An In Vitro Study: Construction, Expression Of Recombinant Eukaryotic Vector Of KiSS-1 Gene And The Relationship Between It And Hepatoma Carcinoma Cell In Proliferation, Adhesion And Invasion

Objective By constructing a recombinant eukaryotic expression vector containing the functional region of human KiSS-1 gene and transfecting this vector into human hepatoma carcinoma cell line MHCC97-H with high metastasis potential to abtain transient-expression cell line, we meant to investigate the impact of the expression of KiSS-1 gene on the proliferation, adhesion and invasion of hepatoma carcinoma cells, which settled the foundation for the father study of the inhibitory effect of KiSS-1 gene on invasion and metastasis of human hepatocellular carcinoma(HCC).Methods KiSS-1 gene was amplified using reverse transcription polymerase chain reaction ( RT-PCR ).Then the gene was cloned to pcDNA3. 1/ HisC and amplified in E. coli Dh5α. The recombinant eukaryotic expression vector was obtained and detected using PCR, digestion identification and sequencing. Liposome was used to transiently transfer KiSS-1 into MHCC97-H cells,a human hepatoma carcinoma cell line with high metastasis potential and the result was detected by RT-PCR, immunocytochemistry ( ICC )and Western blot. The effect of KiSS-1 on the proliferation of MHCC97-Hs was detected by flow cytometry ( FCM ) and MTT assay and their adhesive abilities to matrigel and fibronectin were also detected by MTT assay. The effects of KiSS-1 on invasion and motility of the transfected cells were investigated by reconstituted matrigel invasion and polycarbonate filters migration experiments.Results1.The recombinant eukaryotic expression vector pcDNA3. 1/ HisC/ KiSS-1 was obtained and it was confirmed that KiSS-1 cDNA was inserted into the eukaryotic expression vector correctly using PCR, digestion identification and sequencing. 2. It was showed that KiSS-1 gene had been transfected into MHCC97-H cells using liposome and the result was detected by RT-PCR, ICC and Western blot.3.The expression of KiSS-1 obviously reduced the adhesion of MHCC97-H cells to matrigel and fibronectin (P<0.01 ), when compared with cells of other two groups.4.The invasion of the cells expressing KiSS-1 gene was significantly retarded in matrigel-coated invasion chambers(P<0.01), when compared to cells of control and parental groups.5.The motility of the cells expressing KiSS-1 gene was significantly retarded in invasion chambers(P<0.01), when compared with cells of other two groups.6.There was no significant cell cycle blockage in MHCC97-H cells expressing KiSS-1, when compared with cells of control and parental groups, analyzing by FCM.7. The proliferation of MHCC97-H cells expressing KiSS-1 gene was a little inhibited and with no significance, while compared with cells of control and parental groups.Conclusion1. The recombinant eukaryotic expression vector pcDNA3. 1/ HisC/ KiSS-1 and the transient-expression KiSS-1 cells were successfully obtained, which settled the foundation for the father study of founding out the impact on proliferation, adhesion and invasion of KiSS-1 in hepatoma carcinoma cells through vitro experiment.2. It was showed that the abilities of adhesion to Matrigel and Fibronectin , the invasion and motility of hepatoma carcinoma cells could be suppressed by KiSS-1 through vitro experiment.3. The ability of proliferation of hepatoma carcinoma cells wasn′t affected by KiSS-1, which showed by MTT assay and FCM.4. It was showed that the abilities of adhesion, invasion and motility of hepatoma carcinoma cells could be suppressed by KiSS-1, and it implied that KiSS-1 might be a tumor metastasis suppressor gene and a new candidate for gene therapy against human hepatocellular carcinoma.