Fig 1: PLAGL2 expression and the association between CNV and PLAGL2 expression in CRC. (A) PLAGL2 expression in CRC samples and normal samples was analysed using starBase v2.0. (B) PLAGL2 expression in CRC tissues and normal tissues (n=20) was detected by reverse transcription-quantitative polymerase chain reaction. (C) The main somatic variation of the PLAGL2 gene and correlation analysis between CNV and PLAGL2 expression in CRC, through analyzing The Cancer Genome Atlas data. (D) CNV does not fully explain the high expression of PLAGL2 in CRC, as determined through analysing COSMIC data. Data are presented as the mean ± standard deviation. *p<0.05 and **p<0.01, with comparisons indicated by lines. PLAGL2, pleomorphic adenoma gene like-2; CNV, copy number variation; CRC, colorectal cancer.
Fig 2: HuR protein may stabilize PLAGL2 mRNA. (A) Correlation analysis between PLAGL2 expression and HuR expression in CRC was analysed using starBase v2.0. (B) HuR expression in CRC samples and normal samples was analysed using starBase v2.0. (C) HuR protein expression in 6 randomly selected, paired CRC tissues and normal tissues, was detected by western blotting. (D) Co-precipitated RNAs put down by HuR protein and IgG (negative control) were detected by RT-qPCR. (E) Following transfection with empty vector (negative control) and pCMV6-HuR (HuR-expressing plasmid), the mRNA expression of HuR and PLAGL2 was detected by RT-qPCR and the protein expression of PLAGL2 were detected by western blotting. Data are presented as the mean ± standard deviation. **p<0.01, with comparisons indicated by lines. HuR, human antigen R; PLAGL2, pleomorphic adenoma gene like-2; CRC, colorectal cancer; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
Fig 3: The effects of PLAGL2 3′-UTR on tumor growth in vivo. (A) Growth curve. The tumor volume was measured every 3 days from 10 days after inoculation. (B) Representative images of tumors, with (C) quantification. Tumors were removed and weighed on the 30th day following inoculation (n=5 for each group). Data are presented as the mean ± standard deviation. *p<0.05 and **p<0.01, with comparisons indicated by lines. PLAGL2, pleomorphic adenoma gene like-2; 3′-UTR, 3′-untranslated region.
Fig 4: Bioinformatics analysis of the potential miRNAs of PLAGL2. (A) A venn diagram shows the potential miRNAs of PLAGL2, which were predicted by Starbase, TargetScan, miRTarBase and Tarbase software. (B) Correlation analysis and the expression of miRNAs in CRC were provided by Starbase v2.0. miRNA, microRNA; PLAGL2, pleomorphic adenoma gene like-2.
Fig 5: C-MYC and CD44 expression were regulated by PLAGL2 3′-UTR. (A) A chart shows the potential candidate ceRNA genes of PLAGL2 3′-UTR. The mRNA expression of candidate ceRNAs in (B) SW620 and (C) HCT116 cells were detected by reverse transcription-quantitative polymerase chain reaction following transfection with lentivirus. (D) Protein expression of C-MYC and CD44 in SW620 and HCT116 cells were detected by western blotting following transfection with lentivirus. Data are presented as the mean ± standard deviation. *p<0.05 and **p<0.01, with comparisons indicated by lines. PLAGL2, pleomorphic adenoma gene like-2; 3′-UTR, 3′-untranslated region; ceRNA, competing endogenous RNA.
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