| ObjectIn this study,Lentivirus method was used to construct PAQR3 overexpressing gastric cancer stably transfected cell line.To study the effect of PAQR3 overexpression on the biological behavior of gastric cancer.Whether PAQR3 overexpression affects the characteristics of gastric cancer stem cells has been discussed.MethodsFirstly,we used the gene cloning technology to obtain and amplify the PAQR3 gene,select and cut the gene vector,link the PAQR3 gene with the vector,and screen the recombinant vector into competent cells and recombinant DNA to construct the eukaryotic expression plasmid PAQR3-CD513.PCR test,double enzyme digestion and gene sequencing were used to determine whether the recombinant plasmid was constructed correctly.The PAQR3-CD513 plasmid and the control plasmid GFP-CD513 were co transfected into 293 T cells with adjuvant plasmids respectively.PAQR3 and GFP overexpressing lentivirus infect BGC823 cells respectively.Fluorescence microscope observation and Puro screening confirmed whether BGC823 stable transfected cell lines were successfully constructed.Western blot experiment verified whether PAQR3 gene was successfully expressed in the stable transfected cell line.After confirming the successful establishment of the stable cell line,the stable PAQR3-BGC823 cells were named PAQR3group(experimental group),and GFP-BGC823 stable transfected cells werethe control group.The proliferation ability of PAQR3 and control cells was detected by MTT.Cell scratch test was used to compare the cell migration area ratio between PAQR3 group and control group.After subcutaneous injection of PAQR3 and control cells,nude mice were examined for tumor size,tumor size and tumor volume.The nude mice were killed and weighed after the death of the tumor.Western blot was used to detect the expression of CSC marker ALDH1A1 protein in PAQR3 and control groups.RT-PCR test was used to detect the relative expression of OCT4 and SOX2 in two groups.Results1.PAQR3 DNA fragment was cloned and amplified by PCR.Electrophoretic analysis of double enzyme products showed that the cloned 1clone had obvious positive bands at the same size as the PAQR3 DNA molecule.Sequence analysis of clone 1 was carried out,and the sequence covered in the credible region was consistent with the standard sequence.Clone 1 is a positive recombinant of PAQR3-CD513.The results showed that the recombinant expression plasmid was constructed successfully.PAQR3-CD513 plasmid and control plasmid GFP-CD513 were transfected into 293 T cells respectively.PAQR3 and GFP overexpressed lentivirus infected BGC823 cells respectively.Under fluorescence microscope,GFP-BGC823 cells in the control group had strong green fluorescence signals,while PAQR3-BGC823 cells had no GFP gene and no fluorescence signal.Puromycin(Puro)screening can confirm the success of PAQR3 virus infection.Western blot results showed that the expression level of PAQR3 protein in PAQR3-BGC823 cells was significantly higher than that in GFP-BGC823 stable cells.Successfully constructed lentivirus PAQR3 gene over expression stable transformation cell line.2.MTT test results showed that the growth rate of PAQR3 group was lower than that of control group(P < 0.05).3.The results of cell scratch test showed that the migration area ratio(0.48 + 0.013)% of PAQR3 group was less than that of the control group(0.63 + 0.012)%,and thedifference was statistically significant(P<0.05).4.PAQR3 overexpression group and control group were injected subcutaneously in nude mice.The growth rate of tumor in group PAQR3 was lower than that of the control group,the difference was statistically significant(P < 0.05),two groups of tumor were stripped and the tumor mass of group PAQR3 was less than that of the control group;the weight of the tumor block in group PAQR3 was less than the weight of the group,and the difference was statistically significant(P < 0.05).5.the results of Western Blot showed that the gray value of the control group was(0.673 + 0.021),and the gray value of the PAQR3 group was(0.582 + 0.015),and the difference was statistically significant(P < 0.05).Compared with the control group,the expression of ALDH1A1 protein in PAQR3 group decreased,and the difference was statistically significant(P < 0.05).6.RT-PCR results showed that the expression of dry related genes OCT4 and SOX2 in PAQR3 group decreased compared with the control group(P < 0.05).Conclusion1.A lentivirus mediated PAQR3 overexpressed cell line was successfully constructed.2.PAQR3 gene inhibits the proliferation and migration of gastric cancer cells and the ability of nude mice to be tumorigenic.The PAQR3 gene can be used as a target gene for tumor.3.Up-regulation the expression of PAQR3 gene to reduce the protein of tumor stem cell marker ALDH1A1.PAQR3 gene inhibits the expression of OCT4 and SOX2 related to stem cell related genes.PAQR3 gene plays a role in inhibiting the "stem cells" of gastric cancer. |
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