NOB1影响胶质瘤细胞增殖、凋亡的实验研究

目的 利用RNA干扰（RNAi）在人胶质瘤U251和U87-MG细胞中沉默NOB1基因的表达,探讨NOB1对恶性胶质瘤细胞增殖以及凋亡的影响.方法 构建NOB1基因的短发卡（shRNA）慢病毒表达载体,包装成病毒颗粒并感染人胶质瘤U251和U87-MG细胞,采用实时定量聚合酶链反应检测NOB1在恶性胶质瘤细胞系中的表达.采用甲基噻唑基四唑（MTT）比色法和克隆形成实验检测细胞增殖和克隆形成能力情况;同时利用PI染色流式细胞仪检测细胞凋亡情况,观察NOB1基因对U251和U87-MG细胞增殖、凋亡的影响.结果 NOB1基因在人胶质瘤U251、U87-MG细胞系中均明显高表达.NOB1-shRNA慢病毒能有效感染胶质瘤细胞,实验结果显示感染后U251和U87-MG细胞的增殖能力明显下降;U251克隆形成能力明显受到抑制;细胞周期在G0/G1停滞,而且细胞出现显著性凋亡;上述差异均具有统计学意义（P＜0.05）.结论 NOB1在人胶质瘤U251和U87-MG细胞中高表达;降低NOB1基因的表达,可以显著降低胶质瘤细胞的增殖,并促进胶质瘤细胞凋亡;NOB1基因在体外对胶质瘤细胞的发生发展可能具有癌基因的调节作用.     

Long non coding RNA UCA1 contributes to the autophagy and survival of colorectal cancer cells via sponging miR-185-5p to up-regulate the WISP2/β-catenin pathway

The estimated number of new cases of colorectal cancer (CRC) will increase to 140 250 in 2018 worldwide. The long non-coding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) has recently been shown to be dysregulated in CRC, which plays an important role in the progression of CRC. However, the biological role and the underling mechanism of UCA1 in the carcinogenesis of CRC remain unclear. Herein, we found that UCA1 was aberrantly upregulated in two CRC cell lines (SW620 and HT29) compared to colorectal cell CCD-18Co. UCA1 knockdown inhibited the apoptosis, growth and autophagy of CRC cell lines in vitro. Furthermore, UCA1 could act as an endogenous sponge by directly interacting with miR-185-5p and downregulation miR-185-5p expression. In addition, UCA1 could reverse the inhibitory effect of miR-185-5p on the growth and autophagy of CRC cells, which might be involved in the derepression of member 1 (WNT1)-inducible signaling pathway protein 2 (WISP2, a target gene of miR-185-5p) expression and the activation of the WISP2/β-catenin signaling pathway. In vivo, the present study elucidates a novel UCA1-miR-185-5p-WISP2-Wnt/β-catenin axis in CRC, which may help us to understand the pathogenesis and the feasibility of lncRNA-directed diagnosis and therapy of CRC.

The estimated number of new cases of colorectal cancer (CRC) will increase to 140 250 in 2018 worldwide.     

Combination therapy of malignant glioma cells with 2-5A-antisense telomerase RNA and recombinant adenovirus p53

Malignant gliomas of astrocytic origin have commonly expressed several features such as alterations in the tumor-suppressor gene p53 or p16 or the acquisition of telomerase activity, which are distinctive from astrocytes. Therefore, restoration of the tumor-suppressor gene or telomerase inhibition is expected to provide a cure for malignant gliomas. We have recently demonstrated that the treatment with a 19-mer antisense oligonucleotide against human telomerase RNA linked to a 2',5'-oligoadenylate (2-5A-anti-hTR) inhibited the growth of malignant glioma cells. From a therapeutic point of view, it is very important to investigate the antitumor efficacy of 2-5A-anti-hTR combined with the restoration of p53 or p16 gene. In this study, we evaluated the antitumor effect of 2-5A-anti-hTR in combination with recombinant adenoviruses bearing p53, its associated p21WAF1/CIP1, or p16CDKN2 gene (Ad5CMV-p53, Ad5CMV-p21, or Ad5CMV-p16) against malignant glioma cells in vitro and in vivo. Five malignant glioma cell lines expressing the mutant p53 gene (A172, GB-1, T98G, U251-MG and U373-MG) were more sensitive to the combination of 2-5A-anti-hTR and Ad5CMV-p53 than to other combinations. The additive effect of the combination therapy was due to induction of caspase-dependent apoptosis and cell growth arrest. Furthermore, the 2-5A-anti-hTR treatment when combined with Ad5CMV-p53 showed greater efficacy against subcutaneous U251-MG tumors in nude mice. In contrast, U87-MG cells expressing the wild-type p53 gene were insensitive to Ad5CMV-p53, although the treatment with 2-5A-anti-hTR was significantly effective. These results indicate that combining 2-5A-anti-hTR with Ad5CMV-p53 has the most therapeutic potential for malignant gliomas with mutant p53. For tumors exhibiting wild-type p53, it may be useful to treat with 2-5A-anti-hTR. Gene Therapy (2000) 7, 2071-2079.     

Eupalmerin acetate, a novel anticancer agent from Caribbean gorgonian octocorals, induces apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase pathway

The marine ecosystem is a vast but largely untapped resource for potential naturally based medicines. We tested 15 compounds derived from organisms found in the Caribbean Sea (14 gorgonian octocoral-derived compounds and one sponge-derived compound) for their anticancer effects on human malignant glioma U87-MG and U373-MG cells. Eupalmerin acetate (EPA) was chosen as the lead compound based on its longer-term stability and greater cytotoxicity than those of the other compounds we tested in these cell types. EPA induced G(2)-M cell cycle arrest and apoptosis via the mitochondrial pathway; it translocated Bax from the cytoplasm to the mitochondria and dissipated the mitochondrial transmembrane potential in both cell types. EPA was found to increase phosphorylated c-Jun NH(2)-terminal kinase (JNK) by >50% in both U87-MG and U373-MG cells. A specific JNK inhibitor, SP600125, inhibited EPA-induced apoptosis, confirming the involvement of the JNK pathway in EPA-induced apoptotic cell death. Furthermore, 7 days of daily intratumoral injections of EPA significantly suppressed the growth of s.c. malignant glioma xenografts (P < 0.01, on day 19). These results indicate that EPA is therapeutically effective against malignant glioma cells in vitro and in vivo and that it, or a similar marine-based compound, may hold promise as a clinical anticancer agent.     

Retracted Article: Overexpression of circ_0034642 contributes to hypoxia-induced glycolysis,cell proliferation,migration and invasion in gliomas by facilitating TAGLN2 expression via sponging miR-625-5p

Glioma is an aggressive brain cancer with poor prognosis and high invasiveness. Dysregulation of circular RNAs (circRNAs) has been widely discovered in various cancers, including glioma. However, the molecular mechanism of circ_0034642 in glioma is still unclear. The expression of circ_0034642, microRNA (miR)-625-5p and transgelin-2 (TAGLN2) in glioma tumors and cells was detected by performing a quantitative real-time polymerase chain reaction (qRT-PCR). The stability of circ_0034642 was determined by carrying out RNase R treatment. Cell proliferation was evaluated by performing the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Glycolysis was analyzed by measuring the extracellular acidification rate (ECAR) using glucose detection and lactic acid detection kits. Cell migration and invasion were determined by performing the transwell assay. Protein expression levels of the proteins hexokinase 2 (HK2), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9) and TAGLN2 were analyzed using western blots. The interaction between miR-625-5p and circ_0034642 or TAGLN2 was proved using a dual-luciferase reporter system. Animal models were established by subcutaneously injecting glioma cells stably transfected with sh-NC or sh-circ_0034642. Circ_0034642 and TAGLN2 were overexpressed whereas miR-625-5p was expressed at low levels in glioma tumors and cells. Moreover, circ_0034642 and TAGLN2 were upregulated while miR-625-5p was downregulated under hypoxic conditions in a time-dependent manner. Next, elimination of circ_0034642 was shown to inhibit cell glycolysis, proliferation, migration and invasion under hypoxic conditions in gliomas. Then, we found that circ_0034642 acted as a “sponge” of miR-625-5p while TAGLN2 acted as a target of miR-625-5p. In addition, recovery of circ_0034642 attenuated the repression mediated by miR-625-5p on glioma cell glycolysis and progression under hypoxic conditions. Meanwhile, an inhibitor of miR-625-5p alleviated TAGLN2 deficiency-induced inhibition of glioma cell development under hypoxic conditions. We also discovered that circ_0034642 could interact with miR-625-5p and further alter the expression of TAGLN2. Lastly, a circ_0034642 knockdown hindered tumor growth in vivo by regulating the miR-625-5p/TAGLN2 axis. Enhanced expression of circ_0034642 was found to promote cell glycolysis, proliferation, migration and invasion under hypoxic conditions in gliomas by sponging miR-625-5p to improve TAGLN2 expression, providing prospective biomarkers for the diagnosis of glioma.

Circ_0034642 was upregulated under hypoxic conditions in gliomas.     

氯化甲基汞对人SHG44胶质瘤细胞抗肿瘤活性的体外实验研究

目的探讨氯化甲基汞(MMC)对人SHG44胶质瘤细胞株的增殖、杀伤和细胞凋亡的影响,为胶质瘤治疗提供理论依据。方法体外培养SHG44胶质瘤细胞,分为空白对照组和MMC实验组(按氯化甲基汞浓度分组)。采用MTT比色法检测不同浓度MMC对体外培养SHG44细胞的增殖抑制和杀伤作用。用流式细胞仪测定MMC对SHG44细胞凋亡/坏死的影响。结果在体外1.25、2.50、5.00和10.00μmol.L-1的MMC均可抑制SHG44胶质瘤细胞的增殖,SHG44胶质瘤细胞存活率明显低于对照组(P0.05),增殖抑制作用随浓度的增加而增强。SHG44胶质瘤细胞接触2.50、5.00和10.00μmol.L-1的MMC后4、8、16和32 h的细胞存活率明显低于对照组(P0.05),细胞杀伤作用随浓度的增加和时间的延长而增强。SHG44胶质瘤细胞接触0.075、0.15、0.3和1.2μmol.L-1氯化甲基汞12 h后细胞凋亡/坏死率呈现剂量依赖性增高趋势(P0.01)。结论MMC具有杀伤SHG44胶质瘤细胞、抑制其增殖、诱导凋亡的抗肿瘤活性,有应用于胶质瘤治疗的潜在价值。     

Inhibition of autophagy enhances the effects of E1A-defective oncolytic adenovirus dl922-947 against glioma cells in vitro and in vivo

Oncolytic viruses represent a novel therapeutic approach for aggressive tumors, such as glioblastoma multiforme, which are resistant to available treatments. Autophagy has been observed in cells infected with oncolytic viruses; however, its role in cell death/survival is unclear. To elucidate the potential therapeutic use of autophagy modulators in association with viral therapy, we analyzed autophagy induction in human glioma cell lines U373MG and U87MG infected with the oncolytic adenovirus dl922-947. dl922-947 infection triggered an autophagic cellular response, as shown by the development of acidic vesicular organelles, LC3-I→LC3-II conversion, and reduction of p62 levels. However, on infection, the Akt/mTOR/p70s6k pathway, which negatively regulates autophagy, was activated, whereas the ERK1/2 pathway, a positive regulator of autophagy, was inhibited. Accordingly, MEK inhibition by PD98059 sensitized glioma cells to dl922-947 effects, whereas autophagy induction by rapamycin protected cells from dl922-947-induced death. Treatment with two inhibitors of autophagy, chloroquine and 3-methyladenine, increased the cytotoxic effects of dl922-947 in vitro. In vivo, the growth of U87MG-induced xenografts was further reduced by adding chloroquine to the dl922-947 treatment. In conclusion, autophagy acts as a survival response in glioma cells infected with dl922-947, thus suggesting autophagy inhibitors as adjuvant/neoadjuvant drugs in oncolytic virus-based treatments.     

Retracted Article: MiR-182-5p and miR-96-5p increased hepatocellular carcinoma cell mobility,proliferation and cisplatin resistance partially by targeting RND3

We investigated whether miR-182-5p or miR-96-5p could increase hepatocellular carcinoma (HCC) development by targeting Rho Family GTPase 3 (RND3) gene expression. The expression levels of miR-182-5p, miR-96-5p and mRNA/protein of RND3 in non-HCC liver tissue, HCC tissue and adjacent tissue specimens were evaluated by RT-qPCR and western blot. Patient-derived HCC cell culture was established, and miR-182-5p or miR-96-5p agomir or antagomir treatment was performed to mimic the overexpression or knockdown of the two miRNAs. HCC cell mobility in vitro was monitored by trans-well migration and invasion assay, while HCC cell growth in vitro was evaluated by cell viability, proliferation and apoptosis assay. HCC cell apoptosis was further investigated by caspase-3/-8/-9 activity assay. MiR-182-5p and miR-96-5p were significantly upregulated in HCC tissue specimens compared with non-HCC or adjacent tissue specimens, inversely correlating to RND3 mRNA expression level. Treatment with miR-182-5p or miR-96-5p agomir significantly reduced RND3 mRNA/protein expression level in HCC cells. MiR-182-5p- or miR-96-5p-targeting RND3 mRNA was verified by luciferase reporter assay and AGO2-RNA immunoprecipitation assay. MiR-182-5p or miR-96-5p agomir treatment significantly rescued HCC cell migration and invasion in vitro that were repressed by RND3 overexpression, during which ROCK1 and ROCK2 inhibition were involved. MiR-182-5p or miR-96-5p agomir treatment also increased HCC cell proliferation and cisplatin resistance in vitro, which could be antagonized by RND3 overexpression or ROCK inhibition. Thus, miR-182-5p and miR-96-5p increased HCC cell mobility, proliferation and cisplatin resistance in vitro partially by targeting RND3.

We investigated whether miR-182-5p or miR-96-5p could increase hepatocellular carcinoma (HCC) development by targeting Rho Family GTPase 3 (RND3) gene expression.     

Enhanced apoptosis following treatment with TRA-8 anti-human DR5 monoclonal antibody and overexpression of exogenous Bax in human glioma cells

Specific activation of apoptosis in tumor cells offers a promising approach for cancer therapy. Induction of apoptosis leads to activation of specific proteases. Two major pathways for caspase activation in mammalian cells have been described. One apoptotic pathway involves members of the tumor necrosis factor family of cytokine receptors (eg death receptor 5 (DR5)). The other pathway is controlled by the Bcl-2 family of proteins. The purpose of this study was to investigate whether increased apoptosis occurs in human glioma cells following infection with a recombinant adenoviral vector encoding the human Bax gene under the control of human vascular endothelial growth factor (VEGF) promoter element (AdVEGFBax) in combination with an anti-human DR5 monoclonal antibody (TRA-8). Specific overexpression of exogenous Bax protein induced apoptosis and cell death in glioma cell lines, through activation of both caspase-8 and -9, leading to activation of downstream caspase-3. The relative sensitivity to AdVEGFBax for the glioma cell lines was U251MG>U373MG>U87MG>D54MG. The recently characterized TRA-8 monoclonal antibody induces apoptosis of most TRAIL-sensitive tumor cells by specific binding to DR5 receptors on the cellular membrane. TRA-8 induced rapid apoptosis and cell death in glioma cells, but did not demonstrate detectable cytotoxicity of primary normal human astrocytes. The efficiency of TRA-8-induced apoptosis was variable in different glioma cell lines. The relative sensitivity to TRA-8 was U373MG>U87MG>U251MG>D54MG. The combination of TRA-8 treatment and overexpression of Bax overcame TRA-8 resistance of glioma cells in vitro. Cell viability of U251MG cells was 71.1% for TRA-8 (100 ng/ml) alone, 75.9% for AdVEGFBax (5 MOI) alone and 41.1% for their combination as measured by MTS assay. Similar enhanced apoptosis results were obtained for the other glioma cell lines. In vivo studies demonstrated that the combined treatment significantly (P<0.05) suppressed the growth of U251MG xenografts and produced 60% complete tumor regressions without recurrence. These data suggest that the combination of TRA-8 treatment with specific overexpression of Bax using AdVEGFBax may be an effective approach for the treatment of human malignant gliomas.     

MiR-139-5p通过靶向NFAT抑制卵巢癌SKOV3细胞的生长、集落形成、侵袭和迁移

目的:探究miR-139-5p对卵巢癌SKOV3细胞的生长、集落形成、侵袭能力以及迁移能力的影响及其机制。方法:采用qRT-PCR检测卵巢癌患者癌组织和卵巢癌细胞SKOV3中miR-139-5p表达情况。MiR-139-5p mimic转染SKOV3细胞,WST比色实验、集落形成实验和Transwell实验分别检测细胞的活性、集落形成、侵袭和迁移能力。采用TargetScan在线软件筛选miR-139-5p的潜在靶基因NFAT,并进一步验证。结果:MiR-139-5p在卵巢癌组织和SKOV3细胞中表达异常降低。MiR-139-5p过表达显著抑制SKOV3细胞的生长、集落形成、迁移及侵袭能力。NFAT是miR-139-5p的靶基因。过表达NFAT能逆转miR-139-5p过表达对SKOV3细胞集落形成、侵袭能力以及迁移能力与侵袭的抑制作用。结论:MiR-139-5p通过抑制靶基因NFAT来抑制卵巢癌SKOV3细胞的生长、集落形成、侵袭和迁移能力。     

Retracted Article: Long non-coding RNA KCNQ1OT1 regulates cell proliferation,apoptosis and chemo-sensitivity through modulating the miR-186-5p/NCAM1 axis in acute myeloid leukemia cells

Recent studies show that lncRNA KCNQ1OT1 and microRNA-186-5p (miR-186-5p) are involved in various human cancers. Moreover, it is reported that KCNQ1OT1 expression is upregulated in acute myeloid leukemia (AML). However, their roles in AML remain unknown. This study aimed to reveal the functional mechanism of KCNQ1OT1 and miR-186-5p in AML development. Quantitative real time polymerase chain reaction (qRT-PCR) was performed to detect the levels of genes. Cell proliferation and apoptosis were assessed by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry analysis respectively. A western blot assay was carried out to examine the protein levels. In addition, the interaction between miR-186-5p and KCNQ1OT1 or neural cell adhesion molecule 1 (NCAM1) was predicted by bioinformatics analysis tool starbase2.0 and confirmed by the dual luciferase reporter assay. KCNQ1OT1 and NCAM1 expressions were increased and miR-186-5p expression was decreased in AML samples and cells. The depletion of KCNQ1OT1 inhibited cell proliferation, and promoted apoptosis and chemo-sensitivity in AML. In addition, the upregulation of miR-186-5p suppressed AML cell proliferation, and induced apoptosis and chemo-sensitivity. Interestingly, KCNQ1OT1 directly downregulated miR-186-5p expression and miR-186-5p decreased NCAM1 expression by binding to the 3′ untranslated region (UTR) of NCAM1 mRNA. Furthermore, miR-186-5p knockdown or NCAM1 overexpression reversed the effects of KCNQ1OT1 depletion on AML cell progression. Our results firstly revealed a linear relationship between KCNQ1OT1, miR-186-5p, and NCAM1, and demonstrated that KCNQ1OT1 mediated AML cell progression via regulating the miR-186-5p/NCAM1 axis, revealing functional mechanisms of KCNQ1OT1 and miR-186-5p in AML development.

Recent studies show that lncRNA KCNQ1OT1 and microRNA-186-5p (miR-186-5p) are involved in various human cancers.     

Overexpression of WW domain-containing oxidoreductase WOX1 preferentially induces apoptosis in human glioblastoma cells harboring mutant p53

Purpose

Human WWOX gene encoding WW domain-containing oxidoreductase, named WWOX, FOR, or WOX1, has been studied in various types of cancer cells and shown to be a tumor suppressor with pro-apoptotic properties. Mutation or gain-of-function of p53 in glioma cells is associated with resistance to radiation therapy and poor prognosis. In this study, we overexpressed WOX1 to examine the pro-apoptotic activity against human glioblastoma cells harboring mutant p53.

Methods

Overexpression of WOX1 in glioblastoma cell lines and apoptosis-related assays were performed.

Results

Our results showed that overexpressed WOX1 induced apoptosis of glioblastoma U373MG harboring mutant p53 by causing hypoploidy and DNA fragmentation. However, ectopic WOX1 had no effect with U87MG possessing wild type p53. Unlike temozolomide, WOX1 induced apoptosis of U373MG cells via a mitochondria-independent and caspase-3-independent pathway.

Conclusions

Overexpression of WOX1 preferentially inhibited viability and induced apoptosis in human glioblastoma cells expressing mutant p53 via a mechanism independent of the intrinsic apoptotic pathway. Conceivably, the survival of human glioblastoma cells depends upon interactions between the gain-of-function of p53 and WOX1. This suggests that modulation of WOX1 expression may be a novel strategy for treating human glioblastoma cells with mutant p53.     

MiR-873-5p regulated LPS-induced oxidative stress via targeting heme oxygenase-1 (HO-1) in KGN cells

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women. Increasing evidence reveals that PCOS may be associated with an increased level of oxidative stress. This study aimed to explore the role and potential mechanism of microRNA-873-5p (miR-873-5p) in PCOS. Quantitative real time-PCR (qRT-PCR) analysis was performed to evaluate the miR-873-5p levels in both clinical follicular fluid samples from patients with PCOS and cultured human ovarian granulosa cell-like KGN cells. The results indicated that miR-873-5p was up-regulated in the follicular fluid from patients with PCOS, as well as in the LPS-induced KGN cells. MTT assay showed that miR-873-5p inhibitor attenuated the LPS-induced inhibition of KGN cell viability. Flow cytometry indicated that miR-873-5p inhibitor suppressed cell apoptosis in LPS-induced KGN cells. Besides, miR-873-5p inhibitor resulted in a decrease in oxidative stress, which was evidenced by the reduced production of reactive oxygen species (ROS) and malondialdehyde (MDA). Further luciferase reporter assay proved that miR-873-5p directly targeted the 3′UTR of HO-1 mRNA. Small-interfering RNA (siRNA) targeting heme oxygenase-1 (HO-1) attenuated the effect of miR-873-5p inhibitor on oxidative stress in KGN cells. Besides, we also found that miR-873-5p inhibitor activated the p38/Nrf2/HO-1 signaling pathway in KGN cells. The findings may provide insightful evidence for preventing and treating PCOS by targeting miR-873-5p.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women.     

microRNA-181b影响U87胶质瘤细胞干细胞对替莫唑胺化疗耐受性的实验研究

目的 本研究旨在建立miR-181b慢病毒载体感染U87胶质瘤干细胞模型后,检测其对TMZ化疗耐受性的变化,并探讨miR-181b对于恶性脑胶质瘤（GBM）辅助化学治疗的意义.方法 通过miR-181b慢病毒表达载体转染U87胶质瘤干细胞,提高miR-181b在U87胶质瘤干细胞中的表达.进一步通过RT-PCR、二次神经球形成实验、琼脂糖集落形成实验、MTT实验来分析miR-181b对于U87胶质瘤干细胞在TMZ化疗耐受性方面的作用.结果 miR-181b慢病毒表达载体转染U87胶质瘤干细胞后,U87胶质瘤干细胞中的miR-181b明显上调,而且U87胶质瘤干细胞的生长受到明显抑制;miR-181b可以加强TMZ抑制U87胶质瘤干细胞的生长的作用.结论 miR-181b可以加强TMZ抑制U87胶质瘤干细胞的生长,miR-181b可能对于提高U87胶质瘤干细胞对TMZ的敏感性,降低化疗耐受性起到一定作用.