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| The Influence of Downregulation of DEPTOR Expression by RNA Interference on the Proliferation and Apoptosis of Human Multiple Myeloma Cells in vitro |
| ZHANG Hao-ran, ZENG Zhi-yong, CHEN Jun-min |
| Department of Hematology, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, China |
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Abstract Objective: To construct short hairpin RNA (shRNA) lentiviral vectors targeting human DEPTOR gene, detect its effect of gene silence and investigate the effect of DEPTOR on the proliferation and apoptosis in human multiple myeloma (MM) RPMI-8226 cells. Methods: Four human DEPTOR siRNA sequences were designed and then cloned into hU6-MCS-CMV-EGFP(GV115)-shRNA vector. The above recombinants were transfected into 293T cells by means of lipofectamine mediation. The gene silencing efficacy of these 4 siRNA sequences was compared in Western blot analysis. The GV115-shRNA was co-transfected into 293T cells. The most effective GV115-shRNA was screened out by Western blot. GV115-shRNA and lentiviral packaging plasmid were co-transfected into packging cell line 293T. Culture supernatants were harvested and concentrated to generate the lentivirus encoding DEPTOR-RNAi. The lentivirus particles were packaged and DEPTOR specific shRNA was transmitted into RPMI-8226 cells after screening for the valid shRNA. DEPTOR silencing efficiency was determined by Real-time PCR at mRNA level and Western blot at protein level. MTT method was used to detect the proliferation of the cells. Flow cytometry was used to detect the cell apoptosis. Changes of cleaved caspase-3 and cleaved PARP were analyzed by Western blot. Results: The constructed revealed shRNA plasmids were proved to be correct by PCR and sequencing. shRNA plasmid (GV115-shRNA2), which efficiently silenced DEPTOR, was screened out. GV115-shRNA2 and lentiviral packaging plasmid were successfully packaged with a titer of 1?109 TU/ml. EGFP expression could be detected in RPMI-8226 after infection of the recombinant lentivirus. The expression of DEPTOR decreased obviusly detected by Real-time PCR and Western blot. Down-regulation of DEPTOR expression distinctly decrease the proliferation rates of MM line (P<0.05), induce tumor cell apoptosis (P<0.01). DEPTOR shRNA could increase expression of cleaved caspase-3 and cleaved PARP and Bax. The expression of protein Bcl-2 was decreased, while the PI3K/Akt signaling pathway was blocked (P<0.01). Conclusion: The DEPTOR shRNA recombinant lentivirus vectors was successfully constructed, and the shRNA can significantly inhibit the expression level of DEPTOR in RPMI-8226 cells. DEPTOR shRNA will induce MM cell apoptosis and inhibit its proliferation. In addition, PI3K/Akt pathway may be involved in the process of apoptosis.
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Received: 17 December 2012
Published: 25 May 2013
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