MiR-145 expression accelerates esophageal adenocarcinoma progression by enhancing cell invasion and anoikis resistance

Abstract

Background: Carcinoma of the esophagus has a high case fatality ratio and is now the 6th most common cause of cancer deaths in the world. We previously conducted a study to profile the expression of miRNAs in esophageal adenocarcinoma (EAC) pre and post induction therapy. Of the miRNAs differentially expressed post induction chemoradiation, miR-145, a known tumor suppressor miRNA, was upregulated 8-fold following induction therapy, however, its expression was associated with shorter disease-free survival. This unexpected result was explored in this current study.

Methods: In order to study the role of miR-145 in EAC, miRNA-145 was overexpressed in 3 EAC cell lines (OE33, FLO-1, SK-GT-4) and one ESCC cell line (KYSE-410). After validation of the expression of miR-145, hallmarks of cancer such as cell proliferation, resistance to chemotherapy drugs or anoikis, and cell invasion were analyzed.

Results: There were no differences in cell proliferation and 5 FU resistance between miR145 cell lines and the control cell lines. miR-145 expression also had no effect on cisplatin resistance in two of three cell lines (OE33 and FLO-1), but miR-145 appeared to protect SK-GT-4 cells against cisplatin treatment. However, there was a significant difference in cell invasion, cell adhesion and resistance to anoikis. All three EAC miR-145 cell lines invaded more than their respective controls. Similarly, OE33 and SK-GT-4 miR-145 cell lines were able to survive longer in a suspension state.

Discussion: While expression of miR-145 in ESCC stopped proliferation and invasion, expression of miR-145 in EAC cells enhanced invasion and anoikis resistance. Although more work is required to understand how miR-145 conveys these effects, expression of miR-145 appears to promote EAC progression by enhancing invasion and protection against anoikis, which could in turn facilitate distant metastasis.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. MiR-145 inhibited cell proliferation, delayed wound closure and enhanced anoikis in ESCC cells.

(A) Cell proliferation and (B) wound healing assay of KYSE-410 pcmv and KYSE-410 miR-145 cells. miR-145 expression in KYSE-410 led to decreased numbers of colonies after cell suspension culture (C) and enhanced PARP and caspase 3 cleavage (D).*: p

Figure 2. MiR-145 expression did not affect EAC cells proliferation.

Cell proliferation assay of the pcmv and miR-145 EAC cell lines, n = 3

Figure 3. MiR-145 enhanced cell adhesion to fibronectin but not vitronectin.

OE33 (A), SK-GT-4 (B) and FLO-1 (C) were plated onto plates pre-treated with either fibronectin or vitronectin. After 30 mins, the detached cells were counted. *: p

Figure 4. MiR-145 accelerated wound closure and enhanced cell invasion.

Wound healing assay (A) with pcmv and miR-145 cell lines. The wound length was assessed after 8 h culture. *: p

Figure 5. MiR-145 enhanced the clonogenic potential of OE33 and SK-GT-4 after suspension culture.

Photo images of clonogenic assay after 72 h suspension culture with OE33 and SK-GT-4 (A). Results showed the average percentage of colonies formed after suspension culture compare to the number of colonies formed in monolayer (B). *: p

Figure 6. MiR-145 protected EAC cells against anoikis.

pcmv and miR-145 EAC cell lines were cultured in suspension for 72 h. The levels of cleaved PARP and cleaved caspase 3 were assessed by Western Blotting.

Figure 7. MiR-145 did not generally affect the EAC resistance to chemotherapy drugs.

OE33, FLO-1 and SK-GT-4 (pcmv and miR-145) cells were cultured for 72 h with either (A) cisplatin (5 µM) or (B) 5-fluorouracil (35 µM) and their respective control. Live cell number was then assessed. Results show the percentage of live cells compared to the control treatment. *: p