The Mechanism Of CircPIK3R1 In Regulating Metastasis Of Non-small Cell Lung Cancer

Part 1 CircPIK3R1 inhibits metastasis in non-small cell lung cancerObjective:Metastasis of lung cancer is the main cause of poor prognosis of lung cancer,so it is very important to study the molecular mechanism of lung cancer metastasis to improve the prognosis of lung cancer patients.Non-small cell lung cancer(NSCLC)makes up the majority of lung cancers,Currently,the function of circRNA circPIK3Rl in NSCLC and its molecular mechanism have not been reported.This project first detected the characteristics of circPIK3R1 in cells,and then explored the effect of circPIK3R1 on the metastasis of NSCLC,and elaborated its mechanism.Methods:(1)The relative expression of circPIK3R1 in NSCLC tissues and cell lines was detected by qRT-PCR;(2)The structure of circPIK3R1 was analyzed,and the divergent and convergent primers of circPIK3R1 were designed for RT-PCR and agarose gel electrophoresis was performed.The product of CircPIK3R1 divergent primer was collected and send for Sanger sequencing.Total RNA was treated with RNase R,and the mRNA expression levels of circPIK3R1 and PIK3R1 were detected by qRT-PCR.After A549 cells were treated withα-amanitin(AMA),the mRNA expression levels of CircPIK3R1 and PIK3R1 were detected by qRT-PCR at different time points.Cell nucleus and cytoplasm were extracted from A549 and H460 cells,respectively,and the content of circPIK3R1 in cell nucleus and cytoplasm was detected by qRT-PCR.The localization of circPIK3R1 in cells was detected by FISH assay.(3)CircPIK3R1 was overexpressed in A549 and H460 cells,respectively,and cell migration and invasion were detected by Transwell assay.(4)The RIP assay was used to enrichment circPIK3R1 and detect miRNAs binding to circPIK3R1.The dual luciferase report assay was used to detect the binding possibility of miRNA to circPIK3R1.Western Blot result showed that circPIK3R1 inhibited the expression of EMT-associated proteins via the miR-301a/PTEN axis.(5)SiRNAs targeting circPIK3R1 were designed,and circPIK3R1 was knocked down in A549 and H460 cells respectively to detect the expression level of related proteins;After the overexpression of miR-301a or co-overexpression of circPIK3R1 in A549 and H460 cells.Western Blot and Transwell assay were used to detect whether circPIK3R1 could inhibit the oncogene function of miR-301a.(6)After overexpressed circPIK3R1 in A549 cells,nude mice were injected into the cells through tail vein.Six weeks later,lung tissues were taken out and H&E staining was performed on pathological sections.Count the number of tumor metastatic in the lungs.Results:(1)CircPIK3R1 was low expressed in NSCLC tissues and cell lines;(2)CircPIK3R1 consists of the first exon and part of the 5’-UTR of PIK3R1 mRNA,and circPIK3R1 can only be amplified in cDNA template,while there is no product amplified in genomic DNA(gDNA)due to PIK3R1 mRNA primer across the exons.Sanger sequencing results showed the presence of the predicted back splicing site of CircPIK3R1.After total RNA was treated with RNase R,circPIK3R1 was not reduced significantly,while the linear PIK3R1 mRNA was significantly reduced,indicating that circPIK3R1 was resistant to RNA enzyme degradation.The half-life of circRNAs was significantly longer than that of linear RNAs after treatment with α-amanitin(AMA).The localization of circPIK3R1 in NSCLC cells was detected by qRT-PCR and FISH assay,and the results showed that circPIK3R1 was mainly localized in the cytoplasm of NSCLC cells.(3)Overexpression of circPIK3R1 significantly inhibit cell migration and invasion;(4)RIP showed that circPIK3R1 could bind miR-301a;The dual luciferase report system results showed that miR-301a could bind to the predicted sequence sites of circPIK3R1.Western Blot result showed that circPIK3R1 inhibited EMT-related protein expression through miR-301a/PTEN axis.Knockdown circPIK3R1 significantly inhibited the expression of PTEN protein and promoted EMT-related proteins in NSCLC cells.miR-301a promoted EMT of NSCLC through the PTEN/AKT pathway,while circPIK3R1 inhibited the oncogenic effect of miR-301a.(5)The number of lung tumor metastases in the circPIK3R1 group was significantly less than that in the control group.Conclusion:CircPIK3R1 is localized in the cytoplasm of cells and is low expressed in NSCLC tissues and cells.Overexpression of circPIK3R1 inhibits the migration and invasion ability of NSCLC cells.Further studies showed that circPIK3R1 inhibits the expression of EMT-associated proteins through the miR-301a/PTEN axis,and thus plays a role in inhibiting NSCLC metastasis.Part 2 Exosomal circPIK3R1 inhibits metastasis of NSCLC cells by inhibiting M2 macrophage polarizationObjective:Tumor Microenvironment(TME)plays an important role in the progression of NSCLC,and tumor-associated macrophages(TAMs),as an important component of TME,promote Tumor metastasis through various mechanisms.This project will further study the relationship between circPIK3R1 and TAMs,and reveal the role of circPIK3R1 in TME.Methods:(1)The cells were treated with RNase A or Triton X-100 at the same time,and the expression of circPIK3R1 in cell medium was detected by qRT-PCR.Exosomes were extracted,observed by electron microscopy,the size of exosomes was analyzed by particle size,and the related markers of exosomes were detected by Western Blot.After overexpression of circPIK3R1 in NSCLC cells,the content of circPIK3R1 in exosomes was detected.(2)The exosomal circPIK3R1 treated macrophages,and the expression of circPIK3R1 in macrophages was detected by qRT-PCR.The uptake of exosomes by macrophages was observed by fluorescence tracer assay.QRT-PCR and Western Blot were used to detect the expression of marker proteins and mRNA in M2-type macrophages.(3)After the treatment of macrophages with the exosome CircPIK3R1,macrophage conditional medium(TCM)was collected.After the treatment of NSCLC cells with TCM,Transwell assay was performed to observe the metastasis ability of NSCLC cells,and Western Blot was used to detect NSCLC EMT-related proteins.MiR-301a overexpressed or circPIK3R1 co-overexpressed in macrophages(M0),and then TCM was collected to treat NSCLC cells for Transwell assay,and Western Blot was used to detect NSCLC EMT-related proteins.(4)CircPIK3R1 was knocked down in macrophages to detect the PTEN/AKT and STAT3 signaling pathways protein by Western Blot;P110γ was knocked down after knocked down circPIK3R1 in macrophages,and PTEN/AKT and STAT3 signaling pathway proteins was detected by Western Blot.CircPIK3R1 or mir-301a were transfected into macrophages,then macrophages were induced with IL4 and IL13,and the related proteins were detected by Western Blot.(5)The macrophages were treated with exosomal miR-301a or treated with exosomal circPIK3R1 simultaneously,and then the macrophages and A549 cells were injected through the tail vein of nude mice.After 6 weeks,lung tissues were taken out and H&E staining was performed on pathological sections.Count the number of tumor metastatic in the lungs.Results:(1)When the cells were treated with RNase A alone,the expression of circPIK3R1 in the cell medium did not change much.However,when the cells were treated with RNase A and Trion X-100,the expression of circPIK3R1 in the cell medium significantly decreased,indicating that the extracellular circPIK3R1 was mainly wrapped in membrane rather than released into the medium directly.Electron microscopy directly observed the extracted exosomes;Particle size analysis showed that the extracted exosomes were in the range of 30-70 nm,which was in line with the size range of exosomes.Western Blot detected the related markers of exosomes,indicating that the exosomes extracted in this study met the requirements.(2)The expression of circPIK3R1 increased significantly after macrophages were treated with exosome circPIK3R1,and the exosome uptake by macrophages was observed in fluorescence tracer assay.The mRNA levels of Arginase-1,CD206,CD 163 and IL-10 were increased and the protein expressions of Arginase-1 and CD206 were increased in the exosome miR-301a treatment group.However,the mRNA levels of Arginase-1,CD206,CD 163 and IL-10 in the treatment group with both exosome miR-301a and exosome circPIK3R1 decreased,and the protein expression levels of Arginase-1 and CD206 decreased.These results indicated that circPIK3R1 inhibited M2-type macrophages polarization.(3)After exosomal miR-301a promoted M2 macrophages polarization,M2 macrophages further promoted the migration and invasion ability of NSCLC,while exosomal circPIK3R1 inhibited M2 macrophages polarization and then inhibited the migration and invasion ability of NSCLC.MiR-301a promoted the promotion of NSCLC migration and invasion by promoting M2 macrophages polarization,while circPIK3R1 inhibited the promotion of NSCLC migration and invasion abilities by inhibiting M2 macrophages polarization.(4)The expression of circPIK3R1 was inhibited by siRNA in macrophages.Western Blot showed that p-AKT and p-STAT3 increased while M2 macrophage marker protein CD206 were also increased.In macrophages,circPIK3R1 was knocked down and p110γwas further knocked down.The results showed that p-AKT,p-STAT3 and CD206 were decreased,suggesting that circPIK3R1 inhibited M2 macrophages polarization through the PI3Kγ/AKT-STAT3 pathway.Overexpression of circPIK3R1 in macrophages inhibits the polarization of M2-type macrophages induced by IL4 and IL13.Knockdown of PTEN protein in macrophages also inhibits the polarization of M2-type macrophages induced by IL4 and IL13.These results indicate that circPIK3R1 inhibits the PI3Ky/AKT-STAT3 pathway through mir-301a/PTEN axis and thus inhibits the polarization of M2-type macrophages.(5)The number of tumor metastases in the exosomal miR-301 a group was significantly higher than that in the exosomal NC group,while the number of tumor metastases in the exosomal miR-301a and circPIK3R1 group was significantly lower than exosomal miR-301a group.Conclusions:CircPIK3R1 exists in exosomes and can be taken up by macrophages.CircPIK3R1 inhibits M2 macrophages polarization through the PI3Ky/AKT-STAT3 pathway,thereby inhibiting the role of M2 macrophages in promoting NSCLC cell metastasis.ConclusionsIn this study,the characteristics of circPIK3R1 in cells were firstly analyzed.Divergent primer PCR could detect circPIK3R1,and Sanger sequencing proved the existence of back splicing site.The half-life of circRNAs in cells is longer than that of linear RNAs because of their covalently closed circular structure,which is resistant to RNA enzyme degradation.Most of the circRNAs composed of exons are localized in the cytoplasm,as is circPIK3R1.The expression of circPIK3R1 in NSCLC tissues and cell lines was analyzed,and the expression of circPIK3R1 was significantly low,suggesting that circPIK3R1 may play a role as a tumor suppressor gene.After overexpression of circPIK3R1 in NSCLC cells,it was found that circPIK3R1 inhibited the migration and invasion ability of NSCLC cells,suggesting that circPIK3R1 plays a role as a tumor suppressor gene in NSCLC.CircPIK3R1 inhibits NSCLC metastasis by inhibiting the expression of EMT-associated proteins through the miR-301a/PTEN axis..On the other hand,we found that circPIK3R1 exists in exosomes,and it was found that exosomal circPIK3R1 inhibiting PI3Ky/AKT-STAT3 pathway through miR-301a/PTEN axis in macrophages,and preventing M2 macrophages polarization,thereby weakening the role of M2 macrophages in promoting NSCLC cell metastasis.In summary,the role and molecular mechanism of circPIK3R1 in NSCLC metastasis was reported for the first time in this project,and the research results will contribute to a deeper understanding of the mechanism of NSCLC tumor metastasis and provide a new reference for the treatment of NSCLC.