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    • br To further predict the miRNA regulating ADAM

    2020-08-18


    To further predict the miRNA regulating ADAM9, five miRNA-mRNA relation prediction databases (TargetScan, miRSearch, miRTarBase, miRWalk, and mirDIP) were used to predict the target miRNA, after which the results obtained were compared with the 
    predicted results. The detailed results are displayed in Table S2. In accordance with the Venn diagram (Figure 1H), three intersection miRNAs, hsa-miR-26b-5p, hsa-miR-126-3p, and hsa-miR-373-3p, were found by comparison of five miRNA prediction results, indi-cating that these three miRNAs were highly likely to regulate ADAM9. Furthermore, through screening of the miRNA LDN193189 chip GEO: GSE28955 of pancreatic cancer by R, among the three aforementioned miRNAs that targeted ADAM9, only hsa-miR-126-3p was poorly expressed in pancreatic cancer tissues (Figure 1I), suggesting that miR-126 could target ADAM9 in pancreatic cancer. The specific expression data of ADAM9 and hsa-miR-126 are de-picted in Table S3.
    miR-126-3p Is Poorly Expressed in Pancreatic Cancer Cells
    In order to ascertain whether the expression of miR-126-3p is altered in pancreatic cancer cells, its expression in 28 patients with pancreatic cancer, as well as among 32 patients with pancreatitis, was determined through the application of qRT-PCR. The expression of miR-126-3p in the pancreatic cancer tissues was markedly lower than the results of the patients with pancreatitis (Figure 2A), suggesting that miR-126-3p is downregulated in pancreatic cancer cells. In addition, qRT-PCR was employed to detect the expression of miR-126-3p in the normal human pancreatic cell line HPC-Y5, with six pancreatic can-cer cell lines (PANC-1, SW1990, Capan-1, AsPC-1, PC-3, and MIAPaCa-2 cell lines) screened in order to detect the cell line with the poorest expression of miR-126-3p. The results obtained distinctly indicated that the expression of miR-126-3p was decreased among all the pancreatic cancer cell lines when compared with the normal pancreatic cell line HPC-Y5 (Figure 2B), with the expression of miR-126-3p in PANC-1 found to be the lowest (p < 0.05) among all tested pancreatic cancer cell lines. Thus, PANC-1 was selected for subsequent cell experiments.
    miR-126-3p Inhibits Proliferation, Migration, and Invasion of Pancreatic Cancer Cells and Promotes Their Apoptosis
    In order to examine the influence of miR-126-3p on pancreatic cancer cells, PANC-1 was treated with a mimic and inhibitor of miR-126-3p. The results (Figures 3A–3D) revealed that when compared with the mimic-negative control (NC) group, the cell proliferation, migration, and invasion abilities were decreased, whereas the apoptotic rate was elevated in the miR-126-3p mimic group (p < 0.05). In contrast, when compared with the inhibitor-NC group, the miR-126-3p inhibitor group exhibited increased cell proliferation, migration, and invasion abilities along with a diminished rate of apoptosis (p < 0.05). Restored miR-126-3p was associated with inhibited proliferation, migration, and invasion of pancreatic cancer cells in addition to stimulated rates of apoptosis, whereas a contradictory trend was observed when miR-126-3p was silenced and opposite outcomes to the above mentioned were exhibited.
    Based on the online software analysis, miR-126-3p was detected to have a specific binding region for ADAM9 within its 30 UTR (Fig-ure 4A). In order to ascertain as to whether miR-126-3p regulates
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    A
    B
    Expression 8
    normal tumor
    G Overall Survival 1.0
    Low ADAM9 TPM
    High ADAM9 TPM
    Months
    type 14 type C
    type 14 type
    EPO
    normal
    normal
    AQP12B PTX3
    C12orf39 tumor TWIST2 12 tumor
    GSTA2
    KDM6B
    GRPR
    NFATC1
    ACADL BRE−AS1
    SELE
    SLC5A9 SERPINB5
    KRT7
    KIRREL2
    YIF1B
    PEX5L FGD6 6
    KAAG1
    FBXO22OS CYB5D1
    ARHGDIG
    GUCA1C TMPRSS4
    GLS2 FXYD3
    DNAH12L
    ABHD17C
    BRSK2
    MLPH
    AMY2B
    SFN
    MYLK2 MSLN
    ADAM9 GJB2
    OSBPL10
    NUCB2
    TUFT1
    ANPEP
    CGN
    CUZD1
    SDC1
    ALB MTMR11
    PNLIPRP2 BAIAP2L1
    ITGA2
    KLK1 TSPAN1
    CTRL
    LAMC2
    PDIA2 AHNAK2
    SYCN
    CST1
    AQP8 ANO1
    SERPINI2
    ADAM9
    SPACA3
    RBPJL SOX4
    AQP12A RTP4
    LIPH
    TEX11 FERMT1
    NRG4
    PVRL4
    TRHDE
    TMPRSS3
    PHYHIPL
    PTK6
    AMHR2
    CEACAM5
    FGL1
    MYC
    MT1H
    MAL2
    TMED6
    SLPI
    CCKBR
    CELA2B
    NET1
    PTF1A
    AGR2
    GNMT RAB25