Study On Pediatric Acute Leukemia Of GATA-3Gene Polymorphism And LAPTM5Gene Function

Part one. The effect of GATA-3SNP rs3824662on Chinese Childrenwith Acute lymphoblastic LeukemiaObjective： Compare the GATA-binding protein-3(GATA-3) SNP rs3824662difference between control and Chinese children with Acute lymphoblastic leukemia(ALL), and research the relationship between GATA-3SNP rs3824662and pediatricsAcute lymphoblastic leukemia of Chinese.Methods: Peripheral blood was collected from137patients of Chinese children withALL and389people without hematologic malignancies. Genomic DNA was extracted andnested PCR was performed to amplify the specific band contains GATA-3SNP, rs3824662then sequencing was done by the company. Company to help sequencing. SPSS18.0software was employed for statistic analysis.Results: For GATA-3SNP rs3824662, the frequencies of genotypes C-A, C-A, A-Aand alleles C, A didn’t show any difference between Chinese children with ALL andcontrol, and also with clinical indicators.Conclusion: Current evidence suggests that there was no association between theGATA-3SNP rs3824662and Chinese children with ALL which indicated that GATA-3SNP rs3824662might work in a racial way. Part two. The Expression and Clinical significance of LAPTM5in Children Acute lymphoblastic LeukemiaObjective：In this study, we investigated LAPTM5expressions and its clinicalimplications in children with acute lymphoblastic leukemia (ALL).Methods:144patients at different stage of ALL were enrolled in this study whichincludined82cases at initial stage,54cases at complete remission (CR), and8cases atrelapse from the Children’s Hospital of Soochow University during2012to2013;40ITPpatients and cell lines were used as controls. Expression levels of LAPTM5were detectedin bone marrow samples using real-time quantitative RT-PCR analysis. SPSS18.0softwarewas employed for statistic analysis.Results: The relative expression levels of LAPTM5in initial and relapse patientswere higher than that in control (P<0.001, P=0.033). In ALL patients, the relativeexpression level of LAPTM5in initial and relapse patients revealed no significantdifference (P=0.171), but were both higher than that in CR (P<0.001，P=0.006). There wasno significant difference between CR and control (P=0.985) for the relative expressionlevel of LAPTM5. The relative expression level of LAPTM5in initial group of B-ALLwas higher than that in T-ALL (P=0.002). LAPTM5’s relative expression level had positivecorrelation with BM blast (P=0.034，r=0.303), but didn’t show any correlation with WBC,HB, PLT, CRP, LDH, PB blast, early treatment response, risk group and MRD.Conclusion: LAPTM5gene expressed higher in initial ALL and relapse, which wasespecially prominent in B-ALL.LAPTM5’s relative expression level had positivecorrelation with BM blast. Part three. Construction of Venus Vector Carrying LAPTM5Gene and Its Expression in K562CellsObjective：Venus vector carrying lysosomal-associated protein transmembrane5gene（LAPTM5） was constructed to explore its function in leukemia cells.Methods: LAPTM5CDS was amplified from NB4cell line by RT-PCR with theprimers containing EcoR Ⅰa nd BamHⅠrestriction sites, and then inserted intopLVX-IRES-Puro lentiviral expression vector and sequencing analysis was employed.Lentiviruses were produced in293T cells with the help of plasmids lentiviral packagingsystem (pLVX, pLP-VSVG, pLP1, pLP2) by the method of calcium phosphateprecipitation. Lentiviral vector overexpressed LAPTM5and its control were transfectedinto K562cells and confirmed with RT-PCR and Western blot.Results: Sequencing analysis showed that the amplified coding region of LAPTM5gene was same as Gene Bank provided and successfully inserted into the lentivirusesvector which named as pLVX/LAPTM5. Identification showed LAPTM5gene transfectedinto K562was confirmed either in DNA level and the levels of mRNA and protein whichwas high expressed compared to the parental strain and empty vector transfected cells.Conclusion: We successfully constructed pLVX/LAPTM5vector which containsLAPTM5gene and transfected into K562which lays a foundation for exploration ofLAPTM5functions in leukemia cells. Part four. The relationship between LAPTM5gene and autophagy,apoptosis in K562cellsObjective： LAPTM5gene is located in lysosomes which are responsible forautophagy. In this study we observed autophagy and apoptosis of K562overexpressed withLAPTM5gene to explore the function of LAPTM5gene.Methods: K562cells transfected with LAPTM5and empty vector were cultured in1640conditional medium in which serum was replaced with HBSS. After treatment of0h,3h,6h cells, cells were collected for Western blot to detecte the expression of LC3-IIandLC3-I, and Flow cytometry using FITC-labeled LC3antibody for autophagy andLysoSensor Green DND-189labeled lysosomes for its PH value, and AnnexinV-FITC/PI staining for autophagy and apoptosis.Results:1. Cells were starved after0h,3h,6h, the ratios of LC3-II/LC3-I in LAPTM5gene transfected K562cells are much lower than that in pLVX empty vector.2. Multiplefluorescent spots in K562transfected with LAPTM5gene was57%which was much lowerthan that in empty vector (83.1%.) which indictated that LC3expression is lower inLAPTM5transfected than that of empty vector’s.3. Fluorescence intensity of K562cellstransfected with LAPTM5gene was much weaker than that with empty vector whichindicated that PH value in LAPTM5transfected is lower than that of empty vector’s.4.Apoptosis rate of K562cells transfected with LAPTM5gene was33.1%, which was muchsimilar as that in empty vector which was34.2%. There was no significant differencebetween two group cells.Conclusion: The gene of LAPTM5can inhibit autophagy of K562cells withoutaffecting apoptosis.