| BackgroundObstructive sleep apnea(OSA)is a common clinical sleep breathing disorder,due to the recurrent complete/incomplete collapse of the upper airway during sleep,resulting in repeated hypoxic-reoxygenation(intermittent hypoxia).Intermittent hypoxia is the main pathophysiological mechanism in the pathogenesis of OSA.In recent years,more and more epidemiological studies have found that OSA is associated with tumor morbidity and mortality.Molecular basic studies have found that OSA related intermittent hypoxia can enhance the epithelial mesenchymal conversion,angiogenesis and stem cell characteristics of tumor cells,thus promoting tumor proliferation,invasion,chemoradiotherapy resistance,immune escape and so on,leading to poor tumor prognosis.Lung cancer is one of the most common tumor types worldwide and the leading cause of cancer-related death worldwide.Lung adenocarcinoma is the most common subtype of lung cancer,and its incidence is increasing gradually.Due to the lack of typical clinical symptoms in the early stage,most patients are diagnosed with advanced tumors.Although the prognosis of tumor patients has improved with the development of targeted therapy and cellular immunotherapy,the survival rate of patients is still very low.Therefore,new targets of targeted therapy and cellular immunotherapy for lung adenocarcinoma need to be further explored.hypoxia is common in solid tumors.Hypoxia acts on a variety of hypoxia response elements(HRE)by activating the transcription factor HIF1α,thereby regulating the transcription and activation of related genes and participating in tumor proliferation,metabolism,angiogenesis,invasion and drug resistance.Studies have shown that tumor hypoxia is not a static state and oxygen level fluctuates.Compared with persistent hypoxia,intermittent hypoxia can better simulate the pattern of tumor hypoxia in vivo.A large number of abnormally expressed mirnas exist in the anoxic microenvironment of tumors.These mirnas are involved in the regulation of tumor cell proliferation,apoptosis,autophagy and other processes to affect tumor progression,and are potential new targets.Mirnas are involved in various cellular activities,including cell growth,differentiation,and apoptosis,by binding to the 3 ’UTR segment of m RNA targeting,silencing and inhibiting gene expression.Studies have found that miR-142-3p is low expressed in breast cancer,lung cancer and colorectal cancer,and miR-142-3p can inhibit the proliferation,migration and invasion of tumor cells.In addition,studies have shown that miR-142-3p can alleviate hypoxia/reoxygenation damage in hepatocytes and cardiomyocytes.However,studies on miR-142-3p and OSA-related intermittent hypoxia have not been reported so far,and the mechanism of miR-142-3p in the progression of pulmonary adenocarcinoma associated with intermittent hypoxia remains to be clarified.Objective1.To investigate the correlation between miR-142-3p and OSA related hypoxia parameters and prognosis of lung adenocarcinoma;2.To confirm the effects of OSA related intermittent hypoxia on the biology of lung adenocarcinoma cell lines;3.To clarify the effect of miR-142-3p on the malignant biology of lung adenocarcinoma promoted by intermittent hypoxia;4.Explore the specific molecular mechanism of miR-142-3p in relieving intermittent hypoxia and promoting malignant biology of lung adenocarcinoma.MethodIn the first part,we analyzed the correlation between OSA hypoxia related parameters and prognosis of lung adenocarcinoma by PSG detection and collection of hypoxia related parameters in patients with lung adenocarcinoma.Secondly,we conducted progressive sequencing analysis of lung adenocarcinoma patients with different degrees of OSA to find target molecules of miR-142-3p targeting HIF1α.Finally,p RT-PCR was used to detect the expression level of miR-142-3p in lung adenocarcinoma patients with different degrees of OSA,and to explore the correlation between miR-142-3p and sleep parameters and prognosis of lung adenocarcinoma.In the second part,we constructed an intermittent hypoxic cell model and detected the expression of miR-142-3p in normal oxygen/hypoxia and different cell lines by p RT-PCR.In the third part,we constructed a stable cell line overexpressing miR-142-3p by lentivirus transfection,and tested the transfection efficiency by fluorescence microscopy and p RT-PCR.The effects of overexpression of miR-142-3p on the proliferation,migration and invasion of lung adenocarcinoma cell lines were analyzed by CCK-8,scratch assay and Transwell assay.Finally,the effect of miR-142-3p on the growth of lung adenocarcinoma in vivo was explored by constructing a transplanted tumor model in nude mice,HE and immunohistochemical staining.In the fourth part,the binding site of miR-142-3p and HIF1α was predicted by Target Scan,and the targeted regulatory relationship was verified by dual luciferase assay.Subsequently,the expression level of HIF1 α in lung adenoma patients and overexpressed miR-142-3p cell lines was detected by p RT-PCR and WB assay.Next,the effects of HIF1α on cell proliferation,migration and invasion were investigated by response experiment.Finally,we detected the protein expression changes of angptl4 after HIF1α recovery through JASPAR and GEPIA databases,as well as WB experiment,to explore the potential downstream target genes of the mechanism.ResultIn the first part,we found that compared with simple lung adenocarcinoma,the prognosis of lung adenocarcinoma complicated with OSA was worse.As the severity of OSA increased,the TNM stage was later and metastasis was more likely.OSA related anoxic parameters AHI and ODI were different in patients with different tumor stages.The expression of Mi R-142-3p was decreased in lung adenocarcinoma patients with OSA,and the later the TNM stage was,the lower the expression level of miR-142-3p was.The more severe OSA was(the higher AHI and ODI index were),the lower miR-142-3p expression level was.In the second part,we found that intermittent hypoxia promoted the proliferation,migration and invasion of lung adenocarcinoma cells.Mi R-142-3p lung cancer mat cell lines and decreased expression after intermittent induction.In the third part,we successfully constructed overexpressing miR-142-3p cell lines.Cell function experiments showed that overexpression of miR-142-3p could alleviate the promoting effect of intermittent hypoxia on the proliferation,migration and invasion of lung adenocarcinoma cells.In addition,animal experiments showed that overexpression of miR-142-3p could reduce the ability of transplanted tumor formation and inhibit the expression of HIF1α in nude mice.In the fourth part,the results of Target Scan database and dual luciferase reports suggest that HIF1 α has a targeted regulatory relationship with miR-142-3p,and overexpression of miR-142-3p can reduce the expression of HIF1 α m RNA and protein,and there is a negative correlation between the two expressions in the tumor tissues of lung adenoma patients with OSA.The response experiment found that the up-regulation of HIF1 α expression level could partially reverse the effect of miR-142-3p on the malignant biology of lung adenocarcinoma cells.Through JASPAR database,we found that there were targeted binding sites between HIF1 αtranscription factor and angptl4 promoter region,and their expressions were positively correlated in GEPIA lung adenocarcinoma database.Finally,we found that intermittent hypoxia could promote angptl4 protein expression.Overexpression of HIF1α further promoted the expression of angptl4.Conclusion:The expression of Mi R-142-3p was decreased in lung cancer tissues and cells,and was further decreased after OSA and intermittent induction in lung adenocarcinoma,and the expression level of Mi R-142-3p was correlated with tumor TNM staging and OSA sleep parameters AHI and ODI.Mi R-142-3p can alleviate the promoting effect of intermittent hypoxia on the malignant biology of lung adenocarcinoma cells by inhibiting HIF1α.Angptl4 was positively regulated by HIF1α under intermittent hypoxia condition. |
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