Sponging of five tumour suppressor miRNAs by lncRNA-KCNQ1OT1 activates BMPR1A/BMPR1B-ACVR2A/ACVR2B signalling and promotes chemoresistance to hepatocellular carcinoma

Diverse mechanisms have been established to understand chemoresistance of hepatocellular carcinoma (HCC), but the contribution of non-coding RNAs are not surveyed well. We aimed here to explore the lncRNA/miRNA axis in hepatitis C and B virus infected HCC to investigate the mechanism of chemoresistance and to classify a potential therapeutic target for HCC. The small RNA transcriptome, and qRT-PCR validation with the liver tissues of both HCV and HBV infected HCC patients revealed that miR-424-5p/miR-136-3p/miR-139-5p/miR-223-3p/miR-375-3p were the most downregulated five miRNAs in HCC compared to normal (log 2 fold change ≤-1.5, P adj ≤0.05). In-silico pathway analysis with the validated targets of each miRNA depicted that the signalling pathways regulating pluripotency of stem cells commonly targeted by the all five miRNAs. Subsequent validation by 3’UTR-luciferase assays and western blot analysis disclosed that these five miRNAs impeded either same or diverse genes, but all from BMP signaling pathways including BMPR1A/BMPR1B by miR-139-5p, miR-136-5p & miR-375-3p and ACVR2A/ACVR2B by miR424-5p & miR223-3p. Furthermore, restoration of each miRNA in Huh7/SNU449 cells inhibited phosphorylation of downstream SMAD1/5 and ERK1/2, and attenuated EMT/stemness/sphere formation/chemoresistance/invasion/migration of cells. To investigate the mechanism of suppression of these miRNAs, “DIANA” prediction tool was employed and lncRNA-KCNQ1OT1 was retrieved as interacting partner of all five miRNAs. Ago2-RNA-immuno-precipitation/in vitro RNA pull-down assays revealed that lncRNA-KCNQ1OT1 physically interacted and sequestered all the five miRNAs in the cytoplasm. Hence, KCNQ1OT1 was deleted in Huh7/SNU449 cells using CRISPR-technology and observed regression of oncogenic properties with enhanced chemosensitivity and reduced metastasis of cells. Shrinkage of tumor size/volume in NOD-SCID mice injected with KCNQ1OT1-K/O cells further strengthened our observations. Thus, lncRNA-KCNQ1OT1 is the main regulator, which reduces the level of beneficiary miRNAs in the tumor milieu and modulates BMP signaling to promote chemoresistance to HCC suggesting lncRNA-KCNQ1OT1 might have robust potential to be a therapeutic target for HCC.