MAP4K4在原发性肝细胞癌中的表达及生物学意义
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摘要
原发性肝癌(Primary hepatic carcinoma,PHC)绝大多数是肝细胞癌(HCC),素有“癌中之王”之称,现有的治疗手段效果均不佳,因此越来越多的学者致力于肝细胞癌基因靶向治疗的研究。肝细胞癌的发生是多因素、多途径、多步骤长期作用的结果,包括外环境致癌因素(病毒、寄生虫、细菌的感染、黄曲霉毒素的摄入、水源污染以及吸烟、饮酒)和自身遗传因素(癌基因的激活和抑癌基因的失活)。目前已经发现与人类肝细胞癌的发生有关的基因有:N-ras、c- fos、p53、C- myc、IGF-Ⅱ、IGF-ⅡR、p16、p21、DCC、nm- 23、c- erB-b- 2、TGF-α、CSF- IR、raf等。癌基因和抑癌基因的突变是肝细胞癌发生的分子基础,肝细胞的癌变是一个多基因参与多阶段的过程。
丝裂原激活蛋白激酶激酶激酶激酶4(MAP4K4)系丝/苏氨酸激酶亚家族STE20中的一员,位于染色体2q11.2,含有33个外显子,编码区包括九个可供选择的剪接位点。人MAP4K4最早克隆于巨噬细胞cDNA库,不同来源的组织可分离出不同的由cDNA编码的MAP4K4亚型。MAP4K4属于丝裂原激活蛋白激酶(MAPK)信号传导通路的一个上游激活因子,MAPK是一组可以被多种细胞外信号(包括生长因子,激素,紫外线辐射,DNA损伤剂,炎性细胞因子和环境应激等)激活的丝/苏氨酸激酶。MAPK信号转导通路非常保守,自膜受体到MAPK激酶激酶(MAPKKK)再到MAPK激酶(MAPKK)然后到MAPK,在细胞信号转导通路中MAPK处于细胞质部分的终末位置,活化后可以转到核内作用靶点,调节基因的表达。这种激活模型存在于酵母至哺乳类动物。它参与细胞的多种生物学行为,包括细胞凋亡,分化和增殖、细胞周期调控,细胞生存的维持以及细胞恶性转化等。STE20家族是MAPKKK的上游激酶,哺乳类动物STE20/丝裂原蛋白激酶激酶激酶激酶(MAP4K)家族由其催化区相关的28种丝氨酸/苏氨酸激酶组成。这些激酶可分为两种结构类别,P21活性蛋白激酶(PAKs)和生发中心激酶(GCKs)。GCK激酶缺乏PAKs激酶中调节Cdc42/Rac的作用区,而代之以N-末端激酶区和不同长度的C-末端延伸。C-末端是一个枸椽同源区(CNH),称为枸椽rho相互作用激酶(CRIK),该区域对于激酶活性的调节起着重要作用。GCK激酶在激酶区域外显示出低度同源性,并分为九个亚家族。MAP4K4是GCK IV组中的一个成员,它对细胞的作用包括调节细胞的运动性,细胞骨架重排以及细胞增殖等。研究发现MAP4K4在多种肿瘤中有高表达并证明了它在加速肿瘤细胞转化,促进细胞侵袭,降低细胞黏附性方面起着一定的作用。近期又有研究提出MAP4K4与卵巢癌、乳腺癌、前列腺癌和恶性黑色素瘤的侵袭和转移有着密切的关系,通过siRNA敲减这些细胞系的MAP4K4基因可抑制它们的侵袭和转移。最新研究报道MAP4K4在胰腺癌中也有高表达,并与其不良预后有关。以上提示MAP4K4信号通路很可能与肿瘤的发生,发展有着密切的关系。而目前对于MAP4K4在肝细胞癌中的研究尚未见报道。
我国为肝细胞癌高发国家,大多数肝细胞癌的发生与乙肝病毒感染密切相关,HBV是我国肝细胞癌发生的最直接危险因素。本实验首先应用cDNA芯片对1例HBV相关肝细胞癌进行检测,发现MAP4K4基因在肝细胞癌组织较癌旁组织显著高表达,受此启发,本课题拟探讨MAP4K4在肝细胞癌的生物学活性中是否也发挥一定的作用。据此,本课题的研究思路为:
首先,继续采用cDNA芯片技术对5例HBV相关肝细胞癌基因表达谱进行初步研究,对比癌和癌旁组织的差异基因表达,明确MAP4K4基因在肝细胞癌组织中确实较癌旁组织显著高表达。
然后,采用分子生物学方法和组织芯片技术进一步从转录和翻译水平检测肝癌细胞株及肝细胞癌组织中MAP4K4的表达情况,验证基因芯片的结果,并分析不同临床病理参数中MAP4K4蛋白表达水平的差异,以初步证实MAP4K4在肝细胞癌发展中的临床意义。
最后,再通过RNAi技术研究MAP4K4基因敲减对肝癌细胞生物学活性的影响,并通过基因芯片技术初步探讨其生物学作用产生的可能机制。
第一部分肝细胞癌基因表达谱的初步研究目的:
应用含20228个基因的人cDNA芯片研究5例HBV相关肝细胞癌及配对癌旁肝组织的基因表达差异。
方法:
随机选择HBV相关肝细胞癌及其癌旁肝组织样本5例分别抽提标本的总RNA,反转录为cDNA与芯片进行杂交,对比差异表达的基因。
结果:
HBV相关肝细胞癌组织相对于癌旁组织明显上调表达基因有52个(>3倍),其中上调大于5倍的基因为ASPH,FACL4,LAPTM4B,PEX11B,MAP4K4,SQSTM1,CCT3,CPD,CKS1B,HMGA1,ENO1;明显下调表达的基因有37个,下调大于10倍的基因为CYP2E1,MT2A,MT1X,CPS1,EXTL1,FCN3,CYP2A7,NOLA2,CYP3A4,CYP2B6,CYP4A11,HSD11B1。
结论:
通过cDNA芯片技术,发现肝细胞癌广泛存在的异常表达基因,其中MAP4K4基因明显上调,综合考虑它作为MAPK信号途径的一个上游因子可能参与了细胞分化、增殖、凋亡及恶性转化等生物学功能,因此进一步对MAP4K4基因进行分析将可能有助于了解它在肝细胞癌中的生物学作用,为肝细胞癌的基因靶向治疗开辟一条新途径。
第二部分MAP4K4在肝癌细胞株及肝细胞癌组织中的表达
目的:
从转录和翻译水平检测肝癌细胞株及肝细胞癌组织中MAP4K4的表达情况,以验证基因芯片结果,并分析不同临床病理参数中MAP4K4蛋白表达水平的差异,初步证实MAP4K4在肝细胞癌发展中的临床意义。
方法:
选取20例新鲜肝细胞癌组织和配对癌旁肝组织标本及四种肝癌细胞(Hep3B,HepG2,SMMC7721,Huh-7)进行RT-PCR,qRT-PCR和Western-blot分别检测MAP4K4 mRNA和蛋白水平的表达;对400例肝细胞癌蜡块标本构建肝细胞癌组织芯片,采用免疫组织化学技术检测MAP4K4蛋白在含400例配对肝细胞癌及癌旁组织的表达活性,分析不同临床病理参数中MAP4K4蛋白表达水平的差异。
结果:
1.肝细胞癌组织中MAP4K4 mRNA的表达
在癌旁肝组织,MAP4K4 mRNA表达为0.001339～0.018747(平均0.007900),而大部分癌组织表达高水平MAP4K4 mRNA,0.007367～0.080935(平均0.029500);与配对癌旁肝组织相比,肝细胞癌组织MAP4K4 mRNA上调1.062～28.520倍(平均6.852倍)。配对t-检验证实肝细胞癌组织和癌旁肝组织差异有显著性(P < 0.001)。
2.肝癌细胞株中MAP4K4 mRNA的表达
经RT-PCR检测,MAP4K4 mRNA在各细胞株的表达为:HepG2> Hep3B> Huh-7 > SMMC7721;qRT-PCR检测MAP4K4 mRNA在HepG2、Hep3B、Huh-7、SMMC7721四种肝癌细胞株中相对表达量依次为(0.016008±0.002358)、(0.015629±0.001389)、(0.011674±0.001456)、(0.004895±0.000947),结果得到qRT-PCR检测证实。
3.肝癌细胞株MAP4K4蛋白表达
经Western-blot检测,MAP4K4蛋白在四种肝癌细胞株中均有表达,在HepG2细胞中表达最高,其次是Hep3B细胞,而在SMMC7721中表达最低。
4.MAP4K4在肝细胞癌组织中的定位和表达
在正常肝、肝炎和肝硬化组织的肝细胞MAP4K4呈一致阴性或弱阳性表达,周围结缔组织、血管和胆管无MAP4K4阳性着色,Kuffer细胞和淋巴细胞生发中心可见阳性染色,与相应的癌旁肝组织相比,异型增生结节的肝细胞MAP4K4的表达明显强于正常肝、肝炎和肝硬化组织的肝细胞。肝细胞癌组织MAP4K4表达显著增强。肝细胞癌组织MAP4K4呈异质性表达,其中过度表达194例(48.5%),灶性阳性或阴性206例(51.5 %)。HBsAg阳性患者MAP4K4阳性率51.5%(157/305)显著高于阴性的38.8% (37/95)(P = 0.033);肿瘤直径≤2cm的MAP4K4阳性率31.6%(18/57)显著低于肿瘤直径> 2cm的51.3%(176/343) (P = 0.006);MAP4K4在不同组织学分级中的阳性率差异有显著性(P<0.001);肝内转移病例的MAP4K4阳性率55.5%(152/274)显著高于未转移者的33.3%(42/126)(P = 0.002)。MAP4K4表达在患者年龄、血清甲胎蛋白浓度、肝硬化、包膜浸润方面差异无显著性。
结论:
MAP4K4在肝细胞癌中普遍高表达,并且其表达水平与HBV感染状态,肿瘤大小、组织学分级及转移情况可能有一定关系。
第三部分MAP4K4对肝癌细胞生物学功能的影响
目的:
检测MAP4K4基因敲减后肝癌细胞生物学活性(细胞增殖、细胞克隆形成、细胞周期、细胞凋亡)的改变并初步探讨其作用机制。
方法:
检测HepG2、Hep3B、Huh7、SMMC-7721肝癌细胞株中MAP4K4蛋白表达,选择表达活性最强者进行siRNA实验。设计一个空载体质粒转染组及三对靶向MAP4K4的特异性siRNA核苷酸序列质粒转染组(0—HepG2-pGCSil-U6,1—pSilMAP4K4-1,2—pSilMAP4K4-2,3—pSilMAP4K4-3),用Lipofectamine 2000法分别转染到选取的肝癌细胞,qRT-PCR和Western-blot方法检测干扰效率,并对转染后肝癌细胞进行WST-8细胞增殖实验,平板克隆形成实验,PI染色法细胞周期检测,Annexin V法细胞凋亡检测,然后,选择TOLL样受体信号途径相关芯片对干扰效果最好转染组及空载体转染组细胞进行基因检测,以进一步探究MAP4K4基因在肝癌细胞中可能的作用机制。
结果:
依据Western-blot检测四种肝癌细胞株中MAP4K4蛋白表达情况,我们选择其中表达该蛋白最高的HepG2肝癌细胞株作为MAP4K4-siRNA的研究靶标。
1.qRT-PCR和Western-blot检测干扰效率
转染干扰片段1,2,3后与对照组相比,MAP4K4 mRNA抑制率分别为68.7%,25.9%和53.7%;MAP4K4蛋白的抑制率分别为:61.4%,27.8%和45.8%。
2.细胞生长形态改变
MAP4K4 RNA干扰后,对数生长期的转染组细胞数目减少,体积变大,贴壁性增强,较对照组比较,圆形M期细胞比例减少。
3. WST-8细胞增殖实验结果
pSilMAP4K4-1、pSilMAP4K4-3组,细胞增殖速度明显减慢( p<0.01),而转染pSilMAP4K4-2组与对照组相比差异无显著性(p>0.05)。
4.平板克隆实验结果
各转染组细胞克隆形成能力的检测结果:HepG2-pGCSil-U6 (56.83±8.51%)、pSilMAP4K4-1组(13.75±4.26%)、pSilMAP4K4-2组(50.71±7.47%)、pSilMAP4K4-3组(28.11±6.36%)。pSilMAP4K4-1和pSilMAP4K4-3组细胞的平板克隆形成率明显低于对照组(p<0.01),而pSilMAP4K4-2组与对照组间的差异无显著性(p>0.05)。
5.细胞周期实验结果
与对照组比较, pSilMAP4K4-1和pSilMAP4K4-3组均出现S期阻滞(p<0.01),M/G1期细胞比例减少(p<0.01),S、G2期细胞比例增多(P<0.05)。而pSilMAP4K4-2组与对照组相比差异无显著性(p>0.05)。
6.细胞凋亡检测结果
经无血清诱导,与对照组比较,pSilMAP4K4-1、pSilMAP4K4-3组的HepG2细胞凋亡比例增多(p<0.05),而pSilMAP4K4-2组与对照组比较差异无显著性(p>0.05)。
7.基因芯片检测结果
干扰前后肝癌细胞样本的基因芯片检测结果显示MAP4K4基因敲减后,有一定数目与其相关的基因的表达有明显改变(上调2倍以上或下调1/2以上),参照TOLL样信号传导途径通路图进行分析,我们发现MAP4K4基因在肝细胞癌中可能是通过NF-kB及JNK信号传导途径发挥作用的。
结论:
MAP4K4基因敲减可以阻滞细胞周期的进程,抑制肝癌细胞的恶性增殖,其机制可能是通过NF-kB信号传导途径、JNK信号传导途径发挥作用。
Hepatocellular carcinoma(HCC) is the most common primary malignancy of the liver, which is also known as“the king of cancers”. Due to the fact that there is no available therapeutic means for HCC, more and more scientists have been devoting themselves to the research of gene targeted therapy. The genesis of HCC is the result of multi-factors, multi-approaches and multi-steps, including carcinogenes in the external environment (virus, parasite, bacterial infection, intake of aflatoxin, water pollution, smoking and drinking) and inherent genetic factors(activation of oncogenes and inactivation of tumor suppressor genes). So far, several HCC-associated genes have been discovered, consisting of N-ras, c- fos, p53, C- myc, IGF-Ⅱ, IGF-ⅡR, p16, p21, DCC, nm- 23, c- erB-b- 2, TGF-α, CSF- IR, raf, et al. The molecular basis of HCC is the mutation of oncogenes and tumor suppressor genes which is a process with multi-genes participation and a multi-steps.
Mitogen-activated protein kinase kinase kinase kinase 4(MAP4K4) is a member of the serine/threonine kinase subfamily, Ste20. It lies in the chromosome 2q11.2 which includes 33 exons with the coding region containing nine optional spliced sites. MAP4K4 was firstly cloned from cDNA library of macrophage . Different subtypes of MAP4K4 could be separated from different tissues. MAP4K4 is an upstream stimulating factor of mitogen-activated protein kinase (MAPK) signaling. As a kind of Ser/Thr kinase, MAPK can be activated by many extracellular signals(including growth factors, hormones, ultraviolet radiation, DNA damage agents, proinflammato- ry cytokines,environmental stress, et al). The signal transduction pathway of MAPK is very conservative. Transferred from membrane receptors to MAPKKK, then to MAPKK, finally to MAPK, MAPK lies in the end location of the cytoplasm. When activated, MAPK could be transferred into intranuclear target and regulate the expression of genes. This kind of activated pattern resides from yeast to mammalian, which plays a pivotal role in a variety of processes, such as apoptosis,cell differentiation and cell proliferation, regulation of cell cycle, sustain of cell survival and the malignant transformation, and so on. STE20 is an upstream kinase of MAPKKK. In mammalians, STE20/MAP4K is composed of 28 Ser/Thr kinases which locate in its catalytic domain. These kinases can be divided into two structure categories,P21 PAKs and GCKs. GCKs lack the acting region which PAKs posses that could regulate the Cdc42/Rac, but with N-terminus kinase region and C-terminus extension of different length instead. C-terminus is a CNH region, which is called CRIK and plays an important role in regulating the kinase activity. GCK kinases are divided into nine subfamilies and show low-grade homology. As a member of GCK IV, MAP4K4 participates in the regulation of cellular mobility, cystoskeleton rearrangement and cell proliferation. Previous researches have demonstrated that MAP4K4 is highly expressed in many tumors, and confirmed that it plays a certain role in accerlerating the transformation of tumour cells, promoting the cellular invasion as well as reducing the cell adhesion. Recently, it has also been proposed that MAP4K4 is closely associated with the invasion and diversion of ovarian cancer, breast cancer, prostate carcinoma and malignant melanoma. Knockdown of MAP4K4 by siRNA can inhibit the cell invasion and metastasis. The latest study has shown that MAP4K4 is also overexpressed in pancreatic cancer which is related to its poor prognosis. The findings above indiacated that MAP4K4 signaling may have close relationship with the genesis and development of cancer. But up to now, there is no report about MAP4K4 in HCC.
There is a high incidence of HCC in China, and most patients are associated with the infection of hepatitis B virus (HBV) which is the most directly risk factor of HCC in our country. The cDNA array was firstly applied in our research to detect one case of HBV associated HCC. We discovered that MAP4K4 was significantly highlier expressed in HCC compared to adjacent tissues. Inspired by it, we plan to investigate the biologic activity of MAP4K4 in HCC. And as follow is our research strategy:
Firstly, the gene expression profile of 5 cases of HBV associated HCC will be analysed preliminarily through cDNA array to compare the differential gene expression between HCC and adjacent tissues, and to ascertain whether MAP4K4 is significantly highier expressed in HCC than adjacent tissues.
Secondly, further detection for the expression levels of MAP4K4 in hepatoma cells and hepatocellular carcinoma tissues will be carried out using RT-PCR and Western-blot to verificate the results of gene array and analyze the expression level of MAP4K4 protein in different clinicopathological parameters, thus to confirm preliminarily the clinical significance of MAP4K4 in the development of HCC.
Finally, the effect of MAP4K4 knockdown on the biological activity of hepatoma cells will be investigated by RNA interference, and the possible mechanism involved will also be researched through gene chip technology.
Part I The preliminary research of gene expression in hepatocellualr caicinoma tissue
Objective:
Compared the gene expression between HCC and adjacent tissues in five HBV associated HCC genes through cDNA array that contain 20228 genes.
Method:
5 cases of samples were randomly selected from HBV associated HCC and adjacent tissues to extract their total RNA which were transcripted reversely to cDNA and then hybridized with gene chip to compare the differentially expressed genes.
Results:
Comparing the HBV associated HCC to adjacent tissues, there were 52 expressed genes obviously up-regulated. Genes more than 5-fold up-regulation: ASPH, FACL4, LAPTM4B, PEX11B, MAP4K4, SQSTM1, CCT3, CPD, CKS1B, HMGA1, ENO1. And there are 37 genes obviously down-regulated. Genes more than 10-fold down-regulation: CYP2E1, MT2A, MT1X, CPS1, EXTL1, FCN3, CYP2A7, NOLA2, CYP3A4, CYP2B6, CYP4A11, HSD11B1.
Conclusion:
According to the result of cDNA array, we have discovered that abnormally expressed genes widely existed in HCC among which the expression of MAP4K4 was obviously up-regulated. Also, as an upstream activity factor of MAPK signal conduction pathway, MAP4K4 participates in the cell apoptosis, differentiation and proliferation as well as the malignant transformation and so on. In conclusion, further analysis on MAP4K4 may contribute to understand its biological behavior on HCC, thus provides us a new approach for the gene targeted therapy of HCC.
PartⅡThe expression of MAP4K4 in hepatoma cells and hepatocellular carcinoma tissue
Objective:
To detect the expression of MAP4K4 at transcriptional and translational levels in hepatoma cells and hepatocellular carcinoma tissues to verificate the results of gene array, and analyze the expression level of MAP4K4 protein in different clinicopathological parameters, thus to preliminarily confirm the significance of MAP4K4 in the development of HCC.
Methods:
20 cases of samples selected from fresh HCC and adjacent tissues as well as four hepatoma cells(Hep3B, HepG2, SMMC7721, Huh-7)were detected respectively for the expression of MAP4K4 mRNA and protein through RT-PCR, qRT-PCR and Western-blot. We detected the expression activity of MAP4K4 protein in 400 paired samples of HCC and adjacent tissues through tissue microassay which constructed by 400 paraffin embedded blocks of hepatocellular carcinoma and analyzed the expression level of MAP4K4 protein in different clinicopathological parameters.
Results:
1. MAP4K4 mRNA expressed in HCC tissue
In adjacent tissues, MAP4K4 mRNA was expressed from 0.001339 to 0.018747(average 0.007900), but from 0.007367 to 0.080935(average 0.029500) in HCC tissues. Compared to the adjacent tissues, MAP4K4 mRNA in HCC tissues was upregulated from 1.062 to 28.520 folds (average 6.852). Paired t-test demonstrated that the difference was significant between HCC and adjacent tissues(P < 0.001).
2.The expression of MAP4K4 mRNA in hepatoma cells
According to RT-PCR result, the expressions of MAP4K4 mRNA in the four cell lines were as follows: HepG2> Hep3B> Huh-7> SMMC7721. The result of qRT-PCR showed the relative quantity in the four hepatoma cells were HepG2(0.016008±0.002358), Hep3B(0.015629±0.001389, Huh-7(0.011674±0.001456), SMMC7721(0.004895±0.000947).
3. The expression of MAP4K4 protein in hepatoma cells
Through Western-blot, the expression of MAP4K4 protein could be detected in all the four hepatoma cells among which the expression in HepG2 was the highest, with Hep3B secondary and the SMMC7721 lowest.
4.The location and expression of MAP4K4 in hepatocellular caicinoma tissue
The expression of MAP4K4 was uniformly negative or weakly positive in normal, hepatitis and cirrhosis tissue. There was no positive staining of MAP4K4 in surrounding connective tissue, blood vessel and biliary ducts. Positive staining could be seen in germinal center in Kupffer cells and lympholeukocyte. Compared to the corresponding adjacent tissues, the expression of MAP4K4 was obviously overexpressed in dysplasia nodule cells than in normal, hepatitis and cirrhosis cells. The expression of MAP4K4 for hepatocellular carcinoma which increased significantly was heterogeneity with the number of overexpressed sample 194(48.5%) and the focus positive or negative 206(51.5%). The positive rate of MAP4K4 in HBsAg carriers was 51.5%(157/305), significantly higher than those in control group at 38.8% (37/95)(P = 0.033). The positive rate of MAP4K4 in tumor diameter≤2cm was 31.6%(18/57), notablly lower than tumor diameter > 2cm which was 51.3%(176/343) (P = 0.006). The difference of MAP4K4 was significant among different histological grading(P<0.001). The positive rate of MAP4K4 in metastasis was 55.5%(152/274), higher than non-metastasis which was 33.3%(42/126)(P = 0.002). No significant difference was found in the expression of MAP4K4 with different age, serum AFP concentration, degree of liver cirrhosis and capsular infiltration.
Conclusion:
MAP4K4 is generally highly expressed in hepatocellular carcinoma, which may have a certain relationship with HBV infection state, tumor size , histological grade and metastasis status.
PartⅢThe influence of MAP4K4 on the biological behavior of hepatoma cells
Objective:
To detect the change of biological activity (cell proliferation,cell clone,cell cycle and cell apoptosis) of hepatoma cells when MAP4K4 is knocked down and discuss the mechanism of this act preliminarily.
Methods:
In the first place, the expression of MAP4K4 protein in HepG2、Hep3B、Huh7、SMMC-7721 was detected and the highest expressed cell was selected for RNAi experiment. Secondly,a control group and three groups of specific MAP4K4 targeted siRNA(0—HepG2-pGCSil-U6,1—pSilMAP4K4-1,2—pSilMAP4K4-2,3—pSilMAP4K4-3)were designed. After that, we transfected them to hepatoma cells and detected the interfering efficiency through qRT-PCR and Western-blot, and then carried out the next tests: WST-8 cell proliferation test, flat plate clone formation test, cell cycle test and apoptosis test. Finally, we compared the differentially expressed genes between the best transfection group and the control group to further explore the possible mechanism of MAP4K4 in hepatoma cells by TOLL-like receptor signaling associated gene chip.
Results:
According to the expression of MAP4K4 protein in the four cells by Western-blot,we chose the highest expression cell HepG2 as the research target of MAP4K4-siRNA.
1. Detect the interference efficiency by qRT-PCR and Western-blot
By comparing the interference fragments from 1 to 3 after transfecting with the control group,we found that the inhibition rates of MAP4K4 mRNA were 68.7%, 25.9% and 53.7% and the inhibition rates of MAP4K4 protein were 61.4%, 27.8% and 45.8%.
2. The change of cellular shape
After interference, the population of transfected cells in logarithmic growth phase reduced with the volume becoming larger, cell adherence strengthened and the percentage of stage M cells decreasing .
3. Result of WST-8 cell proliferation test
In groups of pSilMAP4K4-1 and pSilMAP4K4-3, the speed of cell proliferation slowed down obviously compared to the control group(p<0.01). But there was no significant difference between pSilMAP4K4-2 group and control one(p>0.05).
4. result of Flat plate clone test
As follow was the detection result of cell clonality: HepG2-pGCSil-U6 (56.83±8.51%), pSilMAP4K4-1 (13.75±4.26%), pSilMAP4K4-2 (50.71±7.47%), pSilMAP4K4-3 (28.11±6.36%). The cloning efficiencies of pSilMAP4K4-1 and pSilMAP4K4-3 group were lower than the control group(p<0.05). But there was no significant difference between pSilMAP4K4-2 group and control one(p>0.05).
5. Result of cell cycle test
Compared with the control group, we could observe arrest of S phase(p<0.01), decrease of cell ratio in phase M/G1 (p<0.01) as well as increase of cell ratio in S and G2 phase (p<0.05) in group pSilMAP4K4-1 and pSilMAP4K4-3. But there was no significant difference between pSilMAP4K4-2 group and control one(p>0.05).
6. Result of apoptosis test
Induced by serum-free culture, the percentage of apoptotic HepG2 cells in pSilMAP4K4-1 and pSilMAP4K4-3 group increased compared with control one(p<0.05). But there was no significant difference between pSilMAP4K4-2 group and control one(p>0.05).
7. Result of gene chip
Detection result of gene chip for hepatoma cells before and after interference showed that the expression of considerable associated genes changed obviously (more than 2 folds up-regulated or 50% down-regulated). By referring to the map of TOLL-like signal pathway, we discovered that the possible mechanism of MAP4K4 acts in hepatoma cells may be associated with NF- kB and JNK signal pathway.
Conclusion:
MAP4K4 gene knockdown can arrest cell cycle and then inhibit proliferation of hepatoma cells. The mechanism may be associated with NF- kB and JNK signal pathway.
丝裂原激活蛋白激酶激酶激酶激酶4(MAP4K4)系丝/苏氨酸激酶亚家族STE20中的一员,位于染色体2q11.2,含有33个外显子,编码区包括九个可供选择的剪接位点。人MAP4K4最早克隆于巨噬细胞cDNA库,不同来源的组织可分离出不同的由cDNA编码的MAP4K4亚型。MAP4K4属于丝裂原激活蛋白激酶(MAPK)信号传导通路的一个上游激活因子,MAPK是一组可以被多种细胞外信号(包括生长因子,激素,紫外线辐射,DNA损伤剂,炎性细胞因子和环境应激等)激活的丝/苏氨酸激酶。MAPK信号转导通路非常保守,自膜受体到MAPK激酶激酶(MAPKKK)再到MAPK激酶(MAPKK)然后到MAPK,在细胞信号转导通路中MAPK处于细胞质部分的终末位置,活化后可以转到核内作用靶点,调节基因的表达。这种激活模型存在于酵母至哺乳类动物。它参与细胞的多种生物学行为,包括细胞凋亡,分化和增殖、细胞周期调控,细胞生存的维持以及细胞恶性转化等。STE20家族是MAPKKK的上游激酶,哺乳类动物STE20/丝裂原蛋白激酶激酶激酶激酶(MAP4K)家族由其催化区相关的28种丝氨酸/苏氨酸激酶组成。这些激酶可分为两种结构类别,P21活性蛋白激酶(PAKs)和生发中心激酶(GCKs)。GCK激酶缺乏PAKs激酶中调节Cdc42/Rac的作用区,而代之以N-末端激酶区和不同长度的C-末端延伸。C-末端是一个枸椽同源区(CNH),称为枸椽rho相互作用激酶(CRIK),该区域对于激酶活性的调节起着重要作用。GCK激酶在激酶区域外显示出低度同源性,并分为九个亚家族。MAP4K4是GCK IV组中的一个成员,它对细胞的作用包括调节细胞的运动性,细胞骨架重排以及细胞增殖等。研究发现MAP4K4在多种肿瘤中有高表达并证明了它在加速肿瘤细胞转化,促进细胞侵袭,降低细胞黏附性方面起着一定的作用。近期又有研究提出MAP4K4与卵巢癌、乳腺癌、前列腺癌和恶性黑色素瘤的侵袭和转移有着密切的关系,通过siRNA敲减这些细胞系的MAP4K4基因可抑制它们的侵袭和转移。最新研究报道MAP4K4在胰腺癌中也有高表达,并与其不良预后有关。以上提示MAP4K4信号通路很可能与肿瘤的发生,发展有着密切的关系。而目前对于MAP4K4在肝细胞癌中的研究尚未见报道。
我国为肝细胞癌高发国家,大多数肝细胞癌的发生与乙肝病毒感染密切相关,HBV是我国肝细胞癌发生的最直接危险因素。本实验首先应用cDNA芯片对1例HBV相关肝细胞癌进行检测,发现MAP4K4基因在肝细胞癌组织较癌旁组织显著高表达,受此启发,本课题拟探讨MAP4K4在肝细胞癌的生物学活性中是否也发挥一定的作用。据此,本课题的研究思路为:
首先,继续采用cDNA芯片技术对5例HBV相关肝细胞癌基因表达谱进行初步研究,对比癌和癌旁组织的差异基因表达,明确MAP4K4基因在肝细胞癌组织中确实较癌旁组织显著高表达。
然后,采用分子生物学方法和组织芯片技术进一步从转录和翻译水平检测肝癌细胞株及肝细胞癌组织中MAP4K4的表达情况,验证基因芯片的结果,并分析不同临床病理参数中MAP4K4蛋白表达水平的差异,以初步证实MAP4K4在肝细胞癌发展中的临床意义。
最后,再通过RNAi技术研究MAP4K4基因敲减对肝癌细胞生物学活性的影响,并通过基因芯片技术初步探讨其生物学作用产生的可能机制。
第一部分肝细胞癌基因表达谱的初步研究目的:
应用含20228个基因的人cDNA芯片研究5例HBV相关肝细胞癌及配对癌旁肝组织的基因表达差异。
方法:
随机选择HBV相关肝细胞癌及其癌旁肝组织样本5例分别抽提标本的总RNA,反转录为cDNA与芯片进行杂交,对比差异表达的基因。
结果:
HBV相关肝细胞癌组织相对于癌旁组织明显上调表达基因有52个(>3倍),其中上调大于5倍的基因为ASPH,FACL4,LAPTM4B,PEX11B,MAP4K4,SQSTM1,CCT3,CPD,CKS1B,HMGA1,ENO1;明显下调表达的基因有37个,下调大于10倍的基因为CYP2E1,MT2A,MT1X,CPS1,EXTL1,FCN3,CYP2A7,NOLA2,CYP3A4,CYP2B6,CYP4A11,HSD11B1。
结论:
通过cDNA芯片技术,发现肝细胞癌广泛存在的异常表达基因,其中MAP4K4基因明显上调,综合考虑它作为MAPK信号途径的一个上游因子可能参与了细胞分化、增殖、凋亡及恶性转化等生物学功能,因此进一步对MAP4K4基因进行分析将可能有助于了解它在肝细胞癌中的生物学作用,为肝细胞癌的基因靶向治疗开辟一条新途径。
第二部分MAP4K4在肝癌细胞株及肝细胞癌组织中的表达
目的:
从转录和翻译水平检测肝癌细胞株及肝细胞癌组织中MAP4K4的表达情况,以验证基因芯片结果,并分析不同临床病理参数中MAP4K4蛋白表达水平的差异,初步证实MAP4K4在肝细胞癌发展中的临床意义。
方法:
选取20例新鲜肝细胞癌组织和配对癌旁肝组织标本及四种肝癌细胞(Hep3B,HepG2,SMMC7721,Huh-7)进行RT-PCR,qRT-PCR和Western-blot分别检测MAP4K4 mRNA和蛋白水平的表达;对400例肝细胞癌蜡块标本构建肝细胞癌组织芯片,采用免疫组织化学技术检测MAP4K4蛋白在含400例配对肝细胞癌及癌旁组织的表达活性,分析不同临床病理参数中MAP4K4蛋白表达水平的差异。
结果:
1.肝细胞癌组织中MAP4K4 mRNA的表达
在癌旁肝组织,MAP4K4 mRNA表达为0.001339～0.018747(平均0.007900),而大部分癌组织表达高水平MAP4K4 mRNA,0.007367～0.080935(平均0.029500);与配对癌旁肝组织相比,肝细胞癌组织MAP4K4 mRNA上调1.062～28.520倍(平均6.852倍)。配对t-检验证实肝细胞癌组织和癌旁肝组织差异有显著性(P < 0.001)。
2.肝癌细胞株中MAP4K4 mRNA的表达
经RT-PCR检测,MAP4K4 mRNA在各细胞株的表达为:HepG2> Hep3B> Huh-7 > SMMC7721;qRT-PCR检测MAP4K4 mRNA在HepG2、Hep3B、Huh-7、SMMC7721四种肝癌细胞株中相对表达量依次为(0.016008±0.002358)、(0.015629±0.001389)、(0.011674±0.001456)、(0.004895±0.000947),结果得到qRT-PCR检测证实。
3.肝癌细胞株MAP4K4蛋白表达
经Western-blot检测,MAP4K4蛋白在四种肝癌细胞株中均有表达,在HepG2细胞中表达最高,其次是Hep3B细胞,而在SMMC7721中表达最低。
4.MAP4K4在肝细胞癌组织中的定位和表达
在正常肝、肝炎和肝硬化组织的肝细胞MAP4K4呈一致阴性或弱阳性表达,周围结缔组织、血管和胆管无MAP4K4阳性着色,Kuffer细胞和淋巴细胞生发中心可见阳性染色,与相应的癌旁肝组织相比,异型增生结节的肝细胞MAP4K4的表达明显强于正常肝、肝炎和肝硬化组织的肝细胞。肝细胞癌组织MAP4K4表达显著增强。肝细胞癌组织MAP4K4呈异质性表达,其中过度表达194例(48.5%),灶性阳性或阴性206例(51.5 %)。HBsAg阳性患者MAP4K4阳性率51.5%(157/305)显著高于阴性的38.8% (37/95)(P = 0.033);肿瘤直径≤2cm的MAP4K4阳性率31.6%(18/57)显著低于肿瘤直径> 2cm的51.3%(176/343) (P = 0.006);MAP4K4在不同组织学分级中的阳性率差异有显著性(P<0.001);肝内转移病例的MAP4K4阳性率55.5%(152/274)显著高于未转移者的33.3%(42/126)(P = 0.002)。MAP4K4表达在患者年龄、血清甲胎蛋白浓度、肝硬化、包膜浸润方面差异无显著性。
结论:
MAP4K4在肝细胞癌中普遍高表达,并且其表达水平与HBV感染状态,肿瘤大小、组织学分级及转移情况可能有一定关系。
第三部分MAP4K4对肝癌细胞生物学功能的影响
目的:
检测MAP4K4基因敲减后肝癌细胞生物学活性(细胞增殖、细胞克隆形成、细胞周期、细胞凋亡)的改变并初步探讨其作用机制。
方法:
检测HepG2、Hep3B、Huh7、SMMC-7721肝癌细胞株中MAP4K4蛋白表达,选择表达活性最强者进行siRNA实验。设计一个空载体质粒转染组及三对靶向MAP4K4的特异性siRNA核苷酸序列质粒转染组(0—HepG2-pGCSil-U6,1—pSilMAP4K4-1,2—pSilMAP4K4-2,3—pSilMAP4K4-3),用Lipofectamine 2000法分别转染到选取的肝癌细胞,qRT-PCR和Western-blot方法检测干扰效率,并对转染后肝癌细胞进行WST-8细胞增殖实验,平板克隆形成实验,PI染色法细胞周期检测,Annexin V法细胞凋亡检测,然后,选择TOLL样受体信号途径相关芯片对干扰效果最好转染组及空载体转染组细胞进行基因检测,以进一步探究MAP4K4基因在肝癌细胞中可能的作用机制。
结果:
依据Western-blot检测四种肝癌细胞株中MAP4K4蛋白表达情况,我们选择其中表达该蛋白最高的HepG2肝癌细胞株作为MAP4K4-siRNA的研究靶标。
1.qRT-PCR和Western-blot检测干扰效率
转染干扰片段1,2,3后与对照组相比,MAP4K4 mRNA抑制率分别为68.7%,25.9%和53.7%;MAP4K4蛋白的抑制率分别为:61.4%,27.8%和45.8%。
2.细胞生长形态改变
MAP4K4 RNA干扰后,对数生长期的转染组细胞数目减少,体积变大,贴壁性增强,较对照组比较,圆形M期细胞比例减少。
3. WST-8细胞增殖实验结果
pSilMAP4K4-1、pSilMAP4K4-3组,细胞增殖速度明显减慢( p<0.01),而转染pSilMAP4K4-2组与对照组相比差异无显著性(p>0.05)。
4.平板克隆实验结果
各转染组细胞克隆形成能力的检测结果:HepG2-pGCSil-U6 (56.83±8.51%)、pSilMAP4K4-1组(13.75±4.26%)、pSilMAP4K4-2组(50.71±7.47%)、pSilMAP4K4-3组(28.11±6.36%)。pSilMAP4K4-1和pSilMAP4K4-3组细胞的平板克隆形成率明显低于对照组(p<0.01),而pSilMAP4K4-2组与对照组间的差异无显著性(p>0.05)。
5.细胞周期实验结果
与对照组比较, pSilMAP4K4-1和pSilMAP4K4-3组均出现S期阻滞(p<0.01),M/G1期细胞比例减少(p<0.01),S、G2期细胞比例增多(P<0.05)。而pSilMAP4K4-2组与对照组相比差异无显著性(p>0.05)。
6.细胞凋亡检测结果
经无血清诱导,与对照组比较,pSilMAP4K4-1、pSilMAP4K4-3组的HepG2细胞凋亡比例增多(p<0.05),而pSilMAP4K4-2组与对照组比较差异无显著性(p>0.05)。
7.基因芯片检测结果
干扰前后肝癌细胞样本的基因芯片检测结果显示MAP4K4基因敲减后,有一定数目与其相关的基因的表达有明显改变(上调2倍以上或下调1/2以上),参照TOLL样信号传导途径通路图进行分析,我们发现MAP4K4基因在肝细胞癌中可能是通过NF-kB及JNK信号传导途径发挥作用的。
结论:
MAP4K4基因敲减可以阻滞细胞周期的进程,抑制肝癌细胞的恶性增殖,其机制可能是通过NF-kB信号传导途径、JNK信号传导途径发挥作用。
Hepatocellular carcinoma(HCC) is the most common primary malignancy of the liver, which is also known as“the king of cancers”. Due to the fact that there is no available therapeutic means for HCC, more and more scientists have been devoting themselves to the research of gene targeted therapy. The genesis of HCC is the result of multi-factors, multi-approaches and multi-steps, including carcinogenes in the external environment (virus, parasite, bacterial infection, intake of aflatoxin, water pollution, smoking and drinking) and inherent genetic factors(activation of oncogenes and inactivation of tumor suppressor genes). So far, several HCC-associated genes have been discovered, consisting of N-ras, c- fos, p53, C- myc, IGF-Ⅱ, IGF-ⅡR, p16, p21, DCC, nm- 23, c- erB-b- 2, TGF-α, CSF- IR, raf, et al. The molecular basis of HCC is the mutation of oncogenes and tumor suppressor genes which is a process with multi-genes participation and a multi-steps.
Mitogen-activated protein kinase kinase kinase kinase 4(MAP4K4) is a member of the serine/threonine kinase subfamily, Ste20. It lies in the chromosome 2q11.2 which includes 33 exons with the coding region containing nine optional spliced sites. MAP4K4 was firstly cloned from cDNA library of macrophage . Different subtypes of MAP4K4 could be separated from different tissues. MAP4K4 is an upstream stimulating factor of mitogen-activated protein kinase (MAPK) signaling. As a kind of Ser/Thr kinase, MAPK can be activated by many extracellular signals(including growth factors, hormones, ultraviolet radiation, DNA damage agents, proinflammato- ry cytokines,environmental stress, et al). The signal transduction pathway of MAPK is very conservative. Transferred from membrane receptors to MAPKKK, then to MAPKK, finally to MAPK, MAPK lies in the end location of the cytoplasm. When activated, MAPK could be transferred into intranuclear target and regulate the expression of genes. This kind of activated pattern resides from yeast to mammalian, which plays a pivotal role in a variety of processes, such as apoptosis,cell differentiation and cell proliferation, regulation of cell cycle, sustain of cell survival and the malignant transformation, and so on. STE20 is an upstream kinase of MAPKKK. In mammalians, STE20/MAP4K is composed of 28 Ser/Thr kinases which locate in its catalytic domain. These kinases can be divided into two structure categories,P21 PAKs and GCKs. GCKs lack the acting region which PAKs posses that could regulate the Cdc42/Rac, but with N-terminus kinase region and C-terminus extension of different length instead. C-terminus is a CNH region, which is called CRIK and plays an important role in regulating the kinase activity. GCK kinases are divided into nine subfamilies and show low-grade homology. As a member of GCK IV, MAP4K4 participates in the regulation of cellular mobility, cystoskeleton rearrangement and cell proliferation. Previous researches have demonstrated that MAP4K4 is highly expressed in many tumors, and confirmed that it plays a certain role in accerlerating the transformation of tumour cells, promoting the cellular invasion as well as reducing the cell adhesion. Recently, it has also been proposed that MAP4K4 is closely associated with the invasion and diversion of ovarian cancer, breast cancer, prostate carcinoma and malignant melanoma. Knockdown of MAP4K4 by siRNA can inhibit the cell invasion and metastasis. The latest study has shown that MAP4K4 is also overexpressed in pancreatic cancer which is related to its poor prognosis. The findings above indiacated that MAP4K4 signaling may have close relationship with the genesis and development of cancer. But up to now, there is no report about MAP4K4 in HCC.
There is a high incidence of HCC in China, and most patients are associated with the infection of hepatitis B virus (HBV) which is the most directly risk factor of HCC in our country. The cDNA array was firstly applied in our research to detect one case of HBV associated HCC. We discovered that MAP4K4 was significantly highlier expressed in HCC compared to adjacent tissues. Inspired by it, we plan to investigate the biologic activity of MAP4K4 in HCC. And as follow is our research strategy:
Firstly, the gene expression profile of 5 cases of HBV associated HCC will be analysed preliminarily through cDNA array to compare the differential gene expression between HCC and adjacent tissues, and to ascertain whether MAP4K4 is significantly highier expressed in HCC than adjacent tissues.
Secondly, further detection for the expression levels of MAP4K4 in hepatoma cells and hepatocellular carcinoma tissues will be carried out using RT-PCR and Western-blot to verificate the results of gene array and analyze the expression level of MAP4K4 protein in different clinicopathological parameters, thus to confirm preliminarily the clinical significance of MAP4K4 in the development of HCC.
Finally, the effect of MAP4K4 knockdown on the biological activity of hepatoma cells will be investigated by RNA interference, and the possible mechanism involved will also be researched through gene chip technology.
Part I The preliminary research of gene expression in hepatocellualr caicinoma tissue
Objective:
Compared the gene expression between HCC and adjacent tissues in five HBV associated HCC genes through cDNA array that contain 20228 genes.
Method:
5 cases of samples were randomly selected from HBV associated HCC and adjacent tissues to extract their total RNA which were transcripted reversely to cDNA and then hybridized with gene chip to compare the differentially expressed genes.
Results:
Comparing the HBV associated HCC to adjacent tissues, there were 52 expressed genes obviously up-regulated. Genes more than 5-fold up-regulation: ASPH, FACL4, LAPTM4B, PEX11B, MAP4K4, SQSTM1, CCT3, CPD, CKS1B, HMGA1, ENO1. And there are 37 genes obviously down-regulated. Genes more than 10-fold down-regulation: CYP2E1, MT2A, MT1X, CPS1, EXTL1, FCN3, CYP2A7, NOLA2, CYP3A4, CYP2B6, CYP4A11, HSD11B1.
Conclusion:
According to the result of cDNA array, we have discovered that abnormally expressed genes widely existed in HCC among which the expression of MAP4K4 was obviously up-regulated. Also, as an upstream activity factor of MAPK signal conduction pathway, MAP4K4 participates in the cell apoptosis, differentiation and proliferation as well as the malignant transformation and so on. In conclusion, further analysis on MAP4K4 may contribute to understand its biological behavior on HCC, thus provides us a new approach for the gene targeted therapy of HCC.
PartⅡThe expression of MAP4K4 in hepatoma cells and hepatocellular carcinoma tissue
Objective:
To detect the expression of MAP4K4 at transcriptional and translational levels in hepatoma cells and hepatocellular carcinoma tissues to verificate the results of gene array, and analyze the expression level of MAP4K4 protein in different clinicopathological parameters, thus to preliminarily confirm the significance of MAP4K4 in the development of HCC.
Methods:
20 cases of samples selected from fresh HCC and adjacent tissues as well as four hepatoma cells(Hep3B, HepG2, SMMC7721, Huh-7)were detected respectively for the expression of MAP4K4 mRNA and protein through RT-PCR, qRT-PCR and Western-blot. We detected the expression activity of MAP4K4 protein in 400 paired samples of HCC and adjacent tissues through tissue microassay which constructed by 400 paraffin embedded blocks of hepatocellular carcinoma and analyzed the expression level of MAP4K4 protein in different clinicopathological parameters.
Results:
1. MAP4K4 mRNA expressed in HCC tissue
In adjacent tissues, MAP4K4 mRNA was expressed from 0.001339 to 0.018747(average 0.007900), but from 0.007367 to 0.080935(average 0.029500) in HCC tissues. Compared to the adjacent tissues, MAP4K4 mRNA in HCC tissues was upregulated from 1.062 to 28.520 folds (average 6.852). Paired t-test demonstrated that the difference was significant between HCC and adjacent tissues(P < 0.001).
2.The expression of MAP4K4 mRNA in hepatoma cells
According to RT-PCR result, the expressions of MAP4K4 mRNA in the four cell lines were as follows: HepG2> Hep3B> Huh-7> SMMC7721. The result of qRT-PCR showed the relative quantity in the four hepatoma cells were HepG2(0.016008±0.002358), Hep3B(0.015629±0.001389, Huh-7(0.011674±0.001456), SMMC7721(0.004895±0.000947).
3. The expression of MAP4K4 protein in hepatoma cells
Through Western-blot, the expression of MAP4K4 protein could be detected in all the four hepatoma cells among which the expression in HepG2 was the highest, with Hep3B secondary and the SMMC7721 lowest.
4.The location and expression of MAP4K4 in hepatocellular caicinoma tissue
The expression of MAP4K4 was uniformly negative or weakly positive in normal, hepatitis and cirrhosis tissue. There was no positive staining of MAP4K4 in surrounding connective tissue, blood vessel and biliary ducts. Positive staining could be seen in germinal center in Kupffer cells and lympholeukocyte. Compared to the corresponding adjacent tissues, the expression of MAP4K4 was obviously overexpressed in dysplasia nodule cells than in normal, hepatitis and cirrhosis cells. The expression of MAP4K4 for hepatocellular carcinoma which increased significantly was heterogeneity with the number of overexpressed sample 194(48.5%) and the focus positive or negative 206(51.5%). The positive rate of MAP4K4 in HBsAg carriers was 51.5%(157/305), significantly higher than those in control group at 38.8% (37/95)(P = 0.033). The positive rate of MAP4K4 in tumor diameter≤2cm was 31.6%(18/57), notablly lower than tumor diameter > 2cm which was 51.3%(176/343) (P = 0.006). The difference of MAP4K4 was significant among different histological grading(P<0.001). The positive rate of MAP4K4 in metastasis was 55.5%(152/274), higher than non-metastasis which was 33.3%(42/126)(P = 0.002). No significant difference was found in the expression of MAP4K4 with different age, serum AFP concentration, degree of liver cirrhosis and capsular infiltration.
Conclusion:
MAP4K4 is generally highly expressed in hepatocellular carcinoma, which may have a certain relationship with HBV infection state, tumor size , histological grade and metastasis status.
PartⅢThe influence of MAP4K4 on the biological behavior of hepatoma cells
Objective:
To detect the change of biological activity (cell proliferation,cell clone,cell cycle and cell apoptosis) of hepatoma cells when MAP4K4 is knocked down and discuss the mechanism of this act preliminarily.
Methods:
In the first place, the expression of MAP4K4 protein in HepG2、Hep3B、Huh7、SMMC-7721 was detected and the highest expressed cell was selected for RNAi experiment. Secondly,a control group and three groups of specific MAP4K4 targeted siRNA(0—HepG2-pGCSil-U6,1—pSilMAP4K4-1,2—pSilMAP4K4-2,3—pSilMAP4K4-3)were designed. After that, we transfected them to hepatoma cells and detected the interfering efficiency through qRT-PCR and Western-blot, and then carried out the next tests: WST-8 cell proliferation test, flat plate clone formation test, cell cycle test and apoptosis test. Finally, we compared the differentially expressed genes between the best transfection group and the control group to further explore the possible mechanism of MAP4K4 in hepatoma cells by TOLL-like receptor signaling associated gene chip.
Results:
According to the expression of MAP4K4 protein in the four cells by Western-blot,we chose the highest expression cell HepG2 as the research target of MAP4K4-siRNA.
1. Detect the interference efficiency by qRT-PCR and Western-blot
By comparing the interference fragments from 1 to 3 after transfecting with the control group,we found that the inhibition rates of MAP4K4 mRNA were 68.7%, 25.9% and 53.7% and the inhibition rates of MAP4K4 protein were 61.4%, 27.8% and 45.8%.
2. The change of cellular shape
After interference, the population of transfected cells in logarithmic growth phase reduced with the volume becoming larger, cell adherence strengthened and the percentage of stage M cells decreasing .
3. Result of WST-8 cell proliferation test
In groups of pSilMAP4K4-1 and pSilMAP4K4-3, the speed of cell proliferation slowed down obviously compared to the control group(p<0.01). But there was no significant difference between pSilMAP4K4-2 group and control one(p>0.05).
4. result of Flat plate clone test
As follow was the detection result of cell clonality: HepG2-pGCSil-U6 (56.83±8.51%), pSilMAP4K4-1 (13.75±4.26%), pSilMAP4K4-2 (50.71±7.47%), pSilMAP4K4-3 (28.11±6.36%). The cloning efficiencies of pSilMAP4K4-1 and pSilMAP4K4-3 group were lower than the control group(p<0.05). But there was no significant difference between pSilMAP4K4-2 group and control one(p>0.05).
5. Result of cell cycle test
Compared with the control group, we could observe arrest of S phase(p<0.01), decrease of cell ratio in phase M/G1 (p<0.01) as well as increase of cell ratio in S and G2 phase (p<0.05) in group pSilMAP4K4-1 and pSilMAP4K4-3. But there was no significant difference between pSilMAP4K4-2 group and control one(p>0.05).
6. Result of apoptosis test
Induced by serum-free culture, the percentage of apoptotic HepG2 cells in pSilMAP4K4-1 and pSilMAP4K4-3 group increased compared with control one(p<0.05). But there was no significant difference between pSilMAP4K4-2 group and control one(p>0.05).
7. Result of gene chip
Detection result of gene chip for hepatoma cells before and after interference showed that the expression of considerable associated genes changed obviously (more than 2 folds up-regulated or 50% down-regulated). By referring to the map of TOLL-like signal pathway, we discovered that the possible mechanism of MAP4K4 acts in hepatoma cells may be associated with NF- kB and JNK signal pathway.
Conclusion:
MAP4K4 gene knockdown can arrest cell cycle and then inhibit proliferation of hepatoma cells. The mechanism may be associated with NF- kB and JNK signal pathway.
引文
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