The Mechanism Study On The Malignant Transformation Of COPD To Lung Squamous Carcinoma Based On Microfluidic Technology

In recent years, chronic lung diseases, including chronic bronchitis, asthma,tuberculosis, pulmonary scarring lesions and pneumoconiosis, as an important factorassociated with lung cancer development are paid more attention. It is reported that theincidence of lung cancer in patients with chronic obstructive pulmonary disease (COPD)is4~5times than that of the general population, therefore, currently COPD has beenconsidered an independent risk factors of lung cancer. In the process of COPD inducedlung cancer, chronic inflammation promotes the occurrence and development of cancermainly by changing the living conditions of the tumor cells, activating a variety ofprotein and endogenous or exogenous inflammatory mediators that involved insignaling pathways. However, it is still not clear what inflammatory factors play a vitalrole in the process of malignant transformation from COPD to lung squamouscarcinoma and what inflammation related molecular pathways or network are working.In order to further reveal the nature of the malignant transformation of chronicinflammation, we should not only pay attention to the research that is associated withthe process of signaling pathways and key factors, but also pay attention to the improvement of research methods. The weakness of the existing fundamental researchmethod is that it fails in simulating the real body inflammatory microenvironment, andthus it leads to unreliable results in vitro. Therefore, in order to investigate themolecular mechanism of COPD malignant transformed to lung cancer, it is urgent toprovide a new technology that can simulate microenvironment in vivo and detect avariety of inflammatory factors at the same time in order to improve the detectionaccuracy and reliability. Fortunately, the developed microfluidic chip technology canmeet the above requirements, is expected to provide reliable technical supports for thetest, as a new technology platform. Therefore, this study aims to investigate the themolecular mechanisms involved in COPD transformed to lung squamous carcinomawith the full use of the microfluidic chip. At the same time, animal disease model wassupplemented in order to verify pulmonary malignant transformation of chronicinflammation in finding potential molecular targets. This study contains the followingfour parts.Methods1. High-throughput screen of carcinogenic inflammation factors based on amicrofluidic chipA high-throughput inflammation factors screening microfluidic chip for lung cancertissues and adjacent tissue was fabricated,57cases of lung squamous carcinomacombined COPD patients were selected and carried on the three-dimensional cellculture in the microfluidic chip. Inflammatory factors were detected in a chip and at thesame time, traditional molecular biology techniques such as Western blot, real-timePCR and immunohistochemical detection was also performed to find the significantdifference between lung cancer tissues and adjacent tissues.2. The malignant transformation study of cigarette smoke re-exposure on COPD basedon a microfluidic chipA cigarette smoke re-exposure on COPD microfluidic chip was fabricated.10Caseslung squamous carcinoma patients with COPD and a long history of smoking wereinvolved and performed on the primary culture of bronchial epithelial cells in microfluidic chip platform. To observe malignant transformation of bronchial epithelialcells that already exposed to cigarette smoke and detect the levels of reactive oxygenspecies (ROS), molecular pathway change, anchor the independence, the transformationof epithelial mesenchymal (EMT) and chromosome aberration.3. The role of NF-κB, COX2, STAT3, GRP78in the malignant transformation ofbronchial epithelial cells based on a bionic lung chipA bionic lung chip was fabricated. Bronchial epithelial cell line16HBE withmacrophage were co-cultured and epithelial cells grow on the gas-liquid interface,benzopyrene carcinogen induced malignant transformation of epithelial cells, NF-κB,COX2, STAT3, GRP78and its related molecules were inhibited. The epithelialmalignant indicators such as cell morphology, cell cycle, anchor into independence,nude mice tumor as well as related molecular pathways were detected to observewhether inflammation promotes the malignant transformation of epithelial cells andverify the carcinogenic potential of inflammation related factors.4. The role of NF-κB, COX2, STAT3, GRP78in the malignant transformation ofbronchial epithelial cells based on an animal lung cancer modelThe rat COPD model was performed with the method of smoking and LPS bronchialperfusion, while the rat lung cancer model was established with carcinogens trimethylbravery anthracene (MCA) and diethyl nitrosamine (DEN). At the same time, NF-κB,COX2, STAT3, GRP78were down-regulated by siRNA in vivo. To observe thedifferences of lung cancer incidence among the simple carcinogenic group, the COPDcarcinogenic group and the siRNA COPD carcinogenic group to verify whether theabove four factors are the key of inflammation to malignant tumor.Results1. High-throughput screen of carcinogenic inflammation factors based on amicrofluidic chipA high-throughput inflammation factors screening microfluidic chip for lung cancertissues and adjacent tissues was successfully fabricated. The primary cell culture of57cases of lung squamous carcinoma combined COPD patients was successfully finished. We found that the expressions of p-IKKβ, p-IκBα, NF-κB, GRP78, p-IRE1α, XBP1,COX2, STAT3, and CyclinD1in the lung cancer tissues were significant higher thanthat in the adjacent tissues (P<0.05). The results assayed by immunohistochemicaldetection, Western blot detection and real-time PCR detection were consistent with theresults obtained from the microfluidic chip.2. The malignant transformation study of cigarette smoke re-exposure on COPD basedon a microfluidic chipA cigarette smoke re-exposure on COPD microfluidic chip was successfullyfabricated. We found that low doses of CSE exposure can lead to the proliferation ofhuman bronchial epithelial cells, at the same time accompanied by cytoplasm ROSrelease and the activation of inflammatory pathways GRP78/IRE1α, IKKα/NF-κB andPI3K/AKT/mTOR. What’s more, low doses of CSE exposure can lead to irregularmitosis bronchial epithelial cells for increasing the height of the atypia state, but alsocan promote epithelial-interstitial transformation, anchor the independence growth andchromosome aberration, whereas the change of various index could be reversed orreduced after the treatments of ROS, GRP78, NF-κB and PI3K inhibitors.3. The role of NF-κB, COX2, STAT3, GRP78in the malignant transformation ofbronchial epithelial cells based on a bionic lung chipA bionic lung chip was successfully fabricated. We found that the levels of IL-1β,IL-6and TNF-α in the medium from the co-culture of bronchial epithelial cells andmacrophages were significant higher than that from single culture of bronchial epithelialcells, and it increased with the development of cell passages (P<0.05). Benzopyrene cancause malignant changes such as bronchial epithelial cell morphology, the shortness ofcell cycle, contact inhibition disappear, anchor the independence into malignantperformance, such as tumor growth in nude mice. Also macrophages can accelerate themalignant transformation of bronchial epithelial cells showing a forward appearance ofthe above malignant indicators, while the various indicators obviously prolonged afterthe inhibition of NF-κB, STAT3, COX2, GRP78and its related inflammatory factors (P<0.05). 4. The role of NF-κB, COX2, STAT3, GRP78in the malignant transformation ofbronchial epithelial cells based on an animal lung cancer modelThe rat COPD model was successfully performed with the method of smoking andLPS bronchial perfusion, while the rat lung cancer model was successfully establishedwith carcinogens trimethyl bravery anthracene (MCA) and diethyl nitrosamine (DEN).We found that the total number of leukocytes and total classification of neutrophils inthe peripheral blood of COPD rat was obviously higher than that in control group, thedifference was statistically significant (P <0.01), the total number of white blood cellsand neutrophils in the bronchoalveolar lavage fluid (BALF) of COPD rat wassignificantly increased (P <0.01), while the proportion of monocyte/macrophagesignificantly decreased (P <0.01); After the establishment of rat lung cancer model, wefound that the incidence of lung cancer in COPD+carcinogenic group (85.7%) wassignificant higher than that in the only carcinogenic group (30.0%)(P <0.05); theincidence of lung cancer in COPD+NF-κB inhibition+carcinogenic group (22.2%)was significant lower than those of COPD+carcinogenic group (85.7%)(P <0.05); theincidence of lung cancer in COPD+COX2inhibition+carcinogenic group (25.0%)was significant lower than those of COPD+carcinogenic group (85.7%)(P <0.05); theincidence of lung cancer in COPD+STAT3inhibition+carcinogenic group (30.0%)was significant lower than those of COPD+carcinogenic group (85.7%)(P <0.05); theincidence of lung cancer in COPD+GRP78inhibition+carcinogenic group (30.0%)was significant lower than those of COPD+carcinogenic group (85.7%), but there wasno statistically significant difference (P>0.05).Conclusions1. The factors NF-κB, COX2, STAT3, GRP78and its related factors like IKKβ, IκBα,IRE1α, XBP1, CyclinD1screened in the microfluidic chip were potentialinflammatory factors involved in the malignant transformation from COPD to lungsquamous carcinoma.2. Long-term low doses of CSE smoking exposure can lead to the malignanttransformation of bronchial epithelial cells in severe COPD patients, ROS and related GRP78, NF-κB and PI3K molecular pathway are the main molecularmechanisms of malignant transformation, it provides new targets of the smokingrelated lung cancer prevention and control.3. Based on the bionic pulmonary malignant transformation model, we found thatNF-κB, STAT3, COX2, GRP78are the key factors of the malignant transformationin bronchial epithelial cells, and TNF-α or IL-1β/NF-κB, IL-6/STAT3, COX2/ERK,GRP78/IRE1are the main molecular pathways of malignant transformation ofbronchial epithelial cells.4. COPD promoted the incidence of lung cancer in rats. NF-κB, COX2, STAT3playeda key role in the malignant transformation from COPD to lung squamous carcinoma.