The Research Of The Interaction Between B7-H3 And ENO1 And Downstream Signaling Pathways In Lung Cancer Cells

The B7-H3 molecule belongs to the immunoglobulin superfamily and is an immune molecule discovered by Chapoval in 2001 using a dendritic cell c DNA library for B7molecular homologous gene analysis.As a member of the B7 family,unlike the known receptors of PD1/PDL1,since the exact receptor of B7-H3 is still not fully understood,its specific role and mechanism（co-stimulation or co-suppression）remains controversial.At the beginning of the discovery,some scholars found that B7-H3 can play an immune activation role by promoting the proliferation of CD4⁺and CD8⁺cells and up-regulating the expression level of IFN-γ.However,with further research,it was found to be highly expressed in many malignant tumor cells.A large number of studies have shown that B7-H3 is closely related to malignant phenotypes such as tumor proliferation,metastasis,invasion and tumor immune escape.In addition,several studies in recent years have reported that B7-H3 is closely related to abnormal energy metabolism of tumor cells.ENO1 belongs to the family of enolases.As an important rate-limiting enzyme in the glycolytic pathway,ENO1 predominantly catalyzes the oxidation of 2-phosphoglycerate to phosphoenolpyruvate during glycolysis and produces the ATP.Apart from this,ENO1 is also involved in various physiological processes in the cell,such as growth regulation,hypoxia tolerance,and autoimmune regulation.A large number of studies have shown that ENO1 is highly expressed in most malignant tumor cells,and in vitro and in vivo experiments have confirmed that ENO1 overexpression is closely related to malignant phenotypes such as tumor cell metabolism,proliferation,metastasis,invasion and drug resistance,and ENO1 is regarded as one of the biomarkers for diagnostic and has prognostic value in a variety of tumors.These studies all suggest that ENO1 is an oncogene closely related to the occurrence and development of malignant tumors and plays a key role.In the early experiments,it was confirmed by Co-immunoprecipitation experiments and mass spectrometry that there was a direct interaction relationship between B7-H3 and glycolytic enzyme ENO1.However the specific interaction mechanism of the two is exactly what this experiment should explore.Objective:To construct B7-H3/ENO1 over-expression lung cancer cell lines by preparing B7-H3/ENO1 over-expression vectors,respectively;to investigate the changes of B7-H3/ENO1 over-expression in lung cancer cells by cell functional assay such as CCK-8 method,scratch-healing assay and flow cytometry.Using RT-PCR and Western-blotting to analyze the correlation of B7-H3 and ENO1 expression,using the ENO1 activity detection kit to analyze the effect of B7-H3 over-expression on ENO1activity;add the ENO1 activity inhibitor to B7-H3 over-expression cells,using cell proliferation experiments and scratch experiments to explore the effect of ENO1 activity inhibition on tumor-promoting role of B7-H3 overexpression;Western-blot experiments were used to examine the changes of signaling pathway molecules caused by over-expression of B7-H3 and the effect of inhibiting ENO1 activity on those molecules.Methods:Part 1:B7-H3/ENO1 over-expression cell model construction（1）Design ENO1 gene amplification primer,and the ENO1 sequences were amplified by PCR.Then connect it to pc DNA3.1 vector,construct ENO1 over-expression vector pc DNA3.1/ENO1.B7-H3 over-expression vector pc DNA3.1/B7-H3 had been stored in the laboratory;（2）ENO1 over-expression vector-pc DNA3.1/ENO1 was transfected into PC14 cells,B7-H3 over-expression vector-pc DNA3.1/B7-H3 was transfected into sbc-5 cells at logarithmic growth phase with liposome Lipofectamine ^TM2000,respectively.RT-PCR and Western-blot experiments were used to identify the over-expression effects of B7-H3 and ENO1.Part 2:The effect of B7-H3/ENO1 on the phenotype of lung cancer cells（1）Take the cells of each group for CCK-8 cell proliferation experiment,including B7-H3 over-expression cell model,ENO1 over-expression cell model and their corresponding control cells plating,draw growth curve according to absorbance value at OD450nm,and evaluate cell proliferation ability in each group;（2）Scratch test was performed on each group of cells,the scratch widths were recorded at 0h and 48h,and the migration ability of each group was compared according to the ratio of width difference;（3）Cell cycle and apoptosis were detected by flow cytometry in each group of cells and analyse these results.Part 3:Exploration of the interaction between B7-H3 and ENO1（1）Extract RNA and protein of lung cancer cell models constructed in the first part,and detect the expression of B7-H3 and ENO1.Analyze the correlation between B7-H3and ENO1 expression;（2）Detect the ENO1 activity by using ENO1 activity detection kit in the B7-H3 over-expression model and the corresponding control cells,to evaluate changes of ENO1 enemye activity in the case of over-expression of B7-H3;（3）Adding ENO1 activity inhibitor to the B7-H3 over-expression cells,and using CCK-8 and scratch healing experiments to explore whether the change of ENO1 activity promotes or inhibits the tumor promoting effect of B7-H3.Part 4:Detection of related molecules in B7-H3 downstream signaling pathway（1）Extract proteins from B7-H3 over-expression cell model and corresponding control group,and detect downstream signaling pathway molecules（PI3K-p85α,PTEN,Akt,m TOR,GSK3β）and protein expression of phosphorylation levels of these pathway molecules（p-Akt,p-m TOR,p-GSK3β）;（2）ENO1 activity inhibitor（ENOBlock）was added to B7-H3 over-expression cells and detect the expression of the above-mentioned signaling pathway molecular proteins.Results:（1）RT-PCR and WB experiments results verified that the B7-H3/ENO1over-expression cell model was successfully constructed;（2）The CCK-8 and scratch healing experiments results showed that over-expressing B7-H3 or over-expressing ENO1significantly enhanced the proliferation and migration ability of lung cancer cells,but had no significant effect on cell cycle and apoptosis;（3）Using RT-PCR and WB detection,it was found that B7-H3 over-expression cells showed no significant difference in ENO1expression compared with the control group;similarly,compared with the corresponding control cell model,ENO1 over-expression cells has no significant B7-H3 expression difference.But ENO1 activity was found to be elevated in the case of B7-H3over-expression using the ENO1 activity assay kit;（4）After the addition of ENO1 activity inhibitor to B7-H3 over-expression cells,the enhancement of cell proliferation and migration ability was weakened;（5）The over-expression of B7-H3 increased the protein expression levels of PI3K-p85α,p-Akt,and p-m TOR,and reduced the protein expression levels of PTEN and p-GSK-3β,but did not significantly affect the total protein levels of these molecules（Akt,m TOR,GSK3β）.After the addition of ENOBlock,the above-mentioned changes were significantly reverted.Conclusion:（1）Over-expression of B7-H3 or ENO1 enhances the proliferation and migration of lung cancer cells;（2）There is a direct interaction between B7-H3 and ENO1,but their expression level have no correlation.Over-expressing B7-H3 upregulates the activity of ENO1;（3）Inhibit the activity of ENO1 can attenuate the tumorigenic effect of B7-H3.It can be concluded that B7-H3 may promote tumor growth and migration by affecting ENO1 activity or maybe ENO1 is downstream molecule of B7-H3;（4）In lung cancer cells,over-expression of B7-H3 can increase the protein expression levels of PI3K-p85α,p-Akt,p-m TOR,and decrease the protein expression levels of PTEN and p-GSK-3β.After adding ENOBlock,the above changes have been greatly reverted.ENO1is probably an intermediate molecule between B7-H3 and these signaling pathways.To sum up,this study preliminarily explored the role of B7-H3 and ENO1 in the occurrence and development of lung cancer cell lines.Further investigation revealed that B7-H3 may play a tumor-promoting role by affecting ENO1 activity.It provides a new idea for the in-depth interpretation of the interaction between the two.