The Mechanism Of Circular RNA Hsa＿Circ＿0006220 Promotes The Progression In Lung Adenocarcinoma Via Suppressing MiR-214-3p Binging To TPD52

Lung cancer is a major cause of malignancy-related death in most countries,and lung cancer incidence in developing countries is increasing quickly.China is a major lung cancer country,with an incidence rate of 1.63%per year,and the total number of lung cancer patients is expected to reach 1 million.Lung adenocarcinoma is the main pathological type of lung cancer with complex pathogenesis,multiple molecular features and clinical manifestations,and very different prognosis of patients.Therefore,it is particularly important to study the molecular mechanisms of lung adenocarcinoma and to find early diagnostic markers and new therapeutic targets for lung adenocarcinoma.Circular RNAs（circRNAs）are a class of widely expressed RNAs that have been found in recent years to play an important regulatory role in the development of tumors.Previous studies found that hsa＿circ＿0006220 was significantly elevated in tumor tissues of lung adenocarcinoma patients and correlated with poor patient prognosis,and these findings suggest that hsa＿circ＿0006220 may have potential as a better diagnostic value and prognostic marker in lung adenocarcinoma.The detailed role and mechanism of hsa＿circ＿0006220 in lung adenocarcinoma is not fully understood.The aim of this study was to investigate the effects of hsa＿circ＿0006220 on proliferation,migration and invasion of lung adenocarcinoma cells and its potential molecular mechanisms in vitro and in vivo,in combination with bioinformatic analysis techniques.Part 1 Identification of hsa＿circ＿0006220 and its differential expression in lung adenocarcinoma tissues and correlation with clinical characteristicsAim1.Screening the differentially expressed circRNAs（DEcircRNAs）between the lung adenocarcinoma（LUAD）tissues and controls.2.Validating the reliability of the obtained data and identifying the molecules most significantly upregulated in LUAD tissues for the subsequent in-depth study.3.Clarifying the relationship between hsa＿circ＿0006220 and clinical indicators in patients with LUAD.Methods1.Data mining was performed on the circRNA expression profile dataset GSE101586 in the GEO database.Subsequently,volcano and heat maps of DEcircRNAs in LUAD and paracancerous tissues were plotted.2.Real-time fluorescence quantitative-polymerase chain reaction（qRT-PCR）assay was conducted for determining the expression levels of the top five upregulated circRNA molecules in 10 LUAD tissues and 10 control tissues.3.Reverse transcription PCR,agarose gel electrophoresis,Sanger sequencing,and exonuclease（RNase R）tolerance assays were performed by specific reverse primers.Subsequently,the loop structure,cyclization sites,and stability of hsa＿circ＿0006220 were examined.4.After expanding the sample size（80 pairs）,hsa＿circ＿0006220 expression was detected again by qRT-PCR to verify its differential expression in LUAD tissues.In addition,the relationship between hsa＿circ＿0006220 and LUAD patients’ clinical characteristics was evaluated.Results1.A total of 34 DEcircRNA molecules were identified,of which the top five downregulated were hsa_circ₀015278,hsa_circ₀049271,hsa_circ₀007518,hsa_circ₀061817,and hsa_circ₀076798,whereas the top five upregulated were hsa_circ₀072088,hsa_circ₀043278,hsa_circ₀000977,hsa＿circ＿0006220,and hsa_circ₀001666.2.The results of the qRT-PCR assay showed that the expression of five molecules,including hsa＿circ＿0006220,were significantly upregulated in LUAD tissues,which was consistent with the analysis in the dataset.Among them,the expression of hsa＿circ＿0006220 was most significantly upregulated in LUAD tissues（P<0.001）.3.In qRT-PCR and gel electrophoresis experiments,amplification products were obtained using reverse primers against hsa＿circ＿0006220,with cDNA as a template.However,no amplification products could be obtained using genomic DNA（gDNA）as a template.These results demonstrate the presence of a loop structure in hsa＿circ＿0006220.Sanger sequencing results demonstrated the gene sequence at the cyclization site;RNase R experiments displayed circular hsa＿circ＿0006220 to be more stable than linear mRNA.4.After expanding the sample size,RT-PCR results validated hsa＿circ＿0006220 upregulation in LUAD tissues.Clinical characterization revealed that the expression level of this gene has a correlation with the degree of tumor differentiation,patient clinical stage,and metastasis.Statistically significant differences were considered as P<0.05.ConclusionThrough data mining of circRNA expression profiles,we screened and validated the DEcircRNAs in LUAD tissues.The expression level of the hsa＿circ＿0006220 gene showed a significant increase in LUAD tissues,and its upregulation amount probably correlates with the tumor size,patient TNM stage.Part 2 Effects of hsa＿circ＿0006220 on the biological function of lung adenocarcinoma cellsAimThe aim of this study was to clarify the influence of hsa＿circ＿0006220 expression level on the proliferation,apoptosis,migration,and invasion levels of human lung adenocarcinoma（LUAD）cells in vitro.Methods1.For this purpose,using qRT-PCR assay,the expression levels of hsa＿circ＿0006220 were detected in five human LUAD cell lines（A549,H1299,PC9,H1975,and H441）as well as human normal lung epithelial cells（BEAS-2B）.The hsa＿circ＿0006220 localization in LUAD cells was also conducted using the FISH assay.2.We silenced the A549 and H1299 cell lines and performed qRT-PCR assays to detect the knockdown efficiency.3.The overexpressed lentivirus was transfected with A549 and H1299 cells to construct stable silenced A549 and H1299 cell lines.Then,using qRT-PCR,the overexpressed efficiency was assessed.4.The proliferative activities of the above four groups of cells were assayed using the CCK8 assay.5.The migration and invasion properties of the abovementioned four groups of cells were measured using Wound Healing and Transwell assays in vitro.6.The apoptosis level in the abovementioned four groups of cells was detected using flow cytometry.Results1.The results from the qRT-PCR assay revealed that hsa＿circ＿0006220 expression level was enhanced in A549 and H1299 cells（human LUAD cell lines）（P<0.01）.However,the BEAS-2B expression level showed a significant decrease in human normal lung epithelial cells compared to LUAD cell lines（P<0.001）.These results suggest that A549 and H1299 cells can be used as efficient cellular models for functional experiments.2.qRT-PCR assay results showed that transfection of both si-hsa＿circ＿0006220 cells resulted in a significant downregulation of hsa＿circ＿0006220 expression level in comparison with the controls（P<0.01）.This finding indicates that we have successfully constructed silenced A549 and H1299 cells,which can be subjected to subsequent functional experiments.3.qRT-PCR assay results showed that both cells transfected with the overexpressed lentivirus expressed significantly higher hsa＿circ＿0006220 in comparison with the controls（P<0.01）.This finding indicates that stably overexpressing A549 and H1299 cells were successfully constructed and can be used for subsequent functional experiments.4.The CCK8 assay proved the significantly inhibited proliferation of both cells in the silenced group（si-circ）compared to that of controls（P<0.05）.Although,the proliferation levels of both cells in the overexpression group（circ）were significantly promoted in comparison with the controls（P<0.05）.5.The findings from the scratch assay displayed the migration potential of both cells in the silenced group（si-circ）was significantly attenuated,whereas the migration ability of both cells in the overexpression group（circ）was significantly enhanced compared to the controls（all P<0.05）.The migration and invasion properties of both cell types in the silenced group（si-circ）were significantly decreased,whereas the migration and invasion properties of both cell types in the overexpression group（circ）were significantly higher compared to the controls（all P<0.05）.6.The data from the flow cytometry assay revealed that there was no significant effect of hsa＿circ＿0006220 on apoptosis rate of lung adenocarcinoma（P>0.05）.Conclusionhsa＿circ＿0006220 is highly expressed in LUAD cells and can promote proliferation,migration,invasion of LUAD cells in vitro.Part 3 Mechanism research of the effect of hsa＿circ＿0006220 on the biological behavior of lung adenocarcinoma cellsAimThe present study was intended to explore the pathways and mechanisms by which hsa＿circ＿0006220 affects the proliferation,migration,and invasion properties of LUAD cells.Methods1.Starbase and mirDB databases were queried for miRNA molecules that could potentially bind to hsa＿circ＿0006220.2.hsa＿circ＿0006220 pull down experiments were conducted to determine the enrichment of potential target miRNAs for hsa＿circ＿0006220.3.A qRT-PCR assay was performed to determine the expression levels of candidate miRNA molecules in LUAD cells and the most expressed molecule was chosen for subsequent experiments.4.The RNA-binding protein immunoprecipitation（RIP）assay was used to verify the interaction of miR-214-3p derived from data screening with hsa＿circ＿0006220.5.A dual-luciferase reporter gene vector was constructed by inserting the hsa＿circ＿0006220 sequence into the dual-luciferase reporter gene plasmid to confirm the binding relationship of hsa＿circ＿0006220 and miR-214-3p.6.qRT-PCR assay was conducted following either silencing or overexpression of hsa＿circ＿0006220 and the alterations in miR-214-3p expression level were measured.7.CCK8 assay was used to assess the miR-214-3p effect on the proliferative capacity of LUAD cells in vitro.8.Wound Healing and Transwell assays were exerted to examine the miR-214-3p effect on the migration and relocation abilities of LUAD cells.9.Rescue assay was performed to evaluate the downregulation of miR-214-3p expression in LUAD cells with silenced hsa＿circ＿0006220.After overexpressing miR-214-3p,we used CCK8,Wound Healing,and Transwell assays to investigate whether the effects of hsa＿circ＿0006220 on the proliferation,migration,and invasion abilities of LUAD cells were reversed after infection by miR-214-3p.10.Targetscan database and Starbase were searched to predict the target protein of miR-214-3p.11.After miR-214-3p insertion to the target protein（TPD52）,a gene vector with wild-type and mutant fragments of the sequence was bound.Subsequently,the miR-214-3p/protein binding was verified via dual-luciferase reporter assay.12.PCR assay was conducted to determine the changes in the TPD52 expression level in LUAD cells following the inhibition and overexpression of miR-214-3p.13.Western blotting and PCR were performed to determine the expression levels of mRNA and protein of TPD52 in the five LUAD cell lines and normal human lung epithelial cells.14.After silencing and overexpression of hsa＿circ＿0006220,the TPD52 expression level was detected in LUAD cells using western blot assay and PCR.Subsequently,the corresponding rescue experiments were performed to determine the TPD52 expression level again.Results1.Five miRNA molecules with potential binding ability to hsa＿circ＿0006220 were screened in two databases.2.The results of RNA pull down and qRT-PCR assays revealed the significant enrichment of the abovementioned five miRNAs by the hsa＿circ＿0006220 probe compared to the control probe,which proved that all five miRNAs could interact with hsa＿circ＿0006220.3.qRT-PCR results proved the maximized expression level of miR-214-3p in both A549 cells and H1299 cells,contributing to its subsequent application in experiments.4.The results of RIP experiments displayed the significantly higher expression of miR-214-3p compared to the controls,indicating the interaction between hsa＿circ＿0006220 and miR-214-3p.5.Dual-luciferase reporter assay results proved a direct interaction between the hsa＿circ＿0006220 and miR-214-3p,which is a target miRNA molecule for hsa＿circ＿0006220.6.PCR assay findings proved that the overexpression of hsa＿circ＿0006220 in LUAD cells significantly decreased the miR-214-3p expression compared to the controls.However,its silencing led to a significant increase in the expression of miR-214-3p.7.The results of the CCK8 assay proved that silencing miR-214-3p could significantly prevent LUAD cells from proliferation,and miR-214-3p inhibitors can partially reverse this effect.In addition,H1299 cells proliferation could be promoted by miR-214-3p overexpression and partially inhibited by miR-214-3p mimic.8.Wound Healing assay results revealed that silencing hsa＿circ＿0006220 had a significant inhibition effect on the A549 cells’ migration ability.While the miR-214-3p inhibitor could reverse this inhibition effect.Similarly,miR-214-3p overexpression enhanced the A549 cells’ migration,whereas transfecting them with miR-214-3p mimic significantly attenuated this migratory-promoting effect.In addition,the Transwell assay revealed a similar phenomenon.9.The Targetscan database identified the 3’UTR of TPD52 as a potential binding target for miR-214-3p.10.Dual-luciferase report test proved miR-214-3p to be capable of directly interacting with TPD52.11.The results of PCR demonstrated that miR-214-3p overexpression could significantly reduce the TPD52 expression compared with the controls,while its inhibition elevated the TPD52 expression levels.12.The results from Western blot assay and PCR displayed that the TPD52 expression level in LUAD cell lines was significantly higher compared to the normal lung epithelial cells.13.Western blotting results also proved that hsa＿circ＿0006220 silencing significantly reduced the TPD52 expression level compared to that of controls.However,hsa＿circ＿0006220 overexpression could significantly elevate the expression levels of TP52.The above results were also demonstrated by rescue experiments.Conclusion1.The miR-214-3p expression in LUAD cells is significantly increased and the hsa＿circ＿0006220 is the molecular sponge of miR-214-3p.2.Interfering with miR-214-3p reverses the hsa＿circ＿0006220 effects on the proliferation,migration,and invasion properties of LUAD cells.3.hsa＿circ＿0006220 has regulatory effects on the expression level of TPD52 through miR-214-3p,and can result in enhanced proliferation,migration,and invasion properties of LUAD cells,contributing to the progression of LUAD.Part 4 Bioinformatics analysis of the role of TPD52 in lung adenocarcinomaAimThe present study investigated the TPD52 expression level in LUAD tissues and its relationship with patient prognosis using bioinformatics analysis.MethodsThe TPD52 expression level in LUAD cells and its correlation with clinicopathological properties were observed by immunohistochemical assays based on GEPIA,TCGA,GEO,and other public databases.The Kaplan-Meier Plotter database was conducted to investigate the correlation of TPD52 expression level to the survival and prognosis of LUAD patients.In addition,the correlation between TPD52 and immune cell infiltration in the microenvironment of LUAD was analyzed by the TIMER database,TISIDB database,and R software.We divided the TCGA data into two groups of low-expression and high-expression based on the median value of TPD52 expression level as the cut-off.Finally,GSEA functional pathway analysis was performed by GSEA software for the two groups of differentially expressed genes.ResultsTPD52 expression level in LUAD tissues was significantly higher than its expression in paracancerous tissues.Also,higher TPD52 expression correlated to the poor prognosis of patients.However,its expression level showed no significant difference in patients at different stages of tumor.Moreover,the TPD52 expression level was weakly associated with immune-related cells presenting in the tumor microenvironment.In addition,findings from GSEA analysis indicated that TPD52 expression could influence epithelial-mesenchymal transition（EMT）in LUAD.ConclusionTPD52 can be considered a potential marker and/or target respectively for diagnosis or treatment of LUAD patients and may have an impact on EMT.Keywords:TPD52,lung adenocarcinoma,biomarker,immune infiltration,EMTPart 5 Effect of hsa＿circ＿0006220 expression on subcutaneous transplantation tumors in nude mice with lung adenocarcinoma cellsAimThis study explores the effect of hsa＿circ＿0006220 expression on the proliferation of subcutaneous transplantation tumors in nude mice.Methods1.Three groups each containing four nude mice were included in the study.The control group mice were inoculated with A549-NC cells.The overexpression group was transplanted with hsa＿circ＿0006220 overexpression cells and the silenced group mice were transplanted with the hsa＿circ＿0006220 silence cells.2.A tumor model was developed by subcutaneously transplanting the LUAD A549 cells in nude mice and the growth dynamics was observed in the transplanted tumors.3.The expression level of miR-214-3p and TPD52 assay in the established tumor models was evaluated by PCR.Results1.The subcutaneously transplanted tumor volume was significantly smaller in the silenced group than that in the control group.Their difference showed statistical significance from day 7.In the overexpression group,the subcutaneously transplanted tumor volume was significantly larger.2.PCR assay results showed that inhibition of hsa＿circ＿0006220 expression significantly attenuated TPD52 expression and promoted miR-214-3p expression,while overexpression of hsa＿circ＿0006220 significantly attenuated miR-214-3p expression and promoted TPD52 expression compared with the controls.ConclusionInhibition of hsa＿circ＿0006220 expression in LUAD cells significantly reduced the of subcutaneously transplanted tumor growth in nude mice.Then,hsa＿circ＿0006220 can serve as a ceRNA for miR-214-3p and competitively bind to miR-214-3p to upregulate the expression of TPD52.