| Summary: | 碩士 === 國立清華大學 === 生物科技研究所 === 97 === Noxa encodes a BH3 domain-only protein of the Bcl-2 family. Ectopically expressed Noxa affects apoptosis in various cells, such as neural and myloma cells. Noxa in human gastric adenocarcinoma AGS cells was not yet fully studied. Besides, the exact molecular mechanisms of Noxa (p53-dependent or p53-independent) activating apoptosis exhibit diversity from cell types and stresses. Thus, the frame of different Noxa expression background of AGS cells was conducted for further molecular mechanism studies on Noxa in this study.
Five pLKO.1 plasmids (No. 1~5), each inserted a special designed 21-nucleotides fragment of Noxa-siRNA gene, and one pLKO.1-Luc plasmid containing Noxa-siRNA mismatch sequences were prepared from corresponding E. coli DH5α strains which were originally purchased from National RNAi Core Facility (NRC) in Academia Sinica. Two marker genes, functioned to against antibiotics, Amp and puromycin, are also located on these lentivirus-based plasmids. Corresponding Noxa siRNA-targeting sequences No. 2~ 5 were confirmed by sequencing. Plasmids (No. 3 and No. 4) containing two different target sequences near the ORF regions of the Noxa gene and plasmid pLKO.1-luc were transfected to AGS cells, respectively (Fig. 1 and 2), using the puromycin selection. In addition, the p53 siRNA containing AGS cells were established by means of transfecting p53 siRNA inserted pLKO-AS1 transfection successfully (Fig. 9-10). Seventy percent reduction of p53 expression in p53 siRNA transfected AGS cells was found in comparison with the mock cells (Fig. 11). Therefore, the different Noxa expression affected by SA-, X irradiated-, or cisplatin-induced apoptosis in P53 knockdown AGS cells now are ready for further investigation.
The cytotoxic effects on cell survival and apoptosis in p53 wild type AGS cells with different Noxa statuses after treatment with IR, sodium arsenite (SA) or cisplatin were examined. Noxa siRNA transfection has no significant influence on the survival rate of X-irradiated AGS cells. However, a significant decrease on apoptosis was detected in Noxa knockdown AGS cells after 5 Gy X-irradiation (Fig. 6). Also, the polyploidy population exhibited obviously only in mock AGS cells after X-irradiation. The reduction of Noxa expression was investigated to decrease the X-ray induced apoptosis in AGS cells. After treating with 3 or 5 μM SA for 24 hours, the AGS cells with Noxa siRNA had about 20 % increase in survival rate and 10 % decreases in apoptosis in comparison with mock AGS cells (Fig. 7). Noxa was found to modulate SA-induced apoptosis in AGS cells. On the other hand, the effects of cisplatin were opposite to those of SA on AGS cells with different Noxa statuses. Noxa siRNA transfected AGS cells had 10 % lesser in survival rate and 10 % more in apoptosis after 2 or 4 μM cisplatin treatments for 24 hours in comparison with mock AGS cells (Fig. 8). It is possible that the reduction of Noxa expression affected the interaction between the members in Bcl-2 family and promoted apoptosis after cisplatin treatment in Noxa knockdown AGS cells.
The p53 knockdown AGS cells had been constructed in this study for examing the effects of both P53 and Noxa proteins on cell apoptosis and survival. Noxa protein has not been confirmed to be a biomarker for detecting cancer. Also, mutations or deletions of Noxa are not always detected in cancer cells. Therefore, my data using siRNA methods in this study supported the possible clinical usage for wild type Noxa gene therapy for Human Gastric Adenocarcinoma (AGS) patients.
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