miR-520a-3p调控宫颈癌细胞因子分泌的信号通路*

目的: 探讨miR-520a-3p调控宫颈癌细胞因子分泌的分子机制。方法: 通过TargetScanHuman分析miR-520a-3p与NF-κB复合体亚基RELA的匹配情况,然后通过荧光素酶报告系统检测miR-520a-3p是否靶向NF-κB复合体亚基RELA;使用LPS刺激宫颈癌HELA细胞后,将miR-520a-3p mimics与转染试剂混合后滴入HELA细胞中,此为过表达组;将miR-520a-3p inhibitor与转染试剂混合后滴入HELA细胞中,此为敲低组,通过酶联免疫吸附试验检测过表达组和敲低组GCSF, GM-CSF, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 p40, IL-12 p70, IL-13, IL-17, IFN-γ, MCP-1, MCP-5, RANTES, TNFα的表达水平。每次实验重复3次。结果: miR-520a-3p靶向RELA的3’UTR;LPS激活NF-kB信号通路后,宫颈癌HELA细胞分泌的细胞因子GCSF, GM-CSF, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 p40, IL-12 p70, IL-13, IL-17, IFN-γ, MCP-1, MCP-5, RANTES, TNFα的蛋白表达水平上升(P＜0.05);过表达组中NF-κB复合体亚基RELA的蛋白表达水平下降,宫颈癌HELA细胞分泌的上述细胞因子的蛋白表达水平下降(P＜0.05);敲低组中NF-κB复合体亚基RELA的蛋白表达水平上升,宫颈癌HELA细胞分泌的上述细胞因子的蛋白表达水平上升(P＜0.05)。结论: miR-520a-3p通过靶向NF-κB信号通路的关键分子RELA抑制宫颈癌HELA细胞的细胞因子分泌。     

Long non-coding RNA LINC00488 facilitates thyroid cancer cell progression through miR-376a-3p/PON2

Objective: Long non-coding RNAs (lncRNAs) recently have been identified as influential indicators in a variety of malignancies. The aim of the present study was to identify a functional lncRNA LINC00488 and its effects on thyroid cancer in the view of cell proliferation and apoptosis.Methods: In order to evaluate the effects of LINC00488 on the cellular process of thyroid cancer, we performed a series of in vitro experiments, including cell counting kit-8 (CCK-8) assay, EdU (5-ethynyl-2′-deoxyuridine) assay, flow cytometry, transwell chamber assay, Western blot and RT-qPCR. The target gene of LINC00488 was then identified by bioinformatics analysis (DIANA and TargetScan). Finally, a series of rescue experiments was conducted to validate the effect of LINC00488 and its target genes on proliferation, migration, invasion and apoptosis of thyroid cancer.Results: Our findings revealed that LINC00488 was highly expressed in thyroid cancer cell lines (BCPAP, BHP5-16, TPC-1 and CGTH-W3) and promoted the proliferation, migration and invasion, while inhibited the apoptosis of thyroid cancer cells (BCPAP and TPC-1). The results of bioinformatics analysis and dual luciferase reporter gene assay showed that LINC00488 could directly bind to miR-376a-3p and down-regulated the expression level of miR-376a-3p. In addition, Paraoxonase-2 (PON2) was a target gene of miR-376a-3p and negatively regulated by miR-376a-3p. Rescue experiment indicated that LINC00488 might enhance PON2 expression by sponging miR-376a-3p in thyroid cancer.Conclusion: Taken together, our study revealed that lncRNA LINC00488 acted as an oncogenic gene in the progression of thyroid cancer via regulating miR-376a-3p/PON2 axis, which indicated that LINC00488-miR-376a-3p-PON2 axis could serve as novel biomarkers or potential targets for the treatment of thyroid cancer.     

MicroRNA-222 alleviates radiation-induced apoptosis by targeting BCL2L11 in cochlea hair cells

Radiation-induced hair cell injury is detrimental for human health but the underlying mechanism is not clear. MicroRNAs (miRNAs) have critical roles in various types of cellular biological processes. The present study investigated the role of miR-222 in the regulation of ionizing radiation (IR)-induced cell injury in auditory cells and its underlying mechanism. Real-time PCR was performed to identify the expression profile of miR-222 in the cochlea hair cell line HEI-OC1 after IR exposure. miRNA mimics or inhibitor-mediated up- or down-regulation of indicated miRNA was applied to characterize the biological effects of miR-222 using MTT, apoptosis and DNA damage assay. Bioinformatics analyses and luciferase reporter assays were applied to identify an miRNA target gene. Our study confirmed that IR treatment significantly suppressed miR-222 levels in a dose-dependent manner. Up-regulation of miR-222 enhances cell viability and alleviated IR-induced apoptosis and DNA damage in HEI-OC1 cells. In addition, BCL-2-like protein 11 (BCL2L11) was validated as a direct target of miR-222. Overexpression of BCL2L11 abolished the protective effects of miR-222 in IR-treated HEI-OC1 cells. Moreover, miR-222 alleviated IR-induced apoptosis and DNA damage by directly targeting BCL2L11. The present study demonstrates that miR-222 exhibits protective effects against irradiation-induced cell injury by directly targeting BCL2L11 in cochlear cells.     

Ligustrazine inhibits the proliferation and migration of ovarian cancer cells via regulating miR-211

Ovarian cancer (OC) is a commonly diagnosed female cancer. Ligustrazine (LSZ), a natural compound, has been reported to exert anti-cancer activity, although the mechanisms underlying the anti-cancer effects are not clear. The present study investigated the impact of LSZ on cell proliferation and migration by regulating microRNA-211 (miR-211) expression using the human ovarian cancer SK-OV-3 and OVCAR-3 cell lines. OC cells were treated with 0, 0.5, 1, and 2 mM LSZ, and quantitative real-time PCR was utilized to measure miR-211 levels in SK-OV-3 and OVCAR-3 cells with different treatment. Moreover, to further confirm the roles of miR-211 in LSZ induced anti-tumor effects, miR-211 expression was inhibited by transfection of miR-211 inhibitors in SK-OV-3 cells. Cell proliferation of transfected cells was evaluated using the CCK-8 and colony formation assay. The scratch assay was employed to assess cell migration and transwell assay was performed for evaluating the cell invasion. Protein levels of epithelial–mesenchymal transition (EMT) markers were determined by Western blotting. We found that LSZ inhibited the viability, proliferation, migration and invasion ability of SK-OV-3 and OVCAR-3 cells in a dose-dependent manner; moreover, LSZ could significantly increase the expression of miR-211 in both SK-OV-3 and OVCAR-3, and knockdown of miR-211 in SK-OV-3 cells partially abrogated the anti-tumor behavior of LSZ by promoting the viability, proliferation, migration, invasion and EMT of SK-OV-3 cells. Thus, we found that LSZ can inhibit the proliferation and migration of OC cells via regulating miR-211. Our study suggests that LSZ might be a potential and effective treatment for OC.     

LncRNA UCA1 elevates the resistance of human leukemia cells to daunorubicin by the PI3K/AKT pathway via sponging miR-613

Acute leukemia is a hematological malignant tumor. Long non-coding RNA urothelial cancer-associated 1 (UCA1) is involved in the chemo-resistance of diverse cancers, but it is unclear whether UCA1 is associated with the sensitivity of acute leukemia cells to daunorubicin (DNR). DNR (100 nM) was selected for functional analysis. The viability, cell cycle progression, apoptosis, and invasion of treated acute leukemia cells (HL-60 and U-937) were evaluated by cell counting kit-8 (CCK-8) assay, flow cytometry assay, or transwell assay. Protein levels were detected with Western blot analysis. Expression patterns of UCA1 and miR-613 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between UCA1 and microRNA-613 (miR-613) was verified by dual-luciferase reporter assay. We observed that UCA1 expression was elevated in HL-60 and U-937cells. DNR constrained viability, cell cycle progression, invasion, and facilitated apoptosis of HL-60 and U-937 cells in a dose-dependent manner, but these impacts mediated by DNR were reverted after UCA1 overexpression. MiR-613 was down-regulated in HL-60 and U-937 cells, and UCA1 was verified as a miR-613 sponge. MiR-613 inhibitor reversed DNR treatment-mediated effects on viability, cell cycle progression, apoptosis, and invasion of HL-60 and U-937 cells, but these impacts mediated by miR-613 inhibitor were counteracted after UCA1 inhibition. Notably, the inactivation of the PI3K/AKT pathway caused by DNR treatment was reversed after miR-613 inhibitor introduction, but this influence mediated by miR-613 inhibitor was offset after UCA1 knockdown. In conclusion, UCA1 up-regulation facilitated the resistance of acute leukemia cells to DNR via the PI3K/AKT pathway by sponging miR-613.     

MiR-29b-3p aggravates cardiac hypoxia/reoxygenation injury via targeting PTX3

Our current research aimed to decipher the role and underlying mechanism with regard to miR-29b-3p involving in myocardial ischemia/reperfusion (I/R) injury. In the present study, cardiomyocyte H9c2 cell was used, and hypoxia/reoxygenation (H/R) model was established to mimic the myocardial I/R injury. The expressions of miR-29b-3p and pentraxin 3 (PTX3) were quantified deploying qRT-PCR and Western blot, respectively. The levels of LDH, TNF-α, IL-1β and IL-6 were detected to evaluate cardiomyocyte apoptosis and inflammatory response. Cardiomyocyte viability and apoptosis were examined employing CCK-8 assay and flow cytometry, respectively. Verification of the targeting relationship between miR-29b-3p and PTX3 was conducted using a dual-luciferase reporter gene assay. It was found that miR-29b-3p expression in H9c2 cells was up-regulated by H/R, and a remarkable down-regulation of PTX3 expression was demonstrated. MiR-29b-3p significantly promoted of release of inflammatory cytokines of H9c2 cells, and it also constrained the proliferation and promoted the apoptosis of H9c2 cells. Additionally, PTX3 was inhibited by miR-29b-3p at both mRNA and protein levels, and it was identified as a direct target of miR-29b-3p. PTX3 overexpression could reduce the inflammatory response, increase the viability of H9c2 cells, and inhibit apoptosis. Additionally, PTX3 counteracted the function of miR-29b-3p during the injury of H9c2 cells induced by H/R. In summary, miR-29b-3p was capable of aggravating the H/R injury of H9c2 cells by repressing the expression of PTX3.     

CCR-NB-LRR proteins MdRNL2 and MdRNL6 interact physically to confer broad-spectrum fungal resistance in apple (Malus × domestica)

Apple leaf spot, a disease caused by Alternaria alternata f. sp. mali and other fungal species, leads to severe defoliation and results in tremendous losses to the apple (Malus × domestica) industry in China. We previously identified three RPW8, nucleotide-binding, and leucine-rich repeat domain CC_R-NB-LRR proteins (RNLs), named MdRNL1, MdRNL2, and MdRNL3, that contribute to Alternaria leaf spot (ALT1) resistance in apple. However, the role of NB-LRR proteins in resistance to fungal diseases in apple remains poorly understood. We therefore used MdRNL1/2/3 as baits to screen ALT1-inoculated leaves for interacting proteins and identified only MdRNL6 (another RNL) as an interactor of MdRNL2. Protein interaction assays demonstrated that MdRNL2 and MdRNL6 interact through their NB-ARC domains. Transient expression assays in apple indicated that complexes containing both MdRNL2 and MdRNL6 are necessary for resistance to Alternaria leaf spot. Intriguingly, the same complexes were also required to confer resistance to Glomerella leaf spot and Marssonina leaf spot in transient expression assays. Furthermore, stable transgenic apple plants with suppressed expression of MdRNL6 showed hypersensitivity to Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot; these effects were similar to the effects of suppressing MdRNL2 expression in transgenic apple plantlets. The identification of these novel broad-spectrum fungal resistance genes will facilitate breeding for fungal disease resistance in apple.     

lncRNA CEBPA-AS1靶向miR-455-3p调控胃癌细胞生物学行为的分子机制研究

摘要 目的：探讨lncRNA CEBPA-AS1对胃癌细胞生物学行为的影响及其可能作用机制。方法：qRT-PCR法检测胃癌组织、癌旁组织与正常人胃上皮GES1细胞和人胃癌SNU-1、AGS、HS-746T细胞系中lncRNA CEBPA-AS1、miR-455-3p的表达量。si-NC、si-lncRNA CEBPA-AS1、miR-NC、miR-455-3p mimics、si-lncRNA CEBPA-AS1与anti-miR-NC、si-lncRNA CEBPA-AS1与anti-miR-455-3p分别转染至SNU-1细胞（分别命名为si-NC组、si-lncRNA CEBPA-AS1组、miR-NC组、miR-455-3p组、si-lncRNA CEBPA-AS1+anti-miR-NC组和si-lncRNA CEBPA-AS1+anti-miR-455-3p组）后,MTT实验与平板克隆形成实验分别检测细胞增殖及克隆形成能力,Transwell小室实验检测细胞迁移及侵袭能力,双荧光素酶报告基因实验与qRT-PCR实验验证lncRNA CEBPA-AS1与miR-455-3p的靶向调控关系,Western blot法检测MMP2、MMP9蛋白表达情况。结果：与癌旁组织比较,胃癌组织中lncRNA CEBPA-AS1的表达量显著升高,miR-455-3p的表达量显著降低,差异均有统计学意义（均P＜0.05）。与GES1细胞比较,SNU-1、AGS、HS-746T细胞中lncRNA CEBPA-AS1的表达量显著升高,miR-455-3p的表达量显著降低,其中SNU-1细胞的lncRNA CEBPA-AS1表达量最高,差异均有统计学意义（均P＜0.05）。与si-NC组比较,si-lncRNA CEBPA-AS1组细胞活力降低,细胞克隆形成数、迁移及侵袭细胞数减少,MMP2、MMP9蛋白表达水平降低,差异均有统计学意义（P＜0.05）。与miR-NC组比较,miR-455-3p组细胞活力降低,细胞克隆形成数、迁移及侵袭细胞数减少,MMP2、MMP9蛋白表达水平降低,差异均有统计学意义（P＜0.05）。lncRNA CEBPA-AS1可靶向结合miR-455-3p,并可负调控miR-455-3p的表达。与si-lncRNA CEBPA-AS1+anti-miR-NC组比较,si-lncRNA CEBPA-AS1+anti-miR-455-3p组细胞活力升高,细胞克隆形成数、迁移及侵袭细胞数增多,MMP2、MMP9蛋白表达水平升高,差异均有统计学意义（P＜0.05）。结论：干扰lncRNA CEBPA-AS1表达可通过靶向调控miR-455-3p而抑制胃癌细胞增殖、克隆形成、迁移及侵袭。