Mir-145 Breast Tumor Growth And New Blood Vessels Generated

Cancer is the No. 1 killer in human. Breast caner is a prevalent carcinoma worldwide with the second highest mortality rate after lung cancer in women. Due to the heterogeneity of tumors, the molecular basis of breast cancer still remains to be elucidated. Thus, it is very important to explore the further mechanism of breast cancer for diagnosis and prevention. MicroRNAs (miRNAs) are evolutionarily conserved, endogenous, small, noncoding RNA molecules of about 22 nuleotides in length that function as posttranscriptional gene regulators. MiRNAs inhibit the expression of their target genes by affecting the translation or stability of mRNAs by binding to a target site in the mRNA 3'-UTR region. Recent study indicates that miRNAs not only are involved in animal development, but also are associated with cancer development. Accumulating evidence suggests that their expressions in cancer tissues are different from the normal tissue. MicroRNAs act as oncogenes or tumor suppressors in human cancers by targeting some special down stream genes correspondingly either tumor suppressors or oncogenes, respectively.MiR-145 is down-regulated in several kinds of human cancers such as colorectal cancer, esophageal cancer, bladder cancer, and breast cancer. Recent studies have revealed some direct targets of miRNA-145. However, the direct role of miR-145 in cancer cell proliferation, tumor growth and angiogenesis is unknown yet.The process of tumor angiogenesis is induced when tumor starts to grow. The vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis. Inhibition of tumor angiogenesis can specifically kill tumor, but not normal tissues. Our study is to explore whether miR-145 suppresses breast cancer angiogenesis, to identify the direct targets of miR-145 in tumor growth and angiogenesis, and to elucidate the mechanism of miR-145 in suppressing tumor growth and angiogenesis. MiR-145 expression levels in breast cancer tissuesThe expression level of miR-145 was examined by stem-loop quantitative real-time PCR analysis. We observed that miR-145 is consistently down-regulated in breast cancer samples.Generation of stable cell lines expressing miR-145To investigate the role of miR-145 in tumorigenesis and angiogenesis in vivo, stable cell lines lenti-miR-145-MCF-7 and lenti-miR-145-MDA-MB-231 were generated. MiR-145 levels in Lenti-miR-145-MDA-MB-231 cells and lenti-miR-145-MCF-7 were significantly higher than those in the control stable cell linesMiR-145 inhibits breast cancer cell line proliferation, tumor growth, and angiogenesisMiR-145 decreased breast cancer cell proliferation, and anchorage-dependent or anchorage-independent colony formation. VEGF mRNA levels were lower in breast cancer cell lines with miR-145 over-expression when compared to the negative control.When the cells were transfected with VEGF reporter vector containing full length promoter, luciferase activities were lower in lenti-miR-145-MDA-MB-231 and lenti-miR-145-MCF-7 cells when compared with those of their control cell lines. To determine whether miR-145 inhibited VEGF transcriptional activation through the HIF-1a binding site, VEGF reporter plasmids, pMAP11wild type and pMAP11mutant containing three base pair substitutions at the HIF-1a binding site, were transfected into the cells as above. MiR-145 inhibited HIF-1a wild type, but not HIF-1a mutant reporter luciferase activities. These results suggest that miR-145 may inhibit tumor growth and angiogenesis through HIF-1a and VEGF expression. Bioinformatics analysis indicated there are paring sites between miR-145 andBCL2L2 or DDR1. We found that miR-145 significantly repressed the activities of BCL2L2 and DDR1 3'-untranslated region reporters. Next we showed that DDR1 and BCL2L2 proteins levels in breast cancer cells were lower with the miR-145 overexpression. MiR-145 also promoted cancer cell apoptosis through inhibiting BCL2L2, which is an anti-apoptosis protein. DDR1 overexpression was detected in a variety of human cancers. A recent study has found that DDR1 was highly expressed in prostate cancer cells and preneoplastic lesions, and contributed to prostate cancer invasion. When we used siRNA knock down DDR1, phosphorylated caspase-3 were elevated. This showed DDR1 is necessary for breast cancer cell survival. To our knowledge, miR-145 is the first microRNA that targets oncogene DDR1.MiR-145 inhibits VEGF, and PI3K signaling targeting IRS1, IGF-1R, and N-RasWe found that miR-145 reduced VEGF mRNA level through HIF-1αand PI3K/AKT pathway. We showed that miR-145 targets IGF-1R and IRS1 that are involved in activating PI3K. IRS1 protein levels were also lower in miR-145 transfected MCF-7 and MDM-MB-231 cells. The sequence search showed that there were some sequence matches between N-Ras and miR-145. We found that N-Ras is also miR-145 target in breast cancer cells.MiR-145 reduced breast cancer tumor growth and angiogenesisWe injected miR-145-expressing MDA-MB-231 cells into nude mice, and found iR-145 reduced tumor growth when compared to the negative control. After 21 days, tumors with Lenti-miR-145-MDA-MB-231 cells had much lower fluorescent intensity than those with Lenti-scr-MDA-MB-231 cells. MiR-145-expressing tumors also had smaller tumor sizes, lower tumor weight and volume. Histological MiR-145 inhibits cell proliferation in vitro by targeting BCL2L2 and DDR1 study showed that tumor derived from miR-145-expressing cells had a lower level of PCNA antigen than the negative control. Meanwhile, miR-145-expressing tumors had lower DDR1 and BCL2L2 levels. MiR-145 reduced breast tumor growth and angiogenesis through decreasing its target protein levels.