| Objective：Studies had indicated that extracellular matrix metalloproteinase inducer ishighly expressed in many tumor cells, and correlated with the tumor malignant degree,but there is no clear relationship between CD147and leukemic. In this study, weconstruct a lentiviral RNAi vector targeteing to human CD147, and investigate theroles of CD147in SHI-1cells proliferation and infiltration.Methods：1. The complementary Oligo DNA targeting CD147were synthesized andcloned to the pGCL-GFP vector. The positive clones containing CD147shRNA wereidentified by PCR amplification and sequencing.293T cells were cotransfected withlentiviral vector pGC-LV, pHelper1.0and pHelper2.0using Lipfectamine2000.Therecombinant lentiviruses were collected and titered by limiting dilution.2. SHI-1cells were infected by PGC-LV/Emmprin (SHI-1/CD147) and negativecontrol (SHI-1/NC) virus, the GFP expressed in SHI-1/CD147and SHI-1/NC weredetected by FCM. The expression of CD147, MMP-2and MMP-9were detected byRT-PCR. The capacity of SHI-1cells proliferation was investigated by MTT.3. Bone marrow stromal cells were cultured from bone marrow of leukemicpatients. BMSCs and SHI-1cells mixed at the ratio of1:10and added to the uppercompartment of the millicell chamber, which was precoated with Matrigel.24hourslater, the cells in the lower compartment were counted and calculated the invasionrate.4.5×106SHI-1cells were inoculated in the left axiillary of BALB/C nude micewithout any pre-treatment. Mice were sacrificed to measure the volumor and weightof tumor. CD147-protein expression was detected by immunohistochemistry.BALB/C nude mice pre-treatment by splenectony, cytoxan intraperitonealinjection, sublethal irradiation (SCI-mice), were transplanted intravenously with1×10~7SHI-1cells. Mice were dissected when they were moribund or deadimmediately; all organs were fixed by10%formalin, routinely processed for histological examination, and CD45immuno-histochemistry was used to detect theinfiltration of leukemic cells in different organs.Results:1. The CD147RNAi lentiviruses was successtully constructed, the positive viraltiter was1×10~9TU/ml,negative viral titer was2×10~9TU/ml.2. The expression of GFP in SHI-1/CD147was94%，SHI-1/NC was82%. Theexpression of CD147was significantly knockdowned in SHI-1/CD147cells than thatin SHI-1cells and SHI-1/NC cells. We also found that the expression of MMP-2、MMP-9in SHI-1/CD147cells were significantly lower than that in SHI-1/NC andSHI-1cells. There was no significantly difference of CD147, MMP-2and MMP-9between SHI-1and SHI-1/NC cells.3. The proliferation capability of SHI-1/CD147cells was significantly ecreasedthan SHI-1and SHI-1/NC cells. SHI-1cells hardly can trans-matrigel invade into thelower chamber, when co-cultured with BMSCs, trans-matrigel invasion rate of SHI-1cells and SHI-1/NC cells enhanced significantly (18.2±2.5%and16.5±2.7%respectively, p＞0.05). The SHI-1/CD147cells which had the lower CD147expression, demonstrated significantly lower invasion rate (4.5±1.2%, p<0.01) thanSHI-1cells and SHI-1/NC cells when co-cultured with BMSCs.（p<0.01）4. When SHI-1cells inoculated in the left axiillary of nude mice, the volume andweight of tumor were significantly larger in SHI-1and SHI-1/NC group than inSHI-1/CD147, Immunohistochemistry found that the expression of CD147issignificantly decreased in tumor founded by SHI-1/CD147cells.When SHI-1cells inoculated via caudal vein, the median survival time of micereceived SHI-1cells was32days (22-33dsys), mice with SHI-1/NC cells was28days (26-30days), and the median survival time of mice received SHI-1/CD147cellswas significantly prolonged to41days (24-47dsys). SCI-mices received SHI-1/CD147cells were featured by higher infiltration than mice received SHI-1andSHI-1/NC cells.Conclusion:1. RNAi lentivirus vector of Emmprin was constructed successfully, and thevector provides a basis for further studying of the roles of CD147in acute leukemia. 2. The expression of CD147mRNA in SHI-1cells could be knockdowned by theCD147RNAi lentivirus vector effectively.3. When the CD147expression of SHI-1cells was knockdowned, theexpressions of MMP-2and MMP-9were down-regulated, and the Proliferative andinfiltrative capacity of SHI-1cells were significantly decreased. The growth andinfiltration of SHI-1/CD147cells in nude mice were significantly inhibited. |
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