Effect And Mechanism Of Silencing AQP5 On Autophagy In Human Colorectal Cancer Cells

ObjectiveTo study the expression and clinical significance of AQP5 in colorectal cancer.To investigate the effect of lentiviral silencing AQP5 on HCT116 and RKO autophagy in colorectal cancer cells,and to explore the possible mechanism of action.MethodsAnalysis of AQP5 expression levels and clinical significance in colorectal cancer by bioinformatics.Through RT-q PCR,Western blot studied AQP5 expression levels in colorectal cancer and adjacent tissues and human colorectal cancer cell lines and human normal colorectal epithelial cells NCM356 at the transcription level and protein level,respectively.The sh-RNA lentivirus targeting AQP5 gene and the negative control lentivirus were transfected with human colorectal cancer HCT116 and RKO cell lines,and the experiments were divided into untransfected blank group(control),negative control group(sh-NC),and interference group(sh-AQP5).Then,the fluorescence expression efficiency after lentiviral transfection was observed by fluorescence microscopy,and the effects of m RNA and protein silencing were verified by RT-q PCR and Western blot,respectively,and then the stable transfected HCT116 and RKO cells were screened.Later,the changes of autophagy-related proteins Beclin 1,p62,LC3II/LC3 I were detected by Western blot,and the changes in the number of autophagic bodies were observed by transmission electron microscopy and MDC staining.The changes of pathway-related proteins T-AMPK,T-m TOR,TULK1,p-AMPK,p-ULK1 and p-m TOR proteins were further detected by Western blot experiments.The AMPK inhibitor Compound C was then added to the cells that silenced AQP5 for a recovery experiment.Western blot experiments,transmission electron microscopy(TEM)detection and MDC staining were carried out to observe the changes of corresponding protein levels and autophagsome changes before and after the addition of AMPK inhibitors.ResultsAQP5 is highly expressed in the cancerous tissue detected,while it is hardly expressed in the normal tissue next to the cancer.Expression in colorectal cancer cell lines is also higher than in normal colorectal epithelial cells NCM356.Bioinformatics analysis showed that AQP5 had a relatively accurate diagnostic value for the diagnosis of colorectal cancer.In colorectal cancer cells HCT116 and RKO,silence of AQP5 led to enhanced autophagy,and TEM and MDC staining showed that the autophagic bodies in the sh-AQP5 group were significantly more than those in the control and sh-NC groups.The results of Western blot’s detection of autophagy-related proteins and pathway proteins showed that compared with control and sh-NC groups,the expression of Beclin1 and LC3II/LC3 I proteins in the sh-AQP5 group was increased,while the expression of p62 was downregulated(P<0.05),while there was no significant difference between control and sh-NC histone expression(P>0.05).There was no significant change in the total protein associated with the pathway in the three groups(P>0.05),while the expression of p-AMPK and p-ULK1 in the sh-AQP5 group was up-regulated compared with the control and sh-NC groups,p-m TOR expression was downregulated(P<0.05),and there was no significant difference between control and sh-NC groups(P>0.05).After adding the AMPK inhibitor Compound C to two cells HCT116 and RKO that silenced AQP5,it was found that: After the addition of Compound C,the expression of LC3II/LC3 I,Beclin 1,p-AMPK,and p-ULK1 proteins decreased,while the expression of p62 and pm TOR increased(P<0.05).TEM and MDC staining results also showed that the number of autophagies decreased compared with the sh-AQP5 group after the addition of inhibitors.ConclusionsSilencing AQP5 upregulates autophagy levels in HCT116 and RKO ce lls,and its mechanism of action may be achieved by regulating the AMPK/m TOR/ULK1 pathway,AQP5 may be a potential biomarker and molecular t arget for the treatment of colorectal cancer.