| Backgroud and ObjectsAccording to UICC World Cancer Congress 2006, malignant tumor is still the main type of diseases that is severely harmful to people?s health and lives in 21st century. Each year, more than 11 million new cases of cancer are diagnosed, and more than 7 million people die from cancer. At current, there are approximately 25 million cases with the disease. In 2020, if current trends continue, new cases will increase to 16 million and more than 10 million people will die every year. In China, There are more than 2 million new cases of cancer, and 1.5 million cases die yearly, and malignant tumor is still the main cause that induces to death in urban dwellers.Many researches indicate that abnormal expression of hepatocyte growth factor (HGF) is highly correlated to occurrence, development and prognosis of malignant tumors. As a cytokine with multiple bioactivities, HGF is produced mainly in paracrine secretion pathway (stroma-cell) or autocrine secretion pathway (tumor cell). HGF activates receptor tyrosine kinase through HGF/Met signaling pathway to phosphorylate the downstream various signal molecules, and then generates related biological effects, acting mainly as a mitogen, a motogen, a morphogen, and angioneogenesis, to prompt the growth, invasion and metastasis of the tumors. It is reported that there is the co-enhancement between HGF and lymphoma according to the findings in Hodgkin lymphoma, non-Hodgkin lymphoma and diffuse large B-cell lymphoma. High level of HGF promotes the occurrence and development of lymphoma; meanwhile, proliferation of lymphoma cells would promote the autocrine secretion of HGF. In addition, HGF is a potent valuable parameter to be investigated further. Therefore, blockage of HGF/Met signaling pathway maybe is a new strategy of antineoplasia treatment. According to current findings, NK4 is an ideal blocking agent of HGF/Met signaling pathway because it can completely block HGF effects competitively.As a matter of fact, NK4 is a 447-amino acid fragment of HGF composed of the N-terminal harinpin domain and four kringle domains of theα-subunit of HGF, and can binds to the HGF receptor c-MET but does not induce tyrosine phosphorylation of the latter, so competitively inhibit the HGF?s bioactivities such as the ability to promote the tumor?s growth, invasion, metastasis and angioneogenesis. Generally, NK4 is considered as a bifunctional molecule: an HGF-antagonist and an angiogenesis inhibitor. Because of its noticeable suppression effect in tumor from findings in lung cancer, colon cancer, pancreatic cancer, mammary cancer, ovarian cancer, prostatic carcinoma, gastric cancer, hepatoma, multiple myeloma and fibrosarcoma, NK4 has been a focal point in HGF related researches and has a potent perspective.Up to now, there are not any reports about NK4?s suppression effect in lymphoma internal and overseas.At current, there are two methods mainly in the researches of NK4 bioactivity: direct-acting pathway with NK4 protein and indirect-acting pathway with NK4 gene transfection mediated by the vector. The latter is more frequent, and contains two techniques: direct introduction and indirect introduction by transfection. The former can perform directly intratumor or intraperitoneal injection and be enhanced repeatedly. However, the time length of continual expression is short. On the contrary, the latter should transfect the target cell firstly, and then carry out the experiments in vivo or in vitro. Although it brings labourious precedures, it achieves stable, highly efficient expression after transfection, and obtains better experimental results.Therefore, we tried to investigate the suppression effect of NK4 in lymphoma by transfecting Raji cells with NK4 gene. The research was applied comprehensive techniques in molecular biology, cell biology and immunohistochemistry, such as cell culture, gene clone, gene transfection, semisolid culture, real-time FQ-PCR, immuocytohistochemistry and enzyme linked immunology assay.MethodsIn the part one, the special target cDNA sequences were obtained by RT-PCR with the primers designed on basis of target sequence, and then recombined with pGEM-T Easy and pMD19-T Simple to construct the recombinant plasmids, respectively, which were used as quantitative standards of real-time FQ-PCR. Having optimized amplification conditions and concentration of main components, the real-time FQ-PCR were established for NK4 mRNA, c-MET mRNA and HGF mRNA, respectively, and methodology evaluations were carried out, too.Keeping 0.5% agarose of underlayer gel unchanged, agarose concentration of upperlayer (0.1%~0.5%) and cell concentration (100 cell/well~5000 cell/well) were screened in order to modify the semisolid culture system.According to the restricted cleavage sites in multiple clone sites 2 of eukaryotic vector pVITRO2, the primers were designed to amplify the target cDNAs of NK4 and HGF whole sequences by RT-PCR, respectively. The recombinant plasmids were screened and sequenced after the cDNA were recombinated with pMD19-T Simple vectors, and then to be performed restricted cleavage to reclaim the target cDNA fragments of NK4 and HGF contained restrict cleavage sequences, respectively. The latters were recombinated with eukaryotic vector pVITRO2 to construct the recombinant eukaryotic expression plasmids pVITRO2-NK4 Whole and pVITRO2-HGF Whole, respectively.In part two, Raji cells were transfected with the recombinant eukaryotic expression plasmids pVITRO2-NK4 Whole and pVITRO2-HGF Whole by cation lipidosome method, respectively. The transfected cells were screened by hygromycin B, RT-PCR, ELISA and immuocytohistochemistry to verify the successful transfection; influences on Raji cell?s biological characters transfected with NK4 gene or HGF gene were observed by growth curve, clone formation and growth features. The changes of c-MET mRNA in transfected Raji cells were detected using established real-time FQ-PCR. The changes of concentration of phosphorylated c-MET protein in cells after transfection were measured by ELISA. Combination with the results as above, the suppression effect of NK4 gene transfection in growth, invasion and metastasis of Raji cells was estimated.Besides, NK4 gene-transfected Raji cell and HGF gene-transfected Raji cell were treated with 10 ng/ml HGF protein, 100 ng/ml NK4 protein, respectively. The suppression effects of NK4 protein in HGF gene-transfected cell and antagonistic action of NK4 gene-transfected cell to HGF protein were estimated by measurement of changes of parameters such as clone formation, growth character, and expression of mRNA, concentration of phosphorylated c-MET protein. ResultsIn part one, pGEM-T Easy-NK4, pGEM-T Easy-c-MET and pMD19-T Simple-HGF plasmids were constructed successfully, and then were quantitated as quantitation standards for real-time FQ-PCR of NK4 mRNA, c-MET mRNA and HGF mRNA, respectively. Real-time FQ-PCRs were established combined with the second pairs of primers, probes and optimization of amplification reaction conditions and concentrations of primers [0.8μmol/L (0.4μmol/L, each), probe (0.4μmol/L), dNTPs (0.8 mmol/L (0.2 mmol/L, each)) and MgCl2 [2.0 mmol/L (NK4)/1.5 mmol/L (NK4 and c-MET)], respectively.According to the slopes of the standard curve (-3.513±0.028, -3.680±0.073, -3.710±0.011) and correlation coefficients (0.999, 0.996, 0.998), amplification efficiencies of the real-time FQ-PCR of NK4 mRNA, c-MET mRNA and HGF mRNA system were 92.6±1.0%, 87.6±1.4%, and 86.0±0.4%, respectively. The coefficient variations of intra-assay (2.1%, 4.0%, 6.8%), inter-assay (1.9%, 3.9%, 7.0%) and inter-day-assay (2.5%,3.1%,8.3%) were confirmed. Meanwhile, the sensitivity of FQ-PCR was 2, 5, 5 copies of recombined plasmid/μl respectively.The modified semisolid culture system was composed of 0.3% agarose of upperlayer gel and 1000 cells/well cell density combined with 0.5% agarose of underlayer gel. In addition, the recombinant eukaryotic expression plasmids pVITRO2-NK4 Whole and pVITRO2-HGF Whole were constructed after gene recombination was performed with whole fragments of NK4 gene cDNA and HGF gene cDNA which were obtained by RT-PCR, respectively. Integrality and correctness of the cDNAs inserted in vectors were confirmed screened by PCR, restrict cleavage and sequencing, and so on.In part two, the targets sequences of NK4 and HGF were confirmed to be recombinated with genome of Raji cell transfected by recombinant plasmids pVITRO2-NK4 whole and pVITRO2-HGF whole screened by homomycin B. Protein concentrations of NK4 and HGF were 4.14±0.19 ng/ml and 5.38±0.31 ng/ml, respectively. The results of immuocytohistochemistry of transfected Raji cells were positive, too. The results as above indicated that the gene transfection was successful.According growth curves, the peak time of NK4, HGF gene transfected cells were 5 days and 3 days, respectively, and were different from the control groups [nontransfected Raji cells (4 days), pVITRO2-transfected Raji cells (4 days)]. The clones of NH4 gene transfected cells were smaller, regular, and the disseminated cells were rare. However, the clones of HGF gene transfected cells were bigger, irregular, and the disseminated cells were obvious. The expressions of c-MET mRNA (5.71±0.29, 6.48±0.18), the concentrations of phosphorylated c-MET protein (3.35±0.38, 44.96±1.18 U/ml) and clone formation (64.39±4.63, 124.61±5.16) were statistical significance compared with control groups (P<0.001). So do between two experiment groups (P<0.001). The Results indicated that NK4 gene transfection suppressed the proliferation of Raji cells, but HGF gene transfection prompted the proliferation of Raji cells.In addition, clonality (89.78±5.38/well) and phosphorylated c-MET protein at 10 min after 100 ng/ml NK4 protein treatment (8.97±1.19 U/ml) were degraded in HGF gene-transfeceted cell group. On the contrary, c-MET mRNA (7.67±0.20) and HGF mRNA (9.16±0.14) were enhanced. However, at same time point in NK4 gene–transfected cell group treated with 10 ng/ml HGF protein, clonality (64.39±4.63/well), c-MET mRNA(8.10±0.22), HGF mRNA(9.73±0.34) and phosphorylated c-MET protein (65.24±2.04 U/ml) were all enhanced. There were statistical significances (P<0.001) between treated groups and control groups as well as nontreated groups, respectively. The results indicated that NK4 protein strengthened the irritable expression of c-MET mRNA, HGF mRNA, but suppressed the clone formation and c-MET protein phosphorylation in HGF gene-transfected Raji cell group. On the other hand, HGF protein induced the clone formation and strengthened the expressions of c-MET mRNA, HGF mRNA and c-MET protein phosphorylation.ConclusionsIn all, we studied the suppression effect of NK4 in lymphoma from molecular level to cellular level comprehensively and the research conclusions are as follows:1. Real-time FQ-PCRs of NK4 mRNA, c-MET mRNA, HGF mRNA are established successfully on the basis of construction of quantitation standards.2. The semisolid culture system is modified and eligible to cellular experiments.3. The recombinant eukaryotic expression plasmids of pVITRO2-NK4 Whole and pVITRO2-HGF Whole are constructed successfully.4. Raji cell lines transfected with NK4 gene or HGF gene are established when Raji cells are transfected with related recombinant plasmids, respectively.5. Although HGF gene transfection has the promotion effect on malignant characters of Raji cell line, NK4 gene transfection shows suppression effect.6. NK4 protein suppresses the biological characters of HGF gene-transfected Raji cell line, and NK4 gene-transfected Raji cell line is obviously antagonistic to HGF protein. |
