The Mechanism Study Of AQP3-mediated ROS Transmembrane Transportation Regulating Lung Adenocarcinoma Autophagy

Objective: Oxidative stress is closely related to autophagy and participates in the occurrence and development of cancer.Previous studies focused on the imbalance between the source and clearance of reactive oxygen species(ROS),while we focused on the abnormal absorption and transport of ROS in the tumor microenvironment.It was previously thought that hydrogen peroxide was transported across the membrane by simple diffusion,but recent researches have shown that the transmembrane transport of hydrogen peroxide is the facilitated diffusion dependent on aquaporins.The progression of lung cancer is also closely related to oxidative stress.Therefore,it is of great significance to explore whether AQP-dependent transmembrane transport of hydrogen peroxide participates in lung cancer progression and which isoform is responsible.Methods: We used bioinformatics to delineate the expression of different AQP subtypes in different cancer species according to TCGA database,and then verified by immunohistochemistry in lung cancer tissue microarray.In vitro,small interfering RNA was used to knock down AQPs expression,and H2DCFP-DA probe was used to detect the intracellular ROS levels.CCK8 and colony formation assays were used to detect the effect of AQPs-dependent hydrogen peroxide facilitation diffusion on the proliferation of lung cancer cells,and western-blotting was used to explore the effect on downstream key proteins and signal pathways.Finally,the effect of oxidative stress induced by AQPs on autophagy was detected by m RFP-GFP-LC3 double labeled fluorescent plasmid.Results: Bioinformatics analysis showed that AQP3 m RNA was highly expressed in lung adenocarcinoma(LUAD)and was closely related to prognosis.KEGG pathway enrichment found that AQP3 was related to peroxisome metabolism and PI3K/AKT/m TOR pathway.The expression levels of AQP3 protein in 191 paired LUAD samples in tissue microarray was detected by immunohistochemistry.It was found that the mean expression of AQP3 protein in cancer tissues was higher than the adjacent normal tissues,and was closely related to the poor prognosis.In vitro,we used small interfering RNA to transiently knock down AQP3 to detect the ability of hydrogen peroxide absorption in A549 and H1299 cells.We found that after adding exogenous hydrogen peroxide of different concentrations to the culture medium,the level of intracellular ROS increased to the peak at 30 mins,and then decreased gradually.Knockdown of AQP3 could significantly reduce the level of intracellular ROS.CCK8 and colony formation assays showed that exogenous hydrogen peroxide had paradoxical effects on the proliferation of LUAD cells,≤ 20 μM promoted proliferation,and ≥ 40 μM inhibited proliferation and induced apoptosis.At 20 μM,the knockdown of AQP3 could eliminate the promoting effect of hydrogen peroxide on the proliferation.Western-blotting showed that 20 μM hydrogen peroxide could inactivate PTEN and activate the downstream AKT/m TOR pathway,while AQP3 knockdown can eliminate the effects of hydrogen peroxide on PTEN activity and AKT/m TOR pathway.Autophagy is the most important biological behaviour regulated by AKT/m TOR pathway.We used rapamycin(m TOR pathway inhibitor)to activate autophagy,and then 20 μM hydrogen peroxide treatment could inhibit autophagy.Knockdown of AQP3 could eliminate the inhibitory effect of hydrogen peroxide on autophagy.Then,we used immunohistochemistry to detect the relationship between AQP3 and 8-oxo(oxidative stress marker)and P62(autophagy marker)in LUAD samples.We found that AQP3 was positively correlated with oxidative stress and negatively correlated with autophagy.Finally,in vivo experiments,we found that AQP3 knockdown could inhibit the subcutaneous tumorigenesis of LUAD cells in nude mice,and the tumor cells had lower levels of oxidative stress and higher levels of autophagy.Conclusion: Taken together,these results indicate that AQP3 is a LUAD oncogene that facilitates hydrogen peroxide uptake and demonstrate that the AQP3-dependent transmembrane transport of hydrogen peroxide inactivates PTEN and activates the AKT/m TOR pathway to subsequently inhibit autophagy and promote proliferation in LUAD.The above molecular mechanism of AQP3 carcinogenesis in LUAD,which may serve as a candidate target for new therapies.