The Detection Of ING4 In Tumorigenesis And Construction Of PNL-ING4 Lentiviral Vector

Objective To observe the ING4 gene express in transcriptional level and mutation in tumorigenesis. To clone the gene of human ING4 drived from human bone marrow-drived mesenchymal stem cells (hBM-MSC). To recombinate the lentiviral vector PNL-EGFP and construct PNL-ING4.Methods The total RNA were extracted from A549, HeLa, SW480, LTEP-a-2, PC3, hBM-MSC, UVEC-304, GC-MSC(MSC isolated from gastric tumor tissue ), F6, leukemia patient bone marrow mononuclearcell and gastric tumor tissue. The expression of ING4 gene in each cell was detected by RT-PCR and Real-time PCR. ING4 gene was cloned respectly from hBM-MSC, LSC, SW480, LTEP-a-2, F6 and CML patient bone marrow mononuclearcell cDNA. The cloned ING4 gene was sequenced. Lentiviral vector PNL-ires was recombinated by restriction enzyme NheI and BsrgI. The cDNA of ING4 was amplified by RT-PCR from the total RNA extracted from hBM-MSC. The PCR product was inserted into PMD19-T vector. The positive recombinant clone was analyzed by digestion of restriction endonuclease and DNA sequencing. The correct sequence was subcloned into lentiviral vector. The identification was performed by analysis of restricting enzyme digestion and DNA sequence. The 293T cells were transfected by lentiviral vectors using lipofectamine 2000. The expression of ING4 gene was observed by RT-PCR.Results RT-PCR showed that LTEP-a-2, SW480, LSC, Hela, PC3, F6, GC-MSC, hBM-MSC, UVEC-304, bone marrow mononuclearcells of patients with ALL and CML express different levels of ING4. The expression level of ING4 in LSC, bone marrow mononuclearcells of patients with ALL and CML was lower than others. And nearly no expression of ING4 gene was detected in A549 and gastric tumor patient tissues. Real-time PCR results showed that A549, LTEP-a-2, SW480, GC-MSC, PC3, Hela, F6, UVEC-304 and hBM-MSC all express ING4. The expression level of ING4 was lowest in A549 and highest in F6. DNA sequencing showed deletion and mismatch were found in hBM-MSC, LSC, SW480, F6 and bone marrow mononuclearcells of patients with CML. Most of these deletion happened in NLS area. Missense mutation and frameshift mutation also be found. And with the increasing passage numbers of hBM-MSC, the frequency of point mutation and deletion were more and more higher. The recombination of the lentiviral vector PNL-ires was successful which increased the multiple clone sites. The cDNA of ING4 was amplified by RT-PCR using the total RNA extracted from hBM-MSC. Restriction enzyme digestion and DNA sequencing revealed that ING4 cloning was successful. The virus particles were produced in 293T cells and green fluorescence which can be observed about 80% or more percents in the visual fields. The expressions of ING4 gene were observed by RT-PCR.Conclusions Our research showed that the expression level of ING4 was different in various tumor cells. And mutation could be detected in some of these tumor cells. These factors participate tumorigenesis by altering the expression level of ING4 gene or changing the biological activity of ING4 proten. The most needed to point out is that the mutation of ING4 is one of the key points in tumorigenesis of MSC invitro. The results demonstrate that the PNL-ING4 lentiviral vector was obtained. And it lays the foundation for further research on the fuction of ING4 in tumorigenesis and the development of new gene therapy method.