The Role And Mechanism Of Aquaporin-9, Negatively Mediated By IGF2, In Regulating Liver Cancer Stem Cell Properties

Hepatocellular carcinoma（HCC）is the fifth most common cancer in the world,accounting for its third cause of tumor death in the world.China is the country with the highest incidence and mortality of HCC in the world.At present,the overall efficacy and patient survival rate are still unsatisfactory,and its high degree of heterogeneity is the main reason that it is more prone to metastasis,relapse and drug resistance than other solid tumors.Liver cancer stem cells（LCSCs）have unlimited self-renewal capacity and can produce differently heterogeneous tumour cell populations.Due to the aggressively behavioral characteristics,LCSCs play a key role in the development of liver cancer because of the association with prognosis.AQP9 is an intrinsic protein located on the cell membrane that transports water across the membrane.It is also reported to transport hydrogen peroxide（H₂O₂）.Recent studies have shown that the expression of AQP9exerts a negatively regulatory effect on HCC invasion by inhibiting epithelial-to-mesenchymal transition（EMT）.In addition,studies have shown that insulin-like growth factor 2（IGF2）is necessary to maintain the characteristics of CSCs.In addition,IGF2 and proinsulin have homologous amino acid sequences.Studies have shown that the expression of AQP9 can be down-regulated by insulin,whether it is regulated by IGF2 and the role and mechanism in LCSCs are still unclear.Part I.Obtainment of LCSCs and the role of AQP9 in LCSCsObjectiveTo explore the expression and role of AQP9 in the LCSCs stemness.Methods1.Flow cytometry was used to aseptically sort CD133⁺/^-liver cancer cells,and serum-free culture assay was used to enrich liver cancer stem cell spheres.RT-qPCR and Western blot were used to detect the expression of AQP9 in adherent cells,stem cell spheres,and CD133⁺/^-cells.Subcutaneous tumorigenesis experiments were conducted to compare the subcutaneous tumorigenic ability of CD133⁺/^-liver cancer cells.2.AQP9 stable overexpression cell lines was established,and RT-qPCR and Western blot were used to detect the effect of AQP9-overexpression on the expression of LCSCs-related genes.Serum-free culture assay was used to evaluate the effect of AQP9 overexpression on LCSCs sphere-forming ability.Immunofluorescence staining was conducted to detect the effect of AQP9 overexpression on LCSCs-related genes.The proliferation assay was used to detect the effect of AQP9 overexpression on the sensitivity of hepatocellular carcinoma to sorafenib.3.SiRNA was synthesized to silence the expression of AQP9 in AQP9overexpression cells,and then RT-qPCR and Western blot were used to examine the effect of AQP9 knockdown on the expression of LCSCs related genes,and the effect of AQP9 knockdown on the sphere-forming capacity.Immunofluorescence staining was used to detect the effect of AQP9knockdown on LCSCs related genes.The proliferation test was used to evaluate the effect of AQP9 knockdown on the sensitivity to sorafenib in HCC cells.Results1.The levels of mRNA and protein expression of specific LCSCs genes in LCSCs enriched by serum-free were significantly increased,while the mRNA and protein expression of AQP9 were significantly reduced.The ability of CD133⁺cells to form spheres in serum-free medium was significantly stronger than that of CD133^-cells.The subcutaneous tumorigenic ability of CD133⁺cells was significantly enhanced than that of CD133^-cells.2.The mRNA and protein expression levels of LCSCs-related stem genes in Huh7-AQP9 overexpression group were significantly lower than those in the control group.Overexpression of AQP9 inhibited the sphere-forming capacity of HCC cells.AQP9 overexpression significantly impaired the proliferation of HCC cells and enhance the sensitivity of HCC cells to sorafenib.3.After AQP9 expression interference,the mRNA and protein levels of LCSCs-related genes were significantly up-regulated.AQP9 inhibitors enhanced sphere-forming ability of HCC cells.AQP9 knockdown restored the inhibitory effect of AQP9 on the proliferation of HCC cells as well as the enhanced effect of sorafenib.ConclusionsThe expression of AQP9 has been found to be significantly decreased in LCSCs-spheres and CD133⁺LCSCs.Overexpression of AQP9 inhibited the expression of specific LCSCs genes,decreased self-renewal ability and enhanced the sensitivity of HCC cells to sorafenib.AQP9 knockdown reversed the inhibitory effect of AQP9 on the stemness of LCSCs.Part II Regulatory mechanism of IGF2 on the stemness of HCC by inhibiting AQP9ObjectiveTo explore the role and mechanism of IGF2 in LCSCs and the effect on AQP9 expression.Methods1.RT-qPCR and Western blot were conducted to assess the influence of IGF2 pretreatment on the expression of LCSCs-related genes and AQP9,and serum-free culture assay was used to detect the effect of IGF2 pretreatment on sphere-forming ability.Immunofluorescence staining was used to detect the effect of IGF2 pretreatment on LCSCs-related genes.2.Serum-free culture was used to examine the sphere-forming ability of H₂O₂ pretreatment,overexpression of AQP9,IGF2 pretreatment,ROS scavenger NAC,and BSO pretreatment.3.H2DCFDA staining was conducted to evaluate the influence of IGF2pretreatment and AQP9 overexpression on intracellular ROS levels.4.To detect the effect of AQP9 overexpression and ROS scavenger NAC on the ability of subcutaneous tumorigenicity and the expression of LCSCs-related genes in the tumors.IP was used to test the interaction ofβ-catenin with TCF4 or FOXO3a in AQP9 overexpression cells with or without NAC.Results1.IGF2 pretreatment inhibited the mRNA and protein expression of AQP9 and promoted the mRNA and protein expression of LCSCs-related genes.IGF2 pretreatment restored the inhibitory effect of AQP9 on the protein expression of LCSCs-gene.The IGF2 pretreatment significantly enhanced the sphere-forming ability and restored the inhibitory effect of AQP9 on the sphere-forming ability.2.H₂O₂ pretreatment significantly inhibited the self-renewal capacity of LCSCs.IGF2 pretreatment enhanced sphere-forming ability in HCC cells and AQP9 overexpression cells,and this enhancement effect was blocked by BSO pretreatment.3.Overexpression of AQP9 increased intracellular ROS levels.IGF2pretreatment can induce the reduction of ROS levels in AQP9overexpression HCC cells,while BSO treatment reversed the effect of IGF2on intracellular ROS levels.4.Overexpression of AQP9 inhibited tumor formation in vivo,and the volume of tumors increased significantly after administration of NAC.Overexpression of AQP9 increased the accumulation of ROS and thus reduced the binding ofβ-catenin to TCF4 and increased the binding ofβ-catenin to FOXO3a,while NAC treatment restored the effects.ConclusionsH₂O₂ pretreatment inhibits the sphere-forming ability of LCSCs.IGF2decreased the intracellular ROS level by inhibiting AQP9 to enhance the stemness of LCSCs.AQP9 raises the ROS level resulting to the reduced binding ofβ-catenin with TCF4 meanwhile the increased interaction ofβ-catenin with FOXO3a,and finally downregulates the stemness of LCSCs by inhibiting theβ-catenin activity.