The Molecular Motor Myosin 5B and Its Folding Chaperone UNC45A Are Decreased in Colorectal Cancer

Figure 1. MYO5B mRNA is decreased in patients with CRC. (A) RNAseq gene expression data of MYO5B throughout the body. (B) Heatmap of MYO5B and UNC45A expression in intestinal cell types. mRNA expression in transcript per million of MYO5B in (C) normal vs primary tumor samples. The observed MYO5B gene expression in transcript per million in samples is based on the following parameters: (D) gender, (E) ethnicity, (F) age, (G) tumor stage, or (H) type. (H) qPCR analysis of MYO5B expression in healthy normal colon tissue compared with CRC tumor samples. (I) Survival analysis of patients with CRC with low to high expression of MYO5B in CRC tissue. ∗P ≤ .05.

Figure 2. MYO5B is methylated not mutated in patients with CRC. (A) Distribution of mutations reported in MYO5B in patients with CRC depicted in a lollipop mutation diagram. (B) MYO5B mutation types reported in patients with CRC. (C) DNA methylation map of chr18:49844194.50219231 (375.04 Kb), which harbors MYO5B. (D) DMRs across MYO5B (±3 kb). Regions of elevated methylation are highlighted in blue. (E) Quantification of the methylation at the MYO5B promoter region. (F) Methylation profile of the MYO5B promoter region (cg26557404) (GSE166427) in colon samples from healthy individuals, normal tissue adjacent to the tumor and colon cancer tumors. (G) qPCR gene expression data for MYO5B in human colonic organoid monolayers or human colon cancer cell lines Caco-2, T84, HT29, and HCT-8 cell. (H) qPCR gene expression data of MYO5B in HCT-8 cells treated with demethylating agent DAC. (I) qPCR gene expression data of MYO5B in Caco-2 cells treated with the demethylating agent DAC. (J) Protein levels of MYO5B as determined by mass spectrometry-based proteomics (z-value) in normal and CRC tumor samples. ∗P ≤ .05.

Figure 3. MYO5B is decreased in CRC tumors. Immunostaining of MYO5B (magenta), alpha smooth muscle actin (yellow), gamma actin (green), and nuclei (blue) in colorectal tumors and normal colon tissue. Scale bars = 100 mM.

Figure 4. UNC45A mRNA is unaltered in patients with CRC but is decreased at the protein level. (A) RNAseq gene expression data of UNC45A throughout the body. mRNA expression in transcript per million of UNC45A in (B) normal vs primary tumor samples. The observed UNC45A gene expression in CRC is irrespective of (C) gender, (D) ethnicity, (E) age, (F) tumor stage, or (G) type. Protein levels as determined by mass spectrometry-based proteomics (z-value) in normal and CRC tumor samples for (H) UNC45A, ∗P ≤ .05.

Figure 5. The UNC45A gene has a conserved miRNA targeting site across diverse species. microScan of UNC45A gene identifies region targeted by miRNAs.

Figure 6. UNC45A targeting miRNAs are elevated in CRC tumors compared with normal tissue. Using the following datasets, GSE18392 and GSE73487, UNC45A targeting genes were examined for their expression in patients with CRC. Data is expressed as miRNA levels in normal and tumor samples. Data presented for: (A) miR-miR-296, (B) miR-423, (C) miR-1249, (D) miR-3129-5p, (E) miR-485-5p, (F) miR-603, (G) miR-31-5p, (H) miR-199b-3pp, (I) miR-449a, (J) miR-449-5p, (K) miR-6510-5p, and (L) miR-34a-5p. ∗P ≤ .05.

Figure 7. CRC cell lines have varying levels of UNC45A targeting miRNAs. (A) hsa-miR-296. (B) hsa-miR-423. (C) hsa-miR-1249. n = 6 replicates ∗P < .05, 1-way ANOVA.

Figure 8. Inhibition of hsa-miR-296-3p in human colon cancer derived T84 cells results in elevated UNC45A protein. (A) Western blot of UNC45A and ponceau S stain of total protein in control and hsa-miR-296-3p inhibited cells. (B) Quantification of UNC45A normalized to total Ponceau protein. n = 3 replicates ∗P < .05, Student’s t-test.