A New Light on Potential Therapeutic Targets for Colorectal Cancer Treatment

Figures

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  Figure 1. Relative expression of colorectal neoplasia differentially expressed (CRNDE) in colorectal cancer (CRC) tissues.(A) Expressions of CRNDE mRNA in 20 common cancers were compared with those in corresponding normal tissues (in the Oncomine Database). The search criteria thresholds for datasets of cancer versus normal analysis were a multiple of change of ≥2, a p value of <0.05, and a gene rank in the top 10%. Red represents gene overexpression in the analyses; blue represents gene under-expression. (B) Relative CRNDE expression in human CRC tissues compared to noncancerous tissues via a GSE21815 data analysis. (C) Relative expression levels of CRNDE in normal colon/rectum tissues and CRC tissues using the TCGA database. (D and E) Data are presented as relative expression levels in tumor tissues. CRNDE expression was significantly increased in patients at a higher pathological stage and with larger tumors. Kaplan–Meier analysis of overall survival (F) and disease-free survival (G) of CRC patients with the corresponding expression profiles: CRNDE (low) and CRNDE (high). Log-rank analysis was used for comparison between groups.* p < 0.05, ** p < 0.01, *** p < 0.001. ns: non-significance.

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  Figure 2. Colorectal neoplasia differentially expressed (CRNDE) regulates the proliferation of colorectal cancer (CRC) cells. (A) Expression levels of CRNDE in 16 CRC cell lines were obtained from the CellExpress database. (B) CRNDE levels in HCT-116 cells after siRNA-mediated knockdown of CRNDE were detected by an RT-qPCR. (C) An MTT assay was performed to determine the proliferation of CRNDE-depleted HCT-116 cells. (D) A colony-forming assay was performed to determine the effects of CRNDE depletion on the growth of HCT-116 cells. (E) Expression levels of CRNDE in green fluorescent protein (GFP)-CRNDE-transfected HCT-15 cells. The GFP-CRNDE-regulated cell proliferation of HCT-15 cells by an MTT assay analysis (F) and colony-forming assay (G).* p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 3. Functional roles of colorectal neoplasia differentially expressed (CRNDE) in regulating colorectal cancer (CRC) cell growth. (A) HCT-116 cells were stained with propidium iodide (PI) and analyzed using a Muse™ Cell Analyzer. (B) The quantification result of PI-positive cells with CRNDE-knockdown. (C) HCT-116 cells were stained with Annexin V-FITC and analyzed using a Muse™ Cell Analyzer. (D) Quantification of results of Annexin V-positive cells with CRNDE-knockdown. Knockdown of CRNDE-induced cytotoxicity is mediated by cell cycle regulators (E) or apoptotic regulators (F). Actin was used as a loading control. * p < 0.05, ** p < 0.01.

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  Figure 4. Colorectal neoplasia differentially expressed (CRNDE) is involved in regulating autophagy. (A) Small interfering (si)CRNDE-transfected red fluorescent protein (RFP)-LC3 reporter U2OS cells showed a shift in the histogram plot indicating autophagy induction (no autophagy in gray versus induced autophagy in red). (B) Quantification results of the autophagy intensity. (C) Western blot analysis of the effects of CRNDE-knockdown on adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and its downstream targets, mammalian target of rapamycin (mTOR) and autophagy markers in HCT-116 cells. (D) Whole-cell lysates were prepared from HCT-116 cells co-treated with siCRNDE and 3-MA and immunoblotted with antibodies against AMPK, mTOR, and p62. (E) CRNDE was silenced for 48 h with 100 nM of specific siRNA, following which cells were treated with chloroquine (CQ; an autophagy inhibitor) at the indicated concentrations for an additional 48 h. Caspase-3/7 activities were measured with a Muse Cell Analyzer. (F) Quantification results of Annexin V-positive cells with CRNDE-knockdown alone or in the presence of CQ. *** p < 0.001.

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  Figure 5. Knockdown of colorectal neoplasia differentially expressed (CRNDE) caused inhibition of lipid metabolism of colorectal cancer (CRC) cells. (A) Glucose consumption was determined in CRNDE-knockdown HCT-116 cells. (B) Analysis of the extracellular acidification rate (ECAR) of HCT-116 cells with CRNDE-knockdown. (C) HCT-116 cells were transfected with CRNDE small interfering (si)RNA for 48 h. Images of BODIPY505/515-stained cells were captured with a fluorescence microscope. (D) BODIPY505/515-stained results were quantified and are represented as multiples of change, considering the control siRNA cell value as 1-fold. Error bars represent the mean ± standard deviation (SD). (E) A gene set enrichment analysis (GSEA) showed enrichment of downregulated adipogenesis gene sets in stable CRNDE-knockdown CRC cells. Heat-map image illustrating gene expression levels of the leading edge subset. (F) Expressions of representative genes (MYLK, GPX3, and ANGPTL4) in CRNDE-knockdown HCT-116 cells. (G) Western blot analysis of the effects of CRNDE-knockdown on the phosphorylation and expression levels of lipid metabolism-associated targets in HCT-116 cells, including the phosphorylation levels of…

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  Figure 6. Colorectal neoplasia differentially expressed (CRNDE) directly interacts with miR-29b-3p. (A) Correlation analysis revealed the positive relationship between CRNDE and angiopoietin-like 4 (ANGPTL4) expressions in 132 colorectal cancer (CRC) tumor tissues. MiR-134-5p (B) and miR-29b-3p (C) expressions were determined by an RT-qPCR in CRNDE-knockdown HCT-116 cells. Expressions of CRNDE (D) and miR-29b-3p (E) in 17 normal/tumor (NT) pairs of CRC resected tumor (T) tissues and corresponding adjacent non-tumor (N) tissues obtained from a public GEO dataset (GSE32323). (F) Correlation analysis revealed a negative relationship between CRNDE and miR-29b-3p expressions in 34 cases of NT pairs of CRC tissues from the GEO dataset (GSE32323). (G) A bioinformatics analysis revealed predicted binding sites between CRNDE and miR-29b-3p. (H) A luciferase reporter assay demonstrated miR-29b-3p mimics significantly decreased the luciferase activity of CRNDE-wild type (WT) in HCT-116 cells, while miR-29b-3p mimics did not affect the luciferase activity of CRNDE-mutant (Mut). ** p < 0.01, *** p < 0.001.

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  Figure 7. High levels of colorectal neoplasia differentially expressed (CRNDE) and angiopoietin-like 4 (ANGPTL4) and a low level of miR-29b-3p in colorectal cancer (CRC) tissues involved in regulating lipid metabolism. (A) Levels of CRNDE, miR-29b-3p, and ANGPTL4 in CRC tissues were examined by IHC and ISH. (B) Percentage of cases are plotted on the y-axis, and the type of molecule is plotted on the x-axis. (C) Validation of miR-29b-3p expression levels after transfection with a miR-29b-3p mimic for 48 h in the HCT-116 cell line. (D) HCT-116 cells were transfected with a miR-29b-3p mimic for 48 h. Images of BODIPY505/515-stained cells were captured with a fluorescence microscope. (E) BODIPY505/515-stained results were quantified and are presented as multiples of change, considering the control miRNA cell value as 1-fold. Error bars represent the mean ± standard deviation (SD). (F) Western blot analysis of the effects of miR-29b-3p overexpression on the phosphorylation and expression levels of lipid metabolism-associated targets in HCT-116 cells. (G) Schematic model showing that CRNDE silencing induced autophagy of CRC cells by the miR-29b-3p-regulated inhibition of ANGPTL4, causing the…