Loss of exosomal miR-148b from cancer-associated fibroblasts promotes endometrial cancer cell invasion and cancer metastasis

Cancer-associated fibroblasts (CAFs) play crucial roles in tumor progression, given the dependence of cancer cells on stromal support. Therefore, understanding how CAFs communicate with endometrial cancer cell in tumor environment is important for endometrial cancer therapy. Exosomes, which contain proteins and noncoding RNA, are identified as an important mediator of cell–cell communication. However, the function of exosomes in endometrial cancer metastasis remains poorly understood. In the current study we found that CAF-derived exosomes significantly promoted endometrial cancer cell invasion comparing to those from normal fibroblasts (NFs). We identified a significant decrease of miR-148b in CAFs and CAFs-derived exosomes. By exogenously transfect microRNAs, we demonstrated that miR-148b could be transferred from CAFs to endometrial cancer cell through exosomes. In vitro and in vivo studies further revealed that miR-148b functioned as a tumor suppressor by directly binding to its downstream target gene, DNMT1 to suppress endometrial cancer metastasis. In endometrial cancer DNMT1 presented a potential role in enhancing cancer cell metastasis by inducing epithelial–mesenchymal transition (EMT). Therefore, downregulated miR-148b induced EMT of endometrial cancer cell as a result of relieving the suppression of DNMT1. Taken together, these results suggest that CAFs-mediated endometrial cancer progression is partially related to the loss of miR-148b in the exosomes of CAFs and promoting the transfer of stromal cell-derived miR-148b might be a potential treatment to prevent endometrial cancer progression.     

MiR-210 in exosomes derived from CAFs promotes non-small cell lung cancer migration and invasion through PTEN/PI3K/AKT pathway

ObjectiveCancer-associated fibroblasts (CAFs) function as a crucial factor in tumor progression by carrying exosomes to neighboring cells. This study was assigned to expound the underlying mechanism of CAFs-derived exosomal miR-210 in non-small cell lung cancer (NSCLC) progression.MethodCAFs and normal fibroblasts (NFs) were isolated and identified. Exosomes secreted from CAFs and NFs were isolated to analyze their effects on tumor volume and epithelial-mesenchymal transition (EMT). Exosomal miR-210 expression level was measured. The effects of exosomal miR-210 and UPF1 on cell viability, EMT, PTEN/PI3K/AKT signal pathway were determined. Dual-luciferase reporter gene assay was utilized to validate the binding of UPF1 to miR-210.ResultsCAFs-derived exosomes (CAFs-exo) were successfully extracted and proven to be uptake by lung cancer cells. Up-regulated expression level of miR-210 was found in CAFs-exo, which was then proved to enhance cell migration, proliferation, invasion abilities and EMT in NSCLC cells. Overexpression of miR-210 can also inhibit UPF1 and PTEN, but activate the PTEN/PI3K/AKT pathway. UPF1 was a target gene of miR-210. MiR-210 can up-regulate UPF1 expression level to activate PTEN/PI3K/AKT pathway.ConclusionMiR-210 secreted by CAFs-exo could promote EMT by targeting UPF1 and activating PTEN/PI3K/AKT pathway, thereby promoting NSCLC migration and invasion.     

Tumor-suppressive microRNA-10a inhibits cell proliferation and metastasis by targeting Tiam1 in esophageal squamous cell carcinoma

Aberrant microRNAs (miRNAs) expressions could contribute to the progression of numerous cancers, including esophageal squamous cell carcinoma, while miR-10a participates in multiple biological processes on cancers. However, the molecular mechanism of miR-10a in esophageal squamous cell carcinoma (ESCC) has not been investigated. Herein, miR-10a was significantly reduced in ESCC clinical tissues and ESCC cell lines (EC109 and TE-3). In addition, immunohistochemistry indicated that the expressions of α-SMA, Ki-67, and PCNA in tumor tissues were higher than that of controls. In vitro, overexpression of miR-10a dramatically suppressed cell proliferation and enhanced cell apoptosis, while the decrease of miR-10a expressed the opposite outcome. Specially, overexpression of miR-10a caused a G0/G1 peak accumulation. Moreover, miR-10a also negatively regulated ESCC cell migration and invasion. Furthermore, targetscan bioinformatics predictions and the dual-luciferase assay confirmed that Tiam1 was a direct target gene of miR-10a. The statistical analysis showed Tiam1 was negatively in correlation with miR-10a in ESCC patient samples. And silencing Tiam1 could lead to a decline on cell growth, invasion, and migration in ESCC cell lines, while it could enhance cell apoptosis and cause a G0/G1 peak accumulation. In vivo, it revealed that miR-10a notably decreased the tumor growth and metastasis in xenograft model and pulmonary metastasis model. And it showed a lower expressions of Tiam1 in the miR-10a mimics group by immunohistochemistry. Taken together the results, they indicated that miR-10a might function as a novel tumor suppressor in vitro and in vivo via targeting Tiam1, suggesting miR-10a to be a candidate biomarker for the ESCC therapy.     

miR-760 exerts an antioncogenic effect in esophageal squamous cell carcinoma by negatively driving fat metabolism via targeting c-Myc

miR-760 is downregulated in various human tumors, and fat metabolism disorder correlates with tumor progression, especially anomalism of key fat metabolic enzymes that are positively modulated by c-Myc. The aim of our study is to elucidate the presumptive molecular mechanisms of miR-760-mediated esophageal squamous cell carcinoma (ESCC) cell function and to assess the therapeutic significance of miR-760 in ESCC patients. Quantitative real-time PCR (RT-qPCR) analysis indicated that miR-760 was significantly downexpressed in ESCC tissues and cell lines. Cell counting kit-8 (CCK-8) assay, colony formation assay, transwell assay, and flow cytometry denoted that induced ectopic overexpression of miR-760 dramatically inhibited ESCC cells proliferation, attenuated migration, and invasion facilitated apoptosis in vitro. Mechanistically, c-Myc predicted using bioinformatics was identified as a potential target gene of miR-760 by luciferase reporter assay. Furthermore, mRNA and protein expression levels of c-Myc and key fat metabolic enzymes were downregulated with miR-760 mimics. The above investigation results, responsible for the antineoplastic properties of miR-760 in ESCC, preliminarily highlighted that the hypothetical signal amongst miR-760, c-Myc, and key fat metabolic enzymes may develop a novel diagnostic marker, therapeutic target, and independent prognostic indicator.     

Cancer-associated fibroblast-derived exosomal microRNA-24-3p enhances colon cancer cell resistance to MTX by down-regulating CDX2/HEPH axis

MicroRNA-24-3p (miR-24-3p) has been implicated as a key promoter of chemotherapy resistance in numerous cancers. Meanwhile, cancer-associated fibroblasts (CAFs) can secret exosomes to transfer miRNAs, which mediate tumour development. However, little is known regarding the molecular mechanism of CAF-derived exosomal miR-24-3p in colon cancer (CC). Hence, this study intended to characterize the functional relevance of CAF-derived exosomal miR-24-3p in CC cell resistance to methotrexate (MTX). We identified differentially expressed HEPH, CDX2 and miR-24-3p in CC through bioinformatics analyses, and validated their expression in CC tissues and cells. The relationship among HEPH, CDX2 and miR-24-3p was verified using ChIP and dual-luciferase reporter gene assays. Exosomes were isolated from miR-24-3p inhibitor–treated CAFs (CAFs-exo/miR-24-3p inhibitor), which were used in combination with gain-of-function and loss-of-function experiments and MTX treatment. CCK-8, flow cytometry and colony formation assays were conducted to determine cell viability, apoptosis and colony formation, respectively. Based on the findings, CC tissues and cells presented with high expression of miR-24-3p and low expression of HEPH and CDX2. CDX2 was a target gene of miR-24-3p and could up-regulate HEPH. Under MTX treatment, overexpressed CDX2 or HEPH and down-regulated miR-24-3p reduced cell viability and colony formation and elevated cell apoptosis. Furthermore, miR-24-3p was transferred into CC cells via CAF-derived exosomes. CAF-derived exosomal miR-24-3p inhibitor diminished cell viability and colony formation and increased cell apoptosis in vitro and inhibited tumour growth in vivo under MTX treatment. Altogether, CAF-derived exosomal miR-24-3p accelerated resistance of CC cells to MTX by down-regulating CDX2/HEPH axis.     

Cancer-associated fibroblasts-derived exosomes upregulate microRNA-135b-5p to promote colorectal cancer cell growth and angiogenesis by inhibiting thioredoxin-interacting protein

ObjectiveThe role of exosomes in human cancers has been identified, while the effect of cancer-associated fibroblasts (CAFs)-derived exosomes (CAF-exos) transmitting microRNAs (miRNAs) on colorectal cancer (CRC) remains largely unknown. We aim to explore the impact of CAF-derived exosomal miR-135b-5p on CRC progression by targeting thioredoxin-interacting protein (TXNIP).MethodsCRC tissues were collected to obtain CAF-exos, which were used to co-culture with LoVo and HT29 cells. The effect of miR-135b-5p and TXNIP on the in vivo growth, in vitro proliferation, apoptosis, migration, invasion and angiogenesis of CRC cells. miR-135b-5p and TXNIP expression in exosomes and CRC cells were detected and their targeting relationship was confirmed.ResultsMiR-135b-5p was upregulated whereas TXNIP was downregulated in CRC tissues and cells. The CAF-exos and CAF-exos upregulating miR-135b-5p promoted in vivo growth, in vitro proliferation, migration and invasion, and suppressed apoptosis of CRC cells, and also promoted the HUVEC angiogenesis. TXNIP was confirmed as a target of miR-135b-5p and overexpression of TXNIP could weaken the pro-CRC effect of exosomal miR-135b-5p,ConclusionCAF-exos upregulate miR-135b-5p to promote CRC cell growth and angiogenesis by inhibiting TXNIP.     

miR-125a/b inhibits tumor-associated macrophages mediated in cancer stem cells of hepatocellular carcinoma by targeting CD90

Cancer stem cells promote tumorigenesis and progression of hepatocellular carcinoma (HCC). Recently, emerging evidence indicates tumor-associated macrophages (TAMs) play an important role in tumor progression. However, TAMs often occurs with unknown mechanisms. As an important mediator in intercellular communications, exosomes secreted by host cells mediate the exchange of genetic materials and proteins, which involves tumor aggressiveness. The aim of the study was to investigate whether exosomes derived from TAMs mediate stem cell properties in HCC. TAMs were isolated from the tissues of HCC. microRNA (miRNA) expression profiles of TAMs were analyzed using miRNA microarray. In vitro cell coculture was further conducted to investigate the crosstalk between TAMs and tumor cells mediated by TAMs exosomes. In this study, we showed that TAMs exosomes promote HCC cell proliferation and stem cell properties. Using miRNA profiles assay, we identified significantly lower levels of miR-125a and miR-125b in exosomes and cell lysate isolated from TAMs. Functional studies revealed that the HCC cells were treated with TAM exosomes or transfected with miR-125a/b suppressed cell proliferation and stem cell properties by targeting CD90, a stem cell marker of HCC stem cells. The study indicated that miR-125a/b targeting CD90 played important roles in cancer stem cells of HCC.     

Cancer-associated fibroblasts–derived exosomes-mediated transfer of LINC00355 regulates bladder cancer cell proliferation and invasion

The aim of this study was to investigate the regulatory mechanism of cancer-associated fibroblasts (CAFs) exosome in bladder cancer (BC) cell proliferation and invasion. CAFs and normal fibroblasts (NFs) were isolated from tumor tissues and adjacent normal tissues of BC patients, and examined by immunocytochemistry for the expression of fibroblast activation protein alpha (FAP) and α-smooth muscle actin (α-SMA). Exosomes were extracted from CAFs and NFs and observed under a transmission electron microscope, and expression of the exosome markers CD9 and CD63 was confirmed by western blotting. The distribution and intensity of fluorescence were observed by confocal laser microscopy to analyze exosomes uptake by BC cell lines T24 or 5367. BC cell proliferation and invasion were detected by MTT and Transwell assays, respectively. LINC00355 levels in CAFs, NFs, CAFs exosome, NFs exosome, and BC cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that CAFs exosome significantly promoted BC cell proliferation and invasion relative to NFs exosome. LINC00355 expression was significantly elevated in CAFs exosome when compared with that in NFs-exosome. Up-regulated LINC00355 expression was observed both in T24 and 5367 cells co-incubated with CAFs exosome. Exosomes derived from LINC00355 siRNA-transfected CAFs observably repressed BC cell proliferation and invasion when compared with control siRNA-CAFs exosome. In conclusion, CAFs exosome–mediated transfer of LINC00355 regulates BC cell proliferation and invasion. Significance of the study. In this study, our data suggest that the exosomes released from CAFs promote BC cell proliferation and invasion. The mechanism of this effect is, at least in part, related to the increased LINC00355. Regulation of LINC00355 expression in exosomes released from CAFs might be a putative therapeutic strategy against the pathogenesis of BC.     

MiR-206 suppresses the deterioration of intrahepatic cholangiocarcinoma and promotes sensitivity to chemotherapy by inhibiting interactions with stromal CAFs

Background: Intrahepatic cholangiocarcinoma (iCCA) is a highly malignant subtype of cholangiocarcinoma (CCA) with poor prognosis. In iCCA, the interplay between the stroma and tumor cells results in resistance to adjuvant chemotherapy. Increasing evidence indicates that miR-206 participates in tumor progression, but its role in iCCA is still unclear. The aim of this study was to identify dysregulated miR-206 expression in iCCA and to further explore the underlying mechanism.Methods: MiR-206 expression was proven to be downregulated in iCCA tissues by qPCR, and its correlation with clinical characteristics and prognosis was investigated. iCCA-derived cancer-associated fibroblast cells (CAFs) and normal fibroblast cells (NFs) were isolated and identified. MiR-206 was knocked in or down in CAFs and CCA cells, respectively, to explore the role of miR-206, and coculture of these treated CCAs and CAFs was conducted to explore the effects of miR-206 on their mutual promoting effects. Exosomes carrying miR-206 and an orthotopic mouse model were used to determine the inhibitory effects of miR-206 on iCCA deterioration in vivo.Results: We confirmed that miR-206 is a suppressor of iCCA. Overexpressing miR-206 in CCA cells inhibited cell proliferation, migration and invasion. When cocultured with CCA cells, NFs downregulated miR-206 expression, and NFs were susceptible to transforming into CAFs. Moreover, CAFs promoted CCA cell malignant behaviors and gemcitabine resistance. Overexpressing miR-206 in CAFs or CCA cells inhibited this mutual promoting effect. Additionally, when delivered by exosomes, miR-206 suppressed tumor deterioration. And combined with gemcitabine, this treatment resulted in a longer survival time.Conclusion: Our study explained that the interaction between CCA cells and CAFs promoted iCCA deterioration. As a suppressive factor, miR-206 inhibited aggressive characteristics and gemcitabine resistance by interfering with this mutual promoting effect. This research elucidated the molecular mechanism underlying the unfavorable chemotherapeutic response of patients with iCCA, which provided a promising target for iCCA treatment.     

MiRNA expression analysis of cancer-associated fibroblasts and normal fibroblasts in breast cancer

Cancer-associated fibroblasts (CAFs) promote tumorigenesis, growth, invasion and metastasis of cancer, whereas normal fibroblasts (NFs) are thought to suppress tumor progression. Little is known about miRNAs expression differences between CAFs and NFs or the patient-to-patient variability in miRNAs expression in breast cancer. We established primary cultures of CAFs and paired NFs from six resected breast tumor tissues that had not previously received radiotherapy or chemotherapy treatment and analyzed with miRNAs microarrays. The array data were analyzed using paired SAM t-test and filtered according to α and q values. Pathway analysis was conducted using DAVID v6.7. We identified 11 dysregulated miRNAs in CAFs: three were up-regulated (miR-221-5p, miR-31-3p, miR-221-3p), while eight were down-regulated (miR-205, miR-200b, miR-200c, miR-141, miR-101, miR-342-3p, let-7g, miR-26b). Their target genes are known to affect cell differentiation, adhesion, migration, proliferation, secretion and cell-cell interaction. By our knowledge it is firstly identify the expression profiles of miRNAs between CAFs and NFs and revealed their regulation on the associated signaling pathways.     

MicroRNA-488-3p inhibits proliferation and induces apoptosis by targeting ZBTB2 in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is the eighth most prevalent cancer and the sixth leading cause for cancer-associated mortality. MicroRNAs (miRNAs) are increasingly reported to exert important regulatory functions in human cancers by regulating certain gene expression. miR-488-3p has been identified to be a tumor suppressor in multiple cancers, but its role in ESCC is yet to be investigated. The present study aimed to uncover the biological role and modulatory mechanism of miR-488-3p in ESCC. We first revealed the downregulation of miR-488-3p in ESCC tissues and cell lines. Gain-of-function assays confirmed that miR-488-3p overexpression abrogated proliferation and accelerated apoptosis. Mechanistically, we identified via bioinformatics tool and confirmed that zinc finger and BTB domain containing 2 (ZBTB2) was a target for miR-488-3p. Moreover, miR-488-3p activated the p53 pathway through suppressing ZBTB2. Finally, rescue assays proved that ZBTB2 was involved in the regulation of miR-488-3p on proliferation and apoptosis in ESCC. Additionally, we verified that miR-488-3p had alternate targets in ESCC by confirming the involvement of protein kinase, DNA-activated, catalytic subunit (PRKDC), a known target for miR-488-3p, in miR-488-3p-mediated regulation on ESCC. In sum, this study revealed that miR-488-3p inhibited proliferation and induced apoptosis by targeting ZBTB2 and activating p53 pathway in esophageal squamous cell carcinoma, providing a novel biological target for ESCC.     

Retracted: MicroRNA-145 performs as a tumor suppressor in human esophageal squamous cell carcinoma by targeting phospholipase C epsilon 1

Esophageal squamous cell carcinoma (ESCC) is the leading pathologic type in China. miR-145 has been reported to be downregulated in multiple tumors. This study was aimed to investigate the role of miR-145 in ESCC. miR-145 expression was investigated in 65 ESCC samples as well as four ESCC cell lines by quantitative real-time polymerase chain reaction (qRT-PCR). Targetscan 6.2 website ( http://www.targetscan.org/ ) was used to predict the targets of miR-145. Expression of phospholipase C epsilon 1 (PLCE1) messenger RNA and protein was detected by qRT-PCR or Western blot. MTT and wound healing assay were conducted to explore the effects of miR-145 on the proliferation and migration of ESCC cell lines, respectively. miR-145 was significantly decreased in ESCC tissues. An inverse correlation between miR-145 and invasion depth and TNM stage were observed. PLCE1 was a direct target of miR-145, and the expression of PLCE1 was inversely correlated with miR-145 expression in ESCC tissues. In addition, overexpression of miR-145 suppressed cell proliferation and migration in ESCC cells. The enforced expression of PLCE1 partially reversed the suppressive effect of miR-145. These results prove that miR-145 may perform as a tumor suppressor in ESCC by targeting PLCE1.     

Exosomal miR-146a-5p and miR-155-5p promote CXCL12/CXCR7-induced metastasis of colorectal cancer by crosstalk with cancer-associated fibroblasts

C-X-C motif chemokine receptor 7 (CXCR7) is a newly discovered atypical chemokine receptor that binds to C-X-C motif chemokine ligand 12 (CXCL12) with higher affinity than CXCR4 and is associated with the metastasis of colorectal cancer (CRC). Cancer-associated fibroblasts (CAFs) have been known to promote tumor progression. However, whether CAFs are involved in CXCR7-mediated metastasis of CRC remains elusive. We found a significant positive correlation between CXCR7 expression and CAF activation markers in colonic tissues from clinical specimens and in villin-CXCR7 transgenic mice. RNA sequencing revealed a coordinated increase in the levels of miR-146a-5p and miR-155-5p in CXCR7-overexpressing CRC cells and their exosomes. Importantly, these CRC cell-derived miR-146a-5p and miR-155-5p could be uptaken by CAFs via exosomes and promote the activation of CAFs through JAK2–STAT3/NF-κB signaling by targeting suppressor of cytokine signaling 1 (SOCS1) and zinc finger and BTB domain containing 2 (ZBTB2). Reciprocally, activated CAFs further potently enhanced the invasive capacity of CRC cells. Mechanistically, CAFs transfected with miR-146a-5p and miR-155-5p exhibited a robust increase in the levels of inflammatory cytokines interleukin-6, tumor necrosis factor-α, transforming growth factor-β, and CXCL12, which trigger the epithelial–mesenchymal transition and pro-metastatic switch of CRC cells. More importantly, the activation of CAFs by miR-146a-5p and miR-155-5p facilitated tumor formation and lung metastasis of CRC in vivo using tumor xenograft models. Our work provides novel insights into CXCR7-mediated CRC metastasis from tumor–stroma interaction and serum exosomal miR-146a-5p and miR-155-5p could serve as potential biomarkers and therapeutic targets for inhibiting CRC metastasis.Subject terms: Cancer microenvironment, Colon cancer     

Tumor-associated macrophages promote prostate cancer progression via exosome-mediated miR-95 transfer

Tumor-associated macrophages (TAMs) are vital constituents in mediating cell-to-cell communication within the tumor microenvironment. However, the molecular mechanisms underlying the interplay between TAMs and tumor cells that guide cell fate are largely undetermined. Extracellular vesicles, also known as exosomes, which are derived from TAMs, are the components exerting regulatory effects. Thus, understanding the underlying mechanism of “onco-vesicles” is of crucial importance for prostate cancer (PCa) therapy. In this study, we analyzed micro RNA sequences in exosomes released by THP-1 and M2 macrophages and found a significant increase in miR-95 levels in TAM-derived exosomes, demonstrating the direct uptake of miR-95 by recipient PCa cells. In vitro and in vivo loss-of-function assays suggested that miR-95 could function as a tumor promoter by directly binding to its downstream target gene, JunB, to promote PCa cell proliferation, invasion, and epithelial–mesenchymal transition. The clinical data analyses further revealed that higher miR-95 expression results in worse clinicopathological features. Collectively, our results demonstrated that TAM-mediated PCa progression is partially attributed to the aberrant expression of miR-95 in TAM-derived exosomes, and the miR-95/JunB axis provides the groundwork for research on TAMs to further develop more-personalized therapeutic approaches for patients with PCa.     

Epigenetic silencing of microRNA-149 in cancer-associated fibroblasts mediates prostaglandin E2/interleukin-6 signaling in the tumor microenvironment

Tumor initiation and growth depend on its microenvironment in which cancer-associated fibroblasts (CAFs) in tumor stroma play an important role. Prostaglandin E2 (PGE2) and interleukin (IL)-6 signal pathways are involved in the crosstalk between tumor and stromal cells. However, how PGE2-mediated signaling modulates this crosstalk remains unclear. Here, we show that microRNA (miR)-149 links PGE2 and IL-6 signaling in mediating the crosstalk between tumor cells and CAFs in gastric cancer (GC). miR-149 inhibited fibroblast activation by targeting IL-6 and miR-149 expression was substantially suppressed in the CAFs of GC. miR-149 negatively regulated CAFs and their effect on GC development both in vitro and in vivo. CAFs enhanced epithelial-to-mesenchymal transition (EMT) and the stem-like properties of GC cells in a miR-149-IL-6-dependent manner. In addition to IL-6, PGE2 receptor 2 (PTGER2/EP2) was revealed as another potential target of miR-149 in fibroblasts. Furthermore, H. pylori infection, a leading cause of human GC, was able to induce cyclooxygenase-2 (COX-2)/PGE2 signaling and to enhance PGE2 production, resulting in the hypermethylation of miR-149 in CAFs and increased IL-6 secretion. Our findings indicate that miR-149 mediates the crosstalk between tumor cells and CAFs in GC and highlight the potential of interfering miRNAs in stromal cells to improve cancer therapy.