Study On The Clinical Significance And Molecular Mechanism Of Exosomal MicroRNA In Lymph Node Metastasis Of Esophageal Squamous Cell Carcinoma

BackgroundEsophageal cancer(EC)is one of the malignant tumors worldwide,which represent the sixth leading cause of cancer related mortality globally.Among the two main histological subtypes of EC,which include esophageal adenocarcinoma(EAC)and esophageal squamous cell carcinoma(ESCC),ESCC accounts for more than 80%of all ECs and is extremely widespread in East Asia,particularly in China.Because the esophagus is anatomically interspersed to the cardiopulmonary organ,ESCC,which has a lymph node(LN)metastasis,causes significantly worse outcomes than do other types of cancers.Consequently,LN metastasis is therefore recognized as being the most significant independent risk factor for ESCC prognosis,with overall survival(OS)rates decreasing from～70 to～18%when LN metastasis occurs.Moreover,appropriate treatment decision-making such as radiotherapy and chemotherapy for patients,surgery involving radical esophagectomy or less invasive endoscopic tumor resection,and determining the region of lymphadenectomy depends primarily on whether the tumor has undergone LN metastasis.Therefore,accurate detection of LN metastasis plays crucial roles in making treatment strategies and patient prognosis.Current LN metastasis detection methods fail from being a gold standard for various reasons.Image methods,such as computed tomography(CT),is often applied to predict LN status preoperatively;however,they also have been observed to be inaccurate in～40%cancer patients in view of they cannot detect micro-metastasis,which often result in false diagnosis and subsequent inadequate therapy.Although high-risk clinical and histopathologic characteristics,including lymphovascular invasion,high T stage,and poor differentiation,are often known as forecasters of LN metastasis,this information can only really be given after operation.Thus,clinicians urgently need novel non-invasive biomarkers that can improve LN metastasis detection for reaching more accurate decisions for optimal treatment and improvement in ESCC patients’prognosis.Exosomes are macrovesicles ranging from 30 to 150 nm in size released into the microenvironment by various cell types,especially in cancer progression.Exosomes contain proteins,microRNAs(miRNAs),and lipids;and their cargo often varies under various pathological conditions,being reflective of the physiological state of the originating host cells,which made exosomes act as one of the crucial projects in precision medicine and liquid biopsy.Hence,exosomes are a promising source of non-invasive biomarkers for diagnosis,prognosis,and recurrence monitoring of ESCC.MiRNAs are small non-coding RNAs(18-26 nt)that target 3’-untranslated regions(3’-UTRs)of mRNAs,leading to posttranscriptional regulation and mRNA destabilization.Moreover,a recent study also suggested that dysregulation of various miRNAs is closely related to tumor formation,progression,and metastasis.Compared with the unstable features of mRNA and long non-coding RNA(lncRNA),methylated modification of cell-free DNA,and low-abundant circulating RNA(circRNA),exosomal miRNAs are stable with reasonably advanced examination methods,allowing to be suitable predictive biomarkers for various illnesses,including cancer.For instance,miR-192,miR-25-3p,miR-17-5p,and miR-122 are enhanced in different tumor tissues and uniformly released into the medium through exosomes.While several experiments have proposed that circulating miRNAs are predictive metastasis biomarkers,relatively few have tried to establish a serum exosomal miRNA based model to predict LN metastasis preoperatively.Previous study demonstrated that cell released miRNAs can be transferred to the other cells in the body,and could communicated with other organ through the distance transport by systemic circulation,which speculated that miRNAs in the secrete exosomes may be mediated by a new way of communication between cells.Exosomal microRNA could regulated the receptor cell’s transcription level and stability of mRNA,and participate in the construction of ESCC microenvironment,which made them play key roles in the process of disease progression of ESCC.In this study,we performed a systematic and comprehensive profiling and analysis of the serum exosomal miRNAs associated with LN metastasis,and then we developed a novel exosomal miRNA model in clinical cohort.The serum exosomal miRNA-based model was subsequently combined with clinical characteristics for constructing a nomogram to predict LN metastasis before surgery.In addition,Lymphatic vessels play a crucial role in the development of tumors.Lymphatic vessels could modulate tumor-host immunity and act as protective niches for tumor stem cells for tumor recurrence and metastasis.The increased lymphatic density(LMVD)around the tumor or in the tumor tissues provides the tumor cells with expanded vectors to enter and spread through lymphatic vessels.Tumor cells underwent vascular requisition,chemotactic migration,adhesion invasion,and finally forming tumor thrombus in the lymphangion and then suffering distant metastasis.More important,lymph vessel space invasion(LVSI)are the key and first steps of lymph node metastasis.Therefore,we verified the predictive performance and clinical usefulness of the nomogram,followed by comprehensive validation in another independent clinical cohort.Besides,we report that an microRNA,termed miR-320b,was overexpressed in ESCC tissues and highly enriched in serum exosomes from patients with ESCC,and correlated positively with LN metastasis.exosomal miR-320b promoted lymphangiogenesis and LN metastasis in vitro and in vivo.Mechanistically,miR-320b was loaded to exosomes by direct interaction with PDCD4 to evoked lymphangiogenesis.Subsequently miR-320b in tumor cells enhance the proliferation,migration and invasion ability of ESCC cells.Our findings highlight a VEGF-C independent mechanism of exosomal miR-320b-mediated LN metastasis and identify miR-320b as a potential therapeutic target for LN metastasis in ESCC.Part 1:The expression level and clinical value of exosomal microRNA in preoperative LN metastasis prediction of esophageal squamous cell carcinomaObjectPreoperative prediction of lymph node(LN)metastasis is accepted as a crucial independent risk factor for treatment decision-making for ESCC patients.Our study aimed to establish a non-invasive nomogram to identify LN metastasis preoperatively in ESCC patients.MethodsA total of 366 patients and 70 healthy donors who underwent esophagectomy for the treatment of ESCC at the Department of General Surgery of Qilu Hospital and The Second Hospital of Shandong University between November 2016 and December 2019 were enrolled in this study.The morphology,size distribution and specificity analysis were performed using the TEM,ZetaView system western Blot assay.In the discovery phase,next-generation sequencing(NGS)assay was used in serum exosome samples collected from 10 patients with LN metastasis(LN+)and 10 patients without LN metastasis(LN-)to identify LN metastasis-related exosomal miRNAs.In the training phase,17 candidate miRNAs were first tested using qRT-PCR assay in 32 LN-and 32 LN+ESCC patients’serum exosome samples.Next,miRNAs with Ct values less than 35 were further examined in additional samples,including 63 LN-and 51 LN+patients.This combination cohort(cohort 1)was used in training phase to establish the model for LN status prediction.Receiver operating characteristic(ROC)curve and the area under the ROC curve(AUC)were used to evaluate the discriminative efficiency of eosomal miRNA-model for the prediction of LN metastasis.To further explore the predictive value,multivariate logistic regression analysis was employed to assess clinical characteristics which were substantially correlated with LN metastasis and incorporate them with the exosomal miRNA-model to establish a LN metastasis prediction nomogram using cohort 1.Subsequently,the performance of the comprehensive nomogram was assessed in cohort 1.In the validation phase,the coefficients of the nomogram from the training set were applied to another independent clinical cohort(cohort 2)consisting of 91 LN-and 97 LN+patients to validate the predictive performance of this clinical nomogram.In addition,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses and Gene Ontology(GO)terms were calculated using the "clusterProfiler" package in R project software.ResultsIn discovery stage,we performed genome-wide miRNA high-throughput sequencing-based miRNA expression profiling results for 20 ESCC patients(10 LN-and 10 LN+cases).Of the total 6959 annotated miRNAs,91 were differentially expressed(absolute log2 fold change>1,corresponding P<0.05,Wilcoxon signed-rank test).To reduce the miRNAs panel for a clinically viable and practical assay,we filtered out all miRNAs with low expression levels(average expression level<50),which resulted in a selection of 17 miRNAs.Four differently expressed miRNAs(chr 8-23234-3p,chr 1-17695-5p,chr 8-2743-5p and miR-432-5p)were screened in the clinical serum exosome samples from ESCC patients with or without LN metastasis using a backward step-wise elimination approach.Subsequently,constructed 4-miRNAs panel was trained in clinical cohort,which effectively distinguished ESCC patients with LN metastasis,and was significantly superior to preoperative computed tomography(CT)scans.Furthermore,a combination nomogram comprising of the 4-miRNAs panel together with CT-reported LN status was constructed in the training set,which performed well in both the training and validation sets(AUC:0.880 and 0.869,respectively).The calibration plot of our nomogram(Hosmer-Lemeshow test)showed the bias-corrected line lay close to the ideal curve(the 45-degree line),implying a good agreement to the model between prediction and observation in the training and validation set.Decision curve analysis demonstrated that if the threshold probability of a patient or a clinician is range from 20%to 80%,using the nomogram to predict LN status adds more benefit than the "treat-all" or "treat-none" scheme.It also showed higher net benefit than CT-reported LN status the clinical application of the nomogram.In addition,compared to CT-reported LN status criteria,our nomogram was successful in identifying true,high-risk,T1 stage ESCC patients with excellent accuracy in training(AUC=0.811)and validation(AUC=0.831)cohorts.Additionally,KEGG and GO analysis showed the top 20 involved signaling pathways,including MAPK and PI3K-Akt signaling pathway,suggesting these four exosomal miRNAs play crucial roles in metastasis and proliferation progression of ESCC.ConclusionIn conclusion,four differently expressed miRNAs(chr 8-23234-3p,chr 1-17695-5p,chr 8-2743-5p and miR-432-5p)were tested and selected in the serum exosome samples from ESCC patients who have or do not have LN metastasis.Subsequently,a clinical nomogram consisting of the 4-miRNA model and CT-report was established in training cohort,which showed high clinical predictive value in both training and validation cohorts.Our results demonstrated that a novel serum exosomal miRNA-based nomogram was developed for the identification of LN metastasis.Our predictive nomogram also has excellent clinical value in non-invasive discrimination of patients with or without LN metastasis,and may be conveniently used to improve overall patient treatment and outcomes in ESCC.Part 2: Exosome transmitted and intracellular miR-320 b promotes lymph node metastasis in esophageal squamous cell carcinomaObjectAs the major spreading route,lymphatic metastasis is an independent risk factor for clinical outcomes.Our study aim to investigate the underlying molecular mechanisms for cancer-secreted exosome miR-320 b and intracellular miR-320 b in regulating lymphangiogenesis and lymphatic metastasis of ESCC,as well as its clinical relevance,to explore the potential clinical applications in diagnosis and therapy.Methods1.Serum exosomal miRNA expression profiles of ESCC patients with 5 N1 stage group,5 N3 stage group and 10 LN-group were sequenced and analyzed using Next generation sequencing(NGS).miR-320 b was select after the overlap between these two comparisons.2.The expression level of exosome miR-320 b in the serum of366 patients with ESCC from two independent sample cohorts was detected by qRT-PCR and analyzed using Mann-Whitney test.3.The expression of miR-320 b in the exosome of each group was detected by qRT-PCR,and the established overexpressed and knockdown ESCC cell exosomes were collected.The morphology,size distribution and specificity analysis were performed using the TEM,ZetaView system western blot assay.Indicated exosomes was co-cultured with HLECs cells for 48 h,and the effects of exosomal miR-320 b on tube foimation and migration ability of HLECs were assessed by 3D culture and transwell assay.Western Blot assay was used to investigate the associated signaling pathway.After the establishment of mouse KYSE150 cell footpad model,exosome of each group was injected into tumor body by multi-point intratumoral injection.Representative bioluminescent images and IHC were used to investigate whether miR-320 b exosome could promote lymphangiogenesis and lymph node metastasis of ESCC tumor cells in vivo.4.After treatment of KYSE150 and EC9706 Cell lines with miR-320 b overexpression,knockdown and empty vector,respectively,the effects of miR-320 b on proliferation and EMT progression of ESCC cells were evaluated by CCK-8,colony formation assay5 EdU,western blot,transwell and wounding healing assay.After the mouse subcutaneous xenograft model established,exosomes of indicated groups were injected into the tumor body using intratumoral multi-point injection,respectively.Representative bioluminescent images and IHC were used to investigate whether miR-320 b could promote the growth of ESCC tumors in vivo.5,The mRNA and protein expression levels of downstream target gene PDCD4 of miR-320 b in each cell and each treatment group were detected by qRTPCR,western blot and IHC respectively.Meanwhile,miR-320b-PDCD4 related rescue experiments and phosphorylation detection of AKT signaling pathway were applied to identify the specific molecular mechanism and related signaling pathways of miR-320 b promoting ESCC lymph node metastasis.The expression levels of pri-miR-320 b,premiR-320 b,and mature miR-320 b in KYSE150 cells after METTL3 overexpression or knockdown were assessed by qRT-PCR assay.Co-DP assay demonstrated the interaction between DGCR8 and METTL3.RIP assay and MeRIP assay were used to investigate the expression level ofDGCR8-bound pri-miR-320 b in KYSE150 cells with METTL3 overexpression or knockdown,as well as the effects of METTL3 on m6 A methylation of pri-miR-320 b.Results1.The different expressed miRNA between LN-and LN+ groups were overlapped with the miRNA differences in the N1 and N3 groups.After verification by the training group,miR-320 b was identified to play an important role in ESCC lymph node metastasis.2.Two independent sample cohorts were used to investigated the expression level of miR-320 b in serum exosorae.Data shows that exosomal miR-320 b expression level of LN+ patients was significantly higher than that of LN-patients.Further analysis revealed that the expression level of serum exosomal miR-320 b in serum samples from N2-3 ESCC patients was significantly higher than that from N1 patients.3.The results of exosome identification experiment indicate that miR-320 b secreted by ESCC cells is not directly released into the external environment,but is protected by a lipid bilayers structure.KYSE150 cells with miR-320 b high expression could efficiently loaded miR-320 b into exosome and secrete exosome miR-320 b into cell culture medium.The expression level of miR-320 b in HLECs cells was markedly upregulated after 48 h co?culture with miR-320 b loaded exosomes.4.In vitro,the exosome with high miR-320 b could promote the lymphangiogenesis and migration ability of HLECs cells.Exosomal miR-320 b has been shown to promote the formation of lymphatic vessels and increase in the density of lymphatic vessel in in-situ tumor tissues.Moreover,exosomal miR-320 b could significant promotes lymph node metastasis of KYSE150 cells in vivo.Western Blot and IHC results showed that the expression of PDCD4 was decreased with exosome miR-320 b treatment in HLECs cells.Moreover,phosphorylation of AKT was significantly increased in exosomes with high miR-320 b groups rather than those with low miR-320 b groups.5,The mechanism experiment of miR-320 b in ESCC cells showed that miR-320 b could significantly enhance the proliferation,migration and invasion ability of ESCC cells as well as the EMT progression.In vivo,exosome miR-320 b could promote the growth of subcutaneous tumor.6.In addition,miR-320 b in ESCC tumor cells inhibits the expression of PDCD4 in ESCC cells by specifically binding to the 3-UTR region of PDCD4 mRNA,thereby promoting the activation of AKT pathways.7.qRT-PCR results showed that overexpression of METTL3 promoted miR-320 b processing,including decreased the expression level ofpri-miR-320 b in cells, and increased the expression levels of pre-miR-320 b and mature miR-320 b.Conversely,METTL3 knockdown will inhibit miR-320 b processing.Co-IP experiments revealed that METTL3 coprecipitates with DGCR8 and ribonuclease treatment weakens their interaction.Consistently,we observed a significant increase in methylated RNA bound by DGCR8 in METTL3-overexpressing cells.RIP and MeRIP experiments showed that METTL3 overexpression significantly increased the binding of pri-miR-320 b to DGCR8 and the amount of pri-miR-320 b modified by m6 A.ConclusionIn summary,the expression level of serum exosomal miR-320 b was significantly increased in the LN+ESCC groups,and was positively correlated with the number of lymph node metastasis.Exosomal miR-320 b transmitted into HLECs and regulate PDCD4-AKT pathways in receptor cells,which promoted lymphangiogenesis ofESCC.Upregulation of miR-320 b in ESCC cells could enhance the proliferation and EMT progression of ￡SCC cells,thus playing a synergistic role in promoting lymph node metastasis of ESCC tumor cells with AKT pathway activation.We have identified a vicious axis of METTL3/m6A-miR-320b-PDCD4-AKT in ESCC lymph node metastasis,which might be implicated in the regulation of tumor microenvironment and EMT progression.