Long non‐coding RNA ANRIL enhances mitochondrial function of hepatocellular carcinoma by regulating the MiR‐199a‐5p/ARL2 axis

Although the roles of long non‐coding RNA (lncRNA) ANRIL (Antisense non‐coding RNA in the INK4A locus) have been established in various tumors, its roles in mitochondrial metabolic reprogramming of hepatocellular carcinoma (HCC) cells are still unclear. This work aims to explore lncRNA ANRIL roles in regulating the mitochondrial metabolic reprogramming of liver cancer cells. First, we found that lncRAN ANRIL expression was significantly increased in HCC tissues or cells compared with the normal adjacent tissues and normal tissues or cells. Functional experiment showed that overexpression of lncRNA ANRIL promoted mitochondrial function in HCC cells, evident by the increased mitochondrial DNA copy numbers, ATP (Adenosine triphosphate) level, mitochondrial membrane potential, and the expression levels of mitochondrial markers, while ANRIL knockdown exerted the opposite effects. Mechanistically, lncRNA ANRIL acted as a competing endogenous RNA to increase ARL2 (ADP‐ribosylationfactor‐like 2) expression via sponging miR‐199a‐5p. Notably, the miR‐199a‐5p/ARL2 axis is necessary for ANRIL‐mediated promoting effects on HCC cell mitochondrial function. This work reveals a novel ANRIL‐miR‐199a‐5p‐ARL2 axis in HCC cell progression, which might provide potential targets for HCC treatment.     

Genistein enhances the effect of trichostatin A on inhibition of A549 cell growth by increasing expression of TNF receptor-1

Our previous study has shown that genistein enhances apoptosis in A549 lung cancer cells induced by trichostatin A (TSA). The precise molecular mechanism underlying the effect of genistein, however, remains unclear. In the present study, we investigated whether genistein enhances the anti-cancer effect of TSA through up-regulation of TNF receptor-1 (TNFR-1) death receptor signaling. We incubated A549 cells with TSA (50 ng/mL) alone or in combination with genistein and then determined the mRNA and protein expression of TNFR-1 as well as the activation of downstream caspases. Genistein at 5 and 10 μM significantly enhanced the TSA-induced decrease in cell number and apoptosis in a dose-dependent manner. The combined treatment significantly increased mRNA and protein expression of TNFR-1 at 6 and 12 h, respectively, compared with that of the control group; while TSA alone had no effect. TSA in combination with 10 μM of genistein increased TNFR-1 mRNA and protein expression by about 70% and 40%, respectively. The underlying mechanism for this effect of genistein may be partly associated with the estrogen receptor pathway. The combined treatment also increased the activation of caspase-3 and ‐10 as well as p53 protein expression in A549 cells. The enhancing effects of genistein on the TSA-induced decrease in cell number and on the expression of caspase-3 in A549 cells were suppressed by silencing TNFR-1 expression. These data demonstrated that the upregulation of TNFR-1 death receptor signaling plays an important role, at least in part, in the enhancing effect of genistein on TSA-induced apoptosis in A549 cells.     

辐射增敏剂Wortmannin促进组蛋白去乙酰化酶抑制剂诱导的MOLT-4细胞凋亡

目的 探讨选择性磷脂酰肌醇激酶相关蛋白激酶家族（PIKKs）的小分子抑制剂Wortmannnin对组蛋白去乙酰化酶抑制剂（HDIs）诱导的细胞凋亡的影响,并探讨DNA损伤功能信号级联在HDIs类药物抗肿瘤效应中的功能机制。方法 流式细胞术分析加药后不同时间点的细胞周期分布和细胞凋亡情况;荧光显微镜下观察用药后的细胞核形态;免疫印迹法检测加药前后Caspase-2,Caspase-3,Caspase-7和Survivin蛋白表达情况。结果 低剂量Wortmannin单独作用对肿瘤细胞生存率未见显著影响,但能够激活G1/S细胞周期阻滞、抑制TSA引发的G2/M阻滞、并显著促进肿瘤细胞凋亡。在此过程中,Wortmannin促进凋亡效应分子Caspase-2,Caspase-3和 Caspase-7的激活并抑制Survivn的表达。结论 PIKKs家族的小分子抑制剂Wortmannnin 通过下调Survivin并促进Caspase信号级联而促进TSA诱导的肿瘤细胞凋亡。     

Therapeutic effect of carnosine in rat model of experimental liver carcinogenesis

The possible anticancer effect of carnosine versus doxorubicin was investigated against hepatocellular carcinoma (HCC) induced by trichloroacetic acid (TCA) (500 mg/kg/day, p.o., for 5 days) in rats. Following induction of HCC, rats treated with either carnosine (10 mg/kg/day, i.p.), or doxorubicin (2.5 mg/kg, i.p., once weekly), for 2 weeks. Carnosine significantly decreased serum alanine aminotransferase, and hepatic lipid peroxidation, nitric oxide, tumor necrosis factor-α, and nuclear factor-κB p65 unit, and significantly increased liver total antioxidant status in TCA-challenged rats. The effects of doxorubicin on oxidative, nitrative, and inflammatory biomarkers were less significant than carnosine. However, both carnosine and doxorubicin significantly induced liver tissue apoptotic biomarkers, Bax, cytosolic cytochrome C, and caspase-3, in a comparable manner. Additionally, carnosine and doxorubicin reduced the histopathological dysplastic changes, and alpha-fetoprotein expression in liver of rats with HCC. It was concluded that carnosine significantly protected against TCA-induced liver carcinogenesis in rats, through its antioxidant, antinitrative, and anti-inflammatory effects, and induction of apoptosis.     

Trichostatin A sensitizes cisplatin-resistant A549 cells to apoptosis by up-regulating death-associated protein kinase

Aim:

To investigate the apoptosis-inducing effect of trichostatin A (TSA) in the human lung adenocarcinoma cisplatin-resistant cell line (A549/CDDP) and to examine whether TSA can enhance sensitivity to cisplatin treatment and the underlying molecular mechanisms of such an enhancement.

Methods:

Cell viability was evaluated using the Neutral Red assay. Apoptosis was assessed using Hoechst 33258 staining and flow cytometry analysis. Protein expression was detected by Western blotting. To determine the role of Death-associated protein kinase (DAPK) in TSA-induced apoptosis in the A549/CDDP cell line, cells were transfected with pcDNA3.1(+)-DAPK, which has a higher expression level of DAPK compared to endogenous expression, and DAPK activity was inhibited by both over-expression C-terminal fragment of DAPK which may competitive binding DAPK substrates to inhibit the function of DAPK and RNA interference.

Results:

TSA induced apoptosis in both A549 cells and A549/CDDP cells. TSA enhanced the sensitivity of A549/CDDP cells to cisplatin, along with concomitant DAPK up-regulation. When DAPK was over-expressed, A549/CDDP cells became sensitive to cisplatin and the cytotoxicity of TSA could be increased. Moreover, the cytotoxicity of TSA could be alleviated by inhibition of DAPK activity by the expression of a recombinant C-terminal fragment of DAPK or RNA interference.

Conclusion:

TSA induced sensitivity to cisplatin treatment in cisplatin-resistant A549 cells. The up-regulation of DAPK is one of the mechanisms mediating sensitization to TSA-induced apoptosis in cisplatin-resistant cells.     

Microarray analysis of altered long non-coding RNA expression profile in liver cancer cells treated by ginsenoside Rh2

Microarray expression profiles of lncRNAs and mRNAs were investigated in HepG2 cells treated with 20?μg/ml ginsenoside Rh2 as well as in ginsenoside Rh2-untreated cells. Microarray analysis showed 618 upregulated lncRNAs and 161 downregulated lncRNAs in HepG2 cells treated with ginsenoside Rh2 compared with the control group. Moreover, three differentially expressed lncRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). This may be beneficial to patients as an anti-cancer treatment and potentially provide novel targets for HCC (hepatocellular carcinoma) therapy.     

人诱导多能干细胞重编程过程长链非编码RNA的差异表达研究

目的 探讨人诱导多能细胞重编程过程中长链非编码RNA的表达改变及其作用。方法 利用Agilent Human lncRNA (4*180K)芯片检测人体细胞、诱导多能干细胞以及胚胎干细胞中的lncRNA表达情况。通过数据分析比较人体细胞诱导为多能干细胞后lncRNA的差异表达,筛选出在重编程过程中可能发挥作用的lncRNA。结果 人诱导多能干细胞lncRNA表达谱类似于胚胎干细胞而与体细胞不同。通过聚类分析发现干细胞与体细胞之间有3 156个差异表达lncRNA。通过生物学分析发现人体细胞诱导为多能干细胞重编程过程中有222个差异表达的lncRNA。结论 lncRNA在人多能干细胞重编程过程中可能发挥着重要的作用。     

Analysis of long non‐coding RNA involved in atrazine‐induced testicular degeneration of Xenopus laevis

Long non‐coding RNA (lncRNA) plays a critical role in male germline development. Atrazine (AZ) as an environmental endocrine disrupting chemical (EDCs) can induce male reproductive toxicity in amphibians. Our previous studies demonstrated that AZ can alter gene and circular RNA (circRNA) expression of damaged testes in Xenopus laevis (X. laevis). We furthered to investigate the lncRNA expression profiling in the testis of X. laevis. Over 3559 lncRNAs were detected by lncRNA sequencing. AZ induced 40 upregulated and 46 downregulated differentially expressed lncRNAs. KEGG analysis showed that AZ‐affected lncRNAs mainly involve in 19 pathways among which 12 pathways are found in circRNA analysis. This study for the first time demonstrated that AZ can alter lncRNAs which may play a role in testicular degeneration through regulating expressions of functional genes in X. laevis. Our data may provide more insights on the mechanism about male reproductive toxicity of EDCs.     

ESET通过lncRNA GMDS-AS1调控肝癌细胞增殖、凋亡、糖酵解的研究

目的 探究组蛋白甲基转移酶集合域分叉1(ESET)、长链非编码RNA甘露糖4,6-脱水酶反义RNA1(lncRNA GMDSAS1)对肝癌细胞增殖、凋亡、糖酵解的调控及二者之间的潜在关系。方法 收集右江民族医学院附属医院和百色市人民医院2019年1月至2020年1月期间手术切除的肝癌组织及癌旁组织49例。脂质体法将空白组（不做任何处理）、空载体组（转染pcDNA或si-con）、敲减ESET组（转染si-ESET）、过表达lncRNA GMDS-AS1组（转染pcDNA-GMDS-AS1）转染至HepG2细胞；实时荧光定量逆转录聚合酶链反应（qRT-PCR）实验、蛋白质印迹法实验检测ESET、lncRNA GMDS-AS1、细胞增殖抗原标志物Ki-67、葡萄糖转运蛋白1(GLUT-1)、乳酸脱氢酶A(LDHA)、活化胱天蛋白酶-3(C-caspase-3)的表达；细胞计数试剂盒（CCK8）法、流式细胞术检测细胞增殖、凋亡；RNA结合蛋白免疫沉淀（RIP）实验检测ESET与lncRNA GMDS-AS1的关系。结果 肝癌组织中ESETmRNA和蛋白表达量为5.18±0.94、0.69±0...     

TNFα and Fas/FasL pathways are involved in 9-Methoxycamptothecin-induced apoptosis in cancer cells with oxidative stress and G2/M cell cycle arrest

9-Methoxycamptothecin (MCPT) has been recently reported to have a strong anticancer activity. However, its detailed mechanism of action in human cancer cells has not been well clarified. The results showed that MCPT induced cytotoxicity in seven human cancer cell lines in a dose dependent manner after 72 h, with A2780 and Hela cell lines more sensitive, so the two cell lines were chosen to do further studies. MCPT induced strong G₂/M arrest in both A2780 cells and Hela cells after 24 h, following by substantial sub-G1 arrest (indicating apoptosis). The apoptosis was verified by staining with Annexin V-FITC and propidium iodide. ROS generation increased significantly in MCPT-induced apoptosis. Meanwhile, the apoptosis appeared to be dependent on caspase-3, -8 and -9 in A2780 cells, and caspase-3 in Hela cells. In addition, MCPT induced up-regulation expression of most of seventeen genes in both cell lines. Western blot verified that changes of TNFα, Fas, P53 and P27 protein level were consistent with their gene expression changes. Taken together, MCPT plays an important role in tumor growth suppression by inducing apoptosis in both cell lines via extrinsic and intrinsic apoptotic pathways, and has the potential to be developed into an antitumor agent.     

Homeobox B5 suppression attenuates proliferation and elevates apoptosis in hepatoma cell lines through ERK/MDM2 signalling

Homeobox B5 (HOXB5), a member of the HOX gene family, is an important gene in tumourigenesis. However, its role in hepatocellular carcinoma (HCC) cell proliferation and apoptosis remains unclear. In this study, we investigated the role and regulation mechanism of HOXB5 in HCC cell lines Hep3B and LM6. The data indicated high expression of HOXB5 in HCC tissues and cell lines. In HCC cells, inhibition of HOXB5 by transfection with HOXB5 siRNA significantly constrained cell viability, and Bcl-2 levels, and it increased cell apoptosis, cytochrome c levels, BAX levels, and caspase-3 activity. On the contrary, HOXB5 overexpression increased proliferation and Bcl-2 levels but inhibited BAX levels and caspase-3 activity in these cells. HOXB5 downregulation attenuated activation of extracellular signal-regulated kinase (ERK) and expression of the murine double minute 2 (MDM2) oncogene. Incubation with the ERK activator, phorbol 12-myristate 13-acetate (40 μmol/L), for 12 hours reversed the effects of HOXB5 inhibition on MDM2 expression, cell proliferation, and apoptosis in HCC cells. Overall, this study demonstrated that HOXB5 inhibition regulated MDM2 expression by controlling ERK activation and that it modulated proliferation and apoptosis in HCC cells.     

FAT10基因在肝细胞癌中的表达和突变分析

目的：探讨FAT10基因在肝细胞癌中的表达和突变情况。方法：应用Western blot 检测60例肝癌患者癌和癌旁组织FAT10蛋白表达水平。采用DNA直接测序方法检测60例肝癌患者（癌和癌旁组织）和5种人肝癌细胞中FAT10基因编码区遗传变异情况。结果：在60例肝癌中,85%（51/60）的肝癌组织FAT10蛋白的表达明显高于癌旁组织。在60例肝癌患者和5种人肝癌细胞中检测到FAT10基因编码区存在7个单核苷酸多态性位点,未发现基因突变。结论：FAT10基因在中国人肝癌中存在表达上调,但未发现基因突变现象,提示FAT10在肝癌中的异常表达并不依赖于该基因突变,可能存在其他机制,有待于进一步研究。     

组蛋白去乙酰化酶抑制剂曲古抑菌素通过上调Slug促进结直肠癌细胞上皮-间质转化的研究

目的 研究组蛋白去乙酰化酶抑制剂曲古抑菌素(TSA)诱导结直肠癌细胞HCT116发生上皮-间质转化(EMT)的潜在分子机制.方法 TSA处理结直肠癌细胞HCT116,观察细胞形态变化;划痕实验观察细胞迁移侵袭能力的变化.实时定量PCR、Western blot检测HCT116细胞EMT标志物以及Slug的表达情况.siRNA敲除Slug表达,Western blot检测HCT116细胞EMT标志物表达变化,以及细胞形态变化.结果 TSA可以促进结直肠癌细胞HCT116的迁移能力,诱导HCT116细胞发生EMT转化,包括细胞形态改变,EMT标志物表达变化.TSA可以促进EMT关键转录因子Slug的表达,包括蛋白和mRNA水平;敲除Slug表达可以抑制TSA诱导的EMT转化过程.结论 组蛋白去乙酰化酶抑制剂TSA通过上调Slug的表达促进结直肠癌细胞HCT116发生EMT转化,从而提高其迁移能力.     

Cationic lipid nanoparticles for therapeutic delivery of siRNA and miRNA to murine liver tumor

miR-122, a liver-specific tumor suppressor microRNA, is frequently down-regulated in hepatocellular carcinoma (HCC). LNP-DP1, a cationic lipid nanoparticle formulation, was developed as a vehicle to restore deregulated gene expression in HCC cells by miR-122 delivery. LNP-DP1 consists of 2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), egg phosphatidylcholine, cholesterol and cholesterol-polyethylene glycol. In vitro, LNP-DP1-mediated transfection of a miR-122 mimic to HCC cells down-regulated miR-122 target genes by > 95%. In vivo, siRNAs/miRNAs encapsulated in LNP-DP1 were preferentially taken up by hepatocytes and tumor cells in a mouse HCC model. The miR-122 mimic in LNP-DP1 was functional in HCC cells without causing systemic toxicity. To demonstrate its therapeutic potential, LNP-DP1 encapsulating miR-122 mimic was intratumorally injected and resulted in ~ 50% growth suppression of HCC xenografts within 30 days, which correlated well with suppression of target genes and impairment of angiogenesis. These data demonstrate the potential of LNP-DP1-mediated microRNA delivery as a novel strategy for HCC therapy.From the Clinical EditorIn this study, LNP-DP1 –a cationic lipid nanoparticle formulation –is reported as a vehicle to restore deregulated gene expression in hepatic carcinoma cells by siRNA and miRNA delivery using a mouse model. Further expansions to this study may enable transition to clinical trials of this system.     

lncRNA SOX21-AS1的表达对卵巢癌细胞增殖和转移能力的影响及机制研究

目的 探讨长链非编码RNA SRY-box转录因子21反义RNA 1(lncRNA SOX21-AS1)对卵巢癌细胞增殖和转移能力的影响及其作用机制。方法 选取卵巢癌组织和其邻近的癌旁组织各75例,另选取卵巢癌细胞株SKOV3、A2780、OVCR3和人卵巢正常上皮细胞HOSE作为实验对象,实时荧光定量PCR法检测lncRNA SOX21-AS1在组织和细胞中的表达。根据是否干扰lncRNA SOX21-AS1表达将细胞分为si-NC组和si-SOX21-AS1组。MTS实验和平板克隆实验检测细胞增殖能力,Transwell实验检测细胞转移能力,TOP/FOP荧光素酶实验和Western blot实验检测细胞中Wnt/β-catenin信号通路活性和相关蛋白表达水平。结果 与癌旁组织相比,lncRNA SOX21-AS1在卵巢癌组织中的表达上调（P<0.05）。与卵巢正常上皮细胞HOSE相比,lncRNA SOX21-AS1在卵巢癌细胞中的表达上调,SKOV3中最高（P<0.05）。与FIGO分期Ⅰ-Ⅱ期、无淋巴结转移的患者相比,FIGO分期Ⅲ-Ⅳ期、有淋巴结转移患者lnc...