SHIP基因在白血病发病机制中作用的研究
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摘要
背景:白血病是原发于造血系统的恶性肿瘤。近年来,其发病率呈增加趋势。在35岁以下人群中,白血病的死亡率占恶性肿瘤第一位,严重危害中青年人的生命,所以值得特别重视。急性白血病的发生包括了复杂的基因突变、缺失、癌基因激活及抑癌基因失活等多种分子生物学水平的异常。尽管许多基因在白血病发病中的作用及其机制已初步明确,但所有的基因异常仍不能解释全部白血病的发生机制,因此,寻找新的白血病相关基因并研究其生物学功能一直是白血病研究领域的热点之一,不仅有助于阐明白血病发生发展的机制,而且可能找到一些治疗靶位点。
SHIP基因是继PTEN之后发现的仅在造血细胞表达的一种新的肌醇磷酸酶,其主要功能是降解PIP3成为PI3,4P2而负调控PI3K/Akt路径。本课题组多年来一直围绕SHIP基因功能与白血病发生发展关系开展研究工作,取得了一系列有意义的研究成果。我们首次报道急性白血病患者存在SHIP基因突变,并证实SHIP基因低表达和表达缺失与人白血病细胞恶性增殖密切相关,通过干扰RNA封闭K562细胞的bcr/abl融合基因使SHIP蛋白重新表达并可以逆转细胞的异常增殖表型,近年来也陆续发现和报道了一些白血病细胞中新的SHIP基因突变。这些都提示SHIP基因具有调控白血病细胞增殖、凋亡的结构基础。但是,有关SHIP基因调控白血病细胞系K562的PI3K/Akt信号传导通路的分子机理及其在抑制和/或逆转K562细胞增殖能力中的地位,以及SHIP基因突变对PI3K/Akt的调控有何改变等问题,目前国内外均无报道。本研究的目的是:(1)探讨野生型SHIP基因对人白血病细胞系K562中PI3K/Akt信号通路相关蛋白表达水平的影响;(2)构建携带突变型SHIP基因的慢病毒载体并转染K562细胞系;(3)探讨SHIP基因突变后在K562细胞中的功能变化,为进一步研究SHIP基因在白血病发病机制中的作用提供实验依据。
第一部分野生型SHIP基因对白血病细胞系K562增殖凋亡的实验研究
目的探讨肌醇5′磷酸酶基因SHIP对人白血病细胞系K562增殖、凋亡及细胞周期的影响,并对其可能的作用机制进行初步探讨。
方法将携带有野生型SHIP基因和空载体的慢病毒表达载体转染人白血病细胞系K562。MTT检测细胞生长情况;流式细胞仪检测细胞转染效率和细胞周期分布;显微镜及透射电镜下观察细胞形态及超微结构改变,TUNEL、Hoechst33342荧光染色、细胞集落形成等实验检测细胞增殖及凋亡情况。实时荧光定量PCR(FQ-PCR)检测转染前后SHIP mRNA表达变化, Western Blot检测各转染组SHIP、Akt、p-Akt308、p-Akt473、Bcl-2、Bax、Bad、Bcl-xL、CyclinD1、p27kip1、p21WAF1、NFκB蛋白水平变化。Caspase活性检测试剂盒检测Caspase-3、Caspase-9活性。NFκB活性检测试剂盒检测NFκB活性变化。
结果
1转染48 h后,流式细胞仪检测慢病毒载体转染K562细胞效率为(74.6±5.8)%。FQ-PCR和Western blot显示,转染SHIP基因后K562细胞中SHIP呈高效表达,表明转染成功。
2转染野生型SHIP基因的K562细胞表现为增殖受抑,增殖指数减低,克隆形成能力降低,DNA合成减少和凋亡增多,与转染空载体组和未转染组相比有统计学差异(P<0.01)。
3野生型SHIP基因过表达导致细胞周期G0/G1期至S期和G2/M期阻滞。转染第5天,K562/wtSHIP细胞的G0/G1期百分数(60.69±8.3)%明显高于不表达SHIP的K562/FIV细胞(37.48±6.78)%和空白对照组(35.5±4.05)%。
4转染野生型SHIP基因的K562细胞Akt308和Akt473磷酸化水平(分别为:0.125±0.016,0.351±0.064)明显低于K562/FIV细胞(Akt308:0.323±0.025,Akt473:0.788±0.102)和未转染组(Akt308:0.324±0.023,Akt473:0.801±0.095)( P<0.01),后两组间Akt-308和Akt473磷酸化水平均无统计学差异(P>0.05)。
5野生型SHIP基因抑制IL-3诱导的K562细胞增殖,发现外源性野生型SHIP蛋白表达组K562细胞p-Akt308和p-Akt473水平增加程度明显低于对照组呈下降趋势,而总Akt表达水平无明显变化;撤除IL-3刺激后,K562/wtSHIP组细胞Akt磷酸化水平快速下降,而转染空载体组细胞Akt磷酸化水平下降缓慢,持续时间较长。
6转染野生型SHIP基因的K562细胞能明显增加p27蛋白(0.302±0.059)和p21蛋白(0.335±0.027)表达,而cyclin D1蛋白表达(0.142±0.019)明显降低,转染空载体组的K562细胞p27、p21和cyclin D1蛋白的表达(p27:0.159±0.023;p21:0.188±0.032;cyclin D1:0.221±0.031)与未转染组(p27:0.152±0.022;p21:0.182±0.034;cyclin D1:0.228±0.033)相比无明显改变。
7野生型SHIP基因对凋亡相关蛋白表达的影响
7.1转染SHIP基因的K562细胞出现caspase-9和caspase-3酶活性增加[caspase-9 : 32.82±5.80 nmol/(h·mg) ; caspase-3 :16.80±1.98nmol/(h·mg)],转染空载体组的K562细胞caspase-9和caspase-3活性[caspase-9: 8.75±2.66nmol/(h·mg);caspase-3:5.48±1.31nmol/(h·mg)]与未转染组[caspase-9 : 8.22±1.25nmol/(h·mg) ; caspase-3 :5.27±0.58nmol/(h·mg)]相比无显著改变;以上结果表明野生型SHIP基因过表达可激活K562细胞caspase-9和caspase-3。
7.2转染野生型SHIP基因能明显下调K562细胞bcl-xL蛋白表达(K562/wtSHIP组:0.109±0.015;K562/FIV组:0.385±0.043;未转染组:0.392±0.049);显著上调促凋亡蛋白bad表达(K562/wtSHIP组:1.018±0.216;K562/FIV组:0.315±0.052;未转染组:0.304±0.037);K562/wtSHIP组bax蛋白表达( 0.348±0.037 )高于K562/FIV组(0.342±0.043)和未转染组(0.339±0.032),但三组间无统计学意义(P>0.05);各组bcl-2表达无显著性差异(P>0.05)。
7.3转染野生型SHIP基因下调K562细胞NF-κB表达(K562/wtSHIP:0.483±0.027;K562/FIV组:0.924±0.081;未转染组:0.937±0.075);各组IκBа表达水平无显著性差异(P>0.05)。K562/wtSHIP组NF-κB的活性随时间延长而降低,K562/FIV组和未转染组细胞无显著性变化。
结论过表达野生型SHIP基因明显抑制白血病细胞系K562增殖,促进K562细胞凋亡,其机制可能通过抑制Akt磷酸化,进一步影响其下游与多种凋亡相关分子家族蛋白如Bcl-2家族、Caspase家族表达,并通过调控细胞周期相关蛋白表达使细胞周期阻滞在G0/G1期。
第二部分SHIP基因突变体的构建
目的急性白血病患者存在SHIP基因突变,可能与白血病发病有关,构建SHIP基因突变体(muSHIPP28L),为研究突变基因的表达和功能与白血病的发病机制奠定基础。
方法以重组表达质粒wtSHIP为模板,用Stratagen Mutant Kit试剂盒通过PCR定点突变技术对SHIP编码序列TTC进行定点突变→CTC,突变体经测序鉴定正确,连接到慢病毒载体Receiver-Lv31(ORF不带标签)。经重组慢病毒包装,病毒滴度测定,感染K562细胞,采用FQ-PCR和Western blot方法检测SHIP mRNA和蛋白表达。
结果
1构建的突变型SHIPP28L经DNA测序验证,其碱基序列与设计序列完全一致。
2感染293T细胞48 h后在荧光显微镜下观察到GFP较强表达。制备的病毒滴度达4.9×106 pfu/ml。
3 FCM分析表达GFP绿色荧光的阳性细胞百分率,计算活细胞转染效率。悬浮培养的人白血病细胞系K562细胞转染效率分别为K562/wtSHIP:74.6%,K562/FIV:82.5%,K562/muSHIP:76.3%。
4 FQ-PCR和Western blot方法检测转染野生型和突变型SIHP基因的K562细胞中SHIP基因高表达,而K562/FIV组和未转染K562细胞中内源性SHIP基因表达极低,说明外源性野生型和突变型SHIP转染成功。
结论运用基因定点突变基因重组技术成功构建了SHIP基因P28L突变体,为进一步探讨SHIP基因与白血病发病机制创造条件。
第三部分SHIP基因突变体在白血病细胞系K562细胞中的表达及其功能的研究
目的将构建的SHIP基因突变体稳定表达于人白血病细胞系K562中,观察SHIP基因突变后在K562细胞中的功能变化,为进一步研究SHIP基因在白血病发病机制中的作用提供实验依据。
方法FQ-PCR和Western blot鉴定野生型和突变型SHIP基因在K562细胞中的表达;MTT比较K562/wtSHIP和K562/muSHIP两组细胞生长情况;流式细胞分析比较野生型和突变型SHIP基因对细胞周期的不同影响;Hoechst33342荧光染色比较细胞凋亡情况。Western Blot比较转染野生型和突变型SHIP基因后对K562细胞Akt、p-Akt308、p-Akt473、Bcl-2、Bax、Bad、Bcl-xL、CyclinD1、p27kip1、p21WAF1、NFκB蛋白表达的调控。Caspase活性检测试剂盒检测各组Caspase-3、Caspase-9活性。利用高压液相色谱分析比较各转染组PIP3和PI3,4P2水平。
结果
1野生型SHIP基因明显抑制K562细胞生长,而SHIP基因突变体丧失了这种功能。流式细胞仪分析结果表明K562/wtSHIP细胞主要表现为G0/G1期细胞增多、G2/M期细胞减少,PI降低。突变型SHIP基因对K562细胞周期无明显影响。
2 Hoechest染色发现转染野生型SHIP基因的K562细胞早期凋亡率[(38.4±3.08)%]明显高于转染SHIP基因突变子(P28L)[(8.4±1.11)%]和空载体组[(8.06±0.95)%]。SHIP基因突变子不能促进细胞凋亡。
3 SHIP基因突变体不能下调K562细胞p-Akt308和p-Akt473的磷酸化水平(p-Akt308:0.334±0.031;p-Akt473:0.826±0.163)。
4转染野生型SHIP基因的K562细胞周期蛋白p21和p27蛋白水平显著增高,cyclin D1水平明显降低,但转染SHIP基因突变体的K562细胞中细胞周期蛋白表达无上述改变。
5 K562/muSHIP组细胞caspase-3和caspase-9酶活性显著低于K562/wtSHIP组(P<0.05),与K562/FIV组和未转染组相比无统计学差异(P>0.05)。
6转染野生型SHIP基因组K562细胞bcl-xL表达降低,而SHIP基因突变体对K562细胞bcl-xL表达(0.367±0.028)无影响;K562/muSHIP基因组和未转染组、K562/FIV组间无显著性差异(P>0.05)。K562/muSHIP组细胞bad蛋白表达水平(0.322±0.063)较K562/wtSHIP组低,与K562/FIV组和未转染组相比无显著性差异(P>0.05)。K562/muSHIP组细胞bcl-2表达水平(0.155±0.035)较K562/wtSHIP组、K562/FIV组和未转染组间无统计学意义(P>0.05)。Bax在K562/wtSHIP组细胞表达水平略高于K562/muSHIP组(0.329±0.025)但无统计学差异(P>0.05)。
7应用高压液相色谱分析结果显示:转染wtSHIP基因组K562细胞中PI(3,4,5P3水平下降,显著低于转染muSHIP组、转染空载体组和未转染组PIP3的水平(P<0.01),后三组间PIP3水平无显著性差异(P>0.05)。K562/wtSHIP组K562细胞PI(3,4)P2水平明显高于K562/muSHIP组、K562/FIV组和未转染组K562细胞PIP2水平(P<0.01),后三组细胞间PI3,4P2水平相比无统计学意义(P>0.05)。
结论
1 SHIP基因突变体SHIPP28L导致其增殖抑制及促凋亡功能丧失。
2与野生型SHIP基因相比,SHIPP28L突变体导致其对K562细胞caspase-3、caspase-9和NF-κB活性的调节作用丢失。
3 SHIP基因调控PI3K/Akt信号通路蛋白表达可能是其抑制人白血病细胞系K562增殖的重要分子机理之一,SHIP基因对K562细胞增殖的负调控作用及促凋亡作用有赖于其基因结构和功能正常。
Background: Leukemia is the most common malignant clone’s disease of hematopoietic stem cells. The incidence rate of leukemia has the tendency of step-up recent years. Leukemia is the consequences of a multistep process resulting from a series of genetic changes and incidence, and its process is related with activation of oncogene or inactivation of antioncogene. Altohugh many genes’function and machanisms in leukemia has been explicit, all novel gene can not explain all pathogenesis of all leukemia. Thereby, it has become one of the hotspots to investigate leukemia by identifying new correlative oncogene or tumor suppressor genes and studying its biologic function,and it is important for elucidate the pathogenesy of leukemia.
SH2-containing inositol 5’-phosphatase (SHIP) is a recently identified lipid phosphatase after phosphatase and tensin homologue deleted on chromosome 10(PTEN) was observed, which hydrolyzes the 5 prime phosphate of the inositol ring from inositol 3,4,5-P3. It is mainly expressed in hematopoietic cells. Its phosphatase activity toward PIP3 has been linked to suppress several signaling pathways, including Akt, leading to the inhibition of hematopoietic cell proliferation and survival. Our previous researching works have proved that low expression and deletion of SHIP gene was closely correlated with the abnormal proliferation of patients with leukemia; lower Akt activation was detected in K562 cell lines, which was caused by the re-expresion SHIP. transfection of wild type SHIP into the K562 cells can regulate the expression of proliferative relative genes and reverse the proliferative phenotype of K562. The molecular mechanism that SHIP gene suppress leukemia may be involved in some important signal pathways. Up to now, However, there are no any reports about the exact mechanisms of SHIP gene on the regulation of PI3K/Akt signal transduction pathway, the effects of SHIP gene on suppression and/or reversion of abnormal proliferation in human leukemia cell line K562 in the world. There are also no any reports about what changes of the PI3K/Akt signal transduction regulation will happened if SHIP has site mutation in K562 cell lines. The aim of this study are as follows: (1)To detect the influence of cell proliferation and apoptosis of K562 cells transfected with wild-type SHIP gene, and to investigate the molecular mechanism of apoptosis of K562 cells transfected with wtSHIP gene; (2) To construct SHIP gene’s mutant (P28L) found in AML patients. (3) To investigate the functional changes of SHIP gene’s mutant by over-expression it into human leukemia cell line K562,and to make a preliminary discussion on the mechanism of SHIP gene abnormality in leukemia.
Part one: Effects of wild-type SHIP on proliferation, apoptosis and cell cycle of K562
Objective To investigate in vitro the effects of SH2-containing inositol 5’-phosphatase gene-SHIP on proliferation, apoptosis, cell cycle of human leukemia cell line K562 and to explore its possible machinesim preliminarily.
Method Based on the results of our previous studies, with different human leumemia cell lines K562 as study samples, including K562, K562/wtSHIP, K562/FIV; the growth of K562 was observed by MTT assay; the transfection efficiency and cell cycle distribution were assessed by flow cytometry (FCM). Morphological characteristics of light microscope,cell colony fomation,TUNEL and Hoechest 33342 fluorescent staining method were also used to test proliferation and apoptosis of K562 cells. The expression level and difference of SHIP mRNA was detected by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR); the the expression level and difference of SHIP, Akt, p-Akt473, p-Akt308 and the member of apoptosis family Bcl-2, Bcl-xL, Bax, Bad, cell cycle related molecular CyclinD1, p21WAF1 and p27Kip1 were detected by Western blot; Caspase-3, Caspase-9 protein activity were detected by caspase activity kits.
Results
1 The transfection efficiency was about 74.6±5.8% in K562 cell line after transfected with lentivirus on 48 h detected by flow cytometry. The recombinant lentivirus pReceiver-LV-SHIP has been successfully transfected into K562 cell line.
2 The decreased ability of proliferation and DNA synthesis, cell colony fomation ability and enhanced apoptosis rate were observed in K562 cells transfected with wild-type SHIP, and the same changes had not been observed in K562 cells transfected with empty vector and untransfected K562(P<0.01).
3 Cell cycle distribution showed the ratio of G0/G1 phase of K562 cells transfected with wild-type SHIP gene increased from (34.15±8.30)% to (60.69±8.30)%, the ratio of G2/M+S phase decreased form(65.85±9.36)% to (38.85±7.09)%, the cell cycle was arrested at G0/G1 phase.
4 After transfection wtSHIP at the third days, western blot results showed the Akt expression levels had no change but p-Akt308 and p-Akt473 expression level (0.125±0.016 and 0.351±0.064 respectively) decreased compared with K562/FIV group ( Akt308 : 0.323±0.025 , Akt473 :0.788±0.102) and untransfected group(Akt308:0.324±0.023,Akt473:0.801±0.095)( P<0.01).
5 wtSHIP gene can inhibit the proliferation of K562
6 After K562 cells transfected wtSHIP, the cell cycle related molecular expression levels showed that the protein expression levels of p27、p21 (0.302±0.059、0.335±0.027 respectively) increased compared with K562 cells transfected with empty vector (p27 kip1:0.159±0.023;p21WAF1:0.188±0.032) and untransfected group (p27Kip1:0.152±0.022;p21WAF1:0.182±0.034) (P<0.05), but the protein expression of CyclinD1 (0.142±0.019)decreased (P<0.05).
7 The effects of wtSHIP on apoptosis related proteins in human leukemia cell line K562
7.1 After transfection wtSHIP at the third days, the caspase-9 and capsase-3 activity[caspase-9:32.82±5.80nmol/(h·mg); caspase-3: 16.80±1.98nmol/(h·mg)] up-regulated compared with K562/FIV group [caspase-9: 8.75±2.66nmol/(h·mg); caspase-3: 5.48±1.31nmol/(h·mg)] and untransfected K562 cells caspase-9: 8.22±1.25nmol/(h·mg);caspase-3: 5.27±0.58nmol/(h·mg)] (P<0.05).
7.2 The bcl-xL protein expression level in K562/wtSHIP group was lower than K562/FIV group and untransfected K562 (K562/wtSHIP: 0.109±0.015; K562/FIV: 0.385±0.043; untransfected group: 0.392±0.049)(P<0.05). The protein expression levels of Bad in K562/wtSHIP was significantly higher than that of K562/FIV and untransfected K562, but no significant difference of protein expression level of Bad was observed among K562/FIV and untransfected group(K562/wtSHIP: 1.018±0.216; K562/FIV: 0.315±0.052; untransfected group: 0.304±0.037). No significant difference of protein expression levels of bax and bcl-2 was observed among K562/wtSHIP、K562/FIV and untransfected K562.
7.3 In untransfected group, FIV-GFP group, and wtSHIP-GFP group the protein expression of NF-κB (p65) was significantly down-regulated than that in K562/FIV and untransfected group(K562/wtSHIP:0.483±0.027;K562/FIV组:0.924±0.081;untransfected group:0.937±0.075)(P<0.05); and no significant difference of IκBаprotein level was observed among K562/wtSHIP、K562/FIV and untransfected K562.
Conclusions
1 This part of study indicated that over expression wild-type SHIP gene could remarkably suppress proliferation of K562 cells, and induce K562 cell apoptosis.
2 The effects of wild type SHIP gene on K562 probably via inhibiting the phosphorylation of Akt, and regulating its downstream target protein such as apoptosis related family expression such as Bcl-2 family, Caspase family and the cell cycle related melbourne expression.
Part two: Contruction of a lentiviral vector carrying human SHIP mutated gene and its expression in K562 cell line
Objective To construct lentiviral vector carrying human SHIP gene and investigate its expression in K562 cell line.
Method Full-length cDNAs of wild type SHIP gene (wt-SHIP) was amplified from the human chondrocytes by RT-PCR, and site-directed mutagenesis was used to obtain full-length cDNAs of the mutated SHIP gene (mut28P/L). The products was cloned into p-Receiver-LV31vector and their sequences was analysed, then subcloned into lentivirus vector pReceiver-LV31, the inserted fragments were analysed and reconfirmed by restriction endonuclease analysis and sequencing. SHIP-lentivirus gene containing green fluorescent protein gene (SHIP-GFP) or the empty vector (FIV-GFP) was transfected into K562 cells. The green fluorescence protein expression of lentivirus plasmids were observed using laser scanning microscopy after 48 hours of transfection. The mRNA expression levels of wtSHIP, muSHIP gene were detected by FQ-PCR. The protein expression levels of wtSHIP and muSHIP were detected by western blot. .
Results
1 By restriction endonuclease analysis and sequencing, the sequences of SHIPP28L was consistent with that in literature, and the opening frames were matched with the vector sequence.
2 Green fluorescent can be seen in transfected 293T after transfection at 48 h. The virus titer was 4.9×106 pfu/ml.
3 Green fluorescence signal was distributed throughout K562 cells transfected with wtSHIP and muSHIP vector.The transfection efficiency was K562/wtSHIP-74.6%、K562/FIV-82.5%、K562/muSHIP-76.3%, respectively after transfected with lentivirus on the second day.
4 SHIP mRNA and protein were at the highly expression levels in K562/wtSHIP and K562/muSHIP, but in K562/FIV and untransfected group SHIP mRNA and protein level was extremely low, which shown that is transfected into K562 successfully.
Conclusions
1 Lentivirus expression vector of SHIP gene’s mutant of P28L was successfully constructed by using site-directed mutagenesis of SHIP cDNA.
2 It was proved that the sequence of the SHIPP28L mutant was correct DNA sequencing.
3 Green fluorescent、FQ-PCR and Western blot can be seen in transfected K562 with muSHIP-GFP and wtSHIP-GFP, which shown that it was transfected into K562 successfully.
Part three:The study of mutation of SHIP in molecular pathogenesis of leukemia
Objective There are two categories of mutations or gene rearrangements which play important role in molecular mechanisms of leukemogenesis. One class of mutations or gene rearrangements involving mostly tyrosine kinase gene such as BCR/ABL of chronic myelogeous leukemia (CML) confer a proliferative and/or survival advantage to hematopoietic progenitors, and second class of mutations involving mostly transcription factor associated with hematopoietic cell differentiation such as PML/RAR mutation serve primarily to impair hematopoietic differentiation and subsequent apoptosis of cells. Though FLT3-ITD mutation and PML/RAR in acute promyelocytic leukemia (APL) patients as well as c-KIT mutation and AML1/ETO in AML-M2b patients have been reported, the molecular defect involving two categories of mutation are largely unidentified. In this part we focused on elucidate the role of SHIP mutation in leukemogenesis, its association with biological characters of leukemia and its potential in developing new target therpy.
Method The recombined lentivirus plasmids muSHIPP28L was transfected stably into human leukemia cells K562. The cell proliferation, cell life cycle and cell apoptosis of K562/muSHIP cells were determined by MTT, fluorescent staining and flow cytometry. Differentially expressed genes were reconfirmed by Western blot.
Results
1 The enhanced ability of proliferation and DNA synthesis, and decreased apoptosis rate was observed in K562 cells transfected with muSHIPP28L, and the same changes had been observed in K562/FIV and untransfected K562 cells. Increased G0/G1 cells was observed in K562/wtSHIP cells by flow cytometry result, but no such a difference was observed in K562/muSHIP.
2 The early apoptosis rate in K562/muSHIP(P28L) group[(8.4±1.11)%] was significantly lower than that in K562/wtSHIP group[(38.4±3.08)%] by Hoechest stainging, no significant difference of the early apoptosis rate was observed between K562/muSHIP and K562/FIV[(8.06±0.95)%]. The SHIP mutant could not induce K562 cell apoptosis.
3 SHIPP28L mutant can’t down-regulate phosphorylation of Akt308 and 473(p-Akt308:0.334±0.031;p-Akt473:0.826±0.163).
4 The cell cycle related molecular expression levels of p21WAF1 and p27KIP1 in K562 cells transfected with wtSHIP were significantly up-regulated, but the protein expression of cyclin D1 down-regulated; no changes indicated above in K562/muSHIP.
5 The activity of caspase-3 and capase-9 in K562/muSHIP group were significantly lower than those in K562/wtSHIP group(P<0.05), no significant difference was seen between K562/FIV group and untranfected group(P>0.05).
6 The protein expression of bcl-xL decreased in K562/wtSHIP group, but no changes indicated above in K562/muSHIP(0.367±0.028). The expression of bad protein in K562/muSHIP group (0.322±0.063) was lower than that in K562/wtSHIP group , no changes indicate among K562/muSHIP、K562/FIV group and untransfected group(P>0.05). There was no significant difference of bcl-2 expression was observed among K562/muSHIP group、K562/wtSHIP group、K562/FIV group and untransfected group ( P > 0.05 ) . The expression of bax protein in K562/wtSHIP group is a little higher than that in K562/muSHIP group(0.329±0.025), but no significant difference between them(P>0.05).
7 High pressure liquid chromatography result shown that the PI3,4,5-P3 level was decreased in K562/wtSHIP,no changes above indicate above in K562/muSHIP group、K562/FIV group and untransfected group. The PI3,4P2 level in K562/wtSHIP group was significantly higher than that in K562/muSHIP group、K562/FIV group and untransfected group(P<0.01), but no significant difference of PIP3 and PI3,4P2 were seen among the last three group(P>0.05).
Conclusions
1 Site-directed mutation of SHIP gene did not influence on the expression level of gene protein related to the PI3K/Akt signal pathway in the human leukemia cell line K562, however, also decrease ability of apoptosis of K562.
2 There are significant difference of caspase-3、caspase-9 and NFκB activity between K562 cells transfected by wild-type SHIP and SHIPP28L mutant.
3 The effects of SHIP gene on the regulation the protein expression in the typical PI3K/Akt signal pathway might be an important molecular mechanism that SHIP gene suppress the proliferation and induce the apoptosis of K562, and it depend on the structure and function of normal.
SHIP基因是继PTEN之后发现的仅在造血细胞表达的一种新的肌醇磷酸酶,其主要功能是降解PIP3成为PI3,4P2而负调控PI3K/Akt路径。本课题组多年来一直围绕SHIP基因功能与白血病发生发展关系开展研究工作,取得了一系列有意义的研究成果。我们首次报道急性白血病患者存在SHIP基因突变,并证实SHIP基因低表达和表达缺失与人白血病细胞恶性增殖密切相关,通过干扰RNA封闭K562细胞的bcr/abl融合基因使SHIP蛋白重新表达并可以逆转细胞的异常增殖表型,近年来也陆续发现和报道了一些白血病细胞中新的SHIP基因突变。这些都提示SHIP基因具有调控白血病细胞增殖、凋亡的结构基础。但是,有关SHIP基因调控白血病细胞系K562的PI3K/Akt信号传导通路的分子机理及其在抑制和/或逆转K562细胞增殖能力中的地位,以及SHIP基因突变对PI3K/Akt的调控有何改变等问题,目前国内外均无报道。本研究的目的是:(1)探讨野生型SHIP基因对人白血病细胞系K562中PI3K/Akt信号通路相关蛋白表达水平的影响;(2)构建携带突变型SHIP基因的慢病毒载体并转染K562细胞系;(3)探讨SHIP基因突变后在K562细胞中的功能变化,为进一步研究SHIP基因在白血病发病机制中的作用提供实验依据。
第一部分野生型SHIP基因对白血病细胞系K562增殖凋亡的实验研究
目的探讨肌醇5′磷酸酶基因SHIP对人白血病细胞系K562增殖、凋亡及细胞周期的影响,并对其可能的作用机制进行初步探讨。
方法将携带有野生型SHIP基因和空载体的慢病毒表达载体转染人白血病细胞系K562。MTT检测细胞生长情况;流式细胞仪检测细胞转染效率和细胞周期分布;显微镜及透射电镜下观察细胞形态及超微结构改变,TUNEL、Hoechst33342荧光染色、细胞集落形成等实验检测细胞增殖及凋亡情况。实时荧光定量PCR(FQ-PCR)检测转染前后SHIP mRNA表达变化, Western Blot检测各转染组SHIP、Akt、p-Akt308、p-Akt473、Bcl-2、Bax、Bad、Bcl-xL、CyclinD1、p27kip1、p21WAF1、NFκB蛋白水平变化。Caspase活性检测试剂盒检测Caspase-3、Caspase-9活性。NFκB活性检测试剂盒检测NFκB活性变化。
结果
1转染48 h后,流式细胞仪检测慢病毒载体转染K562细胞效率为(74.6±5.8)%。FQ-PCR和Western blot显示,转染SHIP基因后K562细胞中SHIP呈高效表达,表明转染成功。
2转染野生型SHIP基因的K562细胞表现为增殖受抑,增殖指数减低,克隆形成能力降低,DNA合成减少和凋亡增多,与转染空载体组和未转染组相比有统计学差异(P<0.01)。
3野生型SHIP基因过表达导致细胞周期G0/G1期至S期和G2/M期阻滞。转染第5天,K562/wtSHIP细胞的G0/G1期百分数(60.69±8.3)%明显高于不表达SHIP的K562/FIV细胞(37.48±6.78)%和空白对照组(35.5±4.05)%。
4转染野生型SHIP基因的K562细胞Akt308和Akt473磷酸化水平(分别为:0.125±0.016,0.351±0.064)明显低于K562/FIV细胞(Akt308:0.323±0.025,Akt473:0.788±0.102)和未转染组(Akt308:0.324±0.023,Akt473:0.801±0.095)( P<0.01),后两组间Akt-308和Akt473磷酸化水平均无统计学差异(P>0.05)。
5野生型SHIP基因抑制IL-3诱导的K562细胞增殖,发现外源性野生型SHIP蛋白表达组K562细胞p-Akt308和p-Akt473水平增加程度明显低于对照组呈下降趋势,而总Akt表达水平无明显变化;撤除IL-3刺激后,K562/wtSHIP组细胞Akt磷酸化水平快速下降,而转染空载体组细胞Akt磷酸化水平下降缓慢,持续时间较长。
6转染野生型SHIP基因的K562细胞能明显增加p27蛋白(0.302±0.059)和p21蛋白(0.335±0.027)表达,而cyclin D1蛋白表达(0.142±0.019)明显降低,转染空载体组的K562细胞p27、p21和cyclin D1蛋白的表达(p27:0.159±0.023;p21:0.188±0.032;cyclin D1:0.221±0.031)与未转染组(p27:0.152±0.022;p21:0.182±0.034;cyclin D1:0.228±0.033)相比无明显改变。
7野生型SHIP基因对凋亡相关蛋白表达的影响
7.1转染SHIP基因的K562细胞出现caspase-9和caspase-3酶活性增加[caspase-9 : 32.82±5.80 nmol/(h·mg) ; caspase-3 :16.80±1.98nmol/(h·mg)],转染空载体组的K562细胞caspase-9和caspase-3活性[caspase-9: 8.75±2.66nmol/(h·mg);caspase-3:5.48±1.31nmol/(h·mg)]与未转染组[caspase-9 : 8.22±1.25nmol/(h·mg) ; caspase-3 :5.27±0.58nmol/(h·mg)]相比无显著改变;以上结果表明野生型SHIP基因过表达可激活K562细胞caspase-9和caspase-3。
7.2转染野生型SHIP基因能明显下调K562细胞bcl-xL蛋白表达(K562/wtSHIP组:0.109±0.015;K562/FIV组:0.385±0.043;未转染组:0.392±0.049);显著上调促凋亡蛋白bad表达(K562/wtSHIP组:1.018±0.216;K562/FIV组:0.315±0.052;未转染组:0.304±0.037);K562/wtSHIP组bax蛋白表达( 0.348±0.037 )高于K562/FIV组(0.342±0.043)和未转染组(0.339±0.032),但三组间无统计学意义(P>0.05);各组bcl-2表达无显著性差异(P>0.05)。
7.3转染野生型SHIP基因下调K562细胞NF-κB表达(K562/wtSHIP:0.483±0.027;K562/FIV组:0.924±0.081;未转染组:0.937±0.075);各组IκBа表达水平无显著性差异(P>0.05)。K562/wtSHIP组NF-κB的活性随时间延长而降低,K562/FIV组和未转染组细胞无显著性变化。
结论过表达野生型SHIP基因明显抑制白血病细胞系K562增殖,促进K562细胞凋亡,其机制可能通过抑制Akt磷酸化,进一步影响其下游与多种凋亡相关分子家族蛋白如Bcl-2家族、Caspase家族表达,并通过调控细胞周期相关蛋白表达使细胞周期阻滞在G0/G1期。
第二部分SHIP基因突变体的构建
目的急性白血病患者存在SHIP基因突变,可能与白血病发病有关,构建SHIP基因突变体(muSHIPP28L),为研究突变基因的表达和功能与白血病的发病机制奠定基础。
方法以重组表达质粒wtSHIP为模板,用Stratagen Mutant Kit试剂盒通过PCR定点突变技术对SHIP编码序列TTC进行定点突变→CTC,突变体经测序鉴定正确,连接到慢病毒载体Receiver-Lv31(ORF不带标签)。经重组慢病毒包装,病毒滴度测定,感染K562细胞,采用FQ-PCR和Western blot方法检测SHIP mRNA和蛋白表达。
结果
1构建的突变型SHIPP28L经DNA测序验证,其碱基序列与设计序列完全一致。
2感染293T细胞48 h后在荧光显微镜下观察到GFP较强表达。制备的病毒滴度达4.9×106 pfu/ml。
3 FCM分析表达GFP绿色荧光的阳性细胞百分率,计算活细胞转染效率。悬浮培养的人白血病细胞系K562细胞转染效率分别为K562/wtSHIP:74.6%,K562/FIV:82.5%,K562/muSHIP:76.3%。
4 FQ-PCR和Western blot方法检测转染野生型和突变型SIHP基因的K562细胞中SHIP基因高表达,而K562/FIV组和未转染K562细胞中内源性SHIP基因表达极低,说明外源性野生型和突变型SHIP转染成功。
结论运用基因定点突变基因重组技术成功构建了SHIP基因P28L突变体,为进一步探讨SHIP基因与白血病发病机制创造条件。
第三部分SHIP基因突变体在白血病细胞系K562细胞中的表达及其功能的研究
目的将构建的SHIP基因突变体稳定表达于人白血病细胞系K562中,观察SHIP基因突变后在K562细胞中的功能变化,为进一步研究SHIP基因在白血病发病机制中的作用提供实验依据。
方法FQ-PCR和Western blot鉴定野生型和突变型SHIP基因在K562细胞中的表达;MTT比较K562/wtSHIP和K562/muSHIP两组细胞生长情况;流式细胞分析比较野生型和突变型SHIP基因对细胞周期的不同影响;Hoechst33342荧光染色比较细胞凋亡情况。Western Blot比较转染野生型和突变型SHIP基因后对K562细胞Akt、p-Akt308、p-Akt473、Bcl-2、Bax、Bad、Bcl-xL、CyclinD1、p27kip1、p21WAF1、NFκB蛋白表达的调控。Caspase活性检测试剂盒检测各组Caspase-3、Caspase-9活性。利用高压液相色谱分析比较各转染组PIP3和PI3,4P2水平。
结果
1野生型SHIP基因明显抑制K562细胞生长,而SHIP基因突变体丧失了这种功能。流式细胞仪分析结果表明K562/wtSHIP细胞主要表现为G0/G1期细胞增多、G2/M期细胞减少,PI降低。突变型SHIP基因对K562细胞周期无明显影响。
2 Hoechest染色发现转染野生型SHIP基因的K562细胞早期凋亡率[(38.4±3.08)%]明显高于转染SHIP基因突变子(P28L)[(8.4±1.11)%]和空载体组[(8.06±0.95)%]。SHIP基因突变子不能促进细胞凋亡。
3 SHIP基因突变体不能下调K562细胞p-Akt308和p-Akt473的磷酸化水平(p-Akt308:0.334±0.031;p-Akt473:0.826±0.163)。
4转染野生型SHIP基因的K562细胞周期蛋白p21和p27蛋白水平显著增高,cyclin D1水平明显降低,但转染SHIP基因突变体的K562细胞中细胞周期蛋白表达无上述改变。
5 K562/muSHIP组细胞caspase-3和caspase-9酶活性显著低于K562/wtSHIP组(P<0.05),与K562/FIV组和未转染组相比无统计学差异(P>0.05)。
6转染野生型SHIP基因组K562细胞bcl-xL表达降低,而SHIP基因突变体对K562细胞bcl-xL表达(0.367±0.028)无影响;K562/muSHIP基因组和未转染组、K562/FIV组间无显著性差异(P>0.05)。K562/muSHIP组细胞bad蛋白表达水平(0.322±0.063)较K562/wtSHIP组低,与K562/FIV组和未转染组相比无显著性差异(P>0.05)。K562/muSHIP组细胞bcl-2表达水平(0.155±0.035)较K562/wtSHIP组、K562/FIV组和未转染组间无统计学意义(P>0.05)。Bax在K562/wtSHIP组细胞表达水平略高于K562/muSHIP组(0.329±0.025)但无统计学差异(P>0.05)。
7应用高压液相色谱分析结果显示:转染wtSHIP基因组K562细胞中PI(3,4,5P3水平下降,显著低于转染muSHIP组、转染空载体组和未转染组PIP3的水平(P<0.01),后三组间PIP3水平无显著性差异(P>0.05)。K562/wtSHIP组K562细胞PI(3,4)P2水平明显高于K562/muSHIP组、K562/FIV组和未转染组K562细胞PIP2水平(P<0.01),后三组细胞间PI3,4P2水平相比无统计学意义(P>0.05)。
结论
1 SHIP基因突变体SHIPP28L导致其增殖抑制及促凋亡功能丧失。
2与野生型SHIP基因相比,SHIPP28L突变体导致其对K562细胞caspase-3、caspase-9和NF-κB活性的调节作用丢失。
3 SHIP基因调控PI3K/Akt信号通路蛋白表达可能是其抑制人白血病细胞系K562增殖的重要分子机理之一,SHIP基因对K562细胞增殖的负调控作用及促凋亡作用有赖于其基因结构和功能正常。
Background: Leukemia is the most common malignant clone’s disease of hematopoietic stem cells. The incidence rate of leukemia has the tendency of step-up recent years. Leukemia is the consequences of a multistep process resulting from a series of genetic changes and incidence, and its process is related with activation of oncogene or inactivation of antioncogene. Altohugh many genes’function and machanisms in leukemia has been explicit, all novel gene can not explain all pathogenesis of all leukemia. Thereby, it has become one of the hotspots to investigate leukemia by identifying new correlative oncogene or tumor suppressor genes and studying its biologic function,and it is important for elucidate the pathogenesy of leukemia.
SH2-containing inositol 5’-phosphatase (SHIP) is a recently identified lipid phosphatase after phosphatase and tensin homologue deleted on chromosome 10(PTEN) was observed, which hydrolyzes the 5 prime phosphate of the inositol ring from inositol 3,4,5-P3. It is mainly expressed in hematopoietic cells. Its phosphatase activity toward PIP3 has been linked to suppress several signaling pathways, including Akt, leading to the inhibition of hematopoietic cell proliferation and survival. Our previous researching works have proved that low expression and deletion of SHIP gene was closely correlated with the abnormal proliferation of patients with leukemia; lower Akt activation was detected in K562 cell lines, which was caused by the re-expresion SHIP. transfection of wild type SHIP into the K562 cells can regulate the expression of proliferative relative genes and reverse the proliferative phenotype of K562. The molecular mechanism that SHIP gene suppress leukemia may be involved in some important signal pathways. Up to now, However, there are no any reports about the exact mechanisms of SHIP gene on the regulation of PI3K/Akt signal transduction pathway, the effects of SHIP gene on suppression and/or reversion of abnormal proliferation in human leukemia cell line K562 in the world. There are also no any reports about what changes of the PI3K/Akt signal transduction regulation will happened if SHIP has site mutation in K562 cell lines. The aim of this study are as follows: (1)To detect the influence of cell proliferation and apoptosis of K562 cells transfected with wild-type SHIP gene, and to investigate the molecular mechanism of apoptosis of K562 cells transfected with wtSHIP gene; (2) To construct SHIP gene’s mutant (P28L) found in AML patients. (3) To investigate the functional changes of SHIP gene’s mutant by over-expression it into human leukemia cell line K562,and to make a preliminary discussion on the mechanism of SHIP gene abnormality in leukemia.
Part one: Effects of wild-type SHIP on proliferation, apoptosis and cell cycle of K562
Objective To investigate in vitro the effects of SH2-containing inositol 5’-phosphatase gene-SHIP on proliferation, apoptosis, cell cycle of human leukemia cell line K562 and to explore its possible machinesim preliminarily.
Method Based on the results of our previous studies, with different human leumemia cell lines K562 as study samples, including K562, K562/wtSHIP, K562/FIV; the growth of K562 was observed by MTT assay; the transfection efficiency and cell cycle distribution were assessed by flow cytometry (FCM). Morphological characteristics of light microscope,cell colony fomation,TUNEL and Hoechest 33342 fluorescent staining method were also used to test proliferation and apoptosis of K562 cells. The expression level and difference of SHIP mRNA was detected by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR); the the expression level and difference of SHIP, Akt, p-Akt473, p-Akt308 and the member of apoptosis family Bcl-2, Bcl-xL, Bax, Bad, cell cycle related molecular CyclinD1, p21WAF1 and p27Kip1 were detected by Western blot; Caspase-3, Caspase-9 protein activity were detected by caspase activity kits.
Results
1 The transfection efficiency was about 74.6±5.8% in K562 cell line after transfected with lentivirus on 48 h detected by flow cytometry. The recombinant lentivirus pReceiver-LV-SHIP has been successfully transfected into K562 cell line.
2 The decreased ability of proliferation and DNA synthesis, cell colony fomation ability and enhanced apoptosis rate were observed in K562 cells transfected with wild-type SHIP, and the same changes had not been observed in K562 cells transfected with empty vector and untransfected K562(P<0.01).
3 Cell cycle distribution showed the ratio of G0/G1 phase of K562 cells transfected with wild-type SHIP gene increased from (34.15±8.30)% to (60.69±8.30)%, the ratio of G2/M+S phase decreased form(65.85±9.36)% to (38.85±7.09)%, the cell cycle was arrested at G0/G1 phase.
4 After transfection wtSHIP at the third days, western blot results showed the Akt expression levels had no change but p-Akt308 and p-Akt473 expression level (0.125±0.016 and 0.351±0.064 respectively) decreased compared with K562/FIV group ( Akt308 : 0.323±0.025 , Akt473 :0.788±0.102) and untransfected group(Akt308:0.324±0.023,Akt473:0.801±0.095)( P<0.01).
5 wtSHIP gene can inhibit the proliferation of K562
6 After K562 cells transfected wtSHIP, the cell cycle related molecular expression levels showed that the protein expression levels of p27、p21 (0.302±0.059、0.335±0.027 respectively) increased compared with K562 cells transfected with empty vector (p27 kip1:0.159±0.023;p21WAF1:0.188±0.032) and untransfected group (p27Kip1:0.152±0.022;p21WAF1:0.182±0.034) (P<0.05), but the protein expression of CyclinD1 (0.142±0.019)decreased (P<0.05).
7 The effects of wtSHIP on apoptosis related proteins in human leukemia cell line K562
7.1 After transfection wtSHIP at the third days, the caspase-9 and capsase-3 activity[caspase-9:32.82±5.80nmol/(h·mg); caspase-3: 16.80±1.98nmol/(h·mg)] up-regulated compared with K562/FIV group [caspase-9: 8.75±2.66nmol/(h·mg); caspase-3: 5.48±1.31nmol/(h·mg)] and untransfected K562 cells caspase-9: 8.22±1.25nmol/(h·mg);caspase-3: 5.27±0.58nmol/(h·mg)] (P<0.05).
7.2 The bcl-xL protein expression level in K562/wtSHIP group was lower than K562/FIV group and untransfected K562 (K562/wtSHIP: 0.109±0.015; K562/FIV: 0.385±0.043; untransfected group: 0.392±0.049)(P<0.05). The protein expression levels of Bad in K562/wtSHIP was significantly higher than that of K562/FIV and untransfected K562, but no significant difference of protein expression level of Bad was observed among K562/FIV and untransfected group(K562/wtSHIP: 1.018±0.216; K562/FIV: 0.315±0.052; untransfected group: 0.304±0.037). No significant difference of protein expression levels of bax and bcl-2 was observed among K562/wtSHIP、K562/FIV and untransfected K562.
7.3 In untransfected group, FIV-GFP group, and wtSHIP-GFP group the protein expression of NF-κB (p65) was significantly down-regulated than that in K562/FIV and untransfected group(K562/wtSHIP:0.483±0.027;K562/FIV组:0.924±0.081;untransfected group:0.937±0.075)(P<0.05); and no significant difference of IκBаprotein level was observed among K562/wtSHIP、K562/FIV and untransfected K562.
Conclusions
1 This part of study indicated that over expression wild-type SHIP gene could remarkably suppress proliferation of K562 cells, and induce K562 cell apoptosis.
2 The effects of wild type SHIP gene on K562 probably via inhibiting the phosphorylation of Akt, and regulating its downstream target protein such as apoptosis related family expression such as Bcl-2 family, Caspase family and the cell cycle related melbourne expression.
Part two: Contruction of a lentiviral vector carrying human SHIP mutated gene and its expression in K562 cell line
Objective To construct lentiviral vector carrying human SHIP gene and investigate its expression in K562 cell line.
Method Full-length cDNAs of wild type SHIP gene (wt-SHIP) was amplified from the human chondrocytes by RT-PCR, and site-directed mutagenesis was used to obtain full-length cDNAs of the mutated SHIP gene (mut28P/L). The products was cloned into p-Receiver-LV31vector and their sequences was analysed, then subcloned into lentivirus vector pReceiver-LV31, the inserted fragments were analysed and reconfirmed by restriction endonuclease analysis and sequencing. SHIP-lentivirus gene containing green fluorescent protein gene (SHIP-GFP) or the empty vector (FIV-GFP) was transfected into K562 cells. The green fluorescence protein expression of lentivirus plasmids were observed using laser scanning microscopy after 48 hours of transfection. The mRNA expression levels of wtSHIP, muSHIP gene were detected by FQ-PCR. The protein expression levels of wtSHIP and muSHIP were detected by western blot. .
Results
1 By restriction endonuclease analysis and sequencing, the sequences of SHIPP28L was consistent with that in literature, and the opening frames were matched with the vector sequence.
2 Green fluorescent can be seen in transfected 293T after transfection at 48 h. The virus titer was 4.9×106 pfu/ml.
3 Green fluorescence signal was distributed throughout K562 cells transfected with wtSHIP and muSHIP vector.The transfection efficiency was K562/wtSHIP-74.6%、K562/FIV-82.5%、K562/muSHIP-76.3%, respectively after transfected with lentivirus on the second day.
4 SHIP mRNA and protein were at the highly expression levels in K562/wtSHIP and K562/muSHIP, but in K562/FIV and untransfected group SHIP mRNA and protein level was extremely low, which shown that is transfected into K562 successfully.
Conclusions
1 Lentivirus expression vector of SHIP gene’s mutant of P28L was successfully constructed by using site-directed mutagenesis of SHIP cDNA.
2 It was proved that the sequence of the SHIPP28L mutant was correct DNA sequencing.
3 Green fluorescent、FQ-PCR and Western blot can be seen in transfected K562 with muSHIP-GFP and wtSHIP-GFP, which shown that it was transfected into K562 successfully.
Part three:The study of mutation of SHIP in molecular pathogenesis of leukemia
Objective There are two categories of mutations or gene rearrangements which play important role in molecular mechanisms of leukemogenesis. One class of mutations or gene rearrangements involving mostly tyrosine kinase gene such as BCR/ABL of chronic myelogeous leukemia (CML) confer a proliferative and/or survival advantage to hematopoietic progenitors, and second class of mutations involving mostly transcription factor associated with hematopoietic cell differentiation such as PML/RAR mutation serve primarily to impair hematopoietic differentiation and subsequent apoptosis of cells. Though FLT3-ITD mutation and PML/RAR in acute promyelocytic leukemia (APL) patients as well as c-KIT mutation and AML1/ETO in AML-M2b patients have been reported, the molecular defect involving two categories of mutation are largely unidentified. In this part we focused on elucidate the role of SHIP mutation in leukemogenesis, its association with biological characters of leukemia and its potential in developing new target therpy.
Method The recombined lentivirus plasmids muSHIPP28L was transfected stably into human leukemia cells K562. The cell proliferation, cell life cycle and cell apoptosis of K562/muSHIP cells were determined by MTT, fluorescent staining and flow cytometry. Differentially expressed genes were reconfirmed by Western blot.
Results
1 The enhanced ability of proliferation and DNA synthesis, and decreased apoptosis rate was observed in K562 cells transfected with muSHIPP28L, and the same changes had been observed in K562/FIV and untransfected K562 cells. Increased G0/G1 cells was observed in K562/wtSHIP cells by flow cytometry result, but no such a difference was observed in K562/muSHIP.
2 The early apoptosis rate in K562/muSHIP(P28L) group[(8.4±1.11)%] was significantly lower than that in K562/wtSHIP group[(38.4±3.08)%] by Hoechest stainging, no significant difference of the early apoptosis rate was observed between K562/muSHIP and K562/FIV[(8.06±0.95)%]. The SHIP mutant could not induce K562 cell apoptosis.
3 SHIPP28L mutant can’t down-regulate phosphorylation of Akt308 and 473(p-Akt308:0.334±0.031;p-Akt473:0.826±0.163).
4 The cell cycle related molecular expression levels of p21WAF1 and p27KIP1 in K562 cells transfected with wtSHIP were significantly up-regulated, but the protein expression of cyclin D1 down-regulated; no changes indicated above in K562/muSHIP.
5 The activity of caspase-3 and capase-9 in K562/muSHIP group were significantly lower than those in K562/wtSHIP group(P<0.05), no significant difference was seen between K562/FIV group and untranfected group(P>0.05).
6 The protein expression of bcl-xL decreased in K562/wtSHIP group, but no changes indicated above in K562/muSHIP(0.367±0.028). The expression of bad protein in K562/muSHIP group (0.322±0.063) was lower than that in K562/wtSHIP group , no changes indicate among K562/muSHIP、K562/FIV group and untransfected group(P>0.05). There was no significant difference of bcl-2 expression was observed among K562/muSHIP group、K562/wtSHIP group、K562/FIV group and untransfected group ( P > 0.05 ) . The expression of bax protein in K562/wtSHIP group is a little higher than that in K562/muSHIP group(0.329±0.025), but no significant difference between them(P>0.05).
7 High pressure liquid chromatography result shown that the PI3,4,5-P3 level was decreased in K562/wtSHIP,no changes above indicate above in K562/muSHIP group、K562/FIV group and untransfected group. The PI3,4P2 level in K562/wtSHIP group was significantly higher than that in K562/muSHIP group、K562/FIV group and untransfected group(P<0.01), but no significant difference of PIP3 and PI3,4P2 were seen among the last three group(P>0.05).
Conclusions
1 Site-directed mutation of SHIP gene did not influence on the expression level of gene protein related to the PI3K/Akt signal pathway in the human leukemia cell line K562, however, also decrease ability of apoptosis of K562.
2 There are significant difference of caspase-3、caspase-9 and NFκB activity between K562 cells transfected by wild-type SHIP and SHIPP28L mutant.
3 The effects of SHIP gene on the regulation the protein expression in the typical PI3K/Akt signal pathway might be an important molecular mechanism that SHIP gene suppress the proliferation and induce the apoptosis of K562, and it depend on the structure and function of normal.
引文
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