Research On The Mechanism Of Colorectal Cancer Development Regulated By Epstein-barr Virus-encoded Mirna

Background and Purpose:Colorectal cancer（CRC）is a major cause of cancer-related mortality globally,which resulted in 1,096,601 incident cases and 551,269 deaths in 2018.The risk factors of CRC include obesity,high-fat diets,as well as infection of other diseases such as viruses.In recent years,some researchers focused on the potential role of viral infections in CRC carcinogenesis,such as human papillomaviruses（HPV）,human polyomaviruses and human herpesviruses.Their studies suggested a potential role of virus infection in CRC development.However,the underlying molecular mechanism remained obscure.Epstein-Barr virus（EBV）is one well-recognized oncogenic virus,it infects over 90%of the world’s population and is the first virus identified as a carcinogen by the International Agency for Research on Cancer（IARC）.In addition to causing epithelial herpes,it is also correlated with various cancers,such as Burkitt’s lymphoma,nasopharyngeal carcinoma（NPC）,gastric cancer（GC）,and the development of most EBV-related cancers is associated with EBV infection in the latency stage.Whether the EBV infection is in the latency stage depends on the differential expression of the surface protein in the infected cell membrane.Furthermore,the virus remains its transcriptional activity even in latent infection stage.Bam HI-A rightward transcripts（BARTs）and EBV-encoded RNAs（EBERs）are the most widely expressed genes in its latent infection stage.Intensive studies have focused on EBERs such as EBNA,which encoded and expressed latent membrane proteins including LMP1,LMP2A,and LMP2B.However,the pathogenesis and progression of EBV-associated tumors are not only regulated by virus-encoded proteins,EBV-encoded miRNAs（EBV-miRs）also play an indispensable role in this process.MicroRNA（miRNA）is a series of non-coding single-brand RNA comprising 17-23 nucleotides.A total of 25 EBV-miR precursors which can produce 48 mature miRNAs have been found.Most of the mature miRNAs are located in the BART region,which contains 22 miRNA precursors,including EBV-miR-BART1-22,and produces 44 mature miRNAs.Several studies have reported that EBV-miR-BARTs were highly expressed in GC and NPC,and facilitated tumor progression through their role in viral latency and immune escape.However,there is a scarcity of relevant research in CRC.Our subject was focused on EBV-miR-BART18-3p,which is encoded by the Epstein-Barr virus,and explore its potential molecular mechanisms regulating the CRC development.Methods:1 Construction and analysis of miRNA differential expression profiles from CRC patients1.1 The CRC tumor tissues and corresponding normal adjacent tissues from 6 CRC patients（had not been treated with radiotherapy and chemotherapy before surgery）,6 cases of colorectal adenoma tumor tissues were used to miRNA microarray,and EBV-miR-BART18-3p was significantly up-regulated in CRC tumor tissues compared with corresponding normal adjacent tissues or colorectal adenoma tumor tissues and selected for further study.Moreover,403 pairs of CRC tumor tissues and corresponding normal adjacent tissues from CRC patients diagnosed by pathological diagnosis were collected and used for RT-qPCR assays to verify the EBV-miR-BART18-3p expression levels in tissues,and the receiver operating characteristic curve（ROC）was used to calculate the area under the curve（AUC）to determine the potential efficacy of tumoral EBV-miR-BART18-3p expression for CRC auxiliary diagnosis.1.2 The CRC tissue microarray and in situ hybridization technology were used to verify the EBV-miR-BART18-3p expression levels in 1,078 cases of colorectal cancer patients with 10-year follow-up information.Multivariate Cox regression analysis was used to assess the potential risk factors（gender,age,tumor location,tumor grade,TNM stage,and EBV-miR-BART18-3p expression levels）that affect the prognosis and survival of CRC patients.Survival analysis was used to study whether the EBV-miR-BART18-3p expression levels can affect the survival time of CRC patients and the relationship between the EBV-miR-BART18-3p expression and CRC development.2 The effects of EBV-miR-BART18-3p on the CRC development2.1 9 CRC cell lines（CT26,DLD-1,HCT-15,HCT116,HT-29,Lo Vo,RKO,SW480,SW620）and human normal intestinal epithelial cell line NCM460 were analyzed by RT-qPCR to detect the EBV-miR-BART18-3p expression levels.After that,RT-qPCR,in situ hybridization and fluorescence in situ hybridization were used to detect the effects of hypoxia on the EBV-miR-BART18-3p expression levels and its subcellular localization after hypoxia treatment in CRC cells.2.2 Constructing EBV-miR-BART18-3p KD SW480,EBV-miR-BART18-3p KD RKO,EBV-miR-BART18-3p OEX DLD-1 and EBV-miR-BART18-3p OEX SW620 cell lines.Transwell assays were used to detect the invasion and migration ability of CRC cell lines.In addition,nude mouse tumor-bearing models constructed by CRC cells were used to study the effects of EBV-miR-BART18-3p expression levels on CRC tumor growth.Simultaneously,the fluorescence intensity of tumor tissue,liver and lung homogenate from nude mouse tumor-bearing models was measured by a microplate reader to study the effects of EBV-miR-BART18-3p expression on the development and tumor metastasis ability of CRC.3 The regulatory mechanism of EBV-miR-BART18-3p on the CRC development3.1 Constructing the WGCNA regulatory network for differentially expressed miRNAs and m RNAs from CRC patients with or without exogenous miRNAs.KEGG enrichment analyses of module genes were performed to identify key downstream molecules and key pathways（LDHA involved in HIF-1αpathway）that regulated the CRC development by EBV-miR-BART18-3p.At the same time,the CRC tissue microarray mentioned above and IHC were used to verify the LDHA expression levels in CRC patients.Multivariate Cox regression analysis was used to assess the potential risk factors（gender,age,tumor location,tumor grade,TNM stage,and LDHA expression levels）that affected the prognosis and survival of CRC patients.Survival analysis was used to study whether the LDHA expression levels could affect the survival time of CRC patients and the relationship between LDHA expression and CRC development.3.2 The m RNA,protein expression levels of key molecules（LDHA,HIF-1α）and the regulatory relationship among EBV-miR-BART18-3p and LDHA,HIF-1αin CRC cells were detected by RT-qPCR and Western Blot.Bioinformatics analysis was used to predict the potential downstream target genes of EBV-miR-BART18-3p,constructing binding site mutant luciferase reporter gene plasmid and luciferase reporter assays were used to verify its binding and regulatory relationship.The regulatory relationship between EBV-miR-BART18-3p,target gene（SIRT1）,and downstream key molecules HIF-1αand LDHA was verified by RT-qPCR,Western Blot,Ch IP assays.Lactate Assay Kit was used to detect the intracellular and extracellular lactate contents,¹³C isotope tracer assays measured by liquid phase tandem mass spectrometer were used to study the effects of EBV-miR-BART18-3p expression on the metabolism in CRC cells.3.3 LDHA NC and KD SW480 cells were analyzed by RNA sequencing,and KEGG pathways enrichment analysis was used to discover the downstream genes of EBV-miR-BART18-3p/SIRT1/HIF-1α/LDHA in CRC development.RT-qPCR and Western Blot were used to detect the expression levels of downstream genes（FASN）and the regulatory relationship among EBV-miR-BART18-3p,LDHA and FASN in CRC cells.The CRC tissue microarray mentioned above and IHC were used to verify the FASN expression levels in CRC patients.Multivariate Cox regression analysis was used to assess the potential risk factors（gender,age,tumor location,tumor grade,TNM stage,and FASN expression levels）that affected the prognosis and survival of CRC patients.Survival analysis was used to study whether FASN expression levels can affect the survival time of CRC patients and the relationship between FASN expression and CRC development.The effects of EBV-miR-BART18-3p on the FASN expression were studied by DNA Pull-Down and further Western Blot assays.The contents of lipids and triglycerides in CRC cells were measured to study the relationship among the EBV-miR-BART18-3p,FASN expression levels and de novo lipogenesis.4 Preliminary exploration of EBV-miR-BART18-3p in CRC auxiliary diagnosis4.1 Using the constructed Patient-Derived tumor Xenograft（PDX）models to establish PDX Antagomir-EBV-miR-BART18-3p mice models,and the relationship among the EBV-miR-BART18-3p expression levels,tumor growth and mouse survival time was analyzed.The effects of EBV-miR-BART18-3p expression on the pathological structures of tumor tissues were analyzed by HE staining.And the expression levels of EBV-miR-BART18-3p,LDHA,FASN,tumor malignant phenotypic marker（Ki67）were analyzed by ISH or IHC staining.At the same time,the lipid and triglyceride contents in single-cell suspensions of PDX tumor tissues were measured to analyze the biological function of EBV-miR-BART18-3p in CRC.4.2 The EB virus ELISA kits were used to detect whether there is a difference in the EB virus infection rate and EB virus titer between CRC patients and healthy donors.The number of cases was determined by the results of the Matched Case-Control Power Analysis.Meanwhile,the total RNA in the serum of CRC patients was extracted,and the serumal EBV-miR-BART18-3p expression levels in CRC patients was detected by RT-qPCR.The serumal EBV-miR-BART18-3p expression levels in CRC patients with different TNM stages were studied by stratified analysis.The area under the curve（AUC）calculated by receiver operating characteristic curve（ROC）was used to determine the potential diagnostic value of serumal EBV-miR-BART18-3p expression in CRC.Results:1 miRNA differential expression profiles analysis of CRC patients1.1 Bioinformatics analysis of miRNA expression in CRC patientsmiRNA microarray data indicated that the EBV-miR-BART18-3p expression levels in CRC tissues was significantly increased compared with adenoma and adjacent normal tissues,and the results of clinical sample verification also confirmed this result（P<0.001）.ROC curve showed that the AUC values of EBV-miR-BART18-3p for CRC were 0.8036,0.8208 and0.8066 in the Testing,Validation,and Combined set,respectively（P<0.0001）.The results indicated that the tumoral EBV-miR-BART18-3p expression levels might be used for the CRC auxiliary diagnosis.1.2 The relationship between EBV-miR-BART18-3p expression levels and the risk of CRCThe TMA samples staining by in situ hybridization showed that the EBV-miR-BART18-3p expression levels in CRC tissues were significantly higher than that in adjacent normal tissues from the Testing,Validation and Combined set.The EBV-miR-BART18-3p expression levels in CRC were related to the tumor grade,lymph node metastasis,distant metastasis and TNM stages（P<0.05）;Over 55 years old,colon tumors,TNM stage III/IV,and high EBV-miR-BART18-3p expression levels were risk factors that affect the survival time of CRC patients,and the survival time of patients was reduced（P<0.05）;The survival time of CRC patients with high EBV-miR-BART18-3p expression was significantly reduced（P<0.0001）.And EBV-miR-BART18-3p expression levels in patients’tumor tissues were significantly higher than that in adjacent tissues at each stage of TNM（P<0.001）.Those results suggested that the high expression levels of EBV-miR-BART18-3p were related to CRC development.2 The function of EBV-miR-BART18-3p on the CRC development2.1 Expression and function of EBV-miR-BART18-3p in CRC cellsAccording to the results of EBV-miR-BART18-3p expression levels in 9 common CRC cell lines（CT26,DLD-1,HCT-15,HCT116,HT-29,Lo Vo,RKO,SW480,SW620）and human normal intestinal epithelial cell line NCM460 by RT-qPCR,we selected the two cell lines SW480 and RKO with the highest EBV-miR-BART18-3p expression,and the two cell lines DLD-1 and SW620 with the lowest EBV-miR-BART18-3p expression for follow-up experiments.Hypoxia is an important feature of the tumor microenvironment.We performed hypoxia treatment on the 4 CRC cells above.The results of miRNA in situ hybridization,fluorescence in situ hybridization and RT-qPCR showed that hypoxia treatment significantly increased the EBV-miR-BART18-3p expression levels（P<0.01）,and EBV-miR-BART18-3p expression could be detected both in cytoplasm and nucleus,and principally in the cytoplasm.And hypoxia treatment could increase EBV-miR-BART18-3p expression levels in the cytoplasm and nucleus.2.2 The relationship between EBV-miR-BART18-3p and the CRC developmentThe transwell results showed that the expression level of EBV-miR-BART18-3p was positively correlated with the invasion and migration ability of CRC cells（P<0.05）.In the nude mouse tumor-bearing model,the highly expressed EBV-miR-BART18-3p could promote the growth of CRC tumors and the occurrence of liver and lung metastases（P<0.05）.3 The regulatory mechanism of EBV-miR-BART18-3p on the CRC development3.1 Constructing the WGCNA network to recognize the key molecule in the process of CRC development regulated by EBV-miR-BART18-3pThe sample connectivity threshold was set as-2.5,and the soft thresholdβ=22 in WGCNA network construction of differentially expressed miRNAs and m RNAs containing exogenous miRNAs,and 10 gene modules were identified.Correlation analysis results showed that Turquoise,Yellow and Black modules had the highest correlation with CRC（Turquoise modules:cor=0.99,P<1E-200;Yellow modules:cor=0.98,P<1E-200;Black modules:cor=0.98,P<8.3E-182）,and genes in Turquoise and Black modules were positively correlated with CRC,genes in Yellow modules were negatively correlated with CRC.And only 4exogenous miRNAs were included in all valid modules:EBV-miR-BART18-3p and EBV-miR-BHRF1-3 included in Black module（ME correlation coefficient=0.7,P=0.001）;hcmv-miR-US25-2-5p and hcmv-miR-US5-2-3p included in Red modules（ME correlation coefficient=-0.17,P=0.5）.After removing the exogenous miRNA from the above miRNA and m RNA data,we reconstructed the WGCNA network,the sample connectivity threshold was set as-2.5,and the soft thresholdβ=23.Finally,39 gene modules were identified.The results of the module analysis showed that 52.5%of the genes in the Black module which contained EBV-miR-BART18-3p in the previous WGCNA analysis including exogenous miRNAs were transferred to the Turquoise module which is less relevant to CRC（ME correlation coefficient=0.16,P=0.5）.Therefore,the genes in the Black module which included EBV-miR-BART18-3p may be related to the CRC development.KEGG gene enrichment analysis of genes in the Black module showed that LDHA participates in multiple pathways of Top 10 KEGG pathways,such as hypoxia-inducible factor pathway,HIF-1αtranscription factor regulatory network,lactate fermentation-reoxidation of cytosolic NADH.Therefore,LDHA and HIF-1α-related pathways may be involved in the regulatory process of CRC development regulated by EBV-miR-BART18-3p.The TMA samples staining by immunohistochemistry showed that the LDHA expression levels in CRC tissues were significantly higher than that in adjacent normal tissues from the Testing,Validation and Combined set.The LDHA expression levels in CRC were related to the tumor grade,lymph node metastasis,distant metastasis and TNM stages（P<0.05）;Over 55 years old,men,colon tumors,TNM stage III/IV,and high expression levels of LDHA were risk factors that affect the survival time of CRC patients,and the survival time of patients was reduced（P<0.05）;The survival time of CRC patients with high LDHA expression was significantly reduced（P<0.0001）.Those results suggested that the high expression levels of LDHA were related to CRC development.3.2 Effects of EBV-miR-BART18-3p on LDHA-mediated metabolism pathway in the CRC developmentWestern Blot results showed that the EBV-miR-BART18-3p expression in hypoxic CRC cells could positively regulate the LDHA and HIF-1αexpression levels（P<0.05）.RT-qPCR results showed that the EBV-miR-BART18-3p expression in hypoxic CRC cells could positively regulate the LDHA expression levels（P<0.05）,but HIF-1αexpression levels have no statistical significance.Luciferase reporter assays showed that only SIRT1 could be negatively regulated by EBV-miR-BART18-3p in several potential target genes.The Ch IP assays verified the regulatory relationship among EBV-miR-BART18-3p,SIRT1,HIF-1αand LDHA.SIRT1expression levels were decreased by knockdown EBV-miR-BART18-3p expression,which could reduce the inhibition effects of SIRT1 to HIF-1αand subsequent LDHA expression,and SIRT1 overexpression can rescue this regulation process.The intracellular and extracellular lactate contents were significantly decreased in EBV-miR-BART18-3p and LDHA KD CRC cells（in intracellular,P<0.01 between EBV-miR-BART18-3p NC and KD,LDHA NC and KD groups;in extracellular,P<0.05 between EBV-miR-BART18-3p NC and KD groups,P<0.001 between LDHA NC and KD groups）.And ¹³C isotope tracer assays showed that the EBV-miR-BART18-3p expression could regulate the metabolism process,promote acetyl-Co A production.3.3 The effects of EBV-miR-BART18-3p on FASN-mediated de novo lipogenesis in the CRC developmentTranscriptome sequencing analysis of hypoxic LDHA NC and KD SW480 cells identified many target genes regulated by LDHA(Fold change>1.5,P_adj<0.05),5 of them were negatively correlated with LDHA expression levels,and 21 genes were positively correlated with LDHA expression levels.KEGG pathways enrichment analysis showed that FASN participated in multiple pathways of the enriched Top 10 pathways results,including AMPK signaling pathway,insulin signaling pathway,fatty acids biosynthesis and fatty acid metabolism,suggesting that FASN may be involved in the downstream of EBV-miR-BART18-3p/SIRT1/HIF-1α/LDHA to regulate the CRC development.The results of RT-qPCR and Western Blotting showed that the FASN expression was positively correlated with the EBV-miR-BART18-3p and LDHA expression at RNA levels and protein levels（P<0.05）.The TMA samples staining by immunohistochemistry showed that the FASN expression levels in CRC tissues were significantly higher than that in adjacent normal tissues from the Testing,Validation and Combined set.The FASN expression levels in CRC were related to lymph node metastasis,distant metastasis and TNM stages（P<0.05）;Over 55 years old,colon tumors,TNM stage III/IV,and high FASN expression levels were risk factors that affect the survival time of CRC patients,and the survival time of patients was reduced（P<0.05）;The survival time of CRC patients with high FASN expression was significantly reduced（P<0.0001）.Those results suggested that the high expression levels of FASN were related to CRC development.DNA Pull-Down and Western Blot showed that EBV-miR-BART18-3p expression could significantly increase the acetylation levels of histones H3K9,H3K14,and H3K27 in FASN promoter region,thereby increasing the FASN expression levels.Further measurement of the lipids and triglycerides contents in CRC cells revealed that the increased EBV-miR-BART18-3p and FASN expression could up-regulate the process of de novo lipogenesis in CRC cells and promote CRC development.4 Preliminary exploration of EBV-miR-BART18-3p in CRC auxiliary diagnosis4.1 The biological function of EBV-miR-BART18-3p expression in CRC PDX model miceThe PDX Antagomir-EBV-miR-BART18-3p mice models showed that the volume of tumor in the Antagomir-EBV-miR-BART18-3p injection group was smaller than that in the Antagomir-Control injection group,and the survival rate was also significantly increased.Immunohistochemistry and in situ hybridization staining results showed that inhibiting EBV-miR-BART18-3p expression in the Antagomir-EBV-miR-BART18-3p injection group could significantly decrease the expression levels of EBV-miR-BART18-3p,LDHA,FASN and tumor malignant phenotype Ki67 compared with the Antagomir-Control injection group（P<0.001）.Subsequently,the lipid and triglyceride contents measurement results of single-cell suspension from PDX tumor tissues showed that the S2850/S2928 ratio and triglyceride contents were significantly reduced in the antigomir-EBV-miR-BART18-3p injection group compared with the Antagomir-Control group.Those results indicated that reducing EBV-miR-BART18-3p expression in CRC tumors could slow the tumors’growth and increase the survival time of PDX mice,inhibit the downstream LDHA and FASN expression.Besides,the de novo lipogenesis of PDX tumor and the malignant degree of tumors were also inhibited.4.2 Relationship among Epstein-Barr virus infection,serumal EBV-miR-BART18-3p expression levels and CRCMatched Case-Control Power Analysis results showed that the cases number of healthy donors and CRC patients were 254,respectively,which can ensure the reliability for further ELISA assays.The EB virus ELISA kits results showed that Epstein-Barr virus infection rates of CRC patients and healthy controls were more than 95%,and there was no statistical difference（97.64%of EB virus infections in healthy controls and 96.46%in CRC patients）.But EB virus titers in CRC patients were higher than those in healthy controls（P<0.001）,indicated that high titers of EB virus may be a risk factor for CRC.RT-qPCR results of serumal RNA samples from CRC patients found the serumal EBV-miR-BART18-3p expression levels in CRC patients were significantly higher than that in the healthy donors（P<0.001）.The ROC calculated AUC value was 0.7317（P<0.0001）.The further stratified analysis revealed that the expression levels of serumal EBV-miR-BART18-3p in CRC patients were significantly higher than in healthy donors of each TNM stage（P<0.001）.The AUC of each TNM stage was 0.7519,0.7162 and 0.7289,respectively（P<0.0001）,suggested that the serumal EBV-miR-BART18-3p might be use for auxiliary diagnosis of CRC patients.Conclusions:1.The EBV-miR-BART18-3p expression levels in CRC tumor tissues were significantly higher than that in adjacent normal tissues,and its expression levels were positive related to the development and survival time of CRC.2.EBV-miR-BART18-3p was significantly elevated in the tumor hypoxia microenvironment,and its high expression levels could enhance malignant phenotypes such as migration,invasion and tumor formation ability of CRC cells.3.In CRC cells,highly expressed EBV-miR-BART18-3p up-regulated acetyl-Co A production by regulating SIRT1/HIF-1α/LDHA pathway;increased acetyl-Co A promotes the FASN expression,enhances de novo lipogenesis and promoted the CRC development.4.The high-titer EB virus infection was one of the potential risk factors for CRC,and the serumal EBV-miR-BART18-3p might be used for auxiliary diagnosis of CRC patients.