Ethical clearance
The Ethics Committee of the Institute of Post-Graduate Medical Education & Research (IPGME&R), Kolkata and the National Centre for Cell Science, Pune had approved the study [Approval ID: Inst/IEC/2015/108; dated 07 July 2015] and [IAEC/2022/B-414] respectively. Written informed consent was obtained from all the participants or legal guardians.
Study Subjects and samples included in the study
Fifty-nine treatment naïve chronic hepatitis patients mono-infected with either HCV or HBV attending the hepatology Clinic of School of Digestive and Liver Diseases, IPGME&R, Kolkata and Indraprastha Apollo Hospital, New Delhi were included in the study and categorized as Chronic Hepatitis B or C (CHB or CHC) (n = 21), Liver Cirrhosis (LC) (n = 17) and HCC (n = 21). Patients co-infected with HEV/HAV/HIV, having co-morbidities like chronic alcoholism, diabetes mellitus or unwilling to enrol in the study were excluded. Normal liver biopsy tissue was obtained from Gall bladder carcinoma patients (n = 11) during cholecystectomy from IPGME&R as routine evaluation of liver metastasis and confirmed after assessment of histology. Both blood and liver tissues were collected for proper disease evaluation and further study.
Details of the study subjects, biochemical, and clinical data is presented in Supplementary Table S1.
Transcriptome profiling
Our small RNA transcriptome profile of liver tissue samples from HCV-HCC vs. normal individuals using Illumina platform has been deposited in the public domain as GSE140370 [18]. After analysis only downregulated miRNAs (log2fold change ≤-1.5, Padj≤0.05) were considered for this study. Public database GSE21362, GSE40744, GSE74618 were used for validation.
Total RNA isolation and cDNA preparation
About 500ng/2.5µg of RNA was used for cDNA synthesis of miRNA and mRNA using miScript PCR Starter Kit (Qiagen, #218193) or miRCURY LNA miRNA PCR Starter Kit (Qiagen, #339320) and RevertAid reverse transcriptase [ThermoFisher, #EP0441] respectively.
Quantitative Real Time PCR (qRT-PCR)
Both miRNA and mRNA were quantified using PowerUp™ SYBR™ Green PCR master mix (ThermoFisher,) and gene specific primers using QuantStudio 7 Flex RT-PCR machine (ThermoFisher). RNU6B/miR-103a-3p and 18s rRNA were used as an internal controls for miRNA and mRNA respectively. Log22−(Ct of gene−Ct of control)x103 was used as fold change in expression of genes.
Bioinformatics analysis
Targets of the miRNAs were analysed using TargetScan (http://www.targetscan.org/vert_80/)/miRDB(http://mirdb.org)/micro-T-CDS (http://diana.imis.athena-innovation.gr/DianaTools). miRNet (https://www.mirnet.ca/) tool was employed to visualize the overall network of miRNAs. Targets were validated in LIHC and subjected to pathway analysis using KEGG (https://www.genome.jp/kegg/pathway.html).
Lncbase V3.0-DIANA Tools (https://diana.e-ce.uth.gr/lncbasev3/home) was used to predict the common lncRNA that binds to multiple miRNAs having 8mer binding capacity with score ≥ 0.95.
Cell lines and plasmid information
Huh7/SNU449 cell lines were STR profiled and tested for Mycoplasma. Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, HiMedia, #AL111) with 10% FBS (GIBCO, #10082139) in 370C incubator. Lipofectamine 2000 (ThermoFisher) was used for transfection using manufacturer’s protocol.
Replication competent plasmids of HCV genotype2a, pS52/JFH1 and pSVNeo2/HBV Dimer were gifted by Jens Bukh, Copenhagen University Hospital, Denmark and Prof. Chiaho Shih, University of Texas Medical Branch, Galveston, USA respectively.
Pre-miRNA and 3’UTR sequences of the genes were cloned in pRNAU6.1RNA/Neo vector and psiCHECKTM−2 (Promega) vector respectively. The binding regions of the respective miRNA to the lncRNA were cloned into the pGEM-T Easy vector (Promega). pAgo2-Flag (Addgene) and pSpCas9(BB)-2A-Puro (PAX459) (Addgene) [referred as pCas9] were gifted by Edward Chan, and Feng Zhang respectively [19, 20]. All primers and oligonucleotides are listed in Supplementary Table S2.
3’UTR Luciferase Assay
Huh7 cells were co-transfected with the 3’UTR-Luciferase construct and control vector/pre-miRNA/pre-miRNA + anti-miRNA oligo independently. Luciferase assay was performed using Dual Luciferase Reporter assay kit (Promega) and Luciferase activity was normalized to the empty vector.
Immuno-blot analysis
Huh7 cells were transfected with the required plasmids. After 48h, cell extract was prepared using RIPA buffer. About 60–80µg of protein was subjected to polyacrylamide gel electrophoresis, transferred on a PVDF membrane and immuno-blotted with anti-ACVR2A/anti-BMPR1B/anti-CD44/anti-OCT4/anti-NANOG/GAPDH-HRP/anti-Histone-3 (Cell Signalling/Abclonal/Bio-Bharti). Anti-mouse/anti-rabbit-HRP-conjugated secondary antibodies (Santa Cruz) were used as required. Enhanced chemiluminescence (ECL) kit (ThermoFisher) was used to detect specific protein. Detailed antibody list is given Supplementary Table S3.
Wound healing assay
Huh7 cells were transfected with the required plasmids. After 24h of transfection, a thin scratch was created at the bottom of the plate and cell migration was monitored using inverted microscope at 0/24/48/72h. The data was analysed using Image J software.
Cell proliferation assay
Huh7 cells were seeded in a 24-well plate and transfected with the required plasmids. Cells were trypsinized and seeded in a 96-well plate in triplicates. 24h post transfection, tetrazolium salt WST-1 (SigmaAldrich) was added and quantified at 0/24/48/72h in microplate reader.
Spheroid formation assay
Huh7 cells were transfected with the desired plasmids. 48h post transfection, cells were allowed to grow on ultralow attachment plates (BD, USA) and cultured for 6 days in DMEM-F12 media (Himedia) supplemented with 2% B27 (ThermoFisher) and 20ng/ml of epidermal growth factor (ThermoFisher). The size and number of the tumour-spheres were documented.
Migration and invasion assay
Transfected Huh7 cells were transferred on the upper layer of the Boyden chamber and cell number was counted after staining with crystal violet from the lower part of the membrane at different time points where 30% FBS was used as chemo-attractant. The upper chamber was coated with matrigel for invasion assay.
Biotinylation of KCNQ1OT1 fragments
LncRNA clones were subjected to in vitro transcription in a single tube using MEGAscript™ T7 Transcription kit (ThermoFisher). RNA was purified by ethanol precipitation and subjected to biotinylation using the Pierce™ RNA 3’ End desthiobiotinylation kit. RNA was incubated with the extract of Huh7 cells co-transfected with miR-424-5p/miR-223-3p/miR-136-3p/miR-139-5p and ribo-complex was pulled down with streptavidin magnetic beads following manufacturer’s protocol (Pierce™ Magnetic RNA-Protein Pull-Down Kit). RNA was then eluted using Trizol and quantified by qRT-PCR.
RNA Immuno-precipitation (RIP) assay
The plasmid pAgo2-Flag was co-transfected with pCas9/pCas9-KCNQ1OT1 in Huh7 cells. Cells were harvested 48 h post transfection and equal amounts of protein was used for immuno-precipitation with Anti-Flag (Sigma)/Anti-IgG (ThermoFisher) antibody separately, overnight at 4°C. Next day, the RNA-protein complex was precipitated with protein A/G Agarose beads (Sigma) and the RNA was isolated using TRIzol. qRT-PCR was used to quantify RNA.
Mice Experiment
Mice experiments were performed with CRISPR-deleted-KCNQ1OT1-Huh7 cells and vector cells injected subcutaneously into the right dorsal flank of eight weeks old NOD/SCID mice (n = 4). The tumour volumes were measured after 4 weeks using the following formula: π/6[(d1*d2) 3/2], where d1 and d2 are two different diameters dimensions of a tumour.
Statistical analysis
Statistical analysis was performed in excel or in the GraphPad prism version 7. All the data were presented as mean ± standard deviation. Unpaired two-tailed Student’s t-test or Mann-Whitney test was done for the data with Gaussian and Skewed distribution respectively. Bonferroni correction (two-way ANOVA) was done for the grouped data analysis. pvalue ˂0.05 was considered as statistically significant.