慢病毒介导bcr/abl基因长期沉默对K562细胞生物学特性与姜黄素抗肿瘤作用影响及其相关机制研究
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摘要
慢性粒细胞白血病(chronic myelogeous leukemia, CML),是以费城染色体(Philadephia, Ph)和bcr/abl融合基因为特征的多能干细胞异常的骨髓恶性增殖性疾病。bcr/abl基因编码的P210bcr/abl蛋白具有很强的酪氨酸激酶活性,与CML凋亡缺失、分化抑制、耐药有密切关系。
RNA干扰(RNAi)是一种转录后基因沉默(PTGS)的基因阻断技术,近年来被广泛应用于哺乳动物细胞抑制基因表达。通过RNase III内切酶Dicer的作用产生有活性的小的21-23nt干扰性RNA(short interence RNA, siRNA),介导其互补同源mRNA序列的特异性降解,沉寂目的基因的表达。
本研究采用慢病毒介导的RNAi技术,设计合成针对bcr/abl的siRNA,感染人慢性粒细胞白血病K562株,构建bcr/abl长期沉默的细胞模型。利用该细胞模型探讨bcr/abl基因长期沉默对K562细胞生物特性、相关信号分子及姜黄素抗肿瘤作用的影响。
一、慢病毒介导bcr/abl基因长期沉默的K562细胞株构建
为研究慢病毒介导的K562细胞bcr/abl基因RNA干扰的效率及稳定度,并为后续研究提供细胞模型,我们建立了bcr/abl RNAi稳定转染的B/A-K562模型。
研究方法(:1)利用RNAi技术和慢病毒转基因技术,构建pNL-EGFP-U6-bcr/abl-I质粒,使它和pVSV,pHelper组成慢病毒三质粒转染系统,共转染293T细胞,生产EGFP- B/A-I病毒;(2)用EGFP、EGFP-B/A-I病毒分别感染K562细胞,获取混合克隆;(3)极限稀释法、集落形成法挑取稳定表达EGFP、EGFP-B/A-I的克隆;(4)Realtime RT-PCR检测bcr/abl mRNA水平;(5)细胞免疫荧光、Western blotting法检测P210bcr/abl蛋白含量。
结果显示:(1)成功包装出具有感染能力的EGFP和EGFP- B/A-I慢病毒;(2)获得稳定表达EGFP和EGFP-B/A-I的EGFP-K562、B/A-K562细胞;(3)体外传代半年后,慢病毒介导RNAi可使bcr/abl mRNA下调63.5 %;(4)P210bcr/abl蛋白含量减少74.5 %。
二、Bcr/abl基因长期沉默对K562细胞生物学特性及相关基因表达的影响
bcr/abl基因是与CML发生、发展、转归密切相关的癌基因。bcr/abl基因长期“缺席”,可能导致K562细胞各种生物学特性及相关信号分子表达的变化。本部分,我们以bcr/abl基因长期沉默的B/A-K562细胞为模型,与正常对照的K562细胞及空载体对照的EGFP-K562细胞进行比较,分析其主要生物学特性及信号分子的变化。
研究方法:(1)锥虫蓝染色法检测细胞增殖能力变化;(2)集落形成法观察RNAi对集落形成能力的影响;(3)联苯胺染色观察细胞分化;(4)流式细胞仪分析细胞周期变化;(5)ELISA法检测酪氨酸激酶活性;(6)AO-EB、Heochst 33342染色观察细胞凋亡;(7)比色法检测Caspase-3、Caspase-9活性;(8)Western blotting法观察RNAi对细胞增殖凋亡相关信号分子的影响。
结果显示:(1)bcr/abl RNAi显著抑制K562细胞增殖,细胞倍增时间明显延长;(2)bcr/abl RNAi抑制细胞集落形成,集落形成抑制率达55.05 %;(3)bcr/abl RNAi诱导K562细胞向成熟红系分化,联苯胺染色阳性率达24.83 %;(4)bcr/abl RNAi使细胞周期阻滞在S期,出现凋亡的亚二倍体峰;( 5)bcr/abl RNAi使酪氨酸激酶活性降低约41.88 %;(6)bcr/abl RNAi诱导K562细胞凋亡,自发凋亡率达26.67 %;(7)bcr/abl RNAi触发线粒体Cyt-C释放,Caspase-3及Caspase-9活性提高,从线粒体途径诱导细胞凋亡;(8) bcr/abl RNAi下调K562细胞Src、P-Src、Raf-1、P-Erk1/2、P-Stat 1、P-Stat 5、P-P38、C-myc、Bcl-2和Hsp 90含量,上调P53、PKC及Bax含量,对Erk1/2、P-Akt、NF-КB、Hsp 70影响不大。
三、慢病毒介导bcr/abl基因长期沉默对K562细胞裸鼠成瘤的影响及相关机制研究
慢病毒介导的RNA干扰,在体外能实现bcr/abl基因的长期沉默,抑制K562细胞增殖,诱导其向成熟红系细胞分化,促进细胞凋亡。本部分我们关注的是:B/A-K562细胞在裸鼠体内能否继续保持对bcr/abl基因的RNA干扰以及成瘤能力和相关信号分子的变化。
研究方法:(1)皮下注射K562、EGFP-K562、B/A-K562细胞,建立裸鼠异种移植瘤模型;(2)观察成瘤情况,比较肿瘤体积;(3)称量离体瘤块质量、计算抑瘤率;(4)肿瘤组织测定Caspase-3、Caspase-9活性;(5)肿瘤组织冰冻切片观察荧光;(6)HE染色光镜下观察肿瘤组织的形态结构;(7)免疫组织化学检测肿瘤组织P210bcr/abl、Bcl-2、Bax蛋白表达;(8)免疫印迹检测肿瘤组织中Src、Erk1/2、P-Stat 1、Hsp 90的蛋白含量。
结果显示:(1)B/A-K562组肿瘤生长速度明显低于K562组及EGFP-K562组, K562组及EGFP-K562组成廇率100 %,B/A-K562组成瘤率80 %;(2)B/A-K562组抑瘤率达78.4 % (P<0.01);(3)B/A-K562组肿瘤组织Caspase-3、Caspase-9活性显著提高;(4)荧光显微镜观察,慢病毒感染的EGFP-K562及B/A-K562细胞形成的移植瘤均可见绿荧光;(5)免疫组化显示,K562组及EGFP-K562组P210bcr/abl、Bcl-2蛋白呈强阳性表达,Bax蛋白呈弱阳性表达;B/A-K562组P210bcr/abl、Bcl-2蛋白表达明显减弱,Bax蛋白表达明显增强;(6)免疫印迹结果显示,B/A-K562组Erk1/2、Src、P-Stat 1、Hsp 90含量低于K562组及B/A-K562组。
四、慢病毒介导K562细胞bcr/abl基因长期沉默对姜黄素抗肿瘤作用的影响及相关机制研究
bcr/abl基因及其相关蛋白是Cur作用的重要靶点。本部分,我们用Cur分别处理K562、EGFP-K562及B/A-K562细胞,探讨K562细胞的bcr/abl基因表达被慢病毒介导的RNA干扰阻断后,Cur针对K562细胞的抗肿瘤作用及作用机制有何改变。
研究方法:(1)MTT法、锥虫蓝染色法观察Cur对细胞增殖的影响;(2)集落形成法观察Cur对细胞集落形成的影响;(3)联苯胺染色法观察Cur对细胞分化的影响;(4)ELISA法观察Cur对酪氨酸激酶活性影响;(5)流式细胞仪分析Cur对细胞周期的影响;(6)AO-EB染色、DNA琼脂糖凝胶电泳检测Cur对细胞凋亡的影响;(7)分光光度法检测Cur对细胞Caspase-3和Caspase-9活性的影响;(8)Western blotting观察Cur对细胞增殖和凋亡有关信号分子及伴侣蛋白的影响。
结果显示:(1)Cur抑制K562、EGFP-K562及B/A-K562细胞增殖,呈时间和剂量依赖关系,Cur作用48小时,IC50分别为6.94、4.93、12.67μg/ml;(2)Cur影响B/A-K562细胞的集落形成,呈量效关系;(3)Cur诱导细胞向成熟红系细胞分化,其促进分化作用被bcr/abl RNAi减弱;(4)Cur降低细胞PTK活性,bcr/abl RNAi可增强其效应;(5)Cur诱导B/A-K562细胞凋亡亚二倍体峰出现,作用呈时间和剂量依赖性;(6)Cur有效诱导B/A-K562细胞凋亡,呈剂量依赖关系;(7)Cur进一步下调B/A-K562细胞P210bcr/abl含量;( 7)Cur促进B/A-K562细胞线粒体细胞色素C释放,活化Caspase-3、Caspase-9,循线粒体途径诱导凋亡;( 8) Cur上调B/A-K562细胞C-fas、P53、P-P38、Bax蛋白含量,下调P-Src、Raf-1、NF-КB、C-myc、PKC、Bcl-2、Hsp 70、Hsp 90蛋白含量。
从以上实验结果得出如下结论:
1.慢病毒介导的bcr/abl干扰能够实现bcr/abl基因长期沉默,bcr/abl mRNA下调63.5 %,P210bcr/abl下调74.5 %;
2. bcr/abl RNAi显著抑制K562细胞增殖,诱导其向成熟红系分化,显著降低PTK活性,循线粒体途径诱导K562细胞自发凋亡;
3. bcr/abl RNAi抑制K562细胞增殖,诱导凋亡,作用机制可能与bcr/abl沉默,Src、P-Src、Raf-1、P-Erk1/2、P-Stat 1、P-Stat 5、P-P38、C-myc、Bcl-2、Hsp 90下调,P53、Bax上调有关;
4.慢病毒介导的RNAi在裸鼠体内继续沉默bcr/abl基因,显著抑制肿瘤生长,诱导肿瘤组织凋亡和坏死,其抗肿瘤作用机制可能与bcr/abl RNAi及由之引起的Erk1/2、Src、P-Stat 1、Hsp 90、Bcl-2下调,Bax上调有关;
5. Cur抑制B/A-K562细胞增殖,诱导分化,降低PTK活性,循线粒体途径诱导凋亡;
6. Cur进一步降低B/A-K562细胞P210bcr/abl含量,上调C-fas、P53、P-P38、Bax,下调P-Src、Raf-1、NF-КB、C-myc、PKC、Bcl-2、Hsp 70、Hsp 90,可能与Cur在bcr/abl沉默的背景下,诱导B/A-K562细胞凋亡,抑制增殖有关。
总之,慢病毒介导的bcr/abl RNAi能够实现bcr/abl基因的长期沉默,抑制CML细胞增殖,诱导凋亡,作为CML的分子靶向治疗具有潜在应用价值。Cur可在bcr/abl RNAi基础上进一步下调P210bcr/abl,抑制增殖,诱导凋亡,是配合bcr/abl基因靶向治疗理想选择。
Chronic Myelogenous Leukemia(CML) originates in a pluripotent hematopoetic stem cell of the bone marrow, which is characterized by the Philadelphia(Ph) chromosome and bcr/abl oncogene. The bcr/abl fusion gene encoded Bcr/Abl protein, which possesses high activity of tyrosine kinase was known to be a resistant factor for apoptosis, cell differentiation and chemotherapy drugs resistance in chronic myelogenous leukemia (CML).
RNA interference is a kind of gene-blocking technology about post-transcriptional gene silence (PTGS), which has been extensively applied to the inhibition of gene expression in the mammal cells in recent years. Compt short interference RNA (21-23nt) is obtained by the incision enzyme RNaseⅢ(Dicer), which mediate specific degradation of its homologous and complimentary mRNA, to silence the expression of target genes.
In this study, K562 cells were infected by lentivirus carrying bcr/abl RNAi fragment, and further bcr/abl stable-silencing CML cellular model was constructed. As a result of long-term absence of bcr/abl, changes of biological characters, correlated signaling molecules and anti-tumor effects of curcumine were detected.
Construction of lentivirus-mediated bcr/abl stable-silencing cellular model on K562 cells
In this part, we constructed bcr/abl RNAi stable-transfecting cellular model B/A-K562, and detected the efficiency and stability of lentivirus-mediated RNA interference.
Methods: (1) Plasmids pNL-EGFP-U6-brcr/abl-I was constructed, which was combined with pVSVG and pHelper to constitute vector system of three plasmids. The lentiviral vector system was transfected into 293T cell to produce EGFP- B/A-I lentivirus. (2) K562 cells were infected with both EGFP and EGFP- B/A-I lentivirus. (3) Ultimate dilution and colony forming methods were used to pick clones stably expressing EGFP and EGFP-B/A-I fragment. (4) bcr/abl mRNA level was detected by Realtime RT-PCR; (5) Cello-immunofluorescence, western blotting were used to measure the contents of P210bcr/abl.
The results showed: (1) EGFP and EGFP- B/A-I lentivirus with processing infection ability were successfully produced; (2) EGFP-K562, B/A-K562 cells stably expressing EGFP and EGFP-B/A-I fragment were successfully picked; (3) After cultured in vitro for six months, bcr/abl mRNA lever was down regulated by lentivirus-mediated RNAi for 63.5 %; (4) Compared with K562 cells, P210bcr/abl content was obviously decreased by 74.5 % in B/A-K562 cells.
The effect of bcr/abl gene stable-silencing on the biological characters and relevant signaling molecules in K562 cell line
Since bcr/abl plays an important role in the generation, development and turnover of human chronic myelogeous leukemia, many changes in biological characters and cell signaling molecules may be detected in human CML cell line K562 as a result of long-term absence of bcr/abl gene. This study focused on the variations of biological characters and cellular signals in bcr/abl stable-silencing cell model B/A-K562 compared with K562 and EGFP-K562 cells as normal control and vector control respectively.
Methods: (1) Trypan blue exclusive staining was used to access the cell growth arrest; (2) Colony assay was used to observe the CFUs; (3) Benzidine staining was used to detect cell differentiation; (4) FCM was used to analyze cell cycle; (5) ELISA method was utilized to detect the activity of PTK; (6) AO-EB and Heochst 33342 fluorescent staining was used to observe apoptosis; (7) Chromometry was to used measure the activation of Caspase-3 and Caspase-9; (8) Western blot was used to observe the effects of RNAi on the signaling molecules which are relevant with cell growth and apoptosis.
The results showed: (1) bcr/abl RNAi significantly inhibited K562 cell proliferation, doubling generation time was obviously prolonged; (2) Colony forming was arrested by RNAi; (3) Positive rate of benzdine staining was 24.83 %, low level of bcr/abl message leads to enhanced erythroid differentiation; (4) RNAi arrested cell cycle in S phase and induced apoptosis in hypodiploid peak; (5) PTK activity was descended by 41.88 % ; (6) Apoptosis was observed with 41.88 % of apoptosis rate; (7) bcr/abl RNAi triggered the release of mitochondrial Cyt-C, increases Caspase-3 and Caspase-9 activities, induces apoptosis in mitochondrial-depended pathway; (8) bcr/abl RNAi down-regulated Src, P-Src, Raf-1, P-Erk1/2, P-Stat 1, P-Stat 5, P-P38, C-myc, Bcl-2 and Hsp 90 level, and up-regulated P53, PKC and Bax level, but had no effect on the concentration of Erk1/2, P-Akt, NF-КB, Hsp 70 in K562 cells.
The effect of lentivirus-mediated bcr/abl stable-silencing on the tumor formation on K562 cell xenograft in athymic mouse
Lentivirus-mediated RNA interference could reduce the expression of bcr/abl gene in vitro, inhibit cell growth, induce differentiation and hasten apoptosis. In this part, we mainly studied RNAi efficiency in B/A-K562 cells athymic mouse transplantation tumor, tumor formation ability and relevant signaling molecules.
Methods: (1) K562, EGFP-K562, B/A-K562 cells was injected subcutaneously in order to establish athymic mouse heterogeneity transplantation tumor models; (2) Volume of the tumors were measured per 5 days and the tumor formation rates were calculated; (3) At the end of time points, the mice were killed, the tumor xenografts were removed and measured; (4) Activation of Capspase 3, Caspase-9 were detected; (5) Green fluorescence was observed in frozen section of tumor tissue by fluorescence microscope; (6) H&E staining was used to study the pathological change of tumor tissue; (7) Immunohistochemistry was used to investigate the P210bcr/abl, Bcl-2, Bax level in tumor tissue; (8) The protein was extracted from the tumor tissues, western blotting was used to analyze Src, Erk1/2, P-Stat1 and Hsp 90 protein level.
The results showed: (1) Growth velocity of B/A-K562 cell xenograft was obviously lower than that of K562 group and EGFP-K562 group; (2) Tumor formation rate of B/A-K562 group was 80 % lower than that of K562 and EGFP-K562 group, which was 100%; (3) The tumor inhibition rate in B/A-K562 group was 78.4 % (compared with K562 group, P<0.01); (4) The activation of Caspase-3, Caspase-9 was elevated in B/A-K562 cell xenograft; (5) Green fluorescence was confirmed in transplantation tumor of lentivirus infected EGFP-K562 and B/A-K562 cells; (6) The results of immunohistochemistry showed decreased P210bcr/abl, Bcl-2 expression and increased Bax expression in B/A-K562 group, compared with K562 and EGFP-K562 group; (7) The results of Western blotting showed Erk1/2、Src、P-Stat 1、Hsp 90 protein level of B/A-K562 group were lower than that of K562 and EGFP-K562 group.
The effect of lentivirus-mediated bcr/abl stable-silencing on anti-tumor action of curcumin and relevat mechanisms in K562 cells
bcr/abl gene and P210bcr/abl protein were important targets of Curcumin. In this part, K562, EGFP-K562 and B/A-K562 cells were treated with Curcumin in different concentration. We investigated anti-tumoral action and relevat mechanisms of curcumine when bcr/abl gene expression was blocked by lentivirus-mediated RNA interference.
Methods: (1) MTT method and trypan blue exclusive staining were used to observe the cell growth arrest; (2) Colony assay was used to the cell colony forming units(CFUs); (3) Benzdine staining was utilized to investigate cell differentiation; (4) ELISA was used to measure the activity of PTK; (5) Flow cytometry (FCM) was used to analyze cell cycle; (6) AO-EB staining and DNA agarose gel electrophoresis were used to detect cell apoptosis; (7) Absorption spectrometry was used to detect activation of Caspase-3 and Caspase-9; (8) Western blotting was used to analyze the impact of Curcumine on Cell proliferation apoptosis-related signaling molecules and partner protein.
The results showed: (1) Curcumin inhibited K562, EGFP-K562 and B/A-K562 cells proliferation in a time-and-dose-depended manner. IC50 values of K562, EGFP-K562 and B/A-K562 cells were 6.94, 4.93, 12.67μg/ml after 48 h of Cur exposure; (2) Cur inhibited B/A-K562 CFUs in an dose-depended manner; (3) Cur induced differentiation towards mature erythroid cells and its role in the promotion of differentiation could be weakened by bcr/abl RNAi; (4) Cur reduced PTK activity in B/A-K562 cells and bcr / abl RNAi may enhance such effect; (5) Cur induced apoptosis in hypodiploid peak in a time-and-dose-depended manner; (6) Cur effectively induced B/A-K562 cells apoptosis in a dose-depended manner; (7) Cur triggered the release of mitochondrial Cyt-C, increased Caspase-3 and Caspase-9 activities, inducesd apoptosis in mitochondrial-depended pathway in B/A-K562 cells; (8) Cur up-regulated C-fas, P53, P-P38 andBax level, and down-regulated P-Src, Raf-1, NF-КB, C-myc, PKC, Bcl-2, Hsp 70, and Hsp 90 level in B/A-K562 cells.
CONCLUSIONS:
1. Lentivirus-mediated RNA interference may stably silence bcr/abl gene, which brings down bcr/abl mRNA and P210bcr/abl by respectively 63.5 % and 74.5 %.
2. Lentivirus-mediated stable-silencing of bcr/abl inhibits proliferation, induces differentiation towards mature erythroid cells, significantly decreases activity of PTK and hastens apoptosis in mitochondrial-depended pathway in K562 cells.
3. Lentivirus-mediated stable-silencing of bcr/abl inhibits K562 cells growth and induces cell apoptosis which may be correlated with the down-regulation of P210bcr/abl, Src, P-Src, Raf-1, P-Erk1/2, P-Stat 1, P-Stat 5, P-P38, C-myc, Bcl-2, Hsp 90 and up-regulation of P 53 and Bax.
4. Effect of bcr/abl RNAi retains in athymic mouse transplantation tumor,which significantly inhibits tumorous growth, induces apoptosis and necrosis in tumor tissue. Its anti-tumor effect may correlated with down-regulation of signaling molecules related with cell growth and apoptosis such as Erk1/2, Src, P-Stat 1, Hsp 90, Bcl-2 and up-regulation of Bax.
5. Cur inhibits cell growth, induces differentiation, reduces PTK activity, and hastens apoptosis in mitochondrial-depended pathway in B/A-K562 cells.
6. Cur decreases concentration of P210bcra/bl in B/A-K562 cells, up-regulates C-fas, P53, P-P38 and Bax, down-regulates P-Src, Raf-1, NF-КB, C-myc, PKC, Bcl-2, Hsp 70 and Hsp 90, which may contribute to growth inhibition and apoptosis in B/A-K562 cells in the background of stably bcr/abl RNAi mediated by letivirus.
In conclusion, lentivirus-mediated RNAi may lead to long-term silencing of bcr/abl, inhibits cell growth, and induces apoptosis in CML cell line, so it might prove to be a promising alternative approach in CML therapy targeting bcr/abl. Curcumin synergized with lentivirus-mediated bcr/abl RNAi may further inhibits cell growth amd hastens apoptosis in K562 cells. Therefore, Curcumin may be an ideal choice to synergize with bcr/abl targeting therapy
RNA干扰(RNAi)是一种转录后基因沉默(PTGS)的基因阻断技术,近年来被广泛应用于哺乳动物细胞抑制基因表达。通过RNase III内切酶Dicer的作用产生有活性的小的21-23nt干扰性RNA(short interence RNA, siRNA),介导其互补同源mRNA序列的特异性降解,沉寂目的基因的表达。
本研究采用慢病毒介导的RNAi技术,设计合成针对bcr/abl的siRNA,感染人慢性粒细胞白血病K562株,构建bcr/abl长期沉默的细胞模型。利用该细胞模型探讨bcr/abl基因长期沉默对K562细胞生物特性、相关信号分子及姜黄素抗肿瘤作用的影响。
一、慢病毒介导bcr/abl基因长期沉默的K562细胞株构建
为研究慢病毒介导的K562细胞bcr/abl基因RNA干扰的效率及稳定度,并为后续研究提供细胞模型,我们建立了bcr/abl RNAi稳定转染的B/A-K562模型。
研究方法(:1)利用RNAi技术和慢病毒转基因技术,构建pNL-EGFP-U6-bcr/abl-I质粒,使它和pVSV,pHelper组成慢病毒三质粒转染系统,共转染293T细胞,生产EGFP- B/A-I病毒;(2)用EGFP、EGFP-B/A-I病毒分别感染K562细胞,获取混合克隆;(3)极限稀释法、集落形成法挑取稳定表达EGFP、EGFP-B/A-I的克隆;(4)Realtime RT-PCR检测bcr/abl mRNA水平;(5)细胞免疫荧光、Western blotting法检测P210bcr/abl蛋白含量。
结果显示:(1)成功包装出具有感染能力的EGFP和EGFP- B/A-I慢病毒;(2)获得稳定表达EGFP和EGFP-B/A-I的EGFP-K562、B/A-K562细胞;(3)体外传代半年后,慢病毒介导RNAi可使bcr/abl mRNA下调63.5 %;(4)P210bcr/abl蛋白含量减少74.5 %。
二、Bcr/abl基因长期沉默对K562细胞生物学特性及相关基因表达的影响
bcr/abl基因是与CML发生、发展、转归密切相关的癌基因。bcr/abl基因长期“缺席”,可能导致K562细胞各种生物学特性及相关信号分子表达的变化。本部分,我们以bcr/abl基因长期沉默的B/A-K562细胞为模型,与正常对照的K562细胞及空载体对照的EGFP-K562细胞进行比较,分析其主要生物学特性及信号分子的变化。
研究方法:(1)锥虫蓝染色法检测细胞增殖能力变化;(2)集落形成法观察RNAi对集落形成能力的影响;(3)联苯胺染色观察细胞分化;(4)流式细胞仪分析细胞周期变化;(5)ELISA法检测酪氨酸激酶活性;(6)AO-EB、Heochst 33342染色观察细胞凋亡;(7)比色法检测Caspase-3、Caspase-9活性;(8)Western blotting法观察RNAi对细胞增殖凋亡相关信号分子的影响。
结果显示:(1)bcr/abl RNAi显著抑制K562细胞增殖,细胞倍增时间明显延长;(2)bcr/abl RNAi抑制细胞集落形成,集落形成抑制率达55.05 %;(3)bcr/abl RNAi诱导K562细胞向成熟红系分化,联苯胺染色阳性率达24.83 %;(4)bcr/abl RNAi使细胞周期阻滞在S期,出现凋亡的亚二倍体峰;( 5)bcr/abl RNAi使酪氨酸激酶活性降低约41.88 %;(6)bcr/abl RNAi诱导K562细胞凋亡,自发凋亡率达26.67 %;(7)bcr/abl RNAi触发线粒体Cyt-C释放,Caspase-3及Caspase-9活性提高,从线粒体途径诱导细胞凋亡;(8) bcr/abl RNAi下调K562细胞Src、P-Src、Raf-1、P-Erk1/2、P-Stat 1、P-Stat 5、P-P38、C-myc、Bcl-2和Hsp 90含量,上调P53、PKC及Bax含量,对Erk1/2、P-Akt、NF-КB、Hsp 70影响不大。
三、慢病毒介导bcr/abl基因长期沉默对K562细胞裸鼠成瘤的影响及相关机制研究
慢病毒介导的RNA干扰,在体外能实现bcr/abl基因的长期沉默,抑制K562细胞增殖,诱导其向成熟红系细胞分化,促进细胞凋亡。本部分我们关注的是:B/A-K562细胞在裸鼠体内能否继续保持对bcr/abl基因的RNA干扰以及成瘤能力和相关信号分子的变化。
研究方法:(1)皮下注射K562、EGFP-K562、B/A-K562细胞,建立裸鼠异种移植瘤模型;(2)观察成瘤情况,比较肿瘤体积;(3)称量离体瘤块质量、计算抑瘤率;(4)肿瘤组织测定Caspase-3、Caspase-9活性;(5)肿瘤组织冰冻切片观察荧光;(6)HE染色光镜下观察肿瘤组织的形态结构;(7)免疫组织化学检测肿瘤组织P210bcr/abl、Bcl-2、Bax蛋白表达;(8)免疫印迹检测肿瘤组织中Src、Erk1/2、P-Stat 1、Hsp 90的蛋白含量。
结果显示:(1)B/A-K562组肿瘤生长速度明显低于K562组及EGFP-K562组, K562组及EGFP-K562组成廇率100 %,B/A-K562组成瘤率80 %;(2)B/A-K562组抑瘤率达78.4 % (P<0.01);(3)B/A-K562组肿瘤组织Caspase-3、Caspase-9活性显著提高;(4)荧光显微镜观察,慢病毒感染的EGFP-K562及B/A-K562细胞形成的移植瘤均可见绿荧光;(5)免疫组化显示,K562组及EGFP-K562组P210bcr/abl、Bcl-2蛋白呈强阳性表达,Bax蛋白呈弱阳性表达;B/A-K562组P210bcr/abl、Bcl-2蛋白表达明显减弱,Bax蛋白表达明显增强;(6)免疫印迹结果显示,B/A-K562组Erk1/2、Src、P-Stat 1、Hsp 90含量低于K562组及B/A-K562组。
四、慢病毒介导K562细胞bcr/abl基因长期沉默对姜黄素抗肿瘤作用的影响及相关机制研究
bcr/abl基因及其相关蛋白是Cur作用的重要靶点。本部分,我们用Cur分别处理K562、EGFP-K562及B/A-K562细胞,探讨K562细胞的bcr/abl基因表达被慢病毒介导的RNA干扰阻断后,Cur针对K562细胞的抗肿瘤作用及作用机制有何改变。
研究方法:(1)MTT法、锥虫蓝染色法观察Cur对细胞增殖的影响;(2)集落形成法观察Cur对细胞集落形成的影响;(3)联苯胺染色法观察Cur对细胞分化的影响;(4)ELISA法观察Cur对酪氨酸激酶活性影响;(5)流式细胞仪分析Cur对细胞周期的影响;(6)AO-EB染色、DNA琼脂糖凝胶电泳检测Cur对细胞凋亡的影响;(7)分光光度法检测Cur对细胞Caspase-3和Caspase-9活性的影响;(8)Western blotting观察Cur对细胞增殖和凋亡有关信号分子及伴侣蛋白的影响。
结果显示:(1)Cur抑制K562、EGFP-K562及B/A-K562细胞增殖,呈时间和剂量依赖关系,Cur作用48小时,IC50分别为6.94、4.93、12.67μg/ml;(2)Cur影响B/A-K562细胞的集落形成,呈量效关系;(3)Cur诱导细胞向成熟红系细胞分化,其促进分化作用被bcr/abl RNAi减弱;(4)Cur降低细胞PTK活性,bcr/abl RNAi可增强其效应;(5)Cur诱导B/A-K562细胞凋亡亚二倍体峰出现,作用呈时间和剂量依赖性;(6)Cur有效诱导B/A-K562细胞凋亡,呈剂量依赖关系;(7)Cur进一步下调B/A-K562细胞P210bcr/abl含量;( 7)Cur促进B/A-K562细胞线粒体细胞色素C释放,活化Caspase-3、Caspase-9,循线粒体途径诱导凋亡;( 8) Cur上调B/A-K562细胞C-fas、P53、P-P38、Bax蛋白含量,下调P-Src、Raf-1、NF-КB、C-myc、PKC、Bcl-2、Hsp 70、Hsp 90蛋白含量。
从以上实验结果得出如下结论:
1.慢病毒介导的bcr/abl干扰能够实现bcr/abl基因长期沉默,bcr/abl mRNA下调63.5 %,P210bcr/abl下调74.5 %;
2. bcr/abl RNAi显著抑制K562细胞增殖,诱导其向成熟红系分化,显著降低PTK活性,循线粒体途径诱导K562细胞自发凋亡;
3. bcr/abl RNAi抑制K562细胞增殖,诱导凋亡,作用机制可能与bcr/abl沉默,Src、P-Src、Raf-1、P-Erk1/2、P-Stat 1、P-Stat 5、P-P38、C-myc、Bcl-2、Hsp 90下调,P53、Bax上调有关;
4.慢病毒介导的RNAi在裸鼠体内继续沉默bcr/abl基因,显著抑制肿瘤生长,诱导肿瘤组织凋亡和坏死,其抗肿瘤作用机制可能与bcr/abl RNAi及由之引起的Erk1/2、Src、P-Stat 1、Hsp 90、Bcl-2下调,Bax上调有关;
5. Cur抑制B/A-K562细胞增殖,诱导分化,降低PTK活性,循线粒体途径诱导凋亡;
6. Cur进一步降低B/A-K562细胞P210bcr/abl含量,上调C-fas、P53、P-P38、Bax,下调P-Src、Raf-1、NF-КB、C-myc、PKC、Bcl-2、Hsp 70、Hsp 90,可能与Cur在bcr/abl沉默的背景下,诱导B/A-K562细胞凋亡,抑制增殖有关。
总之,慢病毒介导的bcr/abl RNAi能够实现bcr/abl基因的长期沉默,抑制CML细胞增殖,诱导凋亡,作为CML的分子靶向治疗具有潜在应用价值。Cur可在bcr/abl RNAi基础上进一步下调P210bcr/abl,抑制增殖,诱导凋亡,是配合bcr/abl基因靶向治疗理想选择。
Chronic Myelogenous Leukemia(CML) originates in a pluripotent hematopoetic stem cell of the bone marrow, which is characterized by the Philadelphia(Ph) chromosome and bcr/abl oncogene. The bcr/abl fusion gene encoded Bcr/Abl protein, which possesses high activity of tyrosine kinase was known to be a resistant factor for apoptosis, cell differentiation and chemotherapy drugs resistance in chronic myelogenous leukemia (CML).
RNA interference is a kind of gene-blocking technology about post-transcriptional gene silence (PTGS), which has been extensively applied to the inhibition of gene expression in the mammal cells in recent years. Compt short interference RNA (21-23nt) is obtained by the incision enzyme RNaseⅢ(Dicer), which mediate specific degradation of its homologous and complimentary mRNA, to silence the expression of target genes.
In this study, K562 cells were infected by lentivirus carrying bcr/abl RNAi fragment, and further bcr/abl stable-silencing CML cellular model was constructed. As a result of long-term absence of bcr/abl, changes of biological characters, correlated signaling molecules and anti-tumor effects of curcumine were detected.
Construction of lentivirus-mediated bcr/abl stable-silencing cellular model on K562 cells
In this part, we constructed bcr/abl RNAi stable-transfecting cellular model B/A-K562, and detected the efficiency and stability of lentivirus-mediated RNA interference.
Methods: (1) Plasmids pNL-EGFP-U6-brcr/abl-I was constructed, which was combined with pVSVG and pHelper to constitute vector system of three plasmids. The lentiviral vector system was transfected into 293T cell to produce EGFP- B/A-I lentivirus. (2) K562 cells were infected with both EGFP and EGFP- B/A-I lentivirus. (3) Ultimate dilution and colony forming methods were used to pick clones stably expressing EGFP and EGFP-B/A-I fragment. (4) bcr/abl mRNA level was detected by Realtime RT-PCR; (5) Cello-immunofluorescence, western blotting were used to measure the contents of P210bcr/abl.
The results showed: (1) EGFP and EGFP- B/A-I lentivirus with processing infection ability were successfully produced; (2) EGFP-K562, B/A-K562 cells stably expressing EGFP and EGFP-B/A-I fragment were successfully picked; (3) After cultured in vitro for six months, bcr/abl mRNA lever was down regulated by lentivirus-mediated RNAi for 63.5 %; (4) Compared with K562 cells, P210bcr/abl content was obviously decreased by 74.5 % in B/A-K562 cells.
The effect of bcr/abl gene stable-silencing on the biological characters and relevant signaling molecules in K562 cell line
Since bcr/abl plays an important role in the generation, development and turnover of human chronic myelogeous leukemia, many changes in biological characters and cell signaling molecules may be detected in human CML cell line K562 as a result of long-term absence of bcr/abl gene. This study focused on the variations of biological characters and cellular signals in bcr/abl stable-silencing cell model B/A-K562 compared with K562 and EGFP-K562 cells as normal control and vector control respectively.
Methods: (1) Trypan blue exclusive staining was used to access the cell growth arrest; (2) Colony assay was used to observe the CFUs; (3) Benzidine staining was used to detect cell differentiation; (4) FCM was used to analyze cell cycle; (5) ELISA method was utilized to detect the activity of PTK; (6) AO-EB and Heochst 33342 fluorescent staining was used to observe apoptosis; (7) Chromometry was to used measure the activation of Caspase-3 and Caspase-9; (8) Western blot was used to observe the effects of RNAi on the signaling molecules which are relevant with cell growth and apoptosis.
The results showed: (1) bcr/abl RNAi significantly inhibited K562 cell proliferation, doubling generation time was obviously prolonged; (2) Colony forming was arrested by RNAi; (3) Positive rate of benzdine staining was 24.83 %, low level of bcr/abl message leads to enhanced erythroid differentiation; (4) RNAi arrested cell cycle in S phase and induced apoptosis in hypodiploid peak; (5) PTK activity was descended by 41.88 % ; (6) Apoptosis was observed with 41.88 % of apoptosis rate; (7) bcr/abl RNAi triggered the release of mitochondrial Cyt-C, increases Caspase-3 and Caspase-9 activities, induces apoptosis in mitochondrial-depended pathway; (8) bcr/abl RNAi down-regulated Src, P-Src, Raf-1, P-Erk1/2, P-Stat 1, P-Stat 5, P-P38, C-myc, Bcl-2 and Hsp 90 level, and up-regulated P53, PKC and Bax level, but had no effect on the concentration of Erk1/2, P-Akt, NF-КB, Hsp 70 in K562 cells.
The effect of lentivirus-mediated bcr/abl stable-silencing on the tumor formation on K562 cell xenograft in athymic mouse
Lentivirus-mediated RNA interference could reduce the expression of bcr/abl gene in vitro, inhibit cell growth, induce differentiation and hasten apoptosis. In this part, we mainly studied RNAi efficiency in B/A-K562 cells athymic mouse transplantation tumor, tumor formation ability and relevant signaling molecules.
Methods: (1) K562, EGFP-K562, B/A-K562 cells was injected subcutaneously in order to establish athymic mouse heterogeneity transplantation tumor models; (2) Volume of the tumors were measured per 5 days and the tumor formation rates were calculated; (3) At the end of time points, the mice were killed, the tumor xenografts were removed and measured; (4) Activation of Capspase 3, Caspase-9 were detected; (5) Green fluorescence was observed in frozen section of tumor tissue by fluorescence microscope; (6) H&E staining was used to study the pathological change of tumor tissue; (7) Immunohistochemistry was used to investigate the P210bcr/abl, Bcl-2, Bax level in tumor tissue; (8) The protein was extracted from the tumor tissues, western blotting was used to analyze Src, Erk1/2, P-Stat1 and Hsp 90 protein level.
The results showed: (1) Growth velocity of B/A-K562 cell xenograft was obviously lower than that of K562 group and EGFP-K562 group; (2) Tumor formation rate of B/A-K562 group was 80 % lower than that of K562 and EGFP-K562 group, which was 100%; (3) The tumor inhibition rate in B/A-K562 group was 78.4 % (compared with K562 group, P<0.01); (4) The activation of Caspase-3, Caspase-9 was elevated in B/A-K562 cell xenograft; (5) Green fluorescence was confirmed in transplantation tumor of lentivirus infected EGFP-K562 and B/A-K562 cells; (6) The results of immunohistochemistry showed decreased P210bcr/abl, Bcl-2 expression and increased Bax expression in B/A-K562 group, compared with K562 and EGFP-K562 group; (7) The results of Western blotting showed Erk1/2、Src、P-Stat 1、Hsp 90 protein level of B/A-K562 group were lower than that of K562 and EGFP-K562 group.
The effect of lentivirus-mediated bcr/abl stable-silencing on anti-tumor action of curcumin and relevat mechanisms in K562 cells
bcr/abl gene and P210bcr/abl protein were important targets of Curcumin. In this part, K562, EGFP-K562 and B/A-K562 cells were treated with Curcumin in different concentration. We investigated anti-tumoral action and relevat mechanisms of curcumine when bcr/abl gene expression was blocked by lentivirus-mediated RNA interference.
Methods: (1) MTT method and trypan blue exclusive staining were used to observe the cell growth arrest; (2) Colony assay was used to the cell colony forming units(CFUs); (3) Benzdine staining was utilized to investigate cell differentiation; (4) ELISA was used to measure the activity of PTK; (5) Flow cytometry (FCM) was used to analyze cell cycle; (6) AO-EB staining and DNA agarose gel electrophoresis were used to detect cell apoptosis; (7) Absorption spectrometry was used to detect activation of Caspase-3 and Caspase-9; (8) Western blotting was used to analyze the impact of Curcumine on Cell proliferation apoptosis-related signaling molecules and partner protein.
The results showed: (1) Curcumin inhibited K562, EGFP-K562 and B/A-K562 cells proliferation in a time-and-dose-depended manner. IC50 values of K562, EGFP-K562 and B/A-K562 cells were 6.94, 4.93, 12.67μg/ml after 48 h of Cur exposure; (2) Cur inhibited B/A-K562 CFUs in an dose-depended manner; (3) Cur induced differentiation towards mature erythroid cells and its role in the promotion of differentiation could be weakened by bcr/abl RNAi; (4) Cur reduced PTK activity in B/A-K562 cells and bcr / abl RNAi may enhance such effect; (5) Cur induced apoptosis in hypodiploid peak in a time-and-dose-depended manner; (6) Cur effectively induced B/A-K562 cells apoptosis in a dose-depended manner; (7) Cur triggered the release of mitochondrial Cyt-C, increased Caspase-3 and Caspase-9 activities, inducesd apoptosis in mitochondrial-depended pathway in B/A-K562 cells; (8) Cur up-regulated C-fas, P53, P-P38 andBax level, and down-regulated P-Src, Raf-1, NF-КB, C-myc, PKC, Bcl-2, Hsp 70, and Hsp 90 level in B/A-K562 cells.
CONCLUSIONS:
1. Lentivirus-mediated RNA interference may stably silence bcr/abl gene, which brings down bcr/abl mRNA and P210bcr/abl by respectively 63.5 % and 74.5 %.
2. Lentivirus-mediated stable-silencing of bcr/abl inhibits proliferation, induces differentiation towards mature erythroid cells, significantly decreases activity of PTK and hastens apoptosis in mitochondrial-depended pathway in K562 cells.
3. Lentivirus-mediated stable-silencing of bcr/abl inhibits K562 cells growth and induces cell apoptosis which may be correlated with the down-regulation of P210bcr/abl, Src, P-Src, Raf-1, P-Erk1/2, P-Stat 1, P-Stat 5, P-P38, C-myc, Bcl-2, Hsp 90 and up-regulation of P 53 and Bax.
4. Effect of bcr/abl RNAi retains in athymic mouse transplantation tumor,which significantly inhibits tumorous growth, induces apoptosis and necrosis in tumor tissue. Its anti-tumor effect may correlated with down-regulation of signaling molecules related with cell growth and apoptosis such as Erk1/2, Src, P-Stat 1, Hsp 90, Bcl-2 and up-regulation of Bax.
5. Cur inhibits cell growth, induces differentiation, reduces PTK activity, and hastens apoptosis in mitochondrial-depended pathway in B/A-K562 cells.
6. Cur decreases concentration of P210bcra/bl in B/A-K562 cells, up-regulates C-fas, P53, P-P38 and Bax, down-regulates P-Src, Raf-1, NF-КB, C-myc, PKC, Bcl-2, Hsp 70 and Hsp 90, which may contribute to growth inhibition and apoptosis in B/A-K562 cells in the background of stably bcr/abl RNAi mediated by letivirus.
In conclusion, lentivirus-mediated RNAi may lead to long-term silencing of bcr/abl, inhibits cell growth, and induces apoptosis in CML cell line, so it might prove to be a promising alternative approach in CML therapy targeting bcr/abl. Curcumin synergized with lentivirus-mediated bcr/abl RNAi may further inhibits cell growth amd hastens apoptosis in K562 cells. Therefore, Curcumin may be an ideal choice to synergize with bcr/abl targeting therapy
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