Long Non-coding RNA LINC02474 Affects Metastasis and Apoptosis of Colorectal Cancer by Inhibiting the Expression of GZMB

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  Figure 1. Identification of expression profile characteristics of LINC02474. (A–E) Expression profile characteristics of LINC02474 from TCGA data sets (TCGA-COAD and TCGA-READ). (A) Volcano plot of DEGs. Red dots (right) represent up-regulated genes; gray dots (middle) represent genes excluded from the threshold (|log2 fold change| >1, p < 0.05); and blue dots (left) represent down-regulated genes. (B) Heatmap of DEGs (http://shinyheatmap.com/). (C) Expression of LINC02474 in 51 normal tissues and 647 tumor tissues. (D) ROC curve of LINC02474 yielded an AUC value of 0.7915 (95% CI, 0.7495–0.8335) in distinguishing 647 CRC tissues from 51 normal tissues. (E) Survival analysis of LINC02474 from TCGA data sets. (F) Expression of LINC02474 in 80 pairs tissues from CRC patients. (G) ROC curve of LINC02474 yielded an AUC value of 0.6118 (95% CI, 0.5243–0.6994) in distinguishing tumor tissues from adjacent tissues in 80 CRC patients. (H) Coding probability prediction of LINC02474 (coding probability score<0 means no coding potential; coding probability score>0 means certain coding potential.) (I) Expression of LINC02474 in CRC cells (normalized to normal cell line, FHC.). (J) Subcellular…

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  Figure 2. Depletion of LINC02474 with siRNAs may impede metastasis and promote apoptosis of CRC cells. (A) qRT-PCR results of LINC02474 expression by siRNAs. (B, C) Migration and invasion abilities of DLD-1 cells by transwell assay. Representative images (original magnification, ×100) are shown. (D, E) Expressions of apoptosis-related proteins (cleaved PARP, cleaved caspase substrate, and cleaved caspase-3) in DLD-1 cells after LINC02474 were depleted. (F, G) Apoptosis after LINC02474 depletion by flow cytometry. Results are means ± SD. * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001.

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  Figure 3. LINC02474, as an oncogene, promotes the abilities of migration and invasion but inhibits the apoptosis of CRC cells. (A, B) qRT-PCR results for knockdown efficiency of LINC02474 in DLD-1 and LOVO cells by shRNAs. Representative images (original magnification, ×200) are shown. (C, D) Migration and invasion abilities of DLD-1 and LOVO cells after LINC02474 was stably depleted. Representative images (original magnification, ×100) are shown. (E, F) Expressions of apoptosis-related proteins (cleaved PARP, cleaved caspase substrate, and cleaved caspase-3) in DLD-1 and LOVO cells after LINC02474 was stably depleted. (G, H) Apoptosis in DLD-1 and LOVO cells after LINC02474 was stably depleted by flow cytometry. Results are means ± SD. * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001.

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  Figure 4. LINC02474 promotes tumor metastasis in vivo. (A) Representative microscopic images of metastatic pulmonary lesions in two group nude mice (n = 7 for each group). Representative images (original magnification, ×50 [up] and ×400 [down]) are shown. (B) The number of metastatic nodules in the lung were counted. ** represents p < 0.01.

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  Figure 5. Identification of target genes for LINC02474 with RNA-seq. (A) Volcano plot and (B) Heatmap after LINC02474 depletion in DLD-1 cells. (C) GO, (D) KEGG, and (E) GSEA gene markers after LINC02474 depletion in DLD-1 cells. (F, G) qRT-PCR results for expression identification of selected genes in DLD-1, LOVO, and HCT116 cells. (H) Protein expression of GZMB in DLD-1, LOVO, and HCT116 cells. Results are means ± SD. * represents p < 0.05 ; ** represents p < 0.01; *** represents p < 0.001; **** represents p < 0.0001.

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  Figure 6. The influence of LINC02474 on CRC cells can be mediated by restricting transcriptional regulation of GZMB. (A) qRT-PCR and (B) Western blotting analysis for the expression of GZMB by siRNAs. (C–F) Migration and invasion after GZMB depletion in DLD-1 and LOVO cells, in which LINC02474 was stably depleted. Representative images (original magnification, ×100) are shown. (G, H) Expressions of apoptosis-related proteins and GZMB after depleting GZMB in DLD-1 cells, in which LINC02474 was down-regulated. (I) Dual-luciferase reporter gene vector of GZMB gene promoter. (J) Relative luciferase activity of GZMB gene promoter after LINC02474 was depleted. Results are means ± SD. * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001; **** represents p < 0.0001.