In Vitro and In Silico Mechanistic Insights into miR-21-5p-Mediated Topoisomerase Drug Resistance in Human Colorectal Cancer Cells

Abstract

Although chemotherapy for treating colorectal cancer has had some success, drug resistance and metastasis remain the major causes of death for colorectal cancer patients. MicroRNA-21-5p (hereafter denoted as miR-21) is one of the most abundant miRNAs in human colorectal cancer. A Kaplan-Meier survival analysis found a negative prognostic correlation of miR-21 and metastasis-free survival in colorectal cancer patients (The Cancer Genome Atlas Colon Adenocarcinoma/TCGA-COAD cohort). To explore the role of miR-21 overexpression in drug resistance, a stable miR-21-overexpressing clone in a human DLD-1 colorectal cancer cell line was established. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell viability assay found that miR-21 overexpression induced drug resistance to topoisomerase inhibitors (SN-38, doxorubicin, and etoposide/VP-16). Mechanistically, we showed that miR-21 overexpression reduced VP-16-induced apoptosis and concomitantly enhanced pro-survival autophagic flux without the alteration of topoisomerase expression and activity. Bioinformatics analyses suggested that miR-21 overexpression induced genetic reprogramming that mimicked the gene signature of topoisomerase inhibitors and downregulated genes related to the proteasome pathway. Taken together, our results provide a novel insight into the role of miR-21 in the development of drug resistance in colorectal cancer.

Keywords: Connectivity Map; autophagy; colorectal cancer; drug resistance; microRNA.

Figure 1

The prognostic impact of microRNA-21-5p (miR-21) on overall (A) and metastasis-free (B) survival in colorectal cancer (CRC). The prognostic impact of miR-21 in CRC was analyzed using the PROGmiR database (http://www.compbio.iupui.edu/progmir). The input “has-mir-21” was queried, and Kaplan–Meier survival plots were generated based on the expression data of colon adenocarcinoma (COAD) from The Cancer Genome Atlas (TCGA; https://tcga-data.nci.nih.gov/tcga). Patients with high (n = 181) and low (n = 180) miR-21 expression were bifurcated at the median value. HR, hazard ratio; PVAL, p value.

Figure 2

Effect of miR-21 overexpression on chemosensitivity. (A) mRNA expression of miR-21 and programmed cell death 4 (PDCD4) in corresponding vector-overexpressing DLD-1 (DLD-1-vector) and miR-21-overexpressing DLD-1 (DLD-1-miR-21) cells were analyzed by qPCR. (B) Protein expressions of PDCD4 in DLD-1-vector and DLD-1-miR-21 cells were analyzed by Western blot analysis. (C) Growth rates of DLD-1-vector and DLD-1-miR-21 cells were measured by cell counts at approximately 1 to 4 days. p < 0.05 (*) indicates significant differences between DLD-1-miR-21 and DLD-1-vector cells. (D) Cell morphology was observed under bright-field microscopy. (E) DLD-1-vector and DLD-1-miR-21 cells were treated with various doses of 5-fluorouracil (5-FU), SN-38, doxorubicin, and VP-16 for 72 h. Cell viability was analyzed by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***) indicates significant differences between DLD-1-miR-21 and DLD-1-vector cells. n.d., not determined. (F) DLD-1-vector and DLD-1-miR-21 cells were treated with various doses of VP-16 for 48 h. Whole-cell lysates were prepared and subjected to a Western blot analysis. (G) DLD-1-vector and DLD-1-miR-21 cells were treated with various doses of VP-16 for 24 h. Whole-cell lysates were prepared and subjected to a Western blot analysis. (H) DLD-1-vector and DLD-1-miR-21 cells were treated with various doses of VP-16 for 1 h. A band-depletion assay was performed as described in “Materials and Methods”.

Figure 3

Effect of autophagy on miR-21-induced drug resistance. (A) DLD-1-vector and DLD-1-miR-21 cells were treated with various doses of VP-16 for 24 and 48 h. Whole-cell lysates were prepared and subjected to a Western blot analysis. (B) DLD-1-vector and DLD-1-miR-21 cells were treated with various doses of VP-16 with or without 1 μM rapamycin (Rapa) for 48 h. Whole-cell lysates were prepared and subjected to a Western blot analysis. (C) DLD-1-vector and DLD-1-miR-21 cells were treated with various doses of VP-16 with or without 0.5 μM rapamycin for 72 h. Cell viability was examined by an MTT assay. p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***) indicates significant differences between rapamycin-treated and control cells. (D) DLD-1-vector and DLD-1-miR-21 cells were treated with various doses of bafilomycin A1 for 72 h. Cell viability was analyzed by an MTT assay. p < 0.001 (***) indicates significant differences between DLD-1-vector and DLD-1-miR-21 cells. (E) DLD-1-vector and DLD-1-miR-21 cells were transfected with ATG7 or non-targeting siRNAs for 48 h before exposure to 5 μM VP-16 for 48 h. Then, whole-cell lysates were prepared and subjected to a Western blot analysis. (F) The untreated whole-cell lysates from DLD-1-vector and DLD-1-miR-21 cells were analyzed by Western blot analysis.

Figure 4

Microarray analysis of DLD-1-miR-21 cells. The total RNA isolated from DLD-1-vector and DLD-1-miR-21 cells was subjected to a microarray analysis as described in “Materials and Methods”. Differentially expressed genes (DEGs) were prepared for the next-generation Connectivity Map (CMap) analysis (A) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (B). In (A), the top 10 most similar perturbational classes to miR-21 overexpression are shown. The gray grids indicate “not measured”. In (B), the top 10 enriched pathways are plotted on the Y-axis versus a measure of significance (negative logarithm of the p value or Q-value) on the X-axis. The Q-value was calculated by Benjamini. Embedded figure in (B): DLD-1-vector and DLD-1-miR-21 cells were treated with or without 10 μM MG132 for 4 h. Whole-cell lysates were prepared and subjected to Western blot analysis.

Figure 5

Proteasome pathway mapping of altered genes in DLD-1-miR-21 cells. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed as described in “Materials and Methods”. Differentially expressed genes (DEGs) in DLD-1-miR-21 cells were mapped to the KEGG proteasome pathway (hsa03050). The genes highlighted in red and green indicated the upregulated and downregulated genes in DLD-1-miR-21 cells. The original data are shown in Table S2.

Figure 6

Prognostic impacts of proteasome gene alterations on overall (A) and metastasis-free (B) survival in colorectal cancer (CRC). The prognostic impact of the miR-21-downregulated proteasome gene signature (PSME3, PSMA1, PSMA2, PSMA3, PSMB2, PSMB3, PSMB4, PSMB5, PSMB8, PSMB10, PSMC2, PSMC3, PSMC4, PSMC5, PSMC6, PSMD3, PSMD4, PSMD11, PSME1, PSMF1, and PSMD6) in CRC was evaluated using the PROGgeneV2 database (http://www.compbio.iupui.edu/proggene/). Two CRC patient datasets (GSE28722 and GSE28814) were employed to generate the Kaplan–Meier survival plots. Patients with high (n = 61 or 63) and low (n = 61 or 62) miR-21 expression in GSE28722 and GSE28144, respectively, were bifurcated at the median value. HR, hazard ratio; PVAL, p value.