Fusobacterium Nucleatum Infection Promoting Colorectal Cancer Metastasis Through LncRNA EVADR

BackgroundsColorectal cancer(CRC)is one of the most common malignant tumors,and the incidence rate and mortality rate rank of CRC among the top five malignant tumors.The5-year survival rate of patients with localized CRC is 90%,while that of patients with distant metastatic diseases is only 14%.Although various headway in the treatment of CRC,metastatic CRC remains challenging to treat.Therefore,it is of great clinical significance to reseach the driving factors and specific mechanisms of colorectal cancer metastasis,and find new intervention targets to prevent colorectal cancer metastasis and reduce mortality.The progression of CRC can be influenced by many factors,such as gene mutations,dietary habits.Importantly,recent studies have revealed that gut microbiota contributes to the tumorigenesis and progression of CRC.Fusobacterium nucleatum(F.nucleatum)is a kind of Gram-negative anaerobic bacteria commonly found in oral mucosa.Large scale epidemiological research,metagenomic and metabonomic analysis found that the abundance of F.nucleatum in colorectal cancer tissues and feces is closely related to tumor invasion,lymph node metastasis and distal liver and lung metastasis.However,there is still lack of direct evidence that F.nucleatum can promote CRC metastasis,and the mechanisms underlying F.nucleatum in CRC metastasis have not yet been fully elucidated.Long non-coding RNA(lncRNA)is a class of RNA molecules involved in chromatin modification,nuclear transport,transcriptional activation,transcriptional interference and other important regulatory processes.Increasing evidence has suggested that lncRNAs are abundantly expressed and widely associated with tumorigenesis,development and metastasis of CRC.However,the link between lncRNA and F.nucleatum-dependent CRC metastasis is poorly understood.ObjectivesIn clinical specimens analysis,CRC orthotopic mouse model and cell models,we aimed to investigate the pro-metastatic effects of F.nucleatum,and explore the role and specific molecular mechanism of lncRNA EVADR in CRC metastasis with F.nucleatum infection.Methods and ResultsPart Ⅰ F.nucleatum infection promotes colorectal cancer metastasis1.The abundance of F.nucleatum in primary CRC tissue with or without metastasis was compared by qPCR and FISH.The results showed that the level of F.nucleatum in primary CRC tissues from metastatic patients was higher than that from non-metastatic patients.2.We used a CRC orthotopic mouse model to further investigate whether F.nucleatum promotes CRC metastasis.IVIS,H&Eand Ki67 staining showed that solid visceral transfer is more likely to occur in F.nucleatum-treated primary CRC,and F.nucleatum-induced distal metastasis could be blocked by metronidazole.Furthermore,we found that F.nucleatum was present in the liver and lung of metastatic mice.3.CRC migration and invasion were investigated by wound healing and Transwell assays.The results revealed that F.nucleatum infection promoted HCT116 and LoVo cell migration and invasion.4.The ability of F.nucleatum to influence the actin cytoskeleton in CRC motility was evaluated by F-actin staining.The results revealed that F.nucleatum infection induced formation of pseudopodium.The expression of EMT hallmark in CRC cells was evaluated by western blot.It was found that F.nucleatum infection resulted in a decrease in E-cadherin,but an increase in N-cadherin and vimentin in orthotopic tumors,as well as in HCT116 and LoVo cells.Part Ⅱ F.nucleatum infection promotes colorectal cancer metastasis through upregulating lncRNA EVADR1.Microarrays were used for expression profiling of lncRNAs in normal and CRC tissues with or without F.nucleatum infection,and further validation was performed using qRT-PCR in primary CRC of orthotopic mice.It was found that only lncRNA EVADR showed higher association with F.nucleatum induced CRC metastasis.2.LncRNA EVADR levels were detected in primary CRC tissues with or without metastasis by qRT-PCR and CISH.Results showed that LncRNA EVADR was up-regulated in the primary CRC tissues with metastasis,and was strongly associated with clinical outcome.In addition,lncRNA EVADR expression waspositively correlated with the abundance of F.nucleatum present.3.To further validate the association between F.nucleatum infection and lncRNA EVADR expression,qRT-PCR was performed after incubating CRC cells and NCM460 cells with F.nucleatum,B.fragilis,or PBS control for 24 h.Consistent with the microarray data,lncRNA EVADR was up-regulated in F.nucleatum infected CRC cells,but not in F.nucleatum infected normal cells.4.The lentivirus-mediated stable overexpression lncRNA EVADR cell lines(LV-EVADR)and their control cell lines(LV-NC)were constructed.The influences of lncRNA EVADR on CRC metastasis were assessed in an experimental lung metastasis model and cell model.The results revealed that overexpression of lncRNA EVADR promoted CRC metastasis in vivo and in vitro.5.The lentivirus-mediated stable lncRNA EVADR knockdown cell lines(sh EVADR)and their control cell lines(sh NC)were constructed.The influences of lncRNA EVADR on F.nucleatum-dependent CRC metastasis were assessed in an F.nucleatum-infected CRC orthotopic mouse model and F.nucleatum-infected cell model.Further more,the expression of EMT hallmark in lncRNA EVADR-knocked down HCT116 cells with or without F.nucleatum infection was evaluated by western blot.The results revealed that knockdown of lncRNA EVADR could blocked F.nucleatum-dependent CRC metastasis in vivo and in vitro.In addition,the F.nucleatum-dependent changes in N-cadherin,E-cadherin and vimentin protein levels were also abolished in lncRNA EVADR-knockdown cells.6.The expression levels of Snail,Slug,and Zeb1 were measured by qRT-PCR and western blot.The results shown that F.nucleatum infection had no significant effect on the mRNA levels of Snail,Slug,and Zeb1,but significantly increased the protein levels of Snail,Slug,and Zeb1 through up-regulating the expression of lncRNA EVADR.7.To evaluate changes in polysome distribution profiles for these mRNAs,the mRNAs distribution across sucrose gradients were analyzed.The results shown that that F.nucleatum infection increased the translational efficiency of Snail,Slug,and Zeb1 mRNAs through lncRNA EVADR.Part Ⅲ F.nucleatum infection promotes colorectal cancer metastasis through the lncRNA EVADR/YBX1 pathway1.The subcellular localization of lncRNA EVADR was detected by FISH and qRT-PCR analysis of nuclear and cytosolic fractions.The results indicated that lncRNA EVADR was mainly localized in the cytoplasm.2.RNA pull down-MS and RIP experiments were used to screen and identify lncRNA EVADR interacting proteins,and found that lncRNA EVADR interacted with YBX1 protein.3.We found that Snail,Slug,and Zeb1 mRNAs also interact with YBX1 by RIP assay.In addition,lncRNA EVADR,YBX1 and Snail/Slug/Zeb1 mRNAs could form a RNA-protein complex.4.Western blot were used to detect the effect of knockdown YBX1 on protein translation mediated by lncRNA EVADR,and found that lncRNA EVADR regulates protein translation through YBX1.5.The levels of YBX1 mRNA and protein were analyzed in lncRNA EVADR-overexpressed LoVo cells by qRT-PCR and western blot.The results shown that overexpression of lncRNA EVADR did not influence YBX1 transcription or protein stability.In addition,an RIP assay was carried out to detected the binding ability between YBX1 and its mRNA partners in lncRNA EVADR-overexpressed LoVo cells.The results indicated that overexpression of lncRNA EVADR did not influence the interaction between YBX1 and its mRNA partners.6.The distribution of YBX1 in the polysome profile was examined,and it was found that F.nucleatum promoted the redistribution of YBX1 on translating polysomes though up-regulation of lncRNA EVADR.7.Knockdown of YBX1 significantly counteracted F.nucleatum-dependent and lncRNA EVADR-dependent CRC metastasis.The results indicated that F.nucleatum promotes CRC metastasis through the lncRNA EVADR/YBX1 pathway.Conclusions1.Using an experimental CRC orthotopic mouse model,we confirmed that F.nucleatum infection could promote distal metastasis of colorectal cancer cells at the first time.In addition,we found that F.nucleatum could colonize in liver and lung metastases in mice.2.We confirmed that F.nucleatum promoted CRC metastasis through up-regulating the expression of lncRNA EVADR.3.In this study,a new mechanism was identified whereby lncRNA EVADR served as a guide for the RNA binding protein YBX1 to recruit its mRNA partners(Snail,Slug,and Zeb1)to the polyribosome,and consequently promoted CRC metastasis.4.We found that F.nucleatum,lncRNA EVADR and YBX1 might be useful for the prevention and treatment of patients with F.nucleatum-associated CRC metastasis.SignificancesThis study may provide new ideas for the diagnosis and prevention of colorectal cancer metastasis,which has important clinical value and social significance for reducing the metastasis rate of CRC and reducing the social burden.