Study On Silence Of Bmi-1Mediated By Lentivirus ShRNA On Biological Characters And Sensitivity To Bortezomib In Multiple Myeloma Cells

AIM：This study was aimed to construct lentivirus-mediated shRNA expressionvector targeting Bmi-1and to establish stable multiple myeloma cell lines U266andRPMI8226, then analyzed the affects on cell growth, clone formation, proliferation,apoptosis and chemosensitivity to Bortezomib.METHODS：One pair of oligonucleotide sequences targeted at human Bmi-1mRNA were designed and synthesized. The annealed oligonucleotide fragments weresubcloned into pLVTHM vector. Virus particles were collected after shRNA vectorwas cotransfected with the psPAX2packaging plasmid and the envelope plasmidpMD2.G into HEK-293T cells. The multiple myeloma cell lines were transdused with5×106recombinant lentivirus-transdusing. Flow cytometer was used to separate GFP+cells, then stable cell lines were established. Real-time PCR and Western blot wereused to detect the expression of Bmi-1and p14after lentivirus transdusion. TheCCK-8assay and clone formation ratio methods were used to evaluate the effect onsituation of cell proliferation in cells. The cell cycle status and apoptosis of cells weredetected by flow cytometry. The AO/EB and Hoechst33258fluorescent stainingmethod were used to observe the situation of cell apoptosis. Western blot were used todetect the protein expression of P21、Bax and Bcl-2. Furthermore, analyzed thechemosensitivity to Bortezomib in multiple myeloma cells after knockdown of Bmi-1from cell growth, clone formation, proliferation, apoptosis, respectively.RESULTS：(1) Plvthm-shBmi-1was constructed successfully: enzyme cutidentification and partial nucleotide sequencing showed that lentivirus vectorexpressing shRNA of bmi-1was correct.(2) Stable transfected multiple myeloma celllines were established: the mRNA and protein level of Bmi-1were reduced significantly in cells after lentivirus transdusion.(3) Effect of shBmi-1on multiplemyeloma cells growth、 clone formation、 cell cycle and apoptosis：○1Bmi-1knockdown could repress cells proliferation： Bmi-1shRNA decreases cellproliferation by22.83～25.1%in U266and13.9～26.9%in RPMI8226at48～96hcompared to control group.○2Bmi-1knockdown could inhibit cells clone formation.○3Bmi-1knockdown could accumulate G1-phase cells：U266： the G1-phase ofcontrol and shBmi-1cells were39.91%±0.26%and45.65%±0.68%；RPMI8226：G1-phase of control and shBmi-1cells were42.7%±0.47%and50.86%±0.38%.○4Bmi-1knockdown could promote cells apoptosis：U266：the apoptosis ratio of controland shBmi-1cells were9.8%and12.12%；RPMI8226：the apoptosis ratio of controland shBmi-1cells were9.6%and15.69%.(4) Knockdown of Bmi-1sensitize cells toBortezomib：○1Bmi-1knockdown could reduce the IC50to Bortezomib: U266:control24.73nmol/L, shBmi-118.59nmol/L；U266: control32.99nmol/L, shBmi-121.56nmol/L.○2Cell cycle distribution combined with40nmol/L Bortezomib after48hours：U266：the G1-phase of control and shBmi-1cells were45.35%±0.77%and56.81%±0.50%； RPMI8226： G1-phase of control and shBmi-1cells were49.27%±0.90%and56.94%±0.61%.○3Bmi-1knockdown could promote apoptosis：U266：the apoptosis ratio of control and shBmi-1cells were20.83%and28.14%,respectively；RPMI8226：the apoptosis ratio of control and shBmi-1cells were22.5%and49.14%.○4Western blot：compared with control, shBmi-1cells’ p21、Bax proteinlevel were increased significantly, Bcl-2protein was reduced significantly, afteradding Bortezomib, this diference was more obviously.CONCLUSION：(1) Stable transfected multiple myeloma cell lines wasestablished, shBmi-1could repress cell proliferation, inhibit clone formation,promotes apoptosis and block cell cycle in G1-phase.(2) shBmi-1could enhancemultiple myeloma cells to Bortezomib, so inhibition of Bmi-1expression could be anew adjuvant therapy for multiple myeloma.