| Background:Despite more and more novel agents(Proteasome inhibitors,Immunomodulatory drugs and anti-CD38 antibodies and et al)improve the outcome of patients,multiple myeloma(MM)remains incurable.Patients relapse or progress at last.Hence,developing a novel treatment strategy is demanding for the clinical management of MM.Non-coding smallRNAs,a cluster ofRNAs that do not encode functional proteins,have been underlined that play a pivotal role in the pathogenesis of MM.Our previous study indicated that the level of miR-15a was down-regulated inMM cells and correlated with inferior outcome of MM patients.Up-regulating miR-15a inhibited the cell proliferation and promoted the apoptosis of MM cells.All of these indicated that miR-15a acted as a tumor suppressor inMM cells.So,to exert the tumor suppression founction of miR-15a,up-regulateing its expression inMM cells may be a new therapeutic method inMM treatment.However,there was no effective drugs to specially up-regulate certain miRNA.At present,the most common method was designing certain miRNA mimic.Then the mimic was transfected into cells to exert its regulating or therapeutic founction.At present,miRNA mimic are all in the form of smallRNA molecule duplexes which could mimick miRNA perfectly.But this kind of mimics themselves has a few disadvantages that is hard to overcome.They are instable and have a short half-life period in vivo and so on.For these reasons,a brand new kind of miRNA mimics with more advantages are demanded.Objectives and significance:In the present study,we innovated a new kind of miRNA mimic,and verified its function.Firstly,we developed an oligo-single-stranded DNA based on the sequence of hsa-miR-15a-5p,and used it as the hsa-miR-15a-5p mimic.Then we evaluated its anti-tumor effects.Whether it could mimick the founction of miR-15a and suppress the target gene of miR-15a were evaluated too.The greatest significance of this study was providing a brand new choice for miRNA mimics.Compared with smallRNA molecule duplexes,oligo-single-stranded DNA has more advantages.Its molecule is smaller with low negative charge and the aromatic nucleobases are on the exterior too.These make oligo-single-stranded DNA better interactions with cell membranes with more easily cell entry.Moreover,oligo-single-stranded DNA is more stable for storage and transport as drugs.Although the native form of oligo-single-stranded DNA would be degraded by circulating and intracellular nucleases and excreted by the kidney quickly after systemic administration,these disadvantages can be overcomed by locked nucleic acid(LNA)modification.Methods:In the present study,we designed the oligo-single-stranded DNA of miR-15a in two forms,the native form and the LNA modified form.The native form was named OMM-15a in this paper.When OMM-15a was modified with 3 LNAs at both ends was named LNA-15a.1.Cell experiment of LNA-15a and OMM-15a:OMM-15a and LNA-15a was transfected into ARP1,OCI-My5 and MCF7 cells in the concentration of 25nM.Cells were collected 24 h,48 h and 72 h after the transfection.Then trypan blue straining and cell counting was used to evaluate cell proliferation and viability.Flow cytometry analysis was used to detecte cell apoptosis.Also,cells that collected 24 h,48 h and 72 h after the transfection,were lysised,then totalRNA and protein were isolated.Next,the miR-15a target gene expression was detected in mRNA and protein level using quantitative real-time polymerase chain reaction(q RT-PCR)and Western blot.2.LNA-15a and BTZ synergism effects:ARP1 and OCI-My5 cells was treated with BTZ(10 nM),then LNA-15a was transfected immediately.24 h after that,we compared the apoptosis between the LNA-15a and blank transfected cells.The lipo 3000 was used for the transfection,and the concentration of LNA-15a was as follows:0 nM,25 nM,50 nM and 100 nM.The Compusyn softeware was used to evaluate the synergism effect between LAN-15a and BTZ.3.In vivo experiment of LNA-15a and OMM-15a:Lentiviral vector carried with EX-h LUC-Lv201 plasmid was used to stable transfect ARP1 cell line.Then 1×10~6 ARP1-GFP~+-Luc~+cells were injected into the NOD/SCID mice.After 7 days of tumor cell transplantation,the mice were treated with LNA-15a,OMM-15a and PBS,respectively,weekly for 3 weeks.The tumor burden was monitored with bioluminescence intensity and tumor volume.The in vivo experiments were done twice.In the first experiment,bioluminescence was used to detect tumor size per week 14 days after the tumor injection.In the second experiment,tumor volume was examined by vernier calipers every other day when tumor could be detected.Once the tumor volume reached at 1500mm~3,the mice was humanized sacrificed and their tumors would be isolated.Tumor volumes were calculated as follow:(length×width~2)/2.In order to confirm the suppression of tumor growth was induced by LNA-15a,the level of target gene of hsa-miR-15a was detected in the tumor sample of mouse model.Results:1.The MM cell line ARP1,OCI-My5 and breast cancer cell line MCF7 were transfected with 25nM of LNA-15a and the proliferation and apoptosis of these cells were examined at different time point(24 h,48 h and 72h).The result showed that:compared with the blank group,the cell proliferation and viability reduced in the 3 cell lines.Moreover,LNA-15a treatment significantly increased the apoptosis of these cell lines,especially in the breast cancer cell line MCF7.Furthermore,LNA-15a treatment suppressed tumor cell growth and induced cell apoptosis in a dosage dependent manner,at least in part.Then the MM cell line ARP1,OCI-My5 and breast cancer cell line MCF7 were transfected with 25nM of OMM-15a and the proliferation and apoptosis of cells were examined at different time point(24 h,48 h and 72 h).The result showed that:there were no significant defferent in cell proliferation,viability and apoptosis between the OMM-15a transfected group and the blank group,even increased the concentration of OMM-15a.2.The MM cell line ARP1,OCI-My5 and breast cancer cell line MCF7were transfected with 25nM of LNA-15a.Then 24 h,48 h and 72 h after the treatment,the level of target gene of miR-15a was detected by q RT-PCR and western blot.Our data indicated that the level of miR-15a target genes was down-regulated in mRNA and protein level.The MM cell line ARP1,OCI-My5and breast cancer cell line MCF7 were transfected with 25nM of OMM-15a.Then 24 h,48 h and 72 h after the treatment,only part of the target gene of miR-15a was down-regulated.In protein level,PHF19 expression only decreased in ARP1 cell line 24 h after the treatment.No difference of BCL2expression was detected in the 3 cell lines between before and after the transfection.3.ARP1 and OCI-My5 cell lines were treated with BTZ and transfected with LAN-15a at the same time.24 h after the transfection,the apoptosis of ARP1 and OCI-My5 cells were significantly increased with the combination treatment of LNA-15a(25nM)and bortezomib(10nM)compared with the BTZ treatment alone.Most important,the results indicated that there was a synergism effects of anti-MM in the combination of LNA-15a and BTZ in OCI-My5 cell line which was analyzed by the Compusyn software.4.The GFP+/Luc+ARP1 cells were injected into the NOD/SCID mice.After that,mice were divided into 3 groups randomly.7 days later,the mice were treated with LNA-15a,OMM-15a and PBS,respectively,weekly for 3weeks.The tumor burden of mice treated with LNA-15a significantly decreased compared with PBS control group and OMM-15a treatment.And there was no significant difference between PBS and OMM-15a group.Furthermore,the survival of mouse models was prolonged significantly after the treatment of LNA-15a while OMM-15a did not.Further more,the level of target gene expression of hsa-miR-15a was detected in the tumor sample of mouse model.The q RT-PCR and western blot data showed that the treatment of LNA-15a suppressed the target gene(BCL2,PHF19 and VEGF-A)expression both in mRNA and protein level compared with PBS and OMM-15a treatment.Conclusion:1.LNA-15a had significant anti-tumor activity.It suppressed the proliferation of ARP1,OCI-My5 and MCF7,and promoted the apoptosis of them.2.OMM-15a had no significant anti-tumor activity.There was no significant suppression of the proliferation of ARP1,OCI-My5 and MCF7,and no signifcant promotion to their apoptosis.3.LNA-15a could mimick the founction of miR-15a well intracellular.It suppressed the target gene expression of miR-15a both in the mRNA level and protein level.4.LNA-15a enhanced the anti-MM activity of BTZ.And there was a synergism effects of anti-MM in the combination of LNA-15a and BTZ in OCI-My5 cell line.5.LNA-15a had significant anti-MM activity in vivo.It no only suppressed the tumor growth,but also prolonged the mice survival.What more,LNA-15a could mimick the founction of miR-15a well in vivo,so could suppresse the target gene of miR-15a in vivo. |
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