Research On Transcription Regulation Induced By Histone Deacetylation Inhibitor Trichostatin A And Cytotoxicity Treated With Anti-sense HDAC1 Upon MCF-7 Cells

It was well known that histone acetylation and deacetylation were the main post-translation modification in cells, they played significant role in gene transcription regulation. Much evidence recently showed that the outbreaks of the balance correlated much more with the formation of tumors, and would be a direct factor contributing to tumorgenesis. In our experiments, we chose histone deacetylation inhibitor trichostatin A and anti-sense HDAC1 as the treatments, inhibited the histone deacetylase activity or down regulated the histone deacetylase gene expression, observed the subsequent cytotoxicity upon the acetylation modification, and further investigated the possible molecular basis.In our observation, we found the proliferation activity of MCF-7 cells decreased after treatment of TSA, as time and dose increased, the inhibition was more manifest, showing a time and dose dependence. Annexin-V/PI double stain analysis showed apoptosis were identified treated with TSA, apoptosis rate increased as the dose rose for 48h. Cell cycle analysis revealed compared to the blank counterpart, MCF-7 cells were arrested in G2 phase at 48h and 72h after treatment of 0.50μmol/L TSA, but no apoptosis identified; semi-quantitate analysis further revealed there was no appearance of 180-200bp DNA ladder, confirming TSA caused a non-late apoptosis; Gel analysis showed ERα, myc-c, cyclin-D and Bcl-2 were all down regulated except P21 gene, all made it to proliferation inhibition, cell arrest and apoptosis, indicating TSA effectively regulated gene transcription, the cytotoxicity may correlate with transcription regulation.The HDAC enzymes could be inhibited by TSA, but would it be done in RNA level? We selected HDAC1 (ID for NM₀04964) as our target gene. After RNA drawn and reverse transcription PCR, gel analysis showed target gene was successfully amplified, exhibiting a clear strap in the electrophoresis, and was the aimed length in contrast to Marks, we took it to construct our plasmid. The plasmid identification displayed the target gene was successfully constructed, electrophoresis showed one was targeted 731bp strap, the other was the pcDNA3.1 vector. Next we amplified it massively in bacteria, and purified them for the subsequent cell transfection. Later transient transfection demonstrated HDAC1 mRNA lever was decreased to 43% in the anti-sense group compaired to negative counterpart for 72h, gel electrophoresis revealed the intensity of HDAC1 gene was apparently weak, suggetting HDAC1 was effectively down regulated. Additionally, we found HDAC1 was also depressed in the positive control of TSA group. Subsequent proliferation activity assay showed anti-sense HDAC1 could decrease activity of MCF-7 cells, inhibit their proliferation, while the blank and negative groups exhibited no markable difference. Further cycle arrest analysis also displayed anti-sense HDAC1 could arrest cells in G1 and G2 phase, indicating HDAC1 gene was an effective histone deacetylase, may play significant role in acetylation modification during the tumorigenesis of MCF-7 cells.Summarize the above experiments, we concluded HDAC were associated with tumorgenesis, they could be inhibited in drugs or RNA levers, resulted gene transcription regulation and subsequent cytocoxicity, suggesting a potential target for the tumor therapy in future.