miR-34b通过靶向AGR2调控口腔癌糖脂代谢的相关性研究

目的 探索miR-34b在口腔癌中的作用机制。方法 检测口腔癌细胞系中miR-34b和AGR2的表达情况。在口腔癌细胞中过表达miR-34b或敲降AGR2后,采用MTT法检测口腔癌细胞的增殖;油红O染色法检测口腔癌细胞中脂肪和葡萄糖含量;双荧光酶报告基因验证miR-34b与AGR2的关系。结果 miR-34b在口腔癌细胞系中低表达,而AGR2在口腔癌细胞系中高表达。过表达miR-34b或敲降AGR2可抑制口腔癌细胞增殖,并降低细胞中脂肪和葡萄糖含量。结论 miR-34b可通过靶向AGR2调控口腔癌的糖脂代谢,对口腔癌的预防和治疗具有重要意义。     

微小 RNA-218-5p靶向 Jagged 1调控人牙周膜干细胞的活性和骨向分化

目的 研究微小RNA-218-5p(miR-218-5p)对人牙周膜干细胞(hPDLSCs)活性和骨向分化的影响并探讨其机制,为牙周炎的治疗提供研究基础.方法 将anti-miR-218-5p组(转染anti-miR-218-5p)、anti-miR-NC组(转染anti-miR-NC)、pcDNA组(转染pcDNA)、pcDNA-JAG1组(转染pcDNA-JAG1)、anti-miR-218-5p+si-NC组(共转染anti-miR-218-5p和si-NC)、anti-miR-218-5p+si-JAG1组(共转染anti-miR-218-5p和si-JAG1),用脂质体法转染至hPDLSCs细胞;运用实时荧光定量逆转录聚合酶链反应(qRT-PCR)法检测细胞中miR-218-5p、Ⅰ型胶原蛋白(Col-1)、骨钙素、Runt相关转录因子2(Runx2)的表达;蛋白质印迹法(Westrn blotting)检测细胞中Jagged 1(JAG1)的蛋白表达;MTT法检测细胞活性;茜素红染色实验检测细胞的矿化结节;双荧光素酶报告基因检测实验检测细胞的荧光活性.结果 与anti-miR-NC组相比,anti-miR-218-5p组hPDLSCs细胞培养48、72 h时,细胞活性升高[48 h:(0.44±0.04)比(0.62±0.06);72 h:(0.53±0.05)比(0.83±0.08);P<0.001],细胞的矿化结节明显升高,Col-1[(0.26±0.03)比(0.74±0.07)]、骨钙素[(0.21±0.02)比(0.54±0.05)]、Runx-2[(0.29±0.03)比(0.61±0.06)]蛋白相对表达量均显著升高(均P<0.001).与pcDNA组相比,pcDNA-JAG1组hPDLSCs细胞培养48、72 h时,细胞活性显著升高[48 h:(0.42±0.04)比(0.59±0.05);72 h:(0.55±0.05)比(0.81±0.08);P<0.001],Col-1[(0.34±0.03)比(0.71±0.07)]、骨钙素[(0.23±0.02)比(0.48±0.04)]、Runx-2[(0.25±0.03)比(0.56±0.05)]蛋白表达量均显著升高(均P<0.001).miR-218-5p可靶向调控JAG1的表达.抑制JAG1可逆转抑制miR-218-5p对hPDLSCs的活性和骨向分化作用.结论 抑制miR-218-5p可促进人牙周膜干细胞活性和骨向分化,其机制与靶向JAG1有关.     

MiR-195 regulates cell apoptosis of human hepatocellular carcinoma cells by targeting LATS2

Recently, we have reported tissue- and stage-specific expression of miR-195 in human hepatocellular carcinoma cells and so far, not many reports discuss the function of this microRNA (miRNA). Expression profiling of miRNAs revealed a limited set of miRNAs with altered expression in drug resistant hepatocellular carcinoma cell line BEL-7402/5-FU compared to its parental BEL-7402 cell line. Real-time PCR confirmed down-regulation of miR-195 in BEL-7402/5-FU cells. Western blots were performed to determine protein levels of LATS2, P53 and CDK2. MTT analysed the cell proliferation activity. Flow cytometry were performed to determine apoptosis rate. Up-regulation of miR-195 increased expression of LATS2 and increased apoptosis of HCC cells, while Anti-miR-195 treatment inhibited expression of LATS2. miR-195 over-expression inhibited the luciferase activity of a LATS2 3' untranslated region-based reporter construct in BEL-7402/5-FU cells. These results indicate that miR-195 could increase cell apoptosis by targeting LATS2 in hepatocellular carcinoma cells.     

MicroRNA-126 suppresses inflammation in endothelial cells under hyperglycemic condition by targeting HMGB1

MicroRNA-126(miR-126) targets involved in inflammation need to be identified. In this study, we aim to investigate whether high-mobility group box 1(HMGB1), an inflammation-related gene, is the target of miR-126 in diabetic vascular endothelium. The diabetic apoE^−/− mice model, a classical diabetic atherosclerosis model, was established. The aorta of diabetic apoE^−/− mice showed decrease of miR-126 and elevation of HMGB1 and inflammation. Next, we employed several in vitro experiments to address the role of miRNA-126 on the regulation of HMGB1 in endothelial cells under hyperglycemic and inflammatory conditions. Manipulation of miRNA levels in human umbilical vein endothelial cells (HUVECs) was achieved by transfecting cells with miR-126 mimic and antagomir. Overexpression of miR-126 could decrease the expression of downstream components of HMGB1 including TNF-α, ROS, and NADPH oxidase activity in HUVECs under hyperglycemic condition. Nevertheless, such phenomenon was completely reversed by miR-126 antagomir. The expression of HMGB1 protein rather than HMGB1 mRNA was down-regulated after transfection with miR-126 mimic, which indicated the modulation of HMGB1 mediated by miR-126 was at the posttranslational level. Luciferase reporter assay confirmed the 3′-UTR of HMGB1 gene was a direct target of miR-126. Western blot analysis also indicated that overexpression of miR-126 contributed to the elevation of p-eNOS, eNOS and p-AKT expressions, respectively. In summary, our findings suggest that miR-126 may suppress inflammation and ROS production in endothelial cells treated by high glucose through modulating the expression of HMGB1. Our study provides a novel pathogenic link between dysregulated miRNA expression and inflammation in diabetic vascular endothelium.     

MicroRNA-99b-3p promotes angiotensin II-induced cardiac fibrosis in mice by targeting GSK-3β

Cardiac fibrosis is a typical pathological change in various cardiovascular diseases. Although it has been recognized as a crucial risk factor responsible for heart failure, there is still a lack of effective treatment. Recent evidence shows that microRNAs (miRNAs) play an important role in the development of cardiac fibrosis and represent novel therapeutic targets. In this study we tried to identify the cardiac fibrosis-associated miRNA and elucidate its regulatory mechanisms in mice. Cardiac fibrosis was induced by infusion of angiotensin II (Ang II, 2 mg·kg⁻¹·d⁻¹) for 2 weeks via osmotic pumps. We showed that Ang II infusion induced cardiac disfunction and fibrosis accompanied by markedly increased expression level of miR-99b-3p in heart tissues. Upregulation of miR-99b-3p and fibrotic responses were also observed in cultured rat cardiac fibroblasts (CFs) treated with Ang II (100 nM) in vitro. Transfection with miR-99b-3p mimic resulted in the overproduction of fibronectin, collagen I, vimentin and α-SMA, and facilitated the proliferation and migration of CFs. On the contrary, transfection with specific miR-99b-3p inhibitor attenuated Ang II-induced fibrotic responses. Similarly, intravenous injection of specific miR-99b-3p antagomir could prevent Ang II-infused mice from cardiac dysfunction and fibrosis. We identified glycogen synthase kinase-3 beta (GSK-3β) as a direct target of miR-99b-3p. In CFs, miR-99b-3p mimic significantly reduced the expression of GSK-3β, leading to activation of its downstream profibrotic effector Smad3, whereas miR-99b-3p inhibitor caused anti-fibrotic effects. GSK-3β knockdown ameliorated the anti-fibrotic role of miR-99b-3p inhibitor. These results suggest that miR-99b-3p contributes to Ang II-induced cardiac fibrosis at least partially through GSK-3β. The modulation of miR-99b-3p may provide a new approach for tackling fibrosis-related cardiomyopathy.     

Potential role of store-operated Ca2+ entry in Th2 response induced by histamine in human monocyte-derived dendritic cells

Store-operated calcium entry (SOCE) is the main Ca(2+) influx pathway of dendritic cells (DCs). DCs primed with histamine facilitate Th2 immune response via different types of histamine receptors. Histamine induces DCs to release Ca(2+) from internal store. Therefore, we wonder that whether histamine could activate SOCE in DCs through its receptors, and what's the functional relevance of the Ca(2+) influx through SOCE induced by histamine in Th(2) response. We certificate that histamine induced a transient Ca(2+) release followed by pronounced Ca(2+) influx after re-addition of external Ca(2+) which could be inhibited by SOCE blockers SKF-96365 and BTP-2. Moreover, the percentages of DCs that showed an obvious Ca(2+) release response to histamine were decreased in the presence of histamine 1 (H1) receptor antagonist pyridylethylamine (Pyr) or histamine 4 (H4) receptor antagonist JNJ7777120 (JNJ). Histamine up-regulated the mRNA expression of STIM1 in DCs, one of the two major proteins of SOCE channel. SOCE blocker BTP-2 and histamine receptor antagonists JNJ and Pyr inhibited the increase of CD86 induced by histamine on DCs. Histamine increased the level of IL-10 and decreased the level of IL-12p70 secreted by DCs. SOC blockers SKF and BTP-2 inhibited the level of both IL-10 and IL-12p70 secreted by DCs. Pretreatment of SOC blockers and H1, H4 receptor antagonists with DCs inhibited the Th2 polarization of T helper cells induced by histamine in mixed lymphocyte responses (MLR). We demonstrated that SOCE was involved in histamine-induced maturation and Th(2) response of DCs which was through histamine 1 and 4 receptor.     

Suramin has adjuvant properties and promotes expansion of antigen-specific Th1 and Th2 cells in vivo

Aluminum hydroxide and incomplete Freund's adjuvant (IFA) are the only adjuvants approved for human use. Both are T helper 2 (Th2) adjuvants, however, T helper 1 (Th1) immunity is induced if microbial products such as mycobacteria, CpG's, or bacterial toxins are included in the adjuvant preparation. The usefulness of bacterial toxins, such as Pertussis toxin (PT) or Cholera toxin (CT), as adjuvants for human vaccination is limited by toxic side effects and high immunogenicity. Hence, we asked whether or not the adjuvant activity of bacterial toxins on Th1 and Th2 immunity could be mimicked by chemical compounds of small molecular weight and less immunogenicity. In the present study, we show that Suramin, a small molecular weight naphthylurea, which mainly acts on G-proteins and on P2X/P2Y receptors, promotes expansion of hen eggwhite lysozyme (HEL)-specific Th1 and Th2 cells upon immunization of BALB/c mice with HEL in aluminum hydroxide (alum). The results indicated that the adjuvant effects of Suramin on T cell responses were mediated by enhancing the expression of MHC class II and costimulatory molecules on antigen presenting cells (APCs), and by increasing their pro-inflammatory cytokine production. Together, the results suggest that small molecular weight compounds such as Suramin could be used as alternative vaccine adjuvants.     

布鲁氏菌病患者外周血树突状细胞、Th1／Th2细胞因子含量的检测及意义

目的：观察布鲁氏菌病患者外周血树突状细胞表型、Th1／Th2细胞含量的检测及意义。方法选取诊治的布鲁氏菌病患者50例为病例组,另选取同期进行健康体检的正常人50例作为正常组。采用Real time-PCR测定2组Th1相关转录因子T细胞表达的T盒（T-bet）、GATA连接蛋白3（GATA-3）、维A酸相关核孤儿受体γt（RORγt）、叉头蛋白3（Foxp3）及Th1／Th2细胞因子肿瘤坏死因子α（TNF-α）、干扰素γ（IFN-γ）、白介素6（IL-6）、白介素10（IL-10）mRNA含量。酶联免疫吸附实验（ELISA）测定TNF-α、IFN-γ、IL-6、IL-10蛋白表达及补体C3、C4含量。流式细胞术测定树突状细胞表型、Th1、Th2及T淋巴细胞亚群（ CD4＋T细胞、CD8＋T细胞、NK细胞）含量。结果病例组外周血Th1细胞相关转录因子T-bet、RORγt、Foxp3及Th1细胞、Th1／Th2、TNF-α、IFN-γ含量较对照组显著降低,Th2细胞及IL-6、IL-10含量较对照组显著升高,差异有统计学意义（ P ＜0．05）;病例组CD8＋3、CD8＋0、CD8＋6阳性的树突状细胞比例、CD4＋T细胞、NK细胞及补体C3、C4含量较对照组显著降低,CD8＋T细胞含量较对照组显著升高,差异有统计学意义（ P ＜0．05）;TNF-α、IFN-γ含量与CD4＋T细胞、NK细胞、C3、C4含量呈正相关性（ r值分别为2．879、3．214、3．255和2．978, P ＜0．05）,与CD8＋T细胞含量呈负相关性（ r值分别为－3．146和－3．011, P ＜0．05）。 IL-6、IL-10含量与CD4＋T细胞、NK细胞、C3、C4含量呈负相关性（ r值分别为－2．124、－2．343、－3．423、－2．789、－2．993、－2．566、－3．758, P ＜0．05）,与CD8＋T细胞含量呈正相关性（ r值分别为3．465、3．129, P ＜0．05）。结论布鲁氏菌病患者外周血成熟树突状细胞数目减少,同时Th1／Th2细胞及相关细胞因子失衡,且与机体天然免疫和细胞免疫功能降低有关,这可能在布鲁氏菌病发生发展过程中发挥重要作用。     

微小 RNA-335-3p靶向小鼠双微体基因调控骨髓瘤细胞的增殖和凋亡

目的 探讨微小RNA(miR)-335-3p对骨髓瘤细胞增殖和凋亡的影响及作用机制.方法 2018年5月至2019年1月,培养正常骨髓细胞MNC和骨髓瘤细胞KM3和U266,实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测细胞中miR-335-3p表达水平.将KM3细胞分为miR-NC组(转染模拟对照序列)、miR-335-3p组[转染miR-335-3p模拟物(miR-335-3p mimics)]、pcDNA3.1+miR-335-3p组(共转染空载体和miR-335-3p mimics)和pcDNA3.1-MDM2+miR-335-3p组[共转染小鼠双微体基因(MDM2)过表达载体和miR-335-3p mimics],MTT法检测细胞增殖,流式细胞仪检测细胞凋亡的影响,蛋白质印迹法(Western blotting)检测细胞周期蛋白D1(cyclin D1)、细胞周期蛋白依赖性激酶抑制剂1A(P21)、B淋巴细胞瘤-2(Bcl-2)蛋白和Bcl-2相关X(Bax)蛋白表达.双荧光素酶报告基因实验验证KM3细胞中miR-335-3p与MDM2调控关系.结果 骨髓瘤细胞KM3和U266中miR-335-3p表达水平分别为0.24±0.01、0.38±0.02,显著低于正常骨髓细胞MNC中的miR-335-3p表达水平0.77±0.03(均P<0.05).与miR-NC组比较,miR-335-3p组KM3细胞吸光度[48 h:(0.60±0.03)比(0.99±0.06);72 h:(0.82±0.05)比(1.67±0.07)]、细胞中cyclin D1和Bcl-2蛋白表达均降低(均P<0.05),细胞凋亡率[(24.31±0.99)％比(8.13±0.83)％]、细胞中P21和Bax蛋白表达均升高(均P<0.05).miR-335-3p在KM3细胞中负调控MDM2表达.与pcDNA3.1+miR-335-3p组比较,pcDNA3.1-MDM2+miR-335-3p组KM3细胞吸光度[48 h:(0.80±0.05)比(0.63±0.04);72 h:(1.28±0.06)比(0.88±0.05)]、细胞中cyclin D1和Bcl-2蛋白表达均升高(均P<0.05),凋亡率[(15.34±0.66)％比(23.98±1.41)％]、细胞中P21、Bax蛋白表达均降低(均P<0.05).结论 miR-335-3p可能通过下调MDM2表达抑制骨髓瘤细胞的增殖,并诱导细胞凋亡.     

Radiation-induced decrease of CD8 + dendritic cells contributes to Th1/Th2 shift

Exposure to ionizing radiation (IR) often reduce the helper T (Th) 1 like function, resulting in a Th1/Th2 imbalance, which could affect the efficacy of cancer radiotherapy. As the most potent antigen presenting cells, dendritic cells (DC) can be divided into several subsets with specialized function. However, there is no literature covering the changes of DC subsets and their roles in immune regulation in response to IR. In the present study, we were aimed to investigate the changes of DC subsets after IR and its relationship with Th1/Th2 immunity. We found a significant decrease of BDCA3 + DC in the blood of patients treated with radiotherapy. CD8 + DC, a mouse equivalent of human BDCA3 + DC, was also found decreased in mice spleen, peripheral blood and lymph node tissues after irradiation. As CD8 + DC mainly induce Th1 immunity, we tested the changes of Th1/Th2 response and found that IR caused a repression of Th1 immunity, indicating a possible role of CD8 + DC in radiation-induced Th1/Th2 imbalance. We also found that a CD8 + DC-inducing cytokine, Fms-like tyrosine kinase 3 ligand (FLT3 ligand), restored CD8 + DC and reversed Th1/Th2 shift. And then we found that bone marrow cells from irradiated mice differentiated into less CD8 + DC, which was also protected by FLT3 ligand. In conclusion, our data showed that IR induced a decrease of CD8 + DC and Th1/Th2 shift, which was reversed by Flt3 ligand treatment, suggesting a novel mechanism for radiation-induced immunosuppression.     

Lead effects on development and function of bone marrow-derived dendritic cells promote Th2 immune responses

Although lead (Pb) has significant effects on the development and function of macrophages, B cells, and T cells and has been suggested to promote allergic asthma in mice and humans, Pb modulation of bone marrow (BM)-derived dendritic cells (DCs) and the resultant DC effects on Th1 and Th2 development have not been examined. Accordingly, we cultured BM cells with murine granulocyte macrophage-colony stimulating factor (mGM-CSF)+/-PbCl(2). At day 10, culture supernatant (SN) and non-adherent cells were harvested for analysis. Additionally, day 10 non-adherent BM-DCs were harvested and recultured with mGM-CSF+LPS+/-Pb for 2 days. The day 10 Pb exposure significantly inhibited BM-DC generation, based on CD11c expression. Although fewer DCs were generated with Pb, the existing Pb-exposed DCs had significantly greater MHC-II expression than did the non-Pb-exposed DCs. However, these differences diminished upon LPS stimulation. After LPS stimulation, CD80, CD86, CD40, CD54, and MHC-II were all up-regulated on both Pb-DCs and DCs, but Pb-DCs expressed significantly less CD80 than did DCs. The CD86:CD80 ratio suggests a Pb-DC potential for Th2 cell development. After LPS stimulation, IL-6, IL-10, IL-12 (p70), and TNF-alpha levels significantly increased with both Pb-DCs and DCs, but Pb-DCs produced significantly less cytokines than did DCs, except for IL-10, which further supports Pb-DC preferential skewing toward type-2 immunity. In vitro studies confirm that Pb-DCs have the ability to polarize antigen-specific T cells to Th2 cells. Pb-DCs also enhanced allogeneic and autologous T cell proliferation in vitro, and in vivo studies suggested that Pb-DCs inhibited Th1 effects on humoral and cell-mediated immunity. The Pb effect was mainly on DCs, rather than on T cells, and Pb's modification of DC function appears to be the main cause of Pb's promotion of type-2-related immunity, which may relate to Pb's enhanced activation of the Erk/MAP kinase pathway.     

Effects of Th1 and Th2 cells balance in pulmonary injury induced by nano titanium dioxide

To explore the potential immunoregulatory mechanisms linking nano TiO₂ and pulmonary injury, Sprague Dawley rats were exposed by intra-tracheal instillation to nano TiO₂ with the individual doses of 0.5, 4.0 and 32 mg/kg b.w., micro TiO₂ with 32 mg/kg b.w. and 0.9% NaCl, respectively. The exposure was conducted twice a week, for four consecutive weeks. The results of lung histology demonstrated increased macrophages accumulation, extensive disruption of alveolar septa, slight alveolar thickness and expansion hyperemia. Mitochondria tumefaction organelles dissolution, endoplasmic reticulum expansion and the gap of nuclear broadening were shown. The changes of IFN-γ and IL-4 level showed no statistical difference. The mRNA expression of GATA-3 was up-regulated, whereas T-bet was significantly down-regulated. The protein expression of T-bet decreased and there were significant differences in nano 4 and 32 mg/kg groups. The imbalance of Th1/Th2 cytokines might be one of the mechanisms of immunotoxicity of respiratory system induced by nano TiO₂ particles.