| Objective：To construct the lentiviral siRNA vector which can specific knockdown theexpression of CXCR4gene, and investigate the role of CXCR4in the proliferation,adhesion and infiltration of SHI-1cells.Methods：1. The complementary Oligo DNA targeting CXCR4were synthesized andcloned to the GV248-EGFP vector. The positive clones containing CXCR4shRNAwere identified by PCR amplification and sequencing.293T cells were cotransfectedwith lentiviral vector GV248-LV, pHelper1.0and pHelper2.0using Lipfectamine2000. The recombinant lentiviruses were collected and titered by limiting dilution. Anunrelated oligonucleotide was used as negative control.2. SHI-1cells were infected by CXCR4shRNA lentivirus and negative controllentivirus.Differential SHI-1cells to3group. The expression of GFP was used todetect the efficiency of infection by FCM. CXCR4mRNA and protein expressionwere examined by PCR and FCM. When the CXCR4expression was knockdowned,the MMP-2, MMP-9mRNA expressions in SHI-1cells was detected by real-timePCR, and the capability of proliferation ability was investigated by MTT.3. BMSCs were cultured from the bone marrow of ANLL patients. SHI-1cellswere co-cultured with BMSCs,24hours later, the SHI-1cells adherent to the BMSCswere conted in the microscope to investigate the adherent capability.4. The trans-matrigel invasion assay was used to detect the incasion ability ofSHI-1cells. SHI-1cells were mixed with BMSCs at the ratio of10:1and added to theupper compartment of the millicell chamber, which was precoated with Matri-gel.24hours later, SHI-1cells in the lower compartment were counted and calculated theinvasion rate.5.1×107SHI-1cells were inoculated to the left axillary of BALB/C nude micepretreated by cyclophosphamide to investigate the influence of CXCR4on SHI-1 cells growth in vivo.Results:1. The CXCR4RNAi lentivirus was successtully constructed, the positive viraltiter was8×108TU/ml,negative viral tite was1×109TU/ml。2. The expression of GFP in SHI-1/CXCR4was93%,SHI-1/NC was92%. Theexpression of CXCR4mRNA and protein in SHI-1/CXCR4decreased by76%and69.6%than SHI-1cell. There was no significantly difference between SHI-1andSHI-1/NC cells.3. We also found that the expression of MMP-2and MMP-9mRNA in SHI-1/CXCR4decreased63%and62%than SHI-1cell respectively.4. The adhesion ability of SHI-1/CXCR4group cell has decreased significantlyobserved under100times microscope after co-cultured24hours with BMSCs. Tocalculate the number of SHI-1cell adhesion to BMSCs, SHI-1group was(56.1±5.1), SHI-1/NC group was (57.6±5.4) and SHI-1/CXCR4group was (25.1±5.5).The adhesion ability of SHI-1/CXCR4cells were significantly decreased (p<0.01) compared to the SHI-1and SHI-1/NC cells, no significant difference betweenthe SHI-1and SHI-1/NC cells (p>0.05).5. In trans-matrigel invasion system, SHI-1cells can hardly trans-matrigelinvade to the lower compartment alone.When co-cultured with BMSCs, trans-matrigel invasion of SHI-1cells and SHI-1/NC cells enhanced significantly. SHI-1cells in the lower compartment were counted and calculated the invasion rate after24hours,SHI-1+BMSCs was (20.3±3.7)%, SHI-1/NC+BMSCs was (19.6±4.2)%and SHI-1/CXCR4+BMSCs was (9.2±2.1)%, the invasion rate of the SHI-1andSHI-1/NC cells were significantly increased than SHI-1/CXCR4cells (p <0.01).6. When innoculated in nude mice, SHI-1/CXCR4cells didn't growth andformed tumor. However SHI-1and SHI-1/NC cells formed obvious tumor in the nudemice, and no significant difference were found in the volume and weight of tumors.(p>0.05). Conclusion:1. RNAi lentivirus vector of CXCR4was constructed successfully, and thevector provides a basis for further studying of the roles of CXCR4in acute leukemia.2. The expression of CXCR4in SHI-1cells was successfully knockdowned,together with MMP-2and MMP-9mRNA expression. The proliferative capacity ofSHI-1/CXCR4cells had no significant change in vitro, but the adhesion abilitydecreased significantly.3. The elevated invasion of SHI-1cells induced by the leukemia BMSCs wasinhibited by the decreased CXCR4mRNA expression.4. When knockdowned the expression of CXCR4, SHI-1/CXCR4cells couldn'tgrowth to form tumor in nuce mice subcutaneous.To sum up: The ability of adhesion, invasion and subcutaneous tumor formationof SHI-1cell lines decreased significantly when silencing the expression of CXCR4mRNA, these results suggest that CXCR4play an important role in nleukemia cellsmigration and invasion. CXCR4may be a target for the treatment of acute leukemia. |
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