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Cancer-associated fibroblasts-derived extracellular vesicles carrying lncRNA SNHG3 facilitate colorectal cancer cell proliferation via the miR-34b-5p/HuR/HOXC6 axis

ABSTRACT: Cancer-associated fibroblasts (CAFs)-derived extracellular vesicles (EVs) can mediate tumorigenesis. Long noncoding RNA (LncRNA) SNHG3 is implicated in colorectal cancer (CRC) progression. The current study sought to clarify the role of CAFs-EVs carrying SNHG3 in CRC cell proliferation. Firstly, CAFs and normal fibroblasts (NFs) were cultured and identified, followed by isolation and characterization of CAFs-EVs and NFs-EVs. CRC cells were cultured with CAFs-EVs or CAFs-EVs overexpressing SNHG3. The effects of SNHG3 on CRC cell proliferation was evaluated using CCK-8, colony formation, and EdU staining assays. The binding relationships among SNHG3, miR-34b-5p, and HuR were validated, in addition to analyzing the binding between HuR and HOXC6. Lastly, xenograft tumor model was established to verify the role of CAFs-EVs carrying SNHG3 in vivo. SNHG3 was highly expressed in CRC cells and CAFs-EVs, whereas CAFs-EVs facilitated CRC cell proliferation. Mechanically, CAFs-EVs carried SNHG3 into CRC cells to upregulate HuR expression by competitively binding to miR-34b-5p, promote the binding of HuR and HOXC6, and enhance HOXC6 transcription. miR-34b-5p over-expression or HOXC6 silencing annulled the effect of CAFs-EVs. SNHG3 carried by CAFs-EVs facilitated CRC proliferation via the miR-34b-5p/HuR/HOXC6 axis in vivo. Collectively, our findings indicated that CAFs-EVs carried SNHG3 into CRC cells to upregulate HuR expression by sponging miR-34b-5p and finally enhance HOXC6 transcription, thereby facilitating CRC cell proliferation.

SUBMITTER: Zhao J

PROVIDER: S-EPMC9349187 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Project description:Background: Metastasis is the leading cause of death in patients with breast cancer (BC). Primary tumors create a premetastatic niche (PMN) in secondary organs for subsequent metastases. Cancer-associated fibroblasts (CAFs) are a predominant stromal component in the tumor microenvironment and serve as a major contributor to tumor metastasis. However, the function and mechanism of primary CAFs in the premetastatic niche of secondary organs remain unclear in BC. Methods: We investigated the expression profiles of lncRNAs in pairs of CAFs and NFs derived from breast tumor tissues using lncRNA microarray. The expression levels of lncSNHG5, ZNF281, IGF2BP2, CCL2 and CCL5 were assessed by qRT-PCR; the protein levels of related genes (e.g., ZNF281, IGF2BP2, CCL2, and CCL5) were analyzed using western blotting and/or ELISA in primary and immortalized CAFs and clinical samples. Tubule formation and three-dimensional sprouting assays and tissue fluorescence staining were conducted to investigate angiogenesis. In vitro permeability assays, trans-endothelial invasion assays, in vivo permeability assays and tissue fluorescence staining were conducted to examine vascular permeability. The regulatory mechanism of lncSNHG5 was investigated by RNA sequencing, fluorescent in situ hybridization, cellular fractionation assay, mass spectrometry, RNA pull-down, RNA immunoprecipitation, gene-specific m6A assay, chromatin immunoprecipitation, dual luciferase reporter assay and actinomycin D treatment in CAFs and NFs. Results: LncSNHG5 was highly expressed in breast CAFs and played an essential role in premetastatic niche formation by promoting angiogenesis and vascular leakiness through regulation of ZNF281 in CAFs. lncSNHG5 enhanced ZNF281 mRNA stability by binding with the m6A reader IGF2BP2. Enhanced ZNF281 transcriptionally regulated CCL2 and CCL5 expression to activate P38 MAPK signaling in endothelial cells. High CCL2 and CCL5 expression was associated with tumor metastasis and poor prognosis in BC patients. The inhibitors RS102895, marasviroc and cenicriviroc inhibited angiogenesis and vascular permeability in the PMN by blocking the binding of CCL2/CCR2 and CCL5/CCR5. The lncSNHG5-ZNF281-CCL2/CCL5 signaling axis plays an essential role in inducing premetastatic niche formation to promote BC metastasis. Conclusions: Our work demonstrates that lncSNHG5 and its downstream signaling ZNF281-CCL2/CCL5 in CAFs play a crucial role in premetastatic niche formation in breast cancer and may serve as potential targets for the diagnosis and treatment of BC metastasis.

Project description:Ovarian cancer (OC) is a lethal gynecological malignancy. Extracellular vesicles (EVs) are crucial media in cell-to-cell communication by carrying microRNAs (miRs). The current study aims to investigate the underlying mechanism of miR-630 carried by OC cell-derived EVs in regard to invasion and metastasis of OC cells. miRs related to OC metastasis were searched and screened. The expression patterns of screened miRs in human normal fibroblasts (NFs) and carcinoma-associated fibroblasts (CAFs) were detected using RT-qPCR. miR-630 related to OC metastasis and CAFs activation was analyzed further. The levels of FAP and α-SMA were detected using Western blotting and immunofluorescence. The migration of NFs was measured using Transwell assay. OC cell-derived EVs were isolated and identified. Uptake of EVs by NFs was observed using immunofluorescence staining. The culture supernatant of NFs was collected and used to culture the low metastasis cell line OVCAR8. The migration and invasion of OC cells and epithelial mesenchymal transition (EMT) were measured. Moreover, a xenograft model was established by injecting OVCAR8 cells of different groups into nude mice. Lastly, the effect of EV-pretreated NFs on invasion and metastasis of OC cells was observed in vivo. miR-630 was upregulated in OC cells and CAFs, and further associated with CAF activation and OC metastasis. miR-630 overexpression increased the levels of FAP and α-SMA in NFs, resulting in the transformation of NFs into CAFs. EVs carried miR-630 into NFs and EVs promoted CAF activation. miR-630 targeted KLF6. miR-630 inhibition or KLF6 overexpression attenuated EVs-induced CAF activation. EVs activated the NF-κB pathway via the miR-630/KLF6 axis. The conditioned medium of NFs pretreated with EVs promoted the invasion and metastasis of OVCAR8 cells, while downregulating miR-630 in EVs partially inhibited the promotive effect of NFs. EV-pretreated NFs promoted invasion and metastasis of OC in vivo. In conclusion, EVs carried miR-630 into NFs, thereby facilitating CAF activation and promoting invasion and metastasis of OC by inhibiting KLF6 and activating the NF-κB pathway. Our findings might offer a novel mechanism of invasion and metastasis of OC from the perspective of tumor microenvironment.

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Cancer-associated fibroblasts-derived extracellular vesicles carrying lncRNA SNHG3 facilitate colorectal cancer cell proliferation via the miR-34b-5p/HuR/HOXC6 axis

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