Transcription factorIRX5 promotes hepatocellular carcinoma proliferation and inhibits apoptosis by regulating the p53 signalling pathway

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most frequent cause of cancer‐related death. The IRX5 transcription factor plays a different role in multiple cancers and contributes to the development of many tumours. However, little is known about the molecular mechanisms of IRX5 in HCC. In this study, we found that IRX5 was abnormally upregulated in HCC tissues compared with adjacent normal tissues. IRX5 promoted HCC cell proliferation and upregulated the expression of cyclin D1 and knockdown of IRX5 suppressed tumorigenicity in vivo. Furthermore, knockdown of IRX5 increased p53 and Bax expression and decreased Bcl‐2 expression. Thus, IRX5 suppressed apoptosis in HCC cells by inhibiting the p53 signalling pathway, indicating its role as a treatment target for HCC. Significance of the study Our study demonstrated that IRX5 was abnormally upregulated in HCC tissues compared with adjacent normal tissues. IRX5 promoted HCC cell proliferation and upregulated the expression of cyclin D1, and knockdown of IRX5 suppressed tumorigenicity in vivo. Furthermore, knockdown of IRX5 increased p53 and Bax expression and decreased Bcl‐2 expression. IRX5 suppressed apoptosis in HCC cells by inhibiting the p53 signalling pathway, indicating its role as a treatment target for HCC.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most frequent cause of cancer-related death. The IRX5 transcription factor plays a different role in multiple cancers and contributes to the development of many tumours. However, little is known about the molecular mechanisms of IRX5 in HCC.
In this study, we found that IRX5 was abnormally upregulated in HCC tissues compared with adjacent normal tissues. IRX5 promoted HCC cell proliferation and upregulated the expression of cyclin D1 and knockdown of IRX5 suppressed tumorigenicity in vivo. Furthermore, knockdown of IRX5 increased p53 and Bax expression and decreased Bcl-2 expression. Thus, IRX5 suppressed apoptosis in HCC cells by inhibiting the p53 signalling pathway, indicating its role as a treatment target for HCC.
Significance of the study: Our study demonstrated that IRX5 was abnormally upregulated in HCC tissues compared with adjacent normal tissues. IRX5 promoted HCC cell proliferation and upregulated the expression of cyclin D1, and knockdown of IRX5 suppressed tumorigenicity in vivo. Furthermore, knockdown of IRX5 increased p53 and Bax expression and decreased Bcl-2 expression. IRX5 suppressed apoptosis in HCC cells by inhibiting the p53 signalling pathway, indicating its role as a treatment target for HCC.

| INTRODUCTION
Liver cancer was the sixth most commonly diagnosed cancer and the fourth leading cause of cancer-related death worldwide in 2018, 1 with approximately 841 000 new cases and 782 000 deaths annually. 2 Hepatocellular carcinoma (HCC) accounts for 75-85% of primary liver cancers and is one of the most common malignant tumours in the world. 3 Surgery is the most effective therapy for HCC at present. 4 The vast majority of HCC patients present with advanced-stage disease that ultimately leads to poor prognosis 5 and a 5-year survival rate of less than 20% 4,6 ; the recurrence rate is as high as 80%. 7 The mechanism of HCC development has not been clarified, particularly the mechanism underlying the regulation of the proliferation and apoptosis of HCC.
Iroquois homeobox (Irx) genes play pivotal roles in normal embryonic cell patterning and the development of malignancies. 8 The Irx family is composed of six genes in humans and mice, including two clusters, IrxA (Irx1, Irx2 and Irx4) located on chromosome 5 and IrxB (Irx3, Irx5 and Irx6) located on chromosome 16. 9 IRX5 encodes a transcription factor and is a highly conserved member of the Iroquois homeobox gene family. 8 IRX5 plays a different role in multiple cancers, contributing to the development of many tumours by acting as an important transcription factor regulating key regulatory genes that control cell growth, invasion, migration and apoptosis. Recent data suggest that IRX5 is a transcription factor that remarkably promotes tongue squamous cell carcinoma tumour growth by targeting the osteopontin (OPN) promoter and activating the NF-κB pathway. 8 Our previous study showed that CRNDE acted as a tumour oncogene by promoting the oncogenic properties of human HCC and revealed a novel CRNDE-miR-136-5p-IRX5 regulatory network in HCC. 3 CyclinD1 regulates cell cycle progression, forms complexes with cyclin-dependent kinase 4 or 6 in the cytoplasm 10,11 and promotes progression from G1 to S-phase. 12 Previous findings have indicated that cyclin D1 is a downstream target of IRX5. 9 P53 is a tumour suppressor gene, 13 and the p53 protein is a transcription factor that is involved in cell cycle arrest, DNA repair and apoptosis. 14 In the p53 signalling pathway, p53 positively regulates the proapoptotic proteins Bax and p53 negatively regulates the transcription of Bcl-2. 15 In this study, we explored the role ofIRX5 in HCC proliferation and apoptosis. Next, we will discuss the properties of IRX5 in the p53 signalling pathway.    All experiments were performed in triplicate.

| Western blot analysis
We extracted total proteins from the HCC cells using RIPA buffer and protease inhibitors (Solarbio, Beijing, China) according to manufacturer's protocol, and the protein concentrations were determined using a BCA assay kit

| IRX5 promoted HCC cell proliferation in vitro
To further understand the correlation between IRX5 expression and the proliferation and apoptosis capacities of HCC cells, we overexpressed IRX5 (pcDNA3.1-IRX5) and knocked down (sh-IRX5) via transient transfection in SMMC7721 and HepG2 cells. Western blotting confirmed that transfection with the IRX5 overexpression plasmid significantly increased IRX5 expression (Figure 2A,B). The efficiency of sh-RNA-mediated IRX5 knockdown was confirmed (Figure 2A,B).MTS assays showed that the overexpression of IRX5increased the proliferative capacities of SMMC7721 and HepG2 cells compared with control cells containing empty vector, whereas the opposite result was found when IRX5 expression was silenced ( Figure 2C). Moreover, IRX5 overexpression enhanced the colony formation capacities of SMMC7721 and HepG2 cells, whereas knockdown of IRX5 reduced them ( Figure 2D,E).
Together, these data suggest that IRX5 enhanced HCC cell proliferation.

| IRX5 induced cell cycle progression by upregulatingcyclinD1 and suppressing apoptosis via inhibiting p53 signalling in HCC cells
To gain insights into the mechanism by which IRX5 enhances HCC cell proliferation and induces apoptosis, fluorescence-activated cell sorting Knockdown of IRX5 reduced the expression of the oncogenic cell cycle regulator cyclinD1 but increased the expression of the cyclindependent protein kinase inhibitor p53 ( Figure 3E,F).
To investigate the role of IRX5 in the p53 signalling pathway, the expression levels of Bax and Bcl-2 were detected. As shown in Figure 3E

| DISCUSSION
Our study demonstrates that IRX5 is upregulated in HCC cell lines and HCC tissues. Furthermore, IRX5 promoted cell proliferation in vitro and in vivo. In addition, IRX5 suppressed apoptosis in vitro.
IRX5 plays a different role in multiple cancers, contributing to the development of many tumours by acting as an important transcription factor regulating key regulatory genes that control cell growth, invasion, migration and apoptosis. IRX5 activity is also present in the stromal and proliferative late blastemal/early epithelial cells in developing kidneys and Wilms tumours. 16 Myrthue et al had revealed that knockdown of IRX5 by RNA interference significantly reduction in LNCaP cell viability, which resulted in increased LNCaP cell apoptosis and was partially mediated by p53. 17 However, the role of IRX5 in HCC is unknown. In this study, we provided the first evidence that IRX5 is significantly upregulated in HCC cell lines and tissues. We further identified the effects of IRX5 on the biological behaviours of HCC cells, showing that IRX5 promoted HCC cell proliferation and inhibited apoptosis. The results indicated that IRX5 acts as an oncogene in HCC. Moreover, the in vivo studies also confirmed that knockdown of IRX5 suppressed tumour growth in nude mice, suggesting that IRX5 could potentially be applied in the treatment of HCC. However, the underlying mechanisms by which IRX5 promotes tumour cell proliferation and inhibits apoptosis remain unclear.
We provided evidence that IRX5 promoted HCC cell proliferation in vitro and in vivo. Furthermore, our study found that a reduction in the G1 population and an increase in the S and G2/M populations were observed in SMMC7721 cells overexpressing IRX5. Mechanistically, IRX5 was shown to induce cell cycle progression by upregulatingcyclinD1. It has been well defined that tumour-associated cell cycle defects are often mediated by the accumulation of cyclins (CCNs). 18 Cyclins are divided into two groups known as the G1/S cyclins, which are essential for the control of the cell cycle at the G1 to S transition, and the G2/M cyclins, which control the cell cycle at the G2 to M transition phase. 19 Cyclin D1 is a major regulator of the cell cycle and is responsible for the G1/S-phase transition. 20 Ao et al reported that cyclin D1 was overexpressed in liver cancer cells and promoted migration and invasion by regulating several enzymes. 21 Chen et al also reported that HCC tissues and HCC cells exhibited elevated expression levels of cyclin D1 and its expression levels were found to be correlated with tumour size and tumour staging. 22 A previous study reported that IRX5 promotes NSCLC cell proliferation by means of regulating the CCND1 promoter. 9 In this study, cyclin D1 expression was positively correlated with the expression of IRX5 in HCC cells.
In addition to enhanced proliferation, resistance to apoptosis is also a hallmark of cancer cells. 23 The p53 gene is located on human chromosome 17p13 and is a tumour suppressor and pro-apoptosis gene. Wild-type p53 plays a key role in cell gene transcription, cell cycle regulation, apoptosis, proliferation and differentiation. [24][25][26] The amount of wild-type p53 protein is low in normal cells. 27 The dominant components of p53 signalling, including p53, Bcl-2 and Bax, have been extensively studied in carcinomas. 28,29 A previous study reported that p53 positively regulates Bax expression 30 and negatively regulates the transcription of Bcl-2. 15,30 Thus, p53 is likely to affect upstream pro-apoptotic proteins to modulate their functions in the cytoplasm. We also found that the downregulation of IRX5 significantly increased the expression of p53 and Bax and decreased the expression of Bcl-2 in HCC cells. However, in our study, we did not study the mechanism by whichIRX5 inhibits the p53 signalling pathway. Such mechanisms are left to be investigated in future studies.
In summary, our study demonstrated that IRX5 is a potential tumour promoter gene in HCC. IRX5 promotes to the promoter of carcinogenesis by facilitating cell proliferation and suppressing cell apoptosis by inhibiting the p53 signalling pathway. As a result, IRX5 might act as a novel molecular target for the detection and treatment of HCC. However, further studies are needed to investigate the effect of IRX5 on other cellular processes in HCC, such as cell adhesion and differentiation.

| CONCLUSION
Our results showed that IRX5 was upregulated in HCC and cells. Overexpression of IRX5 promoted cell proliferation and tumourigenicity.
Knockdown of IRX5 promoted cell apoptosis through the p53 signalling pathway in HCC.