| Objective:Acute lymphoblastic leukemia(ALL) is a malignancy of hematopoietic stem cell origin. In recent years, targeted killing therapy has become the development trend of ALL treatment, analyse the mechanism, searching for new therapeutic targets become a research hotspot of ALL. The solute transport protein family (SLC) is one of the largest class of intracellular transport protein family. Its main function is to mediate the solute transport between mitochondrial matrix and film clearance or film clearance and cytoplasm, to ensure the material needed for the normal operation of mitochondria. SLC25A38gene, which is located on chromosome3p22, is considered to be an amino acid carrier. Preliminary studies confirmed that SLC25A38is closely related to apoptosis and degeneration of nerve cells. The overexpression of SLC25A38protein in hematopoietic organs (embryonic rat liver and bone marrow), speculated that it is closely related to the physiological functions of hematopoietic stem cells. To study the SLC25A38gene expression in ALL and its relationship with the clinical features of patients with ALL, and to explore the SLC25A38protein expression influence to the raised and apoptosis of ALL cells, we have designed this experiment.Methods:1. Collection55cases of patients with initial acute lymphoblastic leukemia (including23cases of children and32cases of adults) as experimental group,20cases of patients with non hematologic malignancies (including10cases of children and10cases of adults) as control group. By real-time fluorescence quantitative RT-PCR and Western blot method for detecting SLC25A38genetic changes in mRNA level and protein expression. Analysis the relationship between SLC25A38protein expression and the clinical features, chemotherapy response and curative effects of ALL patients. Follow-up part of the adult ALL patients with positive SLC25A38protein expression, testing the SLC25A38protein expression level under different disease states.2. RPMI8226and U266(the MM cell lines), and K562, Molt-4and Jurkat (leukemia cell lines) cells were cultured at RPMI-1640medium, supplemented with10%fetal bovine serum (FBS). By Western blot method to detect the SLC25A38protein expression level in each cell lines, while detecting the Notch1protein expression in cell lines and ALL patients samples.3. With high SLC25A38protein expression Jurkat cells as the model, build the shRNA for SLC25A38gene, transfecting Jurkat cells and screening stable transfection strains by G418. Experiments divide into4groups:SLC25A38-shRNA1group (Sh1group), SLC25A38-shRNA2group (Sh2group), scramble-shRNA group (NC group) and Jurkat blank control group (Ctrl group). Detection each SLC25A38protein expression and mRNA levels by Western blot and RT-PCR. Detect the cell proliferation by MTT method, detecting the cell cycle and cell apoptosis by flow cytometry. Given different adriamycin concentrations, after that detect the apoptosis changes under the action of different concentration of adriamycin by flow cytometry.Results:1. The SLC25A38gene expression in ALL patient samples:In mRNA level group, set the relative SLC25A38mRNA expression level of control group is1. In infant ALL patient samples, the relative expression level of SLC25A38mRNA was0.2659±0.2874in<1group, while that was3.1418±1.0607in>1group. There was significant difference of statistical significance between the two groups (P<0.0001). In adult ALL patient samples, the relative expression level of SLC25A38mRNA was0.3200±0.3382in<1group, while that was1.6707±0.6094in>1group. There was significant difference of statistical significance between the two groups (P=0.0003). In protein level group, the positive rate of SLC25A38protein were34.78%(8/23) in infant ALL patient group;46.9%(15/32) in adult ALL patient group. There were no SLC25A38protein expression was detected in20cases of control group. 2. Clinical characteristics, treatment and curative effect of response:In infant and adult mRNA level group, the patient samples in clinical characteristics such as gender, age, immune classification, white blood cell count in peripheral blood, peripheral blood hemoglobin, platelet and primitive bone marrow cells in less than control group (<1group) and greater than control group (>1group) were no statistical significance (P>0.05). In infant ALL patient samples, there were statistically significance in gender, immune classification, white blood cell count in peripheral blood and lactate dehydrogenase between SLC25A38-positive group and SLC25A38-negative group (P＜0.05), but there were no significance in age, number of peripheral blood hemoglobin, platelet, primitive bone marrow cells, the hepatomegaly, splenomegaly, lymphadenectasis, bleeding, CNSL/TL, prednisone reaction, complete remission rate and recurrence rate between the two groups (P>0.05). While in adult ALL patient samples, the while blood cell count in peripheral blood were higher in SLC25A38-positive group compared with SCL25A38-negative group (P<0.05), but no significance in gender, immune classification, number of peripheral blood hemoglobin, platelet and primitive bone marrow cell number between the two groups.3. Protein levels and tumor burden:Two adult ALL patients that were positive for SLC25A38were analyzed and the level of SLC25A38significantly reduced or disappeared following combined chemotherapy, however, reappeared upon ALL recurrence. The expression level was identified to be associated with the proportion of blast cells in the bone marrow.4. Expression of SLC25A38protein in leukemia cell lines: Overexpression of SLC25A38protein was observed in four cell lines.5. The impact on the Notchl protein expression:Notchl protein expression was analyzed in the cell lines and the patient samples. Notchl and SLC25A38proteins were co-expressed in the cell lines and ALL patient samples.6. SLC25A38gene silencing effects of the proliferation of leukemia cells: 7. MTT results showed that the OD490value of Sh1group and Sh2group were obviously higher than that of negative control group and blank control group, the difference is statistically significant. Silence SLC25A38gene accelerate the proliferation of Jurkat cell lines.8. SLC25A38gene silencing effect on leukemia cell cycle:Flow cytometry showed that the G0/G1phase rates of Sh1group and Sh2group decreased; S phase and G2/M phase rates rised, showing that SLC25A38gene silencing promote the cell growth of Jurkat cell lines.9. SLC25A38gene silencing effect on leukemia cells apoptosis:There were no statistical significance for apoptosis rate in different groups (P>0.05). The apoptosis rate only have a little bit increased in Sh1group and Sh2group after given different concentrations of adriamycin (P>0.05).Conclusion:1. Abnormal expression of SLC25A38gene is a common phenomenon in infant and adult ALL patients, but SLC25A38protein expression and mRNA level were inconsistent.2. High SLC25A38protein expression level were related to the clinical characteristics such as peripheral blood leukocyte number of ALL patients, and with the tumor load of ALL.3. ShRNA technology can successfully targeting SLC25A38gene and can promote the proliferation of Jurkat cell lines, but had no significant effect on cell apoptosis process.4. SLC25A38gene silencing does not affect the doxorubicin chemotherapy sensitivity of Jurkat leukemia cell lines.5. SLC25A38gene play a role of tumor suppressor genes in proliferation mediation of leukemia cells. |
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