HDAC 6 Suppresses Let‐7i‐5p to Elicit TSP 1/ CD 47‐mediated Anti‐tumorigenesis and Phagocytosis of Hepatocellular Carcinoma

: Histone deacetylase 6 (HDAC6) uniquely serves as a tumor suppressor in hepatocellular carcinogenesis, but the underlying mechanisms leading to tumor suppression are not fully understood. To identify comprehensive microRNAs (miRNAs) regulated by HDAC6 in hepatocellular carcinogenesis, differential miRNA expression analysis of HDAC6-transfected Hep3B cells was performed. Using integrative analyses of publicly available transcriptome data and miRNA target prediction, we selected five candidate miRNAs and, through in vitro functional validation, showed that let-7i-5p specifically suppressed thrombospondin-1 (TSP1) in hepatocellular carcinoma (HCC). Ectopic expression of antisense let-7i-5p (AS-let-7i-5p) inhibited in vitro tumorigenesis of HCC cells. In addition, treatments of partially purified TSP1 from culture cell media (ppTSP1) and recombinant TSP1 (rTSP1) exhibited similar effects with AS-let-7i-5p treatment on the same HCC cells, whereas TSP1 neutralizing antibody treatment significantly attenuated these effects. Notably, treatments of HDAC6 plasmid, AS-let-7i-5p, ppTSP1, and rTSP1 significantly suppressed in vitro angiogenesis and metastatic potential of HCC cells, but the co-treatment of TSP1 antibody specific to cluster of differentiation 47 (CD47) binding domain successfully blocked these effects in the same cells. Furthermore, we demonstrated that recovery of HDAC6 elicited let-7i-5p suppression to de-repress TSP1 expression; therefore, it occupied the CD47 receptor to block CD47-SIRPα-mediated anti-phagocytosis of macrophage in HCC. We also observed that HCC-derived exosomal let-7i-5p suppressed TSP1 of recipient hepatocyte cells. Treatments of HDAC6 plasmid, AS-let-7i-5p, and rTSP1 suppressed tumor incidence as well as tumor growth rates in a spontaneous mouse HCC model. Conclusion: Our findings suggest that the HDAC6-let-7i-5p-TSP1 regulatory pathway suppresses neoplastic and antiphagocytic behaviors of HCC by interacting with cell surface receptor CD47 in HCC and neighboring cells of tumor microenvironment, providing a therapeutic target for the treatment of liver malignancy and metastasis.