The Inhibition Effect Of Highly Expressed Gene P02 Antisense Oligonucleotide On Hepatocellular Carcinoma In Vitro

Hepatocellular carcinoma (HCC) is one of the most common malignancies in China. At present, there are many limitations in investigating its mechanism which it happens, diagnosis and therapy. In order to investigate the mechanism and develop the way for early diagnosis and gene therapy, the Institute of Digestive Disease constructed complement DNA (cDNA) library and got 93 clones by using suppression subtractive hybridization (SSH) technology. By using Northern hybridization and RT-PCR technique, these clones were further screened and analyzed and the EST P02 of differential expression gene was found highly expressed in HCC. P02 is homogenized with translationally controlled tumor protein (TCTP) by homological analysis in the Genbank database, but the funtion of P02 is unknown. Antisense technique, a kind of advanced biological technology, is a kind of effective means to investigate gene function by designing blocking gene fragment to inhibit specific gene replication, transcription or translation. Antisense technique includes antisense oligonucleotide(AS-ODN), antisense RNA and ribozyme among which the former is noticed greatly because of its wide application. AS-ODN refers to synthesized short-fragment nucleotide which hybridize with specific target sequence to inhibit gene expression according to base pair rules. AS-ODN is widely used to investigate gene function because of its being synthesized conveniently, designed simply, modified easily, and its high selectivity and high affinity.AIM To investigate the gene funtion and find new target for HCC gene therapy by observing the changes of cell biological behavior after AS-ODN targeting to P02 mRNA initiation codon site was introduced into HCC cell line SMMC-7721 with lipofectin.Methods The experiment was divided into four groups: AS-ODN group, S-ODN group, lipofectin group and empty control group. 1.To introduce AS-ODN into SMMC-7721 cell by using lipofectin transfection technique, then detect absorbance value of 24,48,72,96h after transfection and calculate cell growth inhibition rate. 2. To collect the cells of 72h after transfection and fix the cells with 70% ethanol, then detect the cell cycle with the flow cytometer. 3. To collect the cells of 72h after transfection and extract the total RNA, then detect mRNA expression using reverse transcriptase PCR.Results 1.The results showed AS-ODN obviously inhibited the growhth of HCC cell SMMC-7721. The cell growth inhibition rate of 24, 48, 72, 96h was 53%, 56%, 85% and 78% respectively. However, S-ODN and lipofectin cann' t inhibit cell growth.2. The results of cell cycle showed G0/G1 phase cells increased, however, S phase and M phase cells decreased, after adding AS-ODN to SMMC-7721, compared to S-ODN group, lipofectin group, empty control group.3. RT-PCR showed P02 specific amplification band decreased obviously after adding AS-ODN to SMMC-7721, compared to S-ODN group, lipofectin group and empty control group.Conclusion 1. P02 probably plays an important role in the genesis and development of HCC and may be a new target for HCC gene therapy2. AS-ODN technique is a new kind of means for gene therapy of HCC3. Computer-assisted design for AS-ODN is important means...