Anoctamin 5 regulates the cell cycle and affects prognosis in gastric cancer

Figure 1 Anoctamin 5 expression in gastric cancer cells. A: Real-time quantitative reverse transcription-quantitative polymerase chain reaction (PCR) showed the expression of anoctamin 5 (ANO5) in various cell lines in gastric cancer; B: Western blotting showed the expression of ANO5 in various cell lines in GC; C: Real-time quantitative PCR revealed that ANO5 small interfering RNA (siRNA) effectively reduced ANO5 mRNA levels in NUGC4 and MKN45 cells; D: Western blotting revealed that ANO5 siRNA effectively reduced ANO5 protein levels in NUGC4 and MKN45 cells. n = 3, mean ± standard error of the mean. ^aP < 0.05 (significantly different from control siRNA). ANO5: Anoctamin 5; ACTB: β-actin; siRNA: Small interfering RNA.

Figure 2 Anoctamin 5 controlled the proliferation and the cell cycle in gastric cancer cells. A: The downregulation of anoctamin 5 (ANO5) inhibited the proliferation of NUGC4 and MKN45 cells. Cell numbers were counted 48 h and 72 h after small interfering RNA transfection (left panel). The proliferative ability of NUGC4 and MKN45 cells was significantly suppressed following ANO5 downregulation (right panel); B: The downregulation of ANO5 increased the number of cells in the G₀/G₁ phase in NUGC4 and MKN45 cells. Cells transfected with control or ANO5 small interfering RNA were stained with propidium iodide and analyzed by flow cytometry. n = 3, mean ± standard error of the mean. ^aP < 0.05 (significantly different from control small interfering RNA). ANO5: Anoctamin 5; siRNA: Small interfering RNA; Cont.: Control.

Figure 3 Anoctamin 5 controlled apoptosis, migration, and invasion in gastric cancer cells. A: The downregulation of anoctamin 5 (ANO5) increased the early and late apoptotic cell proportions of NUGC4 and MKN45 cells. Control or ANO5 small interfering RNA-transfected cells were stained with propidium iodide and annexin V and subjected to flow cytometry; B: The downregulation of ANO5 significantly decreased NUGC4 and MKN45 cell migration and invasion, which were evaluated using a Boyden chamber assay. Magnification: × 40. n = 3, mean ± standard error of the mean. ^aP < 0.05 (significantly different from control small interfering RNA). ANO5: Anoctamin 5; PI: Propidium iodide; siRNA: Small interfering RNA; Cont.: Control.

Figure 4 Network analyses by microarray analysis and ingenuity pathway analysis. A: The signaling map of “Cell Cycle: G₁/S Checkpoint Regulation,” one of the top-ranked canonical pathways related to the depletion of anoctamin 5 (ANO5) according to ingenuity pathway analysis. Red and green colors indicate genes with expression levels that were higher or lower, respectively, than reference RNA levels; B: To verify gene expression profiling data, CDK2, CDK4, CDK6, CDKN1A, CCNE2, and E2F1 were examined by real-time reverse transcription-quantitative polymerase chain reaction. The downregulation of ANO5 effectively reduced CDK2, CDK4, CDK6, CCNE2, and E2F1 mRNA levels and increased CDKN1A/p21 mRNA levels in NUGC4 and MKN45 cells; C: The downregulation of ANO5 effectively reduced the phosphorylation levels of Rb protein in NUGC4 and MKN45 cells; D: The downregulation of ANO5 increased the phosphorylation of the c-Jun N-terminal kinase protein in NUGC4 and MKN45 cells. n = 3, mean ± standard error of the mean. ^aP < 0.05 (significantly different from control small interfering RNA). ANO5: Anoctamin 5; siRNA: Small interfering RNA; Cont.: Control; JNK: c-Jun N-terminal kinase; ACTB: β-actin.

Figure 5 Immunofluorescent analysis of intracellular chloride concentration using MQAE reagent, a chloride-sensitive fluorescence probe. The downregulation of anoctamin 5 increased the fluorescence intensity of MQAE in NUGC4 and MKN45 cells. A: NUGC4 cell; B: MKN45 cell. n = 3, mean ± standard error of the mean. ^aP < 0.05 (significantly different from control small interfering RNA). ANO5: Anoctamin 5; siRNA: Small interfering RNA; Cont.: Control.

Figure 6 Anoctamin 5 protein expression levels in human gastric cancer tissues and a survival analysis based on anoctamin 5 expression. A: Non-cancerous gastric epithelia were immunohistochemically stained using an anti-anoctamin 5 (ANO5) antibody. Magnification: × 100; B: Primary human gastric cancer samples were immunohistochemically stained using an anti-ANO5 antibody. Magnification: × 100; C: The immunohistochemical staining results of ANO5 are shown as intensity 0. Magnification: × 400; D: Intensity 1. Magnification: × 400; E: Intensity 2. Magnification: × 400; F: Intensity 3. Magnification: × 400; G: Gastric cancer patients were classified into two groups based on ANO5 expression: A low ANO5 expression group (< 1.3, n = 137) and high ANO5 expression group (≥ 1.3, n = 58). ^aP < 0.05 (significant difference). ANO5: Anoctamin 5.