miR-155-TP53INP1调节轴在结直肠癌化疗药物敏感性中的作用机制

摘要 目的：初步揭示miR-155通过靶向调节TP53INP1表达水平影响结直肠癌细胞对5-FU化疗敏感性。方法：将人结肠直肠癌细胞系HCT116进行培养,提取细胞总RNA后,采用miR-155逆转录特异性引物构建反转录体系进行PCR扩增,通过qRT-PCR检测miR-155在5-FU耐药细胞HCT116/FU及敏感细胞株HCT116中的表达情况;取对数生长期细胞,分别转染miR-155mimics、miR-155抑制剂、miR-155阴性对照后,采用CCK-8法检测miR-155对细胞5-FU药物敏感性的影响,双荧光素酶报告基因系统验证miR-155与TP53INP1的靶基因关系,Western blot检测miR-155对 TP53INP1表达的影响。结果：miR-155在HCT116 /Fu细胞中的表达量是HCT116细胞的7.25倍;在相同5-FU浓度时,HCT116+阴性对照的细胞生长抑制率均高于HCT116+mimics、半数抑制浓度显著低于HCT116+mimics,差异均具有统计学意义(P＜0.05);TP53INP1是miR-155的靶基因,能显著降低野生型TP53INP1 3''-UTR的荧光素酶活性;转染miR-155 mimics后,TP53INP1的相对表达量显著下降,转染miR-155抑制剂后,TP53INP1的相对表达量显著升高,差异均具有统计学意义(P＜0.05)。结论：miR-155水平升高使HCT116细胞对5-FU的敏感性降低,miR-155可能通过靶向调节TP53INP1的表达水平,从而影响结直肠癌细胞对5-FU的敏感性。     

Mir-34a is upregulated during liver regeneration in rats and is associated with the suppression of hepatocyte proliferation

Background

MicroRNAs are a class of small regulatory RNAs that modulate a variety of biological processes, including cellular differentiation, apoptosis, metabolism and proliferation. This study aims to explore the effect of miR-34a in hepatocyte proliferation and its potential role in liver regeneration termination.

Methodology/Principal Finding

MiR-34a was highly induced after partial hepatectomy. Overexpression of miR-34a in BRL-3A cells could significantly inhibit cell proliferation and down-regulate the expression of inhibin βB (INHBB) and Met. In BRL-3A cells, INHBB was identified as a direct target of miR-34a by luciferase reporter assay. More importantly, INHBB siRNA significantly repressed cell proliferation. A decrease of INHBB and Met was detected in regenerating liver.

Conclusion/Significance

MiR-34a expression was upregulated during the late phase of liver regeneration. MiR-34a-mediated regulation of INHBB and Met may collectively contribute to the suppression of hepatocyte proliferation.     

Down-Regulation of miR-126 Is Associated with Colorectal Cancer Cells Proliferation,Migration and Invasion by Targeting IRS-1 via the AKT and ERK1/2 Signaling Pathways

Background

Colorectal carcinoma (CRC) is one of the leading causes of cancer-related mortality worldwide. MicroRNAs (miRNAs, miRs) play important roles in carcinogenesis. MiR-126 has been shown to be down-regulated in CRC. In this study, we identified the potential effects of miR-126 on some important biological properties of CRC cells and clarified the regulation of insulin receptor substrate 1 (IRS-1) and its possible signaling pathway by miR-126.

Methods

The effect of miR-126 on IRS-1, AKT, and ERK1/2 expression was assessed in the CRC cell lines HT-29 and HCT-116 with a miR-126 mimic or inhibitor to increase or decrease miR-126 expression. Furthermore, the roles of miR-126 in regulation of the biological properties of CRC cells were analyzed with miR-126 mimic or inhibitor-transfected cells. The 3′-untranslated region (3′-UTR) of IRS-1 regulated by miR-126 was analyzed by using a dual-luciferase reporter assay.

Results

We found that IRS-1 is the functional downstream target of miR-126 by directly targeting the 3′-UTR of IRS-1. Endogenous miR-126 and exogenous miR-126 mimic inhibited IRS-1 expression. Furthermore, gain-of-function or loss-of-function studies showed that over-expression of miR-126 down-regulated IRS-1, suppressed AKT and ERK1/2 activation, CRC cells proliferation, migration, invasion, and caused cell cycle arrest, but had no effect on cell apoptosis. Knockdown of miR-126 promoted these processes in HCT-116 cells and promoted AKT and ERK1/2 activation by up-regulating the expression of the IRS-1 protein.

Conclusions

MiR-126 may play roles in regulation of the biological behavior of CRC cells, at least in part, by targeting IRS-1 via AKT and ERK1/2 signaling pathways.     

TP53INP1 3′-UTR functions as a ceRNA in repressing the metastasis of glioma cells by regulating miRNA activity

Objectives

To explore the effects of the competitive endogenous RNA (ceRNA) network between TP53INP1 and E-cadherin on the invasion and migration of glioma.

Results

TP53INP1 and E-cadherin mRNA and protein were significantly overexpressed in normal brain tissues compared with glioma tissue specimens and correlated with the grades of glioma negatively. The expression of TP53INP1 and E-cadherin were correlated positively. Patients with higher TP53INP1 or E-cadherin expression had longer overall survival. Moreover, TP53INP1 3′-UTR inhibited glioma cell proliferation, invasion and proliferation; Furthermore, the 3′-UTRs of TP53INP1 and E-cadherin harboured seven identical miRNAs binding sites, and TP53INP1 3′-UTR could increase the expression of E-cadherin and decrease the expression of vimentin thus repressing the epithelial-mesenchymal transition (EMT). However, the coding sequence of TP53INP1 could not increase the expression of E-cadherin and the inhibitory effect on EMT of TP53INP1 3′-UTR was reversed by the siRNA against Dicer.

Conclusions

TP53INP1 3′-UTR could inhibit the EMT, thus hindering the migration and invasion of glioma via acting as a ceRNA for E-cadherin.

     

HCV Core Protein-Induced Down-Regulation of microRNA-152 Promoted Aberrant Proliferation by Regulating Wnt1 in HepG2 Cells

Background

Hepatitis C virus (HCV) has been reported to regulate cellular microRNAs (miRNAs). The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma (HCV-HCC), but HCV core-regulated miRNAs are largely unknown. Our preliminary experiments revealed significant down-regulation of microRNA-152 (miR-152) by HCV core protein in HepG2 cells. Through target gene prediction softwares, Wnt1 was predicted to be a potential target of miR-152. The present study was initiated to investigate whether miR-152 is aberrantly regulated by the HCV core protein, and involved in the regulation of the aberrant proliferation of HCV-HCC cells.

Methods

MiR-152 levels were examined by stem-loop real-time RT-PCR (SLqRT-PCR). Cell proliferation was analyzed by MTT and colony formation assay. Cell cycle analysis was performed by flow cytometry. Luciferase reporter assay was conducted to confirm miRNA-target association. Wnt1 expression was determined by real-time qPCR and Western blotting.

Results

HCV core protein significantly suppressed miR-152 expression, and led to significant Wnt1 up-regulation with a concomitant aberrantly promoted proliferation. Moreover, we validated that miR-152 inhibition promoted, while miR-152 mimics inhibited cell proliferation. Using, qRT–PCR and western blot, Wnt1 was demonstrated to be regulated by miR-152. Luciferase activity assay showed that while miR-152 mimics significantly reduced the luciferase activity by 83.76% (P<0.0001), miR-152 inhibitor showed no effect on luciferase reporter. Most notably, salvage expression of miR-152 after Ad-HCV core infection for 24 h almost totally reversed the proliferation-promoting effect of the HCV core protein, and meanwhile, reduced the expression of both Wnt1 mRNA and protein to basal levels.

Conclusion

These findings provide important evidence that the reduced miR-152 expression by HCV core protein can indirectly lose an inhibitory effect on Wnt1, which might, at least partially lead to cell proliferation of liver cancer cells. MiR-152 may have a therapeutic potential to suppress liver cancer proliferation.     

Evaluating the Potential Bioactivity of a Novel Compound ER1626

Background

ER1626, a novel compound, is a derivate of indeno-isoquinoline ketone. This study was designed to evaluate the biological activity and potential anti-tumor mechanism of ER1626.

Method

MTT assay, scratch assay and flow cytometry were used to determine cell proliferation, cell migration and cell cycle distribution as well as cell apoptosis on human breast cancer MCF-7 cells and endometrial cancer Ishikawa cells. We also explored the antiangiogenic effect of ER1626 on HUVEC cells and chicken embryos. The expression of estrogen receptor protein was investigated with western-blot analysis.

Results

ER1626 down-regulated the expression of estrogen receptor α protein and up-regulated β protein in MCF-7 and Ishikawa cells. The value of IC₅₀ of ER1626 on MCF-7 and Ishikawa cells were respectively 8.52 and 3.08 µmol/L. Meanwhile, ER1626 decreased VEGF secretion of MCF-7 and Ishikawa cells, disturbed the formation of VEGF-stimulated tubular structure in HUVEC cells, and inhibited the angiogenesis on the chicken chorioallantoic membrane. Scratch assay revealed that ER1626 suppressed the migration of MCF-7, Ishikawa and HUVEC cells. In addition to induction tumor cell apoptosis, ER1626 arrested cell cycle in G1/G0 phase in MCF-7 cells and G2/M phase in Ishikawa cells.

Conclusion

In conclusion, our results demonstrated that ER1626 has favorable bioactivities to be a potential candidate against breast cancer and angiogenesis.     

Molecular Characterization of Chronic Lymphocytic Leukemia Patients with a High Number of Losses in 13q14

Background

Patients with chronic lymphocytic leukemia and 13q deletion as their only FISH abnormality could have a different outcome depending on the number of cells displaying this aberration. Thus, cases with a high number of 13q- cells (13q-H) had both shorter overall survival and time to first therapy. The goal of the study was to analyze the genetic profile of 13q-H patients.

Design and Methods:

A total of 102 samples were studied, 32 of which served as a validation cohort and five were healthy donors.

Results

Chronic lymphocytic leukemia patients with higher percentages of 13q- cells (>80%) showed a different level of gene expression as compared to patients with lower percentages (<80%, 13q-L). This deregulation affected genes involved in apoptosis and proliferation (BCR and NFkB signaling), leading to increased proliferation and decreased apoptosis in 13q-H patients. Deregulation of several microRNAs, such as miR-15a, miR-155, miR-29a and miR-223, was also observed in these patients. In addition, our study also suggests that the gene expression pattern of 13q-H cases could be similar to the patients with 11q- or 17p-.

Conclusions

This study provides new evidence regarding the heterogeneity of 13q deletion in chronic lymphocytic leukemia patients, showing that apoptosis, proliferation as well as miRNA regulation are involved in cases with higher percentages of 13q- cells.     

Activation of calcium-sensing receptor increases TRPC3 expression in rat cardiomyocytes

Tumor protein p53-induced nuclear protein 1 (TP53INP1) is a well known stress-induced protein that plays a role in both cell cycle arrest and p53-mediated apoptosis. Loss of TP53INP1 expression has been reported in human melanoma, breast carcinoma, and gastric cancer. However, TP53INP1 expression and its regulatory mechanism in esophageal squamous cell carcinoma (ESCC) remain unclear. Our findings are in agreement with previous reports in that the expression of TP53INP1 was downregulated in 28% (10/36 cases) of ESCC lesions, and this was accompanied by significant promoter methylation. Overexpression of TP53INP1 induced G1 cell cycle arrest and increased apoptosis in ESCC cell lines (EC-1, EC-109, EC-9706). Furthermore, our study showed that the oncoprotein c-Myc bound to the core promoter of TP53INP1 and recruited DNA methyltransferase 3A to methylate the local promoter region, leading to the inhibition of TP53INP1 expression. Our findings revealed that TP53INP1 is a tumor suppressor in ESCC and that c-Myc-mediated DNA methylation-associated silencing of TP53INP1 contributed to the pathogenesis of human ESCC.     

Chronic Alcohol-Induced microRNA-155 Contributes to Neuroinflammation in a TLR4-Dependent Manner in Mice

Introduction

Alcohol-induced neuroinflammation is mediated by pro-inflammatory cytokines and chemokines including tumor necrosis factor-α (TNFα), monocyte chemotactic protein-1 (MCP1) and interleukin-1-beta (IL-1β). Toll-like receptor-4 (TLR4) pathway induced nuclear factor-κB (NF-κB) activation is involved in the pathogenesis of alcohol-induced neuroinflammation. Inflammation is a highly regulated process. Recent studies suggest that microRNAs (miRNAs) play crucial role in fine tuning gene expression and miR-155 is a major regulator of inflammation in immune cells after TLR stimulation.

Aim

To evaluate the role of miR-155 in the pathogenesis of alcohol-induced neuroinflammation.

Methods

Wild type (WT), miR-155- and TLR4-knockout (KO) mice received 5% ethanol-containing or isocaloric control diet for 5 weeks. Microglia markers were measured by q-RTPCR; inflammasome activation was measured by enzyme activity; TNFα, MCP1, IL-1β mRNA and protein were measured by q-RTPCR and ELISA; phospho-p65 protein and NF-κB were measured by Western-blotting and EMSA; miRNAs were measured by q-PCR in the cerebellum. MiR-155 was measured in immortalized and primary mouse microglia after lipopolysaccharide and ethanol stimulation.

Results

Chronic ethanol feeding up-regulated miR-155 and miR-132 expression in mouse cerebellum. Deficiency in miR-155 protected mice from alcohol-induced increase in inflammatory cytokines; TNFα, MCP1 protein and TNFα, MCP1, pro-IL-1β and pro-caspase-1 mRNA levels were reduced in miR-155 KO alcohol-fed mice. NF-κB was activated in WT but not in miR-155 KO alcohol-fed mice. However increases in cerebellar caspase-1 activity and IL-1β levels were similar in alcohol-fed miR-155-KO and WT mice. Alcohol-fed TLR4-KO mice were protected from the induction of miR-155. NF-κB activation measured by phosphorylation of p65 and neuroinflammation were reduced in alcohol-fed TLR4-KO compared to control mice. TLR4 stimulation with lipopolysaccharide in primary or immortalized mouse microglia resulted in increased miR-155.

Conclusion

Chronic alcohol induces miR-155 in the cerebellum in a TLR4-dependent manner. Alcohol-induced miR-155 regulates TNFα and MCP1 expression but not caspase-dependent IL-1β increase in neuroinflammation.     

Endoplasmic Reticulum Stress Stimulates p53 Expression through NF-κB Activation

Background

Induction of apoptosis by endoplasmic reticulum (ER) stress is implicated as the major factor in the development of multiple diseases. ER stress also appears to be a potentially useful major response to many chemotherapeutic drugs and environmental chemical compounds. A previous study has indicated that one major apoptotic regulator, p53, is significantly increased in response to ER stress, and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood.

Principal Findings

In this report, we demonstrate that induction of p53 expression is mediated through NF-κB signaling pathways during ER stress in MCF-7 cells. Tunicamycin or brefeldin A, two ER stress inducers, increased p53 expression in MCF-7 and Hela cells. We found p53 nuclear localization, activity, and phosphorylation at serine 15 on p53 increased during ER stress. Nuclear translocation of NF-κB and activity of NF-κB were also observed during ER stress. ER stress-induced p53 expression was significantly inhibited by coincubation with the NF-κB inhibitor, Bay 11-7082 and downregulation of NF-κB p65 expression. The role of p53 in mediating Brefeldin A-induced apoptosis was also investigated. Induction of p53 expression by Brefeldin A was correlated to Brefeldin A-induced apoptosis. Furthermore, downregulation of p53 expression by p53 siRNA significantly reduced Brefeldin A-induced apoptosis in MCF-7 cells.

Significance

Taken together, NF-κB activation and induction of p53 expression is essential for ER stress-induced cell death which is important for therapeutic effects of clinical cancer drugs. Our results may provide insight into the mechanism of cancer chemotherapy efficacy that is associated with induction of ER stress.     

Global microRNA expression profiling identifies MiR-210 associated with tumor proliferation, invasion and poor clinical outcome in breast cancer

Purpose

Aberrant microRNA (miRNA) expression is associated with cancer and has potential diagnostic and prognostic value in various malignancies. In this study, we investigated miRNA profiling as a complementary tool to improve our understanding of breast cancer (BC) biology and to assess whether miRNA expression could predict clinical outcome of BC patients.

Experimental Design

Global miRNA expression profiling using microarray technology was conducted in 56 systemically untreated BC patients who had corresponding mRNA expression profiles available. Results were further confirmed using qRT-PCR in an independent dataset of 89 ER-positive BC patients homogeneously treated with tamoxifen only. MiR-210 functional analyses were performed in MCF7 and MDA-MB-231 BC cell lines using lentiviral transduction.

Results

Estrogen receptor (ER) status, tumor grade and our previously developed gene expression grade index (GGI) were associated with distinct miRNA profiles. Several miRNAs were found to be clinically relevant, including miR-210, its expression being associated with tumor proliferation and differentiation. Furthermore, miR-210 was associated with poor clinical outcome in ER-positive, tamoxifen-treated BC patients. Interestingly, the prognostic performance of miR-210 was similar to several reported multi-gene signatures, highlighting its important role in BC differentiation and tumor progression. Functional analyses in BC cell lines revealed that miR-210 is involved in cell proliferation, migration and invasion.

Conclusions

This integrated analysis combining miRNA and mRNA expression demonstrates that miRNA expression provides additional biological information beyond mRNA expression. Expression of miR-210 is linked to tumor proliferation and appears to be a strong potential biomarker of clinical outcome in BC.     

MiR-20a Is Upregulated in Anaplastic Thyroid Cancer and Targets LIMK1

Background

There have been conflicting reports regarding the function of miR-20a in a variety of cancer types and we previously found it to be dysregulated in sporadic versus familial papillary thyroid cancer. In this study, we studied the expression of miR-20a in normal, benign and malignant thyroid samples, and its effect on thyroid cancer cells in vitro and in vivo.

Methodology/Principal Findings

The expression of miR-20a in normal, benign and malignant thyroid tissue was determined by quantitative RT-PCR. Thyroid cancer cells were transfected with miR-20a and the effect on cellular proliferation, tumor spheroid formation, and invasion was evaluated. Target genes of miR-20 were determined by genome-wide mRNA expression analysis with miR-20a overexpression in thyroid cancer cells and target prediction database. Target genes were validated by quantitative PCR and immunoblotting, and luciferase assays. MiR-20a expression was significantly higher in anaplastic thyroid cancer than in differentiated thyroid cancer, and benign and normal thyroid tissues. MiR-20a significantly inhibited thyroid cancer cell proliferation in vitro (p<0.01) and in vivo (p<0.01), tumor spheroid formation (p<0.05) and invasion (p<0.05) in multiple thyroid cancer cell lines. We found that LIMK1 was a target of miR-20a in thyroid cancer cell lines and direct knockdown of LIMK1 recapitulated the effect of miR-20a in thyroid cancer cells.

Conclusions/Significance

To our knowledge, this is the first study to demonstrate that miR-20a plays a role as a tumor suppressor in thyroid cancer cells and targets LIMK1. Our findings suggest the upregulated expression of miR-20a in anaplastic thyroid cancer counteracts thyroid cancer progression and may have therapeutic potential.     

MicroRNA-155 as a proinflammatory regulator via SHIP-1 down-regulation in acute gouty arthritis

Introduction

Gout is characterized by episodes of intense joint inflammation in response to intra-articular monosodium urate monohydrate (MSU) crystals. miR-155 is crucial for the proinflammatory activation of human myeloid cells and antigen-driven inflammatory arthritis. The functional role of miR-155 in acute gouty arthritis has not been defined. Therefore, the aim of this study was to examine the role of miR-155 in pathogenesis of acute gouty arthritis.

Methods

Samples from 14 patients with acute gouty arthritis and 10 healthy controls (HCs) were obtained. Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were cultured in vitro with MSU crystals, and gene expression (human miR-155 and SHIP-1) were assessed by real-time PCR. THP-1 cells were stimulated by MSU crystals and/or miR-155 transfection and then subjected to Western blot analysis. Levels of human tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1β in cell culture supernatants were measured by Luminex. Immunohistochemistry was performed on formalin-fixed gout tissues with anti–SHIP-1 antibody. A C57BL/6 J male mouse model of gout was used to analyze the expressions of miR-155, SHIP-1, and inflammatory cytokines.

Results

The samples from gouty arthritis were highly enriched in miR-155, with levels of expression being higher than those found in PBMC from HC. Treatment of the cells with MSU crystals strongly induced miR-155. In addition, overexpression of miR-155 in the cells decreased levels of SHIP-1 and promoted production of MSU-induced proinflammatory cytokines, such as TNF-α and IL-1β. Consistent with in vitro observations, miR-155 expression was elevated in the mouse model of gout. The production of inflammatory cytokines was markedly increased in MSU crystal induced peritonitis mice.

Conclusions

Overexpression of miR-155 in the gouty SFMC leads to suppress SHIP-1 levels and enhance proinflammatory cytokines.