MicroRNA-873 inhibits the proliferation and invasion of endometrial cancer cells by directly targeting hepatoma-derived growth factor

Abstract

An accumulation of evidence has demonstrated that abnormal microRNA (miRNA or miR) expression is associated with different types of cancer, including endometrial cancer (EC). The dysregulation of miRNAs may serve important roles in the development and progression of EC by regulating multiple aggressive biological behaviors, including cell proliferation, apoptosis, metastasis and angiogenesis. An in-depth understanding of the miRNAs associated with EC initiation and progression may be crucial for identifying successful therapeutic techniques. miR-873 has been demonstrated to be dysregulated in different types of cancer. However, the expression status and regulatory roles of miR-873 are yet to be elucidated in EC. In the present study, reverse transcription-quantitative PCR was carried out to detect miR-873 expression in EC tissues and cell lines. Cell Counting Kit-8 and in vitro invasion assays were utilized to determine the influence of miR-873 on the proliferation and invasion of EC cells. miR-873 expression was revealed to be downregulated in EC tissues and cell lines. Decreased miR-873 expression was significantly associated with International Federation of Gynecology and Obstetrics stage and lymph node metastasis of patients with EC. Functional assays revealed that resumed miR-873 expression suppressed the proliferation and invasion of EC cells. Additionally, hepatoma-derived growth factor (HDGF) was indicated to be a direct target gene of miR-873 in EC cells. HDGF was highly expressed in EC tissues and inversely correlated with miR-873 expression. HDGF silencing also imitated the tumor-suppressor activity of miR-873 overexpression in EC cells. A series of rescue experiments identified that recovered HDGF expression hindered the anti-proliferative and anti-invasive roles of miR-873 upregulation in EC cells. In conclusion, the present study indicated that miR-873 serves an important role as a tumor suppressor in EC development by directly targeting HDGF. The results may provide a novel insight into clinical treatments, which can be used to prevent EC aggression.

Keywords: endometrial cancer; hepatoma-derived growth factor; invasion; microRNA-873; proliferation.

Figure 1.

miR-873 is downregulated in EC tissue and cell lines. Reverse transcription-quantitative PCR analysis of miR-873 expression in (A) 47 pairs of EC tissue and adjacent normal endometrial tissues, and (B) four human EC cell lines (AN3CA, HEC-59, HEC-1B and KLE) and normal endometrial epithelial cells. *P<0.05. miR, microRNA; EC, endometrial cancer.

Figure 2.

The inhibitory roles of miR-873 overexpression in EC cells. (A) Reverse transcription-quantitative PCR analysis of miR-873 expression in HEC-59 and HEC-1B cells transfected with miR-873 mimics or miR-NC. (B) Cell Counting Kit-8 assay analysis of miR-873 mimics or miR-NC-transfected HEC-59 and HEC-1B cell proliferation. (C) In vitro invasion assay analysis of the effect of miR-873 upregulation on the invasive ability of HEC-59 and HEC-1B cells (magnification, ×200). *P<0.05 vs. miR-NC. miR, microRNA; NC, negative control; OD, optical density.

Figure 3.

miR-873 directly targets HDGF in EC cells. (A) The miR-873 wild-type binding site in the 3′-UTR of HDGF wt and its mut form. The mut sequence is underlined. (B) Luciferase reporter assay analysis in HEC-59 and HEC-1B cells following the co-transfection of pmirGLO-HDGF-3′-UTR wt and miR-873 mimics. *P<0.05 vs. miR-NC. (C) RT-qPCR analysis of HDGF mRNA expression in 47 pairs of EC tissues and adjacent normal endometrial tissues. *P<0.05 vs. normal endometrial tissues. (D) Spearman's correlation analysis of miR-873 and HDGF mRNA in EC tissues. (E) RT-qPCR analysis of HDGF mRNA and (F) western blot analysis of protein expression in HEC-59 and HEC-1B cells after transfection with miR-873 mimics or a miR-NC. *P<0.05 vs. miR-NC. miR, microRNA; HDGF, hepatoma-derived growth factor; EC, endometrial cancer; UTR, untranslated region; NC, negative control; RT-q, reverse transcription-quantitative; wt, wild-type; mut, mutant.

Figure 4.

HDGF knockdown inhibits the proliferative and invasive abilities of endometrial cancer cells. (A) Western blot analysis of HDGF protein expression in HEC-59 and HEC-1B cells treated with HDGF siRNA or NC siRNA. (B) cell counting kit-8 analysis of HDGF inhibition on the proliferation of HEC-59 and HEC-1B cells. (C) In vitro assay analysis of the invasion of HEC-59 and HEC-1B cells following HDGF siRNA or NC siRNA transfection (magnification, ×200). *P<0.05 vs. NC siRNA. HDGF, hepatoma-derived growth factor; siRNA, small interfering RNA; NC, negative control; OD, optical density.

Figure 5.

HDGF restoration rescues miR-873-induced cellular phenotypes in endometrial cancer cells. (A) Western blot analysis of HDGF protein expression in HEC-59 and HEC-1B cells transfected with miR-873 mimics and the HDGF overexpression plasmid pcDNA3.1-HDGF or an empty pcDNA3.1 plasmid 72 h post transfection. (B) Cell counting kit-8 and (C) in vitro invasion assay analysis of the proliferation and invasion of HEC-59 and HEC-1B cells transfected with miR-873 mimics and the HDGF overexpression plasmid, pcDNA3.1-HDGF, or an empty pcDNA3.1 plasmid (magnification, ×200). *P<0.05 vs. miR-NC. ^#P<0.05 vs. miR-873 mimics + pcDNA3.1. HDGF, hepatoma-derived growth factor; miR, microRNA; NC, negative control; OD, optical density.