Short hairpin RNA-mediated knockdown of nuclear factor erythroid 2-like 3 exhibits tumor-suppressing effects in hepatocellular carcinoma cells

Abstract

Background: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with high mortality-to-incidence ratios. Nuclear factor erythroid 2-like 3 (NFE2L3), also known as NRF3, is a member of the cap 'n' collar basic-region leucine zipper family of transcription factors. NFE2L3 is involved in the regulation of various biological processes, whereas its role in HCC has not been elucidated.

Aim: To explore the expression and biological function of NFE2L3 in HCC.

Methods: We analyzed the expression of NFE2L3 in HCC tissues and its correlation with clinicopathological parameters based on The Cancer Genome Atlas (TCGA) data portal. Short hairpin RNA (shRNA) interference technology was utilized to knock down NFE2L3 in vitro. Cell apoptosis, clone formation, proliferation, migration, and invasion assays were used to identify the biological effects of NFE2L3 in BEL-7404 and SMMC-7721 cells. The expression of epithelial-mesenchymal transition (EMT) markers was examined by Western blot analysis.

Results: TCGA analysis showed that NFE2L3 expression was significantly positively correlated with tumor grade, T stage, and pathologic stage. The qPCR and Western blot results showed that both the mRNA and protein levels of NFE2L3 were significantly decreased after shRNA-mediated knockdown in BEL-7404 and SMMC-7721 cells. The shRNA-mediated knockdown of NFE2L3 could induce apoptosis and inhibit the clone formation and cell proliferation of SMMC-7721 and BEL-7404 cells. NFE2L3 knockdown also significantly suppressed the migration, invasion, and EMT of the two cell lines.

Conclusion: Our study showed that shRNA-mediated knockdown of NFE2L3 exhibited tumor-suppressing effects in HCC cells.

Keywords: Epithelial-mesenchymal transition; Hepatocellular carcinoma; Nuclear factor erythroid 2-like 3; Short hairpin RNA; The Cancer Genome Atlas.

Figure 1

Association of nuclear factor erythroid 2-like 3 expression with clinicopathological features of hepatocellular carcinoma patients in The Cancer Genome Atlas dataset. A: Nuclear factor erythroid 2-like 3 (NFE2L3) expression in G1/2 (n = 213) and G3/4 (n = 131) hepatocellular carcinoma (HCC) patients; B: NFE2L3 expression in T1 (n = 171), T2 (n = 87) and T3/4 (n = 86) HCC patients; C: NFE2L3 expression in stage I (n = 169), stage II (n = 85), and stage III/IV (n = 90) HCC patients. ^bP < 0.01, ^cP < 0.001. NFE2L3: Nuclear factor erythroid 2-like 3; HCC: Hepatocellular carcinoma.

Figure 2

Expression of nuclear factor erythroid 2-like 3 mRNA and protein in hepatocellular carcinoma cell lines. A: Nuclear factor erythroid 2-like 3 (NFE2L3) mRNA levels in BEL-7404 and SMMC-7721 cells infected with shNFE2L3 (NFE2L3 short hairpin RNA) were measured by qPCR; B: NFE2L3 protein levels in BEL-7404 and SMMC-7721 cells infected with shNFE2L3 were measured by Western blot. Data shown are the mean ± SEM, ^aP < 0.05, ^bP < 0.01 vs shCtrl. NFE2L3: Nuclear factor erythroid 2-like 3; shCtrl: Negative control cells; shNFE2L3: Nuclear factor erythroid 2-like 3 short hairpin RNA.

Figure 3

Role of nuclear factor erythroid 2-like 3 in apoptosis, clone formation, and proliferation of hepatocellular carcinoma cells. A and B: Apoptotic cells were stained with Annexin V-APC and measured using flow cytometry. The abscissa represents red fluorescence (RED-R-HLog), and the ordinate represents green fluorescence (GRN-B-HLog) and cell count; C and D: Cell clones were stained with Giemsa and photographed with a digital camera. The number of clones was accurately calculated and statistically analyzed; E and F: Successfully infected cells were green fluorescent protein positive and cell images were taken with a Celigo cytometer for a continuous 5 d. Cell count-fold represents cell count at each time point relative to the average of day 1; G and H: OD values were measured at 490 nm after the treatment of MTT. Cell growth curves were plotted based on OD 490-fold at different time point. Data shown are the mean ± SEM. Student’s t test was used to analyze significant differences, ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs shCtrl. shCtrl: Negative control cells; shNFE2L3: Nuclear factor erythroid 2-like 3 short hairpin RNA.

Figure 4

Short hairpin RNA-mediated knockdown of nuclear factor erythroid 2-like 3 suppresses migration, invasion, and epithelial-mesenchymal transition of hepatocellular carcinoma cells. A and B: Cell migration assay showed that migrated cells of the shNFE2L3 group were less than that of the shCtrl group in BEL-7404 and SMMC-7721 cells; C and D: Cell invasion assay showed that invaded cells of the shNFE2L3 group were less than that of the shCtrl group in BEL-7404 and SMMC-7721 cells. Both cell migration and invasion were measured with transwell assays. Migrated and invaded cells were stained with Giemsa and imaged and counted under a microscope. Scare bar = 150 μm; E and F: Snail 1, N-cadherin, Snail 2, and Vimentin protein levels were analyzed using Western blot. GAPDH was a loading control. Data shown are the mean ± SEM. Student’s t test was used to analyze significant differences, ^cP < 0.001 vs shCtrl. shCtrl: Negative control cells; shNFE2L3: Nuclear factor erythroid 2-like 3 short hairpin RNA.