Study On MicroRNA15a Enchance The Sensitivity To Bortezomib In Multiple Myeloma Cells By Targeting Bmi-1

AIM: This study was aimed to construct lentivirus-mediated microRNA15a highexpression vector targeting Bmi-1and to establish stable multiple myeloma cell lineU266and RPMI8226， and then analyzed the effects on cell growth,proliferation,apoptosis and chemosensitivity to Bortezomib.METHODS: One pair of oligonucleotide sequences of microRNA15a wasdesigned and synthesized. The annealed oligonucleotide fragments was subclonedinto GV217vector. Virus particles were collected after microRNA15a vector wascotransfected with the psPAX2packing plasmid and the envelope plasmid pMD2.Ginto HEK-293T cells.The multiple myeloma cell lines were transdused withrecombinant lentivirus-transdusing. Flow cytometer was used to separate GFP+cells,then stable cell lines were established. Real-time PCR was used to detect theexpression of microRNA15a、Bcl-2and Bmi-1after lentivirus transdusion. Westernblot were used to detect the protein expression of Bmi-1.The CCK8assay was used toevaluate the effects of cell proliferation in cells. The cell cycle status and apotosis ofcells were detected by flow cytometry. The AO/EB and Hoechst33258fluorescentstaining method were used to observe the situation of cell apoptosis. Furthermore, weanalyzed the chemosensitivity to Bortezomib in multiple myeloma cells afterupregulated microRNA15a from cell growth、proliferation、cell cycle and apotosis.RESULTS:(1) GV217-mir-15was constructed successfully: enzyme cutidentification and partial nucleotide sequencing showed that lentivirus vectorexpressing microRNA15a was correct.(2) Stable transfected multiple myeloma celllines were established: the level of microRNA15a was upregulated and the protein ofBmi-1was reduced significantly in cells after lentivirus transdusion.(3) Effect ofupregulated microRNA15a on multiple myeloma cells growth、cell cycle and apotosis: α1Upregulated microRNA15a could repress cells proliferation: upregulatedmicroRNA15a decreases cell proliferation by9.65～31.81%in U266and12.11～26.59%in RPMI8226at48~96h compared to control group. α2UpregulatedmicroRNA15a could accumulate G1-phase cells: U266: the G1-phase of control andmicroRNA15a cells were37.88%±0.60%and41.50%±0.64%; RPMI8226: G1–phaseof control and microRNA15a cells were37.87%±0.60%and45.31%±0.77%. α3Upregulated microRNA15a could promote cells apotosis: U266: the apotosis ratio ofcontrol and microRNA15a cells were37.08%and90.52%; RPMI8226: the apotosisratio of control and microRNA15a cells were44.17%and59.40%.(4) UpregulatedmicroRNA15a sensitize myeloma cells to Bortezomib: α1UpregulatedmicroRNA15a could reduce the IC50to Bortezomib：U266: control35.95nmol/L,microRNA15a25.92nmol/L; RPMI8226： control25.23nmol/L,microRNA15a19.08nmol/L. α2Cell cycle distribution combined with40nmol/L Bortezomib after48hours: U266the G1-phase of control and microRNA15a cells were44.85%±0.77%and61.23%±0.83%; RPMI8226: G1-phase of control and microRNA15a cells were56.41%±0.84%and68.96%±0.85%.α3Upregulated microRNA15a could promoteapotosis:U266: the apotosis ratio of control and microRNA15a cells were60.21%and90.57%;RPMI8226: the apotosis ratio of control and microRNA15a cells were69.80%and86.17%.CONCLUSION:(1)Stable transfected multiple myeloma cell lines wasestablished, upregulated microRNA15a could repress cell proliferation,promotesapotosis and block cell cycle in G1-phase.(2) Upregulated microRNA15a couldenchance multiple myeloma cells to Bortezomib, so the oncogene Bmi-1could be atargeting site of a new therapy for multiple myeloma.