CAJ | 학술논문

背景与目的:宫颈癌是常见的妇科恶性肿瘤,其中80%的宫颈癌可以检测到HPV16基因.本研究观察HPV16 E6基因miRNA干扰对宫颈癌CaSki细胞顺铂敏感性的影响.方法:利用分子生物学技术,将本实验室构建的针对HPV16 E6基因的miRNA表达载体pcDNA?6.2-GW/EmGFPmiR,分别转染至CaSki细胞和C-33A细胞内.倒置荧光显微镜观察转染效率,RT-PCR测定HPV16 E6 mRNA变化,蛋白质印迹法(Western blot)检测转染前后E6蛋白表达,MTT法检测转染前后对宫颈癌细胞顺铂敏感性的影响.结果:经杀稻瘟菌素(blasticidin S HCl)选择培养液筛选2周后,将获得的稳定转染细胞分别命名为CaSki-M3和CaSki-N、C-33A-M3和C-33A-N.RT-PCR显示,CaSki-M3细胞内的E6 mRNA转录水平比未转染组降低了99%,E6蛋白的表达抑制率为87.5%.用0.005、0.05、0.5、5、50和500μg/mL的顺铂处理未转染CaSki和C-33A以及转染后CaSki-M3、CaSki-N、C-33A-M3和C-33A-N细胞,CaSki-M3细胞对顺铂的敏感性显著升高(P<0.01).结论:HPV16 E6 miRNA有效下调HPV16 E6 mRNA水平,抑制HPV16 E6蛋白的表达,可明显提高CaSki细胞对顺铂的敏感程度.
배경여목적:궁경암시상견적부과악성종류,기중80%적궁경암가이검측도HPV16기인.본연구관찰HPV16 E6기인miRNA간우대궁경암CaSki세포순박민감성적영향.방법:이용분자생물학기술,장본실험실구건적침대HPV16 E6기인적miRNA표체재체pcDNA?6.2-GW/EmGFPmiR,분별전염지CaSki세포화C-33A세포내.도치형광현미경관찰전염효솔,RT-PCR측정HPV16 E6 mRNA변화,단백질인적법(Western blot)검측전염전후E6단백표체,MTT법검측전염전후대궁경암세포순박민감성적영향.결과:경살도온균소(blasticidin S HCl)선택배양액사선2주후,장획득적은정전염세포분별명명위CaSki-M3화CaSki-N、C-33A-M3화C-33A-N.RT-PCR현시,CaSki-M3세포내적E6 mRNA전록수평비미전염조강저료99%,E6단백적표체억제솔위87.5%.용0.005、0.05、0.5、5、50화500μg/mL적순박처리미전염CaSki화C-33A이급전염후CaSki-M3、CaSki-N、C-33A-M3화C-33A-N세포,CaSki-M3세포대순박적민감성현저승고(P<0.01).결론:HPV16 E6 miRNA유효하조HPV16 E6 mRNA수평,억제HPV16 E6단백적표체,가명현제고CaSki세포대순박적민감정도.
Background and purpose:Cervical cancer is a common kind of malignant tumor. HPV16 gene could be found in about 80% of the cervical cancer patients. This study aimed to observe the effection of HPV16 E6 gene microRNA (miRNA) on cisplatin sensitivity of cervical cancer cells. Methods:The pairs of miRNA oligonucleotide fragments were designed and synthesized based on the sequence of HPV16 E6 gene and inserted into pcDNATM 6.2-GW/EmGFPmiR vectors. This recombinant (M3) was transformed into CaSki and C-33A cell lines. The transfection efficiency of recombinants was evaluated by calculating the ratio of fluorescent cells to total cells. The mRNA level of HPV16 E6 before and after recombinants transfection was measured by RT-PCR, and protein level of HPV16 E6 was measured by Western blot. Cell survival rates after the treatment of cisplatin were evaluated by MTT detection. Results:The mRNA level of HPV16 E6 reduced by 99% after transfection, respectively, but the mRNA level of GAPDH, as internal control, had no change. The inhibition rate of HPV16 E6 protein was 87.5% after transfection, but the protein level of GAPDH, as internal control, had no change. After the treatment of 0.005, 0.05, 0.5, 5, 50 and 500 μg/mL cisplatin, CaSki-M3 cells’ survival rates were decreased significantly (P<0.01). Conclusion:HPV16 E16 miRNA effectively reduced the level of HPV16 E6 mRNA and inhibit the expression of HPV16 E6 protein. The degree of sensitivity to cisplatin can be significantly enhanced after transfection.