Cell Culture
Primary human pancreatic stellate cells (hPSCs) (3830, ScienCell Research Laboratories Carlsbad, California) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, 11965092, Life Technologies, Carlsbad, CA) supplemented with 10% FBS in a humidified 5% CO2 incubator at 37°C. Cells used were less than passage 9.
Transfection
To overexpress miR-29a, hPSC cells were seeded at 1 X 105 cells/well in 6 well-plates for 24 hrs and transfected with control (CN-001000-01, GE Dharmacon, Lafeyette, CO) or miR-29a mimic (C-300504-07, GE Dharmacon, Lafeyette, CO) using Dharmafect®1 Reagent (T-2001-01, GE Dharmacon, Lafeyette, CO) following manufacturer’s instructions. Total protein or RNA was isolated 48 hrs post-transfection for western blot or qPCR analyses respectively.
RNA Extraction
Total RNA from cultured cells were extracted using the RNeasy plus Mini kit (74134, Qiagen, Venlo, Netherlands) following manufacturer’s protocol. The concentration and purity of the extracted RNAs were measured using a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, Carlsbad, CA).
RNAseq
For RNAseq, the quality and integrity of the extracted RNA were evaluated by a Bioanalyzer 2100 (Agilent technologies, CA). Samples with RNA Integrity Number (RIN) >7.0 were used in RNAseq. cDNA libraries were prepared using the TruSeq RNA library kit (Illumina Inc., San Diego, CA). The libraries were amplified and then sequenced on an Illumina Hiseq.2000 instrument (San Diego, CA) with 100bp paired end reads per sample. The quality of the sequence data was analyzed using FastQC [22]. The reads were mapped to the human genome (hg38) using STAR (v.2.5) [23]. Uniquely mapped sequencing reads were assigned to genes based on Gencode 25 using featureCounts (v1.6.2) [24]. Genes with read count per million (CPM)<0.5 in two or more samples were filtered out and gene expression profiles were normalized using trimmed mean of M values (TMM) method. Differentially expressed genes (DEGs) were assessed by cutoff p-value of less than 0.05 after false discovery rate (FDR) adjustment and amplitude of fold change (FC) of gene expression greater than 2 linear FC.
Target Prediction, Functional Enrichment and Network Analysis
Conserved miR-29a target genes were obtained using TargetScan (v7.1). The hypergeometric model was adopted to identify the overlap between DEGs and miR-29a predicted targets.
Functional enrichment analysis of the gene ontology (GO) terms and KEGG pathway analysis to investigate the biological functions and pathways were performed using the R package. The protein-protein interaction networks of the genes was explored using the STRING (http://www.string-db.org/) database.
Quantitative Real time PCR (qRT-PCR)
RNA was reverse transcribed to cDNA using High capacity cDNA Reverse Transcription kit (4368814, Thermo Fisher Scientific, Carlsbad, CA) with random primers for genes or custom primer pool for miRNA (Thermo Fisher Scientific, Carlsbad, CA). To measure mature miR-29a expressions, TaqMan qRT-PCR reactions were set up using TaqMan Fast Advanced Mastermix (4444557, Applied Biosystems Foster City, CA) with TaqMan probe and primers for mature miR29a (002112, Applied Biosystems, Foster City, CA) or U6 snRNA (001973, Applied Biosystems, Foster City, CA). To assay the mRNA levels of genes, qRT-PCR was performed with PowerUp SYBR Green Mastermix (A25742, Applied Biosystems, Foster City, CA) and custom primers Table S1). miRNA and mRNA qRT-PCR were normalized to U6 and ACTB respectively. Samples were run in triplicates in a 10µl final volume using ABI 7500 Real-Time PCR machine with standard settings. Relative expressions were analyzed using ΔΔCT method.
Western Blot
Protein lysates were prepared with RIPA Buffer (PI-89900, Thermo Fisher Scientific, Carlsbad, CA) and quantified using BCA Protein Assay Kit (23225, Pierce Biotechnology, Waltham, CA). Equal amounts of total protein were loaded onto NuPAGE 4-12% Bis-Tris Gels (NP0323, Invitrogen, Carlsbad, CA). After electrophoresis, the gels were electrotransferred onto polyvinylidene fluoride membranes, blocked with 5% dry non-fat milk and incubated overnight at 4°C with specific primary antibodies. The membranes were washed and then probed with corresponding HRP conjugated goat anti-mouse (31430, Thermo Fisher Scientific, Carlsbad, CA) or goat anti-rabbit (31460, Thermo Fisher Scientific, Carlsbad, CA) antibodies at 1:5000 dilution. To develop the blots, ECL detection kit (34096, Thermo Fisher Scientific, Carlsbad, CA) was utilized and the images were captured on an Amersham Imager 600 (GE Healthcare, Chicago, IL). Densitometry analysis was performed using Image J software to quantify each protein band, which were then normalized against loading control GAPDH. The primary antibodies used in this study were anti-IGF-1 (ab9572, Abcam, Cambridge, MA), anti-COL5A3 (PA5-77257, Thermo Fisher Scientific, Carlsbad, CA), anti-CLDN1 (4933S, Cell Signaling Technology, Danvers, MA), anti-E2F7 (ab56022, Abcam, Cambridge, MA), anti-MYBL2 (PA546845, Thermo Fisher Scientific, Carlsbad, CA), anti-ITGA6 (3750, Cell Signaling Technology, Danvers, MA), anti-ADAMTS2 (3485, Cell Signaling Technology, Danvers, MA), and anti-GAPDH (MA5-15738, Thermo Fisher Scientific, Carlsbad, CA).
Statistical Analysis
All data were expressed as mean ± standard error of the mean (SEM) of three independent experiments. Statistical analysis was performed by ANOVA or Student’s t test. Statistical significance is indicated as *p< 0.05 or **p<0.01 or ***p<0.001.