Primers, antibodies, plasmids and reagent Primary antibodies
Targeted protein Host Clone Provider Catalogue number
PTEN Mouse A2B1 Santa Cruz Biotechnology,
Inc., Dallas, Texas, USA
SC-7974
IRS1 Rabbit Santa Cruz Biotechnology,
Inc., Dallas, Texas, USA
SC-559
HCV core Mouse C7-50 Covalab (Lyon France) mab51009
β-cytoplasmic actin Mouse C4 A gift from C. Chaponnier, Geneva, Switzerland
Plasmids and HCV constructs
pFK-J6/C3 (Jc1) Full lengh HCV A gift from R.
Bartenschlager, Heidelberg, Germany (4)
phCMV 1b9.9 HCV E1/E2 A gift from B. Bartosch,
Lyon, France (5) pLuc-PTEN3’UTR
Luciferase-PTEN3’UTR
pCMV-luc REF : LR-1001 Signosis, Inc. (CA, USA)
pCMV-luc-miR21(P) mir21(P) targets pCMV-luc A gift of B. Cullen (6) (Addgene plasmid # 20382) pCMV-luc-random random sequence
targets
pCMV-luc A gift from B. Cullen (6) (Addgene plasmid # 20877) pTKrenilla_Luc Renilla Luciferase D. Garcin, Geneva,
Switzerland
Control mimic : miRIDIAN
HCV Jc1 Primers KK30 and KM3 (7).
HCV core TCCTAAACCTCAAAGAAA
Hs_SNORD61 Hs_SNORD61_11 miScript Primer Assay (MS00033705, Qiagen AG, Hombrechtikon, Switzerland)
Other reagents:
Name Provider Catalogue number
Jetprime® Polyplus, Berkeley, CA, USA 114-01 Interferin® Polyplus, Berkeley, CA, USA 409-10 Lipofectamine 2000 Invitrogen AG, Basel Switzerland 11668-027
ECL reagent Amersham, Switzerland RPN2135
Dual-Luciferase Reporter Kit Promega, Dübendorf, Switzerland E1910 BODIPY™ 493/503
(4,4-
Difluoro-1,3,5,7,8- Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene)
Invitrogen AG, Basel Switzerland D3922
Oil-Red O Sigma-Aldrich Chemie GmbH, Buchs, Switzerland
75087
Nucleospin RNA II Macherey-Nagel AG, Oensingen, Switzerland
740.955.50
QIAamp Viral RNA Mini Kit Qiagen AG, Hombrechtikon, Switzerland
miRNeasy Micro Kit Qiagen AG, Hombrechtikon,
Switzerland 217084
miScript II RT Kit Qiagen AG, Hombrechtikon,
Switzerland 218161
miScript SYBR Green PCR kit
Qiagen AG, Hombrechtikon,
Switzerland 218073
triglyceride kit Roche Diagnostics AG, Rotkreuz,
Swizerland 12016648-122
Cholesterol/cholesteryl ester
quantitation kit Calbiochem, San Diego, CA, USA 428901-1
AAV8 production and infection
Adeno-associated virus serotype 8 (AAV8) carrying either the HCV-3a core or GFP genes under the control of albumin promoter (pAAV-ALB-core3a-IRES-GFP and pAAV-ALB-GFP) were produced by Vector Biolabs (Malvern, PA, USA) using the plasmid pIRES-EGFP-core 3a as template (1). Mice were injected retro-orbitally with 5x1011 genome copies and sacrificed 10 days later for histopathological analyses of liver tissues.
Lentivirus production
The lentiviral particles were produced and collected as reported (8). Viral titers were estimated by real-time PCR (9). Cells transduced with a GFP-encoding lentivector were used as controls.
Cell transduction and transfection
Cells were seeded at 1.5x104 cells/well in a 24-well plate and cultured 6 hours before lentiviral transduction. For transfection, cells were plated at 3.5x104 cells/well in a 24-well plate and
Interferin® (Polyplus) 6 hours prior either lentiviral transduction or HCVcc infection. Plasmid transfection was performed using JetPrime® (Polyplus), according to the manufacturer protocol.
RNA isolation, reverse transcription and real-time PCR
Total intracellular RNA was extracted using the Nucleospin RNA II Kit (Macherey-Nagel AG, Oensingen, Switzerland). Extracellular RNA level of HCV particles was extracted with a QIAamp Viral RNA Mini Kit (Qiagen AG, Hombrechtikon, Switzerland). Relative quantification of RNA transcripts was then performed by real-time RT-PCR as described (10).
For microRNA measurement, RNA extraction, reverse transcription and real-time PCR were performed using miRNA-dedicated kits (miRNeasy Micro, miScript II RT, miScript SYBR Green PCR, Qiagen AG), according to the manufacturer instructions.
miR16/Sno234/SNORD61 were used to normalize miR-21 expression.
Immunoblot analyses
Cells were lysed in ice-cold RIPA containing 2 mM sodium orthovanadate,10 mM sodium fluoride, 2 mM EDTA and protease inhibitors. Equal amounts of proteins were separated by 10% SDS-PAGE, blotted to nitrocellulose membranes and proteins of interest blotted with specific antibodies (see above table) were revealed with ECL. Quantifications were performed using the ImageJ software.
In vitro transcription
Chimeric JFH1-J6 (Jc1) full length RNA was generated from pFK-J6/C3 (Jc1) (gift from R.
Bartenschlager, Heidelberg, Germany; (4)), by using the T7 Ribo Max Express Large scale RNA Production System (Promega, Dübendorf, Switzerland). In vitro transcripts were purified using the Nucleospin RNAII kit (Macherey Nagel), and their quality verified using the Agilent 2100 bioanalyzer.
HCV particles production, titration
HCV infectious particles were produced by electroporating Huh-7.5 cells (7.5 x 106) with 5 µg of HCV RNA transcribed from pFK-J6/C3 (Jc1) using Amaxa cell line nucleofector kit T (260 V, 950 µF; Lonza, Basel, Swizerland).. Particles were collected in culture supernatant 48 hours after transfection, filtered through 0.45 µm pore-sized PVDF membrane and titered by infecting Huh-7.5 cells with serial dilutions. Cells were fixed after 72 hours with -20°C methanol and immunostained using an anti-HCV-core (C7-50) antibody. TCID50/ml was calculated as reported (4). Huh-7 cells were infected with 2-5 MOI for 48-72 hours. Specific infectivity of
HCV particles produced by Huh-7 cells was calculated as ratio of infectivity titer (TCID50/ml) to viral load (extracellular RNA).
Cells/tissues Immunochemistry and lipid staining
Paraformaldehyde-fixed cells were incubated with anti-HCV core antibody for 30 minutes at RT, and subsequently with Alexa488-conjugated anti-mouse antibody and DAPI for nuclear staining, for 30 minutes at RT. Neutral lipids were stained with Oil-Red-O (ORO).
For mouse tissue, fresh livers were embedded and frozen in OCT using liquid nitrogen and 2-methyl-butane, and then stored at -80°C. Five µm-thick liver cryosections were fixed with 4%
of paraformaldehyde for 15 minutes at RT. Microwave-citrate antigen retrieval was performed for 10 minutes at 98°C, followed by blocking with AffiniPure Fab fragment goat anti-mouse IgG (H+L) and bovine serum. Liver cryosections were then incubated with anti-HCV core antibody for 30 minutes at RT, and subsequently with Alexa555-conjugated anti-mouse antibody and DAPI, for 30 minutes at RT. Neutral lipids were stained with Bodipy 493/503.
Images were acquired with a confocal LSM700 microscope (Carl Zeiss AG, Feldbach, Switzerland). The surface area of individual LD was calculated using the MetaMorph®
software (Molecular Devices, San Jose, CA, USA).
Meta-analysis
The NCBI Gene Expression Omnibus (GEO) and EMBL-EBI were searched for relevant with the search string “(microrna OR miRNA) AND (hepatitis C OR HCV)” on September 13th 2016. The primary outcome of interest was the difference in expression of miR-21-5p in hepatitis C positive human samples versus uninfected controls. A study was considered eligible if it performed a whole genome miRNA assessment in hepatitis C positive samples and uninfected controls, if miR-21-5p was measured and if the study was performed in human liver or serum/plasma samples. The full text of potential articles was retrieved and assessed for inclusion, any disagreements were resolved by the senior author FN.
We retrieved the individual studies in GEO or Array-Express. As multiple array platforms were used within the difference studies we used the data pre-processed as deposited in GEO or Array-Express, we collapsed all probes to the corresponding gene and scaled the observations of each gene to the z-score. To account for the difference in scales, we performed meta-analysis on the standardized mean difference using random effects meta-analysis due to expected heterogeneity of measures. Meta-analysis was performed using the metafor R package, and all statistical analyses were performed using the R statistical language.