miR‐26b‐5p helps in EpCAM+cancer stem cells maintenance via HSC71/HSPA8 and augments malignant features in HCC

Background Targeting cancer stem cells (CSCs) in hepatocellular carcinoma (HCC) is difficult because of their similarities with normal stem cells (NSCs). EpCAM can identify CSCs from EpCAM+AFP+HCC cases, but is also expressed on NSCs. We aimed to distinguish the two using integrated protein, mRNA and miRNA profiling. Methods iTRAQ based protein profiling and Next Generation Sequencing (NGS) was performed on EpCAM+/EpCAM- cells isolated from HCC (Ep+CSC, Ep- HCC) and EpCAM+ cells from non-cancerous/non-cirrhotic control liver tissues (Ep+NSC). Validations were done using qRT-PCR, flowcytometry and western blotting followed by in vitro and in vivo functional studies. Results 11 proteins were overexpressed (>3 fold) in Ep+CSCs compared to Ep- HCC and Ep+NSC cells. However, RNA-sequencing confirmed the Ep+CSC specific up-regulation of only HSPA8, HNRNPC, MPST and GAPDH mRNAs among these. Database search combined with miRNA profiling revealed Ep+ CSC specific down-regulation of 29 miRNAs targeting these four genes. Of these, only miR-26b-5p was found to target both HSPA8 and EpCAM. Validation of HSPA8 overexpression and miR-26b-5p down-regulation followed by linear regression analysis established a negative correlation between the two. Functional studies demonstrated that reduced miR-26b-5p expression increased the spheroid formation, migration, invasion and tumourigenicity of Ep+ CSCs. Furthermore, anti-miR-26b-5p increased the number of Ep+ CSCs with a concomitant overexpression of stemness genes and reduction of proapoptotic protein BBC3, which is a known substrate of HSPA8. Conclusion miR-26b-5p imparts metastatic properties and helps in maintenance of Ep+ CSCs via HSPA8. Thus, miR-26b-5p and HSPA8 could serve as molecular targets for selectively eliminating the Ep+ CSC population in human HCCs.