Novel feedback inhibition of the let-7 microRNA regulator and stem cell marker Lin 28 homolog A by tumorsuppressor miR-622 in hepatocellular carcinoma

Introduction:

Hallmarks of cancer development, progression and emergence of therapy resistance are associated with embryonic and/or stem cell characteristics. Lin 28 homolog A (LIN28A) is a proto-oncogene and RNA binding protein, which represses the let-7 family of miRNAs in embryonic stem cells. The microRNA (miR) let-7 family comprises the most prominent tumorsuppressive microRNAs. However, the exact role of the embryonic stem cell marker LIN28A is unclear in hepatocellular carcinoma (HCC) progression and acquired sorafenib-resistance. Moreover, it is unknown whether LIN28A is only an upstream-regulator of miRs, or if LIN28A itself could also be regulated by miRs.

Methods:

Primary human hepatocytes and several human HCC cell lines (PLC, Hep3B, HepG2 and Huh 7) as well as sorafenib-resistant HCC cells were used for expression and functional analysis. In vivo expression analysis was performed using paired tumor and corresponding non-tumor liver tissues from HCC patients. Gene expression and cellular localization was analyzed qRT-PCR, Western blot, and Fluorescence microscopy. A microRNA mimic/inhibitor for miR-622 was used for functional analysis. For exploration of stem cell properties, clonogenicity assays were performed.

Results:

LIN28A expression was increased in HCC cell lines as compared to primary human hepatocytes. Tissue micro array analysis revealed enhanced LIN28A protein expression in HCC in vivo and correlated with advanced tumor stages. Fluorescence-based microscopic analysis showed that LIN28A can both localize to the cytoplasm and the nucleus in HCC cells and tissues. Furthermore, LIN28A levels were higher in sorafenib-resistant HCC cell lines as compared to non-resistant cells. Clonogenicity assays revealed that LIN28A induces stem cell properties in HCC cells. Multi-step based in silico and experimental analysis revealed a novel feedback regulation of LIN28A expression in HCC mediated by the tumorsuppressive microRNA-622.

Conclusions:

We found that the embryonic stem cell marker LIN28A is overexpressed in HCC in vitro and in vivo and correlated with tumor stages. LIN28A levels were also enhanced in sorafenib-resistance and LIN28A was sufficient to induce stem cell properties. Further, we detected a novel feedback inhibition of LIN28A expression which was mediated by the tumorsuppressive microRNA-622 in hepatocellular carcinoma.