| Objective:Lung cancer is the main cause of cancer-related death worldwide,and the incidence of lung cancer is increasing year by year,among which non-small cell lung cancer accounts for 85%of all lung cancer cases,and the 5-year survival rate is less than 20%.For patients with early NSCLC,surgery is still the first choice of treatment,but up to 45%of patients with early NSCLC will have postoperative recurrence,so the dynamic evaluation and prognosis analysis of patients is helpful for clinicians to make treatment decisions,which has important clinical significance.Circulating aneuploidy cells(CAC)monitoring,as a non-invasive and repeatable method,can provide a large amount of information for individual treatment and prognosis assessment.It plays an important role in tumor diagnosis,treatment evaluation and prognosis prediction.The purpose of this study was to conduct a comprehensive and in-depth analysis of CAC cells and their phenotypes and/or karyotypes in patients with resectable non-small cell lung cancer(NSCLC)by using the new SE-iFISH platform,and to explore the relationship between CAC cells and prognosis.In addition,as global smoking rates have decreased,the proportion of non-smoking-related lung cancer has increased.The pathological subtype of lung adenocarcinoma(LUAD)is the main component of non-smoking-related lung cancer,which is characterized by strong metastatic ability and poor prognosis.Therefore,the pathogenesis,treatment and prognosis evaluation of lung adenocarcinoma have gradually attracted extensive attention.Epigenetic modification,especially non-coding RNAs,was widely reported recently and was considered playing an important role in a wide variety of tumors.In this study,bioinformatics analysis was used to screen prognosis related ncRNAs in patients with LUAD.Real-world patient tissue samples were then used to verify the efficacy of specific ncRNAs in evaluating patient outcomes.Furthermore,the effects of specific ncRNAs on the biological characteristics of lung adenocarcinoma,as well as the interaction relationship between microRNA(miRNA),upstream circRNA and downstream target gene mRNA and the molecular regulation mechanism were further studied.The results of this study are expected to provide new biomarkers for the recurrence,metastasis and prognosis of LUAD,and provide a new theoretical evidence basis for the regulatory mechanism of ncRNAs.Method:7.5mL peripheral blood samples from 50 preoperative and 35 postoperative patients with resectable NSCLC were collected.The CACs were detected by subtractive enrichment and immunostaining fluorescence in situ hybridization(SE-iFISH)detection platform by identifying the aneuploid karyotype of chromosome 8,and then CACs were classified by size and karyotype.Combined with clinicopathological and prognostic data,the relationship between CAC subtypes and clinical data and progression-free survival(DFS)of NSCLC patients was further analyzed.RNA-seq and clinical data packets from the Cancer Genome Atlas(TCGA)were downloaded and analyzed to screen for miRNAs related to overall survival(OS)of LUAD patients.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression levels of miR-421,circ?0000567 and TMEM100 in lung adenocarcinoma cell lines,73 lung adenocarcinoma tissues and corresponding normal tissues.Wound Healing and Transwell assays were used to detect the ability of cell migration and invasion.The upstream competitive endogenous RNA(ceRNA)circ?0000567 and downstream target gene TMEM100 were found out by miR-421 target prediction using multiple bioinformatics databases.The circular structure and stability of circ?0000567 were verified by RNase R digestion assay and qRT-PCR assay using Oligo(dT)or random primers.The binding relationship between miR-421 and circ?0000567 or TMEM 100 was identified by dual luciferase reporter gene assay.Result:Our study obtained the following three main results:First,when CAC diameter was used to classify CACs of resectable NSCLC patients,small cell size CACs(<5?m)accounted for less than 20%,while large CACs(?5m)accounted for more than 80%of the total number of CACs.CAC karyotypes include triploid,tetraploid and multiploid.When large and small cell size CACs were further divided into subtypes of different karyotypes,it was found that triploid subtype accounted for the majority of small cell size CACs,while multiploid subtype accounted for the majority of large cell size CACs.Patients with pre-surgery small cell size CACs and triploid small cell size CACs had shorter DFS compared with those patients without,and both these two kinds of CACs were significantly associated with higher TNM stage and recurrence.Postoperative CAC does not have the above clinical significance.The number of CAC and each subtype increased slightly after operation,but the variation of CACs before and after operation was not related to DFS.Second,bioinformatics analysis was performed on the RNA-seq and clinical data of LUAD patients downloaded from TCGA database,and a total of 61 miRNAs related to OS in LUAD patients were sequenced from high to low according to hazard ratios(HR).Further validation by Kaplan-Meier method revealed that six miRNAs,including miR-3940,miR-873,miR-550-2,miR-1293,miR-421 and miR-212,may be closely related to the overall survival of patients with LUAD.Based on the HR and relative expression level,miR-421 was selected for further study.MiR-421 was highly expressed in 73 LUAD tissue specimens in our department,and was an independent risk factor for patients with short OS.Third,in 73 LUAD tissue specimens,the relative expression level of miR-421 was inversely proportional to TMEM100 and directly proportional to circ?0000567.Bioinformatics analysis,dual luciferase reporter gene assay and cell function assay showed that circ?0000567 can act as a ceRNA to bind miR-421,preventing it from directly targeting the 3 '-untranslated region(3'-UTR)of TMEM100 mRNA and further degrading it.Cell function assays confirmed that miR-421 promoted the migration and invasion of LUAD cell lines,while circ?0000567 inhibited the migration and invasion.Recovery experiments further confirmed that the inhibitory effect of circ?0000567 could be partially neutralized when miR-421 was co-transfected with circ?0000567 overexpressing plasmid in LUAD cells.In LUAD cells,miR-421 can negatively regulate the expression of TMEM 100,and circ?0000567 overexpression can up-regulate the expression of TMEM100.Conclusions:Our results suggest that preoperative small CAC,especially its triploid karyotype,may serve as a potential prognostic biomarker for resectable NSCLC patients;miR-421 can be used as a potential prognostic biomarker in patients with LUAD;moreover,miR-421 could be "sponged" by circ?0000567,so that miR-421 could not target and inhibit the function of downstream target genes,and downstream target gene TMEM100 was released,thus promoting the migration and invasion of lung adenocarcinoma.Our study may become an important element in the clinical prognostic model of non-small cell lung cancer. |
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