| Lung cancer is featured with highest morbidity,aggressive tumor biology and younger onset age in China.The annual increase in incidence rate of lung cancer is 26.7%in 2017,and the cancer-related death is predicted to be one million by 2025[1].Lung cancer currently serves as the first leading cause of cancer-related death in China.Radiotherapy is the major local treatment for cancer.Currently,Stereotactic body radiation therapy(SBRT)markedly improves the local control as compared with traditional radiatherapy.SBRT even becomes a radical treatment for early stage lung cancer[2].However,the local and regional relapse rates are 10-25%after SBRT[3].Radioresistance is to blame,which impairs the therapeutic effect.It is urgent to explore the molecular mechanisms of radioresistance and find an effective sensitizer.Previous studies have shown that cancer radioresistance was not only associated with endogenous resistance of tumor cells,but also depended on tumor microenvironment(TME).Cancer-associated fibroblasts(CAFs),a critical regulator of resistant maintainance,are the most abandunt cells in TME,which exert a crucial role in cancer relapse[4].A greater understanding of the biology of CAFs is necessary to develop effective therapeutic strategies.Recent studies have shown that exosomes,secreted menbrane vesicles that range in size from 30-200 nm in diameter,mediate "cross-talk" between cells.Exosomes are stable in various body fluids and cellular mediums,which become a hot spot in research.Exosomes precisely modulate biological functions of receptor cells via delivery of exosomal-derived information materials.Studies showed that CAFs exposed to X ray significantly increased the release of exosomes[5],which are absorbed effectively via CD29/CD81 compounds on the surface of cancer cells[6].Studies on pancreatic cancer found that CAFs exposed to chemotherapy played an active role in regulating the survival and drug resistance of cancer cells via exosomal-derived chemoresistance-inducing factors[7].As far as we concern,no studies have examined the effects of exosomes derived from CAFs exposed to SBRT.Exosomes contain mRNA,DNA,proteins and are enriched with long non-coding RNA(lncRNA),which is transferred stably without being degraded by RNA enzyme[8].lncRNA is a class of RNA molecules without a protein coding region,and the length of its transcript is more than 200 bp.lncRNA is involved in multiple physiological and pathological processes,such as cancer progression and therapeutic resistance,via widespread modulation of genetic transcription and protein translation[9].Therefore,we speculated that exosomal-derived lncRNAs released by X ray-treated CAFs promoted cancer relapse.Objectives:To explore the possibility and potential molecular mechanism of exosomal-derived IncRNAs KCNQ1OT1 released by cancer-associated cells in radioresistance of lung cancer.Methods:1.Primary culture of CAFs was performed via immediate resected lung cancer tissues.Flow cytometry and immunofluorescence techniques were used to identify the expression of special markers of CAFs.Then,a co-culture system of CAFs and lung cancer cells A549 was established.Some experimental methods,including CKK-8 assay,PI assay,transwell asssay,and clone survival assay,were used to evaluate the influence of CAFs on proliferation,cell cycle,migration and invasion,and radiosensitiveness of co-cultured A549 cells,respectively2.Ultrahigh speed centrigation was used to isolate CAFs-derived exosomes Electronic microscopy and particle size analysis were performed to identify the morphology,average size and main peak diameter.CAFs-released exosomes were added into the mediums of A549,and the real-time uptake of exosomes by A549 was observed via PKH67 staining.Then four groups,including A549/A549 group,CAF-EXO/A549 group,CAFs(GW4869)/A549 group,and CAF-14Gy-EXO/A549 group,were set to evaluate the influence of exosomes on biological functions of A549.To explore the potential mechanisms of radioresistance,we identified the differential expression of lncRNAs in exosomes exposed to X ray by lncRNA assay.3.siRNA against IncRNA KCNQ10T1 was transfected into CAFs,and the expression of KCNQ1OT1 was examined by qPCR.Two groups,including CAFs-NC/A549 group and CAFs-KCNQ10T1-siRNA/A549 group,were set to evalute the role of KCNQ1OT1 in biological functions of co-cultured A549.Results:1.CAFs cells were isolated successfully via primary culture of lung cancer tissues,and identified with a typical expression of a-SMA(+),Vimentin(+),SDF-1(+),and CK-18(-).CAFs significantly promoted the proliferation in co-cultured A549 cells without and with treatment of 14 Gy irradiation(P<0.05).CAFs signifianctly enhanced the colonal formation after irradiation in co-cultured A549 cells,with an increased percentage of S phase cells.In addition,CAFs also increased the migration and invasion in-vitro.2.Exosomes were extracted from CAFs media via ultrahigh speed centrifuge,which showed as cup-shaped enveloped bodies ranging from 30nm-200nm in diameter as shown by transmission electron microscope.Particle size analysis showed the average diameter and the main peak diameter of particles were 135.7nm and 141.8nm,respectively.The percentage of particles with a diameter from 20nm-200nm was 72.3%.The surface markers of CD63 and CD81 were positively expressed by exosomes,while the expression rates were 59.1%and 51.7%,respectively.The exosome transferal analysis showed CAFs exosomes labeled with PKH67 were absorbed by A549 cells in a time-dependent manner,and the highest fluorescent intensity of uptake was at 240 min after incubation.Then,CCK-8 assay,clone survival assay,and cell cycle assay showed that treatment of CAFs with an inhibitor of exosome release,GW4869,significantly reduced the proliferation in co-cultured A549 cells(P<0.05),which also significantly enhanced radiation response with low colony formation abilities(P<0.05),and increased the percentage of A549 cells at G1 phase,respectively.Whereas,as compared with CAFs-exosomes,exosomes from irradiation-exposed CAFs significantly promoted the proliferation and radioresistance with a high percentage of co-cultured A549 cells at S phase(P<0.05).We demonstrated that the expression of lncRNAs in exosomes from irradiation-exposed CAFs was obviously diversified,with 9 molecules upregulated and 12 downregulated as compared to CAFs’ exosomes without irradiation.Moreover,the expression of lncRNA KCNQ10T1 increased more than 3 folds,which was also confirmed by qPCR assay(P<0.05)3.KCNQ1OT1-siRNA was transfected into CAFs via lipofectamine,which markedly downregulated the KCNQ1OT1 expression as shown by qPCR assay.Moreover,the decreased proliferation and enhanced radiosensitivity with low percentage of S phase A549 cells was observed in KCNQIOT1-siRNA-transfected CAFs(P<0.05).Conclusions:1.CAFs with typical markers is obtained successfully via primary culture of lung cancer tissues;2.CAFs enhance proliferation and radioresistance in co-cultured A549 cells,which might be associated with an increase of S phase due to G1/S checkpoint failure;3.CAFs promote the migration and invasion of co-cultured A549 cells;4.Ultrahigh speed centrifuge is a useful method to isolate typical exosomes from CAF media;5.CAFs exosomes absorbed by A549 cells are time-dependent,with the highest uptake speed at 240 min after incubation;6.GW4869,an inhibitor of exosome release,significantly abolished the enhancement of proliferation and radioresistance in co-cultured A549 cells,suggesting the important role of CAFs in radioresistance mediated by CAFs,exosomes,7.CAFs exposed to irradiation lead to further proliferation and radioresistance of co-cultured A549 cells,which may be associated with the upregulation of IncRNA KCNQ1OT1.8.The downregulation of lncRNA KCNQ10T1 in CAFs inhibites proliferation in co-cultured A549 cells,which also increased radiosensitivity and percentage G1 phase cells,suggesting exosomal lncRNA KCNQ1OT1 is a key modulator of radioresistance in A549 cells.In summary,our study explored the radioresistant mechanisms of lung cancer from tumor microenvironment,showing the mechanisms for the influence of radioresistance of lung cancer by lncRNA KCNQ10T1 in CAFs’ exosomes,which provides a novel strategy for the improvement in radiosensitivity via targeting CAFs’exosomes. |
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