MicroRNA-30a-5p (miR-30a) regulates cell motility and EMT by directly targeting oncogenic TM4SF1 in colorectal cancer

Abstract

Purpose: Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide, and many oncogenes and tumor suppressor genes are involved in CRC. MicroRNAs (miRNAs) are small non-coding RNAs that can negatively regulate gene expression. Previous studies have revealed that miRNAs regulate the development and progression of many cancers. In this study, we investigated the role of microRNA-30a-5p (miR-30a) in CRC and its unknown mechanisms.

Methods: qRT-PCR was used to detect miR-30a and TM4SF1 mRNA expression in CRC specimens and cell lines. CRC cell migration and invasion were assessed after transfection with miR-30a or TM4SF1 using wound healing and trans-well migration and invasion assays. Transmembrane-4-L-six-family protein (TM4SF1) was validated as a target of miR-30a in CRC through luciferase reporter assay and bioinformatics algorithms. Moreover, two EMT regulators, E-cadherin and VEGF, were also identified using Western blotting and immunohistochemistry.

Results: We found that miR-30a was down-regulated in CRC tumor tissues and cell lines, and miR-30a was inversely associated with advanced stage and lymph node metastatic status compared with normal tissues. miR-30a decreased migration and invasion in CRC cell lines, and miR-30a overexpression not only down-regulated TM4SF1 mRNA and protein expression, but also inhibited the expression of VEGF and enhanced expression of E-cadherin. We also showed that TM4SF1 was up-regulated in CRC tumor specimens compared with adjacent normal tissues, and TM4SF1 expression was significantly associated with advanced stage and lymph node status compared with adjacent normal tissues.

Conclusions: These results suggest that miR-30a is an important regulator of TM4SF1, VEGF, and E-cadherin for CRC lymph node metastasis, a potential new therapeutic target in CRC.

Keywords: Colorectal cancer; Metastasis; Transmembrane-4-L-six-family protein 1; miR-30a.

Fig. 1

Analysis of miR-30a expression in human CRC specimens and cell lines by qRT-PCR. a The relative expression levels of miR-30a in human CRC tissues (n = 80) and paired adjacent normal tissues, and normalized to RNU48, small nuclear RNA. b The relative expression of miR-30a of TNM stage compared with the normal tissues. c The relative expression of miR-30a in the CRC tissues of different tumor lymph node status compared with normal tissues. d Expression level of miR-30a in four CRC cell lines (Caco-2, LOVO, SW480 and DLD-1). All the data were presented as mean ± SD from three independent experiments. *P < 0.05, ***P < 0.001

Fig. 2

miR-30a suppresses migration and invasion of CRC cells. a Wound-healing assay was used to measure the effect of miR-30a on the motility of CRC cells. A sterile 200 μl pipette tip was used to scratch the cells. The wound width was photographed and measured. b Effects of miR-30a on cell migration and invasion in SW480 and DLD-1 cells in vitro using trans-well assay. All the data were presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

miR-30a directly targeted TM4SF1. a Sequence of the miR-30a binding site within the human TM4SF1 3′-UTR and schematic diagram of the reporter construct showing the wild type (WT) of TM4SF1 3′-UTR sequence and the mutated (MT) TM4SF1 3′-UTR sequence. The WT and MT of TM4SF1 3′-UTR was transfected into SW480 and DLD-1 cell lines with or without synthetic miR-30a mimic. Luciferase activity was determined 24 h after transfection. Data were normalized to the luciferase activity transfected with miR-negative control (miR-NC). b Analysis of TM4SF1 expression level in SW480 and DLD-1 cells transfected with miR-30a by qRT-PCR. c SW480 and DLD-1 cells were transfected with the miR-30a using Lipofectamine for 72 h and analyzed using Western blotting. The experiment were repeated at least three independent times. Values represent the mean ± SD. *P < 0.05, calculated using Student’s t test

Fig. 4

miR-30a modulated E-cadherin and VEGF by regulating TM4SF1. a Western blot analysis of EMT marker including E-cadherin, β-catenin, Slug, α-SMA, Twist and VEGF after transfection with miR-NC (NC) and miR-30a in CRC cell lines. b Relative TM4SF1 and VEGF mRNA expression after transfection with si-TM4SF1 in SW480 and DLD-1 cells. The relative mRNA expression levels were normalized B2M and revealed as mean ± SD from triplicate experiments. c E-cadherin and VEGF levels were measured by Western blotting in si-TM4SF1 expression. Data represent mean ± SD of three replicates. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

TM4SF1 mediated the effect of miR-30a on CRC cell lines. DLD-1 or Caco-2 cells were co-transfected with the indicated miRNA and TM4SF1 plasmid DNAs. a DLD-1 and Caco-2 cells were seeded into the upper chamber of a trans-well for 48 h and counted under a microscope after staining. b 48 h after transfection, DLD-1 and Caco-2 cells were lysed and analyzed by qRT-PCR. c SW480, DLD-1 and Caco-2 cells were collected 72 h after transfection and then subjected to Western blot analysis. Experiments were repeated at least three independent times

Fig. 5

TM4SF1 mediated the effect of miR-30a on CRC cell lines. DLD-1 or Caco-2 cells were co-transfected with the indicated miRNA and TM4SF1 plasmid DNAs. a DLD-1 and Caco-2 cells were seeded into the upper chamber of a trans-well for 48 h and counted under a microscope after staining. b 48 h after transfection, DLD-1 and Caco-2 cells were lysed and analyzed by qRT-PCR. c SW480, DLD-1 and Caco-2 cells were collected 72 h after transfection and then subjected to Western blot analysis. Experiments were repeated at least three independent times

Fig. 6

Analysis of TM4SF1 expression in human CRC specimens. a qRT-PCR analysis of TM4SF1 mRNA expression in 80 paired CRC tissues and adjacent normal tissues (NT). TM4SF1 expression level was calculated by the 2^−△△Ct method and normalized to B2M. b The relative expression of TM4SF1 mRNA in stage I + II and III + IV compared with NT. c qRT-PCR was used to detect the expression of TM4SF1 in N0 (no spread to regional lymph nodes), N1 (1 to more regional lymph nodes) and NT. d The relative expression between the expression level of TM4SF1 and miR-30a in human CRC tissues. e Western blotting analysis of TM4SF1, E-cadherin and VEGF protein levels in CRC tissues and NT. f The expression of TM4SF1, E-cadherin and VEGF in CRC specimens compared with NT by immunohistochemistry. Each bar represents the mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001

Fig. 6

Analysis of TM4SF1 expression in human CRC specimens. a qRT-PCR analysis of TM4SF1 mRNA expression in 80 paired CRC tissues and adjacent normal tissues (NT). TM4SF1 expression level was calculated by the 2^−△△Ct method and normalized to B2M. b The relative expression of TM4SF1 mRNA in stage I + II and III + IV compared with NT. c qRT-PCR was used to detect the expression of TM4SF1 in N0 (no spread to regional lymph nodes), N1 (1 to more regional lymph nodes) and NT. d The relative expression between the expression level of TM4SF1 and miR-30a in human CRC tissues. e Western blotting analysis of TM4SF1, E-cadherin and VEGF protein levels in CRC tissues and NT. f The expression of TM4SF1, E-cadherin and VEGF in CRC specimens compared with NT by immunohistochemistry. Each bar represents the mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001

Fig. 7

An assumed diagram of miR-30a for migration and invasion through TM4SF1/E-cadherin/VEGF in CRC cells