| Background and objectives: To construct and identify the RNAi lentiviral expression vector of Nodal gene,to investigate the expression of MNK-45 in human gastric adenocarcinoma cell line MNK-45,to silence Nodal in human gastric cancer cell line MNK-45,and to explore the effect of Nodal gene on human gastric adenocarcinoma cell line MNK-45 Death and angiogenesis.Methods: 1)Three siRNAs were designed and synthesized for Nodal mRNA,and one negative control siRNA was designed.Annealing to form double-stranded DNA,ligated with the vector(GV248),and the three recombinant plasmids were sequenced.If the sequence was consistent with the target sequence,it was suggested that the RNAi sequence of Nodal gene was inserted correctly.2)packaging and identification of recombinant lentiviral vector,The plasmid was extracted and the293 T cells were co-transfected with three plasmid vectors(recombinant plasmid and pHelper 1.0,pHelper 2.0),96 h after transfection of 293 T cells,observe the cells by fluorescence microscopy and determine the titer of the virus.3)Lentivirus was infected with gastric adenocarcinoma cell line MNK-45.After 72 hours of infection,the fluorescence was taken.Real-time qPCR was used to detect the expression of Nodal gene in MKN-45 cells after Nodal RNAi lentivirus infection.4)shRNA lentivirus was infected with MKN-45 cells.After 5 days of culture,the cells were detected by eBioscience apoptosis detection kit and elicited cytology.5)HUVEC cells were resuscitated in MKN-45 cells infected with hRNA lentivirus.Cell viics was used to observe the vascular area,mean vessel length,mean vessel width and vascular node count of human umbilical vein endothelial cells(HUVECs)to evaluate angiogenesis.Results:(1)Sequencing of Recombinant Lentiviral Vector: The three recombinant plasmids were DNA sequenced.The results showed that the sequence was consistent with the sequence of interest.Suggesting that the inserted Nodal gene RNAi sequence is correct.(2)Packaging and identification of recombinant lentiviral vector: Three kinds of plasmid vector were transfected into 293 T cells for 96 h,andthe cells were observed by fluorescence microscopy.The cells were well grown and the fluorescence intensity was strong.The virus titer was determined to be 5 x 108 TU / ml.(3)The expression of Nodal gene after RNA interference was detected by qPCR: The expression level of Nodal gene in MKN-45 cells was significantly down-regulated after RNAi lentivirus infection.LV-NODAL-RNAi(44789-1)infection was reduced by 80.3% and 84.9%,respectively.(4)RNA interference Nodal gene on MKN-45 cell apoptosis: shRNA lentivirus infected MKN-45 cells,cultured for 5 days,the interference group apoptosis rate was significantly higher than the control group(P <0.05).(5)Effects on the angiogenesis of HUVEC cells in vitro:Matrigel in 96-well plates,HUVEC cells were resuspended with the collected supernatant of the target cells,and 96-well plates were plated.Calcein AM was added and incubated at room temperature.Compared with the control group,there were no significant differences in blood vessel area,mean vessel length,mean vessel width and number of vascular nodes in KD group(P> 0.05).Conclusions: This study successfully constructed the RNAi lentiviral expression system of human Nodal gene.The expression of Nodal gene in human gastric cancer cell line MNK-45 was successfully inhibited by RNAi technique,and it was found that Nodal gene could significantly inhibit the proliferation,invasion and migration of gastric cancer cells and significantly increase the apoptosis of gastric cancer cells.It is suggested that interfering with Nodal gene expression can affect the biological behavior of gastric cancer cells and reduce its malignant tumor Behavior,it is speculated that Nodal gene may play a certain role in the process of metastasis and recurrence of gastric cancer.Nodal Gene is expected to be a new target for the treatment of gastric cancer. |
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