| Background Diffuse large B-cell lymphoma(DLBCL)is the most common malignant lymphoma among adults,accounting for about 48% of lymphoma cases.As the first-line treatment for DLBCL,R-CHOP(rituximab,cyclophosphamide,doxorubicin,vincristine,prednisone)plan can cure about 60% of the patients.However,the therapeutic benefit of R-CHOP for the patients with refractory / recurrent DLBCL and the elderly patients over60 years old is still limited.The abnormal expression of myc accounts for about 20% of DLBCL,which is the main reason leading to the refractory and relapse of DLBCL.Hence,myc signaling pathway is an important target for the treatment of DLBCL.miR34 a and let7 were low expression in DLBCL,and those two genes can regulate myc gene,in particular,the interaction between myc and miR34 a easily forms a vicious circle,which may aggravate the poor prognosis of DLBCL.And improving the expression of these two microRNAs in DLBCL cells may provide a new method for the treatment of DLBCL with abnormal expression of myc gene.Oncolytic virus therapy as a promising approach of biological immunotherapy,can elicit enormous anti-tumor effects via direct oncolysis,inducing anti-tumor immunity or carrying tumor suppressor genes.Purpose We propose to construct a novel oncolytic vaccinia virus that co-expressing miR34 a and let7 genes for the treatment of DLBCL,which could effectively up-regulate the expression of miR34 a and let7 in tumor cells while oncolytic virus plays a killing role,so as to achieve a powerful attack on DLBCL.Methods 1.Construction and identification of OVV-mir34a-IRES-let7,OVV-mir34 a,OVV-let7,OVV-GFP: TK sequence of vaccinia virus vector was deleted by gene recombination technology,and foreign gene mir34a-ires-let7 or single genes mir34 a,let7were inserted respectively.And then the recombinant oncolytic virus was identified through PCR/nucleic acid gel electrophoresis,sequencing and Real-Time Quantitative Reverse Transcription PCR(qRT-PCR).2.To investigate the killing effect of novel recombinant oncolytic vaccinia virus on lymphoma cells(in vitro): 2.1 lymphoma cell lines were infected with a series of oncolytic vaccinia viruses,including OVV-mir34 a,OVV-let7,OVV-mir34a-IRES-let7 and OVV-GFP,virus production in lymphoma cells was detected by TCID50 method,and their effects on DLBCL cells were observed and compared by CCK-8 assay.2.2 Flow cytometry was used to determine the effect of oncolytic viruses on apoptosis of lymphoma cells.2.3 The expression of miR34 a and let7 in virus-treated lymphoma cells was detected by qRT-PCR.3.Research on molecular mechanism of anti DLBCL based on miR34 a and let7 signaling pathway: the expression of miR34 a,let7 downstream genes including bcl-2/Notch1/Sirt-1/Fox P1 and c-myc/K-ras/HMGA2/lin28 were detected by qRT-PCR.4.To explore the anti-lymphoma effect of OVV-mir34a-IRES-let7 in lymphoma bearing mice model: OCI-ly3 cells were used to construct the subcutaneous xenograft lymphoma model in NCG mice.After treating with the recombinant oncolytic vaccinia virus ovv-mir34a-IRES-let7 and the control virus OVV-mir34 a,OVV-let7 and OVV-GFP,observed the changes on size of tumor and the survival time of mice.The qRT-PCR assay was used to detect the level of the virus gene in peripheral blood,spleen,liver,lung and kidney of virus-treated group.Results 1.We successfully constructed a novel oncolytic vaccinia virus carrying miR34 a and let7.2.In vitro experiments: 2.1 The results of TCID50 virus titer test showed that recombinant oncolytic vaccinia virus OVV-mir34a-IRES-let7 and control virus OVV-mir34 a,OVV-let7,OVV-GFP could replicate in DLBCL cells and produce virus progenies,and there was no significant difference in the replication efficiency of each virus in lymphoma cells.2.2 CCK-8 results showed that OVV-mir34a-IRES-let7 elicited the strongest killing effect on lymphoma cells,followed by single gene virus,but stronger than empty oncolytic virus group.The killing effect of all virus treatment groups was higher than PBS control group.2.3 The results of flow cytometry showed that OVV-mir34a-IRES-let7 could significantly promote the apoptosis of lymphoma cells.3.qRT-PCR results showed that after OVV-mir34a-IRES-let7 treatment,the target genes of mir34 a and let7 were significantly down-regulated at the m RNA level.4.Animal experiments showed that the OVV-mir34a-IRES-let7 exhibited the best anti-lymphoma effect and significantly prolonged the survival time of mice compared with the single gene and empty control virus group.qRT-PCR results confirmed that the mir34 a and let7 were up-regulated in tumor tissues which treated with OVV-mir34a-IRES-let7 virus.In addition,no virus expression was detected in peripheral blood and normal tissues.Conclusions We successfully constructed a novel recombinant oncolytic vaccinia virus named OVV-mir34a-IRES-let7 for the first time.Experiments,both in vitro and in vivo,have confirmed the strong anti-lymphoma activity of OVV-mir34a-IRES-let7.Our findings provide a preclinical basis for the combination of OVVs and miRNAs(mir34a and let7)in the treatment of DLBCL patients,especially those with high expression of myc and bcl-2. |
