407004

MIR22

microRNA 22;MIR22;microRNA 22

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17p13.3

ncRNA

metastasis in breast cancer carries a disproportionately worse prognosis than localized primary disease. To identify microRNAs (miRNA) involved in metastasis, the expression of 254 miRNAs was measured across the following cell lines using microarray analysis: MDA-MB-231 breast cancer cells, cells that grew as a tumor in the mammary fat pad of nude mice (TMD-231), metastatic disease to the lungs (LMD-231), bone (BMD-231) and adrenal gland (ADMD-231). A brain-seeking variant of this cell line (231-BR) was used additionally in validation studies. Twenty miRNAs were upregulated and seven were downregulated in metastatic cancer cells compared with TMD-231 cells. The expression of the tumor suppressor miRNAs let-7 and miR-22 was consistently downregulated in metastatic cancer cells. These metastatic cells expressed higher levels of putative/proven miR-22 target oncogenes ERBB3, CDC25C and EVI-1. Introduction of miR-22 into cancer cells reduced the levels of ERBB3 and EVI-1 as well as phospho-AKT, an EVI-1 downstream target. The miR-22 primary transcript is located in the 5'-untranslated region of an open reading frame C17orf91, and the promoter/enhancer of C17orf91 drives miR-22 expression. We observed elevated C17orf91 expression in non-basal subtype compared with basal subtype breast cancers. In contrast, elevated expression of EVI-1 was observed in basal subtype and was associated with poor outcome in estrogen receptor-negative breast cancer patients. These results suggest that metastatic cancer cells increase specific oncogenic signaling proteins through downregulation of miRNAs. Identifying such metastasis-specific oncogenic pathways may help to manipulate tumor behavior and aid in the design of more effective targeted therapies.

tumor cells metastasize to distant organs through genetic and epigenetic alterations, including changes in microRNA (miR) expression. Here we find miR-22 triggers epithelial-mesenchymal transition (EMT), enhances invasiveness and promotes metastasis in mouse xenografts. In a conditional mammary gland-specific transgenic (TG) mouse model, we show that miR-22 enhances mammary gland side-branching, expands the stem cell compartment, and promotes tumor development. Critically, miR-22 promotes aggressive metastatic disease in MMTV-miR-22 TG mice, as well as compound MMTV-neu or -PyVT-miR-22 TG mice. We demonstrate that miR-22 exerts its metastatic potential by silencing antimetastatic miR-200 through direct targeting of the TET (Ten eleven translocation) family of methylcytosine dioxygenases, thereby inhibiting demethylation of the mir-200 promoter. Finally, we show that miR-22 overexpression correlates with poor clinical outcomes and silencing of the TET-miR-200 axis in patients. Taken together, our findings implicate miR-22 as a crucial epigenetic modifier and promoter of EMT and breast cancer stemness toward metastasis.

Breast cancer is the most common cancer in women for which the metastatic process is still poorly understood. CD147 is upregulated in breast cancer and has been associated with tumor progression, but little is known about its regulatory mechanisms. In this study, we demonstrated that CD147 was overexpressed in breast cancer tissues and cell lines, and the high expression correlated with tumor invasion and metastasis. We also found that the transcription factors Sp1 and c-Myc could bind to the CD147 promoter and enhance its expression. The CD147 mRNA has a 748-bp 3'-untranslated region (UTR) with many miRNA target sites, suggesting possible regulation by miRNAs. We discovered that miR-22 repressed CD147 expression by directly targeting the CD147 3'UTR. We also determined that miR-22 could indirectly participate in CD147 modulation by downregulating Sp1 expression. miR-22 could form an autoregulatory loop with Sp1, which repressed miR-22 transcription by binding to the miR-22 promoter. Together with the c-Myc-mediated inhibition of miR-22 expression, our investigation identified a miR-22/Sp1/c-Myc network that regulates CD147 gene transcription. In addition, miR-22 overexpression suppressed breast cancer cell invasion, metastasis, and proliferation by targeting CD147 in vitro and in vivo. Furthermore, we found that miR-22 was significantly downregulated in breast cancer tissues and that its expression was inversely correlated with the tumor-node-metastasis stage and lymphatic metastasis in patients. Our study provides the first evidence that an miR-22/Sp1/c-Myc network regulates CD147 upregulation in breast cancer and that miR-22 represses breast cancer invasive and metastatic capacities.

OBJECTIVE: expression of microRNA-22 (miR-22) and ezrin protein (a membrane-cytoskeleton linking protein) in hepatocellular carcinoma (HCC) was investigated. METHODS: Specimens of HCC and paracancerous tissue (control; ~5 cm away from tumour tissue) were collected from 192 patients. miR-22 expression was detected by real-time polymerase chain reaction; ezrin protein expression in tumour tissue was assessed immunohistochemically. Associations between miR-22 expression and clinicopathological features of HCC and ezrin expression were analysed. RESULTS: miR-22 expression was lower in HCC tissue than in paracancerous tissue samples (median relative expression 0.676 versus 1.000 for control tissue). expression of miR-22 was significantly associated with histological differentiation (relative expression 0.431 for lower grades of differentiation versus 0.918 for higher grades), and was associated with lymphatic metastasis (relative expression 0.518 if metastasis was present, 0.919 if absent). Survival time was shorter in patients with low miR-22 expression than in those with high expression (31.0 +/- 2.6 versus 52.2 +/- 5.1 months). There was a significant negative correlation between the expression of miR-22 and that of ezrin. CONCLUSIONS: miR-22 is downregulated in HCC and its expression is associated with the differentiation, metastasis and prognosis of the carcinoma. Ezrin is a potential regulatory protein of miR-22.

OBJECTIVE: to screen microRNA (miRNA) that inhibit expression of the metastasis-related gene ezrin in ovarian cancer cells and explore their correlation to the invasion and metastasis of ovarian cancer. METHODS: the differential expression of ezrin in two paired high-metastatic and low-metastatic cell lines were examined by real time reverse transcription (RT)-PCR and western blot. A functional screen with microarray was employed to identify miRNA that were differentially expressed between SKOV3 and SKOV3ip cell lines. Three programs, TARGETSCAN (http://www.targetscan.org), MICROCOSM (http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/) and PICTAR (http://www.pictar.mdc-berlin.de), were employed to identify all miRNA, which may inhibit the expression of ezrin and were differentially expressed between SKOV3 and SKOV3ip cells. To test the repressive potential of these miRNA, synthetic mimetics were transfected individually into SKOV3ip cells and endogenous ezrin expression levels monitored by western blot and real-time RT-PCR. RESULTS: (1) the mRNA average level of ezrin were (81.74 +/- 5.34)-fold higher expression level in SKOV3ip versus SKOV3 cells (P < 0.01), while (2.61 +/- 0.14)-fold in HO-8910PM versus HO-8910 cells (P < 0.01). Elevated protein level of ezrin were observed in SKOV3ip cells compared with that in SKOV3 cells, and the same that in HO-8910PM cells compared with HO-8910 cells. Paired SKOV3 and SKOV3ip cells were employed to study the more significant difference in ezrin expression between them. (2) By a functional screen using miRNA microarray combined with bioinformatics analysis, the miR-183 and miR-22 were indentified as two candidate miRNA, which may have the potential regulatory role in ezrin expression. Real time RT-PCR assays revealed that miR-183 and miR-22 were, respectively, an average of (5.84 +/- 0.66)-fold and (6.67 +/- 0.67)-fold higher expression level in SKOV3ip versus SKOV3 cells (P < 0.01), which were in agreement with the microarray data. A subsequent validation by western blot and real time RT-PCR revealed that over-expression of miR-183 and miR-22 could both lead to an obvious decrease in ezrin protein level, while there were not signicant difference in the level of ezrin mRNA (P > 0.05). CONCLUSION: increased expression of miR-183 and miR-22 may both repress the protein level of ezrin, indicating that miR-183 and miR-22 may bear a potential role in inhibiting ovarian cancer metastasis in a ezrin-mediated way.