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

AbstractDiverse 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 (log2fold change ≤-1.5, Padj≤0.05).In-silicopathway 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.