CAJ | 학술논문

目的：研究载脂蛋白B mRNA编辑酶催化多肽样3（APOBEC3s）脱氨酶对HPV（＋）人类永生化上皮细胞（W12）基因组稳定性的影响。方法利用 W12细胞为模型，通过基因转染APOBEC3A（A3A）、APOBEC3G（A3G）和GFP表达质粒，获得转染后24 h和48 h的两组样品。采用Western blot方法检测细胞DNA损伤反应（DDR）通路中经典蛋白激酶的表达情况。此外，利用绿色荧光蛋白（GFP）标记的方式，观察A3A和A3G的细胞定位。结果Western blot结果显示， A3A过表达W12细胞样品中，经典DNA双链断裂的标记物γH2AX表达明显上调。DDR通路中ATM和Chk2激酶的磷酸化水平有所提高。此外，同源重组（HR）修复中的细胞因子pNBs1的表达也被上调。相反A3G的作用却不很明显。另外，GFP标记蛋白显示A3A分布在细胞核和细胞质，而A3G只定位在细胞质中。结论相比A3G，A3A的过表达可以明显影响W12细胞基因组的稳定性，进而激活宿主细胞DDR通路中多种蛋白激酶的活化。
목적：연구재지단백B mRNA편집매최화다태양3（APOBEC3s）탈안매대HPV（＋）인류영생화상피세포（W12）기인조은정성적영향。방법이용 W12세포위모형，통과기인전염APOBEC3A（A3A）、APOBEC3G（A3G）화GFP표체질립，획득전염후24 h화48 h적량조양품。채용Western blot방법검측세포DNA손상반응（DDR）통로중경전단백격매적표체정황。차외，이용록색형광단백（GFP）표기적방식，관찰A3A화A3G적세포정위。결과Western blot결과현시， A3A과표체W12세포양품중，경전DNA쌍련단렬적표기물γH2AX표체명현상조。DDR통로중ATM화Chk2격매적린산화수평유소제고。차외，동원중조（HR）수복중적세포인자pNBs1적표체야피상조。상반A3G적작용각불흔명현。령외，GFP표기단백현시A3A분포재세포핵화세포질，이A3G지정위재세포질중。결론상비A3G，A3A적과표체가이명현영향W12세포기인조적은정성，진이격활숙주세포DDR통로중다충단백격매적활화。
ObjectiveTo investigate the effect of APOBEC3s on genomic instability in HPV(+) human keratinocyte (W12 cell).Methods In W12 cells, Western blot and GFP tagged APOBEC3s microscopy observation were performed to evaluate DNA damage response related protein expression levels and localization.ResultsBy overexpression A3A, γH2AX, one double strand breakage marker, was significantly increased. Moreover, ATM-Chk2 pathway was also activated in W12 cells. pNBs1 was enhanced by overexpression A3A, suggesting human recombination repair pathway was involved. Rather than cytoplasmic distribution in A3G, cellular localization showed ubiquitin distribution of A3A. Conclusion Overexpression of A3A increases the expression levels of DDR related protein, suggesting A3A might impair cell genomic instability in HPV(+) cells. In addition, previous studies showed A3s hypermutated HPV viral DNA in W12 cells. All of these evidence imply that A3s might play crucial role in HPV viral-host integration process, which is a key step in HPV induced tumorgenesis.