Inhibition of miR-20a-5p Suppresses Epithelial-Mesenchymal Transition of Colorectal Cancer Cells Through GJA1

Abstract

This study was designed to clarify the role of GJA1 in colorectal cancer. qPCR was adopted to detect the GJA1 and miR-20a-5p expression levels in tumor tissues and cells; and EdU, Transwell assay, Scratch test to examine the migration, invasion, and proliferation of colorectal cancer cells. The EMT-related protein expression was measured by immunofluorescence and western Blot. The binding relationship between GJA1 and miR-20a-5p was examined using dual luciferase reporting subsystem. In situ hybridization was utilized to examine the miR-20a-5p expression in tumor tissues and metastases. Rescue experiments were performed by simultaneous transfection of sh-GJA1 inhibitor and miR-20a-5p inhibitor. The miR-20a-5p expression was high and the GJA1 expression was low in colorectal cancer tissues and cells. A targeting relationship was found in GJA1 and miR-20a-5p targets. The invasion, migration, and proliferation of colorectal cancer cells can be inhibited by overexpression of GJA1. Meanwhile, overexpression of GJA1 markedly elevated the e-cadherin expression, but reduced the levels of vimentin, α-SMA and n-cadherin expression. miR-20a-5p inhibitor + sh-GJA1 promoted the invasion, migration, and proliferation of colon cancer cells and EMT process. Overall, miR-20a-5p could target GJA1 to down-regulate the GJA1 expression, thereby regulating the EMT response, and ultimately promoting the progression of colorectal cancer.

Keywords: Colorectal cancer; EMT; GJA1; Invasion; Metastasis; MiR-20a-5p.

Fig. 1

Expression of GJA1 in colorectal cancer tissue and cells. A The expression level of GJA1 mRNA in cancer tissue by qPCR. B The expression of GJA1 in cancer tissues. C The content of GJA1 in different cell lines. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001

Fig. 2

Overexpression of GJA1 to detect the proliferation, migration and invasion of colorectal cancer cells. A GJA1 protein expression after transfection. B Effect of overexpression of GJA1 on cell proliferation. C Effect of overexpression of GJA1 on cell migration. D Effect of overexpression of GJA1 on cell invasion. ** indicates P < 0.01, and *** indicates P < 0.001

Fig. 3

Overexpression of GJA1 can inhibit EMT process in colon cancer cells. A The immunofluorescence detection of the expression of EMT-related proteins. B The expression of the four proteins detected by Western blot. ** indicates P < 0.01, and *** indicates P < 0.001

Fig. 4

The targeting relationship between miR-20a-5p and GJA1. A The three online tools, DIANA, miRWalk, and miRDB are used to search for miRNA upstream of GJA1. B The binding site of miR-20a-5p and GJA1. C The detection of the binding relationship between GJA1 and miR-20a-5p using dual luciferase reporter system. D The expression of GJA1 after miR-20a-5p was inhibited. E The expression of miR-20a-5p in tumor tissues and metastatic tissues were detected by in situ hybridization. F The expression of miR-20a-5p in normal tissues and colorectal cancer tissues. G. The expression of miR-20a-5p in FHC, SW480, HCT116, HT29, Caco2 cells. ** indicates P < 0.01, and *** indicates P < 0.001

Fig. 5

The rescue experiment verified the function of miR-20a-5p in migration and invasion of colorectal cancer. A The transfection efficiency was verified by qPCR. B The proliferation of SW480 cells after inhibitor addition and knockdown. C The migration of SW480 cells after inhibitor addition and knockdown. D The invasion of SW480 cells after inhibitor addition and knockdown. *** indicates P < 0.001,^### indicates P < 0.001

Fig. 6

The rescue experiment verified the function of miR-20a-5p in regulating EMT. A The expression of four proteins related to EMT by immunofluorescence. B The expression of these proteins by Western blot. ** indicates P < 0.01, *** indicates P < 0.001, ^# indicates P < 0.05, ^## indicates P < 0.01, and ^### indicates P < 0.001