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  • Meropenem br Fig A and B Knockdown of the CLDN


    (Fig. 4A and B). Knockdown of the CLDN7 gene in the MIA PaCa-2-A cells significantly suppressed proliferation (Fig. 4C). The morphology of the MIA PaCa-2-A cells in which CLDN7 was knocked down did not differ significantly and was almost the same as that of the control siRNA-transected MIA PaCa-2-A cells (Fig. 4D). The expression of p-Erk1/2 in the MIA PaCa-2-A cells was sup-pressed by transfection with CLDN7 siRNA (Fig. 4E). The activation status of NF-kB in the MIA PaCa-2-A cells was not markedly affected by transfection with CLDN7 siRNA (Fig. 4E).
    CLDN7 knockdown induces G1 Meropenem arrest
    The cell cycle distribution of the CLDN7-specific siRNA-transfected MIA PaCa-2-A cells was examined by flow cytometry. After 48 h serum starvation, a higher frequency of cells at the G1 phase and a lower frequency of cells at the G2 phase were observed in the MIA PaCa-2-A cells transfected with CLDN7-specific siRNA (Fig. 5C) than in those untreated or transfected with control siRNA (Fig. 5A and B). Following incubation for 24 h, the frequency of cells in the G1 phase increased in the CLDN7 siRNA-transfected cells (Fig. 5F) compared with the untreated or control siRNA-transfected cells (Fig. 5D and E). These results indicate that G1 arrest and the resultant G1/S block were induced by the knockdown of CLDN7.
    In this study, we isolated morphologically different sub-populations derived from single MIA PaCa-2 cells, namely MIA PaCa-2-A cells with an epithelial morphology and MIA PaCa-2-R 
    cells with a non-epithelial Meropenem morphology. The MIA PaCa-2-A and MIA PaCa-2-R cells had the same DNA STR pattern as the parental MIA PaCa-2 cells, indicating that they were not contaminants derived from other cell lines. Apart from our isolation, Walsh et al. [39] described that several subpopulations of cells with different morphologies were isolated from MIA PaCa-2 cells. Each subpop-ulation exhibited different capacities for invasion, adhesion, anoikis and anchorage-independent growth, and these functional differ-ences were associated with the types of integrins expressed by each subpopulation [39]. According to the results of the in vivo and in vitro proliferation studies and gemcitabine sensitivity, the MIA PaCa-2-A and MIA PaCa-2-R cells represent populations of the MIA PaCa-2 cells with high and low malignant phenotypes, respectively.
    DNA microarray analysis demonstrated that CLDN7 was highly expressed in the MIA PaCa-2-A cells, but not in the MIA PaCa-2-R cells. The CLDN family has 27 members and shows specific distri-bution. Proteins of the CLDN family are important components of tight junctions regulating paracellular permeability, cell polarity and barrier function permanence [21,22]. CLDN7 has been reported as one of the impermeable CLDNs and interacts directly with epithelial cell adhesion molecule (EpCAM) to strengthen the junction of epithelial cells [40]. CLDN-associated cell-to-cell adhe-sion is an important factor for the proliferation, transformation and metastasis of tumor cells [23]. It is conceivable that the MIA PaCa-2-A cells showing tight epithelial feature express high levels of CLDN7 and that the MIA PaCa-2-R cells showing round shape and loosely attached features do not.
    It has been reported that the expression of CLDN family proteins is deregulated and is closely associated with malignant phenotypes
    Fig. 3. Different gene expression profiles between the MIA PaCa-2-A and MIA PaCa-2-R cells. (A) A scatter plot of the gene expression of the MIA PaCa-2-A and MIA PaCa-2-R cells by DNA microarray analysis. The vertical axis indicates the intensity of gene expression in the MIA PaCa-2-R cells, while the horizontal axis indicates the intensity of gene expression in the MIA PaCa-2-A cells. (B) mRNA expression levels of CLDN7 in the MIA PaCa-2-A and MIA PaCa-2-R cells as determined by RT-PCR analysis; *P < 0.01. (C) Western blot analysis of CLDN7 protein expression in the MIA PaCa-2, MIA PaCa-2-A, MIA PaCa-2-R and other human pancreatic cancer cell lines. MCF7 breast cancer cells were used as positive controls for CLDN7 expression.