The oncogenic impact of RNF2 on cell proliferation,invasion and migration through EMT on mammary carcinoma

Mammary carcinoma (MC) is one of most common malignancy in women, and ring finger protein 2 (RNF2) possesses various roles in vast human tumors. In MC tissues as well as in cell lines RNF2 exhibited high expression, had significant association with tumor size, lymph node status, TNM stage, patients’ poor survival, and promoted cell proliferation, colony formation, cell migration and invasion of MC cell lines which was mediated by downregulation of E-cadherin protein. These data reveal that RNF2 protein plays a vital role in the development of MC and may be a potential therapy target of MC.     

miR-30c Impedes Glioblastoma Cell Proliferation and Migration by Targeting SOX9

miR-30c has been acknowledged as a tumor suppressor in various human cancers, such as ovarian cancer, gastric cancer, and prostate cancer. However, the role of miR-30c in glioblastoma (GBM) needs to be investigated. In our study, we found that the expression of miR-30c was significantly downregulated in GBM tissues and cell lines. We found that overexpression of miR-30c inhibited cellular proliferation of GBM cells in vitro and in vivo. More GBM cells were arrested in the G₀ phase after miR-30c overexpression. Moreover, we showed that miR-30c overexpression suppressed the migration and invasion of GBM cells. Mechanistically, we found that SOX9 was a direct target of miR-30c in GBM cells. Overexpression of miR-30c inhibited the mRNA and protein levels of SOX9 in GBM cells. Moreover, there was a negative correlation between the expression of miR-30c and SOX9 in GBM tissues. Finally, we showed that restoration of SOX9 in GBM cells reversed the proliferation, migration, and invasion of GBM cells transfected with miR-30c mimic. Collectively, our results demonstrated that miR-30c suppressed the proliferation, migration, and invasion of GBM cells via targeting SOX9.     

FBXL3 is regulated by miRNA-4735-3p and suppresses cell proliferation and migration in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is the most common type of primary lung cancer and regarded as cancer killer. The aim of this study was to discover the detailed function and molecular mechanism of F-box and leucine rich repeat protein 3 (FBXL3) in NSCLC. In this study, the expression level of FBXL3 in NSCLC tissues and cell lines was firstly examined and identified. Moreover, the relationship between FBXL3 and the overall survival rate of NSCLC patients was analyzed by Kaplan–Meier survival curve. Functionally, MTT, colony formation assay and transwell assays were performed to determine the role of FBXL3 in regulating NSCLC cell proliferation, migration and invasion. The proliferation and migration were suppressed by overexpression of FBXL3, indicating the potential tumor suppressive role of FBXL3 in NSCLC. In addition, the dual-luciferase reporter and RNA pull-down assays revealed that miR-4735-3p was a novel upstream modulator of FBXL3. Further study showed that miR-4735-3p was upregulated in NSCLC tissues and cell lines. Finally, rescue assays and function assays revealed that miR-4735-3p exerted oncogenic function in NSCLC, and this function can be attenuated by FBXL3. Taken together, FBXL3 was regulated by miR-4735-3p and suppressed cell proliferation and invasion in non-small cell lung cancer.     

Musashi1基因调控结肠癌HCT116细胞恶性生物学行为的机制研究

背景与目的：Musashi1（Msi1）属于RNA结合蛋白家族中的一员，是RNA转录后表达的关键调控者，其是否参与肿瘤的发生、发展，以及具体分子机制仍不十分清楚。探讨沉默Msi1基因对结肠癌HCT116细胞恶性生物学行为的影响及可能的机制。方法：采用慢病毒载体构建稳定低表达Msi1的细胞株，细胞计数试剂盒（cell counting kit-8，CCK-8）实验检测细胞增殖能力，克隆形成实验检测细胞克隆形成能力，流式细胞术（flow cytometry，FCM）检测细胞周期的变化，裸鼠移植瘤模型观察沉默Msi1对裸鼠成瘤的影响。实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）和蛋白质印记法（Western blot）检测沉默Msi1基因后p27基因的mRNA表达和蛋白水平，双荧光素酶实验验证Msi1基因与目的基因p27 3’-非编码区（3’-untranslated region，3’-UTR）的相互作用。结果：沉默Msi1基因后，HCT116细胞的增殖能力显著下降，克隆集落数明显减少，G ₀ /G ₁ 期细胞增多，S期细胞明显减少，裸鼠移植瘤生长明显受到抑制。沉默Msi1基因后p27 mRNA表达未见明显变化，而p27蛋白水平明显上调，双荧光素酶实验证实Msi1基因能与p27 3’-UTR区域直接结合，抑制其翻译。结论：沉默Msi1基因通过靶向上调p27，导致结肠癌HCT116细胞G ₀ /G ₁ 期阻滞，从而抑制肿瘤细胞体内及体外增殖能力。     

Inhibition of fatty acid synthase (FASN) affects the proliferation and apoptosis of HepG2 hepatoma carcinoma cells via the β-catenin/C-myc signaling pathway

Introduction and objectivesResearch in the last few years has proven that inhibition of fatty acid synthase (FASN) suppresses the migration and invasion of hepatoma carcinoma cells. This study aims to explore the effect of fatty acid synthase knockdown on the apoptosis and proliferation of HepG2 cells.Materials and methodsThe human liver cancer cell line HepG2 was cultured and then transfected with FASN-specific siRNA and negative control RNAi. After 48 h, cells and protein lysates were used for western blotting, CCK-8 (cell counting kit-8) assays, flow cytometry and other tests. To assess cell apoptosis, Bax, Bcl-2 and caspase-3 were detected; to assess proliferation, CDK4 (cyclin-dependent kinases 4) and P21 were detected; and to determine the signaling pathway involved, β-catenin and C-myc were also detected.ResultsInhibition of FASN in HepG2 cells can decrease proliferation and promote apoptosis. Flow cytometry and CCK-8 assays demonstrated that the apoptosis rate of FASN-specific siRNA-transfected cells was significantly increased compared to that of the control cells (p < 0.01). In addition, the cell cycle analysis revealed that FASN-specific siRNA-transfected cells induced G1 phase arrest (p < 0.05), but an increasing trend in G2 (p < 0.05).Compared with expression in negative RNAi-transfected cells, the expression of Bcl-2 and CDK-4 was reduced and the expression of Bax, caspase-3 and P21 was increased in FASN-specific siRNA-transfected cells (p < 0.05). Regarding the signaling pathway, the expression of β-catenin and C-myc was significantly reduced when compared to that in negative control cells (p < 0.05).ConclusionsInhibition of FASN suppressed the cell survival of HepG2 cells by inhibiting the β-catenin/C-myc pathway. This result suggests the potential treatment value of FASN for hepatoma carcinoma (HCC).     

新加归肾丸通过调节mTOR信号通路促进体外培养大鼠卵巢颗粒细胞增殖

目的:观察补肾活血中药复方新加归肾丸对体外培养大鼠卵巢颗粒细胞增殖的影响。方法:体外培养大鼠卵巢颗粒细胞,分为空白组,模型组,中药低、中、高剂量组共五组,空白组用1%胎牛血清培养基培养,其余四组滴加雷帕霉素,20 min后中药各组加入不同浓度的中药水提醇沉液。分别在培养24、48、72 h后采用流式细胞仪检测细胞周期各时相的变化,并计算细胞增殖指数(PI); 免疫印迹法检测磷酸化-哺乳动物雷帕霉素靶蛋白(p-mTOR)、细胞周期蛋白D₂(Cyclin D₂)的表达水平。结果:用药24、48、72 h 这3个时段,中药各组S期细胞比率、PI均高于模型组(P<0.05),其中中药中剂量组最为明显(P<0.05)。用药24、48、72 h这3个时段,中药各个剂量组p-mTOR、Cyclin D₂表达水平均高于模型组(P<0.05)。中药各个剂量中以中药中剂量组效果最明显(P<0.05)。结论:补肾活血中药复方新加归肾丸能通过激活mTOR信号通路活性,上调Cyclin D₂蛋白表达,促使颗粒细胞由G₁期向S期转化,加速卵巢颗粒细胞周期进程,促进颗粒细胞增殖来实现改善卵泡发育。     

腺病毒介导的Hes1基因抑制肝细胞癌细胞的增殖与迁移CSCD

目的探讨Hes1基因对肝细胞癌细胞系Hep1-6细胞增殖、迁移与侵袭的影响。方法通过重组腺病毒载体介导Hes1基因在Hep1-6细胞系中过表达,随后测定细胞克隆形成率、增殖速率及体外迁移与侵袭能力的改变。结果Hep1-6细胞分别感染Ad-Hes1和阴性对照Ad-GFP后,感染Ad-Hes1的细胞系克隆形成数显著少于对照组(P<0.001);感染病毒6天后,CCK-8检测感染Ad-Hes1的细胞系在OD450 nm的吸光度值显著低于对照组(P<0.01);感染Ad-Hes1的细胞系24 h和48 h的划痕愈合率显著低于对照组(P<0.001);感染Ad-Hes1的细胞系在Transwell小室培养48 h后迁移进入Transwell下室的细胞数量显著低于对照组(P<0.001);感染Ad-Hes1的细胞系在铺有Matrigel基质胶的Transwell小室培养48 h后侵袭进入Transwell下室的细胞数量显著低于对照组(P<0.01)。结论Hes1有抑制Hep1-6细胞增殖、迁移与侵袭的作用。     

沉默信息调节因子过表达或谷氨酰胺剥夺对肾透明细胞癌细胞凋亡、增殖的影响

背景与目的：肾透明细胞癌（clear cell renal cell carcinoma，ccRCC）是最常见的肾癌类型，它与代谢密切相关。探讨沉默信息调节因子4（silent information regulator 4，SIRT4）过表达或谷氨酰胺（glutamine，Gln）剥夺对ccRCC细胞增殖、凋亡的影响。方法：慢病毒构建SIRT4和突变体H161Y过表达的Caki-2细胞株，利用无Gln的培养基来构建Gln剥夺模型，并通过体外增殖活力实验［细胞计数试剂盒-8（cell counting kit-8，CCK-8）］和克隆形成实验来分析两者对Caki-2细胞增殖和生长能力的影响；利用DCFH-DA荧光探针检测细胞内活性氧自由基（reactive oxygen species，ROS）水平进而评估Gln代谢对细胞ROS含量的影响；进一步通过线粒体膜电位检测、凋亡检测和蛋白质印迹法（Western blot）检测凋亡相关分子，分析SIRT4过表达以及Gln剥夺对Caki-2细胞凋亡的影响。结果：过表达SIRT4可抑制Gln代谢从而抑制Caki-2细胞增殖，另外还原性物质还原型烟酰胺腺嘌呤二核苷酸磷酸（reduced nicotinamide adenine dinucleotide phostate，NADPH）的生成减少能够增加细胞内ROS含量，促进细胞凋亡。而Gln剥夺抑制细胞增殖和促进细胞凋亡的效果均比过表达SIRT4明显，但长期缺乏Gln将导致细胞无法生长。结论：无论是过表达SIRT4还是Gln剥夺均能抑制ccRCC细胞增殖，促进凋亡。     

The Proportion of Vδ1T Cells in Peripheral Blood Correlated with Prognosis of Sepsis

Background: Sepsis is a serious condition with a high mortality rate, and septic patients often have organ dysfunction, low tissue perfusion and hypoxia, lactic acidosis, oliguria, or functional brain changes.Objective: To observe the number and the function of Vδ1T cells in peripheral blood of septic patients, to analyze the clinical significance of detecting Vδ1T cells, and to clarify the correlation of their presence with the prognosis of sepsis.Methods: The basic data of the septic patients were recorded at admission. The immunosuppressive function–related molecules on the surface of Vδ1T cells were detected, and the immunosuppressive function of Vδ1T cells was also evaluated.Results: Compared with the healthy controls, the proportion of Vδ1T cells in the blood of septic patients significantly decreased (P<0.01). The proportion of Vδ1T cells in septic patients correlated with the patients’ condition (P<0.05). The expression of glucocorticoid-induced tumor necrosis factor receptor (GITR), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) on the surface of Vδ1T cells in the blood of septic patients significantly increased (P<0.01). The increase of Vδ1T cells in septic patients had inhibitory effects on T cell proliferation and interferon (IFN)-γ secretion. These findings implied that the immunosuppression of Vδ1Tcells in the peripheral blood of septic patients was significantly higher than that of the healthy controls (P<0.01).Conclusion: Changes in Vδ1T cells in septic patients were closely related to the patient’s condition and prognosis.     

miR-513a-3p靶向鼠双微体基因2对胃癌细胞增殖迁移侵袭的影响

目的探讨miR-513a-3p靶向鼠双微体基因2(MDM2)对胃癌细胞的增殖、迁移和侵袭的影响及其作用机制。方法采用脂质体法将miR-NC、miR-513a-3p、anti-miR-NC、anti-miR-513a-3p、si-NC、si-MDM2、miR-513a-3p+pcDNA3.1和miR-513a-3p+pcDNA3.1-MDM2转染至BGC-823细胞中,采用实时荧光定量聚合酶链反应(qRT-PCR)检测miR-513a-3p的表达水平,采用Western blot检测cyclin D1、MMP-2、p21、E-cadherin和MDM2蛋白的表达水平,四甲基偶氮唑蓝法检测各组胃癌细胞BGC-823的活性,Transwell法检测各组胃癌细胞BGC-823的迁移和侵袭能力,双荧光素酶报告基因检测实验检测miR-513a-3p与MDM2的靶向关系。结果胃癌细胞BGC-823、MGC-803中miR-513a-3p的表达水平分别为0.21±0.02和0.34±0.03,与胃上皮细胞GES-1(0.76±0.08)比较,差异均有统计学意义(均P<0.05)。培养24、48和72 h后,miR-NC组细胞的吸光度(A)值分别为0.57±0.05、1.03±0.10和1.43±0.14,miR-513a-3p组细胞的A值分别为0.36±0.03、0.48±0.05和0.63±0.06,差异均有统计学意义(均P<0.05);miR-NC组细胞的迁移和侵袭数分别为(130±11.80)个和(117±10.60)个,miR-513a-3p组细胞分别为(58±5.64)个和(50±5.13)个,差异均有统计学意义(均P<0.05)。培养24、48和72 h后,si-NC组细胞的A值分别为0.53±0.05、0.95±0.10和1.36±0.14,si-MDM2组细胞的A值分别为0.39±0.04、0.57±0.06和0.80±0.08;si-NC组细胞的迁移和侵袭数分别为(141±12.02)个和(109±10.60)个,si-MDM2组的迁移和侵袭数分别为(66±6.67)个和(61±6.18)个,差异均有统计学意义(均P<0.05)。培养24、48和72 h后,miR-513a-3p+pcDNA3.1组细胞的A值分别为0.34±0.03、0.46±0.05和0.61±0.06,miR-513a-3p+pcDNA3.1-MDM2组细胞的A值分别为0.48±0.05、0.82±0.08和1.17±0.12,差异均有统计学意义(均P<0.05);miR-513a-3p+pcDNA3.1组细胞的迁移和侵袭数分别为(56±5.71)个和(51±5.16)个,miR-513a-3p+pcDNA3.1-MDM2组分别为(113±10.28)个和(104±10.02)个,差异均有统计学意义(均P<0.05)。结论miR-513a-3p可抑制胃癌细胞的增殖、迁移和侵袭能力,其机制可能与靶向调控MDM2的表达有关,可为胃癌的预防和治疗提供新靶点。