Copy Number Deletion Related Long Non-coding RNA-MIR99AHG Promote Autophagy And Inhibit The Malignant Progression In Lung Adenocarcinoma

Objective The morbidity and mortality of lung cancer are the highest among all malignant tumors.The incidence of lung adenocarcinoma(LUAD)is much higher than that of lung squamous cell carcinoma(LUSC)in China,which has become the most common pathological subtype of lung cancer.In recent years,a large number of studies have found that long non-coding RNA plays a key regulatory role in tumor genesis and malignant progression.The study aimed to explore the role of lnc RNA-MIR99 AHG in the occurrence and development of lung adenocarcinoma,and provide new targets and new approaches for the diagnosis and prevention of lung adenocarcinoma.Methods By digging up the transcriptome expression profile data and Copy Number Variations(CNV)in The lung adenocarcinoma data set of The Cancer Genome Atlas(TCGA),an lnc RNA-MIR99 AHG with differential expression and unknown function in lung adenocarcinoma was screened.MIR99 AHG expression and its correlation with clinical characteristics were verified through 58 pairs of LAC clinical samples and LAC cell lines.Potential biological functions of MIR99 AHG were investigated both in vivo and in vitro.Western Blot,fluorescence quantitative PCR(RT-PCR),RNA-protein pull-down assay,RNA immunoprecipitation(RIP),immunostaining,transmission electron microscopy and other techniques were used to explore the molecular mechanism of MIR99 AHG.Results Based on the TCGA database,CNV regions of non-small cell lung cancer were analyzed.By establishing relevant models of CNV,expression and prognosis,a down-regulated lnc RNA-MIR99 AHG with unknown function in lung adenocarcinoma was screened.In 58 pairs of LAC tissue samples and LAC cell lines,MIR99 AHG was verified to be low expression in cancer tissues,and its expression was negatively correlated with TNM stage and lymph node metastasis.In vitro cellular functional experiments showed that over expression of MIR99 AHG significantly inhibited the proliferation,invasion and migration of LAC cells,while knock down of MIR99 AHG resulted in the opposite results.The model of tail vein metastasis in nude mice showed that pulmonary metastatic nodules were significantly inhibited after over expression of MIR99 AHG.The results of Western Blot assay for autophagia-associated protein,autophagic flow and transmission electron microscopy suggested that over expression of MIR99 AHG could promote autophagia in LAC cells.Fluorescence in situ hybirdization(FISH)and nucleus/cytoplasm separation assay confirmed that MIR99 AHG is mainly distributed in the cytoplasm.Through RNA-Protein Pull-Down assay,mass spectrometry,and RIP assay,it was revealed that MIR99 AHG can bind to Annexin A2(ANXA2).Taking ANXA2 as the rescue node,Western Blot experiment,fluorescence staining experiment and other experiments confirmed that MIR99 AHG could prevent ANXA2 from entering the nucleus and promote the formation of autophagic vesicle.Conclusion 1.MIR99 AHG was significantly down-regulated in lung adenocarcinoma tissues and cell lines,and its expression was negatively correlated with TNM stage and lymph node metastasis.In vivo and in vitro function experiments confirmed that MIR99 AHG plays a tumor suppressive role in lung adenocarcinoma.2.MIR99 AHG enhances the formation of autophagic vesicle by binding and blocking Annexin A2(ANXA2)into the nucleus,and promotes autophagy to inhibit the malignant progression of lung adenocarcinoma.