| Aim:Kinectin,an endoplasmic reticulum binding protein,was identified as a receptor of kinesin firstly.It can interact with integrins,RhoG and translation elongation factor respectively,which played an important role in cell processes.Our previous study has identified that Kinectin was one of the tumor associated antigens of HCC by SEREX method.There were four alternative splicing region D1-D4 in Kinectin gene in HCC tissues,but only D2 and D3 were located in the open reading frame(ORF).And Kinectin had four transcripts predicted by NCBI,which may translate into three kinds of isforms,D2+D3+,D2+D3-,D2-D3+.In previous study,we found that Kinectin mRNA expression levels in gene conserved regions showed no significant difference in HCC tissue,cancer adjacent tissue and normal liver tissue.But,it is interesting the ratio of Kinectin mRNA expression levels in gene D2L/D2S regions were significant higer in HCC tissue than cancer adjacent tissue and normal liver tissue.The major purpose of this study was to elucidate the role of Kinectin isforms containing the D2 splicing region(hereafter referred as Kinectin D2 isoforms)in hepatocellular carcinoma.That would provid a new point for the study of HCC development mechanism,and a new basic data for the Kinectin used in the clinical diagnosis and treatment of HCC.Methods:1.To prepare polyclonal antibody against short peptides(28AA)from Kinectin splicing region D2,and detected the protein expression of Kinectin D2 isforms containing D2 splicing region and Kinectin conserved region protein expression in hepatocellular carcinoma tissues and cancer adjacent tissues by immunohistochemistry.2.To construct three recombinant lentiviral vectors of LV5-Kinectin D2+and LV5-Kinectin D2-and LV5NC(Negative control),and transfect 293T cells to aquire virus and determin virus titer respectively.3.LV5-KinectinD2+,LV5-KinectinD2-and LV5NC were transfected into hepatoma cells HepG2 respectively,and the best multiply of infection were explicited,and established stable transfection cell lines HepG2/LV5-KinectinD2+and HepG2/LV5-KinectinD2-and HepG2/LV5NC using puromycin.4.To compare the biological behavior of cells in the three groups HepG2/LV5NC and HepG2/LV5-KinectinD2-and HepG2/LV5-KinectinD2+:the CCK8 assay and colony formation assay was used to detect cell proliferation,the flow cytometry was used to detect the change of apoptosis and cycle of HepG2 cells,the scratch-wound assay and transwell migration assay was used to test cell migration capabilities,.5.Tumorigenicity in vivo in nude mice were performed by three groups HepG2/LV5-KinectinD2+ and HepG2/LV5-KinectinD2-and HepG2/LV5NC respectively,by observing the change of tumor size and nude mice weight.Results:1.The immunohistochemistry results showed the total optical density values(IOD)and Mean Optical Density(MOD)of Kinectin D2 protein in hepatocellular carcinoma tissues were significantly higher than adjacent paired tissues(75142±16663vs54079±10825,0.09518±0.013vs0.0777±0.011,P<0.05).2.The immunohistochemistry results showed the IOD and MOD of Kinectin conserved region protein in hepatocellular carcinoma tissues was significantly higher than adjacent paired tissues(76320±15904 vs 57897±14877,0.0982±0.017 vs 0.0819±0.015,<0.05).3.The best MOI values of HepG2 cells transfection were 100/70/70 in LV5-KinectinD2+/LV5-KinectinD2-/LV5NC.The stable transfection cell lines HepG2/LV5-KinectinD2+ and HepG2/LV5-KinectinD2-and HepG2/LV5NC were established.4.The CCK8 assay showed the proliferation of HepG2/LV5-KinectinD2+group was higher than HepG2/LV5-KinectinD2-and HepG2/LV5NC group(2.823±0.256vs2.349±0.076&2.232±0.093,P<0.05).The colony formation assay showed the rate of colony.forming in the HepG2/LV5-KinectinD2+ group was higher than the HepG2/LV5-KinectinD2-and HepG2/LV5NC group(57.00±3.61 vs48.66±5.11&40.33±3.21,P<0.05).5.The cell cycle assayed by flow cytometry showed that the HepG2/LV5-KinectinD2+ group had less G1 phase cells(%)and more S phase cells(%)than the HepG2/LV5-KinectinD2-group and HepG2/LV5NC group(56.973±3.587 vs66.070±2.099&77.850±1.515,25.547±1.638 vs20.120±3.852&17.223±2.140,P<0.01).6.The apoptotic cells assayed by flow cytometry showed that the HepG2/LV5-KinectinD2+ group had less apoptotic cells(%)than the HepG2/LV5-KinectinD2-group and HepG2/LV5NC group(24.633±1.931 vs31.493±0.791&32.793±1.068<0.01).7.The scratch-wound assay showed the migration were no significantly difference among HepG2/LV5-KinectinD2 group and HepG2/LV5-KinectinD2-and HepG2/LV5NC group(28.667±8.327 vs26.343±8.505&23.333±7.095,P>0.05).The transwell migration assay showed the cell that passed the transwell membrane Were no significantly difference among HepG2/LV5-KinectinD2+ group and HepG2/LV5-KinectinD2-and HepG2/LV5NC group(51.010±7.937 vs48.667±5.859&41.001±6.557,P>0.05).8.The size of subcutaneous tumor in nude mice in the HepG2/LV5-KinectinD2+ group were larger than HepG2/LV5-KinectinD2-group and HepG2/LV5NC group from the first week to the fourth week(57.300±34.59 vs30.300±31.66&23.563±30.34/1w,198.870±106.92vs63.701±29.12&49.736±51.02/2w,650.97±142.97 vs440.93±272.33&350.76±192.64/3w,966.14±225.53 vs559.06±239.59&406.45±322.73/4w,P>0.05).9.Weight of nude mice increased from 1-2 weeks,while subcutaneous tumor were formed,but weight was declining after three weeks.The fourth weeks HepG2/LV5-KinectimD2+nude mice weighed was less than the weight of nude mice in HepG2/LV5NC(17.938±1.891vsl9.000±0.927,P<0.05)·Conclusions:1.Kinectin D2 isoforms protein and Kinectin conserved region protein were higher expressed in HCC tissues than in adjacent paired HCC tissues.2.Kinectin D2 isoforns can significantly enhence cells proliferation and clonogenic capacity in HepG2 cells,promote cellular from DNA synthesis early(G1 phase)into the DNA synthesis phase(S phase),and reduce apoptosis,but had no effect on cell migration.3.Kinectin D2 isoforms can significantly promoted the tumorigenicity and development of tumors in nude mice.4.Kinectin D2 isoforms were involved into the development of liver cancer. |
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