Effect Of CircPUM1 On The Development And Progression Of Lung Adenocarcinoma And Its Molecular Mechanism

Objective:Non–small cell lung cancer?NSCLC?accounts for about 80%of all lung cancer,and the major two types are lung adenocarcinoma?LUAD?and lung squamous cell carcinoma?LUSC?.LUAD is the most common histological type of lung cancer.Lung cancer is one of the most common cancers worldwide and is thus a global cancer burden.According to the cancer statics 2017,lung cancer is the second most common cancer and the first-leading cause of cancer-related deaths.Despite the rapid development in the diagnosis and treatment of lung cancer,the prognosis remains poor.This is because the molecular mechanisms for the development and progression of lung cancer remain unclear.Therefore,exploration of the molecular mechanisms underlying lung cancer offers new orientation to improve diagnosis and treatment.The development and progression of cancer depends on multiple genes.mRNA and its encoded proteins are known to play an important role in cancer.In recent years,non-coding RNAs?ncRNAs?gained wide attention.Non-coding RNAs refer to RNA that does not code proteins.More than 90%of human genes transcribe to RNA but don't code proteins.Non-coding RNA were classified into two classes,housekeeper ncRNAs and regulatory ncRNAs.MicroRNA?miRNA?which belongs to regulatory ncRNAs are capable of regulating gene expression.Through inhibiting tumor associtaed genes,miRNA play an important role in the development and progression of cancer.Long non-coding RNA?lncRNA?which also belong to regulatory ncRNAs were reported to have differential expression in tumor,and play an important role in tumor development and progression by interacting with miRNA.Circular RNAs?circRNAs?are a novel type of ncRNA which form a covalently closed loop.CircRNAs are usually abundant,stable,conserved in human and mouse,and often exhibit tissue/developmental-stage specific expression.With the development of technology,it is reported that some circRNAs have differential expression between tumor tissue and adjacent normal tissue,and further research demonstrated that circRNAs are involved in tumor development.Besides,circRNAs are promising biomarkers for the diagnosis and prognosis of cancer.CircRNAs have been reported to serve as miRNA sponges thus eliminating the inhibition on its target genes,and also act as RNA-binding protein?RBP?sequestering agents as well as transcriptional regulators.Acting as miRNA sponges to regulate gene expression may be the most important function of circRNAs as we know by now.Research on circRNAs in lung cancer is just beginning.Few circRNAs were reported to be involved in the development and progression of lung cancer.Cir-ITCH is down-regulated in lung cancer tissue and plays an important role in inhibiting lung cancer development.In addition,hsa_circ₀014130,hsa_circ₀000064,hsa_circ₀007385,hsa_circ₀013958 were reported to act as oncogenes to promote the development of lung cancer.A recent study showed that circPUM1 was upregulated in ovarian cancer and contributes to promote tumor progression.However,the function of circPUM1 in LUAD is not yet known.Therefore in this present study,we aimed to evaluate the expression of circPUM1 in lung adenocarcinoma tissue and cell lines,then determine the role of circPUM1 in lung adenocarcinoma cells proliferation,apotosis,migration and invasion.Besides,we also investigated the potential molecular mechanisms of circPUM1 in regulating the behaviors of lung adenocarcinoma cells.Methods:1.Tissue specimens'collection70 paired LUAD and adjacent non-tumor lung tissues from patients who underwent surgery were obtained at the First Affiliated Hospital of China Medical University.These patients were diagnosed with LUAD based on histopathological evaluation by at least 2 experienced pathologists.All fresh tissue specimens were stored at-80?C until total RNA extraction.RNA was extracted according to the manufacturer's instructions after adding 1ml trizol reagent.The concentration and purity of RNA were measured at260nm and 280nm.RNA was reverse transcripted into cDNA using pomega reverse transcription kit.The study protocol was approved by the ethics committee of the First Affiliated Hospital of China Medical University.2.Detection of circPUM1 expressiomQuantitative polymerase chain reaction?qPCR?was performed on Roche Lightcycler480 with SYBR Premix Ex Taq kit?Takara?to analyze circPUM1 expression level in lung cancer tissues and adjacent non-tumor lung tissues.Positive and negative controls were included in each reaction.18s rRNA served as internal control for normalization and data were analyzed using the comparative cycle threshold?CT?(2^-??CT)method.Primer sequences for circPUM1 are as follows:forward primers,5'-AGTGTACTGGGAGGAGG-3';reverse primers,5'-ATAAGTCCGTGCGTCC-3'The human lung adenocarcinoma cell lines A549,H1975,SPC-A1 and 16 HBE cells were inoculated on 6-well plate,then the next day RNA was extracted according to the instructions.The expression of circPUM1 in cell lines were detected with Takara real-time quantitative kit.3.Cell Culture and Cell transfectionSeveral NSCLC cell lines?A549,SPC-A1,and H1975?and the normal bronchial epithelial cell line,16HBE,were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences?Shanghai,China?.A549,SPC-A1,and H1975 cells were cultured in Roswell Park Memorial Institute?RPMI?-1640,16HBE and HEK-293 T cells were cultured in Dulbecco's modified Eagle's medium?DMEM?supplemented with 10%fetal bovine serum,100U/mL penicillin sodium,and100mg/mL streptomycin sulfate at 37°C in a humidified incubator with 5%CO₂.The cicrPUM1 lower-expressed lung cell line was stably transfected with pLCDH-circ₀000043 plasmid?Geneseed Biotech,China?,while cicrPUM1 higher-expressed lung cell line was transfected with short-hairpin RNA?sh-circPUM1,Hanbio Biotech,China?targeting the backsplice sequences to break up its circular structure.Empty vector was also transfected with lipofectamine 3000?Thermo Fisher,USA?to act as a control.Stable cell lines were selected applying puromycin.The transfection efficiencies were evaluated by qPCR.4.MTT assay3000 cells/well were seeded in 96-well culture plates.At given time points?0,24,48,and 72 h?,MTT was added to each well and incubated at 37°C for an additional 1-4 h.Subsequently,the media was replaced with dimethylsulfoxide?DMSO?to dissolve the formazan crystals.The absorbance at 490 nm was detected with Tecan infinite 200spectrophotometer.5.EdU incorporation assayThe EdU incorporation assay was performed with Click-iT?EdU Alexa Fluor?488Imaging Kit?Thermo Fisher,USA?.Cells were seeded in each well of 24-well plates.After incubation with 10 mM EdU for 6-8 h,the cells were fixed in 4%paraformaldehyde and stained according to the manufacturer's instructions.Hoechst33342 was used to stain the nuclus within the cells.Images were obtained and the percentage of EdU-positive cells was counted using Image J software.6.Flow cytometric analysisWhen the cells had grown to 70–80%confluence,they were harvested and washed with PBS.Subsequently,they were stained with annexin V-PE and 7AAD for 15 min in dark following the instructions of the manufacturer.Then cells were acquired by flow cytometry and analyzed.7.Cell migration assaysCells were cultured in six-well plates;linear wounds were created using a 200?l pipette tip,washed with PBS and cultured in FBS free medium.Cells were photographed after0h,24h and 48h and the wound area was measured using Image J software.Wound healing percentage was calculated as:?the area of the original wound-the area of the actual wound at different times?/the area of original wound×100%.8.Cell invasion assayTranswell chambers?BD Bioscience,USA?coated with matrigel on the upper surface were used.600?l F12 or RPMI-1640 with 10%FBS was added to the lower chamber while 200?l F12 or RPMI-1640 containing 5×10⁴ cells was resuspended in the upper chamber.After incubation for 48 h,cells invaded through the membrane were fixed and stained with 0.1%crystal violet.9.Western blotProtein was isolated from cell lysis using RIPA lysis buffer containing protease inhibitors.Equivalent amount of protein was subjected to 10%SDS-PAGE gel and then transferred onto polyvinylidene difluoride?PVDF?membranes.Then they were incubated with 3%BSA.After that specific antibodies and secondary HRP-goat anti-rabbit/mouse antibodies were incubated.Signals were detected using ECL detection reagent and visualized by Tanon Image Analysis System.10.Luciferase reporter assay HEK-293T cells?5×10³?were seeded into 96-well plates and were cotransfected with wild-type or mutated-type dual luciferase plamid?Hanbio Biotech,China?and the indicated miRNAs.After 48 h of incubation,the firefly and Renilla luciferase activities were quantified through dual-luciferase reporter system?Promega,Madison,WI,USA?.The relative luciferase signal was showed as firefly luciferase activity normalized to renilla luciferase activity.11.Tumor growth in nude miceFour-week-old female immunodeficient nude BALB/c mice were purchased from Charles River Laboratories and bred at the animal center of China Medical University.A549 cells transfected with sh-circPUM1 and sh-NC were harvested,washed and re-suspended in PBS.Subsequently,1×10⁷ cells?0.1 ml?were implanted subcutaneously into the mice.Tumor volume was measured every 3 days and calculated using the following formula:volume?mm³?=length×width²/2.The mice were executed 40days after injection.12.Statistical analysisAll data were analyzed using SPSS 20.0 software?IBM?.The Student's t test,and Chi square test were used to evaluate the significance of differences between two groups.P<0.05 was considered statistically significant.Results:1.Expression of circPUM1 is elevated in lung adenocarcinoma cancer tissues and cell linesWe first evaluated the expression of circPUM1 by qPCR analysis in a cohort of patients with lung adenocarcinoma cancer.Results showed that circPUM1 expression was up-regulated in lung adenocarcinoma cancer tissues compared with matched normal tissues.Further analysis showed that circPUM1 expression is associated with TNM stage.Also,we found that circPUM1 was up-regulated in lung adenocarcinoma cell lines compared to normal bronchial epithelial cell line.The cicrPUM1 higher-expressed lung cell line A549 was transfected with sh-RNA while cicrPUM1 lower-expressed lung cell line SPC-A1 was stably transfected with the overexpression plasmid.2.CircPUM1 promotes the proliferation of lung adenocarcinoma cells The MTT assay showed that the proliferation of A549 cells was declined in the sh-circPUM1 group than that in the sh-NC group?p<0.05?.It showed a significant increase in the proliferation ability?p<0.05?after overexpression of circPUM1 in SPC-A1 cells.Furthermore,EdU incorportion assay was performed to assess the effect of circPUM1on DNA replication.Results demonstrated that DNA synthesis of A549 cells was markedly impaired upon the knockdown of circPUM1?p<0.05?.The percentage of EdU positive cells increased significantly?p<0.05?after the overexpression of circPUM1 in SPC-A1 cells.Collectively,these findings suggest that circPUM1 promotes the proliferation of lung adenocarcinoma cells.3.CircPUM1 inhibits the apotosis of lung adenocarcinoma cellsWe further explored the effect of circPUM1 on cell apotosis.Flow cytometry analysis implied that silencing of circPUM1 obviously increased the apoptosis of A549 cells.In contrast,the apoptosis rate was decreased in SPC-A1 cells stably transfected with circPUM1.4.CircPUM1 promotes the migration and invasion of lung adenocarcinoma cellsWould healing percentage at 48h was significantly lower in the sh-circPUM1 group than in the sh-NC group in A549 cells.Similarly,transwell assays also exhibited that cells invaded to the lower chamber decreased significantly after silencing of circPUM1in the A549 cells.In contrast,opposite results were obtained in SPC-A1 cells stably transfected with circPUM1.5.Silencing of circPUM1 inhibits the tumorienesis in vivoNude mice xenograft model was employed to further validate the above findings in vivo.The nude mice were divided into two groups:sh-circPUM1 group and sh-NC group.It revealed that compared with the sh-NC group,tumorigenicity in the sh-circPUM1 mice were much slower,and given the same duration of observation,the tumor volumes were smaller?p<0.05?.6.CircPUM1 serves as a miRNA spongeCircular RNA Interactome website predicts that circPUM1 has a complementary sequence of miR-326,suggesting a possible interaction between circPUM1 and miR-326.Dual luciferase reporter gene assay showed that miR-326 significantly decreased the relative fluorescence activity of wild circPUM1 luciferase plasmid?p<0.05?,but did not change the relative fluorescence activity of mutant circPUM1 luciferase plasmid.These results suggest that circPUM1 can act as a miR-326 sponge.7.Detection of miR-326 associated proteinWestern blot results showed that circPUM1 overexpression significantly increased the protein expression levels of Cyclin D1?CCND1?and Bcl-2 compared with the control group.In contrast,the expression levels of CCND1 and Bcl-2 decreased after silencing of circPUM1.Conclusion:1.The expression of circPUM1 was higher in cancer tissue than that in adjacent normal tissue;The expression level was also related to TNM stage.2.In vitro functional results showed that circPUM1 can promote the proliferation,invasion and migration ability,and also inhibit the apoptosis of adenocarcinoma cells.3.In vivo experiment showed that silencing of circPUM1 significantly inhibited tumor formation in nude mice.4.CircPUM1 can act as miR-326 sponge,which regulates the expression of cyclin D1and Bcl-2,thus involved in the development and development of lung adenocarcinoma.