Activation of TLR7 increases CCND3 expression via the downregulation of miR-15b in B cells of systemic lupus erythematosus

Systemic lupus erythematosus （SLE） is an autoimmune disease characterized by B-cell hyperreactivity. The Toll-like receptor 7 （TLR7） signaling pathway is abnormally activated in SLE B cells. CyclinD3 （CCND3） plays an important role in B-cell proliferation, development, and differentiation. Although previous studies focused on the B cell-intrinsic role of TLR7 for the development of spontaneous germinal centers, the influence of TLR7 on CCND3 in SLE B cells is still not clear. Here, we used a B-cell profiling chip and found that CCND3 was related to SLE and significantly elevated in SLE B cells. Moreover, we determined that the expression level of CCND3 was higher, while miR-15b was significantly lower in the B cells from SLE patients and B6.MRL-Faslpr/J lupus mice compared to normal subjects. Furthermore, we demonstrated that the activation of TLR7 dramatically increased CCND3 expression but significantly decreased miR-15b in B cells in vitro and we identified that CCND3 is a direct target of miR-15b. To further confirm our results, we established another lupus model by topically treating C57BL/6 （B6） mice with the TLR-7 agonist imiquimod （IMQ） for 8 weeks according to the previously described protocol. Expectedly, topical treatment with I MQ also significantly increased CCND3 and decreased miR-15b in B cells of B6 mice. Taken together, our results identified that the activation of TLR7 increased CCND3 expression via the downregulation of miR-15b in B cells; thus, these findings suggest that extrinsic factor-induced CCND3 expression may contribute to the abnormality of B cell in SLE.

基于网络药理学、分子对接及细胞实验验证探讨二至丸治疗多发性骨髓瘤的作用机制

【目的】基于网络药理学、分子对接及细胞实验验证探讨二至丸治疗多发性骨髓瘤(MM)的潜在作用机制。【方法】应用网络药理学、分子对接筛选药物-疾病-靶标-通路关键指标。培养多发性骨髓瘤细胞株RPMI-8226,给予二至丸干预,采用膜联蛋白V(Annexin V)-异硫氰酸荧光素(FITC)染色法检测细胞凋亡,Transwell实验测定细胞侵袭能力,实时定量聚合酶链反应(qRT-PCR)法检测蛋白激酶B(Akt)、细胞周期蛋白D1(CCND1)、糖原合酶激酶3β(GSK-3β)、c-MycmRNA表达,Western Blot法检测细胞Akt、磷酸化蛋白激酶B(p-Akt)、CCND1、c-Myc、GSK-3β、磷酸化糖原合酶激酶3β(p-GSK-3β)蛋白表达。【结果】获得二至丸14个有效成分。二至丸治疗MM涉及靶点142个,通路富集分析结果显示关键靶点可能主要集中在癌症通路、脂质与动脉粥样硬化等。分子对接结果显示,木樨草素和槲皮素与GSK-3β有较好的结合活性及稳定性。进一步细胞实验验证发现,与空白组比较,二至丸低、中、高剂量组细胞凋亡率显著升高,细胞侵袭数减少,GSK-3β的mRNA和蛋白水平显著升高,CCND1、Akt和c-Myc的mRNA和蛋白水平显著降低,呈剂量依赖性,其中,中、高剂量组差异均有统计学意义(P<0.05或P<0.01)。【结论】二至丸对MM的治疗作用可能是通过木犀草素和槲皮素等关键活性成分,促进GSK-3β表达,并抑制Akt/GSK-3β/CCND1/c-Myc信号通路来实现的。

去泛素化酶泛素特异性肽酶14促进胰腺癌细胞的增殖和迁移能力

胰腺癌仍然是最致命的癌症之一,而且较少发现有效的治疗方案。虽然在许多肿瘤细胞,包括胰腺癌细胞中观察到泛素特异性蛋白酶14(ubiquitin specific peptidase 14,USP14)的过度表达,但它在胰腺癌中的确切作用仍未得到较好的阐明。我们研究了USP14在胰腺癌中的生物学功能及其分子机制。对癌症基因组图谱数据库(The Cancer Genome Atlas,TCGA)的分析显示USP14在胰腺癌组织中高度表达,进一步探究发现,其表达水平与患者的预后呈负相关。利用shRNAUSP14慢病毒建立了稳定的USP14敲低胰腺癌细胞系,通过CCK8、克隆形成实验、划痕和Transwell试验发现,USP14敲低抑制了胰腺癌细胞的增殖和迁移能力。在胰腺癌细胞SW1990和MIAPaCa2中蛋白质印迹结果表明,下调USP14的表达导致细胞周期蛋白D3(cyclin D3)水平下降,而过表达USP14会增加细胞周期蛋白D3表达水平。此外,免疫共沉淀证明,USP14与细胞周期蛋白D3相互作用,泛素化结果显示,USP14的过表达降低了细胞周期蛋白D3的泛素化水平。并且在SW1990胰腺癌细胞中,CRISPR/Cas9介导的USP14敲除导致细胞周期蛋白D3水平下降。这些发现表明,USP14通过与细胞周期蛋白D3的相互作用促进了胰腺癌细胞的增殖和迁移能力,凸显了USP14作为胰腺癌的潜在治疗靶点。

Investigation of osteosarcoma genomics and its impact on targeted therapy: an international collaboration to conquer human osteosarcoma

Osteosarcoma is a genetically unstable malignancy that most frequently occurs in children and young adults.The lack of progress in managing this devastating disease in the clinic has prompted international researchers to collaborate to profile key genomic alterations that define osteosarcoma.A team of researchers and clinicians from China,Finland,and the United States investigated human osteosarcoma by integrating transcriptome sequencing(RNA-seq),high-density genome-wide array comparative genomic hybridization(a CGH),fluorescence in situ hybridization(FISH),reverse transcription-polymerase chain reaction(RT-PCR),Sanger sequencing,cell culture,and molecular biological approaches.Systematic analysis of genetic/genomic alterations and further functional studies have led to several important findings,including novel rearrangement hotspots,osteosarcoma-specific LRP1-SNRNP25 and KCNMB4-CCND3 fusion genes,VEGF and Wnt signaling pathway alterations,deletion of the WWOX gene,and amplification of the APEX1 and RUNX2 genes.Importantly,these genetic events associate significantly with pathogenesis,prognosis,progression,and therapeutic activity in osteosarcoma,suggesting their potential impact on improved managements of human osteosarcoma.This international initiative provides opportunities for developing new treatment modalities to conquer osteosarcoma.

CCND基因家族成员分子进化分析

目的对细胞周期素（cyclinD）家族成员进行分子生物学进化及功能预测，为临床上检测骨质疏松提供更多分子生物学信息。方法对CCND基因家族成员包括CCND1、CCND2和CCND3序列进行分子进化分析，并在所有科属范围和人型总科范围内，计算了同源CCNDs之间的同义和非同义突变频率。结果CCND1和CCND2具有较高的序列相同性，而CCND3的非同义突变率较高，尤其在人型总科各属形成以后，平均每年的同义和非同义突变频率都高于CCND1和CCND2。结论CCND2与CCND1序列相同程度高．而CCND3在序列和进化速率上都显著不同，所以CCND2可以作为骨密度关联研究的候选基因。

MicroRNA-939在甲状腺癌进展中的作用及机制

目的探讨microRNA-939(miR-939)在甲状腺癌中的作用及机制。方法qPCR分析30对甲状腺癌患者组织中miR-939和CCND3的表达水平。通过CCK-8实验、平板克隆形成实验和Transwell侵袭迁移实验研究miR-939在甲状腺癌中的生物学行为。通过TargetScan预测miR939的靶基因,通过双荧光素酶活性测定、Western blotting和qPCR进行验证。结果miR-939模拟物过表达miR-939显著抑制TPC-1细胞的增殖、迁移和侵袭,而CCND3沉默后可以逆转miR-939过表达引起的TPC-1细胞的生物学变化。结论miR-939/CCND3信号通路是甲状腺癌治疗的新靶点。

miRNA-138靶向调控细胞周期蛋白D3及波形蛋白影响肝癌细胞的增殖及迁移

目的研究肝癌细胞miRNA-138(miR-138)通过调控细胞周期蛋白D3(CCND3)、波形蛋白对肝癌细胞的影响。方法利用RT-PCR对肝癌组织及癌旁组织(对照)、4种肝癌细胞系(HepG2、HHCC、HUH7、BEL-7402)及正常肝细胞系HL-7702(对照)的miR-138的mRNA表达进行检测;设计合成miR-138的模拟物(mimics)及抑制剂(inhibitor)。分别将空载体(对照)、miR-138的模拟物及抑制剂转染至HepG2细胞,与相应对照相比,分析miR-138对CCND3、波形蛋白表达及肝癌细胞活力的影响。应用CCK-8法、细胞创伤愈合试验和Transwell细胞迁移试验分别对转染空载体、miR-138 mimics 及inhibitor后的HepG2肝癌细胞增殖及迁移能力进行检测。Western blot检测上调或下调miR-138后miR-138相关靶蛋白CCND3及波形蛋白的表达。结果与相应对照相比,miR-138的表达水平在肝癌组织及肝癌细胞系中明显降低( P <0.01)。当miR-138 mimics转染肝癌细胞后,HepG2细胞的活力降低,创伤愈合能力较弱,迁移能力明显降低( P <0.01),经Western blot检测,波形蛋白和CCND3的表达水平降低( P <0.01)。转染miR-138 inhibitor 后,HepG2细胞的增殖能力升高,肝癌细胞的创伤愈合能力和迁移能力提升( P <0.01),与此同时,波形蛋白和 CCND3的表达水平升高( P < 0.01 )。结论通过调节波形蛋白和 CCND3的表达,miR-138在肝癌细胞的增殖活力、迁移能力的变化机制中发挥重要的作用。