Date Published: July 27, 2017
Publisher: Public Library of Science
Author(s): Chunyan Zhang, Cuifang Chang, Weiming Zhao, Hang Gao, Qiwen Wang, Deming Li, Fuchun Zhang, Shifu Zhang, Cunshuan Xu, Salvatore V Pizzo.
Our previous study has proved that the chromosome 9 open reading frame 116 (C9orf116) (NM_001106564.1) was significantly up-regulated in the proliferation phase of liver regeneration. To study its possible physiological function, we analyzed the effect of C9orf116 on BRL-3A cells via over-expression and interference technique. MTT results showed that the cell viability of the interference group was significantly lower than the control group at 48h after transfection (P<0.05), whereas it was significantly higher in the over-expression group (P<0.05). The flow cytometry results showed that C9orf116 knockdown or over-expression had little effect on BRL-3A cell apoptosis. However, the number of cells in division phase (G2/M) was significantly reduced in the interference group (P<0.05), but significantly increased in the over-expression group (P<0.01). Furthermore, the expressions of cell proliferation-related genes CCNA2, CCND1 and MYC both at mRNA and protein levels were down-regulated in the interference group and up-regulated in the over-expression group. Therefore, we concluded that C9orf116 may promote cell proliferation by modulating cell cycle transition and the expression of key genes CCNA2, CCND1 and MYC in BRL-3A cells.
Liver is an organ with extraordinary ability to regenerate. After liver injury or partial hepatectomy (PH), the remnant liver tissue can regenerate quickly to restore the original size and weight, to maintain optimum liver weight/body weight ratio, and ultimately to rebuild liver tissue structure and liver function, which is known as liver regeneration (LR) [1, 2]. LR was divided into three key stages [3–6]: the initiation stage (2-6h), in which quiescent hepatocytes were activated and G0/G1 conversion occurred; the proliferative stage or advanced stage (12-72h), in which hepatocytes proliferation occurred; the termination stage (120-168h), in which liver regeneration terminates with the rebuilding of structure and function of tissue . LR involves cell activation, differentiation, proliferation and regulation, re-differentiation, tissue structure and function reconstruction and other physiological activities [1, 8–10]. It is well known that multiple-growth factors, hormones, and other bioactive molecules are involved in the LR through a variety of signaling pathways. Our previous study found C9orf116 was significantly up-regulated in LR proliferation stage [11, 12]. Therefore, we hypothesized that C9orf116 may regulate the proliferation of liver cells.
The liver has a variety of physiological functions. In order to further study the molecular mechanism of liver regeneration, we performed genomics and proteomics studies of liver regeneration and found that C9orf116 was highly expressed in the regenerated liver [11, 12]. Therefore, we hypothesized that it might be related to liver cells proliferation in LR. To further confirm the role of C9orf116 in normal liver cells, we used BRL3A cells as models to study the function of C9orf116 in vitro [20–22]. RNA interference has been developed as an important means to study gene function. Lentiviral vectors have little immune response, and are able to infect non-dividing cells with a high transfection efficiency, therefore, they are also often used to study the function of genes [14, 15, 23–26]. In this study, we used lentiviral vector for over-expression of C9orf116 and siRNA to knockdown the expression of C9orf116, and further analyzed its possible role in BRL-3A cells.