MicroRNA-194 modulates epithelial–mesenchymal transition in human colorectal cancer metastasis

Figures & data

Table 1 Sequence of primers used in this study

Name	Sequence (5′–3′)
MMP-2	Forward: ATTCTGGAGATACAATGAGGT
	Reverse: TTCACGCTCTTCAGACTT
MMP-9	Forward: GAAGATGCTGCTGTTCAG
	Reverse: AAATAGGCTTTCTCTCGGTA
E-cadherin	Forward: GACCAAGTGACCACCTTA
	Reverse: AGAGCAGCAGAATCAGAAT
Vimentin	Forward: CATTGAGATTGCCACCTAC
	Reverse: TCGTTGATAACCTGTCCAT
GAPDH	Forward: GGTATCGTGGAAGGACTC
	Reverse: GGGATGATGTTCTGGAGAG
miR-194	Forward: CGATCTCTCATGTAACAGCAACTC
	Reverse: TATGGTTGTTCTCGTCTCTGTGTC
U6 snRNA	Forward: ATTGGAACGATACAGAGAAGATT
	Reverse: GGAACGCTTCACGAATTTG

Figure 1 Expression status of miR-194 in CRC tissue specimens and cell lines.

Notes: (A) The relative expression of miR-194 in lymph nodes metastasis compared with lymph nodes negative. (B) The relative expression of miR-194 in CRC cell lines. *P<0.05, **P<0.01.
Abbreviations: LNs, lymph nodes; CRC, colorectal cancer.

Table 2 Clinicopathological characteristics of 62 patients with colorectal cancer

Characteristics	Number of patients	Relative expression of miR-194	P-value
Age, years			0.315
≤60	25	92.46
>60	37	68.15
Sex			0.928
Male	34	77.34
Female	28	74.62
Tumor location			0.652
Colon	26	65.83
Rectum	36	75.26
Tumor size			0.589
≤5 cm	37	72.31
>5 cm	25	78.69
Histological type			0.745
Well	15	79.90
Moderately	25	51.32
Poor	22	91.25
Infiltration			0.912
pT1–2	11	69.31
pT3	29	67.36
pT4	22	81.97
LN metastasis			0.027
pN0	29	51.34
pN1–2	33	93.76

Notes: Statistical analysis was carried out using chi-square test. P<0.05 was considered statistically significant.

Abbreviation: LNs, lymph nodes.

Figure 2 MiR-194 reveals no significant effect on CRC cell proliferation.

Notes: (A) The changes in morphology before and after transfection were observed by inverted microscopy (×100). (B) SW480 cells were infected with miR-194 or negative control (miR-NC) or mock transfection control (miR-MT) lentivirus, and the expression of miR-194 was analyzed by qRT-PCR. (C) MTT assays were performed to investigate the proliferation ability of CRC cells. *P<0.01.
Abbreviations: CRC, colorectal cancer; NC, negative control; MT, mock transfection; h, hours; OD, optical density.

Figure 3 Overexpression of miR-194 alters F-actin cytoskeleton distribution and cell morphology.

Notes: (A) SW480 cells showing several protrusions in different directions, apparently smaller lamellipodia. (B) Mock transfection group. (C) Negative control group. (D) Cells overexpressing miR-194 were elongated, featuring one or two predominant lamellipodia. Scale bars: 10 µm.

Figure 4 MiR-194 promotes the invasion and migration of CRC cells.

Notes: Wound healing assays (A) and Transwell without matrigel assays (B) were performed to investigate the migration ability of CRC cells. (C) Transwell with matrigel assays was performed to investigate the invasion ability of CRC cells. (a) SW480; (b) miR-MT; (c) miR-NC; (d) miR-194. *P<0.001. Scale bars: 10 µm.
Abbreviations: CRC, colorectal cancer; NC, negative control; MT, mock transfection; h, hours.

Figure 5 The auxo-action of miR-194 on the EMT of CRC cells.

Notes: qRT-PCR (A) and Western blot (B) were performed to examine the effects of miR-194 overexpression on EMT markers (MMP-2, MMP-9, E-cadherin, and vimentin). *P<0.05, **P<0.01, ***P<0.001.
Abbreviations: CRC, colorectal cancer; EMT, epithelial–mesenchymal transition; NC, negative control; MT, mock transfection; qRT-PCR, real-time quantitative reverse transcription polymerase chain reaction.

Figure 6 MiR-194 increased the gelatin-degrading activity of MMP-2.

Notes: Zymography assay was performed to examine whether miR-194 regulates MMP-2 activity, leading to the invasion and migration of CRC cells. **P<0.01.
Abbreviations: CRC, colorectal cancer; NC, negative control; MT, mock transfection.