The Effect And Mechanism Of Exosomal Hsa＿circ＿0002557 On The Fibroblast Activation

Background and objectivesIt is important to find reliable biomarkers for esophageal cancer which causes a great loss to people’s life.With the rapid development of epigenetics,circular RNA（circRNA）has attracted widespread attention.As a potential molecular biomarker,circRNA plays an important regulatory role in the occurrence and development of tumors.The development of tumor is a complex process involving multiple factors,stages and components.In this process,tumour cells,surrounding cells with their essential components,together form the tumour microenvironment.It has been found that exosome-transmitted circRNAs are involved in intercellular communication and influence the development of other cells in the tumour microenvironment.This study focuses on one of the dominant cellular components of the tumour microenvironment,tumour-associated fibroblasts,and investigates the processes involved in the activation of esophageal fibroblasts into tumour-associated fibroblasts by exosome-transmitted circRNA,and how tumour-associated fibroblasts affect the tumour microenvironment and thus the development of esophageal cancer,providing a basis for the use of exosome-transmitted circRNA in esophageal cancer as an early tumour diagnostic biomarker.diagnostic biomarker for esophageal cancer.Methods1.The PCR product agarose gel electrophoresis and Sanger sequencing of the gel recovery product were used to verify the cyclicity of hsa_circ₀002557.RNA was extracted from 64 pairs of esophageal cancer tissues and tissues adjust to tumour,and the expression level of hsa_circ₀002557 was detected by qRT-PCR.t-test was used to analyze the difference of hsa_circ₀002557 expression in esophageal cancer tissues and tissues adjust to tumour.Logistic analysis was used to analyze the relationship between hsa_circ₀002557 expression and the risk of cancer.Chi-square test was used to verify the association between hsa_circ₀002557 expression and clinicopathological characteristic.2.The exosomes were extracted from the supernatant of esophageal cancer cells by differential centrifugation combined with ultracentrifugation.The purity of exosomes was identified by TEM,NTA and Western blot.The expression of hsa_circ₀002557 in EC109 and EC9706 and their exosomes were detected by qRT-PCR.3.FAM fluorescently labelled hsa_circ₀002557 was transfected in esophageal cancer cells,and the exosomes were extracted and co-cultured with fibroblasts.The delivery process of exosomes in the cells was observed under an Axio Image M2 fluorescence microscope.4.EC 109 and EC9706 were transfected with overexpression plasmids to construct high hsa_circ₀002557 models,and EC109 and EC9706 were transfected with siRNA to construct low hsa_circ₀002557 models.The supernatants of esophageal cancer cell cultures from four experimental groups were collected,including hsa_circ₀002557 high expression,hsa_circ₀002557 low expression,hsa_circ₀002557 high expression control and hsa_circ₀002557 low expression control.Then,the expression levels of hsa_circ₀002557 in the exosomes of the four experimental groups were assayed for the efficiency of exosome delivery of hsa_circ₀002557.5.The exosomes were added to the fibroblast culture system,and Western blot was applied to detect the expression levels of fibroblast activation marker proteins,including a-SMA,FAP and Vimentin in four groups.6.We constructed a three-dimensional cell culture model.Fibroblasts from four groups were seeded into the three-dimensional matrix and observed by two-photon microscopy.The arrangement of collagen fibers was observed by applying two-photon microscopy.The number and angle of collagen fibers were analyzed using CT-FIRE and the MATLAB compiler.7.The supernatant of the four groups of co-culture systems was collected.Then the supernatant was used to culture EC 109 and EC9706.We next use Transwell to observe the migration and invasion of these cells.8.Based on the base complementary pairing prediction of the miRNAs that hsa_circ₀002557 might bind,DLR gene plasmid containing the full-length sequence of hsa_circ₀002557 was constructed separately.Then the DLR plasmid was co-transfected with the miRNA mimic to screen out the bound miRNAs.Western blot was used to detect the effect of hsa_circ₀002557 binding miRNA on fibroblast activation markers and to screen out hsamiR-607 which is involved in the regulation of fibroblast activation.Hsa_circ₀002557 was further mutated on the DLR plasmid with the binding sequence of hsa-miR-607 to validate the relationship between hsa_circ₀002557 and hsa-miR-607.9.Screening for the target gene TGF-βⅡ of hsa-miR-607 based on base complementation analysis of the 3’UTR region.Binding of hsa-miR-607 and TGF-βⅡ was verified by DLR.The complementary sequence on the DLR plasmid was mutated to verify the binding site of hsamiR-607 and TGF-βⅡ.Hsa-miR-607 mimic was transfected in fibroblasts to observe the change of in TGF-βⅡ by qRT-PCR and ELISA.Exosomes of high hsa-_circ₀002557 were added to fibroblasts transfected with hsa-miR-607 mimic,and changes in TGF-βⅡ were detected by applying qRT-PCR and ELISA.10.Hsa-miR-607 inhibitor was co-transfected with TGF-βⅡ siRNA on fibroblasts.Then Western blot was applied to detect the effect of hsa-miR-607 on fibroblast activation markers via TGF-βⅡ.Finally,fibroblasts were treated with exosomes of high hsa_circ₀002557 and TGF-βⅡ siRNA.Western blot was applied to detect the effect of hsa_circ₀002557 on fibroblast activation markers via TGF-βⅡ.ResultsⅠ.Characteristics of hsa_circ₀002557 in esophageal cancer1.Variance analysis of hsa_circ₀002557 in esophageal cancer and paraneoplastic tissues（1）The loop structure of hsa_circ₀002557 was first identified by agarose gel electrophoresis of PCR products and Sanger sequencing.（2）qRT-PCR was performed on samples from 64 patients with esophageal cancer in Huai’an area.The expression level of hsa_circ₀002557 in esophageal cancer tissues was 2.58fold of that in tissues adjust to tumour.t-test showed hsa_circ₀002557 expression in tumour tissues was significantly higher than that in tissues adjust to tumour（P<0.001）,which proved that hsa_circ₀002557 was specifically highly expressed in esophageal cancer.Logistic analysis showed that the risk of tumour development for high hsa_circ₀002557 was 1.344fold of low hsa_circ₀002557（OR:1.344,95%CI:1.116-1.618）.The results suggested that high expression of hsa_circ₀002557 may increase the risk of esophageal cancer.（3）The relationship between the expression level of hsa_circ₀002557 and clinicopathological features was analyzed by chi-square test.The results showed that high expression hsa_circ₀002557 was associated with higher TNM stage of esophageal cancer（P<0.05）.The results suggested that high expression of hsa_circ₀002557 may affect the degree of primary tumour infiltration,regional lymph nodes and distant metastasis.2.Expression levels of hsa_circ₀002557 in esophageal cancer cells and exosomes（1）The exosomes were extracted and identified.Western blot showed positive expression of CD63 and negative expression of GM130 and Calnexin.TEM showed that the extract was a vesicular structure with a diameter of 100 nm.NTA particle size analysis showed the particle size distribution of the extracts was 108.0±8.6 nm.Therefore,the exosome extraction was successful.（2）qRT-PCR assayed the expression levels of hsa_circ₀002557 in esophageal epithelial cells（Het-1A）and esophageal cancer cells（EC109,EC9706）,and the exosomes of these cells.The results showed that the expression of hsa_circ₀002557 in EC109 was 4.11-fold of Het1A,the expression of hsa_circ₀002557 in EC9706 was 1.79-fold of Het-1 A.In the exosomes,the expression of hsa_circ₀002557 of EC109 was 43.78-fold of Het-1A,and the expression of hsa_circ₀002557 of EC9706 was 2.37-fold of Het-1A.t-test showed that all the above differences were statistically significant（P<0.05）.It is suggested that the exosomes secreted by esophageal cancer cells can actively take up hsa_circ₀002557.Ⅱ.Exosome-transmitted hsa_circ₀002557 of esophageal cancer contributes to fibroblast activation1.Intercellular transmission of exosome-transmitted hsa_circ₀002557（1）The uptake of FAM fluorescence-labelled exosomes by fibroblasts was observed using Axio Image M2 fluorescence microscopy.The result showed a gradual increase of fluorescence in fibroblasts with increasing co-culture time.It is suggested that the esophageal cancer-derived exosomes can shuttle hsa_circ₀002557 to be taken up by fibroblasts.（2）The expression levels of hsa_circ₀002557 in EC109 and EC9706 cells and exosomes after transfection with overexpression plasmid and empty vector were detected by qRT-PCR.The results showed that the expression of hsa_circ₀002557 in EC109 was 185.96 times higher than that in the control（P<0.01）,and the expression of hsa_circ₀002557 in EC9706 was 0002557 expression was 20.89-fold higher than control（P<0.01）,hsa_circ₀002557 expression in EC109 exosomes was 16.62-fold higher than control（P<0.001）,and hsa_circ₀002557 expression in EC9706 exosomes was 4.84-fold higher than control（P<0.01）.It was suggested that the hsa_circ₀002557 overexpression esophageal cancer cell model was successfully constructed and the esophageal cancer exosomes could carry a large amount of hsa_circ₀002557.（3）The interference effect of siRNA was screened by qRT-PCR.The results showed that hsa_circ₀002557 expression in EC109 cells transfected with siRNA 3 was 0.30-fold of control cells（P<0.001）,and hsa_circ₀002557 expression in EC9706 cells transfected with siRNA 3 was 0.59-fold of control cells（P<0.05）,hsa_circ₀002557 expression in EC109 exosomes was 0.11-fold of control（P<0.001）and hsa_circ₀002557 expression in EC9706 exosomes was 0.41-fold of control（P<0.05）.It is suggested that the hsa_circ₀002557 interference esophageal cancer cell model was successfully constructed,and hsa_circ₀002557 in esophageal cancer exosomes could be effectively interfered.（4）After extracting exosomes from four experimental groups of hsa_circ₀002557 high expression,hsa_circ₀002557 low expression,hsa_circ₀002557 high expression control and hsa_circ₀002557 low expression control,50 ng/mL of exosomes were added to co-culture with fibroblasts.The results showed that hsa_circ₀002557 expression was 5.92 times higher than that of control cells after adding exosomes from the overexpression group（P<0.001）,and 0.10 times higher than that of control cells after adding exosomes from the low expression group（P<0.05）.It is suggested that hsa_circ₀002557 is better delivered to fibroblasts by exosomes as a delivery medium,which directly affects the expression of hsa_circ₀002557 in fibroblasts.2.Exosome-transmitted hsa_circ₀002557 of esophageal cancer promotes fibroblast activation to tumor-associated fibroblastsA co-culture model was constructed by four experimental groups of exosomes（hsa_circ₀002557 high expression,hsa_circ₀002557 low expression,hsa_circ₀002557 high expression control,and hsa_circ₀002557 low expression control）.Western blot results showed that,after co-culture with exosomes at a concentration of 500 ng/ml,the protein grey values of FAP,α-SMA,Vimentin and FSP1 were significantly higher in the hsa_circ₀002557 high expression group than in the control group,and the protein grey values of FAP,α-SMA,Vimentin and FSP1 were significantly lower in the hsa_circ₀002557 low expression group than in the control group.It was suggested that exosome shuttle hsa_circ₀002557 could promote the activation of fibroblasts into tumor-associated fibroblasts.Ⅲ.The effect of exosome shuttle hsa_circ₀002557 on fibroblast activation on extracellular matrix remodeling and tumor metastasis1.The effect of exosome shuttling hsa_circ₀002557 on fibroblast activation on extracellular matrix remodelingTwo-photon microscopy was used to locate the position and morphology of fibroblasts and collagen fibers.A red fibroblast signal can be excited at 880 nm and a green collagen fiber signal at 1045 nm,while the characteristics of collagen fibers can be analyzed in conjunction with CT-FIRE.（1）In Hsa_circ₀002557High group,the collagen fibers were linearly radiating around the fibroblasts,the fiber arrangement was orderly,and the angle of collagen fibers relative to fibroblasts was 90.8°on average,approximately perpendicular to fibroblasts.While in the control group Over-NC,the collagen fiber morphology was curled,the fiber distribution was disordered,and the angle of collagen fibers relative to fibroblasts was 179.5° on average,approximately parallel to fibroblasts.It is suggested that the exosome shuttle hsa_circ₀002557 contributes to the activation of fibroblasts,which can change the morphology of secreted collagen fibers,order the fiber arrangement,and change the relative angle of collagen fibers to stromal cells.（2）The collagen fiber signal was weaker in the Hsa_circ₀002557Low group,and the average number of collagen fibers in a single field of view was 96.67;whereas the collagen fiber signal was stronger in the control Si-NC group,and a clear green fiber signal was visible the average number of collagen fibers in a single field of view was 983.t-test showed that the Hsa_circ₀002557Low group produced a significantly lower number of collagen fibers than the control Si-NC group（P<0.001）.It is suggested that the reduced level of exosome shuttle hsa_circ₀002557 can inhibit the secretion of collagen fibers and reduce the number of collagen fibers.2.The effect of exosome-transmitted hsa_circ₀002557 on fibroblast activation in esophageal cancer metastasis（1）The effect of exosome-transmitted hsa_circ₀002557 on fibroblast activation on esophageal cancer migrationAfter the fibroblasts were co-cultured with exosomes after three days,we collected the supernatants to culture EC 109 and EC9706 cells.The effect on the migration of esophageal cancer cells was examined by Transwell.It was found that both in EC109 and EC9706 cells,the number of migrated cells in hsa_circ₀002557High group was significantly increased by 50%and 81.36%compared with Over-NC group（P<0.001）.The number of migrated cells in hsa_circ₀002557Low group was significantly decreased by 37.43%and 40.80%compared with Si-NC group（P<0.001）.The results indicate that the activation of fibroblasts could improve the migration ability of esophageal cancer cells,while inhibiting the activation of fibroblasts did the opposite.（2）Effect of exosome-transmitted hsa_circ₀002557 on fibroblast activation on esophageal cancer invasionAfter the fibroblasts were co-cultured with exosomes after three days,we collected the supernatants to culture EC 109 and EC9706 cells.The effect on invasion of esophageal cancer cells was examined by Transwell.It was found that both in EC109 and EC9706 cells,the number of invading cells in hsa_circ₀002557High group was significantly increased by 88.60%and 180%compared with Over-NC group（P<0.001）.The number of invading cells in hsa_circ₀002557Low group was significantly decreased by 48.89%and 73%compared with Si-NC group（P<0.001）.The results indicate that the activation of fibroblasts could improve the invasive ability of esophageal cancer cells,while inhibiting the activation of fibroblasts did the opposite.Ⅳ.Analysis of the mechanism of fibroblast activation by the exosome-transmitted hsa_circ₀002557 in esophageal cancer1.Screening of exosome-transmitted hsa_circ₀002557-regulated miRNAs（1）Screening of hsa_circ₀002557 binding miRNAsDLR was applied to validate the possible binding miRNAs of hsa_circ₀002557,and it was found that among the 14 predicted miRNAs.The Fluc/Rluc of hsa_circ₀002557（WT）+hsa-miR-607 mimic and hsa_circ₀002557（WT）+hsa-miR-767-3p was significantly lower in both mimic groups than in the hsa_circ₀002557（WT）+mimic NC group,by about 55.5%（P<0.001）and 40.5%（P<0.01）.It suggested that hsa_circ₀002557 can bind to hsa-miR607 and hsa-miR-767-3p.（2）Hsa_circ₀002557 regulates fibroblast activation through miRNAWestern blot was applied to detect the effect of hsa_circ₀002557 binding to hsa-miR-607 and hsa-miR-767-3p on fibroblast activation.The results showed that overexpression of hsamiR-607 produced inhibition on fibroblast activation,the FAP,α-SMA,and Vimentin expression were significantly decreased.It is suggested that hsa_circ₀002557 regulates the activation of fibroblasts through hsa-miR-607.（3）Validation of hsa_circ₀002557 binding to hsa-miR-607 locusThe binding sequence AAUUUGAA on the mutant DLR plasmid to hsa-miR-607 was cotransfected with hsa-miR-607 mimic.The Fluc/Rluc values in hsa_circ₀002557（MUT）+hsa-miR-607 mimic group were higher than hsa_circ₀002557（MUT）+mimic NC group.There was no difference in Fluc/Rluc between hsa_circ₀002557（MUT）+hsa-miR-607 mimic group and hsa_circ₀002557（MUT）+mimic NC group.It indicated that hsa_circ₀002557 could no longer bind to hsa-miR-607 after mutating the complementary sequence.It suggested that hsa_circ₀002557 binds to hsa-miR-607 at the AAUUGAA site.2.Validation of hsa-miR-607 target genes（1）Dual luciferase reporter gene validation of Hsa-miR-607 binding to TGF-βⅡThe target gene of hsa-miR-607,TGF-βⅡ,was screened based on base complementation analysis of the 3’UTR region.DLR assays showed that the Fluc/Rluc values in the hsa-miR607 mimic+TGF-βⅡ（WT）group were significantly lower than those in the hsa-miR-NC+TGF-βⅡ（WT）group,with a reduction of 57.3%（P<0.001）.After mutating the UUUGAA sequence within the 3’UTR region of TGF-βⅡ to a complementary sequence and transfecting the hsa-miR-607 mimic,the Fluc/Rluc values in the hsa-miR-607 mimic+TGF-βⅡ（MUT）group were higher than those in the hsa-miR-607 mimic+TGF-βⅡ（WT）group.The Fluc/Rluc values in the hsa-miR-607 mimic+TGF-βⅡ（MUT）group had no significant change in values compared to the hsa-miR-NC+TGF-βⅡ（MUT）group.It suggests that TGF-βⅡ binds to hsamiR-607 via the UUUGAA site.（2）Hsa-miR-607 regulates the expression of TGF-βⅡThe qRT-PCR results showed that the TGF-βⅡ mRNA expression level in the hsa-miR607 mimic group was significantly lower than that in the miR-NC group,with a decrease of about 62.04%（P<0.001）.ELISA results showed that TGF-βⅡ protein levels were significantly downregulated in the hsa-miR-607 mimic group,with a decrease of 61.83%compared to the control group（P<0.001）.It suggests that hsa-miR-607 could regulate the expression of TGFβⅡ.（3）Hsa_circ₀002557 regulates TGF-βⅡ expression through hsa-miR-607The qRT-PCR results showed that TGF-βⅡ mRNA expression in the hsa_circ₀002557 overexpressing exosome group was significantly higher than that in the control Over-NC group,with an increase of about 2.52-fold（P<0.001）.After adding hsa-miR-607 mimic in the hsa_circ₀002557Highexosome group,TGF-βⅡ mRNA expression was significantly lower than that of the exosome group,with a reduction of 81.00%（P<0.001）.The ELISA results showed that the TGF-βⅡ protein level in the hsa_circ₀002557 overexpressing exosome group was significantly higher than that in the control Over-NC group,with an increase of about 44.96%（P<0.001）.After adding hsa-miR-607 mimic in the hsa_circ₀002557 overexpressing exosome group,TGF-βⅡ mRNA expression was significantly lower than that of the exosome group,with a reduction of 34.71%（P<0.001）.It suggested that hsa_circ₀002557 can regulate TGF-βⅡexpression through hsa-miR-607.3.Exosome-transmitted hsa_circ₀002557 promotes fibroblast activation via hsamiR-607/TGF-βⅡIn the first section of this chapter,we have verified that the exosome-transmitted hsa_circ₀002557 can regulate fibroblast activation through competitive binding of hsa-miR607.（1）Hsa-miR-607 regulates fibroblast activation via TGF-βⅡThe role of TGF-βⅡ in the regulation of fibroblast activation by hsa-miR-607 was analyzed by interfering with TGF-βⅡ expression in hsa-miR-607 low-expressing cells.Firstly,the siRNA interference efficiency was examined.qRT-PCR results showed that Si-3 could significantly down-regulate the mRNA level of TGF-βⅡ by 49.11%.ELISA results showed that Si-3 could significantly reduce the protein level of TGF-βⅡ by 47.62%.Next,the levels of fibroblast activation-related proteins were detected by Western bolt.The results showed that the protein levels of FAP,α-SMA and Vimentin in the hsa-miR-607 Inhibitor+si-NC group were significantly higher than those in the Inhibitor-NC+si-NC group,while the protein levels of FAP,α-SMA and Vimentin in the hsa-miR-607 Inhibitor+si-TGF-βⅡ group were significantly lower than those in the hsa-miR-607 Inhibitor+si-NC group.It suggested that hsa-miR-607 is involved in the regulation of fibroblast activation through TGF-βⅡ.（2）Hsa_circ₀002557 regulates fibroblast activation through TGFβⅡThe exosomes of high hsa_circ₀002557 were added to fibroblasts which has been interfered with TGF-βⅡ.The expression levels of fibroblast activation-related proteins FAP,aSMA and Vimentin were detected by Western bolt.The results showed that the protein grey values of FAP,α-SMA and Vimentin in the Hsa_circ₀002557High+si-TGF-βⅡ group were significantly lower than those in the Hsa_circ₀002557High+si-NC group.It suggested that hsa_circ₀002557 is involved in the regulation of fibroblast activation through TGF-βⅡ.Conclusion1.Hsa_circ₀002557 is highly expressed in the esophageal cancer tissue,and have association with tumorigenesis and TNM staging.Hsa_circ₀002557 can be actively taken up by esophageal cancer cell exosomes,possibly,and may regulate the progression of esophageal cancer by influencing the tumor microenvironment.2.The exosome-transmitted hsa_circ₀002557 of esophageal cancer can be taken up by fibroblasts and promote fibroblast activation into tumor-associated fibroblasts.3.The exosome-transmitted hsa_circ₀002557 of esophageal cancer promotes fibroblast activation,promotes the rearrangement of collagen fibers in the extracellular matrix of the tumor microenvironment and promotes tumor migration and invasion.4.The exosome-transmitted hsa_circ₀002557 of esophageal cancer can promote the activation of adult esophageal fibroblasts into tumor-associated fibroblasts via hsa-miR-607/TGF-βⅡ.