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. 2022 Dec 30;25(2):72.
doi: 10.3892/ol.2022.13658. eCollection 2023 Feb.

MUC13-miRNA-4647 axis in colorectal cancer: Prospects to identifications of risk factors and clinical outcomes

Ladislav Sojka  1   2 , Alena Opattova  3   4   5 , Linda Bartu  3 , Josef Horak  3   6 , Vlasta Korenkova  7 , Vendula Novosadova  8 , Vera Krizkova  9 , Jan Bruha  3   5   10 , Vaclav Liska  3   5   10 , Michaela Schneiderova  11 , Ondrej Kubecek  12 , Ludmila Vodickova  3   4   5 , Marketa Urbanova  3 , Jaromir Simsa  1 , Pavel Vodicka  3   4   5 , Veronika Vymetalkova  3   4   5
Affiliations

MUC13-miRNA-4647 axis in colorectal cancer: Prospects to identifications of risk factors and clinical outcomes

Ladislav Sojka et al. Oncol Lett. .

Abstract

MUC13, a transmembrane mucin glycoprotein, is overexpressed in colorectal cancer (CRC), however, its regulation and functions are not fully understood. It has been shown that MUC13 protects colonic epithelial cells from apoptosis. Therefore, studying MUC13 and MUC13-regulated pathways may reveal promising therapeutic approaches for CRC treatment. Growing evidence suggests that microRNAs (miRs) are involved in the development and progression of CRC. In the present study, the MUC13-miR-4647 axis was addressed in association with survival of patients. miR-4647 is predicted in silico to bind to the MUC13 gene and was analyzed by RT-qPCR in 187 tumors and their adjacent non-malignant mucosa of patients with CRC. The impact of previously mentioned genes on survival and migration abilities of cancer cells was validated in vitro. Significantly upregulated MUC13 (P=0.02) in was observed tumor tissues compared with non-malignant adjacent mucosa, while miR-4647 (P=0.05) showed an opposite trend. Higher expression levels of MUC13 (log-rank P=0.05) were associated with worse patient's survival. The ectopic overexpression of studied miR resulted in decreased migratory abilities and worse survival of cells. Attenuated MUC13 expression levels confirmed the suppression of colony forming of CRC cells. In summary, the present data suggested the essential role of MUC13-miR-4647 in patients' survival, and this axis may serve as a novel therapeutic target. It is anticipated MUC13 may hold significant potential in the screening, diagnosis and treatment of CRC.

Keywords: MUC13; colorectal cancer risk and clinical outcomes; microRNA; translation research.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.
Graphical representation of in silico predicted binding microRNA-4647 in the polymorphic seed sequence of MUC13 rs1532602.
Figure 2.
Figure 2.
Expression of MUC13 and related miRNA in patients with colorectal cancer. (A) Expression of MUC13 in tumor and non-malignant tissue. (B) Expression of miR-4647 in tumor and non-malignant tissue (Total n=187). *P<0.05. miR, microRNA.
Figure 3.
Figure 3.
Expression correlation of analyzed genes. (A and B) Negative correlation of MUC13 and miR-4647 in (A) tumor tissue and (B) in non-malignant tissue (Total n=187). miR, microRNA.
Figure 4.
Figure 4.
Kaplan-Meier overall survival curves stratified for high and low expression for MUC13 and miR-4647. (A) MUC13. (B) miR-4647 (Total n=187). Survival analysis was performed using the log-rank test and Kaplan-Meier plot approach. miR, microRNA.
Figure 5.
Figure 5.
Kaplan-Meier overall survival curves stratified for rs1532602 in MUC13 in CRC patients with different genotypes. (A) Homozygous GG genotype (P=0.04), (B) heterozygous GA genotype, (C) homozygous variant AA genotype. Survival analysis was performed using the log-rank test and Kaplan-Meier plot approach.
Figure 6.
Figure 6.
Immunohistochemical staining of MUC13 in different parts of colon. (A) Non-malignant ileum, (B) non-malignant colon, (C) non-malignant rectum, (D) cytoplasm of cancer cells and (E) endothelial cancer cells. (F) Figure from Ellipse software of cancer cells using a point-counting method where yellow points stand for positivity for mucin 13, green points for negative.
Figure 7.
Figure 7.
Determination of MUC13 genotype status in colorectal cancer cell lines. HCT-116 (H line) and DLD-1 (D line) evinced heterozygous GA genotype for MUC13 rs1532602.
Figure 8.
Figure 8.
Transfection efficacy. (A and B) Transfection efficacy by miR-4647 mimics in (A) DLD1 and (B) HCT-116 cells. (C) Western blotting of silenced MUC13 by miR-4647. All presented results are average of 3 independent experiments (Man-Whitney test). *P<0.05 and **P<0.01. miR, microRNA; NC, negative control.
Figure 9.
Figure 9.
Expression levels of MUC13 after overexpression of miR-4647. (A and B) Expression of MUC13 decreased after miR-4647 overexpression in (A) DLD1 and (B) HCT-116 cells. (C and D) The adenylate kinase levels after miR-4647 overexpression in (C) DLD1 and (D) HCT-116 cells. All presented results are average of 3 independent experiments (Man-Whitney test). *P<0.05 and **P<0.01. miR, microRNA; NC, negative control.
Figure 10.
Figure 10.
Effect of overexpression of miR-4647 on growth and invasion behaviour of CRC cells. (A and B) The effect of miR overexpression on the proliferation of (A) DLD1 and (B) HCT-116 cells. (C and D) Effect of miR overexpression on the migratory ability of (C) DLD1 and (D) HCT-116 cells. All presented results are average of 3 independent experiments (Man-Whitney test). *P<0.05 and **P<0.01. miR, microRNA; NC, negative control.
Figure 11.
Figure 11.
Effect of silenced MUC13 on HCT-116 cell proliferation and migration. (A) The control expression of MUC13 after silencing. (B) The western blot analysis of silenced MUC13. (C) The effect of MUC13 silencing on the proliferation of CRC cells. (D) Migratory behaviour of CRC cells after MUC13 silencing. All presented results are average of 3 independent experiments (Man-Whitney test). *P<0.05 and ***P<0.001. NC, negative control; si-, small interfering.
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