NK4Gene Regulates HGF-induced Biological Outcome Of Cholangiocarcinoma Cells

Background:Normal cells have the function of homeostatic regulation. But in cancer cells, itperformed malignant behaviors as uncontrolled proliferation, invasion and metastasisetc, most of the reasons were abnormal regulation of cellular signal transduction, lossof control for cell cycle restriction point, growth factors autocrine and the ability toidentify and adhesion in the surface of cancer cells. In signal transduction pathwaysthat mediating tumor cell proliferation and invasion, the most important is thehepatocyte growth factor (HGF). In normal physiological conditions, HGF plays animportant role in promoting cell proliferation and regeneration; while in tumor tissues,it causes excessive proliferation and angiogenesis, and eventually promotes tumorinvasion and metastasis. Met is the specific receptor for HGF, studies confirmed thatactivation of HGF/Met signaling pathway promotes cell cycle progression, therebypromoting cell survival and proliferation; and participates in the formation of bloodvessels, so it plays an important role in tumor formation, invasion, metastasis andchemotherapy resistance, and thus become the target of tumor therapy. NK4has asimilar structure to HGF α, and competitive binding to the receptor Met, therebyinhibiting the biological effects of HGF/Met signaling pathway.Objective:1. To investigate the growth of HuCC-T1at different times and concentrations,determine the suitable concentration of HGF.2. cholangiocarcinoma cells line HuCC-T1were transfected with NK4gene, then HGF induced cells proliferation, invasion and migration were investigated, cellcycle distribution and the expression of cyclin D1and cyclin A were investigatedtoo.3. To investigate Met activation and the downstreamphosphatidylinositol-3-kinase(PI3K)/Akt/GSK-3β signaling pathway.Methods1. NK4gene expression vector pcDNA3/NK4was converted, amplified, endotoxinextraction, then restriction endonucleaser, sequencing and BLAST on line.2. Cholangiocarcinoma cell line HuCC-T1was transfected with empty plasmidpcDNA3and pcDNA3/NK4respectively, cells were cultured in selective mediumcontaining900μg/ml G418for the selection of resistant colonies asempty-vector-transfected clones of HuCC-T1(Hu-Em), and NK4-transfectedclones of HuCC-T1(Hu-NK4), with HuCC-T1cells serving as the control cells.The expression of NK4was confirmed by RT-PCR and Western blotting analysis.3. HuCC-T1cells were stimulated with different concentrations of HGF (0,2,5,10,20,50,100ng/ml) for24,48and72h, MTT method was used to observe thegrowth curve of the cells, and to determine the suitable concentration of HGF.4. Experimental groups: HuCC-T1group (control), Hu-Em group, Hu-NK4group,HuCC-T1+HGF group, Hu-Em+HGF group, Hu-NK4+HGF group.5. MTT and Transwell method were used to detect biological behavior of cellsproliferation, invasion and migration in each group.6. Flow cytometry was used to analyze cell cycle distribution, the mRNA levels ofcyclin D1and cyclin A were monitored using reverse transcription (RT)-PCR andquantitative PCR, and the corresponding protein levels were monitored usingWestern blotting.7. Met phosphorylation and the activity of its important down-stream signaling targets protein kinase B (Akt) and glycogen synthase kinase (GSK)-3β wereevaluated by Western blotting.Results:1. The pcDNA3/NK4plasmid was cloned, Restriction endonucleaser andsequencing identification suggested that the gene fragment was approximately1361bp in length, matched with the NK4gene in the gene bank.2. HuCC-T1cells were transfected with empty plasmid vector and NK4expressionvector,900μg/ml of G418was determined for screening of3-4weeks, RT-PCRand western blotting results showed that normal cells HuCC-T1and empty vectorcells Hu-Em expressed none or low NK4expression, while the stably transfectedHu-NK4highly expressed NK4protein.3. MTT results showed that HGF stimulate cell proliferation in a time and dosedependent manner,20ng/mL and48h were selected as the best concentration andtime of HGF stimulation.4. Without HGF stimulation, Hu-NK4cell proliferation and invasion showed nosignificant difference compared with the control group, but with20ng/mL HGFstimulation, HuCC-T1and Hu-Em cell proliferation and invasion speed up, andHu-NK4group of value-added rate did not change. And regardless of whether theHGF stimulate or not, cells migration in Hu-NK4group significantly smaller thanHuCC-T1and Hu-Em groups.5. Flow cytometry suggest that without HGF stimulation, each group of cell cycledistribution showed no significant difference; HGF stimulation could increase Sphase and decrease G1phase in the control group and Hu-Em, but not in Hu-NK4group. More over, when stimulated with HGF, the increases in mRNA levels ofcyclin D1and cyclin A were accompanied by corresponding increases in proteinlevels. 6. NK4over-expression can suppress HGF-induced Met, Akt and GSK-3β proteinphosphorylation.Conclusions：These findings suggest that NK4gene therapy inhibits HGF/Met-induced growthof human CCA cells by arresting cell cycle progression. It also interferes with Metactivation and the downstream PI3K/Akt/GSK-3β signaling pathway.