| BackgroundLung cancer is a serious health hazard, which incidence and death rate of lung cancer also has the highest proportion of all the cancers in the world. The biological characteristics of Lung cancer cells are quite complicated, and the malignant degree is very high, about 75% of the patients with lung cancer belong to middle-late stage when diagnosed. So as to look for the measures of early diagnosis and early intervention, an in-depth study of the key etiology and molecular mechanism in the occurrence of lung cancer, have an important significance for improving the prognosis of lung cancer.Smoking is the most important risk factor for lung cancer, a large number of epidemiological investigation confirmed that about 90% of lung cancer related deaths associated with smoking. Compared with never-smokers, smokers can make 30 times increased risk of lung cancer. In addition, smoking is also a poor factor of prognosis of lung cancer patients after surgery. Nicotine,4-methyl nitrosamines-3 pyridine-1 butyl ketone (NNK) and polycyclic aromatic hydrocarbons, such as more than 70 kinds of carcinogens are included in the cigarette smoke, which lead to the occurrence, invasion and metastasis of lung cancer, through influencing the change of cell receptor, cell cycle regulation control factor, signaling pathways, apoptosis regulating factor and angiogenesis factor. But its precise molecular mechanism has not been fully elucidating, the main factor of limiting tobacco study is influenced by too many factors:lung cancer model induced by cigrette is quite difficult, research period is very long, and so on.Notch signaling pathway is one of the important intermediary signaling pathways of direct contact between adjacent cells and cells. It plays an important role in the process of development, growth and differentiation of cells. Notch signaling pathway is composed of four types of receptors,5 kinds of ligands and the effects signal transduction molecules of the downstream.The function of this pathway in tumor has caused the attention of many scholars in recent years. Notch1, Notch2 receptors is closely connected with the incidence, development of tumors. The disorders of Notch pathway in tumor cell growth, metabolism, cell cycle and apoptosis plays an important role in the process of biology. The abnormal activation of Notchl and promotion of the tumor growth were found in cervical cancer, breast cancer and liver cancer. But recently, in bladder cancer research, people found that Notch pathway inhibits tumor growth through inhibiting ERK pathway. It shows that the regulation of Notch pathway is extremely complex, which depends on the tumor genetic background and exogenous stimulus signal. In the early development of lungs, Notch pathway, distributed on epithelial cells, plays a role of promoting the proliferation and inhibiting of cell differentiation, which belongs to the early embryonic development Gene. Notch activation is very vital to maintain self-renewal and multi-directional differentiation potential of embryonic stem cells. If Notch is excessively active in a mature body, it will hinder the normal cell differentiation, make the epithelial cells polarity disorder and transform into tumor cells. Studies have found that a third of non-small cell lung cancer patients (NSCLC) have an unusual Notch pathway, abnormal activation of Notch is closely associated with poor prognosis. Vitro studies also found that the Notch pathway can accelerate the invasion growth of lung cancer cell in the lack of oxygen environment. People found that activating mutations of Notch pathway appeared in acute lymphoma, but a lot of controversies arised in solid tumor. However, as one of the most important exogenous factors, the researches about the influence of smoking on the Notch pathway have not been reported.This study discusses the exact role of Notch pathway and its molecular mechanism in tobacco induced lung cancer in vivo and in vitro experiments. This study provides a key experimental evidence for making tobacco carcinogenesis mechanism clear and the early interventions of high-risk groups.Part1. Establishment of Models of Lung Cancer induced by cigarette[Objective]To establish a malignant transformation by using cigarette smoke condensates(CSC) induce immortalized human bronchial epithelial cells (BEP2D), and inoculation of nude mice subcutaneously into tumor. Meanwhile we utilize the smoking machine of animals to establish a long-term smoke into tumor models of A/J mice;[Method]1. Immortalized human bronchial epithelial cells (BEP2D) were continuously cultured to 70 generation by cigarette smoke condensates (CSC), using JiM dyeing to observe cell morphology, detecting clone formation efficiency, observing growth curve, performing resistance to serum-induced terminal differentiation experiment and soft AGAR cloning formation ability to observe the malignant phenotype.2. P0, alcohol group cells (solvent control), P10, P30, P50 and P70 cells were separately added apoptosis inducers Etoposide, Annexin V-PI double staining was using to detect cell apoptosis after 24 h.3. The expression of anti-apoptosis protein (IAP) family members XIAP, Survivin, c-LAP-1,c-IAP-1 and RARP were detected by Western blot in P0, alcohol group, P10, P30, P50 and P70 cells RARP.4. P0, P50 and P70 cells were inoculated into nude mice to observe the formation of tumor, measure the size of tumors after the formation of tumor, nude mices with tumors were executed, to identify by HE and immunohistochemical staining.5. Taking A/J mice with spontaneous formation of tumors as a model, divided them into two groups (40 per group) randomly, namely normal control group and smoke group. Put the mice into special smoking machine of animals for smoke exposure experiments. Before smoking, take 10 mice lung tissue in group of 1,3 and 5 month respectively, to observe the tumor formation of lung by HE dyeing, to detect the expression of antiapoptotic proteins XIAP, Survivin by immunohistochemical method, situ end labeling (TUNEL) method to detect apoptosis levels of lung and tumor tissue in mice, to observe the effects of smoking on cell apoptosis.Mice tumors will be identified by HE and immunohistochemical staining.[Results]1. With the increase of transformation generation, BEP2D induced by CSC had pathological karyokinesis and the nuclear chromatin thickened. The rate of cell proliferation of P30, P40, P50, P60 and P70 was higher than that of PO and the alcohol control group with significant difference (P<0.05). The ability of colony-forming of P20, P30, P40, P50, P60 and P70 was better than that of PO group and the alcohol control group with significant difference(P<0.05). The resistance to serum-induced terminal differentiation of P30, P40, P50, P60 and P70 was stronger than that of PO group and the alcohol control group with significant difference (P<0.05). The ability of soft agar assay colony forming of P30, P40, P50, P60 and P70 was stronger than that of the control groups with significant difference (P<0.05)2. The apoptosis rate of P0, alcohol, P10, P30, P50 group cells was significantly higher after adding Etoposide (P< 0.05); after adding etoposide into P70 cells, there was no significant difference between the two groups in apoptotic rate (P> 0.05)3. The expression of XIAP, Survivin in P70, P50 cells is higher than other cells, the expression of RARP in P70, P50 cells is lower than other generation cells; the expression level of c-IAP-1, c-IAP-2 in each generation cells without significant difference4. Malignant transformation cells of nude mice inoculated model shows that P70 cell group are all transformed into a tumor after vaccinating four months, PO and P50 has no tumor formation. Tumors type was indicated that poor differentiated adenocarcinoma by HE staining and immunohistochemical staining:CK7 and TTF-1 were strong positive, all of CK56, P63 and CKH were negative. The expression of the above mentioned molecular markers confirmed the formation of tumor in nude mice as adenocarcinoma.5. A/J mice acquired lung cancer in the 9th month, all of the 10 mices form tumors, we cannot observe tumor formation in the control group mice without smoking. Tumor HE dyeing showed:2 cases of adenoma,8 cases of adenocarcinoma; 8 cases of tumors immunohistochemical study showed:TTF-1 was strong positive, P63 and CK56 were negative, confirmed adenocarcinoma; compared with control lung tissue, the expression of XIAP and Survivin in mice lung cancer were significantly higher (P<0.01); TUNEL staining showed:compared with control lung tissue of mice, the apoptosis rate of tumor tissue was significantly lower (P< 0.01)(Conclusion]The identification of tumor malignant phenotype and the tumor formation of nude mice showed that CSC was able to induced BEP2D malignant transformation successfully. A/J mice was occurred lung cancer after been smoked for 9 months, provided an ideal model for further study of tobacco carcinogenic mechanism in the body.Part2. Correlation between Notch Pathway and Lung cancer which Induced by Cigarette[Objective]By adopting the method of Gene Chip prime view human, we try to screen differentially expressed genes between P70 cells induced by CSC and P0 cells, and then to verify these differentially expressed genes by means of Realtime qPCR、 Western blot and immunofluorescent assay; Detecting the Notch pathway molecule expression of smoked A/J mice lung or lung tumors by Immunohistochemistry; Detecting the Notch pathway molecule expression of heavy smokers airway mucosa and lung carcinomas by RT-PCR and Immunohistochemistry, and then analyzing the correlation between the expression of Notch and smoking, to specify the exact role of Notch pathway in lung cancer induced by smoking.[Methods]1. Bronchial epithelial cells of the control group (P0) and malignant transformation cells which have been smoked 70 generation (P70) were both selected. We used Affymetrix Gene expression profile Chip method (Gene Chip prime view human) to analysis the differentially expressed genes.2. According to the results of gene chip screening, we detected 17 major genes expression of Notch pathway in group P0, alcohol, P10, P30, P50 and P70 by using the real-time fluorescent quantitative PCR (Realtime qPCR):Notch1、Notch2、 Notch3、Notch4、JAG1、JAG2、DLL1、DLL3、Hes1、Hey1、Hey2、HeyL、 Hes5、RBPJ、MIB2、DNM1、Numb mRNA;According to the results of Realtime qPCR, the protein expression of Notch 1, Notch2、JAG1、JAG2、RBPJ、Hes1、 Hey1、Hey2、DNM1、Numb in the above generation cells were detected by Western blot.3. Notch1, Notch2、JAG1、Hes1、Hey1、Hey2 of P0 and P70 cells were detected by immunofluorescent assay, them observe the cell fluorescence intensity of expression and protein localization in both groups.4. Immunohistochemical method to detect the expression of Notchl, Notch2 and Numb in A/J mice lung tissue and tumor in control, smoked 1 month, smoked 3 months and smoked 9 months groups, dynamic observation on the influence of smoking on the Notch pathway in vivo.5. Screening 10 patient who has a heavy smoking history and 10 who has no smoking history, through detecting the mRNA expression of Notch1, Notch2、JAG1、JAG2、 Hes1、Hey1、Hey2、RBPJ and Numb in bronchial mucosa tissue obstained by AFI or NBI bronchoscopic biopsy by RT-PCR method. According to the results of RT-PCR, we compared the correlation between the above gene expression level and smoking status.6. Collecting 50 cases of NSCLC (non-small cell lung cancer) surgery paraffin specimens (29 severe-smokers,21 never-smokers), using Immunohistochemistry to detecting the expression of Notch1、Notch2、Hes1、Numb of carcinoma tissues, and then compared the correlation between protein expression and smoking status.[Results]1. MAPK, P53, cell cycle regulation pathways which are closely related to the development of tumor were significantly activated in P70, indicating that the transformed cells have malignant characteristics. We also found that multiple Notch pathway genes were expressed differently by comparison of P70 and PO cells (more than two times differences).2. Compared with PO and alcohol groups of cells, Notch1, Notch2, Hesl, JAG1, Hes1, Heyl,Hey2, RBPJ mRNA expression in P70 were significantly increased(all P<0.05); MIB2?DNM1、Numb mRNA expression in P70 was significantly down-regulated (all P<0.05); Notch3, Notch4, DLL1, DLL3, Hes5 HeyL and JAG2 mRNA levels were not significantly different between PO and P70 (all P>0.05); By observing mRNA expression level of cell, we found that mRNA level of Notch2 induced by CSC (P10 cells) have a significant rise, then declined slightly, in P70 cells rose to peak again. Most Notch pathway genes, such as Notch1、Hes1、 JAG1、Hes1、Hey1、Hey2 and RBPJ were all risen in different degrees in P30, while the up-regulated expression level of P70 was the most significant one.3. The protein expression level of Notch1、Notch2、Hes1、Hey2、JAG1 and RBPJ in P70 cells was higher than PO and alcohol significantly. Notch2 significantly increased in P10 cells. In negative regulation factors, the expression of Numb in PO and alcohol was higher than the rest of the groups. The expression of P10 was significantly decreased, and the expression intensity of P70 cells was poor. The expression of DNM1 and JAG2 were not detected in all cells.4.The cytoplasm fluorescence intensity of Notch2 and Notch1、the nuclei fluorescence intensity of Hes1、Hey1Hey2、the expression of membrane fluorescence intensity of JAG1 in P70 cells are significant higher than PO cells.5. The expression of Notchl positive cells in mice lung cancer tissues was significantly higher than smoked 1 month, smoked 3 months groups (P<0.01), while in the control mice, Notchl expression level has no obvious change. The expression of Notch2 became obviously rise in the lung tissue of mice induced by cigarette after 3 months, Notch2 expression intensity was the highest in tumor, interestingly, even the expression level of Notch2 is high in the adenoma; Compared with the control mice, the expression level of Notch2 in cigarette group had significant difference (P<0.01); Numb protein in the lung tissue after smoking for 1 month was decreased, but there was no statistic difference (P<0.05); The positive cells of Numb in mice lung of smoking for 3 months was significantly lower than the control group (P>0.05); The expression of Numb in tumor was the lowest compared with one in control group (P<0.01).6. Notch 2, Hes1, Hey1, Hey2, JAG1, RBPJ mRNA expression level of heavy smokers’bronchial mucosa was significantly higher than never-smokers (P<0.05); The Numb mRNA expression level was just the reverse(P<0.01); There were no difference in expression level of Notchl and JAG2 between two groups (P>0.05).7. The expression level of Notch1、Hesl in lung cancer tissues from heavy smokers was significantly higher than never-smokers(P<0.05); Numb expression in heavy smokers r was significantly lower than those from never-smoke (P<0.05); Notch2 expression level in the two groups had no difference, but the expression of Notch2 in smokers with adenocarcinoma was significantly higher than never-smokers (P<0.01); In squamous cell carcinoma, Notch2 expression had no significant correlation with smoking history (P>0.05). No matter in adenocarcinoma or in the squamous cell carcinomas, Notchl expression in smokers were higher than non-smokers (P<0.05); Pearson Correlation analysis showed that:Notch1 expression in lung cancer tissue were negatively correlated with Numb expression (r=-0.383, P< 0.05). Expression of Notch 2 was significantly negatively correlated with Numb expression (r=-0.542, P<0.01). The expression of Notchl and Notch2 had no difference in smokers, smokers quitting history less than one year and smokers quitting history over one year in patients with lung cancer (P>0.05).[Conclusion]In summary, we identify that tobacco activate Notch pathway, and closely related with the occurrence and development of smoking lung cancer. Up-regulation of Notch 2 may be an early molecular event in tobacco-induced lung cancer.Part3. Molecular mechanism of tobacco activated Notch pathway[Objective]To clarify molecular mechanism of tobacco induced Notch activation, we observe the regulation effect of Numb overexpression on Notch2 expression and Cell colony formation.we also will compare Notch coding region mutation frequency between never-smokers and smokers by PCR re sequencing method.[Methods]1.The expression of Notch2 and Numb inBEP2D group and P70 were detected by immunofluorescence; Using pIRES2-EGFP-Numb added to cells of P70 by lipidosome 3000, Confocal microscopy imaging system observed Numb, Notch 2 fluorescence intensity.2.By cell cloning experiments to observe the effect on colony formation after transfection pIRES2-EGFP-Numb of P70 cell3.The mutation in coding region of Notch1 and Notch2 which were detected in P0, P50, P70 cell and NSCLC tissue samples of 34 cases by PCR re sequencing method, compared the occurrence frequency of each mutation site between the smoking group and non-smoking group, and then using immunohistochemistry to detect Hesl expression in NSCLC tissues with different frequency of mutation.[Results]1.Immunofluorescence test has shown Notch2 proteinsfluorescence intensity were existed in the normal control group and P70 group, but fluorescence intensity of P70 is higher levels than that in PO group. Compared with the empty vector, Numb overexpression plasmid significantly increased the relative fluorescence intensity values of Notch2 protein (P<0.01)2.Cell colony formation after transfection of Numb overexpression plasmid was significantly lower than empty plasmid group (P<0.01).3.Five kinds of SNPs were found in ex on 3、14、27 and 34 through sequence analysis of the coding region of Notch 1. All the SNPS were synonymous mutations: rs 10521(p.Asp 1698=), rs2229971(p.Asn755=), rs2229974(p.Asp2185=), rs3812596 (p.Pro2216=) and rs4489420(p.Asn104=). By contrast, the smoking NSCLC patients with an average number of SNP occurred in the Notch1 coding region was 3.00±1.00, higher than that of the non-smoking patients 1.13±0.83 (P<0.01). Comparison of incidence between the smoking group and non-smoking group showed that there was not statistically different from the incidence rate of each SNPs (Fisher’s Exact Test, P>0.05).4. Nine mutant sequences were detected in exon 1、4、17、18、20、28 and 34 through sequence analysis of the coding region of Notch2.9 mutant sequences containing 3 synonymous mutations (rs11810554, rs61734328 and rs7543643),3 missense mutations (rs146498360(p.Arg237Gln)、rs2603926(p.Ala21Thr) and rs60854092 (p.Ile1689Phe)) and 3 novel mutations by sequence analysis the coding region of Notch2. Of the 3 novel mutations, one was a synonymous mutation (c.2622T>C (p.Ile874=)), two were missense mutations:c.572A>T(p.His191Leu) and c.2882G>A(p.Gly961Glu). By contrast, the average number of SNP in each of the smoking NSCLC patients occurred in the Notch2 coding region was 1.32±1.16, higher than that of the non-smoking patients 0.33±0.61 (P<0.01).Comparison of frequency between the smoking group and non-smoking group,it showed that was not statistically different from the of each SNPs (P>0.05).5.Six mutations:p.Arg237Gln, p.Ile1689Phe, p.Leu2348=, p.His191Leu, p.Ile874= and p.Gly961Glu, only appeared in smoking patients, non-smoking patients were not detected. Comparison of expression differences of Hesl immunohistochemistry between tissue specimens from patients with the above six mutations and other wild-type patients showed except p.Ile874=, the expression of Hes1 was significantly higher than those in wild type (P<0.01),so gain-of-function mutations of Notch2 gene are present primarily.[Conclusion]Numb negative regulation disorder and Notch coding region mutation may be the key molecular mechanism of tobacco induced malignant transformation of bronchial epithelial cells.Part 4. Role and Molecular Mechanism of Notch Pathway in Promoting Malignant Transformation of Immortalized Human Bronchial Epithelial Cells[Objective]The results of the first part suggested that tobacco induced the activation of Notch pathway and was closely related to smoking lung cancer. This part will demonstrate whether Notch pathway has the function of promoting development of tobacco-induced lung cancer through lentiviral RNA interference test、plasmid over-expression test、nude mice experiment by knock-down Notch pathway. And we will discuss the different role of Notch1 and Notch2 in cellular malignant transformation. To explore the molecular mechanism of Notch pathway in promoting cellular malignant transformation through detecting the anti-apoptotic capacity and IAP family protein expression of bronchial epithelial cells induced by the over-expression and knock-down of Notch in vitro and in vivo.[Method]1. P70 cells were transfected by Lentiviral shRNA-Notchl (Notch1-KD) and shRNA-Notch2 (Notch2-KD) vector, interference efficiency was detected by Real time qPCR, cell proliferation and cell cloning were observed by MTT test and cell cloning formation test.2. P70 cells were transfected by Notch1 over-expression plasmid pIRES2-EGFP-Notchl (pIRES2-Notchl) and Notch2 over-expression plasmid (pIRES2-Notch2) plasmid vector respectively, the expression of Notch1 and Notch2 were detected by western blotting, cell proliferation and cell cloning were observed by MTT test and cell cloning formation test.3. Lentiviral vector Notch1-KD and Notch2-KD were infected by P70, and then added apoptosis-induced agents Etoposide into reaction system respectively, and cell apoptosis were detected by staining test.4. P70 cells were transfected by pIRES2-Notch1、pIRES2-Notch2, then adding Etoposide into reaction system respectively, and cell apoptosis were detected by Annexin V-APC staining method.5. The expression of XIAP、Survivin、Cleaved caspase-3、RARP of all groups were detected by western blot.6. To screen cell lines stably-transfected by Lentiviral Notchl-KD、Notch2-KD through adding Puromycin, expression of Notch1 and Notch2 were detected by Western blot; To build Xenografts of nude mice by adding stably-transfected cell lines, and to observe the formation and the growth of tumors, the expression of Notch1、Notch2、Ki67、XIAP and Survivin by immunohistochemistry test, and then detect the apoptosis of tumor tissue by TUNEL assay.[Results]1. Knock-down efficiency of Notchl-KD is 75.3%, knock-down efficiency of Notch2-KD is 83.6%, and the interference efficiency is so good that it can meet the requirements of the subsequent functional experiments; Notch1-KD and Notch2-KD can significantly inhibit cell growth of P70 cells (P<0.05), and inhibit colonality efficiency of P70 cells (P<0.01), and there was no significant difference between NC and blank control group (P>0.05).2. After PO cells were transfected by pIRES2-Notch1、pIRES2-Notch2 for 72h, expression of Notch 1、Notch2 was significantly increased (P<0.01), compared with liposomes and pIRES2 group. pIRES2-Notch2 can significantly enhance the ability of cell proliferation and cell colony formation (P<0.01).3. Annexin V-APC apoptosis assay has showed:Notch1-KD and Notch2-KD not only increased basal cell apoptosis amount (P<0.05),but also significantly enhance apoptosis of P70 cells induced by Etoposide (P<0.01), Notch 1-KD-induced apoptosis rate was higher than Notch2-KD (P<0.05), NC and control groups showed no significant difference in apoptosis rate (P>0.05).4. After PO cells were transfected by pIRES2-Notchl, pIRES2-Notch2 for 72h, pIRES2-Notch2 significantly inhibited apoptosis of P70 induced by Etoposide (P>0.05), by adding Etoposide into pIRES2-Notchl group, pIRES2, liposome group, the apoptosis rate was significantly increased (P<0.05).5. Nochl-KD and Notch2-KD can promote expression of Cleaved-caspase-3 and PARP, and the up-regulated protein expression was more significant after adding Etoposide, Nochl-KD specially can induce significantly down-regulated expression of Survivin, and Notch2-KD can specially induce significantly down-regulated expression of XIAP.6. Screening the puromycin repeatedly, constructed the stable cell lines of P70 which infected Notchl-KD、Notch2-KD stably. Through verifying Western blot, the target protein was significantly decreased, compared with NC group.We vaccinated nude mice against the cells. Empty virus group(NC) had the highest rate of tumor formation, that was 83%(5/6), which was higher than Notch2-KD group (50%,3/6) and Notch 1-KD group (33%、2/6). Expression of Notch1 and Notch2 in the KD group was significantly lower than NC group. The tumor growth curves showed that the growth speed of Notch 1-KD group and Notch2-KD group was significantly lower than NC group (P<0.05). The growth rate, tumor weight and Ki67 expression level of Notch 1-KD were lower than Notch2-KD group (P<0.05); TUNEL test showed that TUNEL-positive cells of Notch 1-KD group was higher than Notch2-KD (P<0.05), and TUNEL-positive cells of Notch 1-KD and Notch2-KD groups were significantly higher than NC group (P<0.01). Expression of Survivin was significantly down-regulated in Notch 1-KD group (P<0.01), while the expression of XIAP was significantly down-regulated in Notch2-KD group than any other groups (P<0.01).[Conclusion]Notch pathway can regulate cell apoptosis through promoting expression of XIAP, Survivin; it can promote malignant transformation of bronchial epithelial cells induced by CSC. Notch2 mainly plays a leading role in the early period of malignant transformation, while Notchl plays a key role in maintaining the malignant phenotype of transformed cells in late stage. |
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