The Molecular Mechanism Of Propofol Inhibiting Migration,Invasion And Epithelial-Mesenchymal Transition In Hypoxia-Induced Esophageal Cancer Cells

Esophageal cancer is one of the most common malignant tumors of digestive system,and its main pathological type is esophageal squamous cell carcinoma.Early diagnosis and early surgical resection are still the most effective ways to treat esophageal cancer.Successful surgery requires good anesthesia.Common clinical anesthesia methods may have some effects on the prognosis of tumors.It is currently believed that anesthesia may accelerate tumor metastasis and recurrence.On the contrary,because anesthesia can effectively reduce surgical stress,some scholars believe that anesthesia can inhibit tumor progression.Propofol is one of the most widely used intravenous anesthetics in clinical anesthesia,which is used in most perioperative scenes such as anesthesia induction,anesthesia maintenance and intensive care unit use.More and more evidence shows that propofol has a certain inhibitory effect on the growth and metastasis of human tumor cells,which is directly inhibited the growth of tumor cells.In recent years,the literature level of propofol and tumor development is not limited,and the number of reports on propofol and malignant tumor recurrence and metastasis is not uncommon.Propofol has been reported to be associated with migration,invasion and epithelial-mesenchymal transition of esophageal cancer cells.However,the detailed molecular mechanism has not been fully reported.Long non-coding RNA(Lnc RNA)is involved in the progression of various cancers.Lnc RNA-miRNA-m RNA regulatory network is critical for the occurrence and development of tumors.Thymopoietin Antisense Transcript 1(TMPO-AS1)is a special Lnc RNA,which is closely related to DNA replication and cell cycle progression.Abnormal expression can affect the occurrence and development of tumors.TMPO-AS1 is of great significance in the apoptosis,recurrence and metastasis of esophageal squamous cell carcinoma and the immune escape of tumor,and its mechanism of action is still unclear.Hypoxia is one of the characteristics of tumor microenvironment,which can promote tumor cell migration,invasion and epithelial-mesenchymal transition(EMT),and cause tumor cell immune escape.Hypoxia-inducible factor-1α(HIF-1α)has been identified as one of the hypoxia-activated transcription factors,which is closely related to tumor migration,invasion and epithelial-mesenchymal transition.In order to further explore the role of propofol in the migration and invasion of esophageal cancer cells and epithelial-mesenchymal transition,we will further study the specific molecular mechanism of propofol regulating the migration and invasion of esophageal cancer cells and epithelial-mesenchymal transition,the role of Lnc RNA TMPO-AS1 and miR-498 in the regulation of propofol,and whether Lnc RNA TMPO-AS1 and miR-498 can interact.This study intends to analyze from the following four aspects : the first part : propofol inhibits the migration,invasion and epithelial-mesenchymal transition of hypoxic esophageal cancer cells by regulating the expression of HIF-1α;Part II: The mechanism of propofol inhibiting migration,invasion and epithelialmesenchymal transition of hypoxic esophageal cancer cells by regulating the expression of Lnc RNA TMPO-AS1;Part III : The mechanism of propofol inhibiting migration,invasion and epithelial-mesenchymal transition of hypoxic esophageal cancer cells by regulating the expression of miR-498;Part IV : Lnc RNA TMPO-AS1 regulates the migration,invasion and epithelial-mesenchymal transition of hypoxic esophageal cancer cells by regulating the expression of miR-498.Part Ⅰ Propofol inhibits migration,invasion and epithelialmesenchymal transition of hypoxic esophageal cancer cells by regulating HIF-1α expressionBackground: Tumor cells cause hypoxic microenvironment changes by regulating the level of HIF-1α,and promote tumor migration,invasion and epithelial-mesenchymal transition.Propofol has been reported to be associated with migration,invasion and epithelial-mesenchymal transition of esophageal cancer cells.However,the detailed mechanism has not yet been fully reported.Methods: Hypoxic cells were constructed and Transwell assay was used to evaluate cell migration and invasion.The levels of EMT markers and HIF-1α were detected by Western Blot.Results: Transwell migration test results: Compared with the Normoxia group,the number of cells in the hypoxic group was increased,the difference between the two groups was statistically significant(P<0.05).After propofol intervention,the number of cells decreased,and compared with the blank group and the propofol group,the difference was statistically significant(P<0.05).Transwell invasion test results: compared with the Normoxia group,the number of cells in the hypoxic group was increased,and compared with the blank group and the propofol group,the difference was statistically significant(P<0.05).After propofol intervention,the number of cells decreased,and compared with the blank group and the propofol group,the difference was statistically significant(P<0.05).Western Blot results showed that compared with the hypoxia group,the levels of Ecad,Vim and HIF-1α in the Normoxia group were significantly different(P<0.05).After the intervention of esophageal cancer cells,the differences in Ecad,Vim and HIF-1α between the blank group and the propofol group were statistically significant(P<0.05).Conclusion: Propofol inhibits the migration,invasion and epithelial-mesenchymal transition of hypoxic esophageal cancer cells by inhibiting the expression of HIF-1α.Part Ⅱ The Mechanism of propofol inhibiting migration,invasion and epithelial-mesenchymal transition of hypoxic esophageal cancer cells by regulating Lnc RNA TMPO-AS1Background: TMPO-AS1 is found to be highly expressed in tumors and is closely related to the prognosis of patients.However,the mechanism of TMPO-AS1 in esophageal cancer has not been studied in depth.Whether TMPO-AS1 is involved in the molecular mechanism of propofol-regulated migration,invasion and epithelialmesenchymal transition of esophageal cancer cells.Methods: The cell migration and invasion rate are determined by the Transwell invasion test.EMT markers and HIF-1α levels were detected by Western blot.TMPO-AS1 was transfected into esophageal cancer cells,and the level of TMPOAS1 was detected by real-time fluorescent quantitative polymerase chain reaction(q RT-PCR).Then,it was confirmed by double luciferase reporter gene detection and RNA immunoprecipitation(RIP)detection.Results: 1.To detect the expression of TMPO-AS1 in normal cells and esophageal cancer cells.TMPO-AS1 was highly expressed in esophageal cancer cells,and the difference was statistically significant compared with normal cells(P<0.05);2.In the two esophageal cancer cells,the expression level of TMPO-AS1 and TMPO-AS1 in hypoxic esophageal cancer cells were significantly higher than those in the normoxic group,there is a statistically significant difference in the expression of the two genes(P<0.01).After two hypoxic esophageal cancer cells were treated with propofol,the expression level of TMPO-AS1 was measured.The results showed that the expression of TMPO-AS1 was down-regulated,compared with DMSO group,the expression difference of the two genes TMPO-AS1 and DMSO is statistically(P<0.05);3.TMPO-AS1 and its control sequence vector were transfected into esophageal cancer cells,respectively.The expression level of TMPO-AS1 in the two esophageal cancer cells was detected by q RT-PCR.Compared with the cells transfected with vector,which have statistical differences between the two esophageal cancer cells(P<0.05);4.TMPO-AS1 transfection of esophageal cancer cells,after the treatment of propofol,migration and invasion experiments,the number of cells were increased,compared with the number of cells treated with propofol transfected control sequence in Transwell experiments,the difference was statistically significant(P<0.05).In the TMPO-AS1 transfected cells with high expression,the Ecad level was down-regulated,and the expression levels of Vim and HIF-1α were up-regulated.Compared with the esophageal cancer cells transfected with the control sequence,the difference was statistically significant(P<0.05).Conclusion: Propofol exerts migration,invasion and epithelial-mesenchymal transition by down-regulating the expression of TMPO-AS1.TMPO-AS1 overexpression weakens the inhibitory effect of propofol on migration,invasion and epithelial-mesenchymal transition of hypoxic esophageal cancer cells.Part Ⅲ The Mechanism of propofol regulating migration,invasion and epithelial-mesenchymal transition of hypoxic esophageal cancer cells by regulating the expression of miR-498Background: miR-498 is downregulated in esophageal squamous cell carcinoma,and promotes the development of tumor cells.Propofol can inhibit tumor development by regulating miRNA expression.The mechanism of miR-498 regulating the migration,invasion and epithelial-mesenchymal transition of esophageal cancer cells by propofol is still unclear.Methods: Anti-miR-498 was transfected into hypoxic esophageal cancer cells by propofol.Using Transwell to assess cell migration and invasion ability.Quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect the level of miR-498.Western blot was used to detect EMT markers.RNA immunoprecipitation(RIP)was used to detect RNA enrichment by q RT-PCR.Results: 1.The expression levels of miR-498 in normal cells and esophageal cancer cells,whether Het-1A and EC109 or Het-1A and KYSE70,were statistically significant(P<0.05).The expression level of miR-498 in esophageal cancer cells was significantly decreased;2.The expression levels of miR-498 in esophageal cancer cells treated with hypoxia and propofol were determined.The results showed that the expression levels of miR-498 in the two esophageal cancer cells and the expression levels of miR-498 in hypoxic esophageal cancer cells were lower,compared with the normoxia group,the difference between the two groups was statistically significant(P<0.01).The expression level of miR-498 was measured.The results showed that the expression of miR-498 was up-regulated.And there is a statistically difference in the expression of the two groups(P<0.05);3.The expression levels of miR-498 in the two cells were determined.Compared with the expression level of the control sequence cells transfected with anti-miR-498,the expression level of miR-498 in the esophageal cancer cells transfected with anti-miR-498 was significantly downregulated.And there is a statistically difference in the expression of the two groups(P<0.05);4.miR-498 was knocked down and transfected into esophageal cancer cells.After propofol treatment,the number of cells passed through increased in migration and invasion experiments,and the difference was statistically significant compared with the number of cells obtained by Transwell experiment of cells transfected with the control sequence treated with propofol(P<0.05).In the transfected cells knocking down miR-498,the Ecad level was down-regulated,and the expression levels of Vim and HIF-1α were up-regulated.Compared with the esophageal cancer cells transfected with the control sequence have statistical differences between the two esophageal cancer cells(P<0.05).Conclusion: Propofol regulates migration,invasion and epithelial-mesenchymal transition of esophageal cancer cells by up-regulating the expression of miR-498.Part Ⅳ The mechanism of Lnc RNA TMPO-AS1 regulating the migration,invasion and epithelial-mesenchymal transition of hypoxic esophageal cancer cells by inhibiting the expression of miR-498 Background: Starbase predicts that miR-498 is a potential target for TMPO-AS1.To investigate whether TMPO-AS1 can regulate the progression of esophageal cancer cells by down-regulating the expression of miR-498.Methods: Starbase predicted the interaction between TMPO-AS1 and miR-498.Transfection of si-NC,si-TMPO-AS1,si-TMPO-AS1 + anti-NC,si-TMPO-AS1 + anti-miR-498 hypoxic esophageal cancer cells.Quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect TMPO-AS1 and miR-498 levels.Then,it was confirmed by double luciferase reporter gene detection and RNA immunoprecipitation(RIP)detection.Results: 1.TMPO-AS1 acts as the sponge molecule of miR-498 to interact with each other;2.The verification of dual luciferase reporter gene showed that after transfection of miR-498 in tumor cells,the luciferase activity of TMPO-AS1 was significantly lower than that of the negative control miR-NC,without affecting the luciferase activity of TMPOAS1 MUT.The comparison of the two groups of data was statistically significant(P<0.05);3.The RIP experiment confirmed the interaction between TMPO-AS1 and miR-498;4.Transfection of si-TMPO-AS1 significantly reduced TMPO-AS1 level.Subsequently,our data showed that the expression of miR-498 was significantly up-regulated after TMPO-AS1 knockdown(P<0.05),while the overexpression of TMPO-AS1 significantly down-regulated the expression of miR-498;5.Migration and invasion of esophageal cancer cells after transfection with miR-498 were lower than those in miR-NC group,and migration and invasion of esophageal cancer cells after transfection with TMPO-AS1 and miR-498 were higher than those in miR-498 + vector group(P<0.05).After transfection of anti-miR-498,the invasion number of esophageal cancer cells was higher than that of anti-miR-NC group;The number of migration and invasion of esophageal cancer cells transfected with si-TMPO-AS1 and anti-miR-498 was lower than that of anti-miR-498 + si-NC group(P<0.05).Compared with the miR-NC group,the expression level of Ecadherin protein in esophageal cancer cells was increased and the expression level of Vimentin protein was decreased after miR-498 transfection.Compared with miR-498 + vector group,TMPO-AS1 and miR-498 transfection decreased E-cadherin protein expression and increased Vimentin protein expression in esophageal cancer cells(P<0.05).Compared with the anti-miR-NC group,the expression level of Ecadherin in esophageal cancer cells was decreased and the expression level of Vimentin was increased after transfection of anti-miR-498;Compared with the antimiR-498 + si-NC group,the expression levels of E-cadherin and Vimentin in esophageal cancer cells were increased and decreased after transfection of si-TMPOAS1 and anti-miR-49(P<0.05).Conclusion: Overall,our study found that propofol inhibits hypoxia-induced migration of esophageal cancer cells by regulating TMPO-AS1/miR-498/HIF-1α axis,which provides a theoretical basis for the treatment of esophageal cancer.