Circ-HMGA2 Promotes Lung Adenocarcinoma Metastasis Through The MiR-1236-3p/EMT Axis

BACKGROUNDWith the continuous development of sequencing technology in recent years,the research on circRNA is getting more and more in-depth.CircRNA has been found to be a biomarker for diseases and can be used for the diagnosis of diseases.CircRNA can also participate in the occurrence and development of various diseases and can be used as a target for future treatment.However,there are few studies on circRNA and Lung adenocarcinoma,which provides opportunities for our research.Methods1.Take three pairs of Lung adenocarcinoma patients’ cancer tissues and adjacent tissues for gene chip sequencing to obtain differentially expressed circRNA.2.In order to obtain significantly differently expressed circRNA,we set the screening conditions to fold change>2,P<0.05.Then the function of these circRNAs was predicted by bioinformatics,and pre-experimental verification was performed.3.Sanger sequencing and treat Lung adenocarcinoma cells with RNase R and actinomycin D were used to identify circular RNA.4.Through the FISH experiment,we find the position of circ-HMGA2 in the cell.5.Using the transwell and cell scratch experiment,we detect the changes in cell migration and invasion ability under the conditions of knocking down and increasing the circ-HMGA2 expression of Lung adenocarcinoma cells.6.Using western blot to analyze the effect of circ-HMGA2 on EMT.7.Analyze the relationship between circ-HMGA2 and miR-1236-3P by bioinformatics,and then verify the relationship between them by luciferase reporting experiments.8.Co-localize circ-HMGA2 and miR-1236-3P in cells by FISH experiments.9.The effect of miR-1236-3P on the EMT and metastatic ability of lung adenocarcinoma cells was detected by Western blotting and transwell experiments.10.Use transwell assay to detect the effect of co-expressing circ-HMGA2 and miR-1236-3P on the metastasis ability of lung adenocarcinoma cells.11.Analyze the relationship between miR-1236-3P and ZEB1 through bioinformatics,and then verify the relationship between them through luciferase reporting experiments,and then verify the relationship between circ-HMGA2 and ZEB1.12.Analyze the effects of circ-HMGA2 and miR-1236-3P on ZEB1 and EMT by RT-PCR and western blot.13.The lung cancer metastasis model was created by tail vein injection in nude mice,and the effect of circ-HMGA2 on tumor metastasis was verified in vivo.Results1.In lung adenocarcinoma tissues,we found 103 significantly overexpressed circRNAs and 81 significantly underexpressed circRNAs.2.Pre-experimentation of the screened circRNAs revealed that circ-HMGA2 promoted the metastasis of lung adenocarcinoma.3.The result of sanger sequencing is consistent with the sequence on circbase,which proves that circ-HMGA2 is indeed a circular structure.Rnase R and actinomycin D treated cells found that circ-HMGA2 is more stable than its linear form.4.Circ-HMGA2 is mainly located in the cytoplasm.5.When circ-HMGA2 is overexpressed,the ability of migration and invasion is enhanced;when the expression of circ-HMGA2 is knocked down,the ability of migration and invasion is reduced.6.Circ-HMGA2 promotes the development of EMT.7.Circ-HMGA2 is bound to miR-1236-3P,and it will no longer bind when the binding site is mutated.8.Both circ-HMGA2 and miR-1236-3P are located in the cytoplasm.9.MiR-1236-3P inhibits the development of EMT and the metastasis of lung adenocarcinoma.10.MiR-1236-3P can inhibit the effects of circ-HMGA2 on lung adenocarcinoma metastasis.11.MiR-1236-3P binds to ZEB1,and circ-HMGA2 affects ZEB1 by binding to miR-1236-3P.12.Circ-HMGA2 promotes ZEB1 expression,miR-1236-3P inhibits ZEB1 expression,and circ-HMGA2 inhibits the effects of miR-1236-3P on ZEB1 and EMT.13.In the model of lung cancer metastasis,circ-HMGA2 promotes the metastasis of lung adenocarcinoma.ConclusionThrough gene chip sequencing,we found that circ-HMGA2 is highly expressed in lung cancer tissues of patients with lung adenocarcinoma.In vitro and in vivo experiments have demonstrated that circ-HMGA2 promotes the metastasis of lung adenocarcinoma.Through in-depth research,we found that circ-HMGA2 mainly promotes the metastasis of lung adenocarcinoma through the miR-1236-3P/EMT pathway.These suggest that circ-HMGA2 may serve as a biomarker and new therapeutic target for lung adenocarcinoma in the future.