Long non-coding RNA FENDRR inhibits the stemenss of colorectal cancer cells through directly binding to Sox2 RNA

Abstract

Cancer stem cells (CSCs) contribute to malignant features. Long non-coding RNA (LncRNA) FENDRR has been shown to regulate tumor proliferation, migration, and invasion. However, the effects of FENDRR on the CSC-like traits of colorectal cancer cells remain to be elucidated. Here, we identified that lncRNA FENDRR level was remarkably lower in spheres formed by colorectal cancer cells compared to that in parental cancer cells. Further functional experiments showed that FENDRR overexpression attenuated the CSC-like traits of colorectal cancer spheres, while FENDRR knockdown conferred the CSC-like traits for colorectal cancer cells, as characterized by the alteration of ALDH activity, sphere-formation ability, and expression of stemness markers (Oct4, Sox2, and KLF4). RNA-RNA interaction in vitro analysis combined with mRNA stability assay revealed that lncRNA FENDRR directly interacted with Sox2 mRNA 3'UTR, reduced its mRNA stability and thus inhibited Sox2 expression. In addition, lncRNA FENDRR-mediated effects on the CSC-like traits of colorectal cancer cells depended on Sox2 expression. This work suggests that lncRNA FENDRR can block the CSC-like traits in colorectal cancer cells through directly interacting with Sox2 mRNA 3'UTR.

Keywords: Long non-coding rna; RNA–RNA interaction; cancer stem cell; mRNA stability; sox2.

Figure 1.

LncRNA FEDNRR level is significantly downregulated in colorectal cancer spheres. (a) The representative images of spheres formed by colorectal cancer cells. (b) Sphere size was examined in colorectal cancer spheres and cells. (c) Sphere number was measured in colorectal cancer spheres and cells. (d) ALDH activity was evaluated in colorectal cancer spheres and cells. (e and f) The mRNA level of stemness markers (Sox2, Oct4, KLF4) was determined in colorectal cancer spheres and cells. (g) The protein level of stemness markers was detected in colorectal cancer spheres and cells. (h) FENDRR level was examined in colorectal cancer spheres and cells. (i – l) FENDRR expression was detected in data from TCGA using the Tumor, Normal and Metastatic tissues tool (https://tnmplot.com/analysis/). n ≥ 3, **P < 0.01 vs. control

Figure 2.

Overexpression of lncRNA FEDNRR attenuates the CSC-like traits of colorectal cancer spheres. (a) The overexpression efficiency of FENDRR-oe was validated by RT-qPCR. (b and c) Sphere number and size were measured in colorectal cancer spheres with FENDRR overexpression or not. (d) Colorectal cancer spheres with or without FENDRR overexpression were subjected to ALDH activity detection. (e and f) The mRNA levels of stemness markers (Sox2, Oct4, KLF4) were detected in colorectal cancer spheres with or without FENDRR overexpression. (g) The protein levels of stemness markers (Sox2, Oct4, KLF4) were detected in colorectal cancer spheres with or without FENDRR overexpression. n ≥ 3, **P < 0.01 vs. control

Figure 3.

Knockdown of lncRNA FENDRR confers the CSC-like traits of colorectal cancer cells. (a) The knockdown efficiency of FENDRR-kd was confirmed by RT-qPCR. (b and c) Sphere number and size were determined in colorectal cancer cells with FENDRR knockdown or not. (d) ALDH activity was determined in colorectal cancer cells with FENDRR knockdown or not. (e and f) The mRNA levels of stemness markers were detected in colorectal cancer cells with FENDRR knockdown or not. (g) The protein levels of stemness markers were examined in colorectal cancer cells with or without FENDRR knockdown. n ≥ 3, **P < 0.01 vs. control

Figure 4.

LncRNA FENDRR directly interacts with Sox2 mRNA 3’UTR, enhances its stability and expression. (a – c) The interaction between FENDRR and Sox2, Oct4, or KLF4 was examined through the RNA–RNA interaction in vitro assay. (d – f) The mRNA stability of Sox2, Oct4 and KLF4 was determined in colorectal cancer cells with FENDRR overexpression or not. (g and h) The interaction between FENDRR, and Sox2 CDS, Sox2 5’UTR, or Sox2 3’UTR was evaluated in colorectal cancer cells. (i and j) The activity of Luc-Sox2-3’UTR, Luc-Sox2-CDS, and Luc-Sox2-5’UTR was measured in colorectal cancer cells with FENDRR overexpression or not. n ≥ 3, **P < 0.01 vs. control

Figure 5.

LncRNA FENDRR promotes the CSC-like traits of colorectal cancer cells dependent on Sox2. (a and b) The stemness markers’ mRNA levels were examined in cells with FENDRR overexpression as well as Sox2 knockdown or not. (c) The stemness markers’ protein levels were detected in cells with FENDRR overexpression as well as Sox2 knockdown or not. (d and e) Sphere number and size were determined in cells with FENDRR overexpression plus Sox2 knockdown or not. (f) ALDH activity was evaluated in cells with FENDRR overexpression as well as Sox2 knockdown or not. n ≥ 3, **P < 0.01 vs. control, ^##P < 0.01 vs. FENDRR-oe

Figure 6.

LncRNA FENDRR confers chemoresistance of colorectal cancer cells dependent on Sox2. (a) The mRNA levels of FENDRR and Sox2 were detected in HT-29/5-Fu and HT-29 cells. (b) ALDH activity was measured in HT-29/5-Fu and HT-29 cells. (c and d) Sphere number and size were determined in HT-29/5-Fu and HT-29 cells. (e) HT-29/5-Fu cells with FENDRR knockdown as well as Sox2 overexpression or not were subjected to cell viability detection. (f) Cell viability was examined in HT-29 cells with FENDRR overexpression plus Sox2 knockdown or not. n ≥ 3, **P < 0.01 vs. control