Long Non-Coding RNA ucoo2kmd.1 Regulates CD44-Dependent Cell Growth by Competing for miR-211-3p in Colorectal Cancer

Abstract

In addition to protein-coding genes, the human genome makes a large amount of noncoding RNAs. Long non-coding RNAs (lncRNAs) have been described as the largest subclass of the non-coding transcriptome in human noncoding RNAs. In recent years, lncRNAs have been considered to be the key regulators of tumor behavior. In this study, based on previous research, we investigated the expression and biological role of a newly identified cancer-related lncRNA, lncRNA-uc002kmd.1. We analyzed the relationship between lncRNA-uc002kmd.1 and colorectal cancer (CRC) in a total 45 CRC and paired adjacent, non-tumor tissue samples. We found that lncRNA-uc002kmd.1 expression was usually highly expressed in carcinoma compared with the tissue adjacent to the carcinoma. Through a series of experiments, the results showed that lncRNA-uc002kmd.1 regulates CD44 as a molecular decoy for miR211-3p. Our data indicated that the overexpression of lncRNA-uc002kmd.1 enhanced cell proliferation in CRC.

Fig 1. Cellular and molecular characterization of lincRNA-uc002kmd.1.

(A) The levels of nuclear control transcript (U6), cytoplasmic control transcript (GAPDH mRNA) and lincRNA-uc002kmd.1 were assessed by qRT-PCR in nuclear and cytoplasmic fractions. Data are mean ± SEM. (B) Northern blot analysis of lincRNA-uc002kmd.1expression in CRC cells. (C) Maximum CSF scores of lincRNA-uc002kmd.1 by analysis with PhyloCSF. The score is -178.6075. (D) The lincRNA-uc002kmd.1 was expressed at a higher level in CRC tissues compared to match CRC adjacent tissues. The expression level of lincRNA-uc002kmd.1 was analyzed by qRT-PCR normalized to GAPDH. Data are represented as mean±SEM from three independent experiments. (E) The linear correlations between the lincRNA-uc002kmd.1 expression levels and CD44 mRNA were tested. The relative expression value was normalized by GAPDH expression level. (F, G) lincRNA-uc002kmd.1expression significantly affected CD44 mRNA expression. Knockdown of lincRNA-uc002kmd.1 decreased CD44 expression, while ectopic expression of GAPLINC increased CD44 mRNA level. (H) The protein levels of CD44 was assessed in CRC cells (HCT116 cells and SW480 cells) by Western blot.

Fig 2. The relationship between miR-211-3p, lincRNA-uc002kmd.1 and CD44.

(A)Both lincRNA-uc002kmd.1 and CD44 are targeted by miR-211-3p. MiR-211-3p significantly decreased the luciferase signals of both lincRNA-uc002kmd.1 and CD44. (B) The CD44 and lncRNA-uc002kmd.1 levels were significantly decreased. Data are mean±SEM, normalized to GAPDH. (C) The negative correlations between the lincRNA-uc002kmd.1 expression levels and CD44 mRNA were tested.

Fig 3. lincRNA-uc002kmd.1 mediated cell proliferation in CRC cells.

(A) The reporter vector was co-transfected to CRC cells, which were treated by lncRNA-uc002kmd.1 siRNA or control siRNA. The luciferase signal was significantly decreased. (B) Cells were harvested and the stability of lncRNA-uc002kmd.1 mRNA was analyzed by qRT-PCR relative to time 0 after blocking new RNA synthesis with actinomycin D; data are mean±SEM, normalized to GAPDH. (C) HCT116 and SW480 cells were seeded in 96-well plates after been transfected, and cell proliferation was performed daily for 3 days using the CCK-8 assay. Six replicates for each group and the experiment repeated three times. Data aremean±SEM. *P<0.05 compared with controls. (D) The data showed tumor volumes of xenografts in each group 4 weeks after subcutaneously implanted stable CRC cells. Mean tumor volumes from six nude mice of each group are shown at different time points. *P<0.05 compared with controls.