Study On The Antitumor Efficacy And Mechanism Of Up-regulation Of E-cadherin By RNA Activation

Part 1 The design and screening of small activating RNAAim To screen the relatively efficient candidate small activating RNAs (saRNAs) molecules that can activate the target tumor-suppressor genes including p12, p27 and p57.Materials and methods Based on the design rules provided by the foreign literature, we designed 2-3 pairs of small double-stranded RNA (dsRNA) for each target gene, and the control dsRNA is non-homologous to the whole human genome. The candidate dsRNAs were transfected into 5637 or T24 bladder cancer cell lines and PC3 or DU145 prostate cancer cell lines. The expression of target gene was determined with RT-PCR measuring the mRNA levels. The functional saRNA which defined as to increase target gene expression twice was selected.Results All the candidate dsRNAs did not activate the target gene significantly and fulfill the standard of functional saRNAs.Conclusion It is difficult to screen out the functional saRNAs and the design rules of saRNA should be revised. Part 2 The antitumor effects of E-cadherin activation by saRNAAim To evaluate the antitumor effects of confirmed functional saRNA (dsEcad-215) on 5637 bladder cancer cells and PC3 prostate cancer cells.Materials and methods dsEcad-215 which is complementary to the E-cadherin promoter was transfected into 5637 and PC3 cells for 72 h. The mRNA and protein expression levels of E-cadherin were determined with RT-PCR and Western blotting. The invasive and migrative ability of tumor cells were measured using Transwell chamber and scratch test.Results The expression of E-cadherin increased after transfection of dsEcad-215, suggesting that the RNAa was effective. The invasive ability to extracellular matrix and migration capacity of 5637 and PC3 cells decreased significantly.Conclusion Induction of E-cadherin expression by saRNA leads to suppression of migration and invasion of 5637 and PC3 cells in vitro.The RNAa could be used as an effective tool of gene therapy against cancer.Part 3 The mechanism of up-regulation of E-cadherin by saRNAAim To explore the molecular mechanisms of up-regulation of E-cadherin by RNA activation.Materials and methods The E-cadherin promoter-associated non-coding sense or antisense RNA was detected with gene-specific RT-PCR. The histone modification status of E-cadherin promoter region after dsEcad-215 transfection was determined with chromatin immunoprecipitation. Using RT-PCR assay, the OAS1 and OAS3 expression which involved in interferon pathway were measured.Results Both promoter-associated non-coding sense and antisense RNAs were detected, and transfection of dsEcad-215 caused no change of expression. Chromatin immunoprecipitation demonstrated that loss of histone 3 dimethylation at lysine-9 and lysine-4 were associated with gene activation. dsEcad-215 did not induced the expression of OAS1 or OAS3.Conclusion Transfection with dsEcad-215 in 5637 cells did not affect the non-coding sense and antisense RNA expression levels. dsEcad-215 may bind to the non-coding RNAs, and changed its spatial structure and function thus affecting the modification of histone methylation, such as loss of H3K9 and H3K4 dimethylation levels, resulting in transcription activation. dsEcad-215 did not induced the interferon pathways.Part 4 The mechanism of inhibition of cell invasion and migration by E-cadherin activationAim To investigate the molecular mechanisms of inhibition of cell invasion and migration by E-cadherin activation.Materials and methods The relocalization ofβ-catenin was determined by measuring its expression in cell membrane, cytoplasm and nuclei in PC3 cells. The immunofluorescence assay was used to validate the re-distribution ofβ-catenin. The mRNA expression levels of mmp7, and cyclin D1 were measured using RT-PCR.Results After transfection with dsEcad-215 for 72 h, there was a decrease in cytoplasmic and nuclearβ-catenin concentration, and an increase ofβ-catenin concentration in membrane fraction in PC3 cells treated with dsEcad-215 compared with control and mock groups, whereas totalβ-catenin in the cell homogenate was not affected. The immunofluorescence data further demonstrated that dsEcad-215 induced plasma membrane relocation ofβ-catenin. mmp7 and cyclinDl were downregulated by 72 h of exposure to 50 nM dsEcad-215 compared with control and mock groups.Conclusion Our study showed that upregulation of E-cadherin by saRNA caused the relocalization ofβ-catenin from cytoplasm and nucleus to the plasma membrane in PC3 cells, and then suppressed the transactivation of downstreamβ-catenin/TCF target genes, thus inhibited cell invasion and migration.