circ_0001721/miR-198对前列腺癌细胞生物学行为的影响

目的:探讨circ_0001721/miR-198分子轴对人前列腺癌细胞DU145生物学行为的影响及相关作用机制。方法:应用qRT-PCR法检测circ_0001721、miR-198的表达量;DU145细胞转染si-circ_0001721、miR-198 mimics、anti-miR-198以及相应的对照（si-NC、miR-NC、anti-miR-NC）;CCK-8法、平板克隆形成实验、流式细胞术、Transwell实验检测细胞增殖、克隆、凋亡、迁移及侵袭;双荧光素酶报告实验验证circ_0001721与miR-198的靶向关系;Western blot检测E-cadherin、N-cadherin蛋白表达量。结果:与癌旁组织比较,circ_0001721在前列腺癌组织中高表达（P＜0.05）,而miR-198在前列腺癌组织中低表达（P＜0.05）;与转染si-NC或转染miR-NC相比,si-circ_0001721或miR-198 mimics可以提高细胞增殖抑制率、凋亡率和E-cadherin蛋白水平（P＜0.05）,降低克隆形成数、迁移和侵袭数以及N-cadherin蛋白水平（P＜0.05）;circ_0001721可靶向结合miR-198;与共转染si-circ_0001721和anti-miR-NC相比,共转染si-circ_0001721和anti-miR-198降低了细胞增殖抑制率、凋亡率和E-cadherin蛋白水平（P＜0.05）,而增加了克隆形成数、迁移和侵袭数以及N-cadherin蛋白水平（P＜0.05）。结论:敲低circ_0001721可通过上调miR-198抑制前列腺癌细胞的生长和转移。     

miR-17-92基因簇增强前列腺癌DU145细胞的迁移、侵袭能力及对顺铂的耐药性

背景与目的:miR-17-92基因簇与多种疾病的发生密切相关,其在肺癌、肝癌、胃癌和前列腺癌等多种肿瘤细胞中均高表达.本研究利用慢病毒包装系统建立稳定高表达miR-17-92基因簇的DU145细胞株,探讨miR-17-92基因簇对前列腺癌DU145细胞的迁移、侵袭能力及对顺铂耐药性的影响.方法:构建高表达miR-17-92基因簇的表达载体,转染DU145细胞株,同时转染空载体作为对照,并用实时荧光定量聚合酶链反应(real-time lfuorescent quantitative polymerase chain reaction,RTFQ-PCR)进行鉴定.用xCELLigence系统监测细胞的迁移、侵袭能力及顺铂处理后的生长情况;通过划痕实验观察细胞的迁移情况;采用蛋白[质]印迹法(Western blot)、凝胶酶谱实验和RTFQ-PCR检测相关蛋白质和基因的表达以探讨miR-17-92增强DU145细胞的迁移、侵袭能力及对顺铂耐药性的相关机制.结果:DU145-miR-17-92细胞迁移速率和侵袭能力高于DU145-control细胞(P<0.01).DU145-miR-17-92细胞中整合素β1的蛋白质表达水平和基质金属蛋白酶-9(matrix metalloprotein-9,MMP-9)的活性显著高于DU145-control细胞.顺铂处理后,DU145-miR-17-92细胞的生长速度自12 h起快于DU145-control细胞并呈顺铂耐药性(P<0.01).细胞外调节蛋白激酶1/2(extracellular regulated protein kinases,ERK1/2)在DU145-miR-17-92细胞中呈现持续高水平磷酸化,顺铂处理后,其磷酸化水平无明显变化.DU145-miR-17-92细胞中切除修复互补交叉基因1(excision repair cross complementing1,ERCC1)的mRNA和蛋白质表达水平显著高于DU145-control细胞.结论:高表达miR-17-92增强了DU145细胞的迁移、侵袭能力,其机制与整合素β1的表达上调及MMP-9活性增强有关.此外,高表达miR-17-92增强了DU145细胞对顺铂的耐药性,该过程与ERK1/2的磷酸化水平增加和ERCC1的表达水平上调相关.     

人前列腺癌肿瘤干细胞12-LOX表达的初步研究

目的：研究花生四烯酸12-脂氧化酶（12-LOX）在人前列腺癌肿瘤干细胞中的表达情况,并探讨其意义。方法：采用反转录聚合酶链反应（RT—PCR）技术和蛋白质印迹法,检测12-LOX在人前列腺癌细胞系DU145细胞和从其中分离出来的Du145侧群细胞中的表达情况;采用免疫荧光4通道激光扫描共聚焦显微镜技术,检测12-LOX在7例前列腺癌组织肿瘤干细胞和10例正常前列腺组织干细胞中的表达情况。结果：在细胞水平,应用RT—PCR技术和蛋白质印迹法分别证实,12-LOX mRNA和12-L0x蛋白在具有肿瘤干细胞特性DU145侧群细胞中的表达水平明显高于对应的母系DU145细胞;在组织水平,应用免疫荧光4通道激光扫描共焦显微镜检测证实,12-LOX蛋白在所有7例前列腺癌组织的肿瘤干细胞中均为阳性表达,而在所有10例正常前列腺组织的前列腺干细胞中均无表达。结论：12-LOX在人前列腺癌的肿瘤干细胞中有特异性的高表达,从而有望成为前列腺癌肿瘤干细胞一个新的、特异性的标志,并进一步有望成为前列腺癌肿瘤干细胞一个新的治疗靶位。     

The functional significance of microRNA-145 in prostate cancer

Background:

MicroRNAs (miRNAs) are small noncoding RNAs that have important roles in numerous cellular processes. Recent studies have shown aberrant expression of miRNAs in prostate cancer tissues and cell lines. On the basis of miRNA microarray data, we found that miR-145 is significantly downregulated in prostate cancer.

Methods and results:

We investigated the expression and functional significance of miR-145 in prostate cancer. The expression of miR-145 was low in all the prostate cell lines tested (PC3, LNCaP and DU145) compared with the normal cell line, PWR-1E, and in cancerous regions of human prostate tissue when compared with the matched adjacent normal. Overexpression of miR-145 in PC3-transfected cells resulted in increased apoptosis and an increase in cells in the G2/M phase, as detected by flow cytometry. Investigation of the mechanisms of inactivation of miR-145 through epigenetic pathways revealed significant DNA methylation of the miR-145 promoter region in prostate cancer cell lines. Microarray analyses of miR-145-overexpressing PC3 cells showed upregulation of the pro-apoptotic gene TNFSF10, which was confirmed by real-time PCR and western analysis.

Conclusion:

One of the genes significantly upregulated by miR-145 overexpression is the proapoptotic gene TNFSF10. Therefore, modulation of miR-145 may be an important therapeutic approach for the management of prostate cancer.     

circ_0001955靶向miR-149对前列腺癌DU145细胞系放射敏感性的实验研究

目的 探讨circ_0001955对前列腺癌DU145细胞系放射敏感性的影响及分子机制。方法 将si-con、si-circ_0001955、miR-con、miR-149转染至DU145细胞中,分别记为si-con、si-circ_0001955、miR-con、miR-149组;将miR-149与pc-circ_0001955共转染至DU145细胞记为miR-149+pc-circ_0001955组,以未经任何处理的细胞作为空白对照组。实时荧光定量PCR检测circ_0001955和miR-149表达水平;MTT检测细胞活力;流式细胞仪检测细胞凋亡;Transwell检测细胞迁移和侵袭;蛋白质印迹法检测MMP-2、MMP-9、Cleaved caspase-3、Cleaved caspase-9、γ-H2AX蛋白表达;细胞克隆形成实验检测细胞放射敏感性;双荧光素酶报告实验验证circ_0001955和miR-149的靶向关系。结果 细胞中circ_0001955高表达,miR-149低表达。沉默circ_0001955或过表达miR-149后细胞活性、迁移和侵袭数降低,MMP-2、MMP-9表达水平降低,Cleaved caspase-3、Cleaved caspase-9表达水平升高,细胞凋亡率升高(P<0.05)。4Gy X线照射后细胞中γ-H2AX表达水平升高,细胞存活分数降低,敏感性比1.38。circ_0001955靶向调控miR-149,同时过表达circ_0001955和miR-149后细胞增殖活性、迁移和侵袭细胞数升高,细胞凋亡率、细胞存活分数升高,敏感性比0.72。结论 沉默circ_0001955靶向上调miR-149抑制DU145细胞增殖、迁移、侵袭,诱导细胞周期阻滞,诱导细胞凋亡和增加细胞放射敏感性。     

Evidence of oxytocin/oxytocin receptor interplay in human prostate gland and carcinomas

Data on the presence of oxytocin receptors (OTR) within the prostate are still controversial and variable among different species. In the present study, OTR expression and localization has been investigated in human hyperplastic and neoplastic prostate at mRNA and protein levels using in situ hybridization (ISH) and immunohistochemistry (ICC) techniques, respectively. In all the cases studied, epithelial cells expressed OTR mRNA and protein. Interestingly, this expression was more intense in neoplastic epithelial cells compared to the hyperplastic ones. In order to determine whether OTR might mediate a biological effect of oxytocin (OT) in prostate cancer cells, OTR expression was studied by RT-PCR and immunofluorescence technique in the human androgen-independent prostate cancer cell line DU145. In addition, a possible heterotopic production of OT by DU145 cells was studied using RT-PCR. The data obtained showed that DU145 cells expressed OTR, whereas no OT mRNA was detected. When DU145 cells were treated with OT (100 nM) a significant inhibition of cell proliferation was observed, while co-incubation with the OT antagonist OTA (100 nm) abolished such an effect. The involvement of apoptosis in the OT effect contrasting cell proliferation was excluded by ISEL technique, which revealed a similar pattern of DNA fragmentation in either untreated or OT-treated cells. Altogether, the data indicate that the OT/OTR system could be involved in the control of prostate neoplastic pathology.     

miR-133b acts as a tumor suppressor and negatively regulates FGFR1 in gastric cancer

MicroRNAs (miRNAs) are a class of small noncoding RNAs that negatively regulate protein expression by binding protein-coding mRNAs and repressing translation. Accumulating evidence suggests that miRNAs are involved in cancer development and progression, acting as either tumor suppressors or oncogenes. Intriguingly, it has been shown that miR-133b was significantly downregulated in several types of cancers. However, its role and relevance in gastric cancer are still largely unknown. We showed that miR-133b was downregulated in human gastric cancer tissues and cell lines compared with nontumor counterparts by quantitative RT-PCR analysis. Overexpression of miR-133b could inhibit cell proliferation and colony formation of the gastric cancer cell lines MKN-45 and SGC-7901. Bioinformatics analysis indicated two putative miR-133b binding sites in the 3′-untranslated region of fibroblast growth factor receptor 1 (FGFR1) mRNA. In dual-luciferase reporter assay, miR-133b reduced the luciferase activity of Luc-FGFR1-wt, and mutation of miR-133b binding sites abolished the inhibitory effect of miR-133b. In this study, we found that miR-133b reduced the protein but not the mRNA levels of endogenous FGFR1. Furthermore, FGFR1 expression was upregulated in gastric cancer tissues and inversely correlated with miR-133b expression. Finally, knockdown of FGFR1 inhibited the growth of MKN-45 cells in a dose-dependent manner and overexpression of FGFR1 promoted the growth of GES-1 cells. These results indicate that miR-133b targets FGFR1 and inhibits gastric cancer cell growth, suggesting that it may serve as a tumor suppressive target in gastric cancer therapy.     

miR-149通过靶向FOXM1基因抑制前列腺癌细胞的生长和侵袭

  目的  探讨miR-149对前列腺癌细胞生长和侵袭的影响,并研究miR-149的靶基因及其功能。  方法  利用实时荧光定量PCR检测前列腺癌和癌旁组织miR-149和FOXM1 mRNA表达水平。在人前列腺癌细胞系PC3和DU145细胞中瞬时转染miR-149模拟物和阴性对照miRNA,运用平板克隆形成和Transwell侵袭实验检测细胞生长和侵袭。在细胞中分别转染miR-149靶基因FOXM1的siRNA和siRNA阴性对照,利用Western blot检测FOXM1蛋白表达情况,并检测细胞的克隆形成和侵袭能力。  结果  在前列腺癌中miR-149低表达（P < 0.01）,FOXM1 mRNA高表达（P < 0.01）。与对照组细胞相比,转染miR-149模拟物的PC3和DU145细胞克隆数目减少（P < 0.01）,发生侵袭的细胞减少（P < 0.01）;FOXM1蛋白水平在转染miR-149模拟物的PC3（P < 0.01）和DU145细胞（P < 0.05）中较对照组细胞降低。转染FOXM1 siRNA的PC3和DU145细胞中FOXM1蛋白水平较对照组降低,且克隆形成和侵袭能力较对照组明显降低（P < 0.01）。  结论  miR-149通过靶向FOXM1抑制前列腺癌细胞生长和侵袭,发挥着抑癌基因的作用,可作为前列腺癌分子治疗的有效靶点。      

miR-1266-5p and miR-185-5p Promote Cell Apoptosis in Human Prostate Cancer Cell Lines

Objective: Small non-coding RNA molecules are dysregulated in prostate cancer (PCa). In our previous study,downregulation of miR-1266 and miR-185 was demonstrated in PCa tissues and cell lines. The aim of the presentstudy was to investigate whether miR-1266 and miR-185 are involved in the regulation of B-cell lymphoma (BCL) 2and BCL2L1, respectively, and whether transfection of PCa cell lines with miR-1266 and miR-185 mimics can altertumorigenic phenotypes. Methods: In order to investigate the regulation of BCL2 and BCL2L1 mRNA levels bymiR-1266 and miR-185, respectively, a luciferase reporter assay was used. Real-time PCR was also used to analyzechanges in the levels of BCL2 and BCL2L1 mRNAs in PCa cell lines following transfection with synthetic miR-1266and miR-185. Cell apoptosis was determined by Annexin V protein expression analysis via flow cytometry. In additionto the MTT assay, a cell proliferation assay was performed. Result: A luciferase assay confirmed that the BCL2 andBCL2L1 genes may be targeted by miR-1266 and miR-185, respectively, through binding to their 3′UTR regions.Transfection of PC3 and DU145 cells with miR-1266 and miR-185 induced apoptosis and reduced proliferation, whichalso revealed an inverse correlation with BCL2 and BCL2L1 gene expression in the treated cells. Conclusion: Ourdata suggests that miR-1266 and miR-185 may be novel candidates for further research in PCa treatment through theanti-apoptotic pathway.     

Prostate cancer mediates osteoclastogenesis through two different pathways

The present study was undertaken to test the effects of prostate cancer cell lines (LNCaP, DU145, PC3, and MDA PCa 2b) on osteoclastogenesis. Crude conditioned medium (CM) from all four prostate cancer cell lines enhanced expression of the mRNA for receptor activator of NF-kappaB ligand (RANKL) in a mouse osteoblast cell line, MC3T3-E1; however, CM had no effect on expression of osteoprotegerin (OPG) mRNA. Coculture of MC3T3-E1 with prostate cancer cells yielded similar results. The number of mature osteoclasts induced by soluble RANKL increased significantly when osteoclast precursor cells were cultured with CM from LNCaP and DU145 cells. CM from LNCaP and DU145 cells also induced maturation from precursor in the absence of soluble RANKL, and this effect was not blocked by OPG. Addition of CM from DU145 cells increased expression of MMP-9 mRNA by osteoclast precursors. Our findings indicate that prostate cancer mediates osteoclastogenesis through induction of RANKL expression by osteoblasts and through direct actions on osteoclast precursors mediated by some factors other than RANKL.     

lncRNA GTSE1-AS1在前列腺癌组织中的表达及其对LNCaP细胞增殖与侵袭的影响

目的:探讨长链非编码RNA(long non-coding RNA,lncRNA)GTSE1-AS1在前列腺癌组织中的表达及其影响LNCaP细胞增殖和侵袭的机制.方法:收集2017年11月至2018年12月郑州大学附属洛阳中心医院泌尿外科手术切除的68例前列腺癌患者的癌和癌旁组织标本,以及前列腺癌细胞系LNCaP、PC...     

胱硫醚β合成酶在前列腺癌中的异常表达及其对前列腺癌细胞增殖的调控作用

目的:探讨胱硫醚β合成酶(CBS)在前列腺癌中的差异表达情况,及其对前列腺癌细胞增殖的调控作用。方法:利用GEPIA网站分析来源于TCGA和GTEx数据库的492例前列腺癌组织和152例正常组织中CBS mRNA表达情况。利用ULCAN网站分析来源于TCGA数据库的497例前列腺癌组织和52例正常组织中CBS mRNA的表达情况。用RPIM-1640培养基培养前列腺癌细胞系DU145、PC3、LNCaP和C4-2,免疫印迹实验检测CBS蛋白表达情况。shRNA转染DU145细胞,分为对照shRNA组、CBS shRNA#1组或#2组,克隆形成实验检测细胞克隆形成数量,BrdU标记实验检测细胞增殖能力,免疫印迹实验检测AKT、mTOR、S6K蛋白表达情况,以及AKT S473位点、mTOR S2448位点、S6K T421/S424位点的磷酸化水平。将稳定表达对照shRNA、CBS shRNA#1或#2的DU145细胞注射入小鼠皮下,建立小鼠前列腺癌移植瘤模型,观察肿瘤生长情况。结果:数据库分析结果显示,与正常前列腺组织比较,前列腺癌组织中CBS mRNA的表达水平显著上升。CBS蛋白在...     

地塞米松对前列腺癌DU145细胞ERK1/2活性和cyclin D1表达的影响

目的: 〖HT5"SS〗 探讨糖皮质激素地塞米松抑制雄激素非依赖性前列腺癌细胞DU145的机制。〖HT5W〗方法: 〖HT5"SS〗通过细胞培养方法观察地塞米松及其受体阻断剂RU486对前列腺癌DU145细胞增殖的作用;采用流式细胞仪测定地塞米松对DU145细胞细胞周期的影响;利用Western blot技术检测DU145细胞受地塞米松作用后cyclin D1表达水平和ERK1/2活性的变化;用RTPCR技术检测DU145细胞中糖皮质激素受体mRNA的表达。〖HT5W〗结果: 〖HT5"SS〗 地塞米松明显抑制DU145细胞的增殖,并且有剂量依赖效应;地塞米松使细胞周期阻滞于G0/G1期。Western blot结果显示,地塞米松作用DU145细胞3 d后,细胞内ERK1/2的活性降低,cyclin D1表达量下降。RU486可以拮抗地塞米松对DU145细胞的作用效果。RTPCR结果显示DU145细胞有糖皮质激素受体mRNA的表达。〖HT5W〗结论: 〖HT5"SS〗地塞米松具有抑制前列腺癌DU145细胞增殖和阻滞细胞周期的作用,此作用与其降低ERK1/2活性、抑制cyclin D1合成有关,提示糖皮质激素对前列腺癌的治疗有重要意义。