Antiproliferative Effect Induced By Cyclin-dependent Kinase 7 Antisense On Human Hepatoblastoma Hep G2 Cells In Vitro

Objective: Cdk7, a key regulator in cell cycle control and RNA transcription, is overexpressed in neoplastic tissues, and low molecule weight non-target-specific Cdks inhibitors which inhibit the Cdks activity ubiquitously had shown a promising anticancer effect in preclinical and clinical trials but with a dissatisfied adverse reaction profile. Owing to all characteristics mentioned above, we hypothesized that Cdk7 was a potential target for anticancer therapeutics discovery and development. In order to validate Cdk7 as the target for anticancer agents development, we evaluated the antiproliferative effect induced by Cdk7 silencing with antisense strategy, a specific target gene knock-out tool, and tried to find the lead for further anticancer therapeutics development.Methods: Secondary structures of homo sapiens Cdk7 mRNA were predicted by RNAstructure 3.7 software. And the antisense fragments were selected according to the partial single-strand structures such as bulges, hairpins, knots and internal loops. Fragment which should form secondary structure in itself and which be homogeneous to other important genes were out of consideration. After that, the antiproliferative effect of each fragment selected was determined by human hepatoblastoma HepG2 cells in vitro. The antiproliferative effect of the structural-modified fragments based on the most powerful one selected in the first round of screening were evaluated with the HepG2 cell culture again, then the fragment that had shown the highest potencyin antiproliferation test was considered to be the hit.DNA fragmentation was analyzed by agar electrophoresis to see if the DNA ladder was formed, infrastructure of the cells which had been transfected with ASODN was observed under the transmission electron microscope to see if the characteristics of apoptosis appeared. Indexes of apoptotic cell and cell cycle distribution in cells treated with ASODN were determined by flow cytometry. Changes of Cdk7 mRNA levels were assayed by reverse transcriptase polymerase chain reaction and changes of Cdk7 protein levels were assayed by Western blotting respectively. Phosphorylation levels of retinoblastoma and Cdk2 protein in ASODN treated cells were analyzed by Western blotting.Results: 21 fragments were selected according to the Cdk7 mRNA structures predicted by RNAstructure software. Antiproliferative activity for each fragment was evaluated with HepG2 cell culture by MTT method after the fragments those might to form the secondary structure and fragments those had shown homogeneous to other important genes had been eliminated. The results showed that antiproliferative effect of the fragment complementary to 284-303 region was higher than that of the others. Then 10 fragments complementary to the up or down stream 1~5 bases to the complementary region were picked and antiproliferative potency for each was measured with HepG2 cell culture. The results showed that the fragment complementary to 287-306 region was capable of inhibiting cell growth by 68.3 ?2.6%, and the IC50 was l/L.In the DNA fragmentation formation test, the DNA extracted from-9-cells which had been treated with 200, 400nmol/L ASODN for 72 hours showed an obvious characteristic of DNA fragmentation and formed a bright ladder with an about 200 base pairs intervals in the electrophoresis gel. And for the cell treated with constant 200nmol/L ASODN, DNA fragmentation didn't form until the cells had been treated for 36 hours, and the fragmentation was obvious at the 48h. The photographes taken by transmission electron microscope showed that the cells treated with 100, 200, 400 nmol/L ASODN were induced to programmed cell death according to the changes of the ultrastructures. The typical changes in the apoptotic cells were chromatin condensation in the nucleolus, electronic density increasing, blebbing in the cytoplasm and particle with lipid membrane forming and distracted from the cell. In the worst condition, the cell separated into a group of granule without any characteristics of a cell. The apo...