You-Chin Lin

miR-149* induces apoptosis by inhibiting Akt1 and E2F1 in human cancer cells

microRNAs (miRNAs) play vital roles in several biological processes, including apoptosis, by negatively regulating the expression of target genes. The molecular mechanisms of the key survival signal, Akt family, have been widely explored. However, it remains to be ascertained whether Akt1, the predominant isoform in most tissue, is a direct target of miRNA. In this study, we identified Akt1 and E2F1 to be two direct targets of miR‐149* and b‐Myb to be an indirect target by reporter assays and Western blot analyses. Ectopic expression of miR‐149*‐induced apoptosis in Be2C, a neuroblastoma cell line, and in HeLa cells. Silencing of Akt1 or E2F1 expression also led to similar apoptotic changes in these two cell lines, suggesting that the pro‐apoptotic effects of miR‐149* were exerted by repressing Akt1 and E2F1 expressions. Importantly, analysis of primary neuroblastoma samples revealed a significant inverse correlation of miR‐149* with E2F1 expressions (P = 0.026). Interestingly, using the reporter assays, excess miR‐149 introduced by transfection to simulated its preponderance in the in vivo condition, could not overcome the repressive function of miR‐149* on the target genes. This implies that the pro‐apoptotic function of miR‐149* may not be dampened by its predominant cognate, miR‐149, in vivo. Our findings not only provided the first evidence that Akt1 is a direct target of miRNA but also demonstrated that miR‐149* is a pro‐apoptotic miRNA by repressing the expression of Akt1 and E2F1.

c-Myb Is an Evolutionary Conserved miR-150 Target and miR-150/c-Myb Interaction Is Important for Embryonic Development

Human c-Myb proto-oncogene is highly expressed in hematopoietic progenitors as well as leukemia and certain solid tumor. However, the regulatory mechanisms of its expression and biological functions remain largely unclear. Recently, c-Myb has been shown to be targeted by microRNA-150 (miR-150) which thereby controls B cell differentiation in mice. In this study, we demonstrated that c-Myb is an evolutionary conserved target of miR-150 in human and zebrafish, using reporter assays. Ectopic expression of miR-150 in breast cancer and leukemic cells repressed endogenous c-Myb at both messenger RNA (mRNA) and protein levels. Among several leukemia cell lines, primary leukemia cells, and normal lymphocytes, expression levels of miR-150 inversely correlated with c-Myb. The miR-150 overexpression or c-Myb silencing in zebrafish zygotes led to similar and serious phenotypic defects in zebrafish, and the phenotypic aberrations induced by miR-150 could be reversed by coinjection of c-Myb mRNA. Our findings suggest that c-Myb is an evolutionally conserved target of miR-150 and miR-150/c-Myb interaction is important for embryonic development and possibly oncogenesis.

Concordant and Discordant Regulation of Target Genes by miR-31 and Its Isoforms

It has been shown that imprecise cleavage of a primary or precursor RNA by Drosha or Dicer, respectively, may yield a group of microRNA (miRNA) variants designated as “isomiR”. Variations in the relative abundance of isoforms for a given miRNA among different species and different cell types beg the question whether these isomiRs might regulate target genes differentially. We compared the capacity of three miR-31 isoforms (miR-31-H, miR-31-P, and miR-31-M), which differ only slightly in their 5′- and/or 3′-end sequences, to regulate several known targets and a predicted target, Dicer. Notably, we found isomiR-31s displayed concordant and discordant regulation of 6 known target genes. Furthermore, we validated a predicted target gene, Dicer, to be a novel target of miR-31 but only miR-31-P could directly repress Dicer expression in both MCF-7 breast cancer cells and A549 lung cancer cells, resulting in their enhanced sensitivity to cisplatin, a known attribute of Dicer knockdown. This was further supported by reporter assay using full length 3′-untranslated region (UTR) of Dicer. Our findings not only revealed Dicer to be a direct target of miR-31, but also demonstrated that isomiRs displayed similar and disparate regulation of target genes in cell-based systems. Coupled with the variations in the distribution of isomiRs among different cells or conditions, our findings support the possibility of fine-tuning gene expression by miRNAs.

The prognostic significance of RUNX2 and miR-10a/10b and their inter-relationship in breast cancer

BackgroundThe major cancer related mortality is caused by metastasis and invasion. It is important to identify genes regulating metastasis and invasion in order to curtail metastatic spread of cancer cells.MethodsThis study investigated the association between RUNX2 and miR-10a/miR-10b and the risk of breast cancer relapse. Expression levels of RUNX2 and miR-10a/b in108 pairs of tumor and non-tumor tissue of breast cancer were assayed by quantitative PCR analysis and evaluated for their prognostic implications.ResultsThe median expression levels of RUNX2 and miR-10b in tumor tissue normalized using adjacent non-tumor tissue were significantly higher in relapsed patients than in relapse-free patients. Higher expression of these three genes were significantly correlated with the hazard ratio for breast cancer recurrence (RUNX2: 3.02, 95% CI = 1.50 ~ 6.07; miR-10a: 2.31, 95% CI = 1.00 ~ 5.32; miR-10b: 3.96, 95% CI = 1.21 ~ 12.98). The joint effect of higher expression of all three genes was associated with a hazard ratio of 12.37 (95% CI = 1.62 ~ 94.55) for relapse. In a breast cancer cell line, RUNX2 silencing reduced the expression of miR-10a/b and also impaired cell motility, while RUNX2 overexpression elicited opposite effects.ConclusionsThese findings indicate that higher expression of RUNX2 and miR-10a/b was associated with adverse outcome of breast cancer. Expression levels of RUNX2 and miR-10a/b individually or jointly are potential prognostic factors for predicting breast cancer recurrence. Data from in vitro studies support the notion that RUNX2 promoted cell motility by upregulating miR-10a/b.

The synthetic caged Garcinia xanthone cluvenone induces cell stress and apoptosis and has immune modulatory activity

Several caged Garcinia xanthone natural products have potent bioactivity and a documented value in traditional Eastern medicine. Previous synthesis and structure activity relationship studies of these natural products resulted in the identification of the pharmacophore represented by the structure of cluvenone. In the current study, we examined the anticancer activity of cluvenone and conducted gene expression profiling and pathway analyses. Cluvenone was found to induce apoptosis in T-cell acute lymphoblastic leukemia cells (EC50 = 0.25 μmol/L) and had potent growth-inhibitory activity against the NCI60 cell panel, including those that are multidrug-resistant, with a GI50 range of 0.1 to 2.7 μmol/L. Importantly, cluvenone was approximately 5-fold more potent against a primary B-cell acute lymphoblastic leukemia compared with peripheral blood mononuclear cells from normal donors, suggesting that it has significant tumor selectivity. Comparison of cluvenones growth-inhibitory profile to those in the National Cancer Institute database revealed that compounds with a similar profile to cluvenone were mechanistically unlike known agents, but were associated with cell stress and survival signaling. Gene expression profiling studies determined that cluvenone induced the activation of mitogen-activated protein kinase and NrF2 stress response pathways. Furthermore, cluvenone was found to induce intracellular reactive oxygen species formation. Lastly, the modulation in the expression of several genes associated with T cell and natural killer cell activation and function by cluvenone suggests a role as an immune-modulator. The current work highlights the potential of cluvenone as a chemotherapeutic agent and provides support for further investigation of these intriguing molecules with regard to mechanism and targets. Mol Cancer Ther; 9(11); 2869–78. ©2010 AACR.

Abstract 1947: FAM129B regulates cell proliferation and activates NF-κB and Nrf2 by inactivating KEAP1 in cancer cells

The Family with sequence similarity 129 (FAM129) contains three membrane proteins, FAM129A, B and C. FAM129A (also called Niban) and FAM129C (also called BCNP1) have been found to be up-regulated in thyroid tumors and chronic lymphocytic leukemia, respectively. Although phosphorylation of FAM129B has been suggested to play a role in controlling melanoma cell invasion, the molecular mechanism of FAM129B in tumorigenesis remains largely unknown. In this study, we first identified FAM129B as one of proteins with altered phosphorylation status in breast cancer stem cells by the phosphor-mass spectrum technique. Silencing of FAM129B expression in breast cancer cell line, MDA-MB-231, led to decreased proliferation, along with flattened and rounded cell morphology. By mammalian two-hybrid system and FRET (fluorescence resonance energy transfer) assay, KEAP1 (kelch-like ECH-associated protein 1) was detected to interact with FAM129B. KEAP1 has been reported to function as a substrate adaptor protein for Cul3-dependent E3 ligase complex to repress IKKβ and transcription factor Nrf2. We indeed observed that the expression of NF-κB and Nrf2 downstream genes, FN1 and NQO1, was down-regulated in FAM129B-silenced cells by qPCR analysis. On the other hand, FAM129B together with either IKKβ or Nrf2 synergistically activated NF-κB and Nrf2 promoter activity by reporter assays. Taken together, our results suggest that FAM129B might play an important role in the regulation of cell proliferation in cancers by inactivating KEAP1 which in turn activates Nrf2 and IKKβ activities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1947. doi:10.1158/1538-7445.AM2011-1947

Abstract 3983: MiR-31 isoforms differentially regulate expression of dicer in cancer cells

MicroRNAs (miRNAs) are a group of small non-coding RNAs involved in several important biological processes. Recent studies have implied that imprecise cleavage of primary or precursor RNA by Drosha or Dicer respectively could produce miRNAs with variations at 5’-and/or 3’-ends, which were termed “isomiR”. The existence of isomiRs may increase the complexity of miRNA biology, although their functions have not been explored. This is further complicated by the observation of variations in the population of isomer for a given miRNA among different species as well as among different types of cells within the same species (human cells), which raises the issue whether these isomers might exert differential effects on target gene regulation. Here, we utilized three miR-31 isoforms (miR-31-H, miR-31-P, and miR-31-M) which differ from each other only slightly in their sequences at both ends, to compare their capacity to regulate a predicted target, Dicer. Interestingly, we found only miR-31-P was able to directly repress Dicer expression in both breast cancer MCF-7 cells and lung cancer A549 cells, resulting in their enhanced sensitivity to cisplatin, which is known to be associated with Dicer knockdown. In line with this, the amount of miR-31-P bound to the RNA-induced silencing complex (RISC) was greater than that of miR-31-H and -M by RNA-CHIP analysis. Furthermore, evaluation of other known miR-31 target genes showed that expression of some target genes was indeed differentially regulated by miR-31 isoforms. Our findings not only revealed Dicer to be a direct target of miR-31, but also demonstrated the disparate functions of isomiRs for the fist time, providing a novel functional mechanism of fine-tuning of gene expression by miRNAs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3983. doi:10.1158/1538-7445.AM2011-3983

Abstract 2038: miR-149* induces apoptosis by inhibiting Akt1 and E2F1 in human cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC microRNAs (miRNAs) play vital roles in several biological processes, including apoptosis, by negatively regulating the expression of target genes. The molecular mechanisms of the key survival signal, Akt family, have been extensively investigated, but whether is regulated by miRNA remains unknown. In a recent study of neuroblastoma samples, we found the expression of miR-149 to be lower in the high-risk group than the low- and intermediate-risk groups. Upon transfection of Be2C, a neuroblastoma cell line and HeLa cells with miR-149 oligo, no changes in cell growth were observed. On the other hand, ectopic expression of miR-149*, the cognate miRNA of miR-149, inhibited the growth and induced apoptosis in these two cancer cell lines. By TargetScan screening, Akt1, the predominant Akt isoform in most tissue, E2F1, and b-Myb were predicted as possible target genes of miR-149*. In Be2C and HeLa cells transfected with miR-149* oligo, reduced expressions of Akt1, E2F1, and b-Myb at protein level were detected by Western blotting, and decreased mRNA levels of Akt1 and E2F1 were also noted by RT-qPCR. Using luciferase reporter assay, the target sites were verified in the 3′UTRs of Akt1 and E2F1, but not in b-Myb, suggesting that Akt1 and E2F1 were the direct target and b-Myb was an indirect target of miR-149*. Silencing of Akt1 or E2F1 expression also led to similar apoptotic changes as miR-149* transfection in these two cell lines, suggesting that the pro-apoptotic effects of miR-149* was exerted at least in part by repressing Akt1 and E2F1 expressions. Importantly, analysis of primary neuroblastoma samples (n=56) revealed a significant inverse correlation of miR-149* with E2F1 expressions (p=0.026). Furthermore in stage1 samples, the mean expression level of miR-149* was significantly higher than in stage 2/3 (p<0.05), and that of E2F1 appeared to be lower. The results provide further support of an important role of miiR-149* in regulation of E2F1, especially in stage1 neuroblastoma tumors. Interestingly, using the reporter assays, excess miR-149 introduced by transfection to simulated its preponderance in the in vivo condition, could not overcome the repressive function of miR-149* on the target genes. This implies that the pro-apoptotic function of miR-149* may not be dampened by its predominant cognate, miR-149, in vivo. Our findings not only provided the first evidence that Akt1 is a direct target of a miRNA but also demonstrated that miR-149* is a pro-apoptotic miRNA by repressing the expression of Akt1 and E2F1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2038.