Identification and Validation of New Cancer Stem Cell-Related Genes and Their Regulatory microRNAs in Colorectal Cancerogenesis

Abstract

Significant progress has been made in the last decade in our understanding of the pathogenetic mechanisms of colorectal cancer (CRC). Cancer stem cells (CSC) have gained much attention and are now believed to play a crucial role in the pathogenesis of various cancers, including CRC. In the current study, we validated gene expression of four genes related to CSC, L1TD1, SLITRK6, ST6GALNAC1 and TCEA3, identified in a previous bioinformatics analysis. Using bioinformatics, potential miRNA-target gene correlations were prioritized. In total, 70 formalin-fixed paraffin-embedded biopsy samples from 47 patients with adenoma, adenoma with early carcinoma and CRC without and with lymph node metastases were included. The expression of selected genes and microRNAs (miRNAs) was evaluated using quantitative PCR. Differential expression of all investigated genes and four of six prioritized miRNAs (hsa-miR-199a-3p, hsa-miR-335-5p, hsa-miR-425-5p, hsa-miR-1225-3p, hsa-miR-1233-3p and hsa-miR-1303) was found in at least one group of CRC cancerogenesis. L1TD1, SLITRK6, miR-1233-3p and miR-1225-3p were correlated to the level of malignancy. A negative correlation between miR-199a-3p and its predicted target SLITRK6 was observed, showing potential for further experimental validation in CRC. Our results provide further evidence that CSC-related genes and their regulatory miRNAs are involved in CRC development and progression and suggest that some them, particularly miR-199a-3p and its SLITRK6 target gene, are promising for further validation in CRC.

Keywords: cancer stem cells; colorectal cancer; differentially expressed genes; qPCR.

Figure 1

Identification and prioritization of miRNAs. Legend: DEG, differentially expressed genes; DSE, destabilising elements; ΔG, minimum free energy; ID, identification.

Figure 2

Expression (∆Cq) of four genes (L1TD1, SLITRK6, ST6GALNAC1, TCEA3) in normal mucosa, adenoma and adenoma with early carcinoma. Legend: x, mean; ◦, outlier; ** p ≤ 0.01.

Figure 3

Expression (∆Cq) of the investigated genes in carcinoma without (a) and with lymph node metastases (b) and corresponding normal mucosa. Legend: CRC N0, colorectal carcinoma without lymph node metastases; CRC N+, colorectal carcinoma with lymph node metastases; x, mean; ◦, outlier; * p ≤ 0.05; ** p ≤ 0.01.

Figure 4

Expression (∆∆Cq) of the investigated genes (L1TD1, SLITRK6, ST6GALNAC1, TCEA3) in carcinoma without and carcinoma with lymph node metastases. Legend: CRC N0, colorectal carcinoma without lymph node metastases; CRC N+, colorectal carcinoma with lymph node metastases; x, mean; ◦, outlier; *** p ≤ 0.001.

Figure 5

Expression (∆Cq) of the investigated miRNAs in normal mucosa, adenoma and adenoma with early carcinoma. Legend: x, mean; ◦, outlier; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 6

Expression (∆Cq) of the investigated miRNAs in carcinoma without (a) and with lymph node metastases (b) in comparison to corresponding normal mucosa. Legend: CRC N0, colorectal carcinoma without lymph node metastases; CRC N+, colorectal carcinoma with lymph node metastases; x, mean; ◦, outlier; * p ≤ 0.05, ** p ≤ 0.01; *** p ≤ 0.001.

Figure 7

Expression (∆∆Cq) of the investigated miRNAs in carcinoma without and with lymph node metastases. Legend: CRC N0, colorectal carcinoma without lymph node metastases; CRC N+, colorectal carcinoma with lymph node metastases; x, mean; ◦, outlier; ** p ≤ 0.01.

Figure 8

Expression of L1TD1 and miR-1303 in adenoma, adenoma with early carcinoma and carcinoma without and with lymph node metastases. Legend: CRC N0, colorectal carcinoma without lymph node metastases; CRC N+, colorectal carcinoma with lymph node metastases; * p ≤ 0.05; ** p ≤ 0.01.

Figure 9

(a) Expression of SLITRK6, miR-199a-3p and miR-425-5p in adenoma, adenoma with early carcinoma and carcinoma without and with lymph node metastases; (b) Correlation between expression (∆Cq) of miR-199a-3p and target gene SLITRK6. Legend: CRC N0, colorectal carcinoma without lymph node metastases; CRC N+, colorectal carcinoma with lymph node metastases; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 10

Expression of TCEA3, miR-1225-3p and miR-1233-3p in adenoma, adenoma with early carcinoma and carcinoma without and with lymph node metastases. Legend: CRC, colorectal carcinoma; N0, without lymph node metastases; N+, with lymph node metastases; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.