Experimental Study Of A Tissue-Specific APE1 SiRNA Vector On Enhancing The Chemo-Sensitivity Of Multiple Myeloma

Multiple myeloma (MM) is an incurable disease now, to which increasing DNA damage repair gene expression, increasing tumor cell drug resistance, and decreasing tumor cell apoptosis may contribute much. One strategy for tumor gene therapy is to inhibit expression of DNA damage repair gene, selectively enhance radiation and chemosensitivity of MM cells with the purpose of eliminating tumor cell rapidly and effectively. Yet, poor targeting rate and specificity of inducing DNA damage repair gene silence limited this therapy. It is important to enhance chemosensitivity of MM cells specifically, we should select MM tissue-specifically to inhibit DNA damage repair gene expression. As a new technology of gene knock-down, RNA interference (RNA) may be a optimized molecular biological means with following important features: high stability, high efficiency, and high specificity. Hence, it may become a new means of tumor gene therapy. Apurinic apyrimidinic endonuclease (APE1) is also named Redox factor-1 (Ref-1), has two relatively independent functional area that is DNA damage repair and redox, which is an important repair factor of alkylating agent and radiotherapy inducing DNA damage. Therefore, detecting expression of APE1 protein in different clinical effective MM patients, analyzing the relationship between the expression of APE1 and treatment, prognosis of MM are important, and setting up MM-specific APE1 RNAi expression system in enhancing chemosensitivity of MM by targeted inducing APE1 gene silence is necessitous.Objective1. To research the expression of APE1 protein in MM cells, analyze the relationship between APE1 expression and MM clinical effect, and determinate that APE1 is an effective therapeutic target gene of MM.2. To set up MM-specific APE1siRNA expression system, and indentificate its knock down function in APE1 gene of MM cells.3. To explore the effect of enhancing chemosensitivity of MM by inducing APE1 gene silence.Materials and Methods1. The expression of APE 1 protein in MM cells and its significanceBone marrow specimens from MM patients and their medical records were collected. The expression of APE1 in bone marrow specimens of MM patients and MM cell lines (KM3) were detected by immunocytochemical staining, immunofluoresence double staining method and laser cofocal scanning microscopy and Western blot, then analyzed relationship between expression of APE1 and clinical classification, clinical effect of MM.2. Set up MM-specific expression vector APElsiRNA and indentificate its function APElsiRNA gene sequnces were screened, designed and synthsized by submittingGenBank and analyzing active domain sequnce of APE1, which annealed to formed ds-linker, inserted into linear expression vector and got pSilencer APElsiRNA. Immunoglobulin promoter (IgP) sequnces were chemo-synthsized, and then pSilencer APElsiRNA was digested by enzyme EcoRI and BamHI. Linear vector and IgP fragments were conjugated by T4 DNA ligase and got pSilencer IgP-APElsiRNA. Kappa lightchain intronic enhancer (IE) and kappa lightchain 2>' enhancer (K) fragments were produced from murine genomic DNA by PCR means. The PCR product was gel purified and degisted with EcoRI and Xhol respectively. Then pSilencer IgP-APE lsiRNA was digested by enzyme EcoRI. Linear vector and IE fragments were conjugated by T4 DNA ligase and got pSilencer IE-IgP-APE lsiRNA. pSilencer IE-IgP-APE lsiRNA was digested by enzyme Xhol. Linear vector and K fragments were conjugated by T4 DNA ligase and got pSilencer K-IE-IgP-APElsiRNA. The recombinant vector was indentificated according to the DNA sequncing and double enzyme digestions every once. pSilencer K-IE-IgP-APE lsiRNA plasmid was transfected to KM3 cells by liposome, got positive clones by G418-resistance screening. Dose-effect and time-effect relationship of APE 1 gene silence inducing by RNAi were measured using Western blot analysis. With KM3, HOS and MDA-231 cells, konck down APE1 gene effect of MM cells specifically by pSilencer K-IE-IgP-APE lsiRNA vector was observed by Western blot analysis. By using EGFP expression plasmid, the transfection efficiency of RNAi vector was measured with fluorescence microscopy.3. To study the effect of enhancing chemosensitivity of MM by inducing APE 1 gene silence. pSilencer K-IE-IgP-APE lsiRNA plasmid was transfected to KM3 cells by liposome,got positive clones by G418-resistance screening. Induce APE1 gene silence, the cellularproliferation capacity was observed with MTT assay, the cell cycle distribution and the apoptosis rate were measured by flow cytometry, DNA fragmentation rate and TLJNEL were tested after different-dose melphalan dealed. With different concentrated IL-6 simulated bone marrow hematopoiesis microenviroment, the effect of enhancing melphalan chemosensitivity on MM was observed by MTT assay, flow cytometry and DNA fragmentation rate analysis after APE 1 gene silenced. Results1. The expression of APEl protein in MM cells and its significanceThe positive rate of APEl gene expression in bone marrow specimens of 32 MM patients was 65.6%. The degree of APEl expression was related with clinical effect of MM. Beyond II grade positive rate of APEl expression in relapse/refractory group reached 55%, much higher than that of untreated group. No beyond II grade positive results was detected in bone marrow specimens of noncancerous disease. The location of APEl expression was also related with MM clinical effect. Expression of APEl gene in nucleus/cytoplasm in relapse/refractory group were significantly elevated compared to those in untreated and nomal control group (f<0.01). As for MM lines KM3, expression of APEl gene was high in nucleus/cytoplasm, the degree of expression of APEl protein was related with treating. The expression of APEl protein could be induced by alkylating agents melphalan.2. Set up MM-specific expression vector APElsiRNA and indentificate its function After evaluation and sequencing, MM-specific expression vector APElsiRNA wassetted up successfully, and transfected into KM3^0S $i MDA-231 by liposome. The result of western blot showed that, APEl protein in MM cells could be koncked down specifically by pSilencer K-IE-IgP-APElsiRNA vector, and inhibition rate of APEl expression was 80-90%. The best inhibition of expression of APEl gene was 3.0 u g and duration two days using pSilencer K-IE-IgP-APElsiRNA vector. The results of EGFP plasmid was detected by laser cofocal scanning microscopy suggested that the transfected rate of RNAi plasmid was 38-48%.3. To study the effect of enhancing chemosensitivity of MM by inducing APEl gene silence. After evaluation and sequencing, MM-specific expression vector APElsiRNA wassetted up successfully, and achieved continuous siRNA expression in KM3 MM cells bygene transfection. The condition of APE1 gene silence, the apoptosis of KM3 cells was verified by TUNEL. The apoptosis rate in RNAi group was much higher than that of control group by flow cytometry analysis, the apoptosis efficiency was similar to the results of TUNEL. The result of MTT showed that it enhanced melphalan chemosensitivity to MM cells by transfecting APElsiRNA. With different concentrated IL-6 simulated bone marrow hemato- poiesis microenviroment, the apoptosis of MM cells in APElsiRNA group was much more different than that of control group by MMT, flow cytometry and DNA fragmentation rate after melphalan treated. The apoptosis rate of KM3 group, KM3Ctr' group, KM3APElsiRNA group, KM3+Mel group, KM3Ctrl+Mel group and KM3APElslRNA+Mel group was 0.51%, 0.93%, 1.24%, 0.93%, 1.32%, 7.65%, separately. Conclusion1. Patients with MM have increased APE1 protein expression, especially those with relapse/refractory group. Detection of APE 1 gene may help clinical effect and prognosis of MM.2. After alkylating agents melphalan treated, APE1 protein increases remarkably, showing that besides killing tumor cells, chemotherapy drugs also lead to APE1 synthesis and expression in tumor cells as a result of apoptosis tolerance, which may partially contribute to drug resistance of MM cells.3. APE I gene in MM cells could be koncked down specifically by pSilencer K-IE-IgP-APElsiRNA vector. The largest inhibition effect of expression of APE 1 gene was dose 3.0 u g and duration two days with pSilencer K-IE-IgP-APElsiRNA vector.4. RNAi can specifically inhibit expression of mammlian cell gene. The efficiency of gene silence was 60-90% that induced by RNA interfencing technology in the reseach.5. Inhibiting APE1 expression of MM cells, it could improve chemotherapy drug sensitivity remarkably and induce apoptosis of MM cells. APEl-based RNAi could induce apoptosis of MM cells specifically and could serve as an assistant therapy for chemotherapy of multiple myeloma.