Circ-000881 Inhibits Malignant Progression Of Lung Adenocarcinoma By Regulating MiR-665/PRICKLE2 Axis

Background:The latest cancer epidemiology data show that lung cancer incidence rate is the highest in the world.NSCLC is the most important pathological type,accounting for more than 85%.NSCLC is mainly divided into two major histologic types:lung adenocarcinoma and lung squamous cell carcinoma.Lung adenocarcinoma accounts for 40%of all lung cancer and 25%of lung squamous cell carcinoma.In recent years,with the development of molecular targeted drugs for many kinds of driving genes,the survival time of NSCLC patients with specific gene mutation has been prolonged,but the overall five-year survival rate of lung cancer patients is still around 15%.One of the most important reasons is the abnormal proliferation and distant metastasis of tumor cells.Cyclic ribonucleic acid is a new hotspot in the field of tumor biology in recent years.Many cyclic ribonucleic acids have been proved to play an important role in the development of lung adenocarcinoma.In this work,we studied the expression of hsa-circ-000881 in LUAD,the clinical prognostic value,the influence on the invasion and metastasis of lung adenocarcinoma and the mechanism of action.This paper mainly focuses on the following aspects:1.The expression of hsa-circ-000881 in the tissue of LUAD;2.The effect of hsa-circ-000881 on the growth,proliferation,migration and invasion of lung adenocarcinoma cells;3.The miRNA regulated downstream of hsa-circ-000881 was found by bioinformatics analysis,and its expression in LUAD tissue was analyzed;4.The expression of hsa-circ-000881 and target miRNA in LUAD was verified;5.The mRNA regulated downstream of miRNA was found by bioinformatics analysis,and its expression in LUAD tissue was analyzed;6.The expression of miRNA and target mRNA in LUAD was verified;7.The expression of mRNA protein was detected by knocking down hsa-circ-000881;8.The proliferation,migration and invasion of lung adenocarcinoma cells were detected by overexpression of hsa-circ-000881 and miRNA;The expression of hsa-circ-000881 and knockdown mRNA were also detected;9.The expression of PRICKLE2 in the tissues of LUAD and the correlation with clinicopathology and prognosis;10.The effect of PRICKLE2 on the growth,proliferation,migration and invasion of lung adenocarcinoma cells;11.The effect of PRICKLE2 on the cell cycle and metastasis of lung adenocarcinoma;12.The effect of PRICKLE2 on the proliferation of lung adenocarcinoma was studied in animal experiments;13.The mechanism of regulation of Myc signal pathway by PRICKLE2.Materials and methods:1?The expression of the cyclic RNA molecules in lung adenocarcinoma tissues was analyzed by using TCGA database.The relative expression of hsa-circ-000881 in lung adenocarcinoma was detected by RT-qPCR or Western blotting.The effects of hsa-circ-000881 on the proliferation,migration and invasion of LUAD cells were analyzed by ckk-8,plate clone formation experiment,wound healing experiment and Transwell migration experiment.2?The target prediction tool Circular RNA Interactome(https://circinteractome.nia.nih.gov/)was used to predict the target miRNA of hsa-circ-000881,and miR-665 was selected.The expression of miR-665 in LUAD was analyzed by TCGA public database.The results of luciferase report were analyzed to verify the target relationship between hsa-circ-000881 and miR-665.The expression of miR-665 in lung adenocarcinoma and the coexpression with has-circ-000881 were detected by RT-qPCR.3?The target prediction tool Targetscan(http://www.targetscan.org/vert₇2/)was used to predict the mRNA targeting miR-665 and PRICKLE2 was selected.The expression and prognosis of PRICKLE2 in LUAD were analyzed by TCGA public database.The results of luciferase report were analyzed to verify the target relationship between PRICKLE2 and mir-665.The expression of PRICKLE2 in lung adenocarcinoma and the coexpression with mir-665 were detected by RT-qPCR.4?The expression of PRICKLE2 protein was detected after knocking down hsa-circ-000881;After overexpression of circ-000881 and miR-665,the proliferation,migration and invasion of a549 and hcc-827 cells were analyzed by plate clone formation experiment,wound healing experiment and Transwell migration experiment.After overexpressio of circ-000881 and knowcdown of PERICKLE2,the proliferation,migration and invasion of a549 and hcc-827 cells were analyzed by plate clone formation experiment,wound healing experiment and Transwell migration experiment.5?The expression of PRICKLE2 mRNA and its prognostic significance were analyzed by TCGA public database.The expression of PRICKLE2 was detected in LUAD and adjacent tissues by Western blot and immunohistochemistry(IHC).6?The overexpression of PRICKLE2 in two lung adenocarcinoma cell lines(NCIH1395,NCIH1975)with high expression of PRICKLE2,the expression efficiency was verified by Western blot.The growth and proliferation of the cells were detected by ckk-8 and edu experiments.The cell cycle was tested and the changes of cyclin D1 and p21 were detected by Western blot,and the effects of over expression of PRICKLE2 on cell cycle were investigated.7?The NCIH1395 cell lines with stable overexpression of PRICKLE2 were divided into two groups(NCIH1395 empty plasmid group and OE PRICKLE2 cell group).The subcutaneous tumor model was established in nude mice,and the growth rate,tumor volume and the expression of proliferation and apoptosis related indicators were observed.8?The transfer ability of cells was detected by Transwell migration test,and the changes of apoptotic protein index were further detected by Western blot.9?GSEA analysis was carried out on the expression data of LUAD in TCGA,and the downstream channel related to PRICKLE2 was obtained.MYC pathway was selected for subsequent analysis.The expression of myc and PRICKLE2 in the LUAD of TCGA was detected.The expression of PRICKLE2 in NCIH 1395 and NCIH 1975 cells was detected by Western blot method to verify the changes of Myc pathway related genes(MYC,NOLC1,NPM1 and MCM5).10?The expression of myc protein was detected by Western blot.Ckk-8,edu,Transwell migration and cell cycle were used to detect the growth,proliferation,migration and cycle changes of cells.Results:1.It is found that hsa-circ-000881 is significantly down regulated in LUAD tissue.2.The knockdown hsa-circ-000881 enhances the growth,migration and invasion of LUAD cells.3.The results of luciferase report gene analysis and bioinformatics analysis confirmed that hsa-circ-000881 has sponge adsorption on mir-665,while PRICKLE2 is the direct target of miR-665.4.Overexpression of mir-665 or silence of PRICKLE2 can eliminate the inhibition of tumor behavior in the LUAD cells mediated by has circ-000881.5.By analyzing TCGA-LUAD data,we found that PRICKLE2 was down regulated in the LUAD,which was significantly related to the bad survival of the patients with LUAD.The protein expression of PRICKLE2 were significantly down regulated in cancer tissues.The expression of PRICKLE2 was detected by IHC method by using the clinic specimen of lung adenocarcinoma and adjacent to the cancer.The results showed that PRC1 was significantly low in lung adenocarcinoma,6.The expression of PRICKLE2 was detected in lung adenocarcinoma cell lines(NCIH1395,NCIH1975,A549,PC9 and H1299).The results showed that the expression of pricle2 was low in two lung adenocarcinoma cell lines(NCIH1395 and NCIH1975).The expression of PRICKLE2 was over expressed in NCIH1395 and NCIH1975 cells by lentivirus infection,and the transfection efficiency was verified by Western blot.The results showed that the expression of PRICKLE2 was significantly increased.The cell proliferation and cycle test showed that the cell growth ability was significantly decreased after overexpression of PRICKLE2 by MTT and edu test in a54 and nci1975 cell lines;The overexpression of PRICKLE2 in cell cycle experiment resulted in the stagnation of G1 phase cell cycle in NCIH1395 and NCIH1975 cells.In addition,Western blot showed that cyclin D1 was down regulated,while p21 was up regulated when PRICKLE2 was overexpressed.7.The growth of subcutaneous tumor was observed 6 days later from the injection of stable PRICKLE2 overexpressed NCIH1395 cells after plasmid infection.By the 21st day,the volume of subcutaneous tumor in OE PRICKLE2 group was significantly reduced compared with that of the control group.The difference between the two groups increased with the time.The results of H-E staining,Ki67 staining and tunnel staining showed that the proliferation of lung cancer cells was inhibited and the apoptosis rate of the cells increased significantly after the overexpression of PRICKLE2.8.Transwell migration experiment showed that the cell migration ability was significantly reduced after overexpression of PRICKLE2.In addition,Western blot results showed that overexpression of PRICKLE2 led to the increase of E-cadherin protein level,but decreased the levels of N-cadherin and vimentin.9.The results of TCGA-LUAD GSEA data analysis showed that the expression of PRICKLE2 was negatively correlated with the expression of MYC.Western blot results showed that overexpression of PRICKLE2 resulted in the decrease of MYC,NOLC1,NPM1 and MCM5 protein levels.10.Ckk-8,edu,Transwell and cell cycle showed that the overexpression of Myc could eliminate the inhibition of PRICKLE2 on the proliferation,migration and invasion of LUAD cellsConclusion:1.hsa-circ-000881 was down regulated in LUAD tissues.2.hsa-circ-000881 can inhibit LUAD through miR-665/pricle2 axis,which indicates that has-circ-000881 may be a potential therapeutic target for LUAD.3.PRICKLE2 is down regulated in LUAD and it's low expression is closely related to the bad prognosis of LUAD.4.In vitro,the results showed that PRICKLE2 could inhibit the proliferation and migration of lung adenocarcinoma cells and affect the cell cycle.5.In vivo,PRICKLE2 can also inhibit the proliferation of lung adenocarcinoma cells.6.PRICKLE2 can inhibit the proliferation,migration and invision of LUAD cells by inhibiting myc pathway,and then inhibit the progression of LUAD.