The Mechanism Of CBS Promoting Growth Of Laryngeal Cancer Cells Based On PI3K/AKT/mTOR Pathway

BackgroundLaryngeal cancer is the second most common head and neck cancer globally.More than 80,000 patients die of laryngeal cancer every year.Because of the lack of biomarkers for early diagnosis,most patients with laryngeal cancer are not diagnosed until the late stage.Therefore,revealing the potential molecular mechanisms leading to laryngeal cancer is crucial for exploring effective treatment strategies.Hydrogen sulfide（H₂S）is the third gaseous signal molecule after nitric oxide and carbon monoxide.Studies have shown that H₂S is widely involved in the physiological and pathological processes of the body and plays an essential role in cardiovascular,respiratory,and tumor development.In mammals,H₂S is produced by three main enzymatic pathways,cystathionineγ-lyase（CSE）,cystathionineβ-synthase（CBS）,and through the coupling of cysteine aminotransferase（CAT）and 3-mercaptopyruvate sulfurtransferase（3-MST）.In many pathological processes of the body,may be accompanied by abnormal expression of endogenous H₂S synthase,leading to changes in hydrogen sulfide content,thus affecting disease progression.According to the current study,the expression levels of the three enzymes are usually up-regulated in many tumor cells.This abnormal expression will affect the proliferation,migration,invasion,and apoptosis of tumor cells,thus affecting cancer development.There are more and more studies on H₂S and its endogenous synthase in cancer,but related research in laryngeal cancer has not been reported.Therefore,it is significant to study the mechanism of H₂S and its synthase in laryngeal cancer cells.This article mainly expounds on the effect of CBS on the growth and death of laryngeal cancer cells.It discusses some of the mechanisms of action,which have specific theoretical guiding significance for further study of the regulatory role of CBS in laryngeal cancer cells.ObjectiveTo study the effects of CBS on proliferation,migration,invasion,apoptosis,and autophagy of laryngeal cancer cells and to explore the mechanism of CBS in the development of tumor cells.Methods1.The expression of three key enzymes in bronchial epithelial cells and laryngeal cancer cells was verified at the protein and m RNA levels,and the content of H₂S in the cells was detected.2.The laryngeal cancer cell lines with stable overexpression and knockdown of CBS were constructed by lentivirus infection,TU686 and TU212;western blot and q PCR were used to verify whether the stable cell line was successfully constructed,and the intracellular H₂S content was detected.3.The effect of CBS overexpression or knockdown on the proliferation of laryngeal cancer cells was detected by MTT,CCK-8,and Ed U.Cell cloning ability was detected by plate cloning and soft agar colony formation assay.Scratch,transwell,and invasion tests were used to detect cell migration and invasion ability.Tunel detected laryngeal cancer cell apoptosis.4.The mechanism of CBS regulating the growth of laryngeal cancer cells:EMT,apoptosis,and autophagy-related proteins were detected by western blot;the expression of PI3K/AKT/mTOR pathway protein phosphorylation was detected by Western blot.5.In vivo nude mouse tumor carrying experiment:In vivo imaging to dynamically monitor the growth of transplanted tumors;The volume of subcutaneous tumor in nude mice was measured and recorded every day to analyze the volume change of tumor and observe the growth rate of laryngeal cancer tissue.6.The expression of Ki67,CD31,Vimentin,Cleaved caspase-3,Beclin-1 and p-AKT was analyzed by immunohistochemistry.The effects of overexpression and knockdown of CBS on proliferation,apoptosis,autophagy,and pathway-related protein expression were further explored at the tissue level.7.Bioinformatics analysis,q PCR,and dual luciferase reporter assay determined the upstream inhibitors of CBS.Results1.The expression of CBS in laryngeal cancer cells TU686 and TU212 was significantly higher than that in bronchial epithelial cells HBE,and the content of H₂S in laryngeal cancer cells was also significantly higher than that in normal control cells.2.The results of fluorescence observation,Western blot,and q PCR showed that the laryngeal cancer cell lines with stable overexpression and knockdown of CBS were successfully constructed.3.In vitro functional experiments showed that overexpression of CBS could promote the proliferation,migration,and invasion of laryngeal cancer cells and inhibit the apoptosis and autophagy of TU686 and TU212.Knockdown of CBS can achieve the opposite effect.4.It was verified by Western blot that CBS may regulate the growth of laryngeal cancer cells through the PI3K/AKT/mTOR signaling pathway.5.The results of tumor-bearing experiments in nude mice showed that overexpression of CBS could promote tumor growth,while knockdown of CBS inhibited the growth rate of laryngeal cancer cells in nude mice.6.The tissue level test results were consistent with the western blot.7.miR-154-5p was negatively correlated with the m RNA level of CBS in HBE,TU686 and TU212 cells,and the miR-154-5p mimics could inhibit the expression of CBS.Conclusions1.Knockdown of CBS can inhibit the growth of laryngeal cancer cells TU686 and TU212 in vivo and in vitro,while overexpression of CBS can promote the growth of laryngeal cancer cells.2.CBS affects tumor growth by regulating laryngeal cancer cells’proliferation,migration, invasion,apoptosis,and autophagy.3.CBS regulates the growth of laryngeal cancer cells may,be through the intervention of the PI3K/AKT/mTOR pathway.4.miR-154-5p is an upstream inhibitor of CBS.