miR-409-3p靶向CXCL9调控滋养层细胞的增殖、迁移和侵袭

目的探讨微小RNA-409-3p(miR-409-3p)对滋养层细胞增殖、迁移和侵袭影响及作用机制。方法实时荧光定量PCR(RT-qPCR)检测正常妊娠胎盘组织和子痫前期胎盘组织中miR-409-3p和干扰素伽玛诱导的单核细胞因子(CXCL9)mRNA表达水平,蛋白印迹(Western blot)法检测CXCL9蛋白表达水平。转染miR-409-3p模拟物或CXCL9小干扰RNA至滋养层细胞HTR8/SVneo,构建miR-409-3p过表达或CXCL9表达抑制的HTR8/SVneo细胞,四甲基噻唑蓝染色法(MTT)检测细胞增殖,Transwell检测细胞迁移和侵袭,Western blot检测细胞周期蛋白D1(CyclinD1)、p21、基质金属蛋白酶2(MMP-2)和MMP-9蛋白表达水平。双荧光素酶报告基因实验验证miR-409-3p与CXCL9调控关系。结果与正常妊娠胎盘组织比较,子痫前期胎盘组织中miR-409-3p水平升高(P<0.05),CXCL9 mRNA和蛋白水平降低(P<0.05)。miR-409-3p过表达或干扰CXCL9表达后,HTR8/SVneo细胞培养48 h和72 h后OD值、迁移和侵袭数、CyclinD1、MMP-2和MMP-9蛋白表达水平降低(P<0.05),p21蛋白表达水平升高(P<0.05)。miR-409-3p在HTR8/SVneo细胞中靶向负调控CXCL9表达。CXCL9过表达逆转了miR-409-3p过表达对HTR8/SVneo细胞增殖、迁移和侵袭的影响。结论 miR-409-3p抑制HTR8/SVneo细胞的增殖、迁移和侵袭与下调CXCL9表达有关。     

抑制lncRNA LINC01503通过靶向调控miR-335-5p对肺癌细胞活力、迁移和侵袭的影响及其机制研究

目的:探讨长链非编码RNA(lncRNA)LINC01503对肺癌细胞活力、迁移和侵袭的影响及其作用机制。方法:将人肺癌H1299细胞分为si-NC组(转染si-NC)、si-LINC01503组(转染si-LINC01503)、pcDNA组(转染pcDNA)、pcDNA-LINC01503组(转染pcDNA-LINC01503)、miR-NC组(转染miR-NC)、miR-335-5p组(转染miR-335-5p mimics)、si-LINC01503+anti-miR-NC组(共转染si-LINC01503和anti-miR-NC)、si-LINC01503+anti-miR-335-5p组(共转染si-LINC01503和anti-miR-335-5p)、miR-NC+WT-LINC01503组(共转染miR-NC和WT-LINC01503)、miR-NC+MUT-LINC01503组(共转染miR-NC和MUT-LINC01503)、miR-335-5p+WT-LINC01503组(共转染miR-335-5p和WT-LINC01503)和miR-335-5p+MUT-LINC...     

miR-454-3p靶向BPTF表达调控肺癌细胞A549增殖、迁移和侵袭

目的探讨miR-454-3p对肺癌细胞增殖、迁移和侵袭的影响及分子机制。方法采用RT-PCR技术检测miR-454-3p在肺癌组织以及肺癌细胞株中的表达,以表达量最低的肺癌细胞A549为后续分析对象,将miR-454-3p mimic转入A549细胞,RT-PCR验证miR-454-3p的过表达效率;采用CCK-8、迁移、侵袭等实验,观察转染组与对照组肺癌细胞的增殖、迁移和侵袭情况;生物信息学预测BPTF是miR-454-3p的靶标,构建BPTF 3′UTR荧光素酶载体,通过双荧光素酶报告基因验证miR-454-3p和BPTF的靶向关系;应用Western blot法检测BPTF的表达及迁移、侵袭相关蛋白的变化。结果RT-PCR实验表明miR-454-3p在肺癌组织和细胞中表达下调,且在A549细胞中表达最低(P<0.05)。与对照组相比,过表达miR-454-3p的肺癌细胞A549增殖能力明显降低,迁移和侵袭能力均受到抑制(P<0.05)。生物信息学软件分析BPTF为miR-454-3p的潜在靶基因,过表达miR-454-3p后BPTF的表达水平明显受到抑制。同时,迁移相关蛋白MMP-2和MMP-9表达明显下调,E-cadhern表达明显上调,而E-cadherin的负性调控N-cadherin表达下降。结论miR-454-3p可能通过靶向下调BPTF的表达,抑制肺癌细胞的增殖、迁移和侵袭能力,为肺癌的靶向治疗提供潜在靶标。     

miR-483-5p通过上调IGF2基因转录促进肝癌细胞生长、迁移与侵袭

目的:探讨miR-483-5p对人胰岛素样生长因子2(IGF2)基因P3启动子驱动的m RNA(P3 m RNA)表达的影响及其在肝细胞癌发生发展中的作用。方法:(1)采用real-time PCR检测人肝癌细胞株Huh7、Hep3B、Bel-7402、Hep G2和SMMC-7721,人正常肝细胞株HL-7702,83例人肝细胞癌组织和配对的癌旁组织,22例正常肝组织中miR-483-5p和P3 m RNA的表达水平,并应用Pearson相关分析评估P3 m RNA与miR-483-5p表达水平之间的关系。(2)将IGF2基因P3 m RNA的5’端非翻译区(5’UTR)克隆入p GL3启动子载体,构建P3 m RNA 5’UTR野生型(p GL3-P3-5’UTR-WT)及P3 m RNA 5’UTR突变型(p GL3-P3-5’UTR-MUT)重组萤光素酶报告质粒,将其分别与miR-483-5p mimic、miR-483-5p inhibitor及scrambled control共转染He La、293T及Huh7细胞,采用双萤光素酶报告系统检测萤光素酶活性。(3)分别将miR-483-5p mimic、miR-483-5p inhibitor及scrambled control转染Huh7及Hep3B肝癌细胞,应用real-time PCR检测这2种肝癌细胞P3 m RNA表达水平的变化。(4)应用real-time PCR检测肝癌细胞Huh7及Hep3B的细胞核和细胞质中miR-483-5p的表达水平;应用核连缀实验(nuclear run-on assay)分析miR-483-5p对P3 m RNA转录的影响;应用RNA稳定性实验分析miR-483-5p对P3 m RNA稳定性影响。(5)应用体外细胞功能实验研究miR-483-5p对Huh7肝癌细胞生长、凋亡、迁移与侵袭能力的影响。结果:(1)5种肝癌细胞株miR-483-5p及P3 m RNA表达水平均明显高于正常肝细胞株HL-7702(P0.01),肝细胞癌组织中miR-483-5p及P3 m RNA表达水平均明显高于配对的癌旁组织及正常肝组织(P0.01);线性相关分析显示,在5种肝癌细胞株及肝细胞癌组织中,P3 m RNA表达水平均与miR-483-5p水平呈正相关。(2)萤光素酶实验显示,miR-483-5p与P3m RNA 5’UTR的同源位点互补结合可促进P3 m RNA的表达。(3)瞬时转染实验显示,过表达miR-483-5p呈剂量依赖性促进Hep3B和Huh7肝癌细胞P3 m RNA表达水平的增高。(4)miR-483-5p表达实验显示,成熟miR-483-5p存在于肝癌细胞Hep3B和Huh7的细胞质和细胞核中;核连缀实验显示,miR-483-5p诱导Huh7肝癌细胞核中新生P3 m RNA转录;RNA稳定性实验表明,miR-483-5p不改变Huh7肝癌细胞P3 m RNA稳定性。(5)体外细胞功能实验显示,miR-483-5p促进Huh7肝癌细胞增殖,抑制其凋亡,并增强迁移与侵袭能力。结论:miR-483-5p高表达可部分通过上调IGF2基因P3 m RNA转录促进肝癌细胞生长、迁移与侵袭,进而参与肝细胞癌发生。     

Long noncoding RNA LINC00173 is downregulated in cervical cancer and inhibits cell proliferation and invasion by modulating the miR-182-5p/FBXW7 axis

Accumulating evidence has supported the concept that long noncoding RNAs (lncRNAs) participate in the initiation and progression of human cervical cancer (CC). The long intergenic nonprotein-coding RNA 173 (LINC00173) is a recently identified cancer-associated factor. However, the expression and biological role of LINC00173 in CC are poorly understood. Here, for the first time, we found that the expression of LINC00173 was decreased in CC tissues compared with that in nontumor tissues. Data from The Cancer Genome Atlas (TCGA) further revealed that the downregulated expression of LINC00173 in CC tissues was correlated with poor survival. Functionally, LINC00173 overexpression suppressed HeLa cell proliferation via induction of G0/G1 phase arrest. Ectopic expression of LINC00173 also repressed the invasiveness of HeLa cells. Conversely, LINC00173 depletion resulted in the enhanced proliferation and invasiveness of C33A cells. Mechanistically, LINC00173 functioned as a molecular sponge for miR-182-5p and inversely regulated the miR-182-5p level in CC cells. F-box and WD repeat domain-containing 7 (FBXW7) was identified as the target of miR-182-5p. LINC00173 overexpression enhanced the FBXW7 level via regulation of miR-182-5p in HeLa Cells. More importantly, the inhibitory effects of LINC00173 on HeLa cell proliferation and invasiveness were reversed by FBXW7 silencing. Taken together, the results indicate that the LINC00173/miR-182-5p/FBXW7 axis is critical for CC progression, which might offer new insights into effective therapy for CC.     

miR-140-3p通过靶向PD-L1抑制非小细胞肺癌A549细胞的活力、迁移和侵袭

目的:探究微小RNA-140-3p(mi R-140-3p)对程序性细胞死亡配体1(PD-L1)的靶向关系以及对非小细胞肺癌A549细胞的活力、迁移和侵袭的影响。方法:使用RT-qPCR检测HLF-1、A549和H1299不同细胞株中mi R-140-3p的表达水平,选择差异最显著的A549细胞用作后续研究对象; Target Scan软件预测和双萤光素酶报告基因实验验证mi R-140-3p和PD-L1之间的靶向关系; RT-qPCR和Western blot检测转染mi R-140-3p模拟物和抑制剂对PD-L1表达水平的影响; MTT法检测mi R-140-3p和PD-L1对A549细胞活力的影响; Transwell实验检测mi R-140-3p和PD-L1对A549细胞迁移和侵袭的影响。结果:mi R-140-3p在人肺癌A549和H1299细胞的表达中显著下调(P 0. 05); mi R-140-3p高表达能够使PD-L1表达下调,对A549细胞的活力、迁移和侵袭具有抑制作用;转染pc DNA3. 0-PD-L1能够阻断mi R-140-3p对A549细胞活力、迁移和侵袭的抑制作用。结论:mi R-140-3p可通过靶向负调控PD-L1抑制A549细胞的活力、迁移和侵袭。     

MiR-1-5p is down-regulated in gallbladder carcinoma and suppresses cell proliferation,migration and invasion by targeting Notch2

Background

Numerous studies have demonstrated that aberrant microRNAs (miRNAs) are involved in tumorigenesis and tumor progression. Nevertheless, the precise role of miR-1-5p in gallbladder carcinoma cell growth and metastasis remains not fully revealed.

miR-34c-3p inhibits cell proliferation,migration and invasion of hepatocellular carcinoma by targeting MARCKS

Recent studies have shown that microRNA-34c-3p (miR-34c-3p) is down-regulated in various types of cancers and involved in tumor growth, invasion and metastasis. However, the roles of miR-34c-3p in hepatocellular carcinoma (HCC) are poorly understood. In this study, the expression profile of miR-34c-3pin HCC tissues and cell lines were examined by quantitative real-time polymerase chain reaction (qRT-PCR). The correlations of miR-34c-3p expression and clinicopathological characteristics were analyzed. The biological role of MiR-34c-3pin cell proliferation, migration and invasion was examined. In addition, the targets of miR-34c-3p were identified. The results showed that miR-34c-3p expression was significantly down-regulated in HCC tissues and cell lines; low expression level of miR-34c-3p was correlated with vascular invasion and advanced TNM stage. In vitro functional assays showed that overexpression of miR-34c-3pin HepG2 and Huh7 cells significantly reduced cell proliferation, migration and invasion. Furthermore, target analysis and luciferase assay identified myristoylated alanine-rich protein kinase c substrate (MARCKS) as a specific target of miR-34c-3p. Knockdown of MARCKS in HepG2 cells reduced cell migration and invasion, but not cell proliferation. Taken together, our findings implicate the potential application of miR-34c-3p as a tumor suppressor in cancer therapy.     

Exosomal miR-155-5p promotes proliferation and migration of gastric cancer cells by inhibiting TP53INP1 expression

Exosomal microRNA (miRNA) secreted by tumor cells plays an important biological role in tumorigenesis and development. We aimed to explore the effects of exosomal miR-155-5p in gastric cancer (GC) and understand its mechanism of action in GC progression. We isolated exosomes from the human gastric mucosal epithelial cell line GES-1 and gastric cancer cell line AGS, and then identified them according to their surface markers by flow cytometry. Later, we detected the miR-155-5p expression levels in tissues and isolated exosomes using RT-qPCR. Bioinformatics analysis showed that miR-155-5p directly binds to the 3' untranslated region (3'-UTR) of tumor protein p53-induced nuclear protein 1 (TP53INP1) mRNA. We also investigated whether the miR-155-5p-rich exosomes caused changes in cell cycle, proliferation, and migration in AGS cells. In this study, we found that the levels of miR-155-5p were significantly increased in GC tissues and AGS cells, and that the TP53INP1 protein level was downregulated in GC tissues using IHC and IFC. TP53INP1 was found to be directly regulated by miR-155-5p following a dual luciferase-based reporter assay. After co-culturing with the isolated miR-155-5p-rich exosomes, the proliferation and migration capabilities of AGS cells were enhanced. Thus, our results reveal that exosomal miR-155-5p acts as an oncogene by targeting TP53INP1 mRNA in human gastric cancer.     

miR-338-3p inhibits the proliferation and migration of gastric cancer cells by targeting ADAM17

Background: MicroRNAs (miRNA) have been documented playing a critical role in cancer progression. Although miR-338-3p has been implicated in several cancers, its role in gastric cancer is still unknown. The aim of our study was to investigate the role of miR-338-3p in gastric cancer progression. Methods: Expression levels of miR-338-3p in gastric cancer cell lines and tissues were determined by quantitative real-time PCR (qRT-PCR). The effect of miR-338-3p on proliferation was evaluated by MTT assay, cell migration and invasion were evaluated by transwell migration and invasion assays. Furthermore, luciferase reporter assay was conducted to confirm the target gene of miR-338-3p, and the results were validated in gastric cancer cells. Results: In the present study, we found that miR-338-3p was down-regulated in both gastric cancer cell lines and tissues. Enforced expression of miR-338-3p inhibited proliferation, migration and invasion of gastric cancer cells in vitro. Moreover, we identified A disintegrin and metalloproteinase 17 (ADAM17) gene as potential target of miR-338-3p. Importantly, ADAM17 rescued the miR-338-3p mediated inhibition of cell proliferation, migration and invasion. Conclusions: Our study suggested that miR-338-3p is significantly decreased in gastric cancer, and inhibits cell proliferation, migration and invasion partially via the downregulation of ADAM17. Thus, miR-338-3p may represent a potential therapeutic target for gastric cancer intervention.     

Downregulation of miR-101-3p by hepatitis B virus promotes proliferation and migration of hepatocellular carcinoma cells by targeting Rab5a

RAB GTPase 5A (RAB5A), a member of the Rab subfamily of small GTPases, acts as an oncogene and has been associated with various key cellular functions, including cell growth, differentiation, apoptosis and angiogenesis. Recently, it has been reported that the Rab5a gene is involved in the progression of cancer. Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancers, and it is usually associated with persistent hepatitis B virus (HBV) infections. Emerging evidence suggests that HBV alters microRNA (miRNA) expression profiles, but the mechanisms underlying this process have not yet been fully elucidated. Here, we examine how HBV affects the production of miR-101-1, which has been shown to be downregulated in HCC. We found that HBV could repress miR-101-3p by inhibiting its promoter activity. Downregulation of miR-101-3p promoted cancer cell growth and migration, and a specific miR-101-3p inhibitor was able to enhance proliferation and migration. Moreover, we identified Rab5a was one of the target genes of miR-101-3p in HBV-related HCC. Forced expression of miR-101-3p in liver cell lines resulted in a marked reduction of the expression of Rab5a at both the mRNA and protein level by directly targeting the 3′untranslated region of Rab5a. Overexpression of Rab5a resulted in a reversal of the suppression of proliferation and migration of SMMC-7721 cells mediated by miR-101-3p. Taken together, our data show that HBV can downregulate miR-101-3p expression by inhibiting its promoter activity and that downregulation of miR-101-3p promotes HCC cell proliferation and migration by targeting Rab5a. This provides new insights into the mechanisms of HBV-related HCC pathogenesis.     

lncRNA MALAT1靶向调控miR-146b-5p影响膀胱癌细胞侵袭和迁移

目的:探讨长链非编码RNA(lncRNA)肺腺癌转移相关转录因子1(MALAT1)靶向微小RNA-146b-5p(miR-146b-5p)影响膀胱癌细胞侵袭和迁移的机制。方法:在膀胱癌BIU-87细胞中转染MALAT1 siRNA,以real-time PCR方法测定转染效果,Transwell法测定侵袭及迁移能力,Western blot法检测细胞中上皮-间充质转化(EMT)相关蛋白波形蛋白(vimentin)、上皮型钙黏蛋白(E-cadherin)和迁移侵袭相关蛋白基质金属蛋白酶-2(MMP-2)蛋白表达的变化。生物信息学软件预测MALAT1与miR-146b-5p有靶向互补位点,利用双萤光素酶报告系统鉴定靶向关系。用real-time PCR方法检测下调MALAT1后BIU-87细胞中miR-146b-5p表达的变化。将MALAT1 siRNA和miR-146b-5p inhibitor共转染至BIU-87细胞中,用上述方法分析细胞侵袭、迁移及vimentin、E-cadherin和MMP-2蛋白表达的变化。结果:转染MALAT1 siRNA可明显下调BIU-87细胞中MALAT...     

miR-671-5p通过靶向肿瘤坏死因子α诱导蛋白8调控胰腺癌细胞增殖和凋亡

目的：探讨miR-671-5p 靶向肿瘤坏死因子α 诱导蛋白8（ TNFAIP8）对胰腺癌细胞增殖和凋亡的影响。 方法：实时荧光定量PCR（ qRT-PCR）和免疫印迹检测20 例胰腺癌组织和与其配对的癌旁正常组织miR-617-5p、 TNFAIP8 mRNA和TNFAIP8表达水平,验证miR-671-5p 和TNFAIP8的靶向调控关系。将体外培养胰腺癌细胞 capan-1分为miR-NC组、miR-671-5p 组、si-NC 组、si-TNFAIP8 组、miR-671-5p+pcDNA组和miR-671-5p+pcDNATNFAIP8 组。采用四甲基偶氮唑蓝（ MTT）实验检测细胞活力,流式细胞术检测细胞凋亡,免疫印迹检测细胞周 期蛋白D1（ cyclin D1）、p21、B细胞淋巴瘤/ 白血病-2（ Bcl-2）和Bcl-2 相关蛋白（ Bax）的表达水平。结果：与 癌旁正常组织比较,胰腺癌组织miR-617-5p 表达量显著降低,TNFAIP8 的表达量显著升高。miR-671-5p 靶向负向 调控TNFAIP8 表达。与miR-NC组比较,miR-671-5p 组capan-1 细胞活力显著降低,细胞凋亡率显著升高,cyclin D1和Bcl-2 的表达量显著降低,p21 和Bax 的表达量显著升高;与si-NC 组比较,si-TNFAIP8 组capan-1 细胞活力 显著降低,细胞凋亡率显著升高,cyclin D1和Bcl-2 表达量显著降低,p21 和Bax 表达量显著升高;与miR-671- 5p+pcDNA 组比较,miR-671-5p+pcDNA-TNFAIP8 组capan-1 细胞活力显著升高,细胞凋亡率显著降低,cyclin D1和Bcl-2 的表达量显著升高,p21 和Bax 的表达量显著降低。结论：miR-671-5p 通过靶向下调TNFAIP8 抑制胰 腺癌细胞增殖并促进其凋亡。上调miR-671-5p 是胰腺癌潜在治疗靶点。     

冬凌草甲素通过上调miR-204-5p抑制PC-3细胞迁移与侵袭的作用机制研究

目的 探索冬凌草甲素影响PC-3细胞迁移及侵袭的作用及机制,为冬凌草甲素治疗前列腺癌骨转移提供前期体外实验参考。 方法 通过CCK8检测冬凌草甲素对PC-3细胞增殖的影响,Transwell检测冬凌草甲素对PC-3细胞迁移和侵袭的影响,qRT-PCR分析冬凌草甲素溶液处理PC-3细胞后miR-204-5p及NF-κB信号通路表达情况,Western blotting分析冬凌草甲素溶液处理PC-3细胞后NF-κB信号通路蛋白表达情况。 结果 冬凌草甲素溶液以浓度依赖性和时间依赖性方式抑制PC-3细胞的增殖,与对照组相比差异有统计学意义（P<0.01）,冬凌草甲素IC50值为10 μmol/L;冬凌草甲素溶液呈浓度依赖性抑制PC-3细胞的迁移和侵袭,差异较对照组有统计学意义（P<0.01）;冬凌草甲素溶液能促进PC-3细胞表达miR-204-5p并阻断NF-κB信号通路下游基因表达;过表达miR-204-5p和冬凌草甲素均能抑制PC-3细胞NF-κB信号通路下游基因表达。 结论 冬凌草甲素能抑制PC-3细胞的增殖、迁移及侵袭,其作用机制可能与其能上调miR-204-5p阻断NF-κB信号通路有关。     

Long non-coding RNA MIF-AS1 promotes breast cancer cell proliferation,migration and EMT process through regulating miR-1249-3p/HOXB8 axis

Breast cancer (BC) is one of the leading cause of cancer-related death among females worldwide. Mounting evidences indicate that long non-coding RNAs (lncRNAs) were involved in tumor progression by acting as either oncogenes or tumor suppressors in multiple cancers. In this study, we focused on the function and mechanism of lncRNA Migration Inhibitory Factor Antisense RNA 1 (MIF-AS1) in BC. qRT-PCR showed that MIF-AS1 was upregulated in BC tissues and cells. To detect its bio-function, a series of loss-of-function assays were carried out. Thereafter, we found that MIF-AS1 depletion inhibited BC cell proliferation, migration and epithelial-mesenchymal transition (EMT). Recently, increasing studies indicate that lncRNAs can function as competing endogenous RNAs (ceRNAs). Using bioinformatics analysis and luciferase reporter assay, we identified that MIF-AS1 regulated the level of Homeobox B8 (HOXB8) via binding to miR-1249-3p. Taken all together, our findings proved that MIF-AS1 acted as a ceRNA by modulating miR-1249-3p/HOXB8 axis in breast cancer. LncRNA MIF-AS1 might be a new biomarker and therapeutic target for BC patients.     

微小RNA-138-5p抑制肺癌细胞增殖、迁移和侵袭能力的机制研究

目的:探讨微小RNA-138-5p(miR-138-5p)抑制肺癌细胞增殖、迁移和侵袭能力的相关机制。方法:以肺癌细胞A549和H460作为研究对象,分别转染miR-NC(对照组)或miR-138-5p(实验组);生物信息学技术预测miR-138-5p的靶基因;RT-qPCR检测转染后细胞miR-138-5p、叉头框蛋白C1(FOXC1)mRNA和波形蛋白(vimentin)mRNA的相对表达量;Western blot法检测FOXC1、vimentin、E-cadherin、N-cadherin和β-catenin蛋白表达变化;MTS法和集落形成实验分别检测细胞的增殖能力;划痕愈合实验和Transwell法检测细胞迁移和侵袭能力。结果:miR-138-5p过表达显著降低FOXC1和vimentin的mRNA及蛋白的表达(P0.05),E-cadherin和β-catenin蛋白表达上调,N-cadherin蛋白表达下调,显著抑制肺癌细胞的增殖、迁移和侵袭能力(P0.05)。结论:miR-138-5p可以通过靶向干扰FOXC1和vimentin的表达抑制肺癌细胞的增殖、迁移和侵袭,可能是肺癌基因治疗的潜在靶点。