miR-1 suppresses the growth of esophageal squamous cell carcinoma in vivo and in vitro through the downregulation of MET, cyclin D1 and CDK4 expression

Abstract

Several aberrant microRNAs (miRNAs or miRs) have been implicated in esophageal cancer (EC), which is widely prevalent in China. However, their role in EC tumorigenesis has not yet been fully elucidated. In the present study, we determined that miR‑1 was downregulated in esophageal squamous cell carcinoma (ESCC) tissues compared with adjacent non-neoplastic tissues using RT-qPCR, and confirmed this using an ESCC cell line. Using a nude mouse xenograft model, we confirmed that the re-expression of miR‑1 significantly inhibited ESCC tumor growth. A tetrazolium assay and a trypan blue exclusion assay revealed that miR‑1 suppressed ESCC cell proliferation and increased apoptosis, whereas the silencing of miR‑1 promoted cell proliferation and decreased apoptosis, suggesting that miR‑1 is a novel tumor suppressor. To elucidate the molecular mechanisms of action of miR‑1 in ESCC, we investigated putative targets using bioinformatics tools. MET, cyclin D1 and cyclin-dependent kinase 4 (CDK4), which are involved in the hepatocyte growth factor (HGF)/MET signaling pathway, were found to be targets of miR‑1. miR‑1 expression inversely correlated with MET, cyclin D1 and CDK4 expression in ESCC cells. miR‑1 directly targeted MET, cyclin D1 and CDK4, suppressing ESCC cell growth. The newly identified miR‑1/MET/cyclin D1/CDK4 axis provides new insight into the molecular mechanisms of ESCC pathogenesis and indicates a novel strategy for the diagnosis and treatment of ESCC.

Figure 1

miR-1 expression in 34 paired esophageal squamous cell carcinoma (ESCC) tissues and other tumor cells. (A) miR-1 levels were determined in 34 surgical specimens of ESCC tissues and were normalized to those in corresponding adjacent non-neoplastic esophageal tissue specimens. ^*P<0.05. (B) RT-qPCR detection of miR-1 expression in Het-1A (normal esophageal), KYSE-150 (ESCC), QBC939 (cholangiocarcinomca), AGS (gastric adenocarcinoma) and HepG2 (hepaqtocellular carcinoma) cell lines. ^*P<0.05, KYSE-150 vs. Het-1A cells.

Figure 2

Effect of miR-1 overexpression on the growth of esophageal squamous cell carcinoma (ESCC) tumor xenografts. (A) Tumor growth curves after intratumor injection of miR-1 mimics, mimics negative control (mimics-NC), transfection reagent (Entranster) or cisplatin. (B) Tumor weights of the 4 experimental groups when the mice were sacrificed. (C) Mice carrying ESCC tumors following sacrifice on day 21. ^*P<0.05, ^**P<0.05.

Figure 3

The expression of miR-1 in transfected KYSE-150 cells. (A) KYSE-150 cells were transiently transfected with miR-1 mimics (100 nM), (B) miR-1 inhibitor (100 nM), or the respective negative controls. Transfection efficiency was determined by RT-qPCR. Data were normalized to U6 expression. Data are represented as the means ± SEM from 3 independent experiments (^*P<0.05).

Figure 4

Effect of miR-1 overexpression/downregulation on cell proliferation. (A) MTT assay following transfection with miR-1 mimics or mimics-negative control (NC). ^*P<0.05, miR-1 mimics vs. mimics-NC (B) MTT assay following transfection with miR-1 inhibitor and inhibitor-NC. ^*P<0.05, miR-1 inhibitor vs. inhibitor-NC. (C) Living cell counts following transfection with miR-1 mimics or mimics-NC. (D) Living cell counts following transfection with miR-1 inhibitor or inhibitor-NC. Data are represented as the means ± SEM ± SD. ^*P<0.05, ^**P<0.01.

Figure 5

Effect of miR-1 overexpression/downregulation on apoptosis. (A and B) Flow cytometric analysis of apoptosis following transfection with miR-1 mimics, inhibitor and their respective NCs. (C) Rate of apoptosis was determined following transfection with miR-1 mimics, inhibitor and their respective NCs. ^*P<0.05.

Figure 6

miR-1 targets 3′-UTR of MET, cyclin D1 and cyclin-dependent kinase 4 (CDK4). (A) Conservation of the predicted miR-1 binding site in MET/cyclin D1/CDK4 3′-UTR across species. (B) Bioinformatics analysis of the interaction between miR-1 and MET, cyclin D1 and CDK4 3′-UTR binding sites. (C) Effect of miR-1 overexpression/downregulation on MET, cyclin D1 and CDK4 expression by western blot analysis, with GAPDH as the internal control. Proteins were quantified by densitometric analysis, which was performed using Quantity One software (Bio-Rad Laboratories). ^*P<0.05, ^#P<0.05.

Figure 7

Immunohistochemical analysis of MET, cyclin D1 and cyclin-dependent kinase 4 (CDK4) expression patterns in esophageal squamous cell carcinoma (ESCC). (A) Representative images of immunohistochemical staining for MET, cyclin D1 and CDK4 in human ESCC tissues and adjacent non-neoplastic tissues (original magnification, ×100). (B) The expression levels of MET, cyclin D1 and CDK4 in ESCC tissues did not directly correlate with the miR-1 expression levels.

Figure 8

Luciferase activity assay with MET, cyclin D1 and CDK4 3′-UTR construct co-transfected into 293T cells. Schematic representation of luciferase activity assay of MET, cyclin D1 and CDK4 3′-UTR construct or control luciferase construct co-transfected with miR-1, anti–miR-1 or negative controls. ^*P<0.05, ^**P<0.01.