Hypoxia Promotes The Migration Of Human Lung Adenocarcinoma A549 Cells Via Acetyl-CoA Carboxylase 1

BackgroundLung adenocarcinoma currently has the highest incidence in all lung cancer pathology types.It has been demonstrated that hypoxia,an important impact factor of the tumor microenvironment,can regulate the metastasis of tumor cells through lipid metabolism.Because of the lack of effective therapeutic methods for the metastasis of lung adenocarcinoma,it is important to clarify the molecular mechanism of lung adenocarcinoma metastasis driven by hypoxia microenvironment and early intervention for the metastasis of lung adenocarcinoma,which is crucial for the prognosis of lung adenocarcinoma.Acetyl-Co A carboxylase 1（ACC1）,a rate limiting enzyme catalyzing the carboxylation of acetyl-Co A to malonyl-Co A in fatty acid synthesis,plays an important role in fatty acid metabolism in tumor cells.Researches have shown that the drugs targeted ACC1 have certain anti-tumor effects on mouse lung cancer model,however,the effect of ACC1 on lung cancer cell migration is still unclear.ObjectiveTo clarify the mechanism of hypoxia up-regulating the expression of ACC1 in lung adenocarcinoma cells and the mechanism that hypoxia promotes the migration of lung adenocarcinoma cells by up-regulating ACC1.MethodsHuman lung adenocarcinoma A549 cell line was used and lentiviral transfection was conducted to establish control A549 cell line sh-NC and ACC1 knocking down A549 cell line sh-ACC1.Firstly,the Transwell experiment was conducted to detect the effect of hypoxia on lung adenocarcinoma cells,and then Western blot was performed to observe the expression of ACC1,Vimentin and E-cadherin in lung adenocarcinoma A549 cells.The changes in ACC1 and SREBP-1 m RNA levels were detected by RT-q PCR.After normoxia or hypoxia,the Transwell experiment was conducted to detect the migration and the Western blot was used to detect the expression of EMT-related proteins Vimentin and E-cadherin of sh-NC and sh-ACC1A549 cells.Then A549 cells were treated by hypoxia or hypoxia combined with HIF-1αinhibitor PX-478 and Transwell experiment was performed to observe migration changes.Western blot was conducted to observe the levels of HIF-1α,SREBP-1,ACC1 and EMT-related proteins Vimentin and E-cadherin.RT-q PCR was performed to detect the levels of ACC1 and SREBP-1 m RNA;After normoxia or hypoxia treatment,Transwell experiment was performed to examine the migration of A549 cells that knockdown of SREBP-1;Then the expression of ACC1 and EMT-related proteins Vimentin and E-cadherin were detected by Western blot;Finally,sh-NC and sh-ACC1 A549 cells were treated with Linoleic acid（LA）,and the cell migration was examined by Transwell assay and Western blot was conducted to observe the changes of Vimentin and E-cadherin.ResultsHypoxia promotes the migration of lung adenocarcinoma A549 cells and up-regulates the expression of ACC1,which can promote EMT process.A549 cells were dealed with 5%O₂ for 24 h.Then Western blot demonstrated that the expression of ACC1 and Vimentin was up-regulated,and the E-Cadherin’s expression was decreased.A549 cells that knockdown of ACC1 were treated with normoxia or 5%O₂ and the reuslts showed that the hypoxia-induced migration promotion of A549 was inhibited.In normoxia and 5%O₂ groups,the expression of Vimentin in sh-ACC1groups was less,and the E-Cadherin was higher.There was no statistically significant difference in the expression of Vimentin and E-Cadherin proteins in the sh-ACC1groups after normoxia or 5%O₂ conditions.The results showed that the effects of hypoxia on A549 cell migration enhancement and the expression of Vimentin and E-Cadherin disappeared after ACC1 was knocked down.RT-q PCR detected that the levels of ACC1 and SREBP-1 m RNA in A549 cells increased after 5%O₂treatment,and Western blot detected that the expressions of ACC1 and SREBP-1 increased,while the E-cadherin expression decreased and the Vimentin increased.A549 cells were treated with 5%O₂ combined with HIF-1αinhibitor PX-478.Then RT-q PCR detected that ACC1 and SREBP-1 m RNA levels were less than the hypoxia group;Western blot showed that the expression of ACC1 and SREBP-1decreased than the hypoxia group,and the E-cadherin expression was increased but Vimentin expression was decreased.Transwell experiments showed that the migration number of cells in the si-SREBP-1 group was higher than that in the normoxia group.Compared with the hypoxia group,the cell migration number of the si-SREBP-1group decreased compared with the hypoxia group,howerer,the difference was not statistically significant compared with the normoxic control group.Compared with the si-NC group,the si-SREBP-1 group had a decreased expression of ACC1 and Vimentin,while the level of E-cadherin was increased;After hypoxia treatments of the si-SREBP-1 cells,the expression of ACC1 reduced.The E-cadherin was higher but the level of Vimentin reduced.To explore the mechanism of ACC1 promoting the migration of A549 cells under hypoxia conditions,A549 cells that knocked down of ACC1 were dealed with LA.The results showed that supplementation of LA restored the migration promoting effect of hypoxia on A549 cells that knockdown of ACC1.At the same time,it was detected that Vimentin level was higher,however,the level of E-Cadherin was decreased.ConclusionHypoxia up-regulates the expression of ACC1 by HIF-1α/SREBP-1 to cause the occurrence of EMT,and subsequently promotes the migration of lung adenocarcinoma A549 cells.