Hypoxia Influences Colorectal Cancer Progression Through SIRT1 Pathway

Colorectal cancer（CRC）recently has become the third most malignant tumors globally.Although the prognosis of patients with CRC has been dramatically improved by using continuously updated therapeutic methods,the median survival duration is only about 18 to 22 months.Therefore,it is pivotal to explore the mechanisms related to CRC carcinogenesis and progression.CRC belongs a kind of solid tumor.With the growth of tumors,the center region of tumor is usually in a state of scanty vascularization and hypoxia.In order to adapt to hypoxic microenvironment,tumor cells modulate their biological behaviors by the alteration of gene expression profiles.Hypoxic microenvironment promotes tumor progression by inducing epithelial-mesenchymal transition（EMT）,activating autophagy,and promoting inflammatory cell infiltration,etc.Silencing information regulator 1（SIRT1）is a NAD⁺-dependent deacetylase,which regulates multiple signaling pathways through catalyzing histones and non-histones deacetylation,and plays important roles in cell proliferation,differentiation,apoptosis,aging and stress.In addition,it also plays active roles in the process of tumorigenesis and progression.However,there still are controversies on whether SIRT1 acts as an oncogene or a tumor suppressor.Mounting studies have shown that SIRT1 deacetylates the upstream proteins,initiation complexes and autophagy-related proteins（ATGs）in the process of autophagy,thus participating in the regulation of autophagy.In addition,SIRT1 interacts with E-cadherin,vimentin,c-Myc,NF-κB（nuclear factorκB）and TGF-β（transforming growth factorβ）to influence invasion and migration of tumor cells.In hypoxic tissues,SIRT1 is regulated by a series of transcription factors,such as HIF-1α（hypoxia-inducible factor-1α）,Sp1 and NF-κB.Its higher expression level plays a protective role in ischemia-reperfusion in myocardium,liver and brain tissues.Hypoxic microenvironment also affects the biological behaviors of cells by altering the expression levels of SIRT1 In tumor tissues.Therefore,this study aims to investigate the alterations and effects of SIRT1 expression on CRC cells and to explore their mechanisms on tumor invasion and migration,as well as autophagy under hypoxia.In order to study the alterations of SIRT1 expression level under hypoxia,CRC cell lines HCT116 and SW480 were cultured in a hypoxic environment（1%O₂）.The expression levels of SIRT1 mRNA and protein were dectected by Western blot and real-time PCR.The results showed that the expression level of SIRT1 decreased with the prolongation of hypoxic exposure.In cobalt chloride treatment model,the similar result was found,which implied that hypoxia inhibited SIRT1 expression.Analysis of clinical specimens from nine patients with CRC showed that the expression level of SIRT1 in carcinoma tissue was significantly lower than that in normal colorectal and para-carcinoma tissues.For exploring accurate mechanisms,we constructed plasmids with different length of SIRT1 promoter sequence truncations（truncated from upstream-1000 bp）.Reporter gene assay indicated that the core transcriptional regulatory region of SIRT1 expression affacted by hypoxia was located in upstream-100 bp of the promoter sequence.Two transcription factors,namely EGR1（early growth response 1）and USF2（upstream stimulatory factor 2）,which were potentially involved in the regulation of this process were found by bioinformatics.And a known transcription factor Sp1 was exploited as a positive control.The experiment results showed that EGR1 was responsible for the regulation of SIRT1 transcription,even more effective than Sp1.However,USF2 had no effect on SIRT1 transcription.The results from Western blot and chromatin immunoprecipitation（ChIP）showed that the expression of EGR1 was decreased under hypoxic environment,and the binding of EGR1 to the-100 bp sequence of SIRT1promoter was significantly decreased.Finally,by the point mutation of promoter in this region,we found that EGR1 could bind to GC-rich motif at-80 bp to-72 bp and-21 bp to-13 bp in the upstream of SIRT1.In summary,we confirmed that EGR1 regulates SIRT1transcription and affects SIRT1 expression under hypoxia.To clarify influences of altered SIRT1 expression on the biological behaviors of CRC cells under hypoxia,we constructed stably transfected cells with SIRT1 knockdown or overexpression lentivirus（HCT116 sh-SIRT1,SW480 Lenti-SIRT1）.By using Transwell assays,we found that hypoxia promoted the invasion and migration of CRC cells.The abilities of invasion and migration in SIRT1-knockdown cells or SIRT1 inhibitor（EX527）-treated cells were significantly enhanced;while SIRT1-overexpressed cells and SIRT1 agonist（SRT1720）-treated cells were significantly inhibited.In addition,Western blot and real-time PCR analyses were used to detect NF-κB and its downstream molecules.The results showed that under hypoxia,the acetylation of NF-κB p65 Lysine 310 residue was increased,along with the up-regulation of MMP-2 and MMP-9.In SIRT1-downregulated HCT116 cells,the acetylation level of NF-κB as well as MMP-2and MMP-9 were significantly higher than that of parental cells,suggesting that decreased SIRT1 might activate the NF-κB pathway as well as promote the expression of MMP-2and MMP-9,and thus affecting the invasion and migration of CRC cells.Moreover,we conducted a preliminary study to explore autophagic flux in cells under hypoxia.The results of Western blot and transmission electron microscopy showed that hypoxia significantly reduced autophagy-specific substrate p62 and enhanced LC3-I/II conversion,increased the formation of autolysosomes,and induced abnormal organelle morphology.Compared with parental cells,overexpression of SIRT1 inhibited the degradation of p62,decreased LC3-I/II conversion,and reduced the number of autolysosomes.In order to further observe the autophagic flux in tumor cells,GFP-mRFP-LC3 double-labeled adenovirus was used to infect cells.The results showed that the fusion of autophagosome and lysosome of SIRT1-overexpressed cells was inhibited,and autophagic flux was blocked.These results suggested that SIRT1 was involved in the regulation of autophagy in CRC cells under hypoxia.However,the specific mechanism remains to be further studied.In conclusion,we find that SIRT1 influences the progression of CRC under hypoxic conditions.For the first time,the mechanism that EGR1 participates in the regulation of SIRT1 transcription under hypoxia is elucidated.Moreover,our results propose that hypoxia regulates the invasion and migration of CRC cells through an EGR1/SIRT1/NF-κB pathway,and SIRT1 mediates the regulation of cell autophagy under hypoxia.Our studies enrich the roles of SIRT1 in the progression of CRC and might provide a possible therapeutic target for CRC clinical treatment.