LINC01006 and miR-3199 Serve as Novel Markers of Poor Prognosis in Colon Cancer and Regulate Cell Proliferation, Migration and Invasion

Abstract

Purpose: Colon cancer is the most commonly diagnosed gastrointestinal cancer. This research intended to evaluate the prognostic values of LINC01006 and miR-3199 for colon cancer and their effects on cell physiology.

Patients and methods: LINC01006 and miR-3199 expression levels were determined by RT-qPCR. Patients' 5-year cumulative survival rate was analyzed by Kaplan-Meier curves with the Log rank test. Chi-square test and multivariate Cox regression analysis were used to access the clinical significance. CCK-8 assay, transwell assay, and TUNEL assays were used to monitor the change of cell proliferation, invasion, migration, and apoptosis.

Results: The expression level of LINC01006 was increased while miR-3199 was decreased in colon tissues and cells compared to normal ones. This dysregulated expression was correlated with T stage (P = 0.002) and N stage (P = 0.009). High LINC01006 level (HR = 4.048, 95%: 1.502-10.911, P = 0.006) or low miR-3199 level (HR = 3.421, 95% CI: 1.254-9.330, P = 0.016) was outstanding for predicting poor prognosis in patients with colon cancer. Downregulation of LINC01006 reduced cell proliferation, invasion, and migration but induced cell apoptosis (P < 0.05).

Conclusion: LINC01006 knockdown showed anti-proliferative, anti-metastatic, and apoptotic-induced effects on colon cancer cells. This study contributes to research on promising prognostic biomarkers of colon cancer and might give way to further investigation of alternative tumor targets.

Keywords: LINC01006; colon cancer; miR-3199; prognosis.

Figure 1

LINC01006 is highly expressed in colon cancer and its expression in colon cancer tissues correlates with overall survival. (A) Expression of LINC01006 in colon cancer tissues and normal tissues detected by RT-qPCR. The comparison was done by paired t-test. ***P < 0.001. (B) Expression of LINC01006 in colon cancer cell lines (HCT 116, SW480, HT-29, SW620) and normal intestinal epithelial cells (FHC) detected by RT-qPCR. Comparison was done by unpaired t-test. **P < 0.01, ***P < 0.001. (C) Overall survival was estimated using the Kaplan–Meier method. Comparison was done with the Log rank test. P =0.007.

Figure 2

The effect of the knockdown of LINC01006 on cell survival, metastasis and cell death of HCT 116 or SW620 cells in vitro. (A) LINC01006 expression in HCT 116 or SW620 cells transfected with si-NC or si-LINC01006-1 or si-LINC01006-2. Cell proliferation changes of HCT 116 cells (B) or SW620 cells (C) were determined by the CCK-8 assay. (D) HCT 116 or SW620 cell death was detected by TUNEL assay. Transwell assay was used to indicate migratory cells (E) and invasive cells (F). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Expression of miR-3199 is decreased in colon cancer and its reduction predicted poor prognosis. (A) Relative miR-3199 expression level in different human tissues, cancerous and normal by RT-qPCR. ***P < 0.001. (B) Relative miR-3199 expression level in different human cell lines by RT-qPCR. **P < 0.01, ***P < 0.001. (C) Overall survival of the two groups stratified according to median LINC01006 expression value in the colon cancer tissues. Log rank test P =0.005.

Figure 4

MiR-3199 is sponged by LINC01006. (A) Binding sites between miR-3199 and LINC01006. (B) miR-3199 level was negatively correlated with LINC01006 level in colon tissues. (C) Knockdown of LINC01006 increased the level of miR-3199. (D and E) Luciferase reporter assays were carried out to examine the interaction between miR-3199 and LINC01006 in HCT 116 or SW620 cells. **P < 0.01.

Figure 5

LINC01006 knockdown repressed colon cancer cell function via miR-3199. (A) miR-3199 expression in HCT 116 cells transfected with si-NC or si-LINC01006-2 or si-LINC01006-2+anti-miR-3199. (B) miR-3199 inhibition can offset the effect caused by LINC01006 knockdown upon cell proliferation. (C) The induction of HCT 116 cell death was suppressed by anti-miR-3199. (D and E) miR-3199 inhibition can bring down the cell number in migration and invasion assay. *P < 0.05, **P < 0.01, ***P < 0.001.