MicroRNA-485-5p targets keratin17 to regulate pancreatic cancer cell proliferation and invasion via the FAK / SRC / ERK pathway

Abstract

Background: It is crucial to probe into the biological effect and mechanism of miRNA-485-5p regulating keratin 17 (KRT17) in pancreatic cancer (PC) to understand its pathogenesis and identify potential biological targets. Methods: The bioinformatics means were used to evaluate the clinical significance of KRT17 expression in the Cancer Genome Atlas (TCGA) database. TargetScan database analysis in conjunction with dual luciferase and RNA Immunoprecipitation (RIP) experiments was used to probe the interaction relationship of miRNA-485-5p with KRT17. The expression of miRNA-485-5p and KRT17 in PC tissue and cancer cell lines was detected by Q-PCR paired with western blot assay. The biological function of miRNA-485-5p in regulating KRT17 was investigated in the PC cell line via gene silencing/overexpression technique. A western blot experiment was utilized to investigate the regulatory effect of KRT17 on cell cycle-related proteins and the FAK/Src/ERK signal pathway. Results: The level of KRT17 was increased in PC tissues and this significantly decreased the survival rate of PC patients. TargetScan in combination with dual luciferase and RIP experiments verified the miRNA-485-5p target KRT17. The expression of KRT17 was high in the PC cell line, although the expression of miRNA-485-5p was low. Silencing KRT17 or overexpression of miRNA-485-5p significantly inhibited PC cell viability, proliferation, invasion, and colony formation, while promoting apoptosis. Overexpression of KRT17 drastically reversed the function of miRNA-485-5p. The silenced KRT17 remarkably downregulated the expression of cyclinD1, Cyclin Dependent Kinase 1 (CDK1), CDK2, Phospho-Focal Adhesion Kinase (p-FAK), p-Src, and p-ERK proteins in the PC cells. Conclusion: Generally, an essential signaling cascade of miRNA-485-5p/KRT17/FAK/Src/ERK influences the biological functions of PC cells.

Keywords: Cell cycle; KRT17; MiRNA-485-5p; Pancreatic cancer; Proliferation.

Figure 1

Expression level and Clinical significance of KRT17 in Pancreatic Cancer. A The expression of KRT17 in the TCGA database. B Analyses of the Overall Survival of KRT17 expression. C Analyses of the Disease-Free Survival of KRT17 expression. D Detection of KRT17 expression in Pancreatic Cancer and healthy human pancreas by Q-PCR. E Immunoblots of KRT17 and β-actin in cells. F The statistical plot of KRT17 protein expression. ^*P < 0.05 vs. Normal or H6c7.

Figure 2

Silencing efficiency of KRT17-siRNAs in Pancreatic Cancer (PC) Cell Line and its effect on cell viability. A The immunoblots of KRT17 and β-actin in PANC-1 cell. B The statistical plot of KRT17 protein content. C The immunoblots of KRT17 and β-actin in SW1990 cell. D The statistical plot of KRT17 protein content. E The influence of silencing KRT17 on PANC-1 cell viability. F The influence of silencing KRT17 on SW1990 cell viability. ^*P < 0.05 vs. si-NC.

Figure 3

Effects of inhibition of KRT17 gene expression on invasion and apoptosis of cells. A The influence of silencing KRT17 on apoptosis rates of PC cells detected by flow cytometry experiment. B Statistical chart of apoptosis rate. C The effect of silencing KRT17 gene expression on the invasion of PC cells was observed by the Transwell experiment. D The statistical plot of the invasion number of cells. ^*P < 0.05 vs. si-NC.

Figure 4

Influence of silencing KRT17 on the proliferation and colony formation ability of cells. A The EdU experiment was applied to detect the influence of silencing KRT17 on the proliferation ability in the cells. B The statistics plot of cell-associated proliferation rates. C The influence of silencing KRT17 on the clone-forming ability of cells was using the cloning experiment. D The statistical chart of the clone-formation ability of cells. ^*P < 0.05 vs. si-NC.

Figure 5

Analysis of the correlation between KRT17 and cell cycle regulatory molecules. A Scatter plots display the correlation between krt17 and CyclinD1 (CCND1) expression. B Scatter plots demonstrate the correlation between krt17 and CDK1 expression. C Scatter plots exhibit the correlation between krt17 and CDK2 expression. D The immunoblots of cyclinD1, CDK1, CDK2, and β-actin in PANC-1 by the western blot. E The statistical plot of cyclinD1, CDK1, and CDK2 protein expression levels in PANC-1; F The immunoblots of cyclinD1, CDK1, CDK2, and β-actin in SW1990 by the western blot. G The statistical plot of cyclinD1, CDK1, and CDK2 protein expression levels in SW1990. ^*P < 0.05 vs. si-NC.

Figure 6

Effect of silencing KRT17 on FAK / SRC / ERK pathway. A Stand for the immunoblots of p-FAK, FAK, p-Src, Src, p-ERK, ERK, and β-actin in PANC-1 by the western blot. B The statistical plot of p-FAK, p-Src, and p-ERK protein expression levels in PANC-1. C The immunoblots of p-FAK, FAK, p-Src, Src, p-ERK, ERK, and β-actin in SW1990 by the western blot. D The statistical plot of p-FAK, p-Src, and p-ERK protein expression levels in SW1990. ^*P < 0.05 vs. si-NC.

Figure 7

Validation of miR-485-5p association with KRT17. A The binding site of KRT17 was targeted by miR-485-5p in the TargetScan database. B The Q-PCR validation of miR-485-5p expression. C The correlation between miR-485-5p and KRT17 was detected by the anti-Ago2 RIP experiment. D and E A double luciferase reporter test proved the interaction between miR-485-5p and KRT17. F The transfection efficiency of miRNA-485-5p was determined by Q-PCR. G The influence of miRNA-485-5p on KRT17 levels was tested by Q-PCR. ^*P < 0.05 vs. the Normal, miR-NC, anti-NC, or mimic-NC. ^#P < 0.05 vs. miR-485-5p mimic+oe-NC.

Figure 8

Influence of KRT17 regulation by miRNA-485-5p on the level of apoptosis and invasion of cells. A The regulatory apoptosis effect of miRNA-485-5p on KRT17 expression was studied by flow cytometry. B The statistical plot of apoptosis rates of cells. C Effect of KRT17 regulation by miRNA-485-5p on cell invasion. D The statistical plot of the invasion number of cells. ^*P < 0.05 vs. mimic-NC+oe-NC. ^#P < 0.05 vs. miRNA-485-5p mimic+oe-NC.

Figure 9

Regulation of KRT17 by miRNA-485-5p impairs cell proliferation and clonogenic ability in cells. A EdU experiments were performed to test the effect of KRT17 regulation by miRNA-485-5p on the proliferative capacity of cells. B The statistics plot of cell-associated proliferation rates for cells. C The cloning experiments were performed to test the effect of krt17 regulation by miRNA-485-5p on the clonogenic ability of cells. D The statistical chart of the clone formation ability of cells. ^*P < 0.05 vs. mimic-NC+oe-NC. ^#P < 0.05 vs. miRNA-485-5p mimic+oe-NC.