Functions Of miR-128 Research Articles

Identification of miR-128 Target mRNAs That Are Expressed in B Cells Using a Modified Dual Luciferase Vector.

MicroRNAs (miRNAs) are 21-25 nucleotide long non-coding ribonucleic acids that modulate gene expression by degrading transcripts or inhibiting translation. The miRNA miR-128, originally thought to be brain-specific, was later also found in immune cells. To identify a valuable immune cell model system to modulate endogenous miR-128 amounts and to validate predicted miR-128 target mRNAs in B cells, we first investigated miR-128 expression using Northern blot analysis in several cell lines representing different stages of B cell development. The results showed that only primary brain cells showed significant levels of mature miR-128. To study the function of miR-128 in immune cells, we modified dual luciferase vectors to allow easy transfer of 3' UTR fragments with predicted miR-128 binding sites from widely used single to dual luciferase vectors. Comparison of in silico predicted miR-128-regulated mRNAs in single and dual luciferase constructs yielded similar results, validating the dual luciferase vector for miRNA target analysis. Furthermore, we confirmed miR-128-regulated mRNAs identified in silico and in vivo using the Ago HITS-CLIP technique and known to be expressed in B cells using the dual luciferase assay. In conclusion, this study provides new insights into the expression and function of miR-128 by validating novel target mRNAs expressed in B cells and identifying additional pathways likely controlled by this miRNA in the immune system.

The role of miRNA-128 in the development and progression of gastrointestinal and urogenital cancer.

Increasing data have shown the significance of various miRNAs in malignancy. In this regard, parallel to its biological role in normal tissues, miRNA-128 (miR-128) has been found to play an essential immunomodulatory function in the process of cancer initiation and development. The occurrence of the aberrant expression of miR-128 in tumors and the unique properties of miRNAs raise the prospect of their use as biomarkers and the next generation of molecular anticancer therapies. The function of miR-128 in malignancies such as breast, prostate, colorectal, gastric, pancreatic, esophageal, cervical, ovarian and bladder cancers and hepatocellular carcinoma is discussed in this review. Finally, the effect of exosomal miR-128 on cancer resistance to therapeutics and cancer immunotherapy in certain malignancies is highlighted.

Fine-tuning of the PAX-SIX-EYA-DACH network by multiple microRNAs controls embryo myogenesis

MicroRNAs (miRNAs), short non-coding RNAs, which act post-transcriptionally to regulate gene expression, are of widespread significance during development and disease, including muscle disease. Advances in sequencing technology and bioinformatics led to the identification of a large number of miRNAs in vertebrates and other species, however, for many of these miRNAs specific roles have not yet been determined. LNA in situ hybridisation has revealed expression patterns of somite-enriched miRNAs, here we focus on characterising the functions of miR-128. We show that antagomiR-mediated knockdown (KD) of miR-128 in developing chick somites has a negative impact on skeletal myogenesis. Computational analysis identified the transcription factor EYA4 as a candidate target consistent with the observation that miR-128 and EYA4 display similar expression profiles. Luciferase assays confirmed that miR-128 interacts with the EYA4 3′UTR. In vivo experiments also suggest that EYA4 is regulated by miR-128. EYA4 is a member of the PAX-SIX-EYA-DACH (PSED) network of transcription factors. Therefore, we identified additional candidate miRNA binding sites in the 3′UTR of SIX1/4, EYA1/2/3 and DACH1. Using the miRanda algorithm, we found sites for miR-128, as well as for other myogenic miRNAs, miR-1a, miR-206 and miR-133a, some of these were experimentally confirmed as functional miRNA target sites. Our results reveal that miR-128 is involved in regulating skeletal myogenesis by directly targeting EYA4 with indirect effects on other PSED members, including SIX4 and PAX3. Hence, the inhibitory effect on myogenesis observed after miR-128 knockdown was rescued by concomitant knockdown of PAX3. Moreover, we show that the PSED network of transcription factors is co-regulated by multiple muscle-enriched microRNAs.

MiRNA-128 and MiRNA-142 Regulate Tumorigenesis and EMT in Oral Squamous Cell Carcinoma Through HOXA10.

BackgroundOral squamous cell carcinoma (OSCC) accounts for more than 90% of all oral cavity cancers, and the 5-year survival rate for OSCC patients remains unsatisfactory. MiRNA-128/miRNA-142 has been reported to work as a tumor suppressor in diverse tumors. However, the biological function of miR-128/miR-142 in OSCC is still unknown.MethodsThe expression of miR-128/miR-142 and homeobox A10 (HOXA10) in OSCC tissues and cells was measured by quantitative real-time polymerase chain reaction (RT-qPCR). The effects of miR-128/miR-142 or HOXA10 on proliferation, migration, invasion and apoptosis were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), transwell and flow cytometry assays, respectively. The expression levels of epithelial–mesenchymal transition (EMT)-associated proteins (E-cadherin, N-cadherin and Vimentin), proliferation-associated protein ki-67 and HOXA10 were detected by Western blot assay. The interaction between HOXA10 and miR-128/miR-142 was predicted by TargetScan, and then confirmed by dual-luciferase reporter assay.ResultsMiR-128/miR-142 was downregulated in OSCC tissues and cells. Overexpression of miR-128/miR-142 inhibited proliferation, migration, invasion and EMT and induced apoptosis in OSCC cells. HOXA10 as the target of miR-128/miR-142 was verified in OSCC cells. Knockdown of HOXA10 also repressed proliferation, migration, invasion and EMT and boosted apoptosis in OSCC cells. Upregulation of miR-128/miR-142 hindered the expression level of HOXA10, while introduction of HOXA10 weakened the effect.ConclusionMiR-128/miR-142 suppressed OSCC tumorigenesis and metastasis by targeting HOXA10, providing a new promising therapeutic approach for OSCC patient diagnosis and treatment.

MicroRNA-128 inhibits proliferation and invasion of glioma cells by targeting COX-2

MicroRNAs (miRNA), a class of small noncoding RNAs, regulates message RNA (mRNA) by targeting the 3′-untranslated region (3′-UTR) resulting in suppression of gene expression. In this study, we identified the expression and function of miR-128, which was found to be downregulated in glioma tissues and glioma cells by real time PCR. Overexpression of miR-128 mimics into LN229 and U251 cells could inhibit proliferation and invasion of glioma cells. However, the inhibitory effects of miR-128 mimics on the invasion and proliferation of glioma cells were reversed by overexpression of cyclooxygenase-2 (COX-2). Our data showed that COX-2 was a candidate target of miR-128. Luciferase activity of 3′-UTR of COX-2 was reduced in the presence of miR-128. Additionally, miR-128 obviously decreased COX-2 mRNA stability determined by real time PCR. Contrarily, we found that miR-128 inhibitor significantly increased the COX-2 mRNA expression, and elevated the protein expression of MMP9 and ki67, and promoted the proliferation of glioma cells. Furthermore, luciferase activity of the 3′-UTR was upregulated by miR-128 inhibitor. All of these results supported that miR-128 was a direct regulator of COX-2. Further studies proved that COX-2 was elevated in glioma tissues and its expression was negatively correlated with the levels of miR-128. These findings may establish miR-128 as a new potential target for the treatment of patients with gliomas.

Abstract 2668: The exosomes derived from BM-hMSCs home to glioma and deliver synthetic microRNA mimics after systemic administration

Abstract Glioblastoma is the most malignant type of glioma (World Health Organization grade 4) and has a median survival time of only 14.6 months even after the standard treatment of surgical resection followed by radiotherapy with concomitant and adjuvant temozolomide administration. Although some novel and promising anti-glioma agents have been developed, therapeutic advancement has remained disappointing due to the lack of efficient delivery tools. Here we focused on exosomes derived from mesenchymal stem cells (MSCs) as carriers to deliver biological therapeutic agents for the treatment of gliomas. These exosomes are expected to cross the blood-brain barrier and selectively home to the glioma tissue, similar to the parental cells (MSCs). Additionally, MSCs could produce sufficient amounts of exosomes on a clinically applicable scale. Thus, MSC exosomes present a potentially ideal cell-free delivery vehicle for glioma therapy. We hypothesized that exosomes derived from human bone marrow-derived MSCs (BM-hMSCs) can target gliomas and can be used as delivery vehicles following systemic administration. In this study, we employed microRNA (miR-128) as the therapeutic cargo, and exosomes were isolated from a cultured media of BM-hMSCs using ultracentrifugation. At first, exosomes labeled with fluorescent dye were incubated with glioma cells, and exosomes taken up by glioma cells were evaluated by confocal fluorescence microscopy and flow cytometry. Labeled exosomes were also systemically injected into a glioma xenograft model and the ability to home to glioma tissue was evaluated by an in vivo imaging system and histological study. The results showed that MSC exosomes can be taken up by glioma cells and can home to glioma tissues. Next, we cultured miR-128-enriched MSC exosomes with glioma cells. To check the function of miR-128 delivered to glioma cells by MSC exosomes, the down-regulation of the BMI-1 target gene of miR128 was evaluated using real-time polymerase chain reaction and western blot analyses. We also systemically injected these exosomes into glioma xenograft models and evaluated the function of miR-128 by analyzing the downregulation of BMI-1 at the tumor site and the animal survival time in vivo. We confirmed that miR-128 delivered by MSC exosomes could function at the target site both in vitro and in vivo. These results indicate that MSC exosomes may be an ideal delivery vehicle for the treatment of gliomas, especially for microRNA replacement therapy. Citation Format: Shinji Yamashita, Tal Shahar, Anwar Hossain, Brittany Parker Kerrigan, Joy Gumin, Shoudong Li, Feng Gao, Kouji Yamasaki, Yuzaburo Shimizu, Frederick F. Lang. The exosomes derived from BM-hMSCs home to glioma and deliver synthetic microRNA mimics after systemic administration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2668.

MicroRNA-128 regulates the proliferation and differentiation of bovine skeletal muscle satellite cells by repressing Sp1.

MicroRNAs (miRNAs) play essential roles in muscle cell proliferation and differentiation. The muscle-specific miRNAs miR-1 and miR-206 have been shown to regulate muscle development and promote myogenic differentiation; however, it is likely that a number of other miRNAs play important roles in regulating myogenesis as well. microRNA-128 (miR-128) has been reported to be highly expressed in brain and skeletal muscle, and we found that miR-128 is also up-regulated during bovine skeletal muscle satellite cell differentiation using microarray analysis and qRT-PCR. However, little is known about the functions of miR-128 in bovine skeletal muscle satellite cell development. In this study, we investigated the biological functions of miR-128 in bovine skeletal muscle cell development. Using a dual-luciferase reporter assay, we confirmed that miR-128 regulates the Sp1 gene. Over-expression of miR-128 reduced Sp1 protein levels and inhibited muscle satellite cell proliferation and differentiation. Inhibition of miR-128 increased Sp1 protein levels and promoted muscle satellite cell differentiation but also suppressed proliferation. Changes in miR-128 and Sp1 expression levels also affected the protein levels of MyoD and CDKN1A. Sp1, an activator of MyoD and a suppressor of CDKN1A, plays an important role in bovine muscle cell proliferation and differentiation. The results of our study reveal a mechanism by which miR-128 regulates bovine skeletal muscle satellite cell proliferation and myogenic differentiation via Sp1.

MicroRNA-128 suppresses cell growth and metastasis in colorectal carcinoma by targeting IRS1

Evidence has shown that microRNAs play important roles in tumor development, progression, and metastasis. miR-128 has been reported to be deregulated in different tumor types, whereas the function of miR-128 in colorectal carcinoma (CRC) largely remains to be elucidated. The aim of the present study was to investigate the clinical significance, biological effects and underlying mechanisms of miR-128 in CRC using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. It was found that the expression of miR-128 was downregulated in CRC tissues and cell lines as determined by RT-qPCR. Furthermore, the expression of miR-128 in tumor tissues was significantly negatively correlated with TNM stage and lymph node metastasis in CRC patients. Functional assay revealed that the overexpression of miR-128 inhibited CRC cell proliferation, colony formation, migration and invasion and promoted apoptosis invitro, and suppressed CRC xenograft tumor growth invivo. In addition, insulin receptor substrate1 (IRS1), a key mediator in oncogenic insulin-like growth factor (IGF) signaling, was confirmed as a direct target of miR-128 by a luciferase reporter assay. Western blot analysis indicated that the overexpression of miR-128 significantly downregulated IRS1 expression and its downstream Akt signaling in CRCcells. Moreover, miR-128 was negatively associated with IRS1 in CRC tissues compared to adjacent non-tumor tissues. Taken together, these data suggested that miR-128 serves as a tumor suppressor and blocks CRC growth and metastasis by targeting IRS1.

MicroRNA-128 promotes cell-cell adhesion in U87 glioma cells via regulation of EphB2

MicroRNAs (miRNAs) are small, non-coding RNAs which regulate gene expression at the post-transcriptional level. Abnormal expression of miRNAs occurs frequently in human tumors. Despite the fact that reduced expression of miR-128 has been observed in glioma tissues and cells, the role of miR-128 in tumors has not been fully characterized. In the present study, cell adhesion assays indicated that overexpression of miR-128 can promote cell-cell adhesion. Target site prediction algorithms indicated that miR-128 binds the 3'-untranslated regions of erythropoietin-producing hepatocellular receptor (Eph)B1 and EphB2 mRNAs. Luciferase reporter assays confirmed that miR-128 binds and regulates EphB1 and EphB2 mRNAs. Overexpression of EphB2 reduced the ability of miR-128 to promote cell-cell adhesion. The wound-healing assay indicated that miR-128 significantly inhibited cell migration via EphB2. This study revealed the novel functions of miR-128 in cell-cell adhesion and cell migration in glioma cells through the regulation of EphB2, and identified EphB1 and EphB2 as novel miR-128 targets.

Abstract 3170: Comprehensive analysis of the microRNA-128 function in head and neck squamous cell carcinoma

Abstract MicroRNAs are 22∼nucleotide-long endogenously expressed, highly conserved noncoding RNAs with important regulatory functions in proliferation, apoptosis, and metastasis. miR-128 a brain-enriched, which is transcribes from two gene copies (miR-128-1 and miR-128-2) on chromosome 2 and chromosome 3 which produce identical mature forms. miR-128 is most commonly in gliomas that have shown to function as a tumor suppressor which inhibits cell proliferation. Proteomic alterations of prostate cancer show miR-128 to down regulate cell invasiveness. Here we evaluated the role of miRNA-128 in HNSCC, to identify its putative targets and elucidate the possible mechanisms underlining the function of miR-128 in HNSCC. We established HNSCC cell lines that stably expressed individual members of the miR-128 using a lentiviral delivery system. The levels of miR-128 and their targeted proteins were analyzed by qRT-PCR and Western blot. miR-128 affinity was elucidated by MTT, colony formation, flow cytometry, and a tumor xenograft model. Two kinds of stably transfected HNSCC cell lines, including JHU-22miR-128 transfected with miR-128 and a vector control cell line JHU-22vector, were generated as a testing system. The miR-128 level in the JHU-22miR-128 cells was eleven fold higher than in JHU-22vector cells. We found that overexpression of miR-128 enable to effectively suppress cell viability (30%), decrease of proliferation (55%), and significantly reduced solid tumor growth in tumor xenografts models compared to the control groups. Transfected miR-128 cells exhibited astrocyte morphology. The inhibition mechanism of miR-128 was determined by evaluating five putative targets (BMI-1, BAG2, BAX, H3f3b, and Paip2) of miR-128. Overall, miR-128 enables to directly inhibit all these targets, but the binding affinity of miR-128 is dominantly towards to BMI-1 and BAG-2 targets. Conclusions: miR-128 exhibits anti-proliferative effects and phenotypic alteration in HNSCC by targeting multiple predicted conserved targets activity function in apoptosis, cell cycle, transcription, and translation. Our results provide a better understanding of miR-128 function in HNSCC and the potential targets to develop novel diagnostic markers and targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3170. doi:1538-7445.AM2012-3170

Abstract B30: Exogenous miR-128 Enforced HNSCC Inhibits Proliferation

Abstract Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease with complex molecular abnormalities. There are more than 65,000 Americans diagnosed with head and neck cancer and 25,000 die from this disease. microRNAs are 22~nucleotide-long endogenously expressed, highly conserved noncoding RNAs with important regulatory functions in proliferation, apoptosis, and metastasis. miR-128 a brain-enriched microRNA most commonly in gliomas that have shown to function as a tumor suppressor which decreases cell proliferation and cell invasiveness in prostate cancer. miR-128 has been shown to target BMI-1 in squamous cell carcinoma; however we believe miR-128 functions by targeting multiple targets in HNSCC. Methods: We established HNSCC cell lines that stably expressed individual members of the miR-128 using a lentiviral delivery system. The levels of miR-128 and their targeted proteins were analyzed by qRT-PCR and Western blot. miR-128 affinity was elucidated by MTT, colony formation, flow cytometry, and a tumor xenograft model. Results: Enforced transfection of miR-128 in HNSCC (JHU-22) expression was stable in vitro and in vivo. We identified five specific targets of miR-128 (BMI-1, BAG2, BAX, H3f3b, and Paip2). BMI-1 and H3f3b were shown to be a more dominant targeted of miR-128. Overexpression of miR-128, lead to the down regulation of these targets including decrease cell viability (30%), decrease of proliferation (55%), and significantly reduced solid tumor formation in tumor xenografts models. Conclusions: miR-128 exhibits anti-proliferative effects and phenotypic alteration in HNSCC by targeting multiple predicted conserved targets activity function in apoptosis, cell cycle, transcription, and translation. Therefore, screening for differentially expressed targets of miR-128 help elucidate the possible mechanisms underlining the function of miR-128.Our results indicate that miR-128 has the potential to serve as a therapeutic approach. Citation Format: Belinda Hauser, Yubin Hao, Joseph Califano, Xinbin Gu. Exogenous miR-128 Enforced HNSCC Inhibits Proliferation [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B30.