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. 2022 Feb 15;14(2):1361-1375.
eCollection 2022.

Over-expression of miR-193a-3p regulates the apoptosis of colorectal cancer cells by targeting PAK3

Tao Ma  1 , Hai Li  2 , Wenjing Yang  1 , Quanxia Liu  1 , Hui Yan  1
Affiliations

Over-expression of miR-193a-3p regulates the apoptosis of colorectal cancer cells by targeting PAK3

Tao Ma et al. Am J Transl Res. .

Abstract

Although dysregulated expression of microRNAs (miRNA) has been investigated in colorectal cancer (CRC), MiR-193a-3p, as a tumor inhibitor, is less studied. To investigate the function and mechanism of miR-193a-3p in CRC, the potential function of miR-193a-3p in regulating PAK3 in CRC with a series of experimental assays including western blotting, qRT-PCR, bioinformatics analysis, a luciferase reporter assay, flow cytometry, Transwell assay, CCK8 assay and immunofluorescence were performed in this study. The results showed that miR-193a-3p was down-regulated in CRC tissues and cell lines, which was also correlated with tumor progression. PAK3 was predicted as a target gene of miR-193a-3p in CRC cells by TargetScan database, which was confirmed by luciferase assays. Moreover, overexpression of miR-193a-3p suppressed the viability, cell cycle progression, migration, and invasion, and induced apoptosis of CRC cells in vitro by regulating the PAK3 signaling pathway. Therefore, miR-193a-3p may serve as a tumor suppressor and potential target for CRC treatment.

Keywords: PAK3; apoptosis; colorectal cancer; miR-193a-3p; regulate.

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Conflict of interest statement

None.

Figures

Figure 1
Figure 1
Expression of miR-193a-3p and PAK3 in CRC tissues and cell lines. A. MiR-193a-3p was downregulated in CRC tissues (adjacent normal tissues vs. CRC tissues). B. The mRNA expression of PAK3 in CRC tissues was measured by qRT-PCR assay (adjacent normal tissues vs. CRC tissues). C. The mRNA expression of PAK3 was negatively correlated with miR-193a-3p in tumors. D. Low expression of miR-193a-3p was associated with the poor prognosis. E. Representative IHC staining of PAK3 in CRC tissues. F. ISH assay for the expression of miR-193a-3p in CRC tissues. G and H. The expression of miR-193a-3p and PAK3 mRNA in CRC cell lines (normal control cells vs. CRC cells). I and J. The protein expression of PAK3 in CRC cells (normal control cells vs. CRC cells). CRC cell lines: SW480, SW620, HCT116, DLD1, Caco2. Normal cell line: CCD-18Co. Statistical significance: *P<0.05, **P<0.01, ***P<0.005.
Figure 2
Figure 2
PAK3 was proved to be a direct target of miR-193a-3p. A. The first proximal and conserved binding sites of miR-193a-3p on PAK3 mRNA 3’-UTR. B and E. The luciferase activity of the wild type target sequences (WT), but not the mutant type (MUT), was suppressed by miR-193a-3p mimics. C, D, F and G. The mRNA and protein levels of PAK3 were negatively regulated by miR-193a-3p in SW620 and DLD1 cells. H. PAK3 was validated as a direct target of miR-193a-3p in DLD1 cells. CRC cell lines: SW620, DLD1. Statistical significance: *P<0.05, **P<0.01, ***P<0.005.
Figure 3
Figure 3
MiR-193a-3p inhibited cell proliferation, cell cycle and enhanced cell apoptosis. A. MiR-193a-3p inhibited the proliferation of CRC cells in vitro as analyzed using CCK-8 assay. B. Overexpression of miR-193a-3p increased the number of apoptotic cells, while knockdown of miR-193a-3p decreased the apoptotic cells. C. Cell cycle distribution of CRC cells was analyzed by Flow cytometer. CRC cell lines: SW620, HCT116, DLD1. Statistical significance: *P<0.05, **P<0.01, ***P<0.005.
Figure 4
Figure 4
MiR-193a-3p promoted cell apoptosis by regulating apoptosis-related proteins. A. The cell apoptosis was analyzed by Hoechst 33258 staining; nuclear morphological changes were observed by fluorescence microscope. B. Bar charts of the results of Hoechst 33258 staining. C. The protein expression of caspased-3/9 and Apaf-1 was stimulated by miR-193a-3p, while inhibited by miR-193a-3p inhibitor. CRC cell lines: SW620, HCT116, DLD1. Statistical significance: *P<0.05, **P<0.01, ***P<0.005.
Figure 5
Figure 5
MiR193a-3p inhibited migration, invasion and the expression of CD73/CD133 in CRC cells. (A and B) Cell migration (A) and invasion (B) were inhibited by miR-193a-3p mimics and promoted by miR-193a-3p inhibitors. (C) The expression of CD73/133 molecules in CRC cells transfected with miR-193a-3p mimics and inhibitors. (D) Bar graphs of mean fluorescence intensities of CD73/133 in CRC cells. (E) A schematic model showing that miR-193a-3p expression in CRC leads to the activation of its target gene, PAK3, which contributes to the apoptosis of CRC by activating Apaf-1 and caspase-9 pathway and the restriction of metastasis of CRC by inhibiting the expression of CD73 and CD133. CRC cell lines: SW620, HCT116, DLD1. Statistical significance: *P<0.05, **P<0.01, ***P<0.005.
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