To evaluate anti-tumor effects of recombinant adenovirus p53, time-course p53, E6 expression, and cell growth inhibition were investigated in vitro and in vivo using cervical cancer cell lines such as CaSki, SiHa, HeLa, HeLaS3, C33A, and HT3. The cell growth inhibition was studied via cell count assay, MTT assay and neutral red assay. After transfecting AdCMVp53 into SiHa cells-xenografted nude mice, the transduction efficiency and anti-tumor effect were investigated for a month. The results showed that adenoviral p53 expression induced significant growth suppression on the cancer cells, in which E6 transcript was strongly repressed, and that the expression of p53 and E6 were remarkably dependent on each cell type. The transduction efficiency was highly maintained in vivo as well as in vitro, and the size of tumor was remarkably decreased in comparison with AdCMVLacZ control. The results suggest that the adenovirus-mediated p53 gene transfection was done very effectively in vitro and in vivo experiment, and the cell growth was suppressed via p53-dependent apoptotic cell death, and that the anti-tumor effect could be related to E6 and p53 expression pattern.

Background MicroRNA has been also shown to mediate regulation of embryonic stem cell (ESC) division, as well as differentiation in adipocyte, cardiac, neural, and hematopoietic lineages. Compared with these examples examining microRNAs in ESC, relatively few studies have identified microRNA expression patterns in differentiating MSC or specific pathways that could be targeted. Objectives In this study, we tried microRNA microarray analysis with total RNAs from PTHrP treated BMSCs. Four novel microRNAs were detected by microarray analysis, we conformed that one of them can increase the chondrogenic potential of BMSCs in the lentiviral transduction with the miRNA. Methods We performed experiments as follows for this study: miRNA microarray, miRNA overexpression in hBMSCs, Chondrogenic induction of lentiviral transduced hBMSCs, 3′UTR reporter assay, Western blotting, Realtime PCR analysis Results Four novel microRNAs were detected by microarray analysis, we confirmed that one of them can increase the chondrogenic potential of MSCs. In chondrogenic induction of PTHrP-treated hBMSCs, 9 novel miRNAs were up- and down-regulated. 4 miRNAs of them were up-regulated in PTHrP treatment. miR-7, 8 and 9 expression patterns were gradually increased for 21 days. In case of miR-im6, expression pattern was increased by 7 days and then decreased at 21 days in spite of long-term treatment of PTHrP. Each up-regulated miRNA was subcloned into lenti-viral expression vector and successfully overexpressed in hBMSCs by lentiviral infection at MOI=10. Amount of GAG/DNA measured in cultured lentiviral transduced pellets after 4 weeks of culture. GAG/DNA content of lenti-miR-im6 transduced hBMSCs was higher than other groups treated with TGF-β. From safranin-O staining of each chondrogenic hMSC pellet, staining intensity of lenti-miR-im6 transduced hBMSCs was also stronger than TGF-β-treated positive control, similar to PTHrP-treated positive control. Overexpression of miR-im6 in chondrogenic induction of hMSCs enhanced the expressions of chondrogenic markers (Type II collagen, SOX9) and suppressed the expression of hypertrophic markers (ALP, Type X collagen). Desert hedgehog (DHH), Wnt6, and Wnt9b were screened as target genes to miR-im6 in this pathway. PTCH2, WNT6 and WNT9B of these proteins also were down-regulated in lenti-miR-im6- transduced and chondrogenesis-induced hMSCs pellets. Luciferase activities were significantly decreased in Hela cells which were transformed with luciferase reporter vectors harboring PTCH1 or WNT6 3’UTR. This result demonstrates that PTCH1 and WNT6 are target genes of has-miR-im6. We are currently studying about another target gene (KLF10) of miR-im6, can be key regulator in novel PTHrP mediated chondrocyte hypertrophy blocking pathway. Conclusions Our results show that miR-im6 acts as a positive regulator of chondrogenic differentiation as well as a hypertrophy blocker in BMSCs by decreasing the ALP expression through unknown mechanism. To our knowledge, this is the first report showing that a specific...