Expression And Biological Significance Of MAP4K4 In Primary Hepatocellular Carcinoma

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 tissueObjective: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 tissueObjective: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 tissueIn 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 cellsAccording 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 tissueThe 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 cellsObjective: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-blotBy 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 shapeAfter 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 testIn 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 testAs 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 testCompared 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 testInduced 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 chipDetection 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.