| Childhood acute leukemia (AL) is a group of malignant clonal disease derived from hematopoietic stem cells. It is due to immaturity of the lymphatic system or myeloid cells in the maturation process of losing control, which showed clonal expansion, the cells were blocked at certain differentiation. AL is the most common malignancy of children blood diseases, with a high degree of heterogeneity, in addition to the normal function of bone marrow suppression performance, such as anemia, fever, bleeding, often accompanied by the proliferation of leukemia cell infiltrates such as enlarged lymph modes, liver, and/or spleen,, bone pain, joint pain, central nervous system leukemia, which seriously threaten to children's health and lives. AL pathogenesis is not fully understood, many believe that the current environmental factors, oncogenes. tumor suppressor genes and other risk factors combined the result.Phosphatase and tensin homologue deleted on chromosome ten gene (PTEN) is the first tumor suppressor gene ever found in the Phosphatase family. The PTEN protein has the dual activeness of lipid phosphatase and protein phosphatase which regulate the cells'proliferation and apoptosis depend on the activity of lipid phosphatase dephosphorylate the 3 phosphate group of Phosphatidylinositol 3,4, 5-triphosphate(PIP3) to convert it into Phosphatidylinositol 4,5-bisphosphate (PIP2). The activity of protein phosphatase can inhibit focal adhesion kinase (FAK), phosphorylate mitogen-activated protein kinase (MAPK), and regulate the adhesion and migration of cells. Mutations and the absence or low expression of allele of PTEN were frequently found in variety of solid tumors, and different degrees of deletion, low-expression, methylation of PTEN gene were also found in cases of Leukemia. But the function of PTEN gene and its protein in the AL development, angiogenesis, and extramedullary infiltration to be further explored.RNA interference (RNAi) technology can be efficient and specific reduction in target gene expression, is a powerful tool to study the function of endogenous genes and signal transduction pathways. But "off-target effect" often occurs in the RNAi process, resulting in false positive test results, that is a serious impediment to the process of genetic research. In this study, lentiviral vector of RNA interference and RNA interference escape strategy construct was constructed to study PTEN function in lymphocytic leukemia. Human T lymphocytes (T-LC) was used to establish its negative control (T-LC-GFP), PTEN gene knockdown (T-LC-shPTEN) and RESC(T-LC-rrshPTEN) cell models through the knockdown of PTEN gene or RESC concurrent rescue to discuss for the role of PTEN and its downstream signal molecule of AKT pathway effected cell proliferation and cell cycle. Human aortic endothelial immortalized cells (HAEC-hT) was used to establish its negative control (HAEC-hT-GFP), PTEN gene knockdown (HAEC-hT-shPTEN), and RESC (T-LC-rrshPTEN) cell models to observe the migration, wound renovation change of HAEC-hT and test-related signaling pathway molecule expression. This study was to investigate the biological characteristics of PTEN gene and its downstream signaling molecules in children with acute leukemia and investigate the function of RNAi and the RESC concurrent rescue of the lentiviral vector system in gene manipulation and off target effects during RNAi experiments resulted in a miscarriage of justice.Part One. Construction and identification knockdown and RESC concurrent rescue of RNAi lentiviral vector on human PTEN geneObjective:construct and identify the lentiviral expression vectors of human PTEN for RNAi and concurrent rescue of RNAi escape strategy construct (RESC) to provide stable transfection vectors for the research on the function of PTEN gene, also avoid effectively the interference from the false-positive responses caused by "off-target effect".Methods:1. Construction strategy. A pair of single-strand oligonucleotides capable of expressing PTEN shRNA sequece were designed and synthesized, with loop ring(TTCAAGAGA) added in the middle, and endonuclease sites on both ends of each pair of single-strand for the sake of gene operation. The characteristics of the target sequence were as follows:content of GC was 35%-55%, starting from base "G" and ending after 5 consecutive base "T", Function in the experiment:G was the recognition site of RNA polymerase?. Transcription started from G and ended at TTTTT after the transfection of plasmid to cells. Lentiviral expression vector of pFLRu-U6-shPTEN was obtained When double-strand DNA was formed after annealing, then it was directionally cloned to XbaI/XhoI endonuclease site of pFLRu-GFP plasmid. RESC concurrent rescue strategy was subsequently introduced: 3-4 synonymous mutations were introduced to the corresponding site of PTEN gene shRNA to change the genes in nucleotide while its expression of PTEN wild protein code remained unchanged and the exogenous mRNA was free from the function of shRNA. The exogenous mRNA had endonuclease sites on both ends for the convenience of gene operation. After combination with GFP report gene, the exogenous mRNA gene segment was directionally cloned to the EcoRI/BamHI endonuclease site of the vector--- pFLRu-U6-shPTEN plasmid and the lentiviral vectors of human PTEN gene for RNAi and its RESC concurrent rescue----pFLRu-U6-rrshPTEN was obtained. False-positive responses caused by shRNA off-target effect could be obviated if pFLRu-U6-rrshPTEN responded to the changes caused by RNAi after being introduced to RNAi experiment.2. Construction of lentiviral expression vector of PTEN gene pFLRu-U6-shPTEN. Three oligonucleotide fragment:fl containing hU6 promoter (XbaI endonuelease site contained), f2 shRNA fragment (XhoI endonuelease site contained), f3 U6-shPTEN fragment was obtained as the result of amplification after 3 PCR. Afterwards, f3 was taken as inserted fragment and was subcloned to Xba1/Xhol endonuelease site of pFLEu-GFP plasmid. the plasmid of positive clones were extracted after bacterial conversion and ampicillion resistance gene selecting, then identificated by sequencing with U6 promoter. Its homology with other genes was analyzed on Genbank data base-BLAST.3. Construction of lentiviral vectors of human PTEN gene for RNAi and its RESC concurrent rescue:The following was obtained as the result of amplification after 3 PCR reactions:650bp mRNA fragment on the left side of the corresponding shRNA target sequence with synonymous mutation (EcoRI endonuelease site contained) (F1),560bp mRNA fragment on the right side of the corresponding shRNA target sequence with synonymous mutation (BamHI endonuelease site contained) (F2), full-length fragment of PTEN gene mRNA with four synonymous mutants in the corresponding shRNA sequence (F3). The integration of F3 and GFP gene was taken as the inserted fragment and was subcloned to EcoRI/BamHI endonuelease site of pFLRu-U6-shPTEN plasmid. The positive clones of plasmid were extracted after bacterial conversion and ampicillion resistance gene selecting, then identificated by sequencing with U6 promoter. Its homology with other genes was analyzed on Genbank data base.4. Package and titer of lentiviral virus:Under the mediation of TRANSIT, Opti-MEM culture medium and the mixture of slow virus packaging plasmid pCMV-dR8.2?R and pCMV-VSV-G (8:1) were used respectively with lentiviral virus plasmid pFLRu-U6-shPTEN, pFLRu-U6-rrshPTEN to co-transfect 293T cells; 48 hours after the transfection, supernatant was collected, centrifuged and filtered to obtain the lentiviral virus liquid of pFLRu-U6-shPTEN and pFLRu-U6-rrshPTEN. NIH3T3 cell was applied to measure the virus titer by the method of hole-by-hole gradient dilution (10-1?10-6). GFP expression was detected under inverted microscope with the product of the number of cells expressing GFP and the corresponding dilute rate as the virus titer.Results:1. The amplified product of pFLRu-U6-shPTEN and pFLRu-U6-rrshPTEN gene fragment with 1% TAE agarose gel electrophoresis was observed under ultraviolet transilluminator, a clear band was seen at about 420bp in keeping with the length of shRNA. A clear band was observed at about 1200bp, the length was the same with cDNA of the aimed gene PTEN.2. The fact that fragment bands with expected length was obtained from pFLRu-U6-shPTEN after 3 enzyme digestions and electrophoresis confirmed that the amplified fragment had been inserted into the vector. DNA sequencing indicated that shPTEN gene sequence was in accordance with the expected gene sequence.3. The fact that fragment bands with expected length was obtained from pFLRu-U6-rrshPTEN after 4 enzyme digestions and electrophoresis confirmed that the amplified fragment had been inserted into the vector. DNA sequencing indicated that rrshPTEN aim gene sequence was of the same homology with the gene sequence of gene bank at amino acids level.4. Most transfected 293T cells were found emitting green fluorescence under inverted microscop. It indicated that lentiviral vector expression of pFLRu-U6-shPTEN and its RESC concurrent rescue was successfully constructed. The titer of viruses was 6.0×105 pfu/mL and 5.0×105 pfu/mL respectively after the amplification of 293T cells to meet vivo experiments.Conclusions:Human PTEN gene RNAi and its RESC concurrent rescue lentiviral vector were successfully constructed. The interference on the experiment from the false-positive result caused by shRNA off-target effect was avoided and stable transfected vectors were provided for the research on PTEN gene.Part Two. Changes in T-Lymphocytes Proliferation and Signal Pathway after the Knockdown and RESC Concurrent Rescue of RNAi Lentiviral VectorObjective:Construct lentiviral expression vectors of human PTEN gene for RNAi and RESC concurrent rescue by infecting normal human T-Lymphocytes (T-LC) and establish cell models T-LC-shPTEN and T-LC-rrshPTEN. Then observe the changes of signal pathway, cell proliferation and cell cycle in human T-Lymphocytes after PTEN gene knockdown and RESC concurrent rescue, in order to provide an experimental foundation for a further research into the pathogenesis of acute lymphoblastic leukemia in children.Methods:1. Culture and purification of T-LC. Healthy person's peripheral blood was provided by healthy volunteers (consent forms signed). Peripheral blood mononuclear cell (PMNC) was prepared, then T-LC was obtained through purification by magnetic cell sorting system and was cultured in RPMI1640 containing 10% FBS.2. Establishment of PTEN gene knockdown and RESC concurrent rescue T-LC cell models. T-LC was transfected by pFLRu-GFP plasmid, shPTEN and rrshPTEN to establish negative control model of T-LC (T-LC-GFP), PTEN gene knockdown model (T-LC-shPTEN) and PTEN gene RESC concurrent rescue model (T-LC-rrshPTEN).3. Detection the expression of PTEN and pAKT protein by Western blot. Cells were collected and were lysed to obtain total protein, equal amount of protein collected from each group was applied polyacrylamide gel electrophoresis, transmembraned and coated with PVDF membrane, the primary antibodies, PTEN, pAKT, AKT and P-actin etc, were added for incubation, then the secondary antibody was used for incubation, chemiluminescence and radiography. Image J public image processing software was adopted for the quantitative analysis of the results of Western blot.4. Detection of the effects of PTEN knockdown and concurrent rescue of RESC concurrent rescue on T-LC growth by MTT. Log-growth-phase cells were taken from T-LC, T-LC-GFP, T-LC-shPTEN and T-LC-rrshPTEN. When the cell concentration was 1×106/mL, incubated them in 200?L per hole to 96-hole cell culture flask,3 multiple holes for each group, one plate was taken out respectively when culturing 1d,2d,3d,4d,5d, and was continued culturing for 4 hours after MTT solution was added, OD 570 nm value was determined with microplate reader.5. Detection of the effects of PTEN knockdown and concurrent rescue of RESC concurrent rescue on cell cycle by flow cytometry. Cells of T-LC, T-LC-GFP, T-LC-shPTEN and T-LC-rrshPTEN were adjusted to 5×105/50?L, 10mg/mL RNaseA 20?L, and were kept at 37?for 30 minutes, propidium iodide was added, cell cycle was then detected with flow cytometer.6. Statistical analysis:The statistical software SSPS 13.0 was applied for data statistical analysis. The data were expressed with mean±standard deviation(?±s). Using analysis of variance and q test, it was considered statistically significant when P<0.05.Results:1. After T-LC was infected by lentiviral expression vectors pFLRu-GFP, pFLRu-U6-shPTEN and pFLRu-U6-rrshPTEN, GFP expression was observed with inverted microscope and most T-LC were found emitting green fluorescence. This indicated the successful construction of cell models T-LC-GFP, T-LC-shPTEN and T-LC-rrshPTEN.2. Western blot indicated:There was no significant difference between the expression of PTEN gene of T-LC-GFP and T-LC (P>0.05), PTEN gene expression of T-LC-shPTEN was weaker than that of T-LC and T-LC-GFP (P<0.01); positive expression was found in T-LC-rrshPTEN and PTEN gene (together with GFP gene), the difference had statistical significance when compared with T-LC-GFP and T-LC-shPTEN (P<0.05).3. Detection of the expression of pAKT protein by Western blot:Compared with cells of T-LC group and T-LC-GFP group, pAKT expression of the cells of T-LC-shPTEN group increased significantly (P<0.01), pAKT of T-LC-rrshPTEN group was similar to that of the former two groups (P>0.05). This indicated that PI3K/AKT signal pathway of T-LC was activated after PTEN gene knockdown, but RESC concurrent rescue brought the pAKT expression caused by PTEN gene knockdown back to its previous state. 4. The curve of the cell growth detected by MTT indicated:Compared with T-LC group and negative control group T-LC-GFP, cell growth of T-LC-shPTEN group increased and significant differences appeared the second day (P<0.05). But cell growth speed of T-LC-rrshPTEN group was similar to that of T-LC group and T-LC-GFP group (P>0.05), which indicated that RESC concurrent rescue could recover PTEN gene's function in inhibiting cell growth.5. Changes in cell cycle and apoptosis detected by flow cytometer indicated: Cells in G0/G1 phase decreased obviously after PTEN gene knockdown (T-LC-shPTEN group), cells in S phase increased significantly, cells in G2/M phase increased, and apoptosis decreased. Compared with cells of the other 3 groups, the difference was significant (P<0.01). After RESC concurrent rescue (T-LC-rrshPTEN group), cells apoptosis, G0/G1 phase, S phase and G2/M phase recovered respectively, the difference was no statistical significance when compared with T-LC group and T-LC-GFP group (P>0.05).Conclusions:1. The construction of the cell models of T-LC cell negative control (T-LC-GFP), PTEN gene knockdown (T-LC-shPTEN) and RESC concurrent rescue (T-LC-rrshPTEN) was successful.2. Slow virus-medicated RNAi could down-regulate the expression of PTEN gene effectively. After PTEN gene expression was down-regulated, PI3K/AKT pathway was activated, T lymphocytes growth was promoted, cells in G0/G1 phase decreased, cells in S phase and G2/M phase increased, and apoptosis reduced.3. RESC concurrent rescue of PTEN gene RNAi could completely recover the results caused by PTEN gene knockdown. This confirmed that the results obtained from the experiment were all caused by PTEN gene knockdown, but not the false appearance caused by RNAi off-target effect. Part three Prediction of sphingosine 1-phosphate and VEGFstimulated endothelial cell migration rates using RNAi lentiviral vector of human PTENObjective:Construct lentiviral expression vectors of human PTEN gene for RNAi and RESC concurrent rescue by infecting human aortic endothelial cells and establish cell models negative control (HAEC-hT-GFP), PTEN RNAi (HAEC-hT-shPTEN) and RESC (HAEC-hT-rrshPTEN). Then observe the migration, wound renovation change of HAEC-hT and test-related signaling pathway molecule expression.Methods:1. HAEC immortalization and verification. Immortalized HAEC were purchased from Mr. Longmore, Louis, Washington University School of Medicine and cultured with Complete EGM.2. Construct cell models of human PTEN gene for RNAi and concurrent rescue of RESC. Establishment cell models of negative control (HAEC-hT-GFP), PTEN RNAi (HAEC-hT-shPTEN) and RESC (HAEC-hT-rrshPTEN) with pFLRu-GFP, pFLRu-U6-shPTEN and pFLRu-U6-rrshPTEN plasmid transfected to HAEC.3. HAEC-hT single cell migration analysis after PTEN knockdown or rescue. Observed cells not in split process, not in contact with other cells, with Olympus X81 inverted microscope photograph once 4 min continuous 12 h. ImageJ software to track the movement of each cell path. Draw the path of the cell migration to compare migration direction and speed among the control group, PTEN gene knocked down, rescue groups and S1P, VEGF added to the cells at different time.4. Single cell migration analysis after PTEN knockdown or rescue. Production of about 230±32?m wide wound (about to remove 49±7% of the cultured cells) after cells were serum starved 2 h. Then take photos of wound healing every 5 min with the Olympus X71 microscope (10×). Rate of wound healing quantitatively analysed with ImageJ. 5. The expression of AKT protein detected by western blot. PTEN RNAi and RESC concurrent rescue cells were damaged after serum starved 2 h, then added with or without 1?g/mL S1P,50 ng/mL VEGF. Cells were harvested at 0,15,30 respectively. pAKT protein expression was detected by western blot.Results:1. Immortalized endothelial cell morphology and growth rate had no significant differences with primary culture cells under VEGF and S1P stimulation.2. Two control groups were spindle cell morphology, the nucleus had a distinct polarization, cell shape changed with pseudopods stretch and shrink, but the movement toward a pole. After PTEN knockdown endothelial cells became larger, the cells tend to round with no obvious nuclear morphological changes. Cell velocity was significant difference with the control group (P<0.05), and the apparent lack of movement direction. Whether from the shape, velocity and movement cells had no significantly different on speed and direction after RESC rescue (P>0.05)3. Added SIP and VEGF, the cell migration and wound healing of endothelial cells were significantly promoted (P<0.05), but were no significant difference after PTEN knockdown (P>0.05)4. pAKT expression in the control cells had increased gradually by SIP and VEGF stimulation with the time(P<0.05). After PTEN knockdown, pAKT expression in three time points was no significant difference (P>0.05), but they were increased compared with the control group. S1P and VEGF increased pAkt protein to join the activity during the wound healing. pAKT expression in the concurrent rescue cells were similar with the control groups.Conclusions:1. Vascular endothelial cells lost their ability of polar motion after PTEN gene knochdown. The wound healing of endothelial cells was steped down d ue to lack of directional movement tends to the wound.2. SIP, VEGF significantly promoted the migration of endothelial cells and wound healing through activation of PI3K/AKT signaling pathway. However, in PTEN knockdown cells, the wound healing of endothelial cells was slower after S1P and VEGF stimulation, and maybe it was due to other signaling pathway activation and cell motility behavior change.3. PTEN gene may be involved AL pathogenesis including angiogenesis, tumor cell metastasis and invasion at the molecular level. |
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