Knockdown of the differentially expressed gene TNFRSF12A inhibits hepatocellular carcinoma cell proliferation and migration in vitro

Abstract

Human hepatocellular carcinoma (HCC) has been reported to be highly insensitive to conventional chemotherapy. In the current study, the Agilent Whole Human Genome Oligo Microarray (4x44 K) was used in order to identify the differentially expressed genes between HCC and adjacent tissues, and the top 22 differentially expressed genes were confirmed through reverse transcription‑quantitative polymerase chain reaction. Among the identified differences in gene expression, expression of tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) was markedly higher in HCC tissue than in adjacent tissue. Previous studies have suggested that TNFRSF12A may serve a role in tumor growth and metastasis, thus in the current study, TNFRSF12A was knocked down in the SMMC7721 cell line through siRNA. This demonstrated that cells exhibited reduced reproductive and metastatic capacity ex vivo. Thus, the results of the current study suggest that TNFRSF12A may be a candidate therapeutic target for cancer including HCC, and additional genes that exhibited significantly different expression from normal adjacent tissues require further study.

Figure 1.

The heatmap and GO analysis between HCC and adjacent tissue. (A) Cluster results for all genes. (B) GO enrichment results by GSEA. GO, gene ontology; HCC, hepatocellular carcinoma; GSEA, gene set enrichment analysis.

Figure 2.

Gene expression levels between HCC and adjacent tissue. (A) Hierarchical clustering and heat map analysis of the top 22 genes. (B) Gene expression in HCC and adjacent samples detected by RT-qPCR and microarray. The ∆∆Cq method was used to calculate the fold change in the expression of each gene and data were statistically analyzed using a two-tailed t-test. (C) Gene expression of TNFRSF12A in TCGA. *P<0.05; **P<0.01; ***P<0.001 HCC vs. adjacent tissue. HCC, hepatocellular carcinoma; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; TNFRSF12A, tumor necrosis factor receptor superfamily member 12A; TCGA, The Cancer Genome Atlas.

Figure 3.

TNFRSF12A knockdown can reduce SMMC7721 cell viability. (A) TNFRSF12A expression detected in SMMC7721 cells following different siRNA transfection through western blotting, β-actin was used as the loading control. (B) Densitometric analysis was performed using ImageJ software to analyze TNFRSF12A expression. (C) Cell growth curves were increased according to cell viability for three days compared with NC siRNA transfection. **P<0.01; ***P<0.001 si-TNFRSF12A vs. NC control. TNFRSF12A, tumor necrosis factor receptor superfamily member 12A; NC, negative control; siRNA, small interfering RNA.

Figure 4.

Effect of TNFRSF12A knockdown on migration and invasion of SMMC7721 cells. (A) Quantitative differences of cells between NC and si-TNFRSF12A transfection following transfection of different siRNAs and incubation for 24, 48 and 72 h. (B and C) Representative images of the Transwell migration assay show the number of cells passing through the membrane was significantly reduced in the si-TNFSRF12A group compared with the NC group. Magnification, ×10. ***P<0.001. TNFRSF12A, tumor necrosis factor receptor superfamily member 12A; NC, negative control; siRNA, small interfering RNA.