| Objective To explore effect and mechanism of chitosan(CS) as gene carriers of transforming growth factor-β1(TGF-β1) in colorectal cancer(CRC).Methods(1)Transfected mice colon cancer cell line CT26 with different TGF-β1-si RNA, selected the best sclinced TGF-β1-si RNA by western blot, used as a follow-up study.(2) Cross-linked sodium tripolyphosphate(TPP) and CS in ion-gel method into a blank nanoparticles, explored the impact on physical and chemical with changes of properties formulated ingredients.(3) Prepared a certain particle size of nanoparticles suspension by binding the TGF-β1-si RNA and CS / TPP, intravenous injecte mice, verified its liver targeting and investigated the liver targeting influence TPP modification to the CS, to select the best condition of preparation of liver targeted nanoparticles.(4) Dissolved CS in different PH acetic acid solutions to explore the encapsulation efficiency of si RNA; Then dissolved CS / TPP / si RNA nanoparticles again in different PH solutions to explore the release rate of si RNA by changing the PH.(5) Explored the effect on liver metastasis of CRC and its mechanism by injecting the CS / TPP / si RNA nanoparticle suspension into the tail vein of mice CRC liver metastasis models.(6) Transient transfected cell line CT26 with the TGF-β1si RNA, analyzed the expression profiles of TGF-β1-related mi RNAs. Then screened the mi RNAs raised multiple> 2 times with microarray. mi R-493-5p significantly diffidently expressed in cell lines silenced by TGF-β1 si RNA and metastases tissues injected with CS / TPP / TGF-β1 si RNA nanoparticles by q RT-PCR.(7) Transient transfected cell line CT26 with selected mi RNA(mi R-493-5p)- mature body(mimics, M) in vitro, assessed the cell growth inhibition by MTT, assaied migration by Transwell migration assay respectively.(8) The CT26 cells were inoculated into nude mice for in vivo experiments, intratumoral injection with CS/TPP/M, preliminary estimate of the impact of mi R-493-5p on tumorigenicity by measuring the volume of the subcutaneous tumor. 9 Predict mi R-493-5p target genes by bioinformatics technology and network resources western blotting was conducted to validate target gene of mi R-493-5p.Results(1) TGF-β1-mus-1998 si RNA gene was silenced best in CT26 cells.(2) Malvern Zetasizer results showed that major with changes of concentration of CS, the particle size and zeta potential of CS / TPP nanoparticles changed.(3) 250 nm particle diameter CS / TPP nanoparticles had good liver targeting, and TPP modification can increase this performance of the nanoparticles.(4) PH sensitivity test results showed that the highest rate of release of nanoparticles was measured in PH6.8, which was the PH of tumor microenvironment.(5)There was a better suppression in the mouse models of liver metastasis received CS / TPP / si RNA tail vein injection than simply CS / TPP and si RNA.(6) mi RNA microarray results show six mi RNA(mi R-10a-3p, mi RNA-871-3p, mi R-678, mi R-493-5p*, mi R-M1-7-3p and mi R-1983) overexpress in TGF-β1-si RNA treated CT26 cell line; and mi R-493-5p and mi R-1983 were verified by detecting TGF-β1-silenced cell lines and tissues by q RT-PCR.(7) M significantly increased the expression of mi R-493-5p; Cell proliferation and migration declined in CT-26 cell lines transfected by M.(8) Tumor volumes of the mice injected with CS/TPP/M declined.(9) mi R-493-5p candidate target genes follow: SPRK2(Serine/arginine protein-specific kinase 2) Pumilio2(pumilio RNA-binding family member 2) FUBP1(far upstream element binding protein 1)and Pescadillo1, Srpk2 and Pumilio2 are the direct target genes of mi R-493-5p.Conclusions(1) Formulate nanoparticles of different size and zeta potential by changing the concentration of CS, of which the nanoparticles with the size of 250 nm modified with TPP expresse good liver targeting.(2) The nanoparticles release rate in the PH of the solution near the tumor microenvironment PH was significantly increased, which indicates the potential tumor targeting.(3) CRC liver metastases model display that CS/TPP/TGF-β1 si RNA nanoparticles systerm can inhibit tumorigenicity of CT26 cells in vivo.(4) TGF-β1 related mi R-493-5p has been selected as a follow-up study of tumor suppressor genes by mi RNA chips and q RT-PCR.(5) In vitro, mi R-493-5p M significantly increases the expression of mi R-493-5p, and inhibites activity and migration of CT26 cells.(6) In vivo, CS / TPP / M can also inhibit the tumorigenic of CT26 cells.(7) Srpk2 and Pumilio2 are the direct target genes of mi R-493-5p. |
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