Study On The Effect And Mechanism Of Melittin On Bladder Cancer

Objective:(1)Detect the effect of melittin on cell proliferation by CCK8 method and clone formation experiment.(2)Detect the effect of melittin on cell migration and invasion by scratch test and Transwell test.(3)The effects of melittin on the expression of bladder cancer cells mi R-146 a,NUMB and NOTCH2 were detected by q PCR and WB.(4)Exploring the influence of mi R-146 a on NUMB and NOTCH2.(5)Explore the effect of overexpression of NUMB on the proliferation of bladder cancer cells.(6)Explore the clinical significance of mi R-146 a and NUMB through bioinformatics and testing clinical specimens.Methods:(1)CCK8 assay and plate cloning assay were used to explore the proliferation ability of bladder cancer cells.(2)Clone formation assay and Transwell assay were used to detect the effect of melittin on the migration and invasion of bladder cancer cells.(3)QPCR detects the result of melittin on the m RNA levels of bladder cancer cells mi R-146 a,NUMB and NOTCH2;WB detects the protein levels of melittin on bladder cancer cells NUMB,NOTCH2 The mi R-146 a mimic and the control of the mimic were co-transfected with psi CHECK2-NUMB-MUT and psi CHECK2-NUMB-WT,and then the dual luciferase reporter experiment was used to explore the direct binding effect of mi R-146 a and NUMB.(4)Lipo2000 was used to transfect mi R-146 a mimics and control mimics,and CCK8 and clone formation experiments were used to explore the effects of mi R-146 a on the proliferation of bladder cancer cells and the expression levels of NUMB and NOTCH2.(5)Transfect NUMB overexpression plasmid and control plasmid with lipo2000,and use CCK8 and clone formation experiment to explore the effect of mi R-146 a on the proliferation of bladder cancer cells and the expression level of NUMB and NOTCH2.(6)Use bioinformatics technology and q PCR to detect the expression of mi R-146 a in bladder cancer tissues and analyze its clinical significance;use bioinformatics technology,q PCR and WB to detect NUMB m RNA and protein expression levels in bladder cancer tissues.Results:(1)After treatment with 4μg/m L melittin,the absorbance value of bladder cancer cells was significantly reduced(P<0.01),the number of clones formed by the cells was less(P<0.01),and the longer the treatment time,the greater the effect of inhibiting proliferation Obvious;flow cytometry results showed that the ratio of Annexin V/PI double-stained cells in the treatment group was significantly increased after treatment with 4μg/m L melittin 4μg/m L for 72h(P<0.01);WB detected the apoptosis after treatment with 4μg/m L melittin for 48 h Death protein level: The expression level of CASPASE9 increases,the expression level of BAX increases,and the expression level of BCL-2 decreases.(2)After treatment with 4μg/m L melittin for48 h,the ability of bladder cancer cells to migrate and invade was significantly weakened(P<0.01).(3)The RNA level of 4μg/m L melittin treatment was detected by q PCR for 48 h.The level of mi R-146 a was significantly down-regulated(P<0.01),the expression level of NUMB was up-regulated(P<0.01),and the expression level of NOTCH2 was down-regulated(P<0.01);WB detects the protein level of 4μg/m L melittin treatment 48 h,NUMB expression level is up-regulated,NOTCH2 expression level is down-regulated;dual luciferase report experiment found that psi CHECK2-NUMB-MUT and mi R-146 a mimics and mimics were co-transfected The fluorescence value of the control is significantly different(P<0.01).Whether the fluorescence value is different between the co-transfected psi CHECK2-NUMB-WT and mi R-146 a mimic and mimic control.(4)CCK8 method and clone formation experiment found that overexpression of mi R-146 a affected the proliferation inhibitory effect of melittin on bladder cancer cells;q PCR and WB found that overexpression of mi R-146 a affected melittin on bladder cancer cells NUMB and NOTCH2 The regulation of the expression level.(5)Through CCK8 method and clone formation experiment,the results showed that overexpression of NUMB can reverse the proliferation effect of mi R-146a;through WB,it can be found that overexpression of NUMB can reverse the proliferation effect of mi R-146 a.(6)The detection of bioinformatics and clinical samples found that mi R-146 a is highly expressed in bladder cancer,and mi R-146 a is related to lymph node metastasis in patients with bladder cancer;NUMB is low in bladder cancer.Conclusion:(1)Melittin can inhibit the proliferation,migration and invasion of BC cells.(2)Melittin can regulate the mi R-146a/NUMB/NOTCH2 axis to affect the proliferation of bladder cancer cells.(3)Mi R-146 a can be used as a tumor marker and therapeutic target for bladder cancer.