Effect of PKC pathway on G1/S progression control in HeLa cells

The effect of PKC activity on G₁/S progression in HeLa cells has been studied. The result shows that (ⅰ ) PKC activity alteration in G₁ phase affects G₁/S progression in HeLa cells. It has been observed that G₁/S progression is stimulated by PKC agonist TPA and inhibited by PKC inhibitor GF-109203X. ( ⅱ) The expression of c-myc and c-jun is stimulated by TPA and inhibited by GF-109203X treatment in early G₁ phase. (ⅲ ) During G₁/S progression, the expression of CyclinD₁ is stimulated by TPA treatment and inhibited by GF-109203X treatment. There is no effect on the expression of CDK4. It is likely that PKC pathway regulates G₁/S progression through regulating the expression of some early response genes and engine molecules in HeLa cells.     

Effects of antisense CENP-B on the proliferation of HeLa (Tet-off) cells

In order to study the change of the expression of centromere protein B (CENP-B) caused by antisense transfection, proto-eukaryotically expressed fused protein GST-CENP-B (65 ku) was injected into mouse, and a peculiar anti-CENP-B serum MaCenpB was collected. A strain of transfected HeLa Tet-off cell HaCb, which contains antisense CENP-B expressing vector pBI-EGFP-as-CenpB, was prepared. Northern blot and Western blot were used to analyze the repression of internal CENP-B in transfected cells. According to the growth curve, the proliferation of HeLa (Tet-off) is repressed by antisense CENP-B, and the multiplication time is prolonged for 32.81 h. The analysis of flow cytometry revealed that, compared with HeLa (Tet-off), the G₁ cell population of HaCb is increased (ΔG₁= 9%) while S fraction is decreased (ΔS = 11%), but the G₂/M phase is nearly unchanged (ΔG₂/M=3%). In the meanwhile, the mitotic index of HaCb declines greatly compared with that of HeLa (Tet-off). Immunofluorescence showed that the assembling of centromeres in HaCb cell is arrested. These results suggest that a normal expression of CENP-B may be necessary for cell proliferation.     

Correlation of microRNAs responding to high dose γ-irradiation with predicted target mRNAs in HeLa cells using microarray analyses

An increasing data indicates that altered microRNAs （miRNAs） participate in the radiation-induced DNA damage response. However, a correlation of mRNA and miRNA profiles across the entire genome and in response to irradiation has not been thor- oughly assessed. We analyzed miRNA microarray data collected from HeLa cells after ionizing radiation （IR）, quantified the ex- pression profiles of mRNAs and performed comparative analysis of the data sets using target prediction algorithms, Gene Ontol- ogy （GO） analysis, pathway analysis, and gene network construction. The results showed that the altered miRNAs were involved in regulation of various cellular functions, miRNA-gene network analyses revealed that miR- 186, miR- 106b, miR- 15 a/b, CCND 1 and CDK6 played vital role in the cellular radiation response. Using qRT-PCR, we confirmed that twenty-two miRNAs showed differential expression in HeLa cells treated with IR and some of these miRNAs affected cell cycle progression. This study demonstrated that miRNAs influence gene expression in the entire genome during the cellular radiation response and suggested vital pathways for further research.     

Chk1 prevents abnormal mitosis of S-phase HeLa cells containing DNA damage

To explore effects of DNA damage on cell-cycle progression in p53-deficient tumor cells, synchronized HeLa cells at G1, S and G2/M phases were treated with methyl methanesulfnate （MMS）. The results showed that the MMS treatment resulted in the cell-cycle arrest or delay in all 3 phases, while the S-phase cells were the most sensitive to MMS. Further studies demonstrated that ATM-Chk2 and p38 MAPK signaling pathways were activated in all 3 phases when the cells were treated with MMS; whereas Chk1 was activated only in S phase under the drug treatment, indicating that Chk1 specifically participated in S-phase checkpoints. To analyze the role of Chk1 in S-phase checkpoints, we administered a specific Chk1 inhibitor, UCN-01, to the S-phase cells. The results showed that the S-phase cells treated with MMS＋UCN-01 could enter aberrant mitosis without finishing DNA replication, indicating that Chk1 mainly functions in the DNA damage checkpoint rather than in the replication checkpoint. In addition, MMS treatment alone inhibited the accumulation of cyclin B1, a key component of M-phase CDK-cyclin complex, in the S-phase cells, whereas the inhibition of Chk1 activation resulted in the accumulation of cyclin B1 in the MMS-treated S-phase cells. This observation further supports the view that DNA-damaged S-phase cells enter abnormal mitosis when Chk1 activation is inhibited. Our results demonstrate that Chk1 is a specific kinase that plays an important role in the MMS-induced S-phase DNA damage checkpoint. As p53 is not involved in this process, Chk1 may be a potential target for p53-deficient tumor therapy.     

siRNA沉默DNA甲基化转移酶1基因干扰胎牛成纤维细胞增殖和凋亡

为阐明Dnmt1-siRNA对胎牛成纤维细胞（FBFCs）的影响，设计了3种针对Dnmt1的siRNA来转染FBFCs．结果显示：Dnmt1-siRNA3转染FBFCs 24,48,72 h后，Dnmt1 mRNA表达水平显著降低（P<0.01）．在转染后48 h，siRNA3对Dnmt1抑制效率达到近80%，Dnmt1-siRNA3处理的FBFCs增殖也受到显著影响，FBFC细胞活力下降、处于G₀/G₁过渡期细胞数量增多（P<0.05）．但Dnmt1-siRNA3组FBFC细胞的凋亡率也显著增加（P<0.05）．说明通过Dnmt1特异性siRNA能够有效地敲低FBFC中的Dnmt1 mRNA表达量，由于细胞周期分布变化，siRNA处理后FBFC作为核供体能提高SCNT的效率．     

Clinical and pathological features of miR-10b and RHOC gene expression in hepatocellular carcinoma

Aberrant expression of microRNAs （miRNAs） was reported frequently in different human cancers. The major role of miRNA is targeting 31-UTR of coding gene and causing translational repression or mRNA degradation. miR-10b overexpression was reported to promote breast cancer metastasis by up-regulating RHOC expression. But its expression in hepatocellular carcinoma （HCC） remains unclear. Our study indicated that the expression of miR-10b was different in HCC and adjacent tissue samples, and reduced expression of miR-10b in HCC was related tovein invasion. High-level expression of RHOC was also related to vein invasion in HCC. But no correlation was found between miR-10b and RHOC expression. These results suggest that miR-10b and RHOC are independent predictors of HCC invasion and metastasis.     

Identification of common microRNA-mRNA regulatory biomodules in human epithelial cancer

The complex regulatory network between microRNAs and gene expression remains an unclear domain of active research. We proposed to address in part this complex regulation with a novel approach for the genome-wide identification of biomodules derived from paired microRNA and mRNA profiles, which could reveal correlations associated with a complex network of dys-regulation in human cancer. Two published expression datasets for 68 samples with 11 distinct types of epithelial cancers and 21 samples of normal tissues were used, containing microRNA expression and gene expression profiles, respectively. As results, the microRNA expression used jointly with mRNA expression can provide better classifiers of epithelial cancers against normal epithelial tissue than either dataset alone (P=1×10^–10, F test). We identified a combination of 6 microRNA-mRNA biomodules that optimally classified epithelial cancers from normal epithelial tissue (total accuracy = 93.3%; 95% confidence intervals: 86%–97%), using penalized logistic regression (PLR) algorithm and three-fold cross-validation. Three of these biomodules are individually sufficient to cluster epithelial cancers from normal tissue using mutual information distance. The biomodules contain 10 distinct microRNAs and 98 distinct genes, including well known tumor markers such as miR-15a, miR-30e, IRAK1, TGFBR2, DUSP16, CDC25B and PDCD2. In addition, there is a significant enrichment (Fisher’s exact test P=3×10^–10) between putative microRNA-target gene pairs reported in 5 microRNA target databases and the inversely correlated microRNA-mRNA pairs in the biomodules. Further, microRNAs and genes in the biomodules were found in abstracts mentioning epithelial cancers (Fisher’s Exact test, unadjusted P<0.05). Taken together, these results strongly suggest that the discovered microRNA-mRNA biomodules correspond to regulatory mechanisms common to human epithelial cancer samples. In conclusion, we developed and evaluated a novel comprehensive method to systematically identify, on a genome scale, microRNA-mRNA expression biomodules common to distinct cancers of the same tissue. These biomodules also comprise novel microRNA and genes as well as an imputed regulatory network, which may accelerate the work of cancer biologists as large regulatory maps of cancers can be drawn efficiently for hypothesis generation.     

Human endothelial senescence can be induced by TNF-α

TNF-α is one of the most important proinflammatory cytokines in mediating multiple physio-pathological functions during immunological responses. Vascular endothelial cells, when stimulated by TNF-α, can increase the expression of multiple cytokines and cellular adhesion molecules and, in turn, actively promote the inflammatory responses by recruiting and activating of leukocytes to the inflammatory site. In addition to endothelial death induced by TNF-α, we found for the first time that TNF-α can also induce the human endothelial cells senescence. The induced senescent endothelial cells will display SA-β-Gal staining and they were arrested in G0-G1 phase. We found that †Ψ_m would always be up-regulated in response to TNF-α stimulation at early time but when the cells become senescent, †ψ_m shows a tendency to decrease. It may reflect the sthenic function of mitochondria at early time in response to TNF-α stimulation and decay when the endothelial cells were induced senescent. ROS fluctuates at early time and also decreases when the endothelial cells become senescent. Our results show that the change of mitochondrial function may be related to the senescent process.     

Dynamic analysis of DNA damage induced miRNAs in colon cancer cells

It is known that microRNAs （miRNAs） expression profile shows substantial changes in cells under DNA damage. Here, we did miRNA microarray and quantitative real-time PCR to comprehensively identify the differentially expressed miRNAs in colon cancer cell lines HCT116 p53＋/＋ and HCT116 p53-/-. Cluster analysis revealed a panel of differentially expressed miRNAs which are regulated by p53 and/or UV-C induced DNA damage. These altered miRNAs tend to be located in chromosomes 13, X and 17. Moreover, pathways enrichment analysis estimated that MAPK pathway, focal adheren pathway, p53 pathway and Wnt pathway were mediated by these miRNAs to exert their functions in DNA damage response. Additionally, we found that miR- 320a, one of the UV-C induced miRNAs, play a role in protecting cells from DNA damage. Taken together, our results show that miRNAs are dynamic regulated in p53- dependent or -independent manners in different cell contexts and different situations following DNA damage.     

Caveolin-1 re-expression reverses G<Subscript>0</Subscript>/G<Subscript>1</Subscript> arrest in caveolin-1 knockout mesangial cells

Flow cytometry, BrdU incorporation, and western blotting were used to investigate whether caveolin-1 (Cav-1) is involved in cell cycle progression of renal glomerular mesangial cells. Wide type mesangial cells re-entered the cell cycle after 22 h incubation with 20% fetal bovine serum (FBS) and Cav-1 knockout cells remained in G₀/G₁ phase after 48 h, which indicated that Cav-1 knockout mesangial cells were G₀/G₁ arrest. The protein level of cyclin D1 significantly increased after incubation with 20% FBS for 6 h in wide type mesangial cells, and 12 h in Cav-1 knockout cells. It suggested that cyclin D1 upregulation was delayed in knockout mesangial cells. Cav-1 re-expression in Cav-1 knockout mesangial cells increased the ratio of S phase from 4.8% to 15.26%, and decreased the ratio of G₀/G₁ phase from 90.96% to 77.84%, which implied that Cav-1 re-expression can reverse cell cycle arrest. It concludes that Cav-1 may promote the proliferation of mesangial cells.     

Function of resveratrol derived from transgenic plant expressing resveratrol synthase gene

Two genes from grapevine coding for resveratrol synthase, named RS1 and RS2, were cloned by RT-PCR. AnEscherichia coli expression vector was constructed by insertion of RS1 into pBV221. A specific protein with the same molecular weight (42 ku) as the resveratrol synthase was expressed and used to prepare the rabbit antiserum. A plant expression vector was constructed by inserting the RS1 gene into pBin438 downstream of the doubled CaMV 35S promoter and TMV-Ω fragment. PCR-positive transgenic tobacco plants were obtained after transformation withAgrobacterium tumefaciens LBA4404 harboring the plant expression vector. Southern blot analysis demonstrated that the foreign gene was integrated into the tobacco genome. The results of RT-PCR and Western blot indicated that the RS1 gene was transcribed and expressed. Formation of resveratrol in transgenic tobacco was further determined by thin-layer chromatography of silica gel and HPLC. Increased accumulation of human breast adenocarcinoma cells in G₀ and G₁ phases of cell cycle was observed in cells treated with resveratrol purified from transgenic tobacco as compared to the untreated cells.     

MicroRNA及其靶基因在糖尿病肾病小鼠肾脏的表达

 验证与糖尿病肾病小鼠肾脏相关的microRNAs的表达并运用实时荧光定量PCR分析靶基因与糖尿病肾病的关系。以db/db小鼠为模型组（DN组）,db/m小鼠为正常组（NC组）,定期测量小鼠的体重、血糖、甘油三酯、总胆固醇及24 h尿蛋白排泄率。留取DN小鼠与NC小鼠肾脏组织,检测肾脏组织形态学染色及实时荧光定量PCR（qRT-PCR）。qRT-PCR验证差异表达的microRNAs及其靶基因的mRNA表达水平。血糖、24 h尿蛋白排泄率结果表明糖尿病肾病动物模型构建成功。与NC小鼠相比,DN小鼠肾脏miR-196a、miR-21、miR-200b表达明显升高,且差异有统计学意义（P<0.05）。miR-196a、miR-200b、miR-21的表达水平与血糖、甘油三酯、总胆固醇、24 h尿蛋白排泄率存在正相关关系（P<0.05）。利用miRNAs数据库预测miR-196a的靶基因有ANX1、HOXB7、PTEN、FOXO1、HOXB8、HOXA5等。与NC组比较,DN组ANX1、FOXO1的mRNA表达水平降低,且差异有统计学意义（P<0.05）。同时ANX1、FOXO1与24 h尿蛋白排泄率存在正相关（P<0.05）。MiR-196a可能通过调节ANX1、FOXO1的表达水平来参与糖尿病肾病的发生发展。     

The changes in telomerase activity and telomere length in HeLa cells undergoing apoptosis induced by sodium butyrate

The changes in telomerase activity and telomere length during apoptosis in HeLa cells as induced by sodium butyrate (SB) have been studied. After a 48 h SB treatment, HeLa cells demonstrated characteristic apoptotic hallmarks including chromatin condensation, formation of apoptotic bodies and DNA Laddering which were caused by the cleavage and degradation of DNA between nucleosomes. There were no significant changes in telomerase activity of apoptotic cells, while the telomere length shortened markedly. In the meanwhile, cells became more susceptible to apoptotic stimuli and telomere became more vulnerable to degradation after telomerase activity was inhibited. All the results suggest that the apoptosis induced by SB is closely related to telomere shortening, while telomerase enhances resistance of HeLa cells to apoptotic stimuli by protecting telomere.     

MiR-205 inhibits the invasion and migration of esophageal squamous cell carcinoma by modulating SMAD1 expression

Esophageal squamous cell carcinoma （ESCC） is one of the most lethal cancers worldwide. In this study, we aimed to investigate the underlying mechanisms of metastasis inhibition by miR-205 in ESCC. In microRNA （miRNA） array and quantitative RT-PCR analyses, we found that the expression level of miR-205 was significantly lower in patients with lymph node metastasis compared with that in patients without lymph node metastasis. After transfection of miR-205 mimics or inhibitors into ESCC cell lines, a significant negative correlation was observed between the expression level of miR-205 and Smad 1. In luciferase reporter assays, we revealed that miR- 205 inhibited the expression of SMAD1 by targeting the 3＇ untranslated region （3＇-UTR） of SMAD1 mRNA in ESCC cells. Furthermore, our results showed that miR-205 sup- pressed the invasion and migration of ESCC cells, whereas Smadl increased their invasion and migration. Taken together, our study demonstrates that miR-205 functions as a suppressor of tumor metastasis by regulating SMAD1 expression through targeting the 3＇-UTR of SMAD1 mRNAin ESCC. Therefore, miR-205 may be a potential therapeutic target for miRNA-based therapy of ESCC.     

MicroRNAs 103 and 107 regulate insulin sensitivity

Defects in insulin signalling are among the most common and earliest defects that predispose an individual to the development of type 2 diabetes. MicroRNAs have been identified as a new class of regulatory molecules that influence many biological functions, including metabolism. However, the direct regulation of insulin sensitivity by microRNAs in vivo has not been demonstrated. Here we show that the expression of microRNAs 103 and 107 (miR-103/107) is upregulated in obese mice. Silencing of miR-103/107 leads to improved glucose homeostasis and insulin sensitivity. In contrast, gain of miR-103/107 function in either liver or fat is sufficient to induce impaired glucose homeostasis. We identify caveolin-1, a critical regulator of the insulin receptor, as a direct target gene of miR-103/107. We demonstrate that caveolin-1 is upregulated upon miR-103/107 inactivation in adipocytes and that this is concomitant with stabilization of the insulin receptor, enhanced insulin signalling, decreased adipocyte size and enhanced insulin-stimulated glucose uptake. These findings demonstrate the central importance of miR-103/107 to insulin sensitivity and identify a new target for the treatment of type 2 diabetes and obesity.     

高温和姜黄素联合作用诱导HeLa细胞周期阻滞和凋亡

目的:研究不同高温和姜黄素联合作用对肿瘤细胞的生长抑制作用.方法:37~43℃与0~40mol/L姜黄素联合作用4~16h后,MTT比色法检测细胞活性,流式细胞仪测定细胞周期时相变化.结果:高温作用后主要引起细胞凋亡和G0/G1期阻滞,姜黄素能抑制HeLa细胞的体外生长,呈时间与剂量依赖性,细胞周期阻滞于G0/G1期.联合作用后表现出了两者作用的双重特点.结论:应用高温和中药联合治疗可能是提高肿瘤治愈率、减少对正常组织细胞损害的有效方式之一.     

云芝多糖影响人宫颈癌HeLa细胞的增殖和凋亡的研究

研究云芝多糖(CVP)对宫颈癌HeLa细胞系的增殖、凋亡及产生途径。采用MTT法测定了CVP对HeLa细胞增殖的体外抑制作用,流式细胞仪检测凋亡细胞的比例,qRT-PCR检测P53、Bcl-2和Fas基因在细胞处理前后表达量的变化。结果显示：CVP以时间和剂量依赖的方式抑制HeLa细胞的生长。作用时间在24 h,48 h和72 h时对细胞抑制的抑制率分别为92%、95%和98%;当CVP浓度达到1.25 mg/L时,作用24 h、48 h和72 h时的IC50值分别为0.2507±0.01 mg/L、0.2720±0.04 mg/L和0.2736±0.03 mg/L;当CVP浓度为0.5 mg/L时,作用24 h和48 h后细胞凋亡率分别为26.36% 和63.81%;CVP处理HeLa细胞24 h后,Bcl-2基因的表达被显著下调。研究表明：CVP促进HeLa细胞凋亡,其作用机制与Bcl-2基因的表达下调有关,推测是通过Bcl-2介导的途径促进细胞凋亡。图4表1参30     

扭曲椭圆管内超临界CO2冷却换热的数值模拟

为了提高CO₂热泵的传热性能,基于Fluent的数值模拟方法研究了不同质量流量下,扭距为100 mm及无扭曲状态下的水平椭圆管管内超临界CO₂冷却换热特性及二次流的变化规律,并针对竖直椭圆管引入局部换热系数和压降,研究了长短轴比b/a及扭距对扭曲管换热性能的影响。结果表明,低质量流量下扭曲椭圆管内浮升力明显大于椭圆管扭曲结构所产生的浮升力,对于低质量流量G<200 kg/(m²·s²)下的超临界CO₂流体,椭圆管具有更大强度的浮升力所造成的二次流,强化传热更明显;对于高质量流量G>200 kg/(m²·s²)下的超临界CO₂流体时,扭曲椭圆管具有更大强度自身结构所产生的周期性二次流来强化传热;管内的传热系数及压降随着扭曲程度及压扁程度的增大而增大。为扭曲椭圆管在CO₂热泵中的应用提供了重要的理论与数据支持。