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。     

miR-105 Inhibits Prostate Tumour Growth by Suppressing CDK6 Levels

A significant role for micro (mi)RNA in the regulation of gene expression in tumours has been recently established. In order to further understand how miRNA expression may contribute to prostate tumour growth and progression, we evaluated expression of miRNA in two invasive prostate tumour lines, PC3 and DU145, and compared it to that in normal prostate epithelial cells. Although a number of miRNAs were differentially expressed, we focused our analysis on miR-105, a novel miRNA not previously linked to prostate cancer. miR-105 levels were significantly decreased in both tumour cell lines in comparison to normal prostate epithelial cells. To determine its potential role in prostate cancer pathogenesis, we overexpressed miR-105 in both PC3 and DU145 cells and determined its effect on various tumourigenic properties. miR-105 overexpression inhibited tumour cell proliferation, tumour growth in anchorage-independent three-dimensional conditions and tumour invasion in vitro, properties of highly aggressive tumour cells. Of potential clinical significance, miR-105 overexpression inhibited tumour growth in vivo in xenograft models using these cell lines. We further identified CDK6 as a putative target of miR-105 which is likely a main contributor to the inhibition of tumour cell growth observed in our assays. Our results suggest that miR-105 inhibits tumour cell proliferation and hence may represent a novel therapeutically relevant cellular target to inhibit tumour growth or a marker of aggressive tumours in prostate cancer patients.     

MicroRNA-4723 Inhibits Prostate Cancer Growth through Inactivation of the Abelson Family of Nonreceptor Protein Tyrosine Kinases

The Abelson (c-Abl) proto-oncogene encodes a highly conserved nonreceptor protein tyrosine kinase that plays a role in cell proliferation, differentiation, apoptosis and cell adhesion. c-Abl represents a specific anti-cancer target in prostate cancer as aberrant activity of this kinase has been implicated in the stimulation of prostate cancer growth and progression. However, the mechanism of regulation of c-Abl is not known. Here we report that Abl kinases are regulated by a novel microRNA, miR-4723, in prostate cancer. Expression profiling of miR-4723 expression in a cohort of prostate cancer clinical specimens showed that miR-4723 expression is widely attenuated in prostate cancer. Low miR-4723 expression was significantly correlated with poor survival outcome and our analyses suggest that miR-4723 has significant potential as a disease biomarker for diagnosis and prognosis in prostate cancer. To evaluate the functional significance of decreased miR-4723 expression in prostate cancer, miR-4723 was overexpressed in prostate cancer cell lines followed by functional assays. miR-4723 overexpression led to significant decreases in cell growth, clonability, invasion and migration. Importantly, miR-4723 expression led to dramatic induction of apoptosis in prostate cancer cell lines suggesting that miR-4723 is a pro-apoptotic miRNA regulating prostate carcinogenesis. Analysis of putative miR-4723 targets showed that miR-4723 targets integrin alpha 3 and Methyl CpG binding protein in addition to Abl1 and Abl2 kinases. Further, we found that the expression of Abl kinase is inversely correlated with miR-4723 expression in prostate cancer clinical specimens. Also, Abl1 knockdown partially phenocopies miR-4723 reexpression in prostate cancer cells suggesting that Abl is a functionally relevant target of miR-4723 in prostate cancer. In conclusion, we have identified a novel microRNA that mediates regulation of Abl kinases in prostate cancer. This study suggests that miR-4723 may be an attractive target for therapeutic intervention in prostate cancer.     

RP4 通过miR-183-5p.1上调FOXO1抑制乳腺癌细胞增殖

近年来,越来越多的证据表明,长非编码RNAs在肿瘤发生发展中发挥重要作用。位于12号染色体的长非编码RNA RP4-816N1.7（简称RP4）在乳腺癌细胞中的作用未见报道。我们通过实时荧光定量PCR证实,RP4在乳腺癌细胞中的表达量普遍低于其在正常乳腺上皮细胞MCF-10A中的表达量。RP4在MCF-7和MDA-MB-231中表达量分别比其在MCF-10A中的表达量下调21.57%和91.33%。过表达RP4可明显抑制乳腺癌细胞增殖。敲低RP4可显著增加乳腺癌细胞的增殖能力。生物信息学预测,RP4可能与miR-183-5p.1结合,且叉头蛋白O1(FOXO1)可能是miR-183-5p.1的潜在靶标。实时荧光定量PCR结果提示,RP4可下调miR-183-5p.1,而miR-183-5p.1也可下调RP4和FOXO1的表达。双荧光素酶报告基因结果证实,miR-183-5p.1可与RP4结合,下调其表达,也能与FOXO1 3′UTR结合,抑制其mRNA和蛋白质水平的表达量。最后,本文通过BrdU实验证实,RP4通过FOXO1抑制乳腺癌细胞的增殖。总之,RP4通过内源性结合miR-183-5p.1,上调FOXO1表达,进而抑制乳腺癌细胞增殖。     

MicroRNA-223 regulates FOXO1 expression and cell proliferation

In HCT116 colorectal cancer cells, HeLa cervical cancer cells and HuH-7 hepatoma cells, miR-223 is expressed at a low level. Through infection with lentivirus containing miR-223 precursor, miR-233 was overexpressed in all these cells. Interestingly, the expression levels of FOXO1 mRNA and protein, and phosphorylation levels became significantly lower than those of their control. FOXO1 was down-regulated mainly in the cytoplasm, while the nuclear FOXO1 level became relatively high compared to the cytoplasm. As the unphosphorylated active form of FOXO1 increased in the cells, cyclin D1/p21/p27 were up-regulated at either mRNA or protein level. Proliferation of the cells was also greatly inhibited when miR-223 was over-expressed. Therein, our data suggest that miR-223 regulates FOXO1 expression and cell proliferation.     

Regulation of FOXO3a/beta-catenin/GSK-3beta signaling by 3,3'-diindolylmethane contributes to inhibition of cell proliferation and induction of apoptosis in prostate cancer cells

Previous studies from our laboratory have shown anti-proliferative and pro-apoptotic effects of 3,3'-diindolylmethane (DIM) through regulation of Akt and androgen receptor (AR) in prostate cancer cells. However, the mechanism by which DIM regulates Akt and AR signaling pathways has not been fully investigated. It has been known that FOXO3a and glycogen synthase kinase-3beta (GSK-3beta), two targets of activated Akt, interact with beta-catenin, regulating cell proliferation and apoptotic cell death. More importantly, FOXO3a, GSK-3beta, and beta-catenin are all AR coregulators and regulate the activity of AR, mediating the development and progression of prostate cancers. Here, we investigated the molecular effects of B-DIM, a formulated DIM with higher bioavailability, on Akt/FOXO3a/GSK-3beta/beta-catenin/AR signaling in hormone-sensitive LNCaP and hormone-insensitive C4-2B prostate cancer cells. We found that B-DIM significantly inhibited the phosphorylation of Akt and FOXO3a and increased the phosphorylation of beta-catenin, leading to the inhibition of cell growth and induction of apoptosis. We also found that B-DIM significantly inhibited beta-catenin nuclear translocation. By electrophoretic mobility shift and chromatin immunoprecipitation assays, we found that B-DIM inhibited FOXO3a binding to the promoter of AR and promoted FOXO3a binding to the p27(KIP1) promoter, resulting in the alteration of AR and p27(KIP1) expression, the inhibition of cell proliferation, and the induction of apoptosis in both androgen-sensitive and -insensitive prostate cancer cells. These results suggest that B-DIM-induced cell growth inhibition and apoptosis induction are partly mediated through the regulation of Akt/FOXO3a/GSK-3beta/beta-catenin/AR signaling. Therefore, B-DIM could be a promising non-toxic agent for possible treatment of hormone-sensitive but most importantly hormone-refractory prostate cancers.     

FOXO3a inhibits the EMT and metastasis of breast cancer by regulating TWIST-1 mediated miR-10b/CADM2 axis

BackgroundBreast cancer is the most common malignancy and has been considered as a leading cause of cancer death in women. Exploring the mechanism of breast cancer metastasis is extremely important for seeking novel therapeutic strategies and improving prognosis.MethodsClinical specimens and pathological characteristics were collected for evaluating the expression of forkhead box class O 3a (FOXO3a) and twist-related protein 1 (TWIST-1) in breast cancer tissues. CCK-8 assay was used to analyze cell proliferation. Cell invasion and migration were assessed by transwell assays. The expression of FOXO3a, TWIST-1, miR-10b, CADM2, FAK, phosphor-AKT and the epithelial-mesenchymal transition (EMT)-related protein (N-cadherin, E-cadherin and vimentin) were analyzed by RT-qPCR, immunohistochemical staining, immunofluorescence assay or western blot, respectively. Xenograft mouse models were used to analyze the role of the FOXO3a in breast cancer.ResultsFOXO3a was down-regulated and TWIST-1 was up-regulated in breast cancer tissues. Overexpression of FOXO3a or knockdown of TWIST-1 suppressed the proliferation, invasion, migration and EMT of breast cancer cells. Overexpression of TWIST-1 could reverse the effect of FOXO3a on the proliferation, invasion, migration and EMT of breast cancer. Moreover, FOXO3a suppressed the growth and metastasis of breast cancer by targeting TWIST1 in vivo.ConclusionFOXO3a inhibited the EMT and metastasis of breast cancer via TWIST-1/miR-10b/CADM2 axis.     

circEVI5 acts as a miR-4793-3p sponge to suppress the proliferation of gastric cancer

Circular RNAs (circRNAs) are a novel class of endogenous noncoding RNAs (ncRNAs) with a covalently closed loop structure. Accumulating evidence shows that circRNAs play vital roles in the growth, metastasis, treatment and prognosis of various cancers. However, the detailed functions and underlying mechanisms of circEVI5 (hsa_circ_0013162) in gastric cancer (GC) remain undocumented. In this study, the expression levels and prognostic value of circEVI5 were validated in GC tissue samples by using qRT-PCR. circEVI5 was significantly downregulated in GC tissues and cells, and low circEVI5 expression was correlated with poor prognosis. Next, in vitro CCK-8 assay, EdU incorporation assay, PI staining cell cycle assay, and in vivo xenograft mouse models were conducted to assess the functions of circEVI5. Gain of function experiments indicated that circEVI5 could inhibit GC cell proliferation and retard the cell cycle. Moreover, bioinformatics prediction showed that circEVI5 binds to miR-4793-3p, while FOXO1 may be a target of miR-4793-3p. Pull-down assays, RNA immunoprecipitation (RIP) assays, luciferase assays, and western blot were used to confirm the interactions between circEVI5, miR-4793-3p, and FOXO1. Functional assays demonstrated that circEVI5 suppressed the proliferation of GC by sponging miR-4793-3p and increasing FOXO1 expression levels. In conclusion, our study demonstrated that circEVI5 can bind miR-4793-3p as a ceRNA to eliminate the negative regulation of FOXO1, therefore suppressing GC proliferation.Subject terms: Gastric cancer, Oncogenesis     

miR-183-96-182 cluster is overexpressed in prostate tissue and regulates zinc homeostasis in prostate cells

Decreased zinc levels are a hallmark of prostate cancer tumors as zinc uniquely concentrates in healthy prostate tissue. Increased dietary zinc correlates with decreased risk of advanced prostate cancer and decreased mortality from prostate cancer. The mechanisms of prostatic zinc homeostasis are not known. Lower zinc levels in the tumor are correlated directly with decreased expression of the zinc transporter hZIP1. We report identification of a microRNA cluster that regulates multiple zinc transporters, including hZIP1. Screening in laser capture microdissected prostate cancer tumors identified miR-182 as a potential regulator of hZIP1. Regulation of hZIP1 by miR-182 via two binding sites was confirmed in primary prostate cell cultures. miR-96 and miR-183 are expressed as a cluster with miR-182 and share similar sequences. Array profiling of tissue showed that miR-183, -96, and -182 are higher in prostate cancer tissue compared with normal prostate. Overexpression of the entire miR-183-96-182 cluster suppressed five additional zinc transporters. Overexpression of miR-183, -96, and -182 individually or as a cluster diminished labile zinc pools and reduced zinc uptake, demonstrating this miR cluster as a regulator of zinc homeostasis. We observed regulation of zinc homeostasis by this cluster in prostate cells and HEK-293 cells, suggesting a universal mechanism that is not prostate-specific. To our knowledge, this is the first report of a miR cluster targeting a family of metal transport proteins. Individually or as a cluster, miR-183, -96, and -182 are overexpressed in other cancers too, implicating this miR cluster in carcinogenesis.     

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.     

MiR-144 Inhibits Uveal Melanoma Cell Proliferation and Invasion by Regulating c-Met Expression

MicroRNAs (miRNAs) are a group endogenous small non-coding RNAs that inhibit protein translation through binding to specific target mRNAs. Recent studies have demonstrated that miRNAs are implicated in the development of cancer. However, the role of miR-144 in uveal melanoma metastasis remains largely unknown. MiR-144 was downregulated in both uveal melanoma cells and tissues. Transfection of miR-144 mimic into uveal melanoma cells led to a decrease in cell growth and invasion. After identification of two putative miR-144 binding sites within the 3'' UTR of the human c-Met mRNA, miR-144 was proved to inhibit the luciferase activity inMUM-2B cells with a luciferase reporter construct containing the binding sites. In addition, the expression of c-Met protein was inhibited by miR-144. Furthermore, c-Met-mediated cell proliferation and invasion were inhibited by restoration of miR-144 in uveal melanoma cells. In conclusion, miR-144 acts as a tumor suppressor in uveal melanoma, through inhibiting cell proliferation and migration. miR-144 might serve as a potential therapeutic target in uveal melanoma patients.     

miR-96-5p靶向FOXO1调节脑缺血再灌注损伤机制

摘要 目的：探究miR-96-5p在脑缺血再灌注损伤（CIRI）中的作用及机制。方法：通过qRT-PCR检测36例确诊的缺血性脑卒中患者（IS患者组）和30例健康体检者（健康组）的血清miR-96-5p水平。将PC12细胞分为5组：对照组、NC-ag组、miR-96-5p-ag组、NC-an组、miR-96-5p-an组。采用Lipofectamine 2000对PC12细胞进行转染,通过qRT-PCR验证转染效率。将PC12细胞分为6组：对照组、氧糖剥夺/复氧复糖（OGD/R）组、OGD/R+NC-ag组、OGD/R+miR-ag组、OGD/R+NC-an组、OGD/R+miR-an组。根据分组对PC12细胞进行OGD/R处理和转染。通过MTT法检测PC12细胞活力,TUNEL法检测PC12细胞凋亡。采用改良Longa法建立大鼠CIRI模型,然后将大鼠分为假手术组、CIRI组、CIRI+NC-an组和CIRI+miR-an组。假手术组和CIRI组大鼠尾静脉注射生理盐水,CIRI+NC-an组和CIRI+miR-an组大鼠分别尾静脉注射NC-antagomir和miR-96-5p-antagomir。然后检测各组大鼠的神经功能评分、脑梗死体积和脑组织细胞凋亡情况。按照试剂盒说明书测定PC12细胞和大鼠脑组织中MDA、SOD和GSH-Px的含量。通过qRT-PCR检测PC12细胞和大鼠脑组织miR-96-5p和Forkhead box O1（FOXO1） mRNA水平,通过Western blot检测FOXO1、Ac-FOXO1、Bax和Bcl-2的蛋白表达水平。结果：与Health组比较,IS组患者的血清miR-96-5p水平显著升高（P<0.001）。与对照组和NC-ag组比较,miR-96-5p-ag组的miR-96-5p水平升高,FOXO1的mRNA和蛋白表达水平均降低,FOXO1的乙酰化水平升高（P<0.05）。与对照组和NC-an组比较,miR-96-5p-an组的miR-96-5p水平降低,FOXO1的mRNA和蛋白表达水平均升高,FOXO1的乙酰化水平降低（P<0.05）。与OGD/R组和OGD/R+NC-an组比较,OGD/R+miR-an组的相对细胞活力升高,TUNEL阳性率降低,Bax的蛋白相对表达量降低,Bcl-2的蛋白相对表达量升高,MDA水平降低,SOD和GSH-Px水平升高,miR-96-5p水平降低,FOXO1的mRNA和蛋白表达水平升高,FOXO1的乙酰化水平降低（P<0.05）。与CIRI组和CIRI+NC-an组比较,CIRI+miR-an组大鼠的神经功能评分和脑梗死体积降低,TUNEL阳性率降低,Bax的蛋白相对表达量降低,Bcl-2的蛋白相对表达量升高,MDA水平降低,SOD和GSH-Px水平升高,miR-96-5p水平降低,FOXO1的mRNA和蛋白表达水平升高,FOXO1的乙酰化水平降低（P<0.05）。结论：miR-96-5p在CIRI发生过程中表达上调,而下调miR-96-5p表达可能通过负调控FOXO1以减轻CIRI程度。     

MicroRNAs associated with metastatic prostate cancer

Objective

Metastasis is the most common cause of death of prostate cancer patients. Identification of specific metastasis biomarkers and novel therapeutic targets is considered essential for improved prognosis and management of the disease. MicroRNAs (miRNAs) form a class of non-coding small RNA molecules considered to be key regulators of gene expression. Their dysregulation has been shown to play a role in cancer onset, progression and metastasis, and miRNAs represent a promising new class of cancer biomarkers. The objective of this study was to identify down- and up-regulated miRNAs in prostate cancer that could provide potential biomarkers and/or therapeutic targets for prostate cancer metastasis.

Methods

Next generation sequencing technology was applied to identify differentially expressed miRNAs in a transplantable metastatic versus a non-metastatic prostate cancer xenograft line, both derived from one patient''s primary cancer. The xenografts were developed via subrenal capsule grafting of cancer tissue into NOD/SCID mice, a methodology that tends to preserve properties of the original cancers (e.g., tumor heterogeneity, genetic profiles).

Results

Differentially expressed known miRNAs, isomiRs and 36 novel miRNAs were identified. A number of these miRNAs (21/104) have previously been reported to show similar down- or up-regulation in prostate cancers relative to normal prostate tissue, and some of them (e.g., miR-16, miR-34a, miR-126*, miR-145, miR-205) have been linked to prostate cancer metastasis, supporting the validity of the analytical approach.

Conclusions

The use of metastatic and non-metastatic prostate cancer subrenal capsule xenografts derived from one patient''s cancer makes it likely that the differentially expressed miRNAs identified in this study include potential biomarkers and/or therapeutic targets for human prostate cancer metastasis.     

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.