| ObjectiveRenal cell carcinoma?RCC? is a common malignant tumor of the urinary system,and its incidence rate is increasing year by year.In 2018,about 400,000 new RCC cases were diagnosed and around 170,000 deaths were recorded in the world,RCC accounted for approximately 70,000 new cases and about 43,000 deaths in China[1-4].About 30%of RCC patients presented with distant metastases at primary diagnosis,and nearly 25%of localized RCC developed local recurrence or distant metastases after surgery,the most common metastatic site is lung[6-9].The current treatment of metastatic RCC is still limited,the survival prognosis is also poor[7,12],the related molecular biological mechanisms of metastatic RCC remains unclear.Recent studies have shown that exosomes derived from tumor cells play an important role in the metastasis process of tumor through mediating pre-metastatic niche[37,38],however,the roles and related mechanisms of exosomes derived from RCC cells in mediating lung pre-metastatic niche and affecting the lung metastasis of RCC remain to be elucidated.This study intends to explore the function and related molecular mechanisms of exosomes derived from RCC cells in the process of lung metastasis of RCC,and provide theoretical basis for finding new potential diagnostic markers and therapeutic targets of metastatic RCC with lung metastases.Methods1.Exosomes derived from low-metastatic RCC cells 786-O and high-metastatic RCC cells SN12-PM6 were extracted by ultracentrifugation methods,and labeled as 786-O-exo and SN12-PM6-exo,then NTA?nanoparticle tracking analysis?,transmission electron microscopy and Western blot assays were performed to confirm the identify of exosomes.After the exosomes were labeled with fluorescent dyes,the exosome uptake tracking experiments were carried out in vivo and in vitro to explore the receptor cell types of exosomes in the lungs.2.After we found that lung fibroblasts were the main receptor cells of exosomes in the lungs,the expression of fibroblast activation marker a-SMA in the lung metastases of RCC was examined by immunohistochemistry to verify the presence of activated fibroblasts.To verify whether exosomes could participate in the activation of lung fibroblasts,the migration ability and the expression levels of inflammatory genes such as IL-1?and IL-8 of human lung fibroblasts MRC-5 cells were examined after incubating with 786-O-exo and SN12-PM6-exo.3.After discovering that exosomes derived from RCC cells mediated lung fibroblasts activation,we compared lung metastases of RCC in the mice incubated with 786-O-exo or SN12-PM6-exo by in vivo luciferase-based bioluminescence imaging system and lung tissue H&E stained sections to verify whether exosomes could affect the lung metastasis of RCC by mediating lung fibroblasts activation.4.The miRNAs profiles of SN12-PM6-exo and 786-O-exo were examined by miRNAs chips and the differentially expressed miRNAs were analyzed.We then transfected the miRNAs upregulated in SN12-PM6-exo into MRC-5 to explore the functional miRNAs mediating fibroblast activation by RT-q PCR and Transwell migration experiments.5.It was found that miR-6826-5p mediated the activation of lung fibroblasts,then miR-6826-5p was selected as the research object.We downregulated the expression level of HRS,which mediates the sorting of exosomal contents,to explore the correlation between HRS expression in RCC cells and miR-6826-5p levels in exosomes.The change of miR-6826-5p expression level in MRC-5 cells after exosome incubation was detected and fluorescence colocalization experiments were performed to confirm the process of exosomes transmitting miR-6826-5p into lung fibroblasts.6.We transfected lentivirus into RCC cells to obtain miR-6826-5p-overexpressed exosomes.In-vivo and in-vitro experiments were performed to verify the effects of miR-6826-5p overexpression in exosomes on lung fibroblast activation and lung metastasis of RCC.7.We used Target Scan to predict potential downstream target genes of miR-6826-5p and conducted pathway enrichment analysis to narrow down the screening,and then explored the target genes that combined with miR-6826-5p by miRNA pull down and dual luciferase reporter assays.The results showed that TANK was the target gene of miR-6826-5p,and the rescue methods were applied to explore the regulation effects of miR-6826-5p on the target gene TANK.8.Ed U and Transwell migration experiments were conducted to examine the effects of activated lung fibroblasts on the proliferation and migration ability of RCC cells.In vivo,the effects of lung fibroblast activation on tumor growth and lung metastasis of RCC in vivo were explored.9.To explore the clinical application value of miR-6826-5p,tumor tissues and serum samples from localized RCC and metastatic RCC with lung metastases were collected.The expression levels of miR-6826-5p in tumor tissues and serum exosomes were examined to analyze the correlation between miR-6826-5p expression level and lung metastasis of RCC.Results1.The identifies of exosomes derived from 786-O and SN12-PM6 cells were confirmed.Exosomes derived from RCC cells could be significantly enriched in the lungs and were uptaken by lung fibroblasts.2.Activated lung fibroblasts were present in the lung metastases of RCC.The exosomes derived from RCC cells improved the migration ability and the expression of pro-inflammatory genes such as IL-1?,IL-8 and TGF-?of MRC-5,indicating that exosomes mediated the activation of lung fibroblasts,and the activation ability of SN12-PM6-exo was stronger than that of 786-O-exo.3.Compared with mice in the control group,there were significantly more lung metastases of RCC in the mice educated with exosomes derived from RCC cells,and the lung metastases in SN12-PM6-exo group mice were more than that in 786-O-exo group.4.According to the results of miRNA chips,a total of 9 miRNAs were significantly upregulated in SN12-PM6-exo?up-regulation multiple>4 and average normalized signal value>5?,and only miR-6826-5p significantly promoted the expression levels of IL-1?and IL-8 and migration ability of MRC-5.5.After HRS expression of RCC cells was downregulated,miR-6826-5p level decreased in the exosomes and increased in the cells,indicating miR-6826-5p was sorted into the RCC exosomes by HRS.The expression level of miR-6826-5p in MRC-5 increased significantly after MRC-5 cells were incubated with exosomes derived from RCC cells,and colocalization experiments further confirmed that exosomes delivered miR-6826-5p into lung fibroblasts to increase their miR-6826-5p expression.6.RCC exosomal miR-6826-5p overexpression could enhance their ability to activate lung fibroblasts and promote the lung metastasis of RCC.7.It was found that miR-6826-5p could combine with the TANK gene.Rescue experiments showed that miR-6826-5p negatively regulated TANK gene expression to activate the NF-?B signaling pathway,while TANK overexpression reversed the regulation effects of miR-6826-5p.8.Activated lung fibroblasts promoted the proliferation and migration of RCC cells,as well as tumor growth and lung metastasis of RCC in vivo.9.Clinical studies found that high expression of miR-6826-5p in RCC tumor tissues was positively correlated with malignant progression and poor prognosis of RCC,and high expression of miR-6826-5p in serum exosomes of RCC patients was positively correlated with the formation of lung metastasis of RCC.ConclusionExosomes derived from RCC cells were mainly uptaken by lung fibroblasts after being enriched in the lungs.During the uptake,miR-6826-5p was delivered into lung fibroblasts by exosomes,then regulated the target gene TANK and the downstream NF-?B signaling pathway to activate lung fibroblasts to cancer-associated fibroblasts?CAFs?,promoted their expression of pro-inflammatory genes such as IL-1?and IL-8,ultimately,CAFs mediated the formation of lung pre-metastatic microenvironment and promoted the lung metastasis of RCC.In addition,clinical studies found that the high expression of miR-6826-5p in serum exosomes was positively correlated with the formation of lung metastases of RCC.Therefore,this study demonstrated that miR-6826-5p and the related downstream genes may play an important role in the process of lung metastasis of RCC and have the potential to act as diagnosis markers and therapeutic targets for metastatic RCC with lung metastases. |
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