Silencing USP22 by asymmetric structure of interfering RNA inhibits proliferation and induces cell cycle arrest in bladder cancer cells

The ubiquitin specific peptidase 22 (USP22) is a positive regulator of the growth of tumors. However, little is known about the impact of USP22 knockdown on the growth of human bladder cells. In the present study, we designed a series of asymmetric interfering RNAs (aiRNAs) and compared the efficacy of aiRNA and conventional symmetric interfering RNA (siRNA) in the silencing of USP22 expression and the growth of human bladder EJ cells in vitro and in vivo. In comparison with transfection with the USP22-specific siRNA, transfection with 15/21 aiRNA was more potent in down-regulating the USP22 expression and inhibiting EJ cell proliferation in vitro. Furthermore, transfection with 15/21 aiRNA induced higher frequency of EJ cells arrested at the G0/G1 phases, but did not trigger EJ cell apoptosis. Moreover, transfection with either the siRNA or 15/21 aiRNA up-regulated the expression of p53 and p21, but down-regulated the expression of cyclin E and Mdm2 in EJ cells. The up-regulated p53 expression induced by the specific siRNA or aiRNA was abrogated by induction of Mdm2 over-expression. In addition, treatment with the specific siRNA or aiRNA inhibited the growth of implanted human bladder tumors in mice and the aiRNA had more potent anti-tumor activity in vivo. Therefore, our data suggest that knockdown of USP22 expression by the aiRNA may down-regulate the expression of Mdm2 and cyclin E, resulting in the up-regulated expression of p53 and p21 and leading to cell cycling arrest and inhibition of human bladder EJ cell proliferation. Our findings indicate that the USP22-specific aiRNA may be a novel approach for the intervention of human bladder tumors.     

Identification of MicroRNAs controlling human ovarian cell proliferation and apoptosis

Previous studies have shown that microRNAs (miRNAs) can control steroidogenesis in cultured granulosa cells. In this study we wanted to determine if miRNAs can also affect proliferation and apoptosis in human ovarian cells. The effect of transfection of cultured primary ovarian granulosa cells with 80 different constructs encoding human pre‐miRNAs on the expression of the proliferation marker, PCNA, and the apoptosis marker, Bax was evaluated by immunocytochemistry. Eleven out of 80 tested miRNA constructs resulted in stimulation, and 53 miRNAs inhibited expression of PCNA. Furthermore, 11 of the 80 miRNAs tested promoted accumulation of Bax, while 46 miRNAs caused a reduction in Bax in human ovarian cells. In addition, two selected antisense constructs that block the corresponding miRNAs mir‐15a and mir‐188 were evaluated for their effects on expression of PCNA. An antisense construct inhibiting mir‐15a (which precursor suppressed PCNA) increased PCNA, whereas an antisense construct for mir‐188 (which precursor did not change PCNA) did not affect PCNA expression. Verification of effects of selected pre‐mir‐10a, mir‐105, and mir‐182 by using other markers of proliferation (cyclin B1) and apoptosis (TdT and caspase 3) confirmed specificity of miRNAs effects on these processes. This is the first direct demonstration of the involvement of miRNAs in controlling both proliferation and apoptosis by ovarian granulose cells, as well as the identification of miRNAs promoting and suppressing these processes utilizing a genome‐wide miRNA screen. J. Cell. Physiol. 223: 49–56, 2010. © 2009 Wiley‐Liss, Inc.     

p53siRNA therapy reduces cell proliferation, migration and induces apoptosis in triple negative breast cancer cells

p53 protein is probably the best known tumor suppressor. Earlier reports proved that human breast cancer cells expressing mutant p53 displayed resistance to apoptosis. This study is intended to investigate, the potential applications of RNA interference (RNAi) to block p53 expression, as well as its subsequent effect on cell growth, apoptosis and migration on a triple negative human breast cancer cell line (Hs578T). p53siRNA significantly reduced cell index (CI) compared to the control and we observed an inhibition of cellular migration in the interval of time between 0 and 30 h, as shown in the data obtained by dynamic evaluation using the xCELLigence System. Also, by using PCR-array technology, a panel of 84 key genes involved in apoptosis was investigated. Our studies indicate that the knockdown of p53 expression by siRNA modulates several genes involved in cell death pathways and apoptosis, showing statistically significant gene expression differences for 22 genes, from which 18 were upregulated and 4 were downregulated. The present research also emphasizes the important role of BCL-2 pro-apoptotic family of genes (Bim, Bak, and Bax) in activating apoptosis and reducing cell proliferation by p53siRNA treatment. Death receptors cooperate with BCL-2 pro-apoptotic genes in reducing cell proliferation. The limited success may be due to the activation of the antiapoptotic gene Mcl-1, and it may be associated with the resistance of triple negative breast cancer cells to cancer treatment. Thus, targeting p53siRNA pathways using siRNA may serve as a promising therapeutic strategy for the treatment of breast cancers.     

RNA interference in human foreskin fibroblasts within the three-dimensional collagen matrix

The technique of RNA interference (RNAi) was trialed in primary human foreskin fibroblasts, both in monolayer culture and in the fibroblast-populated collagen matrix. Knockdown of lamin A/C, p53, and FAK was possible with low-confluency (<50%) monolayer fibroblasts, a transfection vehicle concentration of 1%, and an siRNA concentration of 25–50 nM. Knockdown also was possible in the collagen matrix using similar reagent concentrations and a cellular density of one million fibroblasts per ml of matrix. Optimization of transfection conditions appeared to be important to increase knockdown efficiency. Consistent with prediction, knockdown of FAK induced apoptosis in the fibroblast-populated collagen matrix.     

siRNA沉默SIRT1基因对宫颈癌细胞HeLa增殖和凋亡的影响

化学合成靶向SIRT1基因的小干扰RNA,脂质体法转染人宫颈癌细胞株HeLa,观察小干扰RNA沉默SIRT1基因对HeLa增殖及细胞凋亡的影响。在优化siRNA SIRT1转染条件的基础上,应用RT-PCR和Western blot分别检测各组SIRT1 mRNA、SIRT1蛋白及凋亡相关蛋白的表达;CCK-8法检测细胞增殖抑制率;Hoechst荧光染色法和流式细胞仪检测细胞凋亡。结果表明,siRNA SIRT1转染细胞组SIRT1 mRNA水平和蛋白表达量明显低于对照组;siRNA SIRT1转染组细胞增殖受抑制,细胞凋亡率明显增加;凋亡相关蛋白P53、P21表达上调,Survivin表达下调。上述结果表明:siRNA SIRT1诱导的HeLa细胞凋亡与P53、P21、Survivin通路关系密切,但siRNA SIRT1诱导HeLa细胞凋亡的详尽机制有待进一步研究。     

Targeting CD24 for treatment of ovarian cancer by short hairpin RNA

Background aimsCD24 is markedly overexpressed in ovarian cancer and plays a critical role in ovarian cancer survival and metastasis, rendering it an interesting target for anti-tumor therapy. Using short hairpin RNA (shRNA) targeting CD24, we aimed to investigate the anti-tumor efficacy of CD24 knockdown in ovarian cancer cells in vitro and in vivo.MethodsCD24 shRNA vector (CD24–shRNA) and empty plasmid vector (EP) were transfected into ovarian cancer SKOV3 cells and the knockdown efficacy assessed by Western blot analysis. The effects of CD24 knockdown in SKOV3 cells in vitro, including cell viability and apoptosis, were determined using methyl thiazolyl blue tetrazolium bromide (MTT), flow cytometry and propidium iodide (PI) staining assays. The effects in vivo of CD24 knockdown on angiogenesis, cell proliferation and apoptosis were assessed using immunohistochemistry against CD31, proliferating cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) assays.ResultsTransfection of CD24–shRNA effectively down-regulated CD24 expression in vitro and in vivo. Administration of CD24–shRNA into nude mice bearing ovarian cancer significantly suppressed tumor volume growth.ConclusionsKnockdown of CD24 expression by CD24–shRNA significantly inhibited cell viability and induced apoptosis of SKOV3 cells in vitro. Administration with CD24–shRNA in vivo suppressed tumor volume increase by microvessel density (MVD) decrease, cell proliferation inhibition and apoptosis induction. All the data suggested that knockdown of CD24 by shRNA might be a potential therapeutic approach against human ovarian cancer.     

Silencing survivin gene expression promotes apoptosis of human breast cancer cells through a caspase-independent pathway

Survivin is recognized as an attractive target in cancer therapy because of its selective overexpression in the majority of tumors. Upregulated expression of this protein correlates with increased tumor grade, recurrence risk and decreased cancer patients survival. In this study, we assessed the efficacy of two survivin-specific small interfering RNA (siRNA) constructs to inhibit T47D human breast cancer cell growth. After siRNA transfection, T47D cells showed a significant reduction in proliferation and survival exhibiting clear signs of apoptosis. pSil_1 that targeted exon 1 exhibited a stronger inhibitory effect on cell growth, and increased cell apoptosis compared to pSil_30 that targeted exon 4. Cell apoptosis was found to be mediated by translocation of the mitochondrial apoptosis inducing factor (AIF), while no changes were observed in caspase-3 activation and Bid cleavage. Thus, silencing survivin expression using siRNA strategies represents a suitable therapeutic approach to selectively modulate the survival and growth of human breast cancer cells.     

半乳糖凝集素-3(galectin-3)基因沉默对Eca109人类食管癌细胞的影响

半乳糖凝集素-3(galectin-3)是一种多功能的β-半乳糖苷结合凝集素,涉及包括细胞生长、粘附、增殖、进展、转移以及凋亡等多种生物学功能,在恶性肿瘤中高表达。以前的研究已经证实了galecin-3过表达在Eca109人食管癌细胞的生物学作用。本研究试图通过进行小干扰RNA(siRNA)介导的galectin-3沉默,以分析galectin-3沉默对食管癌细胞生物学行为的影响。我们采用Western blotting和RT-qPCR被用来证实在蛋白质和mRNA水平上的galectin-3低表达,使用细胞计数试剂盒-8评估细胞增殖,用Annexin V-PE/7-AAD细胞凋亡检测试剂盒和流式细胞术检测Eca109细胞的凋亡。研究结果表明,转染后72 h,si Gal-3组Eca109细胞增殖明显低于siRNA对照组和未处理组(p<0.001)。Transwell实验结果显示,与其他组相比,galecin-3的抑制作用显著降低Eca109细胞的迁移和侵袭能力(p<0.05)。与siRNA-对照组和未处理组相比,galectin-3敲低显著增加Eca109细胞的凋亡率(p<0.05)。敲低Eca109细胞中galecin-3的表达后,细胞增殖、迁移和侵袭能力下降,而细胞凋亡增强,说明galectin沉默可作为食管癌治疗的新策略。     

The effect of obestatin on porcine ovarian granulosa cells

The aim of our in vitro experiments was to investigate the role of obestatin, a newly discovered metabolic hormone produced in the stomach and other tissues, in the direct control of ovarian cell proliferation, apoptosis and secretion. Porcine granulosa cells were cultured in the presence of obestatin (0, 1, 10 and 100ng/ml medium). The expression of intracellular peptides associated with proliferation (PCNA, cyclin B1, MAP kinase), as well as markers of apoptosis (Bax, p53, Caspase 3), were detected using immunocytochemistry and Western immunoblotting. Secretion of progesterone (P(4)), testosterone (T) and estradiol (E(2)) was measured by EIA. Addition of obestatin (1-100ng/ml) to the culture medium significantly stimulated the expression of PCNA and resulted in an increase in expression of cyclin B1 and MAPK. It also significantly increased the percentage of cells containing the apoptotic and anti-proliferating peptides p53, Caspase 3 and Bax. At 10 and 100ng/ml, obestatin promoted the secretion of P(4), but not T or E(2). Our results are the first demonstration that obestatin directly controls porcine ovarian cell functions: it can stimulate proliferation (accumulation of rPCNA, cyclin B1 and MAPK), apoptosis (expression of p53, Caspase 3 and Bax) and the secretion of progesterone.     

Knockdown of integrin beta4-induced autophagic cell death associated with P53 in A549 lung adenocarcinoma cells

Integrin beta4 is a tissue-specific protein, but its role in autophagy of lung adenocarcinoma cells is not clear. In this study, we used microtubule-associated protein 1 light chain 3 processing and acridine orange staining to reveal that knockdown of integrin beta4 by its specific siRNA induced autophagic cell death in A549 lung cancer cells. Next, we investigated the effects of siRNA-mediated downregulation of integrin beta4 on cell death and the level of p53. The proportion of dead cells and level of p53 were significantly increased. Inhibition of autophagy by the inhibitor 3-methyladenine attenuated the cell death induced by integrin beta4 knockdown. To further understand the relationship between p53 and integrin beta4 in autophagic cell death, we inhibited the expression of integrin beta4 by its specific siRNA in p53-mutated H322 lung cancer cells. Knockdown of integrin beta4 could not induce autophagic cell death in H322 cells. The data suggest that integrin beta4 is implicated in and associated with p53 in autophagy of lung cancer cells.     

应用DNA芯片鉴定HPV18 E6-siRNA诱导HeLa 细胞凋亡的分子机制

高危型人乳头瘤病毒（human papillomavirus,HPV）的E6基因在宫颈癌的发生中起关键作用,特异siRNA能有效抑制宫颈癌HeLa 细胞内HPV18 E6基因的表达,诱导肿瘤细胞凋亡.为进一步探讨HPV18 E6-siRNA诱导HeLa 细胞凋亡的分子机制,针对HPV18-E6基因设计siRNA序列,利用人源U6启动子为模板,经PCR表达框架法体外扩增,转染宫颈癌HeLa细胞抑制HPV18 -E6基因表达,从而诱导肿瘤细胞凋亡.对转染前后HeLa细胞总RNA样品进行荧光标记后,与Agilent Human 1A寡核苷酸芯片杂交、扫描、数据分析及标准化处理,确定表达差异的基因并经荧光定量PCR对部分基因进行验证,结合PANTHER数据分析系统,将这些基因按照生物学功能进行归类,查阅GenBank数据库及相关文献,对其结果进行深入分析及讨论.在检测的18 716个基因和EST中,共筛出差异表达基因359个,其中307个基因表达上调,52个基因表达下调,主要包括细胞周期相关基因CCNG1、p21;凋亡相关基因CASP4、CASP6、IGFBP3、DFFA;泛素蛋白酶解途径相关基因E6-AP、UBE2C;角化细胞分化相关基因KRT4、KRT6E、KRT18;抑癌基因RECK、VHL等.研究结果表明,HPV18 -E6基因抑制引起的细胞凋亡效应主要是通过P53信号途径和泛素蛋白酶解信号途径调节细胞周期相关基因和凋亡相关基因的表达,从而抑制HeLa细胞增殖、促进细胞凋亡.同时,抑癌基因的激活,角化细胞分化和免疫相关基因的表达上调,都说明了E6抑制后肿瘤细胞恶性转化程度的下降.     

The effects on cell growth and chemosensitivity by livin RNAi in non-small cell lung cancer

Livin is highly expressed in most tumor tissues and could inhibit the tumor cells apoptosis. Knockdown of endogenous livin expression in non-small cell lung cancer (NSCLC) cells could inhibit cell growth. But it is still unclear if knockdown of endogenous livin expression combined with conventional chemotherapy could play a positive role in NSCLC treatment. In this article, the efficient RNA interferences (RNAi) of livin were constructed, and then we transfected them into A549 cells and 103H cells to study their influence on cell cycle and apoptosis index. At last, we detected the cell's sensitivity to conventional chemotherapeutic drugs after knockdown endogenous livin expression in A549 cells and 103H cells. Our results showed that knockdown livin expression could inhibit cell growth and induce apoptosis in A549 cells and 103H cells. A549 cells and 103H cells had an increased chemosensitivity to adriamycin and cisplatin after transfection of livin RNAi constructs. The results indicated that cell cycle redistribution and increased apoptosis index after knockdown livin expression might provide the main explanation for the enhanced chemosensitivity. Proper combination of livin RNAi and some conventional chemotherapeutic drugs may entail potential benefits in the treatment of NSCLC.     

Downregulation of PCNA potentiates p21-mediated growth inhibition in response to hyperoxia

Prolonged exposure to hyperoxia inhibits cell proliferation in G1 via increased expression of p21. While p21 inhibits proliferating cell nuclear antigen (PCNA)-dependent DNA synthesis, it can also directly lower PCNA abundance; however, it is unclear whether loss of PCNA contributes to growth arrest. Here, we investigate how PCNA loss affects ability of p21 to exert G1 growth arrest of lung epithelial cells exposed to hyperoxia. In A549 cells that express p21 and growth arrest in G1 during hyperoxia, small interfering RNA (siRNA) knockdown of p21 led to G1 checkpoint bypass, increased cell death, and restoration of PCNA expression. Conditional overexpression of the PCNA binding domain of p21 in H1299 cells that do not normally express p21, or exposure to hyperoxia, caused a time-dependent loss of PCNA. Titrating PCNA levels using siRNA to approximate the low amount observed in cells expressing p21 resulted in S phase arrest. While lowering PCNA by itself caused S phase arrest, the combination of hyperoxia and siRNA against PCNA dramatically reduced PCNA abundance resulting in G1 arrest. G1 growth arrest was markedly enhanced upon the addition of p21 to these cells. Our findings suggest a model in which reducing expression of the abundant protein PCNA allows the less abundant protein p21 to be more effective at suppressing the processivity functions of remaining PCNA, thereby fully exerting the G1 checkpoint. Given that high p21 expression is often associated with lower PCNA abundance, our findings are suggestive of a global growth inhibitory mechanism involving p21-mediated PCNA suppression.     

Comparison of effects of protein kinase A, mitogen-activated protein kinase, and cyclin-dependent kinase blockers on rabbit ovarian granulosa cell functions

The aim of the present study was to define the role of protein kinase A (PKA)-, mitogen-activated protein kinase (MAPK)-, and cyclin-dependent kinase (CDK)-dependent pathways in the control of ovarian cell functions. The effects of PKA, MAPK, and CDK blockers (KT 5720, PD 98059, and olomoucine, respectively), given at doses of 0.001-10.0 μg/ml medium on functions of cultured rabbit granulosa cells were examined. Expression of PKA, MAPK/ERK1,2, secretory activity (IGF-I output), and proliferation (proliferating cell nuclear antigen, PCNA) in these cells were determined by RIA, immunocytochemistry and Western blotting. A PKA inhibitor, KT 5720 suppressed the expression of PKA and MAPK/ERK1,2, the IGF-I release, and the ratio of PCNA-positive cells in granulosa cells. A MAPK blocker, PD 98059 reduced the expression of MAPK/ERK1,2 (but not PKA), the IGF-I release, and percentage of PCNA-positive cells. A CDK blocker, olomoucine, increased the PKA expression, decreased the expression of MAPK/ERK1,2 and PCNA, but did not affect the IGF-I release. These observations confirm the involvement of PKs in control of basic ovarian functions and demonstrate the involvement of PKA in stimulation of ovarian cell proliferation and MAPK (but not CDK) and in promotion of ovarian IGF-I release. Different activity and specificity of the PKA, MAPK, and CDK blockers in their effects on PCNA and IGF-I suggests different biological role of these PKs in control of proliferative and secretory functions of rabbit ovarian cells.