MEX3A promotes cell proliferation by regulating the RORA/β-catenin pathway in hepatocellular carcinoma

Figure 1 Analysis of MEX3A expression. A: Differential expression of MEX3A mRNA in hepatocellular carcinoma (HCC) and adjacent non-tumor tissues (P < 0.001); B and C: Differential expression of MEX3A mRNA in HCC according to age (P = 0.013) and tumor-node-metastasis stages (P < 0.001); D: Kaplan-Meier survival analysis of MEX3A in HCC (P < 0.0001).

Figure 2 MEX3A expression in clinical specimens. A: Real-time fluorescent quantitative PCR was used to detect expression of MEX3A mRNA in hepatocellular carcinoma (HCC) and adjacent tissues; B: Hematoxylin and eosin staining in HCC and adjacent tissues; C: Expression of MEX3A, Ki-67 and vimentin in HCC and adjacent tissues was detected by immunohistochemistry. Scale bar = 100 and 50 μm.

Figure 3 Low and high expression of MEX3A, Ki-67 and vimentin in hepatocellular carcinoma tissues. Scale bar = 100 and 50 μm.

Figure 4 Effect of MEX3A on cell proliferation in hepatocellular carcinoma. A and B: Western blotting detection of knockdown efficiency of MEX3A in HepG2 and MHCC-97H cell lines; C and D: Cell counting kit-8 assay was used to detect the effect of knockdown of MEX3A on proliferation of HepG2 and MHCC-97H cells; E and F: Crystal violet staining was used to detect the clone formation ability of the two cell lines. Data are presented as the mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Figure 5 Effects of MEX3A on cell cycle and related protein expression. A and B: Flow cytometry was used to examine the effect of knockdown of MEX3A on the cell cycle in HepG2 cells; C and D: Flow cytometry was used to examine the effect of knockdown of MEX3A on the cell cycle in MHCC-97H cells. Data are presented as the mean ± SD; E and F: Western blotting detection of the effect of MEX3A silencing on levels of proteins in the Wnt/b-catenin and epithelial-mesenchymal transition pathways; G: Immunofluorescence detection of the effect of MEX3A silencing on localization of β-catenin. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. Scale bar = 50 μm.

Figure 6 Effect of MEX3A on cell migration in hepatocellular carcinoma. A: Gene set enrichment analysis (GSEA) of MEX3A expression and key pathways; red color represents statistically significant results. The size of the circle represents the number of genes; B: GSEA of MEX3A expression and epithelial-mesenchymal transition pathway; C and D: Cell scratch assay was used to detect the effect of MEX3A on cell migration; E and F: Transwell assay was used to detect the effect of MEX3A on hepatocellular carcinoma migration. Data are presented as the mean ± SD. ^aP < 0.01, ^bP < 0.001. Scale bar in C-F = 100 μm.

Figure 7 RORA can be regarded as a downstream gene for MEX3A. A: Analysis of transcription factors found a binding motif for the transcription factor RORA at the promoter of the differential genes associated with MEX3A expression; B: Correlation between RORA and MEX3A; C and D: QPCR was used to detect mRNA level of RORA and MEX3A after knockdown of MEX3A and RORA. Data are presented as the mean ± SD. ^aP < 0.001.

Figure 8 Role of RORA in hepatocellular carcinoma as a downstream gene for MEX3A. A-C: Real-time fluorescent quantitative PCR and western blotting detection of the knockdown efficiency of MEX3A and RORA; D: Cell counting kit-8 assay detection of the proliferative ability of the cells divided into four groups of HepG2 cells (“c”represents significant difference between the shNC + siNC and shMEX3A + siNC groups; “d” represents significant difference between the shMEX3A + siRORA and shMEX3A + siNC groups); E and F: Crystal violet staining was used to detect the clone formation ability of the cells divided into four groups; G and H: Western blotting detection of the effect of MEX3A or RORA silencing on expression of proteins in the Wnt/-catenin pathway. Data are presented as the mean ± SD; I: Immunofluorescence was used to detect the accumulation of β-catenin in the nucleus. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.001. Scale bar = 50 μm.

Figure 9 Schematic representation of mechanisms by which MEX3A promotes hepatocellular carcinoma cell proliferation and migration. HCC: Hepatocellular carcinoma.