USP3, A Target Of MiR-26a, Mediated The Difference Between Liver Normal And Tumor Cell Proliferation

Objective:To study USP3as a target of miR-26a mediating the difference between livernormal and tumor cell proliferation and elucidate the molecular mechanism of miR-26a/USP3suppressing liver tumor cell, finally provide experimental basis for finding USP3as a new target for liver cancer treatment.Methods:1. Real-time PCR to detect expression of miR-26a and USP3in mouse normal livercells and liver tumor cells, and further examine the expression level of miR-26a andUSP3in17paired liver cancer tissues and adjacent non-tumor tissues.2. Luciferase assay to identify USP3as a direct target of miR-26a.3. For detecting cell proliferation, BNL CL.2or Hepa1-6were transfected withmiR-26a mimics or LV-shUSP3and then tested with two different measurement methods:a colorimetric assay with CCK8and an isotope incorporation assay with H-thymidine(3H-TdR).4. Western blot for detecting USP3, PCNA, MDM2, Cdc25C, Chk1expression.Results:1. Expression level of miR-26a was down-regulated and USP3was up-regulated inhepatocellular carcinoma cell lines and clinical specimens.2. We used TagetScan online http://www.targetscan. org/to predict the targets ofmiR-26a, we chose USP3for further analysis because of its complementary structure with miR-26a. To validate USP3as a direct target of miR-26a, we cloned the wildtypeand mutant3’UTR of USP3into a vector fused to a luciferase reporter gene and testedthe change of luciferase activity when this construct was co-transfected with the miR-26amimics into HEK293T cells. The results showed miR-26a could significantly suppressthe luciferase activity of wildtype USP3-3’UTR but no much affecting for mutant USP3-3’UTR.3. After transfecting BNL CL.2and Hepa1-6with miR-26a mimics and LV-shUSP3,we used a colorimetric assay with CCK8and an isotope incorporation assay withH-thymidine (3H-TdR) to detect cell proliferation, and it turned out miR-26a/USP3couldsuppress liver tumor cell growth more significantly than normal liver cells.4. Western blot analysis indicated that the level of USP3and MDM2protein weredown-regulated, PCNA was up-regulated and Cdc25C, Chk1did not change significantlyafter transfecting BNL CL.2with miR-26a mimics and LV-shUSP3compared totransfecting with control; in Hepa1-6cells, level of USP3and MDM2protein were stilldown-regulated, PCNA and Cdc25C, Chk1had no significant change.Conclusion:1. USP3is a direct target of miR-26a.2. Overexpression of miR-26a can suppress liver cancer cell proliferation via USP33. PCNA plays an important role in miR-26a/USP3inhibiting liver cancer cellproliferation...