A bioinformatics analysis of the target role of miRNA-431-5p on KLK6 in colorectal cancer

Abstract

Background: Although increasing evidence suggests that microRNAs (miRNAs) play different roles in the occurrence, development, and prognosis of colorectal cancer (CRC), investigations on miRNA-targeted regulation in CRC are sparse. However, the high morbidity and mortality of CRC necessitates exploring this area of research for potential alternative therapeutic approaches to CRC.

Methods: Bioinformatics analysis was used to obtain the key Hub genes related to CRC, and survival analysis was performed to screen out the core genes. Meanwhile, verification was performed using CCK-8, Transwell, qPCR, WB, immunohistochemistry and dual luciferase assays at a cellular level.

Results: This study identified the hub gene KLK6 associated with CRC based on the GEO and TCGA databases. Survival analysis revealed a significant decrease in the survival rate of CRC patients with increasing expression levels of KLK6. Target gene prediction confirmed that miR-431-5p can target KLK6. Cell experimental results demonstrated that the miR-431-5p inhibitor enhanced cell viability and promoted cell migration and invasion while miR-431-5p mimics reduced cell viability and inhibited cell migration and invasion. Both the inhibitor and mimics of miR-431-5p suppressed and promoted the expression of miR-431-5p, as well as promoted and inhibited the KLK6 mRNA and protein expression. Dual luciferase results showed that miR-431-5p targeted KLK6, and cell recovery experiments further verified that miR-431-5p regulated cell viability, migration and invasion by targeting KLK6.

Conclusions: Through target gene prediction, miR-431-5p was found to target KLK6, suggesting its therapeutic potential in CRC. As such, therapies that can inhibit KLK6 via miR-431-5p offer a promising approach to CRC.

Clinical trial number: Not applicable.

Keywords: Bioinformatics; Colorectal cancer; KLK6; WGCNA; miR-431-5p.

Fig. 1

Analysis of Differentially Expressed miRNAs and mRNAs. (A) Differentially expressed miRNA screening. (B) Screening of differentially expressed mRNAs. (C) Bar graph of GO enrichment analysis. (D) Scatter plot of KEGG enrichment analysis

Fig. 2

WGCNA and enrichment analysis. (A) Scale-free index analysis of various soft threshold powers (β). (B) Cluster module tree of DEGs (top) and color tape (bottom). (C) Gene clustering tree based on topological overlap, and assigned module colors. (D) Analysis of correlations between modules and disease traits. (E) Bar graph of GO enrichment analysis. (F) Scatter plot of KEGG enrichment analysis

Fig. 3

Hub genetic screening and the prognosis of survival analysis. (A) PPI Network of DEGs. (B) The Hub Genes of Its Core Module. (C) Targeting relationship prediction. (D) Prognostic analysis and expression verification of KLK6. (E) Prognostic analysis and expression verification of KRT6A. (F) Prognostic analysis and expression verification of KRT6C

Fig. 4

miR-431-5p targets KLK6 validation. (A) qPCR detection of expression of miR-431-5p and KLK6 after transfection. (B) CCK-8 detection of cell viability after transfection. (C) WB detection of expression of KLK6 protein after transfection. * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 5

Transwell detection of HCT116 cell migration and invasion abilities (A) Transwell assay for cell migration after miR-431-5p transfection (100×). (B) Transwell assay for cellular invasion after miR-431-5p transfection (100×). *** p < 0.001

Fig. 6

miR-431-5p targets KLK6. (A) Dual luciferase reporter gene assay was used to verify the relationship between miRNA and target gene; (B) The expression of miR-431-5p in HCT116 cells was detected by qPCR; (C) qPCR was used to detect the expression of KLK6 mRNA in HCT116 cells; (D) CCK-8 assay was performed to detect the cell proliferation of HCT116 cells. * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 7

The expression of KLK6 protein in HCT116 cells is detected by immunohistochemistry. ** p < 0.01, *** p < 0.001

Fig. 8

miR-431-5p targets KLK6 to regulate cell migration and invasion. (A) The migration ability of HCT116 cells was detected by Transwell (200×); (B) The invasion ability of HCT116 cells was measured by Transwell (200×). ** p < 0.01, *** p < 0.001