miR-124和miR-520b下调Eps8表达并抑制HeLa细胞增殖

Eps8是一个多功能的信号分子,参与肌动蛋白重排、受体内吞和肿瘤的发生发展. 为了寻找靶向Eps8的microRNAs (miRNAs)并研究其在宫颈癌中的调控作用,本文采用软件预测获得4个可能调控Eps8表达的miRNAs. 利用双荧光素酶报告系统和Western印迹研究发现,miR-124 和miR-520b结合到人EPS8 mRNA的3′非翻译区(untranslated region, UTR)并有效抑制Eps8蛋白的表达. 进一步细胞存活检测、MTT法和克隆形成实验分析显示,miR-124和miR-520b过表达显著抑制HeLa细胞的生长和增殖.而且,miRNA调节的Eps8下调能提高HeLa细胞对化疗药物顺铂的敏感性. 同时证明,miR-124和miR-520b激活肿瘤抑制基因p53与下游基因p21报告基因转录活性,也相应地上调了p53与p21的蛋白表达. 这些结果提示,miR-124和miR-520b下调癌基因EPS8表达,从而抑制HeLa细胞增殖,负调控宫颈癌细胞生长.     

MicroRNA-21 directly targets MARCKS and promotes apoptosis resistance and invasion in prostate cancer cells

Prostate cancer is one of the most common malignant cancers in men. Recent studies have shown that microRNA-21 (miR-21) is overexpressed in various types of cancers including prostate cancer. Studies on glioma, colon cancer cells, hepatocellular cancer cells and breast cancer cells have indicated that miR-21 is involved in tumor growth, invasion and metastasis. However, the roles of miR-21 in prostate cancer are poorly understood. In this study, the effects of miR-21 on prostate cancer cell proliferation, apoptosis, and invasion were examined. In addition, the targets of miR-21 were identified by a reported RISC-coimmunoprecipitation-based biochemical method. Inactivation of miR-21 by antisense oligonucleotides in androgen-independent prostate cancer cell lines DU145 and PC-3 resulted in sensitivity to apoptosis and inhibition of cell motility and invasion, whereas cell proliferation were not affected. We identified myristoylated alanine-rich protein kinase c substrate (MARCKS), which plays key roles in cell motility, as a new target in prostate cancer cells. Our data suggested that miR-21 could promote apoptosis resistance, motility, and invasion in prostate cancer cells and these effects of miR-21 may be partly due to its regulation of PDCD4, TPM1, and MARCKS. Gene therapy using miR-21 inhibition strategy may therefore be useful as a prostate cancer therapy.     

Retracted: Upregulated microRNA-15b alleviates ovarian cancer through inhitbition of the PI3K/Akt pathway by targeting LPAR3

Ovarian cancer characterizes as the fourth leading consequence of death associated with cancer for women. Accumulating evidence underscores the vital roles of microRNAs (miRNAs) in preventing ovarian cancer development. Besides, induction of the phosphatidylinositol-3 kinase/serine/threonine kinase (PI3K/Akt) pathway associated with the ovarian cancer cell migration and invasion. The study aims to examine the effects of miR-15b on the proliferation, apoptosis, and senescence of human ovarian cancer cells by binding to lysophosphatidic acid receptor 3 (LPAR3) with the involvement of the PI3K/Akt pathway. The positive expression of LPAR3 protein was detected by immunohistochemistry. Then the interaction between miR-15b and LPAR3 was examined. The possible role of miR-15b in ovarian cancer was explored using gain- and loss-of-function experiments. Subsequently, the functions of miR-15b on PI3K/Akt pathway, proliferation, migration, invasion, senescence and apoptosis of ovarian cancer cells were assessed. Furthermore, in vivo tumorigenicity assay in nude mice was performed. LPAR3 was overexpressed, whereas miR-15b was poorly expressed in ovarian cancer tissues. LPAR3 is a direct target of miR-15b. Restored miR-15b promoted Bax expression, apoptosis, and senescence, inhibited expression of LPAR3 and Bcl-2, the extent of PI3K and Akt phosphorylation, as well as ovarian cancer cell proliferation, migration, and invasion. Further, tumor growth was observed to be prevented by miR-15b overexpression. Collectively, our study demonstrates that miR-15b represses the proliferation and drives the senescence and apoptosis of ovarian cancer cells through the suppression of LPAR3 and the PI3K/Akt pathway, highlighting an antitumorigenic role of miR-15b.     

miR-182在前列腺癌组织中的表达及其对前列腺癌细胞增殖和凋亡的影响

目的:观察前列腺癌组织及不同前列腺癌细胞系中miR-182的表达,并探讨下调其表达对前列腺癌细胞增殖和凋亡的影响及机制。方法:采用实时荧光定量PCR(q RT-PCR)检测30例前列腺癌组织和30例相应的癌旁组织以及前列腺正常上皮RWPE-1细胞、前列腺癌PC-3、LNCa P和DU145细胞中miR-182的表达,进一步采用Lipfectamine 2000脂质体转染miRNA-182 inhibitor和阴性对照miRNA于PC-3细胞后,通过噻唑蓝(MTT)比色法检测细胞增殖情况,流式细胞术检测细胞凋亡率,免疫印迹(Western blot)法检测转录因子FOXO1、血管内皮生长因子(VEGF)和抑癌基因p53蛋白的表达。结果:miR-182在前列腺癌组织中的表达明显高于癌旁组织(P0.05);miR-182在前列腺癌细胞系PC-3、LNCa P和DU145中的表达均高于前列腺正常上皮细胞RWPE-1(P0.05),其中PC-3细胞中miR-182表达水平最高。转染miRNA-182 inhibitor至PC-3细胞成功下调miR-182表达后,细胞的增殖能力明显受到抑制,细胞凋亡能力明显增强,FOXO1表达水平显著升高,VEGF和p53的表达明显降低,差异均具有统计学意义(P0.05)。结论:miR-182在前列腺癌组织及细胞中呈高表达,下调miR-182的表达可能通过增加FOXO1的表达并减少VEGF和p53的表达,抑制前列腺癌细胞增殖并诱导细胞凋亡。     

The miR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities

MicroRNAs (miRNAs) are noncoding small RNAs that repress protein translation by targeting specific messenger RNAs. miR-15a and miR-16-1 act as putative tumor suppressors by targeting the oncogene BCL2. These miRNAs form a cluster at the chromosomal region 13q14, which is frequently deleted in cancer. Here, we report that the miR-15a and miR-16-1 cluster targets CCND1 (encoding cyclin D1) and WNT3A, which promotes several tumorigenic features such as survival, proliferation and invasion. In cancer cells of advanced prostate tumors, the miR-15a and miR-16 level is significantly decreased, whereas the expression of BCL2, CCND1 and WNT3A is inversely upregulated. Delivery of antagomirs specific for miR-15a and miR-16 to normal mouse prostate results in marked hyperplasia, and knockdown of miR-15a and miR-16 promotes survival, proliferation and invasiveness of untransformed prostate cells, which become tumorigenic in immunodeficient NOD-SCID mice. Conversely, reconstitution of miR-15a and miR-16-1 expression results in growth arrest, apoptosis and marked regression of prostate tumor xenografts. Altogether, we propose that miR-15a and miR-16 act as tumor suppressor genes in prostate cancer through the control of cell survival, proliferation and invasion. These findings have therapeutic implications and may be exploited for future treatment of prostate cancer.     

Death receptor Fas/Apo-1/CD95 expressed by human placental cytotrophoblasts does not mediate apoptosis

Trophoblasts, the fetal cells that line the villous placenta and separate maternal blood from fetal tissue, express both Fas antigen and the tumor necrosis factor (TNF) receptor p55 (TNFRp55), two members of the TNF receptor family that contain a cytoplasmic "death domain" that mediates apoptotic signals. We show that Fas mRNA expressed by cultured villous cytotrophoblasts isolated from term placentas encodes transmembrane sequences and that the protein is full-length (approximately 45 kDa), suggesting that the product is an active plasma membrane-anchored receptor. Its location on the cell surface was confirmed by cellular ELISA analysis of live cells. Although cytotrophoblast apoptosis was induced by TNFalpha, and both anti-Fas antibody (CH11) and FasL-expressing T lymphocyte hybridoma (activated A1.1) cells induced HeLa cell apoptosis, neither CH11 antibody nor activated A1.1 cells stimulated apoptosis in term or first-trimester cytotrophoblasts or in term syncytiotrophoblasts. We conclude that Fas- but not TNFRp55-mediated apoptosis is blocked in primary villous trophoblasts. These data suggest that the Fas response is specifically inactivated by unknown mechanisms to avoid autocrine or paracrine killing by Fas ligand constitutively expressed on neighboring cyto- or syncytiotrophoblasts.     

miR-181b functions as an oncomiR in colorectal cancer by targeting PDCD4

Programmed cell death 4 (PDCD4) is a RNA-binding protein that acts as a tumor suppressor in many cancer types, including colorectal cancer (CRC). During CRC carcinogenesis, PDCD4 protein levels remarkably decrease, but the underlying molecular mechanism for decreased PDCD4 expression is not fully understood. In this study, we performed bioinformatics analysis to identify miRNAs that potentially target PDCD4. We demonstrated miR-181b as a direct regulator of PDCD4. We further showed that activation of IL6/STAT3 signaling pathway increased miR-181b expression and consequently resulted in downregulation of PDCD4 in CRC cells. In addition, we investigated the biological effects of PDCD4 inhibition by miR-181b both in vitro and in vivo and found that miR-181b could promote cell proliferation and migration and suppress apoptosis in CRC cells and accelerate tumor growth in xenograft mice, potentially through targeting PDCD4. Taken together, this study highlights an oncomiR role for miR-181b in regulating PDCD4 in CRC and suggests that miR-181b may be a novel molecular therapeutic target for CRC.     

Downregulated NOX4 underlies a novel inhibitory role of microRNA-137 in prostate cancer

Prostate cancer is the second highest caused by cancer-related death among males. microRNAs (miRs) have been reported to participate in carcinogenesis, yet their roles in prostate cancer are rarely studied or investigated. Therefore, the present study attempted to explore the effect of miR-137 in prostate cancer via regulating NADPH oxidase 4 (NOX4). Initially, microarray analysis was performed to obtain prostate cancer-related differentially expressed genes and miRs that regulated NOX4, followed by detecting the expression of miR-137 and NOX4 and its target relationship. Moreover, PC-3 cells were transfected with small interfering RNA (siNOX4) and miR-137 mimic for exploring the effect of miR-137 on glycolysis, cell proliferation, and apoptosis in prostate cancer by evaluating lactate production, glucose uptake, adenosine triphosphate (ATP) production, viability rate, and expression of cleaved caspases 3, 8, and 9, cytochrome c, cleaved poly ADP ribose polymerase (PARP), Bax, and Bcl-2. miR-137 was vital to prostate cancer progression via regulating NOX4. Besides, miR-137 expressed poorly while NOX4 expressed highly in prostate cancer. NOX4 was the target gene of miR-137. Additionally, overexpression of miR-137 and silencing of NOX4 were observed to decrease NOX4 and Bcl-2 protein expression, but increase cleaved caspases 3, 8, and 9, cytochrome c, cleaved-PARP, and Bax protein expression. Furthermore, miR-137 overexpression and NOX4 silencing contributed to decreased lactate production, glucose uptake, ATP production, and cell proliferation, but increased apoptosis rate. Collectively, the present study showed that miR-137 repressed glycolysis in prostate cancer through knockdown of NOX4, which might be a potential theoretical target for prostate cancer treatment.     

Fas ligand-independent, FADD-mediated activation of the Fas death pathway by anticancer drugs

Trimerization of the Fas receptor (CD95, APO-1), a membrane bound protein, triggers cell death by apoptosis. The main death pathway activated by Fas receptor involves the adaptor protein FADD (for Fas-associated death domain) that connects Fas receptor to the caspase cascade. Anticancer drugs have been shown to enhance both Fas receptor and Fas ligand expression on tumor cells. The contribution of Fas ligand-Fas receptor interactions to the cytotoxic activity of these drugs remains controversial. Here, we show that neither the antagonistic anti-Fas antibody ZB4 nor the Fas-IgG molecule inhibit drug-induced apoptosis in three different cell lines. The expression of Fas ligand on the plasma membrane, which is identified in untreated U937 human leukemic cells but remains undetectable in untreated HT29 and HCT116 human colon cancer cell lines, is not modified by exposure to various cytotoxic agents. These drugs induce the clustering of Fas receptor, as observed by confocal laser scanning microscopy, and its interaction with FADD, as demonstrated by co-immunoprecipitation. Overexpression of FADD by stable transfection sensitizes tumor cells to drug-induced cell death and cytotoxicity, whereas down-regulation of FADD by transient transfection of an antisense construct decreases tumor cell sensitivity to drug-induced apoptosis. These results were confirmed by transient transfection of constructs encoding either a FADD dominant negative mutant or MC159 or E8 viral proteins that inhibit the FADD/caspase-8 pathway. These results suggest that drug-induced cell death involves the Fas/FADD pathway in a Fas ligand-independent fashion.     

Protein Kinase C�� Modulates Ligand-induced Cell Surface Death Receptor Accumulation: A MECHANISTIC BASIS FOR ENZASTAURIN-DEATH LIGAND SYNERGY*

Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-α-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2–5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCα, PKCβ, PKCϵ, and PKCθ, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCβ shRNA uniquely reversed PMA-induced protection against cell death. The PKCβ-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCβ shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCβ, to tumor necrosis factor-α related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death.     

MicroRNA-216b inhibits heat stress-induced cell apoptosis by targeting Fas in bovine mammary epithelial cells

Heat stress affects milk yield and quality in lactating dairy cows in summer. Bovine mammary epithelial cells (bMECs) play a key role in milk secretion, and microRNAs (miRNAs) regulate numerous functions of bMEC. Previous reports have verified that miR-216b regulated cell apoptosis through repressing target genes in several cancer cells. So, our purpose was to explore the potential involvement of miR-216b in heat stress-induced cell apoptosis in bMECs. Firstly, the heat stress model was constructed and we found that apoptotic rates of bMECs significantly increased under heat stress. The expression of miR-216b, Bax mRNA, and caspase-3 mRNA was upregulated. However, Bcl-2 mRNA level was detected to differentially downregulated. Overexpression of miR-216b remarkably downregulated the expression of caspase-3 and Bax mRNA and protein, and the mRNA and protein level of Bcl-2 was increased. Inhibition of miR-216b increased the activity of caspase-3 and Bax, and the level of Bcl-2 was inhibited. Moreover, Fas was identified as a target gene of miR-216b through bioinformatic analysis and dual-luciferase reporter assay. Fas activity was significantly inhibited and enhanced respectively after transfecting miRNA mimics and inhibitor. Finally, inhibition of Fas via the small interfering RNA (siRNA) also inhibited cell apoptosis induced by heat stress. Taken together, our results indicated that miR-216b exerted as an anti-apoptotic effect under heat stress in bMECs by targeting Fas.     

The microRNA -23b/-27b Cluster Suppresses the Metastatic Phenotype of Castration-Resistant Prostate Cancer Cells

MicroRNAs (miRs) are small, endogenous, non-coding RNAs that regulate the stability and/or translation of complementary mRNA targets. MiRs have emerged not only as critical modulators of normal physiologic processes, but their deregulation may significantly impact prostate and other cancers. The expression of miR-23b and miR-27b, which are encoded by the same miR cluster (miR-23b/-27b), are downregulated in metastatic, castration-resistant tumors compared to primary prostate cancer and benign tissue; however, their possible role in prostate cancer progression is unknown. We found that ectopic expression of miR-23b/-27b in two independent castration-resistant prostate cancer cell lines resulted in suppression of invasion and migration, as well as reduced survival in soft agar (a measure of anoikis). However, there was no effect of miR-23b/-27b on cell proliferation suggesting that these miRs function as metastasis (but not growth) suppressors in prostate cancer. Conversely, inhibition of miR-23b/-27b in the less aggressive androgen-dependent LNCaP prostate cancer cell line resulted in enhanced invasion and migration also without affecting proliferation. Mechanistically, we found that introduction of miR-23b/-27b in metastatic, castration-resistant prostate cancer cell lines resulted in a significant attenuation of Rac1 activity without affecting total Rac1 levels and caused increased levels of the tumor suppressor E-cadherin. Inhibition of these miRs had the opposite effect in androgen-dependent LNCaP cells. These results suggest that miR-23b/-27b are metastasis suppressors that might serve as novel biomarkers and therapeutic agents for castration-resistant disease.     

Inflammatory Cytokines and Survival Factors from Serum Modulate Tweak-Induced Apoptosis in PC-3 Prostate Cancer Cells

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK, TNFSF12) is a member of the tumor necrosis factor superfamily. TWEAK activates the Fn14 receptor, and may regulate cell death, survival and proliferation in tumor cells. However, there is little information on the function and regulation of this system in prostate cancer. Fn14 expression and TWEAK actions were studied in two human prostate cancer cell lines, the androgen-independent PC-3 cell line and androgen-sensitive LNCaP cells. Additionally, the expression of Fn14 was analyzed in human biopsies of prostate cancer. Fn14 expression is increased in histological sections of human prostate adenocarcinoma. Both prostate cancer cell lines express constitutively Fn14, but, the androgen-independent cell line PC-3 showed higher levels of Fn14 that the LNCaP cells. Fn14 expression was up-regulated in PC-3 human prostate cancer cells in presence of inflammatory cytokines (TNFα/IFNγ) as well as in presence of bovine fetal serum. TWEAK induced apoptotic cell death in PC-3 cells, but not in LNCaP cells. Moreover, in PC-3 cells, co-stimulation with TNFα/IFNγ/TWEAK induced a higher rate of apoptosis. However, TWEAK or TWEAK/TNFα/IFNγ did not induce apoptosis in presence of bovine fetal serum. TWEAK induced cell death through activation of the Fn14 receptor. Apoptosis was associated with activation of caspase-3, release of mitochondrial cytochrome C and an increased Bax/BclxL ratio. TWEAK/Fn14 pathway activation promotes apoptosis in androgen-independent PC-3 cells under certain culture conditions. Further characterization of the therapeutic target potential of TWEAK/Fn14 for human prostate cancer is warranted.     

Retracted: MicroRNA-99b suppresses human cervical cancer cell activity by inhibiting the PI3K/AKT/mTOR signaling pathway

Cervical cancer is common cancer among women with high morbidity. MicroRNAs (miRs) are involved in the progression and development of cervical cancer. This study aimed to explore the effect of miR-99b-5p (miR-99b) on invasion and migration in cervical cancer through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway. The microarray-based analysis was used to screen out differentially expressed miRNAs. Expression of miR-99b, PI3K, AKT, mTOR, and ribosomal protein S6 kinase (p70S6K) was determined in both cervical cancer tissues and paracancerous tissues. Next, alteration of miR-99b expression in cervical cancer was conducted to evaluate levels of PI3K, AKT, mTOR, p70S6K matrix metallopeptidase 2, epithelial cell adhesion molecule, and intercellular adhesion molecule 1, as well as the effect of miR-99b on cell proliferation, invasion, migration, cell cycle distribution, and apoptosis. The results demonstrated that miR-99b expression was decreased and levels of PI3K, AKT, mTOR, and p70S6K were elevated in cervical cancer tissues. More important, overexpressed miR-99b repressed the PI3K/AKT/mTOR signaling pathway, inhibited cell proliferation, invasion, and migration, blocked cell cycle entry, and promoted apoptosis in cervical cancer. These results indicate that miR-99b attenuates the migration and invasion of human cervical cancer cells through downregulation of the PI3K/AKT/mTOR signaling pathway, which provides a therapeutic approach for cervical cancer treatment.