Experimental Study Of Effects Of RNA Interference Targeting Livin Mediated By Dextran-Adriamycin-Polyethyleneimine Nanoparticles On Human Osteosarcoma

Objective:Osteosarcoma is the most common primary solid tumor of bone in children and young adults, comprising about 20% of primary bone tumor. A novel member of the inhibitor of apoptosis proteins (IAPs) is Livin. High Livin expression in neoplasms correlates with more aggressive behavior, such as decreased the response to chemotherapeutic agents and shortened survival time. Tumor angiogenesis is regulated by a variety of pro- and anti-angiogenic factors.Among them, PlGF is a dimeric glycoprotein, structurally and functionally related to VEGF. PIGF appears to be crucial for pathological angiogenesis and is not produced by the majority of normal human tissues. PlGF has been shown to be a key molecule in the angiogenic switch in pathological conditions, including cancer. To date, PIGF expression in osteosarcoma and any correlation between PIGF and Livin remains unknown. The first part of this study was to investigate the expression of Livin and PIGF in osteosarcoma tissues and cell lines (MG-63, Saos-2 and U2-OS cells), and analyze the correlation between Livin and PIGF expression and their roles in the prognosis and treatments of osteosarcoma. Another purpose of this part was to analyze the relation between the expression of these two factors in three osteosarcoma cells and the differentiation of these cell lines. Combination of chemotherapy and gene therapy of cancer has synergistic effects on overcoming drug resistance. Biodegradable macromolecular materials such as dextran and PEI had been a potential module for chemotherapeutics and gene delivery. The second part of this study was to develop biodegradable nanoparticles based on the high molecular weight dextran and low molecular weight PEI 1800Da, thereinto dextran and PEI segment are responsible for conjugation with ADM and plasmid, respectively. The physicochemical properties and cytotoxicity were investigated in vitro. The last part of this study was to investigate the synergistic effects of gene therapy and chemotherapeutic on osteosarcoma via delivering both Livin shRNA plasmid and adriamycin into osteosarcoma MG-63 and Saos-2 cells by the DEX-ADM-PEI nanoparticles, which were made in the foregoing study. The ultimate purpose of this study was to explore the possible mechanism of nanoparticles for gene carrier and combination therapy in osteosarcoma therapy. Methods:Part One:To investigate the expression of Livin and PlGF in osteosarcoma tissues and osteosarcoma cell lines (MG-63, Saos-2 and U2-OS cell), and analyze the correlation between Livin and PlGF and their role in the prognosis and treatments of osteosarcoma. Expression of Livin and PlGF in 57 osteosarcoma tissues and 10 normal bone tissues from our hospital were detected by immunohistochemistry (IHC) and the expression of these two proteins were determined by the percentage of positive cells and the intensity of staining. The relation between their expressions and clinical characteristics of osteosarcoma patients were analyzed. The mRNA expressions of these two factors in osteosarcoma cell lines were detected by Real-time PCR and the relation between these expressions and differentiation of these cell lines were analyzed. All statistical analyses were performed by SPSS version 18.0.Part Tow:Herein, we hypothesized the combinational strategy of chemotherapeutic drug and gene therapy in a single nanoplatform. The nanoparticles based on dextran and PEI was developed by two steps. Firstly, dextran was oxidized by sodium periodate and the oxidation degree of oxidized dextran was determined by hydroxylamine hydrochloride titration. The structure of oxidized dextran was measured by ultraviolet spectrum analysis and infrared spectroscopic analysis. The molecular weight of oxidized dextran was determined by aqueous phase gel permeation chromatography. Secondly, the DEX-ADM-PEI nanoparticles were developed by the formation of Skiff base from the aldehyde group on oxidizd dextran and the free amino group on ADM and PEI 1800. The composition of these nanoparticles was determined by ultraviolet spectrum analysis,1H nuclear magnetic resonance and infrared spectroscopic analysis. The coupling efficiency of ADM in DEX-ADM-PEI nanoparticles was calculated by ultraviolet spectrometer method. The morphology and particle size of these nanoparticles was determined by transmission electron microscope. The gene nanoparticles were formed by the electrostatic adhesion between positive charge of nanocarrier and negative charge of DNA and the particle size and Zeta potential of these nanoparticles were determined by laser particle analyzer. The ability to condense the DNA to form nanoparticles was analyzed by agarose gel electrophoresis. The inhibition effect of these nanoparticles on osteosarcoma cell lines was determined by MTT assays in vitro.Part Three:To inverstigae the synergistic effects of genetherapy and chemotherapy on osteosarcoma by these single nanocarriers. Livin gene interference sequence was synthesized and inserted into pGenesil-1 vector, which was confirmed by sequencing. The recombinant RNAi vectors were transfected into osteosarcoma MG-63 cell by Lipofectamine 2000. The cells containing stable transformants were selected by the ability of resistance to G418, and isolated with a limited dilution. The mRNA and protein expression of Livin in the selected clones was detected by Real-time PCR and Western blot respectively to confirm the silencing efficiency of the RNAi vectors. The Livin-shRNA and Adriamycin were delivered into osteosarcoma MG-63 and Saos-2 cell lines by DEX-ADM-PEI nanoparticles according to the optimal transfection efficiency. The transfection efficiency was confirmed by converted fluorescence microscope and flow cytometry and the difference between the transfection efficiency of these nanoparticles, Lipofectamine 2000 and PEI 25K was analyzed. The synergistic effects of Adriamycin and Livin gene silencing mediated by these nanoparticles on the inhibition of osteosarcoma cells were detected by MTT assays.Results:Part one:The expressions of Livin and PIGF in osteosarcoma specimens measured by optical density value and positive cell percentage were significantly higher than that of normal bone tissues (P<0.01). Both Livin and PIGF proteins expressions were consistently detected in 30(52.6%) and 37(64.9%) of the 57 osteosarcoma specimens but in none of the normal bone tissues. Livin and PIGF were predominantly found in a granular pattern in the cytoplasm and few were found in cell nucleus. In 22 cases (38.6%), both markers were present in the cytoplasm, and in 6 cases (10.5%) there was no detection of either protein. In addition, Using Spearman's correlation coefficient analysis, we found no correlation between the expression of Livin and PIGF (P=0.642) in osteosarcoma tissues. Real-time PCR was performed to detect the expressions of Livin and PIGF mRNA in three osteosarcoma cell lines (Saos-2, MG-63 and U2-OS). The expressions of Livin and PlGF mRNA were high in three osteosarcoma cell lines. Results showed that both of the expression of Livin and PlGF mRNA in MG-63 cell were much higher than that in U2-OS cell (2.93 folds for Livin and 8.57 folds for PIGF respectively) and Saos-2 cell line (1.59 folds for Livin and 3.49 folds for PIGF respectively). The IHC results showed that the expressions of Livin and PIGF were associated with pathologic stage and tumor diameter. Livin and PIGF expressions in patients with Stages IB to IIA were significantly lower than those in patients with Stages?B and?(P<0.01 and P=0.008 for Livin and PIGF respectively). Patients were divided into two groups according to tumor diameter (<5cm and >5cm), and the expressions of Livin and PlGF were compared. There were 38 cases in group one, in which all the tumor diameter were less or equal to 5cm, the positive rate of Livin and PlGF expression were respectively 42.1%(16 cases) and 52.6%(20 cases), but were 73.7%(14 cases) and 89.5%(17 cases) in group 2 that all the tumor diameter of 18 cases was bigger than 5cm. There was significant association between Livin or PlGF expressions and tumor diameter (P=0.002 and P=0.013 respectively). On the other hand, there was no significant association between Livin and PlGF expressions and sex, age or site of tumor.Part Tow:In the present study, we succeeded in synthesizing DEX-ADM-PEI conjugation by an aldehyde reaction between oxidised dextran and an free amine group of PEI and ADM. Ultra-violet spectrum and hydroxylamine hydrochloride titration revealed that the oxidation degree of oxidised dextran increased with the increase of the Sodium periodate/Dextran ratios. When this ratio was 2, the aldehyde group content and molecular weight of oxidized dextran were 15.8 mmol/L and 13000 respectively. Compared with pure dextran, noticeable absorption peak was shown at 490 nm in the ultra-violet spectrum, comfirming the existence of ADM moiety in DEX-ADM-PEI conjugation. In addition, the coupling efficiency of ADM in the DEX-ADM-PEI conjugation was 17.43±0.548%. After oxidation, the proton peak of the 2-carbon of dextran was decreased due to cleavage of the carbon-carbon bond by the periodate ion, leading to the formation of aldehyde. The assignment of chemical shifts of PEI and ADM were determined by the same method. As shown in Fig.2, the proton peaks of PEI appeared at 3.2-2.5 ppm and ADM appeared at 4.0-3.9 ppm, indicating that PEI and ADM wERE grafted to the dextran chain.1% agarose gel electrophoresis demonstrated that the migration of DNA was completely retarded when the N/P ratio of complex was around 4. Particle size analyzer indicated that with the increasing N/P ratio, the sizes of the nanoparticles decreased and the zeta potentials increased. The particle size was determined to be 225±17 nm when the N/P ratio was 0.5. However, at a ratio higher than 2, the sizes of the nanoparticles were less than 200 nm. It was shown that the zeta potential was +9.28 mV when the ratio of N/P was 0.5, where the complex could not form completely. With the increase of N/P ratios, the zeta potential rapidly increased (+24.71 mV, at N/P ratio 20). DEX-ADM-PEI conjugations had a well formed, spherical shape and compact structure. All conjugations were about 200 nm, and a relatively homogenous size distribution was observed. MTT assays showed a dose dependent cytotoxicity response that is, decrease in cell survival fraction with increasing concentration of drug. DEX-ADM-PEI conjugations have higher cytotoxicity, compared to DEX-PEI and ADM, in two different cell lines. Furthermore, cell viabilities were a little lower in all cell lines exposured to DEX-ADM-PEI conjugations than to free ADM. However, there was no difference of cell viabilities between free ADM and DEX-ADM-PEI at the same concentration. It should be noted that cell viability of DEX-PEI which maintained over 65% at a concentration of 8 mg/mL was significant lower than free ADM and DEX-ADM-PEI (P<0.01), indicating that these DEX-PEI carrier have low cytotoxicity.Part Three:Sequencing suggested that RNAi eukaryotic expression vectors targeting Livin possessed correct reading frame and nucleotide sequence, and green fluorescence of the stably transfected MG-63 cell could be observed under an inverted fluorescence microscope. Real-time PCR and western blot both revealed that the sequence of Livin shRNA could effectively downregulate the level of Livin mRNA and protein expression in osteosarcoma MG-63 cell, compared to the nonspecific transfection group and control group (P<0.01). To determine the optimal N/P ratio in transfection, GFP fluorescence intensity was examined at varying N/P ratios and Lipofectamine 2000 and PEI 25 kDa were used as the positive control. The tranfection efficiency of Livin shRNA via these nanoparticles was 18.6±0.40% in MG-63 cell and 15.3±0.50% in Saos-2 cell, which was equal to PEI 25K but slightly lower than that of Lipofectamine 2000. Besides, all cells keep the normal morphology. The DEX-ADM-PEI nanoparticles delivered Livin shRNA and Adriamycin into osteosarcoma cells efficiently with low cytotoxicity. The synergistic effects of Livin shRNA and Adriamycin on inhibiting the growth of MG-63 and Saos-2 cells were same to the DEX-ADM-PEI nanoparticles in the early 24h. However, these synergistic effects were significant better than that from Livin-shRNA or Adriamycin solely after 72h (P<0.05).Conclusion:Part one:This study researched the mRNA expression of Livin and PIGF in different osteosarcoma cell lines and the protein expression of PIGF in osteosarcoma tissues for the first time. The results of this study revealed that both the Livin and PIGF were over-expressioned in osteosarcoma tissues and were significantly correlated with the tumor size and Enneking staging of osteosarcoma. On the other hand, there was no significant association between Livin and PIGF expressions and sex, age or site of tumor. There was no correlation between the expression of Livin and PIGF. The expressions of Livin and PIGF mRNA were higher in three osteosarcoma cell lines than normal cell and these high expressions were correlated with the differentiation of these three osteosarcoma cell lines (Saos-2, MG-63 and U2-OS). These two proteins may participate in the pathogenesis and prognosis of osteosarcoma.Part two:DEX-ADM-PEI nanoparticles were combined successfully via the formation of Skiff base between the aldehyde group of oxidized dextran and the free amino-group from PEI and Adriamycin in this study. These nanoparticles can condensed the DNA to form gene nanoparticles efficiently, which could target into the tumor tissue by ERP effect and anchor to osteosarcoma cell cytomembrane selectively through electrostatic adhesion and then were transported into tumor cell via endocytosis. So the DNA and Adriamycin both were delivered efficiently and selectively to osteosarcoma cells with low cytotoxicity by DEX-ADM-PEI nanoparticles.Part three:Livin shRNA eukaryotic expression vectors were successfully constructed and the mRNA and protein expression of Livin were downregulated efficiently by the transfection of this plasmid in osteosarcoma MG-63 cell. The establishment of stably transfected MG-63 cell line laid a solid foundation for uncovering the mechanism of Livin in osteosarcoma cells. This study demonstrated that DEX-ADM-PEI nanoparticles efficiently and selectively delivered both Livin shRNA eukaryotic expression vectors and Adriamycin into osteosarcoma cells (MG-63 cell and Saos-2 cell) with low cytotoxicity. Adriamycin was released from these nanoparticles with the degradation of dextran by dextranase and carry out the anti-tumor effect. Meanwhile, Livin shRNA plasmid downregulated the Livin expressiom via RNA interference and thereby upregulated the sensibility of osteosarcoma cells to Adriamycin. So the chemotherapy and gene therapy mediated by these nanoparticles could generate the synergistic effects and these synergistic effects of these two drugs on inhibiting the growth of osteosarcoma cell lines were significantly better than that of Livin-shRNA or Adriamycin solely.