CAJ | 학술논문

背景:端粒酶反转录酶是端粒酶的活性亚基,已成为肿瘤研究的热点.RNA干扰技术作为一种基因沉默方法,具有高效、特异等优点,现已广泛应用于肿瘤、病毒等研究领域. 目的:构建针对人端粒酶反转录酶的小发夹RNA质粒表达载体,并观察其对乳腺癌T47D细胞人端粒酶反转录酶基因的表达和端粒酶活性的影响. 方法:以Genbank中人端粒酶反转录酶基因的mRNA序列为基础,设计人端粒酶反转录酶基因的小干扰RNA序列,将其连接到具有G418抗性的质粒pBAsi-hU6-Neo(Bam H Ⅰ/HindⅢ)中,应用基因测序加以验证,扩增提取质粒,以脂质体转染表达小发夹RNA的质粒到乳腺癌T47D细胞,抗生素G418筛选出转染成功的各组细胞. 结果与结论:实验所构建的人端粒酶反转录酶的小发夹 RNA 质粒表达载体,经测序验证无误.将 pBAsi-hU6-Neo重组质粒转染入T47D细胞,经G418筛选获得了转染成功的细胞.经RT-PCR和Western blot检测,转染后的人端粒酶反转录酶基因在mRNA和蛋白水平的表达均明显降低(P<0.01),经TRAP-ELISA法检测实验组细胞端粒酶活性出现显著下降(P <0.01).结果证实,实验成功构建人端粒酶反转录酶的小发夹RNA质粒表达载体,实验所设计的小干扰RNA能有效抑制肿瘤细胞人端粒酶反转录酶基因的表达,进而降低细胞的端粒酶活性.
배경:단립매반전록매시단립매적활성아기,이성위종류연구적열점.RNA간우기술작위일충기인침묵방법,구유고효、특이등우점,현이엄범응용우종류、병독등연구영역. 목적:구건침대인단립매반전록매적소발협RNA질립표체재체,병관찰기대유선암T47D세포인단립매반전록매기인적표체화단립매활성적영향. 방법:이Genbank중인단립매반전록매기인적mRNA서렬위기출,설계인단립매반전록매기인적소간우RNA서렬,장기련접도구유G418항성적질립pBAsi-hU6-Neo(Bam H Ⅰ/HindⅢ)중,응용기인측서가이험증,확증제취질립,이지질체전염표체소발협RNA적질립도유선암T47D세포,항생소G418사선출전염성공적각조세포. 결과여결론:실험소구건적인단립매반전록매적소발협 RNA 질립표체재체,경측서험증무오.장 pBAsi-hU6-Neo중조질립전염입T47D세포,경G418사선획득료전염성공적세포.경RT-PCR화Western blot검측,전염후적인단립매반전록매기인재mRNA화단백수평적표체균명현강저(P<0.01),경TRAP-ELISA법검측실험조세포단립매활성출현현저하강(P <0.01).결과증실,실험성공구건인단립매반전록매적소발협RNA질립표체재체,실험소설계적소간우RNA능유효억제종류세포인단립매반전록매기인적표체,진이강저세포적단립매활성.
BACKGROUND:The activation of telomerase is closely related to the occurrence and development of malignant tumor. While telomerase reverse transcriptase is the critical subunit of telomerase and now it is a hot point in the field of cancer research. RNA interference which is a kind of effective and specific gene blocking method has been widely used in cancer and virus research area. @@@@OBJECTIVE:To construct the human telomerase reverse transcriptase-targeted smal hairpin RNA-expressing plasmid (hTERT-targeted shRNA-expressing plasmid) system, and to observe its effect on hTERT expression and telomerase activity in breast cancer cel s T47D. @@@@METHODS:Sequence of hTERT-targeted shRNA was designed based on the mRNA sequence of hTERT which was obtained from the Genbank. They were recombined with the plasmid pBAsi-hU6-Neo (BamHI/Hind to antibiotic G418, and then those plasmids were identified by gene sequencing to make sure they were correctly connected. Then those plasmids were transfected into breast cancer cel s T47D with liposome and those cel s expressing shRNA were selected by G418.Ⅲ)w hich is im m une @@@@RESULTS AND CONCLUSION:The hTERT-targeted shRNA-expressing plasmids were successful y constructed, which was proved by gene sequencing. Then the pBAsi-hU6-Neo recombined plasmids were transfected into T47D cel s, and the successful y transfected cel s were selected with G418. Reverse transcription-PCR and western blot showed that the expression of the trasfected hTERT was significantly decreased both on mRNA and protein levels (P<0.01), and tartrate resistant acid phosphatase-enzyme-linked immunosorbent assay showed that the telomerase activity of the TERT in the experimental group was decreased significantly (P<0.01). It showed that the hTERT-targeted shRNA-expressing plasmids were successful y constructed, and the designed smal interferencing RNA can effectively block the expression of hTERT and then inhibit the telomerase activity.