HCV patients.

Total RNA was isolated from liver biopsies obtained from 10 chronic HCV patients, 10 alcoholic hepatitis patients, 10 NASH patients and 10 normal donor livers (control). Expression of NOTCH1 was determined by Q-PCR. The ΔΔCT value was calculated by normalizing the threshold (CT) values with GAPDH expression and expression of NOTCH1 in controls. The ‘*’ represents p value<0.01 obtained by a two-tailed t test. Error bars represent SD.

Figure 8. Schematic representation of HCV mediated role of miRNA-449a in YKL40 expression.

HCV infection results in downregulation of miRNA-449a that leads to upregulation of NOTCH1 eventually results in nuclear stabilization of P65. P65 upregulates YKL40 expression in co-operation with CEBPα in response to TNFα.

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Hepatitis C virus mediated changes in miRNA-449a modulates inflammatory biomarker YKL40 through components of the NOTCH signaling pathway - PubMed (original) (raw)