中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2013年
2期
322-325
,共4页
刘廷江%杨涛%孙福振%刘学军
劉廷江%楊濤%孫福振%劉學軍
류정강%양도%손복진%류학군
低氧诱导因子-1α%膀胱肿瘤%RNA干扰%增殖细胞核抗原%金属基质蛋白酶
低氧誘導因子-1α%膀胱腫瘤%RNA榦擾%增殖細胞覈抗原%金屬基質蛋白酶
저양유도인자-1α%방광종류%RNA간우%증식세포핵항원%금속기질단백매
Hypoixa inducible factor-1α%Human bladder cancer%RNA interference%Proliferating cell nuclear antigen%Matrix metalloproteinases
目的 采用RNA干扰技术(RNAi)技术观察低氧诱导因子-1α (HIF-1α)对人膀胱癌细胞株BIU-87增殖、侵袭及转移的影响.方法 体外培养BIU-87细胞,实时荧光定量聚合酶链反应(FQ-PCR)和Western blot检测低氧(90％氮气+5％氢气+5％二氧化碳)和常氧下及HIF-1α-siRNA转染前后BIU-87中HIF-1α、增殖细胞核抗原(PCNA)、基质金属蛋白酶(MMP)-2、MMP-9的表达；采用磺酰罗丹明B(SRB)法检测HIF-1α-siRNA转染后的细胞增殖水平,细胞划痕和Transwell试验检测迁移和侵袭能力.结果 低氧8、24h条件下,HIF-1α蛋白(0.56 ±0.08、1.33 ±0.21)、MMP-2 mRNA(1.85±0.21、2.94±0.29)、MMP-2蛋白(0.68 ±0.11、1.37 ±0.22)、MMP-9 mRNA(2.08±0.32、3.49±0.44)、MMP-9蛋白(0.89 ±0.12、1.40±0.23)表达与正常氧条件比较HIF-1α蛋白(0.33±0.05)、MMP-2 mRNA(0.95 ±0.13)、MMP-2蛋白(0.47±0.07)、MMP-9 mRNA(1.21±0.17)、MMP-9蛋白(0.58±0.14)表达均显著升高(均P＜0.05),HIF-1α mRNA(1.05 ±0.09、0.96 ±0.11)与正常氧条件(1.16±0.13)比较无明显变化；HIF-1α-siRNA转染后与转染前比较能有效沉默HIF-1α；与转染前(1.12 ±0.17、315.25±40.94、96.86±9.85)比较,HIF-1 α-siRNA能降低增殖、侵袭及迁移细胞数(0.63 ±0.08、139.62±19.67、69.17±7.39)(均P＜0.05); BIU-87中PCNA mRNA(0.57±0.07)、PCNA蛋白(0.52±0.09)、MMP-2 mRNA(0.64±0.08)、MMP-2蛋白(0.53±0.07)、MMP-9 mRNA(0.49±0.06)、MMP-9蛋白(0.69±0.07)表达与转染前比较显著降低(0.99±0.14、0.80±0.13、1.06±0.13、0.87 ±0.12、0.99±0.12、1.12±0.14) (P ＜0.05).结论 低氧下,HIF-1 α-siRNA通过沉默HIF-1α,降低PCNA、MMP-2、MMP-9表达而抑制膀胱癌细胞增殖、侵袭及转移.
目的 採用RNA榦擾技術(RNAi)技術觀察低氧誘導因子-1α (HIF-1α)對人膀胱癌細胞株BIU-87增殖、侵襲及轉移的影響.方法 體外培養BIU-87細胞,實時熒光定量聚閤酶鏈反應(FQ-PCR)和Western blot檢測低氧(90％氮氣+5％氫氣+5％二氧化碳)和常氧下及HIF-1α-siRNA轉染前後BIU-87中HIF-1α、增殖細胞覈抗原(PCNA)、基質金屬蛋白酶(MMP)-2、MMP-9的錶達；採用磺酰囉丹明B(SRB)法檢測HIF-1α-siRNA轉染後的細胞增殖水平,細胞劃痕和Transwell試驗檢測遷移和侵襲能力.結果 低氧8、24h條件下,HIF-1α蛋白(0.56 ±0.08、1.33 ±0.21)、MMP-2 mRNA(1.85±0.21、2.94±0.29)、MMP-2蛋白(0.68 ±0.11、1.37 ±0.22)、MMP-9 mRNA(2.08±0.32、3.49±0.44)、MMP-9蛋白(0.89 ±0.12、1.40±0.23)錶達與正常氧條件比較HIF-1α蛋白(0.33±0.05)、MMP-2 mRNA(0.95 ±0.13)、MMP-2蛋白(0.47±0.07)、MMP-9 mRNA(1.21±0.17)、MMP-9蛋白(0.58±0.14)錶達均顯著升高(均P＜0.05),HIF-1α mRNA(1.05 ±0.09、0.96 ±0.11)與正常氧條件(1.16±0.13)比較無明顯變化；HIF-1α-siRNA轉染後與轉染前比較能有效沉默HIF-1α；與轉染前(1.12 ±0.17、315.25±40.94、96.86±9.85)比較,HIF-1 α-siRNA能降低增殖、侵襲及遷移細胞數(0.63 ±0.08、139.62±19.67、69.17±7.39)(均P＜0.05); BIU-87中PCNA mRNA(0.57±0.07)、PCNA蛋白(0.52±0.09)、MMP-2 mRNA(0.64±0.08)、MMP-2蛋白(0.53±0.07)、MMP-9 mRNA(0.49±0.06)、MMP-9蛋白(0.69±0.07)錶達與轉染前比較顯著降低(0.99±0.14、0.80±0.13、1.06±0.13、0.87 ±0.12、0.99±0.12、1.12±0.14) (P ＜0.05).結論 低氧下,HIF-1 α-siRNA通過沉默HIF-1α,降低PCNA、MMP-2、MMP-9錶達而抑製膀胱癌細胞增殖、侵襲及轉移.
목적 채용RNA간우기술(RNAi)기술관찰저양유도인자-1α (HIF-1α)대인방광암세포주BIU-87증식、침습급전이적영향.방법 체외배양BIU-87세포,실시형광정량취합매련반응(FQ-PCR)화Western blot검측저양(90％담기+5％경기+5％이양화탄)화상양하급HIF-1α-siRNA전염전후BIU-87중HIF-1α、증식세포핵항원(PCNA)、기질금속단백매(MMP)-2、MMP-9적표체；채용광선라단명B(SRB)법검측HIF-1α-siRNA전염후적세포증식수평,세포화흔화Transwell시험검측천이화침습능력.결과 저양8、24h조건하,HIF-1α단백(0.56 ±0.08、1.33 ±0.21)、MMP-2 mRNA(1.85±0.21、2.94±0.29)、MMP-2단백(0.68 ±0.11、1.37 ±0.22)、MMP-9 mRNA(2.08±0.32、3.49±0.44)、MMP-9단백(0.89 ±0.12、1.40±0.23)표체여정상양조건비교HIF-1α단백(0.33±0.05)、MMP-2 mRNA(0.95 ±0.13)、MMP-2단백(0.47±0.07)、MMP-9 mRNA(1.21±0.17)、MMP-9단백(0.58±0.14)표체균현저승고(균P＜0.05),HIF-1α mRNA(1.05 ±0.09、0.96 ±0.11)여정상양조건(1.16±0.13)비교무명현변화；HIF-1α-siRNA전염후여전염전비교능유효침묵HIF-1α；여전염전(1.12 ±0.17、315.25±40.94、96.86±9.85)비교,HIF-1 α-siRNA능강저증식、침습급천이세포수(0.63 ±0.08、139.62±19.67、69.17±7.39)(균P＜0.05); BIU-87중PCNA mRNA(0.57±0.07)、PCNA단백(0.52±0.09)、MMP-2 mRNA(0.64±0.08)、MMP-2단백(0.53±0.07)、MMP-9 mRNA(0.49±0.06)、MMP-9단백(0.69±0.07)표체여전염전비교현저강저(0.99±0.14、0.80±0.13、1.06±0.13、0.87 ±0.12、0.99±0.12、1.12±0.14) (P ＜0.05).결론 저양하,HIF-1 α-siRNA통과침묵HIF-1α,강저PCNA、MMP-2、MMP-9표체이억제방광암세포증식、침습급전이.
Objective To investigate the effects of silencing hypoixa inducible factor-1α (HIF-1α)gene by RNAi on proliferation,invasion and metastasis of human bladder cancer cell line BIU-87.Methods BIU-87 cells were cultured in vitro.The expression of HIF-1α,proliferating cell nuclear antigen (PCNA),matrix metalloproteinase (MMP)-2,and MMP-9 was detected by using fluorescence real-time quantitative polymerase chain reaction (PCR) and Western blotting under normoxia or hypoxia (90％ N2 + 5％ H2 + 5％CO2).The cell proliferation was tested by SRB assay after transfection of HIF-1α-siRNA.Invasion and metastasis were tested by cell scratch assay and Transwell chambers respectively.The expression of PCNA,MMP-2,and MMP-9 was detected.Results The expression levels of HIF-1α protein (0.56 ±0.08,1.33 ±0.21),MMP-2 mRNA (1.85 ±0.21,2.94 ±0.29),MMP-2 protein (0.68 ±0.11,1.37 ±0.22),MMP-9mRNA (2.08 ±0.32,3.49 ±0.44),and MMP-9 protein (0.89 ±0.12,1.40 ±0.23) were increased under hypoxia for 8,and 24 h than those under normoxia [HIF-1α protein (0.33 ±0.05),MMP-2 mRNA (0.95 ± 0.13),MMP-2 protein (0.47 ± 0.07),MMP-9 mRNA (1.21 ± 0.17),MMP-9 protein (0.58 ±0.14)] (P＜0.05).There was no significant difference in HIF-1α mRNA (1.05 ±0.09,0.96 ±0.11)between hypoxia for 8,24 h and normoxia [(1.16 ± 0.13)] (P ＞ 0.05).HIF-1αt expression was suppressed after HIF-1α-siRNA transfection.The number of proliferation,invasion and metastasis cells (0.63 ± 0.08,139.62± 19.67,69.17 ± 7.39) was decreased after HIF-1 α-siRNA transfection as compared with that before transfection (1.12 ±0.17,315.25 ±40.94,96.86 ±9.85) (P＜0.05).The expression of PCNA mRNA (0.57 ±0.07),PCNA protein (0.52±0.09),MMP-2 mRNA (0.64 ±0.08),MMP-2 protein (0.53 ±0.07),MMP-9 mRNA (0.49 ±0.06),and MMP-9 protein (0.69 ±0.07) was decreased after HIF-1α-siRNA transfection as compared with that after transfection (0.99 ±0.14,0.80 ±0.13,1.06 ± 0.13,0.87 ± 0.12,0.99 ± 0.12,1.12 ± 0.14 respectively) (P ＜ 0.05).Conclusion HIF-1α-siRNA could inhibit proliferation,invasion and metastasis of BIU-87 cells by silencing HIF-1α,and inhibiting the expression of PCNA,MMP-2 and MMP-9.
