LncRNA PVT1 epigenetically silences miR-195 and modulates EMT and chemoresistance in cervical cancer cells

The plasmacytoma variant translocation 1 gene (PVT1) is an oncogenic lncRNA with regulative effect on chemosensitivity in cervical cancer. However, the underlying mechanisms were not fully understood. In this study, HPV16 positive CaSki and SiHa cells were used as in vitro cell model. Knockdown of HPV16 E7 significantly inhibited PVT1 and restored miR-195 expression. PVT1 directly interacts with EZH2 and the complex anchors in the promoter region of miR-195. PVT1 overexpression resulted in increased H3K27me3 levels in the miR-195 promoter region, while PVT1 knockdown decreased H3K27me3 levels in the promoter region. In addition, PVT1 could competitively bind with miR-195. MiR-195 overexpression suppressed PVT1 expression in the cancer cells. Both PVT1 and miR-195 could inhibit paclitaxel (PTX) induced epithelial-to-mesenchymal transition (EMT) and also sensitize CaSki cells to PTX. Based on these findings, we infer that PVT1 could decrease miR-195 expression via enhancing histone H3K27me3 in the miR-195 promoter region and also via direct sponging of miR-195. In addition, the PVT1/miR-195 axis can modulate responses of the cancer cells to PTX via regulating EMT.     

A functional variant in miR-143 promoter contributes to prostate cancer risk

MicroRNAs are important regulators in numerous cellular processes, including cell differentiation, proliferation, and apoptosis. Recently, miR-143 was identified as a tumor suppressor in prostate cancer (PCa). To explore the mechanism of dysregulation and anti-tumor function of miR-143 in PCa, we first found a single-nucleotide polymorphism rs4705342T>C in the promoter region of miR-143 through bioinformatics tools and then performed a case–control study including 608 PCa patients and 709 controls. Results suggested that subjects with TC/CC genotypes had significantly decreased risk of PCa compared with those with TT genotype (adjusted OR 0.68, 95 % CI 0.55–0.85). Further functional assays showed that the risk-associated T allele increased the protein-binding affinity and reduced the activity of the promoter compared with C allele. In addition, restoration of miR-143 by mimics in PCa cells significantly inhibited cell proliferation and migration and down-regulated the expression level of kallikrein-related peptidase 2 (KLK2) mRNA and protein. The miR-143-KLK2 axis was also confirmed by luciferase reporter assay in vitro. In conclusion, our findings demonstrate that there is the significant association between the functional promoter variant rs4705342T>C in miR-143 and PCa risk and newly describe the miR-143-KLK2 interaction which provided another potential mechanism for miR-143 anti-tumor function.     

MicroRNAs and their therapeutic potential for human diseases: microRNAs, miR-143 and -145, function as anti-oncomirs and the application of chemically modified miR-143 as an anti-cancer drug

We examined the expression levels of microRNAs (miRNAs; miRs) in colorectal tumors (63 cancer specimens and 65 adenoma specimens) compared to adjacent non-tumorous tissues. Decreased expression of miR-143 and -145 was frequently observed in the adenoma and cancer samples. As the down-regulation of miR-143 and -145 was observed even in the early phase of adenoma formation, their decreased expression would appear to contribute mainly to the initiation of tumorigenesis. For clinical application, we added aromatic benzene-pyridine (BP-type) analogs to the 3'-overhang region of the RNA-strand and changed the sequences of the passenger strand in the miR-143 duplex (miR-143BPs), leading to greater activity and increased resistance to nuclease. The cell growth inhibitory effect of the chemically modified miR-143BPx in vitro was greater than that of the endogenous miR-143. The modified miR-143BPx showed a significant tumor-suppressive effect on xenografted tumors of human colorectal cancer DLD-1 cells. These findings suggest that miR-143 and -145 are important onco-related genes for the initiation of colorectal tumor development and that chemically modified miR-143BPx may be a candidate for an RNA medicine for the treatment of colorectal tumors.     

微小 RNA-15a-5p靶向蛋白质二硫键异构酶 A6前体蛋白抑制前列腺癌细胞增殖、迁移及侵袭

目的 探讨微小RNA-15a-5p(miR-15a-5p)是否可通过靶向蛋白质二硫键异构酶A6前体蛋白(PDIA6)而抑制前列腺癌细胞增殖、迁移及侵袭.方法 选取2018年2月至2019年3月江南大学附属医院接受治疗的前列腺癌病人50例,采用实时荧光定量PCR(qRT-PCR)与蛋白印迹法(Western blotting)分别检测前列腺癌组织、癌旁组织中miR-15a-5p、PDIA6的表达量;体外培养人前列腺癌DU145细胞,将细胞分为miR-NC组、miR-15a-5p组、si-NC组、si-PDIA6组、miR-15a-5p+pcDNA组、miR-15a-5p+pcDNA-PDIA6组;噻唑蓝(MTT)检测细胞增殖;Transwell小室实验检测细胞迁移及侵袭;双荧光素酶报告实验验证miR-15a-5p、PDIA6的靶向关系.结果 与癌旁组织相比,前列腺癌组织中miR-15a-5p的表达水平降低[(1.00±0.17)比(0.68±0.11)],PDIA6 mRNA[(0.99±0.09)比(1.64±0.18)]和蛋白[(0.44±0.07)比(0.76±0.17)]表达水平升高;转染miR-15a-5p mimics或转染si-PDIA6可降低细胞存活率(P<0.05),减少迁移及侵袭细胞数(P<0.05);双荧光素酶报告实验证实miR-15a-5p可靶向结合PDIA6;PDIA6过表达可降低miR-15a-5p过表达对DU145细胞增殖、迁移及侵袭的抑制作用.结论 过表达miR-15a-5p通过降低PDIA6的表达对列腺癌细胞增殖、迁移及侵袭能力有抑制作用.     

MiR-142-3p enhances chemosensitivity of breast cancer cells and inhibits autophagy by targeting HMGB1

MiR-142-3p has been reported to act as a tumor suppressor in breast cancer. However, the regulatory effect of miR-142-3p on drug resistance of breast cancer cells and its underlying mechanism remain unknown. Here, we found that miR-142-3p was significantly downregulated in the doxorubicin (DOX)-resistant MCF-7 cell line (MCF-7/DOX). MiR-142-3p overexpression increased DOX sensitivity and enhanced DOX-induced apoptosis in breast cancer cells. High-mobility group box 1 (HMGB1) is a direct functional target of miR-142-3p in breast cancer cells and miR-142-3p negatively regulated HMGB1 expression. Moreover, overexpression of HMGB1 dramatically reversed the promotion of apoptosis and inhibition of autophagy mediated by miR-142-3p up-regulation. In conclusion, miR-142-3p overexpression may inhibit autophagy and promote the drug sensitivity of breast cancer cells to DOX by targeting HMGB1. The miR-142-3p/HMGB1 axis might be a novel target to regulate the drug resistance of breast cancer patients.     

Anti-Tumor Effect of AlkB Homolog 3 Knockdown in Hormone- Independent Prostate Cancer Cells

Castrate resistant prostate cancer (CRPC) is a disease that is resistant to both hormone therapy and chemotherapy. At present, no curative therapy for CRPC has been established. Therefore, it is necessary to determine a novel molecular target for the development of therapeutic agents. We previously reported that AlkB homolog 3 (ALKBH3) is highly expressed in prostate cancer but not in benign prostatic hyperplasia or in normal prostate epithelium and that the expression levels of ALKBH3 protein are significantly correlated with the hormone-independent state of prostate cancer. Moreover, ALKBH3 regulates the invasion of prostate cancer cells via the regulation of matrix metalloproteinase 9. Here, we show that ALKBH3 gene silencing markedly induces apoptosis in hormone-independent prostate cancer cell line DU145 but not in the normal prostate epithelial cell line PNT2. Moreover, the in vivo tumorigenicity of DU145 cells was significantly inhibited by the administration of ALKBH3 siRNA. Furthermore, the anchorage-independent growth of DU145 cells was inhibited by ALKBH3 knockdown and promoted by ALKBH3 overexpression, significantly. ALKBH3 shRNA-expressing prostate cancer cells formed significantly smaller tumors than those of control shRNA transfectants in an in vivo xenograft model. These findings suggest that ALKBH3 is a promising target molecule for the development of CRPC therapeutic agents.     

A novel histone deacetylase inhibitor, CG200745, potentiates anticancer effect of docetaxel in prostate cancer via decreasing Mcl-1 and Bcl-XL

We synthesized a novel hydroxamate-based pan-histone deacetylase inhibitor (HDACI), CG200745 {(E)-2-(Naphthalen-1-yloxymethyl)-oct-2-enedioic acid 1-[(3-dimethylamino-propyl)-amide] 8-hydroxyamide]}. Like other inhibitors, for example vorinostat and belinostat, CG200745 has the hydroxamic acid moiety to bind zinc at the bottom of catalytic pocket. Firstly, we analyzed its inhibitory activity against histone deacetylase (HDAC) in hormone-dependent LNCaP cells and hormone-independent DU145 and PC3 cells. CG200745 inhibited deacetylation of histone H3 and tubulin as much as vorinostat and belinostat did. CG200745 also inhibited growth of prostate cancer cells, increased sub-G1 population, and activated caspase-9, -3 and -8 in LNCaP, DU145 and PC3 cells. These results indicate that CG200745 induces apoptosis. Next, we examined the effect of CG200745 on cell death induced by docetaxel in DU145 cells in vitro and in vivo. Compared to mono-treatment with each drug, pre-treatment of DU145 cells with docetaxel followed by CG200745 showed synergistic cytotoxicity, and increased the apoptotic sub-G1 population, caspase activation, and tubulin acetylation. Moreover, the combination treatment decreased Mcl-1 and Bcl-(XL). Docetaxel and CG200745 combination reduced tumor size in the DU145 xenograft model. These preclinical results show that combination treatment with docetaxel and new HDACI, CG200745, potentiated anti-tumor effect in hormone-refractory prostate cancer (HRPC) cells via activation of apoptosis.     

姜黄素上调前列腺癌细胞LNCaP中maspin基因的表达

目的研究姜黄素对前列腺癌细胞LNCaP凋亡的影响和对maspin基因表达的调控作用。方法利用不同浓度的姜黄素处理前列腺癌细胞LNCaP，MTT法和DNA 电泳检测不同时相LNCaP细胞的活性和凋亡情况，RT-PCR和Western blotting检测转录水平和翻译水平maspin基因的表达情况。将包含maspin 5′侧启动子区847 bp(-764～+83)DNA的荧光素酶表达载体pGL3-maspin瞬时转染LNCaP，姜黄素作用细胞48 h后，应用荧光素酶测定系统检测maspin启动子的表达活性作用。结果姜黄素抑制LNCaP细胞的生长活性，诱导LNCaP细胞的凋亡，增强LNCaP细胞中maspin基因的表达。结论姜黄素增强LNCaP细胞中maspin基因的表达是通过促进启动子的转录活性起作用的。     

5-氮-2′-脱氧胞苷对DU145前列腺癌细胞TIMP-3基因去甲基化作用及侵袭能力的影响

目的观察5-氮-2′-脱氧胞苷(5Aza-dc)对DU145前列腺癌细胞金属蛋白酶组织抑制因子3(TI MP-3)启动子去甲基化作用及侵袭能力的影响。方法用5Aza-dc处理DU145前列腺癌细胞,Transwell检测癌细胞的侵袭及运动能力,反转录-聚合酶链反应(RT-PCR)检测TI MP-3 mRNA的表达,Western印迹检测TI MP-3的蛋白表达。结果5Aza-dc作用后的DU145细胞侵袭及运动能力降低;TI MP-3 mRNA及蛋白恢复表达;TI MP-3启动子区去甲基化。结论5Aza-dc可以使DU145前列腺癌细胞TI MP-3启动子区去甲基化,使TI MP-3基因重新表达,恢复其抑制肿瘤侵袭和移动的能力。     

Curcumin inhibits cervical cancer cell proliferation,migration and invasion by suppressing the PI3K/AKT signaling pathway via the miR-29b/KDM2A axis

In this study, we aimed to examine whether curcumin exerted its anti-tumor effects by regulating miR-29b/KDM2A in cervical cancer cells. The cell viability, migration and invasion were estimated in HeLa cervical cancer cells treated with curcumin. The effects of microRNA-29b (miR-29b) on biological behaviors of HeLa SiHa cells were also assessed. Potential target genes of miR-29b were predicted and confirmed using a luciferase reporter assay, and the effects of curcumin and miR-29b on the PI3K/AKTsignaling pathway were analyzed. Curcumin treatment inhibited cell proliferation, migration and invasion of HeLa cells (P<0.05). The miR-29b expression was promoted by curcumin treatment in HeLa cells (P<0.01), and miR-29b depletion could restore the effects of curcumin on cell proliferation, migration and invasion of HeLa cells (P<0.05). KDM2A was proved as a direct target gene of miR-29b, and the activity of the PI3K/AKT signaling could be regulated by curcumin and miR-29b (P<0.05). All the data revealed that curcumin played a protective role in cervical cancer. The proliferation, migration and invasion of cervical cancer cells were inhibited by curcumin through the miR-29b/KDM2A/PI3K/AKT pathway.     

人参皂苷Rg3差异调节前列腺癌细胞PC3和DU145增殖

目的 探讨人参皂苷Rg3对前列腺癌细胞PC3和DU145细胞增殖的调节作用。方法 100 µmol/L人参皂苷 Rg3处理体外培养的前列腺癌细胞 PC3和 DU145,使用 CCK-8实验检测细胞的增殖能力;DCFH-DA活性氧荧光探针试剂盒检测人参皂苷 Rg3处理的 DU145细胞中活性氧（ROS）水平;JC-1法检测 PC3和 DU145细胞线粒体膜电位的去极化;RT-PCR和免疫印迹法检测PC3和DU145细胞中氧化还原相关蛋白的表达差异。结果 CCK-8实验结果表明,100 µmol/L人参皂苷 Rg3能够显著抑制 PC3细胞增殖,而对 DU145细胞的增殖没有显著调节作用;DCFHDA活性氧荧光探针试剂盒检测结果表明,人参皂苷 Rg3能够显著上调 DU145细胞和 PC3细胞中 ROS水平;JC-1检测实验表明,人参皂苷Rg3能够诱导PC3和DU145细胞中线粒体膜电位去极化;RT-PCR和免疫印迹实验表明,抗氧化蛋白谷胱甘肽过氧化物酶-1（GPX-1）和超氧化物歧化酶2（SOD2）在DU145细胞中的表达显著高于PC3细胞。结论 人参皂苷Rg3对PC3和DU145细胞的增殖表现出不同的调节作用,可能与细胞中抗氧化蛋白的表达差异有关。     

miR-21调控HTN1蛋白对胃癌细胞迁移和侵袭能力的影响

目的 探讨miR-21调控HTN1蛋白对胃癌细胞迁移和侵袭能力的作用.方法 定量聚合酶链反应检测胃癌组织和胃癌细胞中miR-21的表达情况,过表达miR-21后蛋白免疫印迹法检测HTN1的表达,双荧光素酶实验检测miR-21和HTN1表达在胃癌细胞中的关系,划痕实验检测过表达miR-21对胃癌细胞迁移能力的影响,Transwell侵袭实验检测过表达miR-21对胃癌细胞侵袭能力的影响.结果 miR-21在胃癌组织及胃癌SGC7901细胞中表达水平均降低(P<0.05).miR-21的表达与病理分期及有无淋巴结转移有关(P<0.05).过表达miR-21胃癌SGC7901细胞株荧光素酶活性、HTN1基因和蛋白表达水平均低于对照组(P<0.05).过表达miR-21胃癌SGC7901细胞的迁移速率较对照组减慢,侵袭细胞数目少于对照组(P<0.05).结论 miR-21在胃癌中表达下调,其可以调控HTN1表达影响胃癌细胞迁移和侵袭能力.     

Ginsenoside 20(S)-Rg3 upregulates HIF-1α-targeting miR-519a-5p to inhibit the Warburg effect in ovarian cancer cells

The Warburg effect, one of the metabolic hallmarks of cancer, is responsible for rapid energy production through a high rate of aerobic glycolysis. Ginsenoside 20(S)-Rg3 antagonizes the Warburg effect in ovarian cancer cells by upregulating some microRNAs, including miR-519a-5p, that target key enzymes involved in aerobic glycolysis. How 20(S)-Rg3-upregulated miR-519a-5p influences the Warburg effect in ovarian cancer cells remains poorly defined, however. Here we report that while overexpression of miR-519a-5p in ovarian cancer cells inhibited the Warburg effect, inhibition of miR-519a-5p negated the suppressive action of 20(S)-Rg3 against the Warburg effect as evidenced by a decrease in glucose consumption, lactate production and HK2 expression. We identified HIF-1α as a direct target of miR-519a-5p via luciferase reporter assays and demonstrated the counteraction by overexpressed HIF-1α of 20(S)-Rg3-suppressed Warburg effect. Further, 20(S)-Rg3 decreased DNMT3A-mediated DNA methylation in the promoter region of its precursor gene, leading to an increase in the level of miR-519a-5p. In conclusion, 20(S)-Rg3 upregulates miR-519a-5p via reducing DNMT3A-mediated DNA methylation to inhibit HIF-1α-stimulated Warburg effect in ovarian cancer.     

卵巢上皮性癌组织中miR-145与PI3K/AKT信号通路相关性的研究

目的研究miR-145、Akt和p-Akt蛋白在卵巢上皮性癌组织中的表达及其相互关系。方法采用RT-PCR和Western blot检测miR-145、AKT和P-AKT蛋白在卵巢组织中的表达水平,分析miR-145、Akt和p-Akt蛋白三者的相关关系。结果(1)miR-145在正常卵巢、良、恶性上皮性卵巢肿瘤组织中的表达逐渐降低,差异有统计学意义(P<0.05);在卵巢上皮性癌中的表达水平与FIGO分期和病理类型无关(P>0.05)。(2)p-Akt在正常卵巢、良、恶性上皮性卵巢肿瘤组织中的表达逐渐升高,差异有统计学意义(P<0.05),在卵巢上皮性癌中的表达水平与FIGO分期有关(P<0.05),而与病理类型无关(P>0.05)。(3)miR-145与p-Akt的表达水平在卵巢组织中表达呈负相关(r=-0.492,P=0.001)。结论 miR-145与PI3K/AKT信号通路的活化可能在卵巢上皮性癌的发生及发展中起重要作用,并且二者呈负相关。     

INPP4B overexpression suppresses migration,invasion and angiogenesis of human prostate cancer cells

Inositol polyphosphate 4‐phosphatase B (INPP4B) has been identified as a tumour suppressor in different human cancers. However, the role of INPP4B in the angiogenesis of human prostate cancer cells remains unclear. In this study, we first compared the expression of INPP4B between prostate cancer tissues and tumour‐adjacent normal prostate tissues using immunohistochemistry. Then, we explored the role of INPP4B in prostate cancer progression via transfection of a Flag‐INPP4B plasmid into PC3 and DU145 cells in vitro and in vivo. Our results showed that reduced INPP4B staining was significantly correlated with the tumour‐node‐metastasis stage. Moreover, transfection with Flag‐INPP4B plasmid suppressed the migration and invasion of prostate cancer cells through inactivating the PI3K/Akt signalling pathway, at the same time decreased vascular endothelial growth factor secretion and suppressed human umbilical vein endothelial cells proliferation and tube formation. Futhermore, it was also found that INPP4B could inhibit tumour growth and angiogenesis in vivo. Altogether, our results supported that INPP4B acted as a tumour suppressor in human prostate cancer, and provided insights into development of a targeted therapy for this disease.     

miR-205 impairs the autophagic flux and enhances cisplatin cytotoxicity in castration-resistant prostate cancer cells

Compelling evidence suggests that epithelial-to-mesenchymal transition is involved in the resistance of human cancer cells to chemotherapy. We previously reported that the expression of miR-205, a miRNA down-regulated in prostate cancer, is further repressed in prostate cancer cells undergoing epithelial-to-mesenchymal transition, suggesting a possible involvement of the miRNA in the acquisition of the chemoresistant phenotype. In the present study, we show that miR-205 replacement in castration-resistant mesenchymal prostate cancer cells caused an enhancement of cisplatin cytotoxic activity in vitro and in vivo, as a consequence of autophagy impairment. Specifically, the constraints on the autophagic flux were associated to the miRNA-dependent down-regulation of the lysosome-associated proteins RAB27A and LAMP3. These findings suggest that miR-205-mediated impairment of the autophagic pathway may interfere with the detoxifying capabilities of prostate cancer cells in their attempt to cope with cisplatin-induced detrimental effects. Overall, our data indicate that (i) loss of miR-205 may indeed contribute to acquire mesenchymal tracts and concomitantly establish a permissive autophagic milieu that confers a chemotherapy resistant phenotype to prostate cancer cells, and (ii) strategies aimed at restoring miR-205 expression levels may represent a successful approach to overcome resistance of prostate cancer to platinum compounds.     

A novel PI3K inhibitor displays potent preclinical activity against an androgen-independent and PTEN-deficient prostate cancer model established from the cell line PC3

Recent studies demonstrated that targeting the phosphatidylinositide 3-kinase (PI3K)/AKT signaling pathway is a major strategy for the treatment of androgen-independent prostate cancer. In the present study, we developed an analog BENC-511 from a recently reported PI3K inhibitor S14161 by structural optimization. Using PC3 and DU145 as the model cell lines, we found PTEN-deficient PC3 cells were more sensitive than PTEN-expressing DU145 ones in terms of cell proliferation, apoptosis, and caspase-3 activation. These findings were consistent with the inhibition on PI3K/AKT signals. BENC-511 preferably suppressed AKT activation in PC3 over DU145 cells. Notably, PTEN restoration attenuated BENC-511 induced apoptosis. Moreover, BENC-511 displayed great therapeutic efficacy in a PC3-derived prostate cancer model in nude mice. With an oral dosage of 50 mg/kg, BENC-511 decreased tumor growth more than 50% in 27 days, which was accompanied with PARP cleavage, but did not show overt toxicity. This study lays a solid rationale for the development of BENC-511 as a drug for the treatment of PTEN-deficient and androgen-independent prostate cancers.     

Curcumin downregulates homeobox gene NKX3.1 in prostate cancer cell LNCaP

AIM: To elucidate the effect and the mechanisms of curcumin on the expression of the human homeobox gene NKX3.1 in the prostate cancer cell LNCaP. METHODS: The expression change of NKX3.1 in cells incubated with varying concentrations of curcumin was observed by Western blotting and RT-PCR. A dual luciferase reporter assay was used to test the effect of curcumin on the activity of the NKX3.1 1040 bp promoter. Curcumin-treated cells disposed to a designated amount of androgen analog R1881 and the androgen receptor (AR) antagonist flutamide, then the expression of NKX3.1 or the activity of the NKX3.1 promoter were investigated by Western blotting or reporter gene assay, respectively. Finally, Western blotting and electrophoretic mobility shift assay were performed to demonstrate the effect of curcumin on the expression of AR and its binding activity to the androgen response element (ARE). RESULTS: Curcumin downregulated the expression of NKX3.1 and the activity of the NKX3.1 1040 bp promoter in LNCaP cells. R1881 increased the expression of NKX3.1, and the AR antagonist flutamide decreased the expression of NKX3.1 in LNCaP cells, while curcumin could inhibit androgen-AR mediated induction of NKX3.1 expression. Curcumin decreased the expression of AR and the binding activity to ARE directly. CONCLUSION: Curcumin could downregulate NKX3.1 expression in LNCaP cells. It could also inhibit the androgen-AR mediated induction of NKX3.1 expression by downregulating AR expression and blocking its DNA binding activity.     

Curcumin up-regulates LDL receptor expression via the sterol regulatory element pathway in HepG2 cells

Plasma low-density lipoprotein-cholesterol (LDL-C) is mainly taken up and cleared by the hepatocellular LDL receptor (LDL-R). LDL-R gene expression is regulated by the sterol regulatory element binding proteins (SREBPs). Previous studies have shown that curcumin reduces plasma LDL-C and has hypolipidemic and anti-atherosclerotic effects. Herein, we investigated the effect of curcumin on LDL-R expression and its molecular mechanism in HepG2 cells. Curcumin increased LDL-R expression (mRNA and protein) and the resultant uptake of DiI-LDL in a dose- and time-dependent manner. Using a GFP reporter system in a transfected HepG2/SRE-GFP cell line, we found that curcumin activated the sterol regulatory element of the LDL-R promoter. In HepG2/Insig2 cells, curcumin reversed the inhibition of LDL-R expression induced by Insig2 overexpression. These data demonstrate that curcumin increases LDL-R protein expression and uptake activity via the SREBPs pathway. These findings contribute to our further understanding of the cholesterol-lowering and anti-atherosclerotic effects of curcumin.