中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2014年
11期
816-822
,共7页
周围%钟妍玉%王洪民%杨思俊%魏文祥
週圍%鐘妍玉%王洪民%楊思俊%魏文祥
주위%종연옥%왕홍민%양사준%위문상
肝肿瘤%非传统型前折叠束RPB5交互因子%RNA干扰%γ射线%细胞周期%细胞凋亡
肝腫瘤%非傳統型前摺疊束RPB5交互因子%RNA榦擾%γ射線%細胞週期%細胞凋亡
간종류%비전통형전절첩속RPB5교호인자%RNA간우%γ사선%세포주기%세포조망
Liver neoplasms%Unconventional prefoldin RPB5 interactor%RNA interferece%Gama rays%Cell cycle%Apoptosis
目的 探讨非传统型前折叠束RPB5交互因子(URI)对肝癌HepG2细胞生物学功能的影响及其分子机制.方法 设计并构建URI真核表达和URI shRNA干扰载体,转染肝癌HepG2细胞并筛选稳定转染细胞株.采用CCK-8实验和软琼脂克隆形成实验检测各组细胞的增殖能力和体外锚定非依赖性生长的能力,采用流式细胞仪检测γ射线照射后各组细胞的细胞周期和细胞凋亡,采用Western blot法检测凋亡相关蛋白的表达量.结果 成功构建了URI表达质粒和干扰质粒,并获得相应的稳定转染细胞株.HepG2组、pCDNA3.1-URI-HepG2组和pGPU6-URIi-HepG2组细胞转染第7天相对于第1天的增殖促进率分别为(588.78±32.12)％、(959.33±58.80)％和(393.93±39.70)％,差异均有统计学意义(均P＜0.05).HepG2组、pCDNA3.1-URI-HepG2组和pGPU6-URIi-HepG2组的克隆数分别为(43±7)个、(85±5)个和(20±4)个,差异有统计学意义(P＜0.05).γ射线照射后,pCDNA3.1-URI-HepG2组细胞凋亡和G2/M期阻滞较HepG2组明显降低(P＜0.05),pGPU6-URIi-HepG2组细胞凋亡和G2/M期阻滞较HepG2组明显升高(P＜0.05).Western blot结果显示,HepG2细胞的URI、Bax和Bcl-2蛋白相对表达水平分别为0.92±0.03、1.11±0.13和0.82±0.01,pCDNA3.1-URI-HepG2组细胞的URI、Bax和Bcl-2蛋白相对表达水平分别为1.79±0.12、0.48±0.01和2.20±0.30,pGPU6-URli-HepG2组细胞的URI、Bax和Bcl-2蛋白相对表达水平分别为0.50±0.04、1.52 ±0.20和0.38±0.01.pCDNA3.1-URI-HepG2组细胞凋亡相关蛋白Bax表达水平低于HepG2组,Bcl-2蛋白表达水平高于HepG2组;pGPU6-URIi-HepG2组细胞Bax蛋白表达水平高于HepG2组,Bcl-2蛋白表达水平低于HepG2组,差异有统计学意义(P＜0.05).结论 URI能够通过促进细胞增殖和抵抗细胞凋亡来促进肝癌细胞的生长.而干扰URI表达后,肝癌细胞的增殖能力被抑制,细胞抵抗γ射线照射的能力降低,URI可能成为肝癌治疗的新靶点.
目的 探討非傳統型前摺疊束RPB5交互因子(URI)對肝癌HepG2細胞生物學功能的影響及其分子機製.方法 設計併構建URI真覈錶達和URI shRNA榦擾載體,轉染肝癌HepG2細胞併篩選穩定轉染細胞株.採用CCK-8實驗和軟瓊脂剋隆形成實驗檢測各組細胞的增殖能力和體外錨定非依賴性生長的能力,採用流式細胞儀檢測γ射線照射後各組細胞的細胞週期和細胞凋亡,採用Western blot法檢測凋亡相關蛋白的錶達量.結果 成功構建瞭URI錶達質粒和榦擾質粒,併穫得相應的穩定轉染細胞株.HepG2組、pCDNA3.1-URI-HepG2組和pGPU6-URIi-HepG2組細胞轉染第7天相對于第1天的增殖促進率分彆為(588.78±32.12)％、(959.33±58.80)％和(393.93±39.70)％,差異均有統計學意義(均P＜0.05).HepG2組、pCDNA3.1-URI-HepG2組和pGPU6-URIi-HepG2組的剋隆數分彆為(43±7)箇、(85±5)箇和(20±4)箇,差異有統計學意義(P＜0.05).γ射線照射後,pCDNA3.1-URI-HepG2組細胞凋亡和G2/M期阻滯較HepG2組明顯降低(P＜0.05),pGPU6-URIi-HepG2組細胞凋亡和G2/M期阻滯較HepG2組明顯升高(P＜0.05).Western blot結果顯示,HepG2細胞的URI、Bax和Bcl-2蛋白相對錶達水平分彆為0.92±0.03、1.11±0.13和0.82±0.01,pCDNA3.1-URI-HepG2組細胞的URI、Bax和Bcl-2蛋白相對錶達水平分彆為1.79±0.12、0.48±0.01和2.20±0.30,pGPU6-URli-HepG2組細胞的URI、Bax和Bcl-2蛋白相對錶達水平分彆為0.50±0.04、1.52 ±0.20和0.38±0.01.pCDNA3.1-URI-HepG2組細胞凋亡相關蛋白Bax錶達水平低于HepG2組,Bcl-2蛋白錶達水平高于HepG2組;pGPU6-URIi-HepG2組細胞Bax蛋白錶達水平高于HepG2組,Bcl-2蛋白錶達水平低于HepG2組,差異有統計學意義(P＜0.05).結論 URI能夠通過促進細胞增殖和牴抗細胞凋亡來促進肝癌細胞的生長.而榦擾URI錶達後,肝癌細胞的增殖能力被抑製,細胞牴抗γ射線照射的能力降低,URI可能成為肝癌治療的新靶點.
목적 탐토비전통형전절첩속RPB5교호인자(URI)대간암HepG2세포생물학공능적영향급기분자궤제.방법 설계병구건URI진핵표체화URI shRNA간우재체,전염간암HepG2세포병사선은정전염세포주.채용CCK-8실험화연경지극륭형성실험검측각조세포적증식능력화체외묘정비의뢰성생장적능력,채용류식세포의검측γ사선조사후각조세포적세포주기화세포조망,채용Western blot법검측조망상관단백적표체량.결과 성공구건료URI표체질립화간우질립,병획득상응적은정전염세포주.HepG2조、pCDNA3.1-URI-HepG2조화pGPU6-URIi-HepG2조세포전염제7천상대우제1천적증식촉진솔분별위(588.78±32.12)％、(959.33±58.80)％화(393.93±39.70)％,차이균유통계학의의(균P＜0.05).HepG2조、pCDNA3.1-URI-HepG2조화pGPU6-URIi-HepG2조적극륭수분별위(43±7)개、(85±5)개화(20±4)개,차이유통계학의의(P＜0.05).γ사선조사후,pCDNA3.1-URI-HepG2조세포조망화G2/M기조체교HepG2조명현강저(P＜0.05),pGPU6-URIi-HepG2조세포조망화G2/M기조체교HepG2조명현승고(P＜0.05).Western blot결과현시,HepG2세포적URI、Bax화Bcl-2단백상대표체수평분별위0.92±0.03、1.11±0.13화0.82±0.01,pCDNA3.1-URI-HepG2조세포적URI、Bax화Bcl-2단백상대표체수평분별위1.79±0.12、0.48±0.01화2.20±0.30,pGPU6-URli-HepG2조세포적URI、Bax화Bcl-2단백상대표체수평분별위0.50±0.04、1.52 ±0.20화0.38±0.01.pCDNA3.1-URI-HepG2조세포조망상관단백Bax표체수평저우HepG2조,Bcl-2단백표체수평고우HepG2조;pGPU6-URIi-HepG2조세포Bax단백표체수평고우HepG2조,Bcl-2단백표체수평저우HepG2조,차이유통계학의의(P＜0.05).결론 URI능구통과촉진세포증식화저항세포조망래촉진간암세포적생장.이간우URI표체후,간암세포적증식능력피억제,세포저항γ사선조사적능력강저,URI가능성위간암치료적신파점.
Objective To explore the effect and molecular mechanism of the unconventional prefoldin RPB5 interactor (URI) in hepatocellular carcinoma HepG2 cells.Methods The cDNA sequence and shRNA of URI were obtained and sub-cloned into eukaryotic expression vectors.Then those vectors were transfected into HepG2 cells to obtain stable transfection cell line.The cell proliferation and anchorindependent growth in URI-overexpressing and knockdown HepG2 cells were determined by CCK-8 and soft agar colony assay.Flow cytometry was applied to detect the cell cycle and apoptosis of γ-ray irradiated cells.Apoptosis related genes were detected by Western blot.Results The pCDNA3.1-URI and pGPU6-URIi eukaryotic expression vectors were constructed successfully and corresponding stable transfection cell lines were obtained.Cell proliferation rates of the HepG2,pCDNA3.1-URI-HepG2 and pGPU6-URIi-HepG2 cells were (588.78 ±32.12)％,(959.33 ±58.8)％ and (393.93 ±39.7)％,respectively (P＜0.05).The number of cell clones of HepG2,pCDNA3.1-URI-HepG2 and pGPU6-URIi-HepG2 cells were 43 ± 7,85 ± 5 and 20 ± 4 (P ＜ 0.05),respectively.After γ-ray irradiation,the URI-overexpressing cell line showed a significantly lower apoptosis rate and G2/M phase arrest than those in the URI-depleted cell line (P ＜ 0.05).In the HepG2 cells,the relative protein expression levels of URI,Bax and Bcl-2 were 0.92 ± 0.03,1.11 ±0.13 and 0.82 ± 0.01 (P ＜ 0.05).In the pCDNA3.1-URI-HepG2 cells,the relative protein expression levels of URI,Bax and Bcl-2 were 1.79 ± 0.12,0.48 ± 0.01 and 2.20 ± 0.30 (P ＜ 0.05),respectively.In the pGPU6-URIi-HepG2 cells,the relative protein expression levels of URI,Bax and Bcl-2 were 0.50 ± 0.04,1.52 ± 0.20 and 0.38 ± 0.01 (P ＜ 0.05),respectively.The expression of Bax was down-regulated and Bcl-2 was up-regulated in the URI-overexpressing cell line.However,on the contrary,expression of Bax was up-regulated and Bcl-2 was down-regulated in the URI-depleted cell line.Conclusions URI may promote the growth of hepatocellular carcinoma cells via inhibition of cell proliferation and reducing the apoptosis in hepatocellular carcinoma cells in vitro.After the impairment of URI expression,the proliferation ability of hepatocellular carcinoma cells is suppressed and the ability to resist γ-ray irradiation is reduced.URI may become a potential new target for cancer therapy of hepatocellular carcinoma.
