Comparison of XTT and Alamar blue assays in the assessment of the viability of various human cancer cell lines by AT-101 (−/− gossypol)

This study compared the two different commercially available in vitro viability assays: XTT and Alamar blue (AB), to detect anti-proliferative effects of AT-101, a cotton plant extract, on six different human carcinoma cell lines including: prostate (PC-3 and DU-145), breast (MCF-7 and MDA-MB-231), and ovary (OVCAR-3 and MDAH 2774) in a time- and dose-dependent manner. Cells were exposed to AT-101 in the concentration range of 2.5–40 µM for 24, 48, and 72?h. The AB assay was slightly more sensitive than the XTT assay in the evaluation of AT-101 at 24?h, suggesting that the AB assay might be used for detecting early changes in cell viability as compared to the XTT assay. Moreover, the AB assay showed less intra-assay variability as compared to the XTT. The non-toxic, non-radioactive AB metabolism assay allows rapid assessment of large numbers of samples, with simple equipment and at reduced cost for continuous monitoring of cancer cell viability, and, thus, should be accepted as a suitable alternative viability method.     

MiR-200c-3p inhibits cell migration and invasion of clear cell renal cell carcinoma via regulating SLC6A1

In this study, we investigated the mechanism of miR-200c-3p and SLC6A1 in regulating cell activity of clear cell renal cell carcinoma (CCRCC). The mRNA and miRNA expressions of tissue specimens were analyzed by CapitalBio Corporation (Beijing, China). The expression of SLC6A1 in CCRCC cells was examined through qRT-PCR and western blot. The migration and invasion ability of 786-O cells was testified by transwell assay after transfected. 786-O cell proliferation ability was detected by MTT assay. Dual luciferase reporter assay verified the association between SLC6A1 and miR-200c-3p. SLC6A1 was high expressed and miR-200c-3p was low expressed in CCRCC tissues and cells. Besides, lower SLC6A1 expression indicated longer survival time and higher survival rate. MiR-200c-3p could directly target at SLC6A1 and reduce its expression. MiR-200c-3p inhibited the proliferation, migration and invasion in 786-O cells by down-regulating SLC6A1 expression. The results suggested that the miR-200c-3p served as a suppressor for CCRCC via down-regulating SLC6A1.     

TIP60-miR-22 axis as a prognostic marker of breast cancer progression

MicroRNAs (miRNAs) are 22- to 24-nucleotide, small, non-coding RNAs that bind to the 3′UTR of target genes to control gene expression. Consequently, their dysregulation contributes to many diseases, including diabetes and cancer. miR-22 is up-regulated in numerous metastatic cancers and recent studies have suggested a role for miR-22 in promoting stemness and metastasis. TIP60 is a lysine acetyl-transferase reported to be down-regulated in cancer but the molecular mechanism of this reduction is still unclear. In this study, we identify TIP60 as a target of miR-22. We show a negative correlation in the expression of TIP60 and miR-22 in breast cancer patients, and show that low levels of TIP60 and high levels of miR-22 are associated with poor overall survival. Furthermore, pathway analysis using high miR-22/low TIP60 and low miR-22/high TIP60 breast cancer patient datasets suggests association of TIP60/miR-22 with epithelial-mesenchymal transition (EMT), a key alteration in progression of cancer cells. We show that blocking endogenous miR-22 can restore TIP60 levels, which in turn decreases the migration and invasion capacity of metastatic breast cancer cell line. These results provide mechanistic insight into TIP60 regulation and evidence for the utility of the combination of TIP60 and miR-22 as prognostic indicator of breast cancer progression.     

MiR-486-3p targeting ECM1 represses cell proliferation and metastasis in cervical cancer

MicroRNAs (miRNAs) regulate gene expression and are involved in cervical cancer. But the molecular mechanism is still unclear. Here, miRNA profile of cervical cancer was performed and demonstrated that miR-486-3p decreased in specimens of cervical cancer patients. In addition, our clinical data show that decreased miR-486-3p was associated with metastasis in cervical cancer patients. ECM1 was predicted and velified as a target gene of miR-486-3p. Overexpression of miR-486-3p inhibited cell growth and metastasis by targeting ECM1. In a conclusion, these findings suggest that miR-486-3p is a tumor suppressor miRNA and induction of miR-486-3p is a potential strategy to inhibit cervical cancer progression.     

5-AZA抑制肝癌细胞BEL7402代谢方式转换

目的研究去甲基化药物5-AZA对miR-34a和miR-34c在肝癌细胞中表达的影响及其抑制肝癌细胞糖酵解的分子机制。方法用real-time PCR法检测肝癌细胞系中miR-34a/c的表达以及糖酵解途径关键酶的表达;在肝癌细胞中过表达miR-34a、miR-34c或用5-AZA处理肝癌细胞,用乳酸检测试剂盒、葡萄糖检测试剂盒分析miR-34a/c及5-AZA对肝癌细胞BEL7402糖酵解的影响;设计拯救实验研究5-AZA、miR-34a/c与肝癌细胞糖酵解调控的关系。结果 5-AZA可以诱导BEL7402肝癌细胞内源性miR-34a、miR-34c的表达上调(P0.01);过表达miR-34a/c可以抑制糖酵解关键酶LDH-A的表达(P0.05);LDH-A是miR-34a/c的潜在靶基因;敲低miR-34a/c的表达可以降低5-AZA对肝癌细胞BEL7302糖酵解途径的抑制作用。结论 5-AZA通过诱导miR-34a/c表达上调,发挥抑制肝癌细胞代谢方式转换的功能。     

MicroRNA-424在肿瘤发生中的作用和机制研究进展

MiR-424是miR-16家族成员。近年研究表明miR-424与肿瘤的发生、发展及治疗预后密切相关。本文对miR-424在乳腺癌、宫颈癌、肺癌、肝癌及结直肠癌等多种肿瘤及白血病中的表达变化、作用及机制进行综述。研究发现miR-424的表达受多种因素的影响，miR-424可作为肿瘤诊断、分期、预后的生物标记物，可用于明确肿瘤范围，也可作为肿瘤的治疗靶物。     

Effects of miR-200a and FH535 combined with taxol on proliferation and invasion of gastric cancer

Gastric cancer is one of the most common cancers in the world; taxol displayed modest efficacy as first-line chemotherapy for gastric cancer, conversely, it has limitations used alone. β-catenin is a multifunctional oncogenic protein and the elevation in expression and activity of β-catenin has been implicated in many cancers. Therefore, we assume that the inhibition of β-catenin can enhanced the efficacy of taxol. The purpose of this study was to investigate the inhibitory effect of miR-200a mimics, FH535 combined with taxol on proliferation and invasion of human gastric cancer cell lines SGC-7901 and BGC-823. In the current study, we identified that the combination of FH535 and miR-200a with taxol had potent growth-inhibitory and pro-apoptotic effects. Further, similar results were also observed in vivo, intratumoral injection of FH535, taxol and miR-200a mimics which also delayed tumor growth in nude mice harboring subcutaneous SGC-7901 xenografts. Collectively, miR-200a and FH535 can enhance the inhibitory effect of taxol on cell proliferation and moderate the invasion of human gastric cancer.     

LncRNA Linc00152靶向miR-193a-3p对胃癌细胞增殖、侵袭和迁移的影响

目的 探讨LncRNA Linc00152靶向调控miR-193a-3p对胃癌细胞增殖、侵袭和迁移的影响。方法 采用qRT-PCR法检测正常胃黏膜细胞GES-1及4种胃癌细胞(MGC803、BGC803、SGC7901、AGS)中Linc00152的表达水平。MGC-803细胞分为pcDNA3.1-GFP-Linc00152组、pcDNA3.1-GFP组;AGS细胞分为si-Linc00152组、si-NC组,分别转染相应的Linc00152过表达和抑制载体。qRT-PCR法检测Linc00152、细胞周期素D1(cyclin D1,CCND1)和miR-193a-3p基因的表达水平,CCK-8检测细胞的活性,细胞克隆试验检测细胞的克隆能力,Annexin VFITC/PI染色后采用流式细胞术检测转染细胞凋亡率,Transwell法检测细胞侵袭,荧光素酶报告试验检测Linc00152和miR-193a-3p靶向关系,Western blotting检测Bcl-2、Bax、CCND1、E-钙黏蛋白(E-cadherin)和波形蛋白(Vimentin)的蛋白表达水平。并将si-Linc00152组、si-NC组细胞接种裸鼠,观察裸鼠体质量和一般状态,处死后称重记录肿瘤体积、质量,qRT-PCR法测定肿瘤组织Linc00152、CCND1和miR-193a-3p的表达水平。结果 与GES-1细胞相比,胃癌细胞MGC803、BGC803、SGC7901、AGS中Linc00152的表达升高,其中Linc00152在胃癌MGC803细胞中表达较低,而在胃癌AGS细胞中的表达较高,选用胃癌MGC803、AGS细胞进行后续试验。与pcDNA3.1-GFP组相比,pcDNA3.1-GFP-Linc00152组细胞的活性显著升高,克隆能力增强,细胞凋亡减少,侵袭能力显著上升,CCND1、Bcl-2、波形蛋白的表达显著升高,miR-193a-3p和E-钙黏蛋白的表达显著降低。双荧光素酶报告基因系统检测结果显示,Linc00152可直接调控miR-193a-3p的转录活性,且Linc00152可显著上调MGC-803细胞CCND1蛋白表达。与si-NC组相比,si-Linc00152组的裸鼠体内的异种移植瘤体积和体质量显著下降,CCND1和Linc00152的表达显著下降,miR-193a-3p的表达显著上升。结论 Linc00152可靶向作用于miR-193a-3p促进胃癌细胞增殖、侵袭和迁移,其作用机制与细胞周期、上皮细胞间充质转化以及细胞凋亡有关。     

miR-16在金黄色葡萄球菌脓毒症中的表达及意义探讨

目的探讨miR-16表达水平与金黄色葡萄球菌脓毒症严重程度的关联性,进一步探讨其潜在的临床意义。方法收集金黄色葡萄球菌脓毒症血液标本共计32例,脓毒性休克、严重脓毒症和一般脓毒症各8例,不同年龄段的健康对照24例;另外,收集革兰氏阴性菌脓毒症血液标本8例。用Trizol液裂解全血后提取microRNA,采用荧光定量PCR测定miR-16在不同组的表达水平(2-△△Ct法),用SPSS 13.0软件分析各组之间的统计学差异。采用SPSS 13.0软件将miR-16定量值与相应CRP和PCT值进行相关性分析。结果 miR-16表达水平与金黄色葡萄球菌脓毒症严重程度呈明显的负关联,各实验组与对照组相比均有统计学差异(P0.001),实验组之间也有统计学差异(P0.01)。miR-16表达水平与CRP和PCT值呈负相关性,相关系数分别为-0.561和-0.769。结论 miR-16表达与金黄色葡萄球菌脓毒症有明显的负关联性,提示其可能作为该菌脓毒症严重程度的标记物。     

Phoenixin-14 regulates proliferation and apoptosis of vascular smooth muscle cells by modulation of KCNQ1OT1/miR-183-3p/CTNNB1 axis

Phoenixin-14 has been reported to be implicated in the process of blood glucose metabolism, reproduction, lipid deposition and cardioprotection. However, the role of phoenixin-14 in vascular smooth muscle cells (VSMCs) remains unkown. In this study, we focused on the effects of phoenixin-14 on VSMCs under oxidized low-density lipoprotein (ox-LDL) treatment. The experimental results demonstrated that phoenixin-14 inhibited mRNA level and nuclear translocation of β-catenin. Functionally, phoenixin-14 inhibited cell proliferation and facilitated apoptosis of VSMCs under ox-LDL stimulation, and CTNNB1 overexpression reversed these effects. Mechanistically, KCNQ1OT1 interacted with miR-183-3p to upregulate CTNNB1 in VSMCs. Furthermore, CTNNB1 expression was negatively correlated with miR-183-3p but positively associated with KCNQ1OT1. Rescue assays indicated that KCNQ1OT1 overexpression or Lithium chloride (LiCl) treatment reversed the effects of phoenixin-14 on proliferation and apoptosis of ox-LDL-stimulated VSMCs. In summary, phoenixin-14 regulates proliferation and apoptosis of ox-LDL-treated VSMCs by regulating the KCNQ1OT1/miR-183-3p/CTNNB1 axis.