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BACKGROUND & AIMS: Nearly 20% of the global cancer burden can be linked to infectious agents. Fusobacterium nucleatum promotes tumor formation by epithelial cells via unclear mechanisms. We aimed to identify microRNAs (miRNAs) induced by F nucleatum and evaluate their ability to promote colorectal carcinogenesis in mice.
METHODS: Colorectal cancer (CRC) cell lines were incubated with F nucleatum or control reagents and analyzed in proliferation and would healing assays. HCT116, HT29, LoVo, and SW480 CRC cell lines were incubated with F nucleatum or phosphate buffer saline (PBS control) and analyzed for miRNA expression patterns and in chromatin immunoprecipitation assays. Cells were incubated with miRNAs mimics, control sequences, or small interfering (si) RNAs; expression of reporter constructs was measured in luciferase assays. CRC cells were incubated with F nucleatum or PBS and injected into BALB/C nude mice; growth of xenograft tumors was measured. C57BL APC(min/+), C57BL miR21a(-/-), and C57BL mice with full-length miR21a (controls) were given F nucleatum by gavage; some mice were given azoxymethane (AOM) and dextran sodium sulfate (DSS) to induce colitis and colon tumors. Intestinal tissues were collected and tumors were counted. Serum samples from mice were analyzed for cytokine levels by ELISAs. We performed in situ hybridization analyses to detect enrichment of F nucleatum in CRC cells. F nucleatum DNA in 90 tumor and matched non-tumor tissues from patients in China were explored for the expression correlation analysis; levels in 125 tumor tissues from patients in Japan were compared with their survival times.
RESULTS: Fusobacterium nucleatum increased proliferation and invasive activities of CRC cell lines, compared with control cells. CRC cell lines infected with F nucleatum formed larger tumors, more rapidly, in nude mice than uninfected cells. APC(min/+) mice gavaged with F nucleatum developed significantly more colorectal tumors than mice given PBS and had shorter survival times. We found several inflammatory factors to be significantly increased in serum from mice given F nucleatum (interleukin 17F [IL17F], IL21, IL22, and MIP3A). We found 50 miRNAs to be significantly upregulated and 52 miRNAs to be significantly downregulated in CRCs incubated with F nucleatum vs PBS; levels of miR21 increased by the greatest amount (more than 4-fold). Inhibitors of miR21 prevented F nucleatum from inducing cell proliferation and invasion in culture. miR21a(-/-) mice had a later appearance of fecal blood and diarrhea after administration of AOM and DSS, and had longer survival times, compared with control mice. The colorectum of miR21a(-/-) mice had fewer tumors, of smaller size, and the miR21a(-/-) mice survived longer than control mice. We found RASA1, which encodes a RAS GTPase, to be one of the target genes consistently downregulated in cells that overexpressed miR21 and upregulated in cells exposed to miR21 inhibitors. Infection of cells with F nucleatum increased expression of miR21 by activating TLR4 signaling to MYD88, leading to activation of the nuclear factor NFκB. Levels of F nucleatum DNA and miR21 were increased in tumor tissues (and even more so in advanced tumor tissues), compared with non-tumor colon tissues from patients. Patients whose tumors had high amounts of F nucleatum DNA and miR21 had shorter survival times than patients whose tumors had lower amounts.
CONCLUSIONS: We found infection of CRC cells with F nucleatum to increase their proliferation, invasive activity, and ability to form xenograft tumors in mice. F nucleatum activates TLR4 signaling to MYD88, leading to activation of the nuclear factor NFκB and increased expression of miR21; this miRNA reduces levels of the RAS GTPase RASA1. Patients with both high amount of tissue F nucleatum DNA and miR21 demonstrated a higher risk for poor outcomes.
Yongzhi Yang,Wenhao Weng,Junjie Peng,Leiming Hong
Sanjun Cai,Ajay Goel,Huanlong Qin,Yanlei Ma
Yongzhi Yang,Wenhao Weng,Junjie Peng,Leiming Hong,Lei Yang,Yuji Toiyama,Renyuan Gao,Minfeng Liu,Mingming Yin,Cheng Pan,Hao Li,Bomin Guo,Qingchao Zhu,Qing Wei,Mary-Pat Moyer,Ping Wang,Sanjun Cai,Ajay Goel,Huanlong Qin,Yanlei Ma