Based On MiR-182 To Explore The Mechanism Of Yiqi Chutan Decoction On Nsclc Autophagy And Apoptosis

ObjectiveBased on the results of pre-clinical studies of miR-182,the expression of miR-182 in the serum of patients with advanced NSCLC was significantly lower than that of healthy volunteers,and it was expressed higher in the serum of patients with advanced NSCLC in the intervention group of Yiqi Chutan Decoction.Based on miR-182,to explore the molecular mechanism and target of Yiqi Chutanfang’s intervention in NSCLC autophagy and apoptosis.MethodsPart 1: miR-182 regulates autophagy and apoptosis(1)Use quantitative real-time PCR to compare the relative expression of miR-182-5p mimic in non-small cell lung cancer A549 cell line transfected with miR-182-5p in the control group;Western blot to detect the relationship of miR-182-5p mimic and the expression of autophagy-related gene protein ATG7.(2)Through proliferation(Edu)and apoptosis(Tunel)experiments,evaluate the effects of miR-182-5p mimic and miR-182-5p inhibitor on the proliferation and apoptosis of A549,95 D,and 95 C cells after 48 hours of intervention.(3)Through cellular immunofluorescence experiments,evaluate the effects of miR-182-5p mimic and miR-182-5p inhibitor on A549,95 D,95C cells in miR-182-5p/ATG7 autophagy pathway and the Bcl-2 family.Part 2: Based on miR-182 to explore the mechanism of Yiqi Chutan Decoction on NSCLC autophagy and apoptosis(1)Using CCK-8 method to detect the effects of Yiqi Chutan Recipe,miR-182 mimics,miR-182 inhibitor on cell viability and drug resistance on non-small cell lung cancer cell lines.(2)Through Ed U and TUNEL experiments,investigate the effects of Yiqi Chutan Recipe,miR-182 mimics,and miR-182 inhibitor on cell proliferation and apoptosis on A549,95 D,and 95 C cell lines.(3)Detect the cell cycle experiment by flow cytometry to investigate the effect of Yiqi Chutan Recipe,miR-182 mimics,and miR-182 inhibitor on the cell cycle of non-small cell lung cancer cell lines.(4)Through cellular immunofluorescence experiment,cellular immunohistochemistry,Western-blot experiment,evaluate the effect of Yiqi Chutan Decoction on A549 cell miR-182-5p/ATG7 autophagy pathway,Bcl-2 family and intrinsic apoptosis after 48 h intervention.(5)Observe autophagy-related indexes such as Atg7,Beclin-1,LC3 by using cellular immunofluorescence,Western-blot,and cellular immunohistochemistry experiments from before,12 h,24h,and 48 h after drug addition.(6)Cellular immunofluorescence experiment,observe the changes of LC3A/B,Beclin-1 at 0min,20 min,40min,60 min,90min,2hour,8hour,10 hour,12hour,24 hour,48hour after adding drugs.ResultsPart 1: miR-182 regulates autophagy and apoptosis(1)Transfection of miR-182-5p mimic 48 h can increase the expression of miR-182-5p in the A549 cell line.(2)The expression of miR-182-5p increased,and the expression of autophagy-related gene protein ATG7 decreased.(3)miR-182-5p mimic inhibits the proliferation of A549 and 95 D cells and promotes apoptosis.(4)miR-182-5p mimic intervention for 48 h can inhibit the expression of autophagy-related proteins such as LC3 in A549 and 95 D cells,and down-regulate the expression of tumor suppressor Bcl-2.Part 2: Based on miR-182 to explore the mechanism of Yiqi Chutan Decoction on NSCLC autophagy and apoptosis(1)The effective concentration of Yiqi Chutan Recipe for non-small cell lung cancer cells is 2mg/ml;(2)Yiqi Chutan Decoction can inhibit the proliferation of non-small cell lung cancer cells,thereby promoting the apoptosis of lung cancer cells;(3)After adding 2mg/ml of Yiqi Chutan Recipe,the formation of A549 cell autophagy membrane(LC3 A/B)is divided into two peaks,one is 20min-40 min after drug addition;the second peak is drug addition After 2 hours to 10 hours.(4)After adding 2mg/ml of Yiqi Chutan Recipe,the first peak of autophagy membrane,the formation of autophagy membrane(LC3 A/B)increased when A549 cells were added for 20 min and 40 min,but Beclin-1 The expression did not increase,which may be related to the release of anti-apoptotic protein bcl-2and Beclin-1.(5)After adding 2mg/ml of Yiqi Chutan Recipe,the autophagy membrane has the second peak.2 hours after the addition,the expression of Beclin-1increases,and the expression of autophagy membrane(LC3 A/B)increases again and continues To 10 hours after dosing,until 12 hours after dosing.(6)Yiqi Chutanfang intervention for 48 hours can inhibit the expression of autophagy-related proteins such as LC3 in A549,95 D,and 95 C cells,and down-regulate the expression of tumor suppressor Bcl-2.Conclusion1.miR-182 regulates the expression of autophagy-related genes Atg7 and LC3 A/B in advanced non-small cell lung cancer,and inhibits autophagy;2.miR-182 inhibits the proliferation of NSCLC,regulates the anti-apoptotic Bcl-2 protein subfamily,and induces late non-small cell apoptosis;3.Yiqi Chutan Decoction up-regulates the expression of miR-182,regulates the expression of autophagy-related genes Atg7,LC3 A/B,etc.in advanced non-small cell lung cancer,and inhibits autophagy;4.Yiqi Chutan Decoction up-regulates the expression of miR-182,regulates the apoptosis-inhibiting Bcl-2 protein subfamily,and induces late non-small cell apoptosis.5.miR-182-5p mimic may be more effective for non-small cell lung cancer with advanced distant metastasis(higher malignancy).miR-182-5p mimic inhibits its autophagy level and promotes its apoptosis.