Research Of Nanoparticles Mediated RNA Interference Target For Colorectal Carcinoma

Colorectal carcinoma is one of the most frequent digestive canal cancers in clinic and the incidence rate goes up year by year.The occurrence of tumorous is as to the disequilibrium between cell proliferation and apoptosis.Apoptosis inhibition is an important mechanism of tumorous occurrence.The regulate mechanism of apoptosis is very complex.Inhibitor of apoptosis proteins(IAPs)are important anti-apoptosis regulatory factors of molecular level and Livin is a novel member of IAPs.Research found that Livin express highly in most tumors but lowly or negatively in most terminal differentiation tissue except for placenta.In most research about Livin expression in cancer,high expression of Livin mRNA and protein is considered the prognostic sign of cancer development,which is related with the incidence,devolepment and prognosis.So many researchs considered Livin as a target for therapy tumor focused on IAP.The cancer therapy may be found a new target by regulate Livin.Though research found that Livin has important significance in some tumors,so far the relationship of Livin with incidence,development,metastasis and prognosis and gene therapy for colorectal carcinoma has not detail report domestic and abroad.RNA interference(RNAi)is a new technology developed in recent years.RNAi can be induced by small interference RNA(siRNA),plasmid or virus carrier that express short hairpin RNA(shRNA).The role of gene silencing of RNAi is specific and effective,which has some merit such as powerful inhibitory action,good stability and easily absorbed by cell,and provide a new and potent method for gene therapy of tumor.RNAi has becaming a widely used tool for gene silencing in current gene therapy research.SiRNA selectively inhibit gene express make gene therapy achieve maximal specificity.But puny introduction capability into cell, definite blood stability and non-specific immunogenicity hinder siRNA development for gene therapy.Researchs both domestic and abroad have not efficiently solved the problem of transfection efficiency low of siRNA.Gene transfection need suitable carrier.An ideal gene transport system should has good gene transfection rate,favourable target and biocompatibility,fine stability and biological degradation.The viral vector and non-viral vector at present have different defect to some extent.So to find a ideal gene carrier is very critical.The research of nanoparticles for gene carrier is an impotant advancing front topic in fields of nano-biology and tumor gene therapy in international.Because of minute volume,nanoparticles can circulate in blood vessel,enter the cells of every organ through vessel wall,mediate DNA transfection efficiently and make it express highly in cell as a novel non-viral carrier.Which provides new technology and method for gene express,research of fuction and therapy.Chitosan(CS)is a natural polycose,which has good biocompatibility and biological degradation.Researchs indicate that CS is a very good gene carrier,which has better target and slow-release if it be modified and reshaped.Through inoculation and copolymerization,the atoxic polyethylene glycol(PEG)is inserted into CS strand to increase target and slow-release,at last increase gene transfection efficience.Based on the background and achievement above,we detected the express of Livin from mRNA and protein level by RT-PCR and western blot and investigated the correspond with characteristic of colorectal carcinoma at first.The result indicated that Livin negative expressed in all para-cancerous tissue and normal tissue,but the express rate attained 45.5%in tumor.It had the same result by two methods.So it hinted that Livin is a marker for cancer of colon,which not only help for the diagnosis but also supply evidence for the biotherapy of it.Analyzed the relationship of Livin express with main clinic parameter we found that there was no significant correlation between livin expression with age, sex,tumor size,tumor location,histological type,lymphnode metastasis and Dukes classification(P＞0.05),however,livin expression was strongly correlated with distant metastasis and radio-/chemotherapy(P＜0.05).It hinted that the express of Livin can affect the biological behaviour of colorectal cancer and make incident of malignant behavior such as metastasis,which is one of the indications of unfavourable prognosis.The express of Livin is also increased remarkably after radiochemotherapy, which indicated that it related with drug resistance for tumor.So that it could supply a new parameter of molecular biology for diagnosis and prognosis.At the same time supplied experiment base for the next research of gene therapy.In the second part,we designed and synthetized Livin shRNA,then linked with pGenesil-1 plasmid vector to construct recombinant plasmid, at last verified that the correct plasmid vector had been construct by enzyme-cutting electro-phoresis and gene sequencing.The plasmid was transfected into HT-29 cell that express highly of Livin and the Livin mRNA was detected to select the optimal shRNA.At last it verified that oligonucleotides were correctly inserted into the recombinant plasmid pGenesil-shRNA by enzyme-cutting electro-phoresis and gene sequencing and the sequence was completely the same as designed,which also illustrated that the eukaryotic expression vector was constructed correctly.After the recombinant plasmid vector was transfected into HT-29 cell,livin mRNA level of cancer cell was remarkably decreased than before transfection and control group(p＜0.01).It verified that the shRNA prepared can inhibit the express of Livin efficiently and the role of livinl is more obvious,the inhibition rate of which was 66 percent.So in the next research,Livinl was selected as experiment template to explore the effect of Livin RNAi for cancer therapy.In the third part is preparing gene-nanoparticle compound.Chitosanpolyethylene glycol particle(CS-PEG)was synthesized firstly through stem grafting and copoly-merizing,then characteristics of nanoparticle was detected by evaluating shape,size and zeta electric potential and cytotoxicity was detected by MTT.Different ratio of gene/nanoparticle was used to prepared gene-nanoparticle compound.Then shape,size,zeta electric potential,envelopment rate and carry drug rate were inspected to evaluate characteristics of drug and expresses of EGFP protein and Livin mRNA were detected to evaluate effect of gene transfection.At last the blank nanoparticle size and zeta electric potential were 60 nm and 37mV respectively,the cytotoxicity was inferior to liposome.In conditions of 1:5 for gene/nanoparticle,the size,zeta electric potential,envelopment rate and carry drug rate of DNA-NP prepared were about 120nm,6.6my, 79.4%and 32.3%respectively.Which also had very good protection to gene.Selected the DNA-NP prepared in accordance with the ratio to continue gene transfection experiment,the persistence time was longer than both of liposome and bare gene,the inbition rate after 72h of transfection was reinforced significantly(p＜0.05 and p＜0.01 respectively).So the next step we selected it to do research of gene therapy. The forth part is research of nanoparticle vector mediated RNAi to therapy colon cancer.At first HT-29 cell was cultivated,then the DNA-NP was transfected into it,after 72h detected inbition rate of Livin mRNA and protein by RT-PCR and western blot,detected apoptosis by flow cytometry and fluorescence microscope to inspect gene therapy efficacy in vitro.Established naked mouse colon carcinoma animal model, injected DNA-NP into tumor multiple,drew the growth curve of tumor,took out the tumor to detect volume inhibition ratio and weight inhibition ratio after 24 d,detected apoptotic index(AI)by histological section and TUNEL dyeing to inspect gene therapy role in animal cancer model.Results indicated that the inhibition ratio of Livin mRNA and protein of test group were about 78 and 76 percent respectively,which increased remarkably than spurious therapy groups(p＜0.01).In test group tumor grew slow remarkably,the mean volume inhibition ratio and weight inhibition ratio were 74 and 80 percent,which also increased significantly than contral groups(p＜0.01),apoptosis cell was significantly more than contral groups in tissue section and AI was increased(p＜0.01).Which indicated that the CS-PEG nanoparticle vector mediated Livin shRNA has very good therapeutic efficacy to colon cancer which Livin express is high.The present research is just a part of National "863" High-technology Project,and supply a primary experiment basis for colorectal carcinoma gene diagnosis and gene therapy new strategy.The exploitation potentiality and clinical application will have more profound research.