Study On The Mechanism Of LPP2 Regulating Endoplasmic Reticulum Stress-mediated Apoptosis In Lung Adenocarcinoma Cells

Objective:Lung cancer is one of the most common malignant tumors with a relatively high mortality rate.According to pathological types,it can be divided into small cell lung cancer and non-small cell lung cancer,which is further divided into lung squamous cell carcinoma,lung adenocarcinoma and large cell lung cancer.Adenocarcinoma of the lung is the most common pathological type of lung cancer.In recent years,with the widespread application of low dose spiral CT and high resolution thin-slice technology in clinical screening of lung diseases,more and more early lung adenocarcinoma has been discovered.Most patients with early lung adenocarcinoma only show ground-glass nodules in imaging examinations without other clinical symptoms,so the degree of good and evil of pulmonary nodules cannot be effectively judged,leading to an increased clinical risk of misdiagnosis or missed diagnosis of early lung adenocarcinoma.In-depth understanding of the molecular mechanism of the occurrence and development of lung adenocarcinoma and finding the landmark molecules in the early diagnosis of lung adenocarcinoma will help to improve the diagnosis rate of early lung adenocarcinoma,which has important clinical significance.In this study,transcriptome sequencing was performed on 34 patients with early stage lung adenocarcinoma to obtain LPP2,Phospholipid Phosphatase 2（LPP2,Phospholipid Phosphatase 2）,a factor associated with early stage lung adenocarcinoma.LPP2 is a member of the phospholipid phosphatase family,which is widely expressed in human tissues and mainly plays physiological functions in cells.LPP2 is distributed in cell membrane,endoplasmic reticulum and early endosomes.It is involved in dephosphorylation of various triglycerides and sphingolipin phosphate esters and is closely related to the formation and maintenance of lung surfactant activity.Studies have shown that lung adenocarcinoma originates from alveolar type II epithelial cells,and the synthesis of lung surfactant is the main biological feature of alveolar type II epithelial cells.Therefore,we consider whether the key molecules involved in the synthesis of surfactant are involved in the malignant process of lung adenocarcinoma in the process of lung adenocarcinoma.Studies have found that LPP2 is highly expressed in a variety of tumor tissues,and inhibition of LPP2 function can reduce the proliferation ability of tumor cells（such as osteosarcoma cells and leiomyosarcoma cells,etc.）.However,whether LPP2 is involved in the occurrence of early lung adenocarcinoma has not been reported in the literature.In this study,LPP2 was taken as the research object to explore the expression of LPP2in lung adenocarcinoma tissues and lung adenocarcinoma cells,further analyze the influence of LPP2 on the malignant phenotype of lung cancer cells,and analyze its possible molecular mechanism of action.Methods:1.m RNA and protein expression levels of LPP2 in lung cancer cells and tumor tissues of lung adenocarcinoma patients were analyzed by Real-time PCR,Western Blot and immunohistochemical experiments.2.Down-regulation of LPP2 expression in lung adenocarcinoma cells by gene recombination and gene intervention techniques.The effects of LPP2 on malignant proliferation of lung adenocarcinoma cells were analyzed by CCK8 cell viability assay,plate cloning assay and flow cytometry3.After downregulating the expression of LPP2 in lung adenocarcinoma cells by gene recombination and gene intervention techniques,the morphological changes of endoplasmic reticulum in lung cancer cells were observed by transmission electron microscopy.4.The distribution and localization of LPP2 in cells were observed by immunofluorescence staining combined with laser confocal microscopy.5.The proteins interacting with LPP2 were found by immunocoprecipitation-binding protein mass spectrometry,and the interactions were verified by Co-IP and immunofluorescence.6.To interfere with the expression of LPP2 in cells,use hydrogen peroxide（H₂O₂）and tunicamycin to induce ER stress,and analyze whether LPP2 mediates ER stress response and the possible mechanism.Results:1.Transcriptome sequencing results showed that the expression level of LPP2 in tumor tissues of patients with early lung adenocarcinoma was higher than that in paracancer tissues.2.m RNA and protein expression levels of LPP2 were increased in lung adenocarcinoma cells（HCC827,NCI-H1975,A549,NCI-H1299）and tumor tissues of lung adenocarcinoma patients.3.Down-regulating the expression of LPP2 with RNAi can significantly inhibit the proliferative ability and anti-apoptotic ability of lung adenocarcinoma cells HCC827,NCI-H1975,A549 and NCI-H1299,but has no significant effect on cell cycle,indicating that LPP2 affects the malignant phenotype of lung adenocarcinoma cells.4.Immunofluorescence results showed that LPP2 was localized in the endoplasmic reticulum when no H₂O₂ treatment was given to lung adenocarcinoma cells（HCC827,NCI-H1975,A549,NCI-H1299）.The colocalization of LPP2 and endoplasmic reticulum marker protein increased after the stress induced by H₂O₂ treatment.5.The results of transmission electron microscopy showed that the length of endoplasmic reticulum swelling became shorter and autophagy occurred after interfering the expression of LPP2.It is suggested that the abnormally high expression of LPP2 in lung adenocarcinoma is closely related to the endoplasmic reticulum.6.In order to search for proteins that interact with LPP2 and are related to the function of endoplasmic reticulum,the protein interacting with LPP2--GRP78 was screened through co-immunoprecipitation technology combined with protein mass spectrometry.The results of immunofluorescence and Co-IP experiments showed that LPP2 was directly related to GRP78 in HCC827 and NCI-H1299 cells.7.Immunofluorescence results showed that the combination of LPP2 and GRP78decreased after H2O2 treatment.Western Blot results showed that there were no significant changes in the expression of endoplasmic reticulum stress marker proteins that interfered with LPP2 expression and were treated with H2O2.8.Interference with the expression of LPP2 Induced endoplasmic reticulum stress response in lung adenocarcinoma cells by ylamycin.Western Blot results showed that the expression level of endoplasmic reticulum stress marker GRP78 was further increased after the expression of LPP2 was inhibited.Conclusions:1.The expression level of LPP2 increased in early lung adenocarcinoma tissues,which was correlated with the early occurrence of lung adenocarcinoma;2.The expression level of LPP2 in lung adenocarcinoma cells is high,which promotes the proliferation and anti-apoptosis ability of lung adenocarcinoma cells;3.Under stress conditions,LPP2 interacts with ER molecular chaperone GRP78 to inhibit ER stress response and resist the occurrence of apoptosis induced by ER stress.