| Colorectal cancer(CRC)is the third most commonly diagnosed cancer and the third leading cause of cancer death worldwide.Despite the rapid advancement in diagnosis and therapy for CRC,conventional treatment such as surgery,radiation and medical therapy could not provide sufficient effect.Furthermore,50%of treated patient would suffer cancer relapse and metastasis.It's urgently desirable to find an alternative way for the treatment of colorectal cancerP21,a multi-functional cyclin-dependent kinases(CDK)inhibitor(CDKI),is a downstream protein of P53.It plays an important role in regulating the proliferation,apoptosis,differentiation,metastasis and stem-ness of cancer cells.The losses of both expression and topological regulation of p21 is commonly detected in colorectal cancer Several observations implicated that cell cycle arrest and apoptosis of colon tumor cells could be stimulated by the induction of p21.Positive p21 expression has been suggested as an indicator for good prognosis in patients with colorectal cancer.Therefore,activation of p21 is a promising way to suppress colorectal cancer.Therefore,activation of p21 might be a promising way to suppress colorectal cancer.According to that,an abundance of efforts has been made to find p21 targeting reagents for the treatment of colorectal cancer,as well as elucidate their molecular mechanisms.However,this attempt was hindered by numerous obstacles.The P21 is a downstream protein of p53,the most frequently mutated cancer suppressor gene in human cancers.Liu et al carried out a comprehensive analysis of the p53 gene and its protein status on a panel of 56 colorectal cancer cell lines.The results showed a relatively high frequency of P53 mutations(76.8%,43in 56)in studied cell lines,with almost half of the mutations being truncating mutations.Thus,in the treatment of colorectal cancer,it is vitally important to find a direct and reliable way for p21 gene inspiration.Small active RNA(saRNAs)is a group of small double strand RNA,which could effectively activate particular gene in a sequence specific manner.It has been demonstrated that the gene-regulatory effect triggered by saRNA was profounder and longer than that caused by siRNA since saRNA could induce epigenetic change which was probably inheritable.SaRNA mediated RNA activation might provide us a new and powerful weapon for the fight against cancer,through inspiring cancer suppressor genes.Since Li et al.synthesized special sequenced saRNA:dsRNA-p21,research has been focused on its tumor suppressing activity and demonstrated that up-regulating p21 via dsRNA-p21 led to inhabitation of cell growth in human cancer cell lines including bladder,prostate,lung,liver,kidney and glioma,in vitro and in vivo.It appears that colorectal cancer could be a potential target for the dsRNA-p21,as p21 down-regulation is a signature abnormality of the cancer and in rectal application might be a suitable approach fordsRNA-p21 delivery.Part.1.Specific up-regulation of p21 by a small active RNA sequence suppresses human colorectal cancer growth.Objective:Study the tumor suppressor gene p21WAF1/CIP1 activation ability of dsRNA-p21 on colorectal cancer and investigate whether this RNA activation process is dependent on transcriptional factor p53 expression.Methods:Three human colorectal cancer cell lines,HCT-116,HCT-116(p53-/-)and HT-29 were transfected with the dsRNA-p21.The expression of P21 protein and p21 mRNA were measured using the Western-blotting and reverse transcriptase polymerase chain reaction(RT-PCR).The effect of dsRNA-p21 on cancer cells was evaluated in vitro;and furthermore,a xenograft colorectal tumor mode in mice was established to estimate the tumor suppressing ability of dsRNA-p21 in vivo.Results:In all three colorectal cancer cell lines,the expression of p21 mRNA and P21 protein were dramatically elevated after dsRNA-p21 transfection.Transfection of p21-saRNA-322 caused apoptosis and cell cycle arrest at the G0/G1.Furthermore,anti-proliferation effect,reduction of colonies formation and cell senescence were observed in dsRNA-p21 treated cells.Animal studies showed that dsRNA-p21 treatment significantly inhibited the HT-29 tumor growth and facilitated p21 activation in vivo.Conclusion:dsRNA-p21-induceded up-regulation of p21 might be a promising therapeutic option for the treatment of colorectal cancer.In the first part,we proved that the sequence specific double strand RNA:dsRNA-p21 could successfully hinder colorectal cancer growth via stimulating p21 gene expression.However,the difficulty of delivering these therapeutic molecules limited their further clinical application.Although amount of efforts has been performed,systemic delivery of nucleic acid faces complicated challenge on conveying sufficient drug molecules to the tissue of interest.A logical way to translate RNA induced gene activation(RNAa)into clinical use is local therapies,in which saRNA is directly delivered to its site of action,ensuring sufficient drug dose reach the target tissue,lessening the potential for off-target side effects,circumventing the substantial systemic delivery barriers and overcoming the bio drug's immunity response.Li et al.indicated that saRNA possessed anti-proliferation activity in bladder cancer via intra-vesical treatment on orthotopic mouse model,which has been considered the most successful exploration of RNA-induced gene activation(RNAa)therapy until now.Thus,we believe in situ application is an applicable approach to delivery saRNA for the treatment of colorectal cancer.Hence,we studied the saRNA in-situ delivery system in the second part.Part.2.Development of tumor-selective and redox sensitive dsRNA-p21 delivery system.Objective:To optimize in-situ dsRNA-p21 delivery system for the treatment of colorectal cancer.Methods:A series of disulfide cross-linked PEI derivatives based on PEI600 were synthesized.After being systematically investigated the cytotoxicity,the optimal disulfide substitute PEI derivation(PEI-SS7)was screened for producing dsRNA-p21 binary complexes.Hyaluronic acid(HA)was introduced to shield the dsRNA-p21 binary complexes to produce supramolecular assemblies.The physicochemical properties,in vitro dsRNA-p21 delivery efficiency,cytotoxicity,intracellular trafficking and mechanism,as well as physiological effect were investigated in colorectal cancer HCT-116p53(-/-)cells.Results:The disulfide cross-linked PEI derivatives with the disulfide substitute of 7 could condense dsRNA-p21 to form a solid sphere.The binary complexes could effectively release dsRNA-p21 via the broken of disulfide under intracellular redox condition.The dsRNA-p21 supramolecular assemblies showed high compatibility and stability in physiological conditions due to the anionic shielding.The dsRNA-p21 supramolecular assemblies had high transfection efficiency in the CD44 abundant HCT-116p53(-/-)cells.In vitro physiological experiment showed that,comparing to the control group,the dsRNA-p21 supramolecular assemblies effectively activated the expression of p21 mRNA and P21 protein,causing blockage of cell cycle at G0/G1 phase and suppression of cancer cell proliferation as well as colony formation.Furthermore,in vivo distribution experiment demonstrated that the dsRNA-p21 supramolecular assemblies could effectively accumulate at rectal wall for up to 10h,following in situ application.Conclusion:These findings indicated that dsRNA-p21 supramolecular assemblies might hold great potential for delivering dsRNA-p21 to treat colorectal cancer.The development of therapeutic strategies for treating colorectal carcinoma requires biologically relevant and adequate animal models.In the third part,we have developed an orthotopic transplant model of human cancer to nude mice utilizing microsurgical techniques.Part.3.The development of orthotopic transplant model of Luciferase expressed human colorectal cancer to nude mice.Objective:To develop orthotopic transplant model of Luciferase expressed human colorectal cancer to nude mice.Methods:HCT-116p53(-/-)cell lines were infected by pseudo-lentivirus with Luciferase.The expressions of Luciferase of the infected cells were observed by fluorescence microscopy.HCT-116p53(-/-)cells clones expressing Luciferase were isolated with cloning cylinders by trypsin/EDTA,then they were transferred and amplified by conventional culture methods.Multiple cell biological characteristics,such as cell morphological,proliferation,invasion abilities and the growth rate of tumors by subcutaneous implantation ere compared between the infected and uninfected cells.The HCT-116p53(-/-)-Luc cells were accurately injected subserosally into the descending colon utilizing microsurgical techniques.The subsequent growth,invasion,metastasis of the transplanted tumors was observed and evaluated with whole-body bioluminescence optical imaging system.Results:HCT-116p53(-/-)cell lines could be infected efficiently by pseudo-lentivirus.We obtained a stable Luciferase cell lines,named HCT-116p53(-/-)-Luc.180 days after consecutive in vitro cell passages,HCT-116p53(-/-)-Luc cell lines expressed Luciferase,stably and intensely.There were no significant differences between HCT-116p53(-/-)-Luc cells and HCT-116p53(-/-)cells in their cell biological characteristics.The growth curves of tumors by subcutaneous implantation were similar.The existence of Luciferase in tumor cells did not interfere on the tumorigenicity.The tumors were detected with bioluminescence in all mice on day 7 after tumor implanted.HCT-116p53(-/-)-Luc cells maintained stably high-level Luciferase expression during their growth in vivo.The resulting transplanted human tumors grow locally in a clinical-like pattern.However,there has been no definitive evidence in colon cancer that the human tumors metastasize in nude mice.Conclusion:he Luciferase tagged colorectal carcinoma models in BALB/c nude mice are superior for the detection and studying relevant patterns of colorectal carcinoma in vivo.These rat models are suitable for the studies about interventional therapy.In the fourth part,we have evaluated the tumor suppressing effect of HA/PEI-SS/dsRNA-p21 in-situ delivery systems on orthotopic nude mice model.Part.4.Tumor suppressing effect of the dsRNA-p21 supramolecular assembly in-situ delivery systems in vivo.Objective:Evaluation of the tumor suppressing effect dsRNA-p21 supramolecular assembly in-situ delivery systems in vivo.Methods:dsRNA-p21 entrapping dsRNA-p21 supramolecular assembly was administered into the rectum 1 cm above the anus using a gavage needle every three days for 5 weeks.Bioluminescence signals and body weight were monitored weekly and death was recorded.Five weeks after treatment,all surviving mice were subsequently euthanized and descending and rectal parts were inspected for cancer.Colorectal tumor tissue was placed in 10%neutral buffered formalin for HE analysis and immune histochemistry(IHC)for P21.RT-PCR and Western-blotting method were used to test the p21 expression in tumor tissues.Results:The dsRNA-p21 supramolecular assembly demonstrated a superior antitumor efficacy and safety in vivo.Conclusion:In situ exposure to dsRNA-p21 supramolecular assembly inhibits orthotopic colorectal tumors growth. |
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