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    2021-03-03


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    (A) The first 400 differentially expressed genes of pancreatic cancer gene ITF2357 (Givinostat) chips (GEO: GSE16515, GSE32676, GSE71989, and GSE101448), with one inter-section gene ADAM9 observed. (B and C) Thermal map of the first 60 genes of the gene expression chips GEO: GSE101448 (B) and GSE32676 (C), respectively. The abscissa was representative of the sample number, whereas the ordinate was reflective of the differentially expressed genes. The upper right histogram is the color order, and each rectangle in the graph corresponds to a sample expression value. (D and E) The expression of ADAM9 in (D) GEO: GSE16515 and (E) GSE71989 gene chips, respectively. (F) The expression of ADAM9 in pancreatic cancer tissues and normal tissues retrieved from the GEPIA database. *p < 0.01. (G) The correlation between ADAM9 expression and total survival rate in patients with pancreatic cancer in the GEPIA database. (H) The comparison of target miRNA of ADAM9 predicted by TargetScan, miRSearch, miRTarBase, miRWalk, and mirDIP. (I) The expression of hsa-miR-126-3p in miRNA expression chip GEO: GSE28955 of pancreatic cancer. ADAM9, a disintegrin and a metalloproteinase-9; GEPIA, Gene Expression Profiling Interactive Analysis; miR-126, microRNA-126.
    Molecular Therapy: Nucleic Acids
    A
    Relative miR-126-3p expression 
    Pancreatitis tissues
    Pancreatic cancer tissues
    B
    Relative miR-126-3p expression 
    MIAPaCa-2
    Capan-1
    Figure 2. Pancreatic Cancer Tissues Exhibited Reduced Expression of miR-126-3p
    human normal pancreatic cell line HPC-Y5 and six pancreatic cancer cell lines detected by qRT-PCR (*p < 0.05, compared with HPC-Y5). The measurement data were expressed as mean ± SD. The data are between two groups compared with the independent sample t test. Data among multiple groups were compared using one-way ANOVA. The experiment was repeated three times. miR-126-3p, microRNA-126-3p.
    ADAM9 by binding to the predicted target site in the 30 UTR, a dual-luciferase reporter gene assay was performed (Figure 4B). Compared with the NC group, overexpression of miR-126-3p inhibited the luciferase activity of 30 UTR of ADAM9-wild-type (WT) (p < 0.05). However, miR-126-3p was found to have no significant effect on the luciferase activity of 30 UTR ADAM9 mutant (MUT) (p > 0.05), indicating that ADAM9 is a target of miR-126-3p.
    qRT-PCR and western blot analysis were then employed to determine the mRNA and protein expression of ADAM9 (Figures 4C and 4D). The expression of ADAM9 gene was decreased when miR-126-3p was overexpressed, while increased levels were noted in the presence of in-hibited miR-126-3p (p < 0.05). Accordingly, ADAM9 was the target of miR-126-3p, whereas overexpressed miR-126-3p was observed to downregulate ADAM9 expression.
    miR-126-3p Inhibits Proliferation, Migration, and Invasion of Pancreatic Cancer Cells and Promotes Their Apoptosis by Negatively Regulating ADAM9
    ADAM9 was interfered with in order to investigate the molecular mechanisms that underlie the miR-126-3p-mediated inhibition on proliferation, apoptosis, migration, and invasion abilities of pancre-atic cancer cells. The results demonstrated (Figures 5A–5D) that compared with the RNA interference of NC (si-NC) group, the cell proliferation, migration, and invasion ability of the si-ADAM9 group were decreased, whereas the apoptotic rate was boosted (p < 0.05). In contrast, compared with the miR-126-3p inhibitor + si-NC group, the proliferation, migration, and invasion ability were reduced, whereas the rate of apoptosis was diminished in the miR-126-3p inhibitor + si-ADAM9 group (p < 0.05). The results of RT-PCR and western blot analysis indicated that the mRNA and protein expression of ADAM9 were decreased (p < 0.05) when compared with the corresponding NC group (Figures 5E and 5F). Taken together, miR-126-3p was ultimately confirmed to inhibit the proliferation, migration, as well as the invasion of pancreatic cancer cells while pro-moting their apoptosis by negatively regulating ADAM9.
    BMSCs Sorting
    After the subculture of BMSCs, the aging cells and a small number of mixed cells were eliminated in a progressive fashion. After three to 
    four generations, relatively homogeneous and vigorous purified cells in shuttle shape along with a swirling arrangement were observed (Figures 6A and 6B). The third-generation cells exhibiting good growth were incubated with fluorescein isothiocyanate (FITC)-labeled mouse anti-human antibodies (CD29, CD34, CD44, CD45, CD71, and histocompatibility leukocyte antigen [HLA]-DR) for iden-tification of surface antigen using flow cytometry. Generally, CD29, CD44, and CD71 are considered to be markers of BMSC expression,25 CD34 and CD45 are regarded as hematopoietic stem cell markers,26 whereas HLA-DR is predominately expressed in certain antigen-pre-senting cells, such as B lymphocyte, macrophage, and activated T lymphocyte.27 CD29 (97.60%), CD44 (98.90%), and CD71 (99%) were found to be positive, whereas HLA-DR (6.11%), CD34 (3.12%), and CD45 (2.41%) were all negative (Figure 6C), supporting that the cultured cells were BMSCs. When cell confluence reached 100%, the BMSCs were cultured with specific culture medium for adi-pogenic differentiation purposes. Next, 72 h postculture, small lipid droplets were observed in the cells. After culturing for an additional 2 h, a large amount of lipid droplets appeared in the cells in addition to the appearance of a long spindle or polygonal shape. The oil red O staining provided verification indicating that lipid composition was deposited, highlighting the BMSC ability of lipid differentiation (Fig-ure 6D). Following the addition of a specific culture inducing liquid, the BMSCs were differentiated into osteoblasts. After 3 days of induc-tion culture, the cells were in short spindle shape with increased vol-ume. On the seventh day of culture, the cells were observed to have a polygonal shape with calcium granules in the cytoplasm. On the 14th day of culture, the whole cells were filled with calcium granules, and the cells grew in a colony-like manner. The cells at the center were observed to gradually fuse in addition to the loss of their typical cell structure with the formation of clear calcium nodules. The cells were stained red using alizarin red staining and stained black using Von Kossa staining (Figure 6E).