miR-193a sensitizes hepatocellular carcinoma cells to sorafenib and impairs their aggressive properties.

HCC (hepatocellular carcinoma) is a challenging malignancy of global importance, it is the sixth most common cancer and the third most common cause of cancer-related mortality worldwide. Its onset is related to hepatitis B and C virus infections, cirrhosis, alcohol, and exposure to aflatoxin. Until now, the best treatment is surgery, but the long-term prognosis after resection of HCC remains unsatisfactory as a result of a high incidence of recurrence. In the last years the multikinase inhibitor Sorafenib has been used for clinical application for advanced HCC patients. Nevertheless, efforts are in the directions to identify early markers for the detection of HCC at the initial stages of carcinogenesis and to study novel molecular therapeutic strategies also in combination with sorafenib. microRNAs are short non coding RNAs able to negatively regulate gene expression in several physiological and pathological conditions. In human cancers they can function as tumor suppressor and oncogenes (onco-miRs, TS miRs) and their dysregulation can play a relevant role in carcinogenesis. Increasing evidence describe miRs as good tools for the molecular targeted therapies. In HCC cells we validated the miR- 23b as negative regulator of both uPA and c-met (uPA: urokinase type plasminogen activator, c-met: a TKR) that have been previously shown as negative prognostic markers for HCC and responsive therapeutic targets 2-4. Here we predicted among others the miR-193a as putative negative regulator of uPA using bioinformatic techniques. Now we have experimentally validated the miR-193a as negative regulator of uPA in 2 HCC-derived cell lines (HA22T/VGH and SKHep1C3) and in the breast cancer cell line MDA-MB-231. In the HA22T/VGH cell line the ectopic expression of miR-193a and the transfection of anti-miR-193a have led to cellular proliferation inhibition and induction respectively. To start the study of the functional role of miR-193a, we have first evaluated by stem-loop Real Time PCR the expression levels of miR-193a in 39 HCC and peritumoral (PT) tissues from 39 HCC patients. We have found miR-193a down-regulation in HCC respect to PT tissues (R=0.59; p<0.05), more in the cirrhotic HCCs than in non-cirrhotic ones. Further, in the class of cirrhotic HCCs stratified on the basis of hepatitis virus infections, we have interestingly found that the class HCV displayed miR-193a down-regulation (R=0.338, p<0.01) while the other 3 classes (HBV, HBV/HCV, -/-) did not show expression level variation between PT and HCC tissues (R=1.29, R=0.64 and R=0.77 respectively). To study novel approaches for multi-target and multi-agent therapies of the HCC, we have co-treated the cells with miR-23b and miR-193a in combination with Sorafenib and we have evaluated the effects on cellular proliferation and apoptosis. The 4 HCC cell lines analyzed have shown different responses to sorafenib treatments. Interestingly, the cell lines displayed an inverse trend between the percentage of the proliferation inhibition and c-met gene copy number. The transfection of miR-23b and miR-193a sensitized respectively the SKHep1C3 and HA22T/VGH cells to the Sorafenib treatment in terms of proliferation inhibition (fold increase: 1.3 ÷ 1.8 in SKHep1C3 and 2.3 ÷ 2.8 in HA22T/VGH). The effects on apoptosis have been evaluated by TUNEL assay. The cells previously transfected with miR-23b (SKHep1C3) and miR-193a (HA22T/VGH) and then treated with the Sorafenib displayed synergic effects on apoptosis mainly at the lower concentration of Sorafenib (fold increase: about 2.6 for both miR-23b and miR-193a). In conclusion, our results present new advances in the posttranscriptionally miR-mediated mechanisms of uPA expression regulation, in the validation of miRs that regulate HCC negative prognostic markers (i.e. uPA) and provide in vitro evidence for new molecular therapeutic strategies for advanced HCC treatment.