lncRNA SBF2-AS1 通过调控miR-140-5p/VEGFA 分子轴促进宫颈癌HeLa细胞上皮间质转化

目的：探讨lncRNA SBF2-AS1 通过调控miR-140-5p/血管内皮生长因子A（VEGFA）分子轴对宫颈癌HeLa 细胞上皮间质转化（EMT）的影响。方法：细胞培养和转染后分为NC、miR-140-5p mimic、miR-140-5p mimic+pcDNA-VEGFA、si-lncRNASBF2-AS1+pcDNA-VEGFA及si-lncRNA SBF2-AS1+miR-140-5p mimic组5 组。采用qPCR检测lncRNA SBF2-AS1 在宫颈癌组织及细胞系中的表达水平,双荧光素酶报告基因验让lncRNA SBF2-AS1、miR-140-5p 与VEGFA的靶向关系,WB检测HeLa细胞中VEGFA及EMT标志物N-cadherin、Vimentin 和E-cadherin 的表达水平,Transwell 实验检测HeLa细胞侵袭和迁移能力。结果：lncRNASBF2-AS1 在宫颈癌组织及细胞系中高表达（P<0.05 或P<0.01）,lncRNA SBF2-AS1 靶向结合miR-140-5p,且VEGFA 是miR-140-5p 的靶基因（P<0.05）。敲降lncRNA SBF2-AS1 抑制HeLa细胞侵袭、迁移及EMT。进一步实验证实,lncRNA SBF2-AS1通过miR-140-5p 上调VEGFA的表达水平,从而促进HeLa 细胞侵袭、迁移及EMT（P<0.05 或P<0.01）。结论：lncRNA SBF2-AS1通过miR-140-5p/VEGFA分子轴促进HeLa细胞EMT。     

Exosomal miR-130a-3p promotes the progression of differentiated thyroid cancer by targeting insulin-like growth factor 1

The aim of the present study was to determine the expression and diagnostic value of exosomal miR-130a-3p in the serum of patients with differentiated thyroid cancer (DTC). Exosomes were isolated from the serum of patients with DTC and were identified using transmission electron microscopy. A novel exosomal miRNA, miR-130a-3p, was found to be significantly decreased in the serum of patients with DTC compared with those with benign thyroid tumors and healthy controls. Further study revealed that exosomal miR-130a-3p was correlated with the malignant characteristics of DTC, including tumor diameter, lymph node metastasis (LNM) and higher TNM stage. Receiver operating characteristic curve analysis demonstrated that the area under the curve of exosomal miR-130a-3p was better compared with that of TgAb and Tg in patients with DTC. More importantly, the combined use of exosomal miR-130a-3p, TgAb and Tg significantly enhanced the sensitivity and specificity, indicating that exosomal miR-130a-3p is a sensitive biomarker for DTC. A dual luciferase reporter assay indicated that insulin-like growth factor (IGF)-1 was a target gene of miR-130a-3p. Pearson''s correlation analysis revealed a negative correlation between serum IGF-1 and serum exosomal miR-130a-3p levels. More importantly, exosomes from patients with DTC increased the expression of IGF-1 and p-PI3K/p-AKT, but these effects were abolished by siRNA targeting IGF-1 in TPC-1 cells. Taken together, the findings of the present study indicated that reduced exosomal miR-130a-3p levels were associated with the risk of DTC and may be used as a biomarker for the diagnosis of DTC.     

miR-133a-5p suppresses gastric cancer through TCF4 down-regulation

BackgroundThe effect of microRNAs (miRNA) on cancer regulations has received a considerable amount of attention recently. MiR-133a-5p has been identified as an anti-tumor miRNA in several types of cancers. However, the effect of miR-133a-5p on gastric cancer (GC) have not been uncovered. In this study, we sought to evaluate the regulation of TCF4 expression by miR-133-5p and the role of the miR-25-3p/TCF4 axis in the progression of GC, with the aim of identifying a potential therapeutic target for GC.MethodsTCGA (The Cancer Genome Atlas), GTEx (The Genotype-Tissue Expression) and GEO (Gene Expression Omnibus) database were used to analyze the expression and prognosis. We performed MTT and EdU assays to elucidate the effect on cell replication. Apoptotic cells were stained with annexin V-fluorescein isothiocyanate and propidium iodide to stain, and then analyzed by flow cytometry. The effect on cell metastasis was investigated in wound healing and transwell assays. A dual-luciferase reporter assay was used to check for the direct targeting of TCF4 by miR-133a-5p. Bioinformatic analysis of the relationship of TCF4 with tumor microenvironment and the signaling cascade of TCF4 was finally performed.ResultsWe found that the level of miR-133a-5p was decreased in both tumor tissues and GC cell lines. MiR-133a-5p inhibited cell growth and metastasis, but promoted cell apoptosis. MiR-133a-5p directly targeted TCF4 and downregulated its expression. TCF4 was highly expressed in tumor and higher level of TCF4 indicated poorer prognosis. Moreover, TCF4 overexpression reversed the aforementioned anti-tumor activity of miR-133a-5p. The expression level of TCF4 was significantly correlated with tumor-infiltrating immune cells.ConclusionsOur findings altogether reveal that miR-133a-5p can serve as a tumor suppressor in gastric cancer via the miR-133a-5p/TCF4 pathway.     

LncRNA HIF1A-AS2 accelerates malignant phenotypes of renal carcinoma by modulating miR-30a-5p/SOX4 axis as a ceRNA

Objective: Several reports have proposed that lncRNAs, as potential biomarkers, participate in the progression and growth of malignant tumors. HIF1A-AS2 is a novel lncRNA and potential biomarker, involved in the genesis and development of carcinomas. However, the molecular mechanism of HIF1A-AS2 in renal carcinoma is unclear.Methods:The relative expression levels of HIF1A-AS2 and miR-30a-5p were detected using RT-qPCR in renal carcinoma tissues and cell lines. Using loss-of-function and overexpression, the biological effects of HIF1A-AS2 and miR-30a-5p in kidney carcinoma progression were characterized. Dual luciferase reporter gene analysis and Western blot were used to detect the potential mechanism of HIF1A-AS2 in renal carcinomas.Results:HIF1A-AS2 was upregulated in kidney carcinoma tissues when compared with para-carcinoma tissues (P < 0.05). In addition, tumor size, tumor node mestastasis stage and differentiation were identified as being closely associated with HIF1A-AS2 expression (P < 0.05). Knockdown or overexpression of HIF1A-AS2 either restrained or promoted the malignant phenotype and WNT/β-catenin signaling in renal carcinoma cells (P < 0.05). MiR-30a-5p was downregulated in renal cancers and partially reversed HIF1A-AS2 functions in malignant renal tumor cells. HIF1A-AS2 acted as a microRNA sponge that actively regulated the relative expression of SOX4 in sponging miR-30a-5p and subsequently increased the malignant phenotypes of renal carcinomas. HIF1A-AS2 showed a carcinogenic effect and miR-30a-5p acted as an antagonist of the anti-oncogene effects in the pathogenesis of renal carcinomas.Conclusions:The HIF1A-AS2-miR-30a-5p-SOX4 axis was associated with the malignant progression and development of renal carcinoma. The relative expression of HIF1A-AS2 was negatively correlated with the expression of miR-30a-5p, and was closely correlated with SOX4 mRNA levels in renal cancers.     

Upregulated lncRNA CASC2 May Inhibit Malignant Melanoma Development Through Regulating miR-18a-5p/RUNX1

This study aimed to investigate the effect and underlying mechanism of lncRNA CASC2 in malignant melanoma (MM). Expression of CASC2 in MM tissues and cells was detected. A375 cells were transfected with pc-CASC2, si-CASC2, miR-18a-5p inhibitor, or corresponding controls, and then cell proliferation, migration, and invasion were detected using MTT assay, colony formation assay, and Transwell analysis, respectively. The relationship of miR-18a-5p and CASC2 or RUNX1 was detected by luciferase reporter assay. The levels of CASC2 and RUNX1 were significantly reduced in MM tissues compared with normal skin tissues or cells, while the miR-18a-5p level was obviously increased (all p<0.01). Cell viability, colony number, migration, and invasion were significantly decreased in cells with pc-CASC2 compared with cells transfected with pcDNA3.1 (all p<0.05). These effects were consistent with the cells transfected with miR-18a-5p inhibitor. The luciferase reporter assay revealed that CASC2 acted as a molecular sponge for miR-18a-5p, and RUNX1 was a target gene of miR-18a-5p. Moreover, CASC2 overexpression promoted the expression of RUNX1, while upregulated miR-18a-5p significantly reversed the effect of CASC2 on the RUNX1 level (all p<0.05). Upregulated CASC2 may inhibit cell proliferation, migration, and invasion through regulating miR-18a-5p and its target gene RUNX1 in MM.     

miR-203a-3p通过靶向PRMT5抑制胃癌细胞增殖

目的:探讨miR-203a-3p在胃癌中的表达及其对胃癌细胞增殖的影响.方法:收集胃癌组织标本44例,采用Real-time PCR方法检测胃癌组织标本中miR-203a-3p的表达,并分析其表达水平与临床病理参数的关系;生物信息学预测miR-203a-3p的靶基因,并采用荧光素酶报告基因实验进行验证;免疫组化检测胃癌组织标本中PRMT5的表达情况,分析其表达水平与miR-203a-3p表达的相关性;利用脂质体介导的瞬时转染方法过表达miR-203a-3p或同时过表达miR-203a-3p和PRMT5,并通过CCK-8实验检测胃癌细胞的增殖情况.结果:胃癌组织中miR-203a-3p的表达水平与正常组织对照相比显著降低(P<0.01),并且miR-203a-3p的表达水平与肿瘤细胞的分化程度显著相关;荧光素酶报告基因实验证实miR-203a-3p可以直接结合在PRMT5 3'-UTR上,即PRMT5是miR-203a-3p的直接靶基因;在胃癌组织标本中,PRMT5表达水平显著高于正常组织对照(P<0.01),并且其表达水平与miR-203a-3p表达呈显著负相关(r=-0.4124,P<0.01);过表达miR-203a-3p后,胃癌BGC823细胞的增殖能力显著低于miR-NC对照组(P<0.01),并且,"挽救"实验表明在过表达miR-203a-3p的细胞中同时过表达PRMT5后会部分恢复miR-203a-3p对细胞增殖的抑制(P<0.01).结论:miR-203a-3p可通过下调靶基因PRMT5的表达,进而抑制胃癌细胞增殖.因此,miR-203a-3p可作为胃癌疾病临床治疗的潜在靶点.     

MiR-361-5p inhibits colorectal and gastric cancer growth and metastasis by targeting staphylococcal nuclease domain containing-1

MicroRNAs (miRs) function as key regulators of gene expression and their deregulation is associated with the carcinogenesis of various cancers. In the present study, we investigated the biological role and mechanism of miR-361-5p in colorectal carcinoma (CRC) and gastric cancer (GC). We showed that microRNA-361-5p (miR-361-5p) was down-regulated in CRC and GC in comparison to the controls. Meanwhile, the expression levels of miR-361-5p negatively correlated with lung metastasis and prognosis in clinical CRC patients. Overexpression of miR-361-5p markedly suppressed proliferation, migration and invasion of cancer cells. Additionally, this phenotype could be partially rescued by the ectopic expression of staphylococcal nuclease domain containing-1 (SND1). SND1 was identified as a target of miR-361-5p using bioinformatics analysis and in vitro luciferase reporter assays. In turn, SND1 bound to pre-miR-361-5p and suppressed the expression of miR-361-5p, thus exerting a feedback loop. Most interestingly, in vivo studies showed that restoration of miR-361-5p significantly inhibited tumor growth and especially the lung metastasis in nude mice. Therefore, it could be concluded that miR-361-5p functions as a tumor-suppressive miRNA through directly binding to SND1, highlighting its potential as a novel agent for the treatment of patients with CRC and GC.     

CircPVT1 promotes progression in clear cell renal cell carcinoma by sponging miR-145-5p and regulating TBX15 expression

Emerging evidence revealed that circular RNAs (circRNAs) play significant roles in regulating tumorigenesis and cancer progression. However, few circRNAs were well characterized in clear cell renal cell carcinoma (ccRCC). We found that circPVT1 was significantly upregulated in ccRCC tissues and positively associated with the clinical stage. The Area Under Curve of tissue and serum circPVT1 expression in ccRCC were 0.93 and 0.86, respectively. Importantly, we demonstrated that circPVT1 promoted ccRCC growth and metastasis in vitro and in vivo. We also found that circPVT1 directly binds to miRNA-145-5p via the Biotin-labelled miRNA pulldown assay and dual-luciferase reporter assay, and miR-145-5p inhibitor significantly attenuated the effect of circPVT1 knockdown on ccRCC cells. Moreover, through RNA sequencing and bioinformatics analysis, we demonstrated that TBX15 was regulated by the circPVT1/miR-145-5p axis and predicted poor prognosis in ccRCC. These findings suggest that circPVT1 promotes ccRCC growth and metastasis through sponging miR-145-5p and regulating downstream target TBX15 expression. The circPVT1/miR-145-5p/TBX15 axis might be a potential diagnostic marker and therapeutic target in ccRCC.     

MicroRNA-30a inhibits cell migration and invasion by downregulating vimentin expression and is a potential prognostic marker in breast cancer

Tumor recurrence and metastasis result in an unfavorable prognosis for cancer patients. Recent studies have suggested that specific microRNAs (miRNAs) may play important roles in the development of cancer cells. However, prognostic markers and the outcome prediction of the miRNA signature in breast cancer patients have not been comprehensively assessed. The aim of this study was to identify miRNA biomarkers relating to clinicopathological features and outcome of breast cancer. A miRNA microarray analysis was performed on breast tumors of different lymph node metastasis status and with different progression signatures, indicated by overexpression of cyclin D1 and β-catenin genes, to identify miRNAs showing a significant difference in expression. The functional interaction between the candidate miRNA, miR-30a, and the target gene, Vim, which codes for vimentin, a protein involved in epithelial-mesenchymal transition, was examined using the luciferase reporter assay, western blotting, and migration and invasion assays. The association between the decreased miR-30a levels and breast cancer progression was examined in a survival analysis. miR-30a negatively regulated vimentin expression by binding to the 3'-untranslated region of Vim. Overexpression of miR-30a suppressed the migration and invasiveness phenotypes of breast cancer cell lines. Moreover, reduced tumor expression of miR-30a in breast cancer patients was associated with an unfavorable outcome, including late tumor stage, lymph node metastasis, and worse progression (mortality and recurrence) (p < 0.05). In conclusion, these findings suggest a role for miR-30a in inhibiting breast tumor invasiveness and metastasis. The finding that miR-30a downmodulates vimentin expression might provide a therapeutic target for the treatment of breast cancer.     

LINC00909靶向miR-365a-5p/FGFBP1分子轴调控结肠癌细胞增殖、迁移及侵袭的实验研究

目的探讨LINC00909对结肠癌细胞增殖、迁移、侵袭的影响及可能机制。方法购买正常结肠上皮细胞NCM460,结肠癌细胞HCT8、Caco-2及DLD-1;用实时荧光定量PCR(RT-qPCR)及蛋白质印迹法(Western blotting)检测NCM460细胞及结肠癌细胞中LINC00909、miR-365a-5p及成纤维细胞生长因子结合蛋白1(FGFBP1)表达水平。将LINC00909小干扰RNA、miR-365a-5p模拟物、FGFBP1小干扰RNA分别转染HCT8细胞,用细胞计数试剂盒(CCK)-8检测细胞活力,Transwell实验检测细胞迁移及侵袭能力。双荧光素酶报告基因实验确定LINC00909与miR-365a-5p、miR-365a-5p与FGFBP1的靶向调控关系。结果与NCM460细胞比较,结肠癌细胞中LINC00909及FGFBP1表达显著升高,miR-365a-5p表达显著降低(P<0.05)。抑制LINC00909表达后,HCT8细胞活力、迁移及侵袭能力显著降低(P<0.05)。过表达miR-365a-5p后,HCT8细胞活力、迁移及侵袭能力显著降低(P<0.05)。抑制FGFBP1表达后,HCT8细胞活力、迁移及侵袭能力显著降低(P<0.05)。LINC00909靶向负调控miR-365a-5p表达,miR-365a-5p靶向负调控FGFBP1表达。抑制miR-365a-5p表达能够逆转抑制LINC00909对HCT8细胞增殖、迁移及侵袭的影响(P<0.05)。结论抑制LINC00909表达可降低结肠癌细胞增殖、迁移及侵袭能力,其机制与靶向调控miR-365a-5p/FGFBP1分子轴有关。     

Excess of miRNA-378a-5p perturbs mitotic fidelity and correlates with breast cancer tumourigenesis in vivo

Background:

Optimal expression and proper function of key mitotic proteins facilitate control and repair processes that aim to prevent loss or gain of chromosomes, a hallmark of cancer. Altered expression of small regulatory microRNAs is associated with tumourigenesis and metastasis but the impact on mitotic signalling has remained unclear.

Methods:

Cell-based high-throughput screen identified miR-378a-5p as a mitosis perturbing microRNA. Transient transfections, immunofluorescence, western blotting, time-lapse microscopy, FISH and reporter assays were used to characterise the mitotic anomalies by excess miR-378a-5p. Analysis of microRNA profiles in breast tumours was performed.

Results:

Overexpression of miR-378a-5p induced numerical chromosome changes in cells and abrogated taxol-induced mitotic block via premature inactivation of the spindle assembly checkpoint. Moreover, excess miR-378a-5p triggered receptor tyrosine kinase–MAP kinase pathway signalling, and was associated with suppression of Aurora B kinase. In breast cancer in vivo, we found that high miR-378a-5p levels correlate with the most aggressive, poorly differentiated forms of cancer.

Interpretation:

Downregulation of Aurora B by excess miR-378a-5p can explain the observed microtubule drug resistance and increased chromosomal imbalance in the microRNA-overexpressing cells. The results suggest that breast tumours may deploy high miR-378a-5p levels to gain growth advantage and antagonise taxane therapy.     

MiR-497 suppresses angiogenesis and metastasis of hepatocellular carcinoma by inhibiting VEGFA and AEG-1

Hepatocellular carcinoma (HCC) is a worldwide malignance and displays marked vascular abnormalities and active metastasis. MicroRNAs (miRNAs) have been shown to play important roles in regulating tumor properties in cancer, however, whether miR-497 contributes to HCC angiogenesis or metastasis remains unclear. In this study, we found that miR-497 was significantly down-regulated in HCC tissue samples and cell lines. Gain-of-function and loss-of-function studies revealed that miR-497 could repress both the pro-angiogenic and metastatic ability of HCC cells. Subsequent investigations disclosed that miR-497 directly inhibited the 3′-untranslated regions (UTRs) of vascular endothelial growth factor A (VEGFA) and astrocyte elevated gene-1 (AEG-1). Furthermore, overexpression of these targets antagonized the function of miR-497. Based on nude mouse models, we demonstrated that overexpression of miR-497 significantly repressed microvessel densities in xenograft tumors and reduced pulmonary metastasis. In conclusion, our findings indicate that miR-497 downregulation contributes to angiogenesis and metastasis in HCC.