| Objective: Lung cancer is one of the most common malignant tumors,accounting for the highest proportion of deaths caused by malignant tumors worldwide.The main type of lung cancer is non-small cell lung cancer(NSCLC),among which lung adenocarcinoma is the main histological type.The low survival rate and poor prognosis of patients with metastatic non-small cell lung cancer have always been a difficult clinical problem.Early diagnosis and treatment will undoubtedly increase the survival rate and improve the prognosis of patients.In case of metastasis,it is necessary to identify the molecules that promote metastasis and give targeted therapy.Circular RNA(circRNA)is a non-coding RNA,which has a unique closed-loop structure and can avoid digestion by endonucleases,so it has good stability.Circ RNA also has good tissue specificity and species conservation,which means that circRNA may become a new diagnostic marker,which also indicates that it may have the value of evaluating the prognosis of diseases.In this study,the highly expressed circRNA in non-small cell lung cancer tissues was screened by high-throughput sequencing,and then its biological characteristics and mechanism were confirmed by a series of experiments,which provided a new perspective for the diagnosis and treatment of non-small cell lung cancer.Methods: 1.We collected cancer tissues and adjacent tissues of patients(n=41)who underwent lung cancer resection from July 2017 to January 2018.These patients were admitted to the Department of Thoracic Surgery,Shengjing Hospital of China Medical University.At the same time,for the follow-up study,we also collected the corresponding clinicopathological data of these patients.2.Three cases of lung adenocarcinoma and three corresponding adjacent tissues were selected for high-throughput sequencing,and differentially expressed circRNA was screened,and bioinformatics analysis was performed.Select the highly expressed circRNA in cancer tissues: hsa?circ?0067582,hsa?circ?0005692;Low expression of circRNAs in cancer tissues were novel?circ-?0008866 and novel?circ?0005280.After enlarging the sample size,it was verified by real-time qPCR,and the clinicopathological correlation between hsa?circ?0067582 and novel?circ?0005280was analyzed according to the expression.3.Hsa-circ-0067582,which is highly expressed in tumor tissues,was selected as the research object,and its cyclic structure was verified by Sanger sequencing and Rnase enzyme tolerance test,and its cyclic site was confirmed.Intracellular localization was determined by nucleoplasm separation experiment.The real-time qPCR in multiple lung cancer cell lines verified its expression level,and A549 cell line with the highest expression level and H1299 cell line with the lowest expression level were selected as tool cells.The interference plasmid hsa?circ?0067582 was constructed and screened out.After transfection,the cells were subjected to colony formation assay and MTT assay to detect their proliferation ability,wound healing assay to detect their migration ability,and transwell invasion assay to detect their invasion ability.4.Using A549 cells as a tool,the protein binding to hsa?circ?0067582 was pulled down by RNA pull-down experiment,and analyzed by protein mass spectrometry.The RNA binding protein hnRNPM with good enrichment was selected as the target protein for further study.The pulled total RNA was verified by real-time qPCR,and the five miRNA predicted by the database were combined with hsa?circ?0067582.RIP experiment and real-time qPCR reverse verification hnRNPM and hsa?circ?0067582 are combined.The m RNA expression and protein expression of hnRNPM were verified by silencing hsa?circ?0067582.The expression of hsa?circ?0067582 was confirmed by silencing hnRNPM.Extract and clean TCGA database data,analyze the expression of hnRNPM in lung adenocarcinoma and lung squamous cell carcinoma,draw receiver operating characteristic curve(ROC)to evaluate its diagnostic value,and draw K-M survival curve to evaluate its prognostic value.Results: 1.The expression of circRNAs in cancer tissues and adjacent tissues of patients with NSCLC were different,which is tissue specific.By analyzing the high-throughput sequencing of cancer tissues and adjacent tissues of three patients with lung adenocarcinoma,the following results were obtained: there were 81 differentially expressed circRNAs in NSCLC cancer tissues and adjacent tissues,of which 17 were highly expressed in cancer tissues and 64 were poorly expressed in cancer tissues.2.Through bioinformatics analysis,this study identified 15 significant functional types.In biological process types,the differential genes are mainly concentrated in cell catabolism,nucleobase regulation,biological process,nucleobase-containing metabolite regulation,nitrogenous compound metabolite regulation and heterocyclic metabolism.In the types of cell component,differentially expressed genes are mainly concentrated in cell parts,intracellular parts,cytoplasm and organelles.Among the functional types of molecules,differentially expressed genes mainly focus on catalytic activity,protein binding and transferase activity.In KEGG pathway analysis,there were differences in phagocyte,actin cytoskeleton regulation and lysine degradation.3.The real-time qPCR experiment was carried out by enlarging the sample size,which verified that novel?circ?0008866 and novel?circ?0005280 were low in NSCLC cancer tissues,and hsa?circ?0067582 was high in NSCLC cancer tissues,which were consistent with the sequencing results.The low expression of hsa?circ?0005692 in NSCLC was inconsistent with the sequencing results.4.Clinicopathological analysis of NSCLC patients showed that the expression level of hsa?circ?0067582 was significantly correlated with lymph node metastasis,and the expression level of patients with lymph node metastasis was higher.However,the expression level of novel?circ?0005280 was significantly correlated with the tumor diameter of patients,and the expression level of patients with large tumor diameter was lower.5.Hsa-circ-0067582 has obvious Rnase R resistance,and its Cyclization site is ACAGTG.6.The real-time qPCR experiments were carried out in various lung cancer cell lines,and the relative expression of hsa?circ?0067582 was the highest in A549 cells and the lowest in H1299 cells.7.Hsa?circ?0067582 is mainly located in the nucleus.8.The silencing of hsa?circ?0067582 weakens the proliferation,migration and invasion of NSCLC.9.Hsa-circ-0067582 binds to RNA binding protein hnRNPM.10.The silencing of hsa?circ?0067582 in A549 cells can significantly down-regulate the protein expression of hnRNPM,but does not affect the m RNA level.The expression of hsa?circ?0067582 decreased after hnRNPM was silenced,and hnRNPM was positively correlated with hsa?circ?0067582.11.TCGA database showed that hnRNPM was highly expressed in non-small cell lung cancer tissues,while it was low expressed in adjacent tissues.12.TCGA database shows that hnRNPM has certain diagnostic accuracy and prognostic ability for non-small cell lung cancer.Conclusion: 1.Novel?circ?0005280 is poorly expressed in human NSCLC,and the expression level is negatively correlated with tumor diameter.Hsa?circ?0067582 is highly expressed in human NSCLC cancer tissues,and the expression level is positively correlated with whether lymph nodes of patients are metastasized.Hsa?circ?0067582 and novel?circ?0005280 may become biomarkers for the diagnosis of human NSCLC.2.The silencing of hsa?circ?0067582 reduced the proliferation,migration and invasion of NSCLC.3.Hsa?circ?0067582 can combine with hnRNPM and regulate its protein expression,which provides a new thinking direction for the diagnosis and treatment of NSCLC. |
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