Long noncoding RNA SNHG6 promotes the progression of colorectal cancer through sponging miR-760 and activation of FOXC1

Figures & data

Table 1 Relationship between SNHG6 expression and clinicopathologic features of colorectal cancer patients (n=40)

Variable	Relative SNHG6 expression		P-value
	Low (n=20)	High (n=20)
Age (years)			NS
<50	8	11
>50	12	9
Gender			NS
Male	14	10
Female	6	10
Lymphatic metastasis			P<0.001
N0/N1	15	4
N2/N3	5	16
Distal metastasis			P<0.05
Negative	19	13
Positive	1	7
Tumor stage			P<0.001
I/II	16	5
III/IV	4	15

Notes: Patients were divided into SNHG6 high group (relative fold change was higher than the median) and low group (relative fold change was lower than the median) according to the analysis of quantitative real-time polymerase chain reaction detection. NS, not significant between different groups. Differences among variables were evaluated by chi-squared or Fisher’s exact chi-squared test.

Figure 1 SNHG6 was overexpressed in CRC tissues and cell lines, and associated with poor prognosis.

Notes: (A) The relative expression of SNHG6 was significantly increased in CRC tissues compared with adjacent normal tissues. (B) The relative expression of SNHG6 in different TNM stages of CRC patients. (C) The relative expression of SNHG6 in CRC cell lines compared with normal IEC. (D) Overall survival of CRC patients by Kaplan–Meier analysis. *P<0.05; **P<0.01.
Abbreviations: CRC, colorectal cancer; IEC, intestinal epithelial cells.

Figure 2 Knockdown of SNHG6 impaired CRC proliferation, invasion and migration in vitro.

Notes: (A) Knockdown of SNHG6 in DLD1 and HCT116 cells by three different shRNA. (B) CCK-8 assays showed that SNHG6 knockdown inhibited cell proliferation in DLD1 and HCT116 cells. (C) Colony formation assay showed that SNHG6 knockdown reduced the number of colonies in DLD1 and HCT116 cells. (D and E) SNHG6 knockdown attenuated cell invasion and migration of DLD1 and HCT116 cells. (F) Wound healing assay indicated that SNHG6 knockdown repressed the migration of DLD1 and HCT116 cells. **P<0.01.
Abbreviations: CRC, colorectal cancer; CCK-8, Cell Counting Kit-8.

Figure 3 Knockdown of SNHG6 impaired CRC proliferation, invasion and migration in vivo.

Notes: (A) Representative image of xenograft using SNHG6 knockdown and control cells. (B and C) SNHG6 knockdown decreased tumor volume and tumor weight of HCT116 cells. (D–F) Representative bioluminescence images and morphology of liver metastasis in SNHG6 knockdown and control cells. **P<0.01.
Abbreviation: CRC, colorectal cancer.

Figure 4 SNHG6 directly interacted with miR-760 to regulate CRC cell proliferation, invasion and migration.

Notes: (A) Bioinformatics analysis of predicted binding sites between SNHG6 and miR-760. (B) Luciferase reporter assay demonstrated that miR-760 mimics significantly decreased the relative luciferase activity of WT-SNHG6 but not Mut-SNHG6. (C) SNHG6 knockdown increased the relative expression of miR-760 in DLD1 and HCT116 cells. (D) The relative expression level of miR-760 in CRC tissues and adjacent normal tissues. (E) Pearson’s correlation analysis of the expression between SNHG6 and miR-760 in CRC tissues. miR-760 inhibition rescued SNHG6 silencing induced cell proliferation (F), colony formation (G), invasion (H) and migration (I) inhibition in HCT116 cells. **P<0.01; ^#Non-significant.
Abbreviations: CRC, colorectal cancer; Mut, mutant; WT, wild type; NC, negative control.

Figure 5 FOXC1 was a direct target of miR-760 and positively regulated by SNHG6.

Notes: (A) Bioinformatics analysis of predicted binding sites between miR-760 and FOXC1. (B) Luciferase reporter assay indicated that miR-760 mimics significantly reduced the luciferase activity of FOXC1 in HEK293T cells. (C) MiR-760 overexpression inhibited the mRNA and protein expression levels of FOXC1 in DLD1 and HCT116 cells. (D) The relative expression levels of miR-760 mRNA in CRC tissues. (E) Pearson’s correlation analysis of the expression between miR-760 and FOXC1 in CRC tissues. (F) SNHG6 knockdown inhibited the mRNA and protein expression levels of FOXC1 and rescued by miR-760 inhibition in DLD1 and HCT116 cells. (G) Pearson’s correlation analysis of the expression between SNHG6 and FOXC1 in CRC tissues. **P<0.01; ^#Non-significant.
Abbreviations: CRC, colorectal cancer; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.