The down-regulation of GNAO1 and its promoting role in hepatocellular carcinoma

Abstract

GNAO1 (guanine nucleotide-binding protein, α-activating activity polypeptide O) is a member of the subunit family of Gα proteins, which are molecular switchers controlling signal transductions and whose deregulation can promote oncogenesis. HCC (hepatocellular carcinoma) is one of the malignant tumours around the world, which summons novel biomarkers or targets for effective diagnosis and treatments. The present study was aimed to investigate the expression of GNAO1 in HCC patient tissues and the possible mechanisms by which it took effects. The expression of GNAO1 was detected by IHC (immunohistochemistry) and real-time qPCR (quantitative PCR). Cell proliferation test and cell senescence test were then performed to explore the role of GNAO1 in the occurrence and development of HCC. It was revealed that the level of GNAO1 was comparably less in HCC tissues than in the adjacent tissues. Furthermore, down-regulation of GNAO1 increased cell proliferation, while suppressing the senescence of HCC cells. In conclusion, our findings revealed and confirmed the importance of GNAO1 in HCC, indicating that GNAO1 is a potential biomarker as well as a promising therapeutic target for HCC.

Figure 1. Expression of GNAO1 in HCC and para-tumour tissue

Relative expression of GNAO1 gene in frozen cancerous tissues and the adjacent non-cancerous tissues is shown in Panel A. mRNA level of GNAO1 gene in cancer was determined with quantitative RT–PCR using human GAPDH as a house-keeping gene control, and expressed in percentage of mRNA level of non-tumour tissue. ‘****’ stands for P value less than 0.0001. Representative immunohistochemical images (brown colour) of GNAO1 are shown in liver cancer tissue (B) and para-tumoural tissue (C), and are marked with black arrows (cancer cells) and white thick arrows (non-tumoural tissue). The tissues were counter-stained with haematoxylin for nuclei. Magnification: ×200.

Figure 2. The inhibitory effect of GNAO1 gene expression by siRNAs in two human liver cancer cell lines: QGY-7703 and SMMC-7721

The cells were transfected with scrambled siRNA (NC) or without siRNA against human GNAO1. The protein level of GNAO1 in these cells 3 days after transfection was determined by Western blot analysis. Compared with scramble RNA, siGNAO1-1 and siGNAO1-2 were shown to remarkably suppress GNAO1 expression at the protein level.

Figure 3. Cell proliferation test of QGY-7703 (upper panel) and SMMC-7721 (lower panel) after transfection with GNAO1 siRNAs (siGNAO1-1 and siGNAO1-2), scrambled siRNA (NC) or without transfection (NTC)

The cell proliferation was determined by CCK-8 and expressed by absorbance. Cells transfected with GNAO1 siRNA were apparently grown faster than control groups, especially 48 h after transfection.

Figure 4. Cell senescence was stained by β-gal staining after transfection with NC, GNAO1 siRNA (siGNAO1-1 or siGNAO1-2) or without transfection

Senescence in both QGY-7703 (upper panel) and SMMC-7721 (lower panel) were inhibited by siRNA transfection when compared with those transfected with scrambled siRNA.