CAJ | 학술논문

目的采用RNA干扰(RNAi)技术抑制人喉癌Hep-2细胞c-Met基因表达,观察对其体外生物学活性和体内生长的影响.方法构建能够表达针对c-Met的小干扰RNA的RNAi质粒和表达不针对任何已知mRNA的siRNA的阴性对照RNAi质粒,采用阳离子脂质体Lipofectamine2000作为转染试剂将其转染人喉癌细胞株Hep-2,通过RT-PCR及Western Blot方法检测转染效率,筛选出抑制效果最好的c-Met-siRNA序列;通过四甲基偶氮唑蓝(MTT)法、运动实验及侵袭实验比较转染前后细胞的生长、运动及侵袭能力.在裸鼠喉癌皮下荷瘤模型模拟c-Met-siRNA基因治疗实验中,采用Western Blot法检测重组质粒对c-Met基因及MMP-9、VEGF基因表达的影响.结果 pSilencer2.0/c-Met-shRNA转染人喉癌细胞株Hep-2后,c-Met的mRNA和蛋白表达水平显著降低(P值分别为0.003和0.02),细胞的生长、运动及侵袭均受到明显抑制(P值分别为0.001,0.004和0.004).肿瘤接种到裸鼠后第35天,重组质粒组肿瘤体积为(138±27)mm~3,与对照组相比差异有统计学意义(P<0.01);重组质粒组瘤体内c-Met、MMP-9、VEGF基因表达率比对照组明显降低(P<0.05).结论 c-Met-siRNA能成功下调靶基因的表达,并能显著抑制喉癌Hep-2细胞的生长、运动、侵袭及移植裸鼠成瘤后的生长,siRNA表达质粒介导的基因治疗有希望成为喉癌靶向性c-Met治疗的新策略.
목적 채용RNA간우(RNAi)기술억제인후암Hep-2세포c-Met기인표체,관찰대기체외생물학활성화체내생장적영향.방법 구건능구표체침대c-Met적소간우RNA적RNAi질립화표체불침대임하이지mRNA적siRNA적음성대조RNAi질립,채용양리자지질체Lipofectamine2000작위전염시제장기전염인후암세포주Hep-2,통과RT-PCR급Western Blot방법검측전염효솔,사선출억제효과최호적c-Met-siRNA서렬;통과사갑기우담서람(MTT)법、운동실험급침습실험비교전염전후세포적생장、운동급침습능력.재라서후암피하하류모형모의c-Met-siRNA기인치료실험중,채용Western Blot법검측중조질립대c-Met기인급MMP-9、VEGF기인표체적영향.결과 pSilencer2.0/c-Met-shRNA전염인후암세포주Hep-2후,c-Met적mRNA화단백표체수평현저강저(P치분별위0.003화0.02),세포적생장、운동급침습균수도명현억제(P치분별위0.001,0.004화0.004).종류접충도라서후제35천,중조질립조종류체적위(138±27)mm~3,여대조조상비차이유통계학의의(P<0.01);중조질립조류체내c-Met、MMP-9、VEGF기인표체솔비대조조명현강저(P<0.05).결론 c-Met-siRNA능성공하조파기인적표체,병능현저억제후암Hep-2세포적생장、운동、침습급이식라서성류후적생장,siRNA표체질립개도적기인치료유희망성위후암파향성c-Met치료적신책략.
Objective The proto-oncogene c-Met was found to express on human laryngeal carcinoma Hep-2 cell line in previous research. In the present study, the author further examined whether inhibition of c-Met by RNA interference(RNAi) might inhibit biologic activity of Hep-2 cell line in vitro and proliferation using a murine laryngeal carcinoma model. Methods RNAi plasmid that can express small interfering RNA targeting c-Met or siRNA that did not match any known human coding mRNA( control siRNA plasmid) was designed,constructed,and transfected into Hep-2 cell line by using cationic liposome Lipofectamine2000 as transfecting agent. In vitro, the transfection efficacy was tested by RT-PCR and Western Blot method, then elected the most inhibitive c-Met-siRNA sequence. Cell proliferation, movement and invasion were studied using MTT, cell migration assay and cell invasion assay, respectively. The Hep-2 cells were transplanted into nude mice, then the time of tumor formation and growth were observed. After tumor formation, c-Met-siRNA was given as the anti-tumor therapy. Expression of c-Met, MMP-9 and VEGF were detected by Western Blot method. Results After the pSilencer2. 0/c-Met-shRNA recombinant plasmid transfeetion into laryngeal carcinoma Hep-2 cells, the expression of mRNA and proteinum of c-Met decreased significantly in Hep-2 cells. On the 35th day after tumor vaccination, the tumor volume was (138±27)mm~3 in c-Met-siRNA transfection group, Which was diminished significantly in contrast with control group( P<0. 01 ). The expression of c-Met, MMP-9 and VEGF in the tumor of experiment group was decreased significantly, respectively (P<0.05 ). Conclusions The results indicated that c-Met-siRNA can down-regulate the expression of c-Met and markedly inhibit laryngeal carcinoma Hep-2 cell proliferation, movement and invasion and the growth of transplantation tumor of nude mice. The siRNA expressing plasmid mediated gene therapy might be a new strategy in targeting molecular therapy of cancer of larynx.