3.1. ASXL2 is significantly elevated in CRC patients and is related to the degree of differentiation
Based on the qRT-PCR assay, the levels of ASXL2 mRNA in CRC tissues were remarkably higher than in normal adjacent tissues (P < 0.001) (Fig. 1A). We also investigated the data from the TCGA database. Among 524 participants, the expression level of AXSL2 was higher in those with tumor, which echoes our results (Fig. 1C). Furthermore, with the improvement of tumor differentiation, ASXL2 levels were gradually decreased (Figure 1B).
3.2. ASXL2 is expressed diversely in CRC at protein level.
We performed IHC analysis to detect the expression of the ASXL2 in TMAs. There were182 cases of CRC and matched normal adjacent tissues in TMAs. Based on the results, ASXL2 was downregulated in 97 (53.3%) of CRC specimns and upregulated in the remaining 85 (46.7%) samples (Fig. 2).
3.3. Relationship between ASXL2 expression and clinicopathological features of CRC patients
The overexpression of ASXL2 in CRC patients was significantly related to AJCC staging (p = 0.020), T classification (p = 0.038), and N classification (p = 0.022). No considerable difference was observed between ASXL2 expression levels and other features, including patient’s, age, gender, tumor size and location and M classification (P > 0.05, Table 1).
3.4. ASXL2 overexpression is an independent risk factor for poor prognosis in CRC
Patients who had high ASXL2 expression exhibited a lower overall survival rate than patients with lower expression of the gene (Figure 3A, log-rank test, P<0.001). Furthermore, the clinicopathological features of CRC patients were considered for the univariate analysis. The results suggested that the AJCC stage, classification of TNM and the expression level of ASXL2 were risk factors of colorectal cancer prognosis. Also, multivariate Cox regression analysis revealed that ASXL2 upregulation was an independent risk factor for poor colorectal cancer prognosis (Table 2).
Besides, the one, three, and five-year survival rates of patients with lymph node metastasis were lower than those in the non-lymph node metastasis group. Among them, the five-year survival rate decreased most significantly. The subgroup analysis among the patients with lymphatic metastasis revealed that high ASXL2 expression was more likely to confer a poor prognosis. (Fig. 3C).
3.5. Effect of ASXL2 on CRC Cell Proliferation in Vitro
To examine the ASXL2 expression in vitro, the level of ASXL2 expression was assessed in CRC cell lines (Caco-2, HT29, SW480, SW1116) of the same source with different colorectal cancer (Fig. 4A), and divided the cell lines into two groups based on ASXL2 expression levels.
Subsequently, Caco-2 and SW1116 were classified into the high expression group, whereas SW480 and HT29 were put in the low expression group. We inhibited ASXL2 expression by transfecting siRNAs-ASXL2 plasmids into the cell lines in the high expression group (Caco-2, SW1116). Cell proliferation was considerably reduced with a decrease in ASXL2 expression (Fig. 4B-4C). For the cell lines in low expression group. We transfected the plasmid overexpressing ASXL2 into HT29 and SW480 to overexpress ASXL2. Relative to the control group (empty vector), overexpression of ASXL2 levels led to a remarkable increase in cell proliferation. (Figures 4E-4F).
Also, we measured the Ki-67 expression because it has been associated with cell proliferation. As we expected, the mRNA levels of Ki-67 had significantly increased with the overexpression of ASXL2 and vice versa, which confirmed our findings. (Fig. 4G).