miR-1 suppresses the proliferation and promotes the apoptosis of esophageal carcinoma cells by targeting Src

Abstract

Nonreceptor tyrosine kinase c-Src, also known as Src, is a potent oncogene involved in a series of biological processes including cell growth, differentiation, and apoptosis; however, its expression pattern and function in esophageal cancer is poorly addressed. In this study, abnormal overexpression of Src protein was observed in esophageal cancer tissues, which fuelled the speculation that microRNA-mediated posttranscriptional regulatory mechanism might be involved. Bioinformatic analyses were applied to identify miRNAs that could potentially target Src. miR-1 was predicted and further validated as a direct repressor of Src. Moreover, we manipulated knockdown and overexpression experiment on TE-1 and TE-10 cells to demonstrate miR-1 suppressed proliferation and promoted apoptosis in esophageal cancer cells by inhibiting Src. Taken together, this study underlines a negative regulatory mechanism in which miR-1 serves as a suppressor of Src in esophageal cancer cells and may provide insights into novel therapeutic approaches for esophageal cancer.

Keywords: Src; apoptosis; esophageal cancer; miR-1; proliferation.

Figure 1

Expression patterns of Src in esophageal cancer tissues. (A) Quantification of Src mRNA expression in 183 human esophageal cancer tissues and 12 esophageal noncancerous tissues. (B) Quantification of Src mRNA expression of esophageal cancer tissues classified by clinical stages. (C and D) Src protein expression in 11 pairs of human esophageal cancer tissues and esophageal noncancerous tissues. GAPDH was used as a loading control. (C) Representative image; (D) Quantitative analysis. (E) Relative Src protein expression of esophageal cancer tissues classified by clinical stages.

Figure 2

Prediction of miR‐1 binding site within Src 3′‐UTR. (A) Heat map of dramatically altered miRNAs from 72 esophageal cancer samples and nine normal esophagus. (B) Schematic description of the conjectural duplex formed by miR‐1 (bottom) and its binding site (top) within Src 3′‐UTR. The seed region of miR‐1 and the seed recognition site within Src 3′‐UTR are indicated in red and blue, respectively. All nucleotides in the seed recognition site were completely conserved among species. The predicted free energy value of the duplex is indicated. (C) Expression levels of miR‐1 in the same 11 pairs of CE and NE tissues. (D) Pearson's correlation scatter plot of the fold change of miR‐1 and Src protein in human esophageal cancer tissues.

Figure 3

Src is a direct target of miR‐1. (A) Expression levels of miR‐1 in TE‐1/TE‐10 cells transfected with equal doses of the miR‐1 mimic (pre‐miR‐1) or scrambled negative control RNA (pre‐miR‐control). (B and C) Src protein expression in TE‐1/TE‐10 cells transfected with equal doses of the miR‐1 mimic or scrambled negative control RNA. GAPDH was used as a loading control. (B) Representative image; (C) Quantitative analysis. (D) Src mRNA levels in TE‐1/TE‐10 cells transfected with equal doses of the miR‐1 mimic or scrambled negative control RNA. (E) Relative luciferase activity of wild‐type (WT) and mutant (MUT) Src 3′‐UTR p‐MIR‐reporter vectors in TE‐1 cells transfected with the control mimic or miR‐1 mimic. Firefly luciferase values were normalized to β‐galactosidase activity.

Figure 4

Roles of miR‐1 and Src in proliferation and apoptosis of esophageal cancer cells. (A) Growth curves of TE‐1 cells transfected with equal doses of the miR‐1 mimics or Src siRNA or scrambled control RNA. (B) Growth curves of TE‐1 cells transfected with the control mimic plus control vector or miR‐1 mimic plus control vector or miR‐1 mimic plus Src overexpression vector (Src vector) or control mimic plus Src overexpression vector. (C) Representative images of ratio of apoptotic TE‐1 cells transfected with equal doses of the miR‐1 mimics or Src siRNA or scrambled control RNA. (D) Representative images of ratio of apoptotic TE‐1 cells transfected with the control mimic plus control vector or miR‐1 mimic plus control vector or miR‐1 mimic plus Src overexpression vector or control mimic plus Src overexpression vector. (E) Quantitative analysis of the flow cytometry analysis of (C and D). (F) Model of miR‐1 enhances apoptosis and suppresses proliferation by targeting Src to restrain tumor growth in esophageal cancer.