Hsa-miR-145对人三阴性乳腺癌细胞侵袭和迁移的影响

目的 探讨Hsa-miR-145对人三阴性乳腺癌细胞增殖、侵袭及迁移的影响,并初步分析其影响三阴性乳腺癌细胞增殖、侵袭及迁移的可能机制.方法 运用脂质体介导的转染方法将miR-145阻遏物(miR-145 inhibitors)转染人三阴性乳腺癌细胞株MDA-MB-231,以inhibitor negative control(inhibitor NC)作为阴性对照,通过MTT法和Transwell侵袭实验检测细胞的增殖能力和侵袭力;采用Transwell迁移实验及划痕试验检测细胞的迁移能力;利用生物信息学方法预测miR-145的靶基因,并对其靶基因进行基因功能分析.结果 (1)miR-145 inhibitors组细胞增殖活性明显高于inhibitor NC组(P<0.05);(2)划痕后miR-145 inhibitors组细胞迁移能力比inhibitor NC组明显增强(P<0.05);(3)Transwell侵袭及迁移实验均显示转染miR-145 inhibitors后,MDA-MB-231细胞的侵袭及迁移能力明显增强(P<0.01,P<0.05);(4)生物信息学方法预测miR-145的靶基因中,部分发挥了促进细胞增殖、侵袭及迁移的生物学功能.结论 (1)miR-145对人三阴性乳腺癌细胞的增殖、侵袭及迁移能力可能存在负性调控作用;(2)miR-145可能通过多种靶基因发挥其对肿瘤的调控作用.     

miR-601 is a prognostic marker and suppresses cell growth and invasion by targeting PTP4A1 in breast cancer

MicroRNAs (miRNA) play important roles in the initiation and progression of breast cancer. Here, we investigated the role of miR-601 in breast cancer and found that its expression was significantly down-regulated in breast cancer tissues compared with matched adjacent non-cancerous breast tissues. Moreover, we found that down-regulation of miR-601 was closely associated with distant metastasis and poor distant metastasis-free survival in breast cancer. In addition, miR-601 levels were inversely correlated with metastatic potential of human breast cancer cell lines. Further experiments showed that ectopic overexpression of miR-601 suppressed breast cancer cell proliferation, migration and invasion, whereas miR-601 knockdown promoted breast cancer cell proliferation, migration and invasion. Furthermore, protein tyrosine phosphatase type IVA 1 (PTP4A1) was identified as a direct target of miR-601. Overexpression of miR-601 repressed PTP4A1 mRNA and protein expression. Conversely, inhibition of miR-601 increased PTP4A1 mRNA and protein expression. Taken together, our data suggest that miR-601 inhibits growth and invasion of breast cancer cells by targeting PTP4A1 and that miR-601 is a potential biomarker for prognosis and therapeutic target in breast cancer.     

miR-425 inhibits melanoma metastasis through repression of PI3K-Akt pathway by targeting IGF-1

miR-425 is a potential tumor suppressor in cancer, but its role in melanoma is still unknown. We aim to investigate miR-425 expression in melanoma tissues and cell lines. Next, cell proliferation, cell cycle, apoptosis and metastasis will be studied using lentivirus-mediated gain-of-function studies. The predicted results are stable miR-425 inhibits cell proliferation and metastasis and induced cell apoptosis. It is predicted that IGF-1 is a potential target gene of miR-495 by bioinformatics analysis. Then luciferase assay analysis identifies IGF-1 as a new direct target gene of miR-425 and miR-425 inhibits melanoma cancer progression via IGF-1. Collectively, our findings suggested that miR-425 may function as a tumor suppressor in melanoma by targeting IGF-1.     

MicroRNA-744 inhibited cervical cancer growth and progression through apoptosis induction by regulating Bcl-2

Growing evidence suggests that microRNA plays an essential role in the development and metastasis of many tumor progressions, including cervical cancer. Aberrant miR-744 expression has been indicated in many growth of tumor, the mechanism of miR-744 inhibits both the proliferation and metastatic ability for cervical cancer remains unclear. Accumulating evidences reported that Bcl-2 signal pathway plays an important role in the cellular process, such as apoptosis, cell growth and proliferation. The goal of this study was to identify miR-744 that could inhibit the growth, migration, invasion, proliferation and metastasis of gastric cancer through targeting Bcl-2 expression. Real-time PCR (RT-qPCR) was used to quantify miR-744 expression in vitro and vivo experiments. The biological functions of miR-744 were determined via cell proliferation. Our study indicated that miR-744 targeted on Bcl-2, which leads to the inactivation of apoptosis signaling and the cell proliferation of cervical cancer cells, ameliorating cervical cancer growth and progression. In addition, both up-regulation of miR-744 and down-regulation of Bcl-2 could stimulate Caspase-3 expression, promoting apoptosis of cervical cancer cells. Therefore, our research revealed the mechanistic links between miR-744 and Bcl-2 in the pathogenesis of cervical cancer through modulation of Caspase-3, leading to the inhibition of cervical cancer cell growth. And targeting miR-744 could be served as a novel strategy for future cervical cancer therapy clinically.     

miR-145通过调控MMP-2、MMP-9的表达对卵巢癌细胞调亡、增殖、迁移的影响

目的研究微小RNA-145(miR-145)通过调控基质金属蛋白酶(MMP)-2、MMP-9的表达对卵巢癌细胞调亡、增殖、迁移的影响。方法将卵巢癌细胞株随机分为3组,其中对照组为单纯卵巢癌细胞株,不做其他处理,进行正常培养;miR-145组为含miR-145质粒的慢病毒转染的卵巢癌细胞株;NC组为含NC质粒的慢病毒转染的卵巢癌细胞株。检测3组卵巢癌细胞的凋亡、增殖、迁移情况。检测3组MMP-2、MMP-9蛋白和mRNA表达水平。结果与对照组、NC组比较,miR-145组卵巢癌细胞凋亡率明显升高,卵巢癌细胞增殖数、迁移数明显下降,差异均有统计学意义(P<0.05);对照组与NC组卵巢癌细胞凋亡率、增殖数、迁移数差异无统计学意义(P>0.05)。miR-145组MMP-2、MMP-9蛋白和mRNA表达水平均低于NC组、对照组,差异均有统计学意义(P<0.05)。对照组与NC组MMP-2、MMP-9蛋白和mRNA表达水平差异无统计学意义(P>0.05)。结论miR-145可抑制卵巢癌细胞的增殖、迁移,促进调亡,其作用机制可能与miR-145能降低MMP-2、MMP-9的表达有关。     

miR-421靶向PDCD4促进前列腺癌DU145细胞增殖的实验研究

目的研究MicroRNA-421(miR-421)促进前列腺癌DU145细胞增殖的作用及潜在的分子机制。方法培养前列腺癌DU145细胞,分为对照组、miR-阴性对照(NC)组、miR-421组、miR-421+pcDNA组、miR-421+pcDNA-细胞程序性死亡基因4(PDCD4),miR-NC组转染miR-NC、miR-421组转染miR-421、miR-421+pcDNA组转染miR-421及pcDNA质粒、miR-421+pcDNA-PDCD4组转染miR-421及pcDNA-PDCD4质粒,MTS法测定细胞增殖活力,荧光定量PCR测定PDCD4的mRNA表达水平,western blot测定PDCD4的蛋白表达水平,双荧光素酶报告基因实验验证miR-421与PDCD4的靶向结合。结果与对照组及miR-NC组比较,miR-421组的增殖活力增加,PDCD4的表达水平及野生型PDCD4双荧光素酶报告基因的荧光活力均降低(P<0.05);与miR-421+pcDNA组比较,miR-421+pcDNAPDCD4组的增殖活力降低,PDCD4的表达水平增加(P<0.05)。结论 miR-421对前列腺癌DU145细胞的增殖具有促进作用,靶向抑制PDCD4是miR-421发挥这一作用的潜在分子机制。     

MiR-522 contributes to cell proliferation of human glioblastoma cells by suppressing PHLPP1 expression

Previous studies have shown that microRNAs play essential roles in cancer growth and progression. Although a number of microRNAs were differentially expressed in glioblastoma (GBM). In this study, we evaluated the miR-522s role in cell proliferation in GBM. Expression of miR-522 is markedly upregulated in GBM tissues and GBM cells compared with the matched non-tumor adjacent brain tissues (TAT) and normal human astrocytes (NHAs). In functional assays, miR-522 promoted GBM cell proliferation, which could be reversed by inhibitor of miR-522. We further identified PH domain leucine-rich repeats protein phosphatase-1 (PHLPP1) as a putative target of miR-522, which is likely a main contributor to the promotion of tumor cell growth observed in our assays. Our results demonstrated that miR-522 promoted tumor cell proliferation and hence may represent a novel therapeutically relevant cellular target to treatment of GBM patients.     

Caprin-1 is a novel microRNA-223 target for regulating the proliferation and invasion of human breast cancer cells

MicroRNAs (miRNAs) are 21–22 nucleotides regulatory small non-coding RNAs that inhibit gene expression by binding to complementary sequences especially the 3’ untranslated region (3’UTR) of mRNA. One miRNA can target many messenger RNAs, leading to a complex metabolic network. Previous studies have shown that miRNA-223 regulates migration and invasion of tumor cells and targets cytoplasmic activation/proliferation-associated protein-1 (Caprin-1). In the present study, we detected the expression of miRNA-223 and Caprin-1 in MCF-7, T-47D and MDA-MB-231 cancer cell lines, and MCF-10A normal breast cell line, and analyzed the role of miRNA-223 in Caprin-1-induced proliferation and invasion of human breast cancer cells. We found that miRNA-223 expression levels are significantly lower in MCF-7, T-47D and MDA-MB-231 cancer cells than in MCF-10A normal breast cells, while Caprin-1 expression is higher in cancer cells than in normal breast cells. The most malignant cancer cell line MDA-MB-231 has the lowest expression of miR-223, but the highest expression of Caprin-1. Further, we found that miR-223 targets the 3’UTR of Caprin-1 miRNA and down-regulates the expression of Caprin-1. We also found that over-expression of Caprin-1 can promote the proliferation and the invasion of breast cancer cells, but miRNA-223 can inhibit the proliferation and the invasion. miRNA-223-induced inhibition can be reversed by ectopic over-expression of Caprin-1. These findings suggest that miR-223 may suppress the proliferation and invasion of cancer cells by directly targeting Caprin-1. Our study also indicates that expression levels of miR-223 and Caprin-1 can be used to predict the state of cancer in breast cancer patient.     

MicroR-760 suppresses cancer stem cell subpopulation and breast cancer cell proliferation and metastasis: By down-regulating NANOG

Background and objectiveEmerging evidences suggest that cancer stem cells are responsible for tumor aggressive, metastasis and therapeutic resistance. To data, the mechanism underlying breast cancer stem cell (BCSC) population within tumor metastasis remains to be fully elucidated. The current study was to investigate the potential role of microRNA-760 (miR-760) and its associated target gene in population and metastasis of BCSC.MethodsCharacteristic BCSCs surface markers (CD44⁺/CD24^−/low) were determined by flow cytometry in breast cancer MCF-7 and BT-549 cells. Quantitative RT-PCR was used to evaluate miR-760 and NANOG mRNA expression. Expression of NANOG protein was determined using western blot. Cell proliferation was determined by MTT assay. The model of breast cancer cell xenograft was used to evaluate the effect of miR-760 on tumor growth.ResultsBT-549 cell has substantially more CD44⁺/CD24^−/low subpopulation than MCF-7 cell. Moreover, BT-549 cell expressed lower level of miR-760 and higher level of NANOG than MCF-7cell. By result from cellular miR-760 modulation, we found that miR-760 overexpression suppressed CD44⁺/CD24^−/low population as well as inhibited cell proliferation and migration of BT-549. On the contrary, knockdown of miR-760 promoted CD44⁺/CD24^−/low population and migration of MCF-7 cells. By luciferase reporter assay, miR-760 was proved to be functional associated with NANOG via regulating its expression. This functional interaction was showed to be involved in controlling proliferation and migration of MCF-7 and BT-549 cell.ConclusionThese data suggest that the target of miR-760/NANOG axis may represent a new therapeutic approach to suppress breast cancer stem cell subpopulation thereby prevent cancer metastasis.     

miR-1184 regulates the proliferation and apoptosis of colon cancer cells via targeting CSNK2A1

MicroRNA (miRNA) exerts an important part in colon cancer cell proliferation and apoptosis. Meanwhile, the dysregulation of some miRNAs is detected in colon cancer cells. However, it remains unclear about the underlying mechanism of their effects on tumor pathogenesis. The current work aimed to examine the miR-1184 effect on colon cancer cells. The differentially expressed miRNAs (DEMs), including miR-9-3p, miR-1184, miR-492, miR-92a-1-5p and miR-20a-3p, were obtained from the GSE115108 and GSE132619 data sets using the ‘GEO2R’ online tool. Based on the findings, miR-1184 was significantly down-regulated within colon cancer cells and tissues. Moreover, the experimental results of CCK8, flow cytometry, colony formation and Western blotting assays showed that, miR-1184 over-expression suppressed colon cancer cell proliferation through inhibiting Ki67 expression and promoted their apoptosis through up-regulating cleaved caspase-3 and down-regulating Bcl-2 expression. By contrast, miR-1184 inhibition exerted the opposite effects. A total of 110 target genes of miR-1184 were predicted using the TargetScan and miRTarBase databases, which were then used to construct the protein-protein interaction (PPI) network based on the DAVID and STRING websites and to perform GO and KEGG pathway enrichment analyses. The MCODE plug-in of cytoscape was utilized to verify that CSNK2A1 was the target gene and key gene in significant modules. MiR-1184 directly targets CSNK2A1 via using RNA immunoprecipitation assay and luciferase reporter gene assay. According to the results, CSNK2A1 over-expression reversed the functions of miR‐1184 over-expression in suppressing colon cancer cell proliferation and enhancing their apoptosis. In conclusion, over-expression of miR-1184 inhibits colon cancer cell proliferation but promotes their apoptosis through down-regulating CSNK2A1 expression.     

miR-1301 promotes prostate cancer proliferation through directly targeting PPP2R2C

Prostate cancer is the leading cause of cancer deaths among men in the worldwide, it’s important to find new prognostic factors and therapeutic targets. microRNAs play critical roles in the development and progression of prostate cancer. Here we revealed miR-1301 promoted prostate cancer progression. miR-1301 was upregulated in prostate cancer tissues and cells, overexpression of miR-1301 promoted anchorage-dependent and -independent growth using MTT analysis, colony formation analysis and soft agar growth analysis, whereas knockdown of miR-1301 suppressed anchorage-dependent and -independent growth. We also found overexpression of miR-1301 inhibited p27 expression and promoted Cyclin D1 expression, whereas knockdown of miR-1301 reduced this effect, suggesting miR-1301 promoted the G1/S transition. These results suggested miR-1301 promoted cell proliferation of prostate cancer. microRNAs can inhibit target mRNA translation or/and induce mRNA degradation, we found tumor suppresser PPP2R2C was the target of miR-1301, simultaneous downregualtion of PPP2R2C and miR-1301 promoted anchorage-dependent and -independent growth. These findings suggested miR-1301 promoted prostate cancer proliferation by inhibiting PPP2R2C, and might a therapeutic target for prostate cancer.     

薄荷醇抑制前列腺癌DU145细胞的增殖和迁移

目的探讨薄荷醇对雄激素非依赖性前列腺癌DU145细胞增殖和迁移能力的影响。方法通过RT-PCR、免疫组织化学和Western blot方法检测TRPM8和TRPA1的表达;MTT和划痕试验检测薄荷醇对DU145细胞的增殖和迁移能力的影响;流式细胞术检测TRPM8对DU145细胞周期和凋亡的影响。结果 RT-PCR、免疫组织化学和Western blot提示TRPM8在DU145细胞中高表达而TRPA1在DU145细胞中不表达;薄荷醇能诱导细胞周期阻滞于G0/G1期,与未经薄荷醇处理的细胞相比,经100μmol/L薄荷醇处理的细胞培养24、48和72h后,G0/G1期细胞显著增加（49.12%±1.92%vs61.71%±2.70%、77.65%±1.63%、71.81%±2.46%,P〈0.05,P〈0.01）,进而抑制细胞增殖（P〈0.05）,并抑制细胞迁移（P〈0.05）,流式细胞术检测显示薄荷醇并不引起细胞凋亡。结论 TRPM8可能成为前列腺癌治疗的一个新靶点,对于高表达TRPM8的雄激素非依赖性前列腺癌针对TRPM8通道的药物治疗可能比TRPM8基因治疗更为实用,因此,薄荷醇作为一个潜在的抗肿瘤药物也拥有很大的发展前景。     

miR-99a promotes proliferation targeting FGFR3 in human epithelial ovarian cancer cells

MiRNAs have been reported as important regulators in normal physiological processes, human cancer, and even their roles as therapeutic targets have been proposed. In epithelial ovarian cancer (EOC), the expression of miRNAs is reported to remarkably deregulate, showing that miRNAs are involved in the initiation and progression of this disease. In this study, we found that miR-99a was obviously decreased in EOC tissues, serums and cell lines SKOV-3. Importantly, fibroblast growth factor receptor 3 (FGFR₃), predicted to be one target gene of miR-99a using computational algorithms, was higher in expression in EOC cells. Subsequently, FGFR₃ was proved to be direct target of miR-99a by dual luciferase assay. Furthermore, overexpression of miR-99a dramatically suppressed expression level of FGFR₃ at both mRNA and protein levels, proving FGFR₃ to be inversely correlated with miR-99a. Finally, overexpression of miR-99a could significantly inhibit EOC cell proliferation in vitro by decreasing the expression of FGFR₃ which also reduced the EOC cell growth after siRNA knockdown. Conclusively, miR-99a expression was remarkably downregulated in serums, tissues and cell and suppresses EOC cell proliferation by targeting FGFR₃, suggesting miR-99a as a prospective prognosis marker and potential tumor suppressor for EOC therapeutics.     

MiR-433 inhibits retinoblastoma malignancy by suppressing Notch1 and PAX6 expression

Retinoblastoma (RB) is the most frequent primary intraocular cancer. It has been demonstrated by previous studies that retinoblastoma is initiated primarily by the inactivation of the retinoblastoma Rb1 gene in retinal cells. However, additional genetic alterations than Rb1 mutation could play important roles in the process of transforming benign retinal cells into retinoblastoma tumor cells. In this study, we identified that microRNA miR-433 is one of such genetic factors. We found that the expression levels of miR-433 were downregulated in RB tissues. We also determined that miR-433 negatively regulated RB cell proliferation, migration and invasion, and induced cell cycle arrest and apoptosis of RB cells. We used bioinformatics method to predict and confirmed that Notch1 and PAX6 were miR-433 target genes in RB cells. Importantly, we demonstrated that restoration of Notch1 and PAX6 expression partially rescued the inhibition of cell proliferation and metastasis induced by miR-433 overexpression, suggesting that miR-433 regulates RB cell proliferation and metastasis through suppressing the expression of Notch1 and PAX6.     

microRNA-21参与调控膀胱癌细胞增殖及多柔比星敏感性的研究

目的膀胱移行细胞癌是一种常见的恶性肿瘤,且复发率很高。由于microRNA-21(miR-21)在多种类型肿瘤中参与肿瘤发生及耐药,故通过此次研究探索其在膀胱移行细胞癌中的功能。方法肿瘤组织及癌旁正常组织中的miR-21及目的蛋白PTEN表达水平分别采用实时qRT-PCR及Western blot方法进行测量。采用MTT法评估miR-21对于癌细胞增殖以及多柔比星敏感性的影响。采用流式细胞术探究T24细胞系中多柔比星引起的细胞凋亡。结果与癌旁正常膀胱组织相比,miR-21在膀胱肿瘤组织中的表达水平显著上调,而PTEN蛋白表达低水平。活体内研究发现miR-21与PTEN表达呈负相关。miR-21过表达可显著促进T24细胞系的细胞增殖与多柔比星敏感性。结论 miR-21可能是膀胱移行细胞癌的致癌因素,有望成为膀胱移行细胞癌的治疗新靶点。     

Over-expression of microRNA-940 promotes cell proliferation by targeting GSK3β and sFRP1 in human pancreatic carcinoma

Increasing study reports that Wnt/β-catenin signaling pathway plays an essential role in numerous cancers growth, progression and metastasis. Aberrant miR-940 expression has been studied in gastric and breast cancer. However, the molecular mechanism of miR-940 enhancing proliferation and metastatic ability in human pancreatic carcinoma is far from to know. Real-time PCR was used to quantify miR-940 expression. Luciferase reporter assays here were performed to verify the activity of Wnt/β-catenin signaling pathway and targeting gene relationships, and immunofluorescence assay was applied to observe β-catenin expressed intensity. Bioinformatics analysis together with in vivo and vitro functional analysis indicated the potential targeting genes of miR-940. Specimens from 15 pairs of patients with human pancreatic carcinoma were involoved to confirm the relationship between miR-940 expression and the GSK3β/sFRP1 through real-time PCR and western blot assays. Bioinformatics combined with cell luciferase function researches determined the possible regulation of miR-940 on the 3′-UTR of the GSK3β and sFRP1 genes, resulting in the Wnt/β-catenin signaling activation. Further, miR-940 knockdown significantly recovered GSK3β and sFRP1 expression and relieved Wnt/β-catenin-mediated cell invasion, migration, metastasis and proliferation. The ectopic up-regulation of miR-940 significantly suppressed GSK3β/sFRP1 expression and promoted pancreatic carcinoma proliferation and invasion. Our study suggested mechanistic relationship between miR-940 and Wnt/β-catenin in the development and progression of pancreatic carcinoma through regulation of GSK3β and sFRP1.     

LINC00346 regulates glycolysis by modulation of glucose transporter 1 in breast cancer cells

Most cancer cells preferentially metabolize glucose by glycolysis rather than oxidative phosphorylation to proliferate efficiently. LncRNAs have been proposed as crucial regulators in pathophysiological processes including cell growth, apoptosis and glucose metabolism. However, little is known regarding the specific role of LINC00346 in regulating glucose metabolism in breast cancer. LINC00346 and miR-148a/b expression in breast cancer cells was detected by qRT-PCR. The relationships between LINC00346, glucose transporter 1 (GLUT1) and miR-148a/b in breast cancer cells were explored by luciferase reporter assay. Cell proliferation and apoptosis were evaluated by CCK-8 and flow cytometry analysis, respectively. Glycolysis was detected by measuring the glucose uptake and lactate production. Results showed that LINC00346 was over-expressed while miR-148a/b was low-expressed in breast cancer cells. miR-148a/b were direct targets of LINC00346 in breast cancer cells. LINC00346 knockdown inhibited cell proliferation and glycolysis, and induced apoptosis by upregulating miR-148a/b in breast cancer cells. Furthermore, we found that LINC00346 knockdown repressed GLUT1 expression in breast cancer cells by upregulating miR-148a/b. In conclusion, LINC00346 knockdown suppressed breast cancer cell glycolysis by upregulating miR-148a/b and repressing GLUT1 expression.