miR-103 Promotes Proliferation and Metastasis by Targeting KLF4 in Gastric Cancer

Abstract

MicroRNAs (miRNAs) play important roles in the cancer development and progression; overexpression of miR-103 has been identified in various tumors. However, its biological function and regulatory mechanism involved in modulation of human gastric cancer (GC) remain largely unknown. This study aimed to confirm clinical significance of miR-103 and investigate its biological role and underlying mechanism in GC. Real-time quantitative PCR (qRT-PCR) revealed miR-103 was highly expressed in GC tissues and cell lines. miR-103 expression was correlated closely with tumor size, Lauren's classification, and lymph node metastasis. Importantly, Kaplan-Meier analysis revealed that high expression of miR-103 was significantly associated with poor overall survival and disease-free survival of GC patients. Downregulation of miR-103 by transfecting with miR-103 inhibitor significantly suppressed cell proliferation, induced apoptosis, inhibited migration and invasion in vitro and in vivo. Furthermore, miRNA target databases and luciferase reporter assay confirmed that Krüppel-like Factor-4 (KLF4) was a direct target of miR-103 in GC, and there was a significant inverse correlation between miR-103 and KLF4 expression in GC tissues. Moreover, KLF4 downregulation could rescue miR-103's oncogenic effect on GC cell proliferation, apoptosis, migration, and invasion. Therefore, these results suggested that miR-103 overexpression could contribute to tumor progression by suppressing KLF4, and it might serve as a promising candidate for the prognosis of GC patients.

Keywords: KLF4; gastric cancer; metastasis; microRNA-103; proliferation.

Figure 1

miR-103 is overexpressed in gastric cancer primary tumor tissues and cell lines and correlates with patient survival. (A) The miR-103 expression in 92 cases of primary GC tissues compared with 20 cases of adjacent nontumorous tissues analyzed by RT-qPCR; (B) The miR-103 expression in primary GC tissues with lymph node metastasis compared with those without lymph node metastasis; (C) miR-103 expression in gastric cancer cell lines; (D) miR-103 expression was positively associated with tumor size by Spearman’s correlation test (n = 92) and (E) The correlation between miR-103 expression and GC patient survival. * p < 0.05; the data represent the mean ± standard deviation (SD) from triplicate measurements.

Figure 2

miR-103 promotes cell proliferation and reduced apoptosis of GC cells. (A) Analyses of miR-103 expression after transfection in SGC7901 and BGC823 cells by real-time PCR; (B,C) Influence of miR-103 downregulation on cell proliferation of SGC7901 and BGC823 cells by CCK-8 (B) and EdU assays (C,D) Influence of miR-103 knockdown on cell apoptosis. * p < 0.05, Scale bar = 100 μm for (C); the data represent the mean ± SD from triplicate measurements.

Figure 3

miR-103 promotes cell migration, invasion and mesenchymal-epithelial transformation (EMT) of GC cells. (A) Effect of miR-103 knockdown on cell migration and invasion ability in SGC7901 and BGC823 cells, Scale bar = 50 μm; (B) E-cadherin and vimentin expression in SGC7901 and BGC823 cells by western blot and (C) epithelial-to-mesenchymal transition (EMT)–associated genes expression in SGC7901 and BGC823 cells by RT-qPCR. * p < 0.05; the data represent the mean ± SD from triplicate measurements.

Figure 4

miR-103 promotes gastric cancer cell growth and metastasis in vivo. (A) Tumor volumes were measured on the indicated days; (B) The number of lung metastasis of indicated SCID mice groups and (C) RT-qPCR analysis of miR-103 expression in implanted tumors. * p < 0.05; the data represent the mean ± SD from triplicate measurements.

Figure 5

KLF4 is a direct target of miR-103 and KLF4 expression is inversely correlated with miR-103 expression in GC tissues. (A) Western blot analysis of KLF4 protein expression after transfection in SGC7901 and BGC823 cells; (B) Luciferase activities of wild-type and the mutant pmirGLO-KLF4-3′-UTR reporter in SGC7901 and BGC823 cells; (C) The predicted miR-103 binding site on the KLF4 mRNA 3′-UTR and the corresponding mutations in 3′-UTR of KLF4; (D,E) KLF4 expression on protein level (D) and mRNA level (E) was determined in GC tissues (T) and adjacent nontumorous tissues (ANT); (F) Spearman’s correlation analysis was performed to detect the association between the expression level of miR-103 and KLF4 in GC tissues; (G,H) KLF4 expression on protein level (G) and mRNA level (H) was determined in three GC cell lines (BGC823, SGC7901 and MKN45) and nontumorous mucosa. Error bars represent mean ± SD from three independent experiments. * p < 0.05.

Figure 6

Downregulation of KLF4 rescues miR-103’s oncogenic effect on GC cell proliferation, apoptosis, migration, and invasion in SGC7901cells. (A) KLF4 protein expression was detected in SGC7901 cells co-transfected with miR-103 inhibitor/miR-NC and KLF4 siRNA or si-NC; (B–E) Cell proliferation, apoptosis, migration and invasion were assessed in SGC7901 cells co-transfected with miR-103 inhibitor/miR-NC and KLF4 siRNA or si-NC. * p < 0.05; the data represent the mean ± SD from triplicate measurements.