AQPs are channel proteins penetrate integral plasma membrane, and belong to MIP (major intrinsic protein). Small uncharged solutes and water can passively permeated these channels [21]. Their highly conserved NPA (asparagine-proline-alanine) boxes structure identify its AQP, meanwhile is vital to formation of pore for water-permeate. All AQPs have not only the special construction of hydrophobic repeated NPA boxes and conserved six transmembrane domains, but minimal different to each other [22, 23]. Besides, the NPA motifs have only poor conservation sequences that are AQP-like [24].
Focusing on apoptotic mechanism related to AQP-9 in SW480 cells, over expression or knockdown of AQP9 was induced. At first, the online software TargetScan (targetscan.org/, version 7.2) was used to predict the target gene of miR-539. The luciferase activity assay was performed, and then AQP9 was verified the potential aim of miR-539. Our results demonstrated the mRNA of AQP9 was the target gene of miR-539. Then, miR-539 was used as the siRNA of AQP9.
Moreover, the data confirmed that growth, proliferation, and mitotic entry of SW480 cells were suppressed by inhibiting expression of AQP-9, however, enhanced by over expression of AQP-9. Furthermore, the apoptosis of SW480 cells was induced by inhibiting expression of AQP-9, and enhanced by over expression of AQP-9. The levels of Bcl-2 increased in SW480 cells treated with over expression of AQP-9, but decreased significantly in cells treated with siRNA of AQP-9 (miR-539). On the contrary, the expression levels of Bax decreased in SW480 cells treated with over expression of AQP-9, but obviously promoted by siRNA of AQP-9 (miR-539). It indicated that the apoptosis induced by down regulation of AQP-9 expression in SW480 cells was associated with Bcl-2 family.
In recent years, there were two human models about metabolism, which could be potentially utilized for patient [25, 26]. The generic models of human metabolism were the base of context models, such as metabolism of cancer and metabolic activities [27, 28]. The vigorous exercise and fasting induced diminishment of glucose level in blood can activate cell gluconeogenesis. Accordingly, the production of glucose restore the homeostasis of glucose. The induction of gluconeogenesis can be achieved by stimulus of hormone that is associated with four gluconeogenesis in these main reactions at transcriptional, post-translational level, even allosteric alterations. The following enzymes of G6Pase (glucose-6-phosphatase), F1, 6BPase (fructose-1, 6-bisphosphatase), PEPCK (phosphoenolpyruvate carboxykinase), and PC (pyruvate carboxylase) can catalyze these reactions. It is critical effect on induction of gluconeogenesis come from these enzymes encoded by these gene through transcriptional regulation [29].
Therefore, focus on the connection between metabolism of glucose and AQP-9, which may be related to CRC tumorigenesis, the investigation about the role of AQP-9 in enhancing de novo synthesis of glucose, gluconeogenesis was conducted. In an attempt to comprehensively confirm the pathways of metabolism associated with AQP-9, over expression and down regulation of AQP-9 were induced in cell line SW480, in the meanwhile, the cell functions and expression levels of these glucogenic gene was studied. We identified that up-regulation of AQP-9 could induce increased expression level of GOT1, GK, and G6PC proteins, but siRNA of AQP-9 (miR-539) reduced these expression.
It had evidences that miR-539 could induce suppression of EMT (Epithelial–Mesenchymal Transition), migration and invasion of prostate cancer cells [30]. Cao et al. Thought that miR-539 might be involved in the progression of ESCC (Esophageal Cancer Cells) and could be a new therapeutic target for this disease [31]. But, in this study, our results indicated that miR-539 promotes development of colorectal cancer through alleviating expression of AQP-9 associated with glucose metabolism.
The aims of this study were to explore associated with glucose metabolism and the methods for treatment on CRC related to AQP-9. For the purpose of identifying the pathways of metabolism at full-scale, which was related to the role of AQP-9 for treatment on CRC, the expression of glucogenic gene was examined. We performed the research on glucose metabolism and the methods for treatment on CRC related to the role of AQP-9 in enhancing de novo glucose synthesis or gluconeogenesis. Our results confirmed that AQP-9 could affect on the genes expression associated with gluconeogenesis. Consistent with these above results, the production of glucose was caused through AQP-9 over-expression in SW480 cells, however, it could be reversed through down expression with AQP-9 siRNA (miR-539).
Our study indicated through knockdown regulation of AQP-9 could cause proliferation and growth suppression of SW480 cells, which was associated with metabolic pathway involved in gluconeogenesis gene GOT1, GK, and G6PC. These genes could enhance the metabolism of glucose. In conclusion, AQP-9 can be served as the potential novel target for treatment on CRC .