CAJ | 학술논문

目的：探讨特异性磷脂酰肌醇蛋白多糖3（ GPC-3）小发夹RNA （ shRNA ）转染对高转移潜能肝癌细胞增殖和肝癌生长的抑制作用。方法将构建与筛选GPC-3 shRNA最有效序列转染MHCC-97H细胞，以荧光定量聚合酶链反应（ PCR）或Western印迹分析mRNA或蛋白表达；以5-乙炔基-2′脱氧尿嘧啶核苷（ EdU）、划痕和Transwell试验评估肝癌细胞增殖、迁移和侵袭能力；以Caspase-Glo?3/7荧光法分析细胞凋亡；以裸鼠移植瘤观察对肝癌形成与生长的影响，免疫组化分析瘤组织GPC-3、β-环连蛋白（catenin）、p-GSK3β及细胞周期蛋白（cyclin）D1表达。结果 shRNA1质粒转染MHCC-97H细胞效率＞80％， GPC-3mRNA下降75.6％（t＝15.473， P＜0.001），蛋白表达受抑；细胞增殖抑制71.1％（t＝10.468，P＜0.001）、迁移抑制80.1％（t＝32.697，P＜0.001）与侵袭抑制69.1％（t＝39.647，P＜0.001）；β-catenin mRNA抑制67.7％（t＝18.4，P＜0.001）；Gli1 mRNA上调53.3％（t＝-4.824，P＝0.008）；干预组裸鼠肿瘤形成平均11.2 d，显著长于阴性组5.5 d和空白组5.3 d （P＜0.001），瘤体积65.5 mm3明显小于空白组404.8 mm3和阴性组365.7 mm3（P＜0.001）；且瘤组织GPC-3、β-catenin、p-GSK3β及cyclinD1等信号分子表达显著下调（ P＜0.01）。结论干预GPC-3转录抑制肝癌细胞增殖、迁移和肿瘤生长，提示GPC-3为潜在的肝癌治疗分子靶目标。
목적：탐토특이성린지선기순단백다당3（ GPC-3）소발협RNA （ shRNA ）전염대고전이잠능간암세포증식화간암생장적억제작용。방법장구건여사선GPC-3 shRNA최유효서렬전염MHCC-97H세포，이형광정량취합매련반응（ PCR）혹Western인적분석mRNA혹단백표체；이5-을결기-2′탈양뇨밀정핵감（ EdU）、화흔화Transwell시험평고간암세포증식、천이화침습능력；이Caspase-Glo?3/7형광법분석세포조망；이라서이식류관찰대간암형성여생장적영향，면역조화분석류조직GPC-3、β-배련단백（catenin）、p-GSK3β급세포주기단백（cyclin）D1표체。결과 shRNA1질립전염MHCC-97H세포효솔＞80％， GPC-3mRNA하강75.6％（t＝15.473， P＜0.001），단백표체수억；세포증식억제71.1％（t＝10.468，P＜0.001）、천이억제80.1％（t＝32.697，P＜0.001）여침습억제69.1％（t＝39.647，P＜0.001）；β-catenin mRNA억제67.7％（t＝18.4，P＜0.001）；Gli1 mRNA상조53.3％（t＝-4.824，P＝0.008）；간예조라서종류형성평균11.2 d，현저장우음성조5.5 d화공백조5.3 d （P＜0.001），류체적65.5 mm3명현소우공백조404.8 mm3화음성조365.7 mm3（P＜0.001）；차류조직GPC-3、β-catenin、p-GSK3β급cyclinD1등신호분자표체현저하조（ P＜0.01）。결론간예GPC-3전록억제간암세포증식、천이화종류생장，제시GPC-3위잠재적간암치료분자파목표。
Objective To explore the silencing glypican-3 ( GPC-3 ) gene transcription by specific small hairpin RNA ( shRNA ) on the inhibition of hepatoma cells with high metastatic potentiality and hepatoma growth.Methods After MHCC-97H cells were transfected with higher effective GPC-3-shRNA, GPC-3 mRNA was analyzed by multiple FG-RT-PCR or protein by Western blot.Cell proliferation was detected by 5-ethynyl-2′-deoxyuridine and sulforhodamine B assay , its migratory metastasis and invasiveness by wound healing or transwell chamber system and cell apoptosis was detected by Caspase -Glo? 3/7 Luminescence assay.Nude mice were subcutaneously injected with stable MHCC-97H cells for observing the forming time or volume of xenograft tumors.And the expressions of GPC-3, β-catenin, p-GSK3βand CyclinD1 were analyzed by immunohistochemistry.Results After shRNA1 transfection with high efficiency (>80%), the expression of GPC-3 was down-regulated to 75.6% ( t=15.473, P<0.001) at mRNA level in accordance with its protein , inhibiting cell proliferation (71.1%, t=10.468, P<0.001)notably, decreasing its migration (80.1%, t=32.697, P<0.001) and invasiveness (69.1%, t=39.647, P<0.001).β-catenin was down-regulated (67.7%, t=18.4, P<0.001) and Gli1 increased (53.5%, t=-4.824, P=0.008) with its protein.The average forming time of subcutaneous tumors was 11.2 days ( d) in the shRNA group and it was significantly longer (P<0.01) than that in the control (5.3 d) or shRNA-neg (5.5 d) group.And the average volume (65.5 mm3 ) of tumors with decreased GPC-3, β-catenin, p-GSK3β, and cyclinD1 expressions in the shRNA group was significantly smaller ( P<0.01) than that in the shRNA-neg (365.7 mm3 ) or control (404.8 mm3 ) group, respectively.Conclusion Specific shRNA might intervene effectively the GPC-3 gene transcription and inhibit invasion and tumor growth.Thus GPC-3 may become a potential molecular target for hepatoma gene therapy.