442904

MIR335

microRNA 335;MIR335;microRNA 335

-

7q32.2

ncRNA

BACKGROUND: microRNA (miRNA) functions broadly as post-transcriptional regulators of gene expression, and disproportionate miRNAs can result in dysregulation of oncogenes in cancer cells. We have previously shown that gastrin-releasing peptide receptor (GRP-R) signaling regulates tumorigenicity of neuroblastoma cells. Herein, we sought to characterize miRNA profile in GRP-R silenced neuroblastoma cells, and to determine the role of miRNAs on tumorigenicity and metastatic potential. METHODS: Human neuroblastoma cell lines, BE(2)-C and SK-N-SH, were used for our study. Stably transfected GRP-R silenced cells were assessed for miRNA profiles. Cells were transfected with miR-335, miR-363, or miR-CON, a nontargeting control, and in vitro assays were performed. In vivo functions of miR-335 and miR-363 were also assessed in a spleen-liver metastasis murine model. RESULTS: GRP-R silencing significantly increased expression of miR-335 and miR-363 in BE(2)-C cells. Overexpression of miR-335 and miR-363 decreased tumorigenicity as measured by clonogenicity, anchorage-independent growth, and metastasis determined by cell invasion assay and liver metastasis in vivo. CONCLUSION: We report, for the first time, that GRP-R-mediated tumorigenicity and increased metastatic potential in neuroblastoma are regulated, in part, by miR-335 and miR-363. A better understanding of the anti-tumor functions of miRNAs could provide valuable insights to discerning molecular mechanisms responsible for neuroblastoma metastasis.

A search for general regulators of cancer metastasis has yielded a set of microRNAs for which expression is specifically lost as human breast cancer cells develop metastatic potential. Here we show that restoring the expression of these microRNAs in malignant cells suppresses lung and bone metastasis by human cancer cells in vivo. Of these microRNAs, miR-126 restoration reduces overall tumour growth and proliferation, whereas miR-335 inhibits metastatic cell invasion. miR-335 regulates a set of genes whose collective expression in a large cohort of human tumours is associated with risk of distal metastasis. miR-335 suppresses metastasis and migration through targeting of the progenitor cell transcription factor SOX4 and extracellular matrix component tenascin C. expression of miR-126 and miR-335 is lost in the majority of primary breast tumours from patients who relapse, and the loss of expression of either microRNA is associated with poor distal metastasis-free survival. miR-335 and miR-126 are thus identified as metastasis suppressor microRNAs in human breast cancer.

Post-transcriptional regulators have emerged as robust effectors of metastasis and display deregulated expression through unknown mechanisms. Here, we reveal that the human microRNA-335 locus undergoes genetic deletion and epigenetic promoter hypermethylation in every metastatic derivative obtained from independent patients malignant cell populations. Genetic deletion of miR-335 is a common event in human breast cancer, is enriched for in breast cancer metastases, and also correlates with ovarian cancer recurrence. We furthermore identify miR-335 as a robust inhibitor of tumor reinitiation. We thus implicate the miR-335 locus on 7q32.2 as the first selective metastasis suppressor and tumor initiation suppressor locus in human breast cancer.

Aberrant expression of miR-335 has been frequently reported in cancer studies, suggesting that there is a close correlation between miR-335 and cancer during its development, progression, metastasis and prognosis. The expression of miR-335 in gastric cancer and its effects are not known. Relative expression of miR-335 in 4 gastric cancer cell lines and in 70 gastric cancer tissues was confirmed by real-time quantitative reverse transcriptase-PCR compared with controls. Transwell cell migration and Matrigel invasion assay in vitro and metastasis formation assay in vivo were used to examine the effects of miR-335 expression on gastric cancer cell invasion and metastasis. The effect of miR-335 expression on gastric cancer cell proliferation was estimated by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Luciferase reporter assay and western blot were used to examine the potential target genes and related pathways. Gene silencing with small-interfering RNA was used to examine the effects of target genes on gastric cancer cell invasion. miR-335 was dramatically downregulated in gastric cancer cell lines than in the normal gastric cell line GES-1. Low expression of miR-335 was significantly associated with lymph-node metastasis, poor pT stage, poor pN stage and invasion of lymphatic vessels. Overexpression of miR-335 suppressed gastric cancer cell invasion and metastasis in vitro and in vivo, but has no significant effects on cell proliferation. Furthermore, miR-335 might suppress gastric cancer invasion and metastasis by targeting Bcl-w and specificity protein 1 (SP1). Taken together, our results provide evidence that miR-335 might function as a metastasis suppressor in gastric cancer by targeting SP1 directly and indirectly through the Bcl-w-induced phosphoinositide 3-kinase-Akt-Sp1 pathway. miR-335 showing altered expression at different stages of gastric cancer could be a target for gastric cancer therapies and could be further developed as a potential prognostic factor.

Dysregulated miRNAs in gastric cancer are usually screened by miRNA microarray from clinical samples, however, reports have indicated that results of each miRNA microarray screening are considerably different, and dysregulated miRNAs, especially downregulated miRNAs were contradictory. In view of this, the Human cancer pathway Finder miRNA PCR array was applied to compare 7 gastric cancer cell lines AGS, SGC-7901, MKN-45, MKN-28, MGC-803, BCG-823, and HGC-27 with an immortalized normal gastric cell line, GES-1 in cancer pathway-related miRNA expression profile, followed by qPCR verification, the clinical significance of downregulated miRNAs and the Enriched KEGG pathways and GO terms of their target genes were analyzed. Thirty-eight miRNAs were upregulated, and four miRNAs were downregulated in gastric cancer cell lines. Clinical significance of 4 miRNAs including mir-124-3p, mir-146a-5p, mir-155-5p and mir-335-5p in gastric cancer tissue compared with adjacent non-tumor tissues of 58 patients indicated that the low-expression group of mir-124-3p, mir-146a-5p, mir-155-5p and mir-335-5p showed more extensive lymph node metastasis, lymphatic invasion, venous invasion, high-stage Borrmann type, lymphatic invasion and poor differentiation than that of the high-expression groups, respectively (P<0.05; chi(2) test). Enriched KEGG pathway analyses showed that most of the targeted genes of the 4 miRNAs concentrated on 37 signaling pathways, and were involved in the same pathways related to cancer. Enriched GO terms showed that targeted genes of the 4 miRNAs concentrated on 339 terms, 24 of 339 terms are associated with cancer tumorigenesis. The Human cancer pathway Finder miRNA PCR array could be used to screen dysregulated miRNAs effectively, and 4 screened miRNAs, mir-124-3p, mir-146a-5p, mir-155-5p and mir-335-5p were found to be downregulated in gastric cancer. Clinical significance and bioinformatic analysis on the target genes of these 4 miRNAs indicated that they were deeply involved in tumorigenesis, suggesting roles such as miRNA tumor suppressors in gastric cancer tumorigenesis which could be applied in gastric cancer diagnosis and prognosis.

Post-transcriptional regulators have emerged as robust effectors of metastasis and display deregulated expression through unknown mechanisms. Here, we reveal that the human microRNA-335 locus undergoes genetic deletion and epigenetic promoter hypermethylation in every metastatic derivative obtained from independent patients malignant cell populations. Genetic deletion of miR-335 is a common event in human breast cancer, is enriched for in breast cancer metastases, and also correlates with ovarian cancer recurrence. We furthermore identify miR-335 as a robust inhibitor of tumor reinitiation. We thus implicate the miR-335 locus on 7q32.2 as the first selective metastasis suppressor and tumor initiation suppressor locus in human breast cancer.

Small cell lung cancer (SCLC) is a rapidly progressing, incurable cancer that frequently spreads to bone. New insights are needed to identify therapeutic targets to prevent or retard SCLC metastatic progression. Human SCLC SBC-5 cells in mouse xenograft models home to skeletal and nonskeletal sites, whereas human SCLC SBC-3 cells only pervade nonskeletal sites. Because microRNAs (miRNA) often act as tumor regulators, we investigated their role in preclinical models of SCLC. miRNA expression profiling revealed selective and reduced expression of miRNA (miR)-335 and miR-29a in SBC-5 cells, compared with SBC-3 cells. In SBC-5 cells, miR-335 expression correlated with bone osteolytic lesions, whereas miR-29a expression did not. Overexpression of miR-335 in SBC-5 cells significantly reduced cell migration, invasion, proliferation, colony formation, and osteoclast induction in vitro. Importantly, in miR-335 overexpressing SBC-5 cell xenografts (n = 10), there were minimal osteolytic lesions in the majority of mice and none in three mice. expression of RANK ligand (RANKL) and insulin-like growth factor-I receptor (IGF-IR), key mediators of bone metastases, were elevated in SBC-5 as compared with SBC-3 cells. Mechanistically, overexpression of miR-335 in SBC-5 cells reduced RANKL and IGF-IR expression. In conclusion, loss of miR-335 promoted SCLC metastatic skeletal lesions via deregulation of IGF-IR and RANKL pathways and was associated with metastatic osteolytic skeletal lesions. IMPLICATIONS: These preclinical findings establish a need to pursue the role of miR-335 in human SCLC with metastatic skeletal disease.