Study On The Role Of Hedgehog Signal System In The Model Of Lung Inflammation To Tumor Transformation

Background:As one of the global malignant tumors,etiology of lung cancer is very complicated.Smoking,air pollution and chronic lung disease are closely related to the occurrence of lung cancer.Studies have shown that COPD is an independent risk factor for lung cancer.Lung cancer and COPD is a homologous disease related to smoking.Lung cancer is even a chronic inflammation related disease.Multiple proteins and cytokines are mainly activated by chronic inflammation and composed complex molecular networks,which can cause tumor-like transform from chronic inflammation through multiple endogenous and/or exogenous signaling pathways.Since EGFR signaling system is one of the important cellular metabolic regulation pathways.Multiple nodes or cytokines have become an important target for clinical therapy.But the clinical efficiency is often less than 20%.It is suggested that the interaction of multiple signal systems may exist in the development of tumor and drug sensitivit y.Therefore,the role of Hedgehog(HH)signal system has been paid more and more highlight.Hedgehog signal transduction pathway is composed of ligand HH,receptor PTCH and SMO,transcription factor Gli,SUFU,and other regulatory factors.An increased Hedgehog signaling protein has been detected in a variety of malignant tumor tissues.According to the progress of the existing research,the possible mechanism of activation of Hedgehog signaling pathway in tumor cells has several situations.Including(1)Hedgehog ligand autocrine or paracrine secretion,may be related to the gene mutation,or secondary to other signal system activity enhancement;(2)PTCH mutation;(3)SMO mutation;(4)Sufu mutation;(5)GLI gene mutation.In addition,the over-expression of the signal transduction protein in this system is possible stimulated by abnormal activation of other related signal systems.Therefore,by so far,the gene mutation,protein expression and interaction mechanism in the process of tumor-like transformation from chronic inflammation need to be further studied.This study was processed with finding out the actual expression of Hedgehog signaling protein in non-small cell lung cancer,through three investigations including the clinical study of gene mutation screening of Hedgehog signaling pathway in NSCLC tissue;the preparation of inflammatory-cancer transformation model from COPD based on microfluidic chip technology and the study of Hedgehog signaling pathway in vitro;and the preparation of tumorigenic nude mice model based on inflammatory-cancer transforming cells and the study of function of Hedgehog signaling pathway in vivo,to investigate gene expression of key nodes of Hedgehog signaling pathway and related regulation measures,and to clarify the role of Hedgehog signaling pathway in the tumor development and lay the foundation for the regulation of related effector proteins.Objective:1.To investigate the gene mutation of HH signaling pathway proteins in non-small cell lung cancer and compared with the classic EGFR signaling system,so as to understand the activity of HH signaling system in non-small cell lung cancer.2.To explore the expression of HH signaling pathway proteins in the process of tumor transformation from COPD,and to study the inhibitory effect of HH signaling pathway proteins related inhibitor on the process of tumor transformation.3.Athymic nude mice was used to investigate tumorigenicity of bronchial epithelial cells after tumor transformation of chronic inflammatory cells,and to examine the inhibitory effect of HH signal pathway related inhibitor on tumorigenesis of transformed cells.4.Establish a microfluidic chip with high throughput,integration,less consumption and more efficient,and use it as a technology platform to jointly monitor various factors under the same system,so as to improve the efficiency,accuracy and reliability of the detection.Method:1.DNA was extracted from the paraffin pathological section of non small cell lung cancer,and the gene mutation of HH signaling pathway protein was detected by gene sequencing after purification.Comprehended the activity of HH signaling pathway in non-small cell lung cancer cells and compared with the activity of EGFR signaling pathway at molecular level.(1)A total of 40 cases of non-small cell lung cancer pathological tissue wax block were selected from the pathological bank of our hospital.Paraffin sections(8 sections/case)were taken?About 30 mg tissue was was scraped down from the sections with a scalpel(as far as possible to remove excess Paraffin).The paraffin genome extraction kit was used to extract DNA to be tested.(2)According to the exons of 8 gene including SHH,PTCH1,PTCH2,SMO,SUFU,EGFR,KRAS,PI3K,we designed corresponding primers.The second generation sequencing method was performed to detect mutations of the PTCH1 and other Hedgehog/EGFR signaling pathway protein genes in non-small cell lung cancer,and then to compare.2.Using PDMS(Polydimethylsiloxane)as the basic material,chip A containing a concentration gradient generator(CGG)and chip B with multi-channels and multicellular culture pool are made by standard soft lithography.Inoculate 10~5 cell/chamber in the chip A.After cell adherence for 24h,complete medium and CSE solution was pumped into the chip at the speed of 7 l/min and 5 l/min respectively.There would be 6 different concentrations of CSE presented by the CGG to stimulate the bronchial epithelial cells.After 48h,Hoechst33342 staining was used for 20 minutes,and then placed the chip under the inverted fluorescence microscope(Olympus IX71,Japan,200 times magnification)to observe the situation of cell apoptosis.The maximum CSE concentration was regard as the best stimulation concentration when the cell survival rate above 80%.The human bronchial epithelial cells were cultured in the microfluidic chip B for a long time with this concentration,and then the model of lung inflammation-cancer transformation was established in vitro.Westernblotting was used to detect the expression of Hedgehog signaling pathway protein in different stages of lung epithelial cell inflammation-cancer transformation.3.On the basis of the model of lung inflammation-cancer transformation established with chip B in vitro.The inhibition of Hedgehog signaling pathway protein antagonist cyclopamine on Hedgehog signaling protein expression and inflammation-cancer transformation process stimulated by CSE on bronchial epithelial cells was investigated.4.Through the tumorigenicity experiments,The transformed cells were subcutanously inoculated into left anterior axillary of athymic nude mice for 40 days to observe the tumorigenicity,examine the impact of the HH signaling pathway related signal inhibitor cyclopamine on tumorigenicity of transformed cells.Result:1.The critical mutation rate is set to 5%levels.Through screening for gene mutation of Hedgehog/EGFR signaling protein in 40 non-small cell lung cancer tissues,a total of138 mutation sites including single nucleoside mutation and insertion/deletion mutation were detected.There were 98 mutations registered rsID in the database,among which86 were single nucleotide mutation,6 were deletion mutation,and 3 were insertion mutation.There are 19 annotated mutations in the clinical mutation database clinvar.4genes are mainly concentrated including EGFR,KRAS,PTCH1 and PTCH2.Among the 19 mutation sites with clinical notes,there are 6 EGFR,4 KRAS,2 PTCH1,4PTCH2,3 SUFU.The mutation related to tumor or targeted therapy is mainly concentrated on EGFR,KRAS and PTCH2.EGFR is seemingly the mainly trend,but there is no statistical significant(P>0.05).2.Through concentration screening,the ultimate concentration of optimal CSE was12.25%.After nearly 8 months stimulation,bronchial epithelial cells morphology has fundamentally changed compared with normal control.The nuclei increased significantly,chromosome thickening,loss of cell polarity.As representative protein of Hedgehog signaling pathway activation,without significant mutation,SMO and GLI expression was significantly higher than normal cells(P<0.05).This phenomenon can be significantly inhibited by the Hedgehog signal protein inhibitor cyclopamine(P<0.05).3.The transformed cells after long-term CSE stimulation can grow into tumors in subcutaneous of athymic nude mice.But this growth can be significantly inhibited by cyclopamine(P<0.05).Conclusion:1.EGFR is seemingly dominant in the trend of Hedgehog/EGFR signal system mutation activity,but in the limited sample size,there is no statistical difference between the two systems(P>0.05).It suggests that the two systems can independently determine the generation and development of lung cancer.2.The mutation of Hedgehog signal pathway protein is mainly concentrated on PTCH2and PTCH1,followed by SUFU.But the latter mutation has no clinical significance,while SHH,SMO and GLI hardly produce effective mutations.Therefore,as the inhibitor of SMO,cyclopamine may effectively inhibit Hedgehog signaling activity.3.Hedgehog signaling pathway protein expression in bronchial epithelial cells was significantly increased during inflammation-cancer transformation process stimulated by long-term CSE.The transformed cells could grow into tumors in subcutaneous of athymic nude mice,indicate the important role of Hedgehog signaling system in development of lung cancer.While this growth process can be significantly inhibited by cyclopamine.4.The chip systems with CGG and multi-cellular culture have characteristics of high-throughput,integrated,less consumption and faster speed,can be used as a platform for detection of continuous biological culture and dynamic detection of biological molecules.The joint monitoring can be combined in the same system for a variety of factors with efficient,accurate and reliable.