Long noncoding RNA LINC00958 suppresses apoptosis and radiosensitivity of colorectal cancer through targeting miR-422a

Abstract

Background: Long noncoding RNAs (lncRNAs) have been elucidated to participate in the development and progression of various cancers. In this study, we aimed to explore the underlying functions and mechanisms of LINC00958 in colorectal cancer.

Methods: LINC00958 expression in colorectal cancer tissues was examined by qRT-PCR. The correlations between LINC00958 expression and clinical characteristics and prognosis were evaluated. The biological functions of LINC00958 were detected by CCK-8, MTT, colony formation and flow cytometric analyses. RNA pulldown, RIP and luciferase reporter assays were used to confirm the regulatory effects of LINC00958 on miR-422a. Rescue experiments were performed to detect the effects of miR-422a on the roles of LINC00958.

Results: LINC00958 was upregulated in colorectal cancer tissues and cell lines. High LINC00958 levels were positively associated with T stage and predicted poor prognosis. Cell experiments showed that LINC00958 promoted cell proliferation and suppressed apoptosis and sensitivity to radiotherapy in vitro and promoted tumor growth in vivo. Bioinformatics analysis predicted the binding site of miR-422a on LINC00958. Mechanistically, RNA pulldown, RIP and luciferase reporter assays demonstrated that LINC00958 specifically targeted miR-422a. In addition, we found that miR-422a suppressed MAPK1 expression by directly binding to the 3'-UTR of MAPK1, thereby inhibiting cell proliferation and enhancing cell apoptosis and radiosensitivity. Furthermore, miR-422a rescued the roles of LINC00958 in promoting MAPK1 expression and cell proliferation and decreasing cell apoptosis and radiosensitivity.

Conclusions: LINC00958 promoted MAPK1 expression and cell proliferation and suppressed cell apoptosis and radiosensitivity by targeting miR-422a, which suggests that it is a potential biomarker for the prognosis and treatment of colorectal cancer.

Keywords: Apoptosis; Colorectal cancer; LINC00958; Radiosensitivity; miR-422a.

Fig. 1

Features of LINC00958 in colorectal cancer tissues. a LINC00958 expression was upregulated in most (82.54%, 52/63) of colorectal cancer tissues. b LINC00958 expression was significantly higher in 63 colorectal cancer tissues than adjacent normal colorectal tissues. c The level of LINC00958 was significantly higher in colorectal cancer tissues at T3 and T4 stages than these at T1 and T2 stages. d and e Kaplan–Meier Plotter analysis revealed that high LINC00958 level predicted a poorer overall survival (d) and disease-free survival (e) for colorectal cancer patients. *p < 0.05, **p < 0.01, ^NSp > 0.05

Fig. 2

LINC00958 promotes cell proliferation, suppresses cell apoptosis and radiosensitization of colorectal cancer. a The expression of LINC00958 in colorectal cancer cell lines. b The efficiencies of siRNA targeting LINC00958 and an overexpression plasmid of LINC00958 were detected. c The flow cytometry assay showed that knockdown of LINC00958 significantly increased the percentage of apoptosis. d, e The CCK-8 (d) and MTT (e) results indicated that si-LINC00958 decreased the ability of cell proliferation. f si-LINC00958 decreased the survival fraction under different irradiation dose. g The flow cytometry assay showed that overexpression of LINC00958 remarkably decreased the percentage of apoptosis. h and i The CCK-8 (h) and MTT (i) results indicated that overexpression of LINC00958 increased the ability of cell proliferation. j Overexpression of LINC00958 increased the survival fraction under different irradiation dose. Data are reported as means ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01

Fig. 3

LINC00958 promotes colorectal cancer cell proliferation in vivo. a The knockdown efficiency of stable SW480/sh-LINC00958 cells was detected by qRT-PCR. b Represent pictures of tumor formation of SW480 cells. c The final tumor weight of SW480 cells was shown. d Tumor volumes of SW480 cells were measured every three days. **p < 0.01

Fig. 4

LINC00958 acts as a miRNA sponge of miR-422a. a Bioinformatics databases predicted that LINC00958 contained the binding site of miR-422a. b Expression of miR-422a after knockdown or overexpression of LINC00958 in colorectal cancer cells. c The expression of miR-422a in 63 paired colorectal cancer tissues. d Pearson’s correlation showed that miR-422a level negatively correlated with LINC00958 level in 63 paired colorectal cancer tissues (R = − 0.5122, p < 0.001). e RNA-FISH assays showed that most of LINC00958 was located in the cytoplasm of SW480 cells. f qRT-PCR results of U6, GAPDH and LINC00958 expressions in cell nucleus and cytoplasm. g The biotinylated RNA pull-down showed that miR-422a was pulled down by LINC00958 probe, but not by mutant LINC00958 probe, in SW480 cells. h RIP assay was performed with AGO2 antibody in SW480 cells transfected with miR-422a mimics or NC, and the enrichment of LINC00958 was detected. i miR-422a mimics significantly decreased the luciferase activity of wild type of LINC00958, but not the activity of mutation of LINC00958. j miR-422a inhibitor evidently increased the luciferase activity of wild type of LINC00958, but not it of mutation of LINC00958. Three independent experiments were performed for each group. All data are reported as the mean ± SD. **p < 0.01

Fig. 5

miR-422a suppresses cell proliferation, increases cell apoptosis and radiosensitization of colorectal cancer through MAPK1. a Bioinformatics databases predicted that MAPK1 was a target of miR-422a. b, c mRNA and protein expression of MAPK1 after knockdown or overexpression of miR-422a in colorectal cancer cells. d miR-422a mimics significantly decreased the luciferase activity of wild type of MAPK1, but not the activity of mutation of MAPK1. In addition, miR-422a inhibitor evidently increased the luciferase activity of wild type of MAPK1, but not it of mutation of MAPK1. e, i The flow cytometry assay showed that miR-422a mimics significantly increased the apoptosis percentage (e), while miR-422a inhibitor remarkably decreased the apoptosis percentage (i). f, g) and j, k The CCK-8 and MTT results indicated that miR-422a mimics decreased the ability of cell proliferation, and miR-422a inhibitor increased it. h, l miR-422a mimics enhanced the radiosensitization of colorectal cancer cell (h), while miR-422a inhibitor decreased the radiosensitization (l). Data are reported as means ± standard deviation of three independent experiments. **p < 0.01

Fig. 6

LINC00958 promotes MAPK1 expression and progression of colorectal cancer through miR-422a. a Pearson’s correlation showed that LINC00958 level positively correlated with MAPK1 level in 63 paired colorectal cancer tissues (R = 0.6412, p < 0.001). b The expression of MAPK1 protein (ERK1/2, pERK1/2), Bcl-2 and Bax after knockdown of LINC00958 and miR-422a in SW480 cells. c The mRNA expression of MAPK1 after knockdown of LINC00958 and miR-422a in SW480 cells. d The relative luciferase activity of wild type of MAPK1 mRNA 3’-UTR after knockdown of LINC00958 and miR-422a in SW480 cells. e The results of flow cytometry assay after knockdown of LINC00958 and miR-422a in SW480 cells. f, g The CCK-8 (f) and MTT (g) results after knockdown of LINC00958 and miR-422a in SW480 cells. h The survival fraction under different irradiation dose after knockdown of LINC00958 and miR-422a in SW480 cells. Data are reported as means ± standard deviation of three independent experiments. **p < 0.01