MiR-196a Promotes Pancreatic Cancer Progression by Targeting Nuclear Factor Kappa-B-Inhibitor Alpha

<div><p>Aberrant expression of miR-196a has been frequently reported in different cancers including pancreatic cancer. However, its function in pancreatic cancer has not been fully elucidated. Here, we investigated the expression pattern and the biological role of miR-196a in pancreatic cancer cell lines, as well as its interaction with a metastasis-related gene, nuclear factor-kappa-B-inhibitor alpha (NFKBIA). We demonstrated that miR-196a was up-regulated in human pancreatic cancer cell lines compared with immortalized pancreatic ductal epithelial cells by means of microRNAs microarray and qRT-PCR. Furthermore, down-regulation of miR-196a in PANC-1 suppressed its proliferation and migration with an increase in G₀/G₁ transition and decreased expression of Cyclin D1 and CDK4/6. Meanwhile, an increased expression in E-cadherin and decreased expression in N-cadherin and Vimentin were also observed. We identified a novel miR-196a target, NFKBIA, and down-regulation of miR-196a enhanced the expression of NFKBIA protein. Luciferase assay confirmed that NFKBIA was a direct and specific target of miR-196a. Silencing NFKBIA in PANC-1 cells enhanced its proliferation and migration. Taken together, our findings indicate that miR-196a is highly expressed in pancreatic cancer cell lines, and may play a crucial role in pancreatic cancer proliferation and migration, possibly through its downstream target, NFKBIA. Thus, miR-196a may serve as a potential therapeutic target for pancreatic cancer.</p></div

Downregulation of miR-196a inhibited proliferation of pancreatic cancer cell line PANC-1.

<p>(A) Transfection of anti-miR-196a and anti-miR-NC in PANC-1. (B) Comparision of transfection rate between anti-miR-196a and anti-miR-NC in PANC-1. The transfection rate of anti-miR-196a was (88.76±2.25)%, while the transfection rate of anti-miR-NC was (91.09±1.77)% (<i>P</i>>0.05). (C) Expression of miR-196a by qRT-PCR. (D) Growth curve among anti-miR-196a, anti-miR-NC and parental PANC-1 cells. Cell proliferation was significantly reduced in anti-miR-196a group compared with that of anti-miR-NC group at 72 h (*, <i>P<0.05</i>). <i>LM</i>: light microscope.</p

MiR-196a directly targets NFKBIA gene.

<p>(A) NFKBIA is a potential target gene of miR-196a predicted by computational analysis. (B) Representative western blot analysis showed the relationship between miR-196a expression and endogenous NFKBIA protein level. The inhibition of miR-196a in PANC-1 cells increased endogenous NFKBIA protein level, whereas overexpression of miR-196a in BxPC-3 cells attenuated endogenous NFKBIA protein level. (C) Insertion of NFKBIA3′UTR target sequences in a luciferase reporter vector leaded to diminished luciferase activity in presence of miR-196a in PANC-1 cells 24 h after co-transfection. Histograms showed the values resulting as the average ± SD from three independent co-transfections (*, <i>P</i><0.05).</p

Effect of NFKBIA on PANC-1 cell proliferation and migration.

<p>(A) Growth curve among si-NFKBIA, si-NFKBIA+anti-miR-196a and their appropriate controls. WST-8 assay showed inhibition of NFKBIA enhanced PANC-1 cells proliferation (*, <i>P</i><0.05). Meanwhile, dual inhibition of NFKBIA and miR-196a promoted cells proliferation. (B) Representative western blot analysis showed up-regulation of Cyclin D1 and CDK4/6 expression after suppression of NFKBIA in PANC-1 cells at 72 h. (C) Representative transwell assay indicated that the migration ability of PANC-1 cells was markedly attenuated by down-regulation of NFKBIA. Moreover, inhibition of NFKBIA blocked the effect of anti-miR-196a on cell migration. (D) Comparison of transmembrane cells among si-NFKBIA, si-NC, si-NFKBIA+anti-miR-196a, and si-NC+anti-miR-NC (*, <i>P</i><0.05).</p

Downregulation of miR-196a suppressed PANC-1 cell migration.

<p>(A) Representative transwell assay indicated that the migration ability of PANC-1 cells was markedly reduced by down-regulation of miR-196a. (B) Comparison of transmembrane cells among anti-miR-196a, anti-miR-NC and Blank (*, <i>P</i><0.05). (C) Morphological changes and immunofluorescence staining of MET markers among anti-miR-196a, anti-miR-NC and blank. (D) Representative western blot analysis revealed that mesenchymal-epithelial transition contributed to suppression of PANC-1 cell migration after silencing miR-196a. After silencing miR-196a, E-cadherin expression increased, as well as expression of N-cadherin and Vimentin decreased.</p

MiR-196a was overexpressed in pancreatic cancer cell lines.

<p>(A) Hierarchical clustering analysis of miRNAs that were either differentially up- or downregulated in pancreatic cancer cell lines and H6C7. MiRNAs that scored a differential value of 1 or greater were categorized as differentially upregulated, and miRNAs that scored a value of −1 or less were categorized as differentially downregulated. The scale bar across the bottom of the heatmap depicts SD change from the mean. MiR-196a expression was significantly higher in pancreatic cancer cell lines in microarray. (B) Validation of miR-196a expression level in pancreatic cancer cell lines by qRT-PCR. MiR-196a was significantly upregulated in pancreatic cancer cell lines. Expression of miR-196a was (706.4±9.4)-fold in PANC-1 cells, (310.1±7.5)-fold in SW1990 cells, (7.6±1.1)-fold in BxPC-3 cells and (204.9±4.8)-fold in Capan-2 cells, compared with H6C7 (*, <i>P</i><0.05).</p

VAMP724 and VAMP726 are involved in autophagosome formation in <i>Arabidopsis thaliana</i>

Macroautophagy/autophagy, an evolutionarily conserved degradative process essential for cell homeostasis and development in eukaryotes, involves autophagosome formation and fusion with a lysosome/vacuole. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play important roles in regulating autophagy in mammals and yeast, but relatively little is known about SNARE function in plant autophagy. Here we identified and characterized two Arabidopsis SNAREs, AT4G15780/VAMP724 and AT1G04760/VAMP726, involved in plant autophagy. Phenotypic analysis showed that mutants of VAMP724 and VAMP726 are sensitive to nutrient-starved conditions. Live-cell imaging on mutants of VAMP724 and VAMP726 expressing YFP-ATG8e showed the formation of abnormal autophagic structures outside the vacuoles and compromised autophagic flux. Further immunogold transmission electron microscopy and electron tomography (ET) analysis demonstrated a direct connection between the tubular autophagic structures and the endoplasmic reticulum (ER) in vamp724-1 vamp726-1 double mutants. Further transient co-expression, co-immunoprecipitation and double immunogold TEM analysis showed that ATG9 (autophagy related 9) interacts and colocalizes with VAMP724 and VAMP726 in ATG9-positive vesicles during autophagosome formation. Taken together, VAMP724 and VAMP726 regulate autophagosome formation likely working together with ATG9 in Arabidopsis.</p