Tumor-associated macrophages promote prostate cancer progression via exosome-mediated miR-95 transfer

Tumor-associated macrophages (TAMs) are vital constituents in mediating cell-to-cell communication within the tumor microenvironment. However, the molecular mechanisms underlying the interplay between TAMs and tumor cells that guide cell fate are largely undetermined. Extracellular vesicles, also known as exosomes, which are derived from TAMs, are the components exerting regulatory effects. Thus, understanding the underlying mechanism of “onco-vesicles” is of crucial importance for prostate cancer (PCa) therapy. In this study, we analyzed micro RNA sequences in exosomes released by THP-1 and M2 macrophages and found a significant increase in miR-95 levels in TAM-derived exosomes, demonstrating the direct uptake of miR-95 by recipient PCa cells. In vitro and in vivo loss-of-function assays suggested that miR-95 could function as a tumor promoter by directly binding to its downstream target gene, JunB, to promote PCa cell proliferation, invasion, and epithelial–mesenchymal transition. The clinical data analyses further revealed that higher miR-95 expression results in worse clinicopathological features. Collectively, our results demonstrated that TAM-mediated PCa progression is partially attributed to the aberrant expression of miR-95 in TAM-derived exosomes, and the miR-95/JunB axis provides the groundwork for research on TAMs to further develop more-personalized therapeutic approaches for patients with PCa.     

过表达微小RNA miR-125b抑制肝癌细胞上皮-间质转换及促进细胞凋亡

微小RNA-125b（miR-125b）在许多恶性肿瘤的增殖、分化和凋亡等过程中具有很重要的作用,但miR-125b是否涉及肝癌的上皮 间质转换过程（EMT）还有待进一步研究。本研究通过构建过表达miR-125b的肝癌稳转细胞株,初步检测miR-125b对于肝癌的EMT过程和相关的TGF-β信号通路的影响,以及对于肝癌细胞凋亡的影响。以慢病毒载体pHRS-1cla EGFP 构建过表达miR-125b的载体质粒(pHRS-1cla-miR125b-CMV-EGFP),并对上述载体进行NheⅠ、XbaⅠ双酶切和测序鉴定,鉴定正确后,在293T细胞中进行慢病毒包装,浓缩病毒后,对MHCC97-H进行慢病毒感染并采用流式分选GFP阳性的细胞。实时定量PCR检测表明肝癌细胞稳转株MHCC97-H-PHRS-miR-125b-EGFP的miR-125b表达量是空载体转染组的6倍。Western印迹检测发现,与空载体对照组相比,MHCC97-H-PHRS-miR-125b-EGFP细胞中间质细胞标志α-SMA表达显著下调,上皮细胞标志E-cadherin表达显著上调,同样的,用Western印迹检测也发现MHCC97-H-PHRS-miR-125b-EGFP细胞中TGF-β信号通路关键下游分子Smad2和Smad4的表达显著下调,细胞凋亡检测结果表明,与对照组相比,过表达miR-125b的稳转株凋亡率增加到19.66%,加入TGF-β1后,过表达miR-125b的稳转株凋亡率进一步增加到74.7%。同样的,在体内治疗实验中,我们采用商品化的体内核酸转染试剂,在皮下肿瘤组织中过表达miR-125b mimics,结果表明miR-125b的过表达与肿瘤组织的凋亡成正相关性（r=0.83463,P < 0.01）,且免疫组化结果也表明,miR-125b过表达后,E-cadherin表达显著上调,α-SMA及Smad2和Smad4的表达显著下调。上述结果表明,我们成功构建了过表达miR-125b的肝癌细胞稳转株,并成功建立了肿瘤组织中过表达miR-125b mimics的动物模型,在体内外均观察到过表达miR-125b后对肝癌细胞EMT过程的抑制作用和对细胞凋亡的促进作用。相关研究结果加深了我们对miR-125b在肝癌中抑制肝癌发展作用机制的理解,及其作为潜在的治疗肝癌的新靶点的重要性。     

miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation

Although various microRNAs regulate cell differentiation and proliferation, no miRNA has been reported so far to play an important role in the regulation of osteoblast differentiation. Here we describe the role of miR-125b in osteoblastic differentiation in mouse mesenchymal stem cells, ST2, by regulating cell proliferation. The expression of miR-125b was time-dependently increased in ST2 cells, and the increase in miR-125b expression was attenuated in osteoblastic-differentiated ST2 cells induced by BMP-4. The transfection of exogenous miR-125b inhibited proliferation of ST2 cells and caused inhibition of osteoblastic differentiation. In contrast, when the endogenous miR-125b was blocked by transfection of its antisense RNA molecule, alkaline phosphatase activity after BMP-4 treatment was elevated. These results strongly suggest that miR-125b is involved in osteoblastic differentiation through the regulation of cell proliferation.     

M2 macrophages contribute to cell proliferation and migration of breast cancer

Breast cancer is a kind of malignant tumor that severely threatens women's lives and health worldwide. Tumor-associated macrophages (TAMs) have been reported to mediate tumor progression, while the mechanism still needs further identification. In this study, we found that M2 macrophages promoted increased cell proliferation and migration as well as reduced expression of interferon regulatory factor 7 (IRF7) and increased the expression of miR-1587 in breast cancer cells. Overexpression of IRF7 or miR-1587 knockdown reversed M2 macrophage-induced cell proliferation and migration as well as tumor growth in vivo. Mechanistically, miR-1587 targeted the 3ʹ-untranslated region (3ʹ-UTR) of IRF7 mRNA to regulate its protein expression leading to tumor progression. Collectively, this study revealed that the miR-1587/IRF7 axis mediates M2 macrophage-induced breast cancer progression, and this sheds light on further clinical therapy for breast cancer by targeting TAMs as well as the miR-1587/IRF7 axis.     

Roles of microRNA-140 in stem cell-associated early stage breast cancer

An increasing body of evidence supports a stepwise model for progression of breast cancer from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). Due to the high level of DCIS heterogeneity, we cannot currently predict which patients are at highest risk for disease recurrence or progression. The mechanisms of progression are still largely unknown, however cancer stem cell populations in DCIS lesions may serve as malignant precursor cells intimately involved in progression. While genetic and epigenetic alterations found in DCIS are often shared by IDC, mRNA and miRNA expression profiles are significantly altered. Therapeutic targeting of cancer stem cell pathways and differentially expressed miRNA could have significant clinical benefit. As tumor grade increases, miRNA-140 is progressively downregulated. miR-140 plays an important tumor suppressive role in the Wnt, SOX2 and SOX9 stem cell regulator pathways. Downregulation of miR-140 removes inhibition of these pathways, leading to higher cancer stem cell populations and breast cancer progression. miR-140 downregulation is mediated through both an estrogen response element in the miR-140 promoter region and differential methylation of CpG islands. These mechanisms are novel targets for epigenetic therapy to activate tumor suppressor signaling via miR-140. Additionally, we briefly explored the emerging role of exosomes in mediating intercellular miR-140 signaling. The purpose of this review is to examine the cancer stem cell signaling pathways involved in breast cancer progression, and the role of dysregulation of miR-140 in regulating DCIS to IDC transition.     

Cancer-associated fibroblasts-derived exosomal miR-3656 promotes the development and progression of esophageal squamous cell carcinoma via the ACAP2/PI3K-AKT signaling pathway

Esophageal squamous cell carcinoma (ESCC) is one of the most common gastrointestinal tumors, accounting for almost half a million deaths per year. Cancer-associated fibroblasts (CAFs) are the major constituent of the tumor microenvironment (TME) and dramatically impact ESCC progression. Recent evidence suggests that exosomes derived from CAFs are able to transmit regulating signals and promote ESCC development. In this study, we compared different the component ratios of miRNAs in exosomes secreted by CAFs in tumors and with those from normal fibroblasts (NFs) in precancerous tissues. The mRNA level of hsa-miR-3656 was significantly upregulated in the former exosomes. Subsequently, by comparing tumor cell development in vitro and in vivo, we found that the proliferation, migration and invasion capabilities of ESCC cells were significantly improved when miR-3656 was present. Further target gene analysis confirmed ACAP2 was a target gene regulated by miR-3656 and exhibited a negative regulatory effect on tumor proliferation. Additionally, the downregulation of ACAP2 triggered by exosomal-derived miR-3656 further promotes the activation of the PI3K/AKT and β-catenin signaling pathways and ultimately improves the growth of ESCC cells both in vitro and in xenograft models. These results may represent a potential therapeutic target for ESCC and provide a new basis for clinical treatment plans.     

过表达微小RNA miR-125b对人胚胎干细胞增殖的影响

目的:通过在人胚胎干细胞(hESC)中有效转染微小RNA miR-125b的真核表达载体,研究过表达miR-125b对hESC增殖的影响。方法:将在无饲养层上培养至第3 d,克隆融合达70%的hESC用Accutase酶消化为单细胞,然后用LipofectAMINE2000对hESC单细胞转染pHRS-1cla-miR125b-CMV-EGFP载体及其对照pHRS-1cla-CMV-EGFP载体,通过实时定量PCR对转染后细胞中成熟miR-125b的表达进行检测;进一步进行细胞计数和克隆计数,对miR-125b表达上调的hESC的增殖情况进行分析。结果:实时定量PCR检测结果表明,细胞转染后72 h,miR-125b的表达上调1.45倍,说明hESC转染成功;克隆计数及细胞计数结果显示过表达miR-125b的hESC增殖受到明显抑制(P<001)。结论:转染miR-125b真核表达载体的hESC能够上调成熟miR-125b的表达,hESC中miR-125b的表达上调能明显抑制hESC的增殖。     

miR-125b在前列腺癌1E8细胞运动转移中的作用及其分子机制的初步探讨

目的 研究 miR-125b在前列腺癌高低转移潜能细胞中的表达差异及其对高转移细胞株1E8细胞的运动转移中的作用和可能的分子机制.方法 realtime PCR法检测前列腺癌高低转移潜能配对细胞系中 miR-125b的表达差异.通过划痕实验及transwell实验观察1E8细胞及转染 miR-125b 抑制剂及其阴性对照后该细胞运动转移能力的变化.结果 realtime PCR结果显示高转移潜能1E8细胞中miR-125b表达水明显高于低转移潜能2B4细胞;下调miR-125b会减弱1E8细胞的运动转移能力.结论 miR-125b可促进前列腺癌细胞的运动转移能力.     

miR-125b靶向MCL1抑制胃癌 MGC-803细胞增殖

探讨mi R-125b对胃癌MGC-803细胞增殖的影响及机制,为阐明胃癌发病的分子机制提供实验依据.采用q RT-PCR和原位杂交,检测mi R-125b在正常胃黏膜(NGM)和胃癌(GAC)组织中的表达.将mi R-125b导入胃癌MGC-803细胞,观察mi R-125b高表达对MGC-803细胞增殖的影响.利用Targetscan 6.2软件及荧光素酶报告基因检测,分析mi R-125b对MCL1基因的靶向性作用.构建MCL1干扰载体,观察干扰MCL1基因表达对MGC-803细胞增殖的影响.结果发现,mi R-125b在胃癌组织中低表达,其表达与胃癌的分化程度及患者预后呈正相关,与TNM分期、淋巴结转移呈负相关(P0.01).mi R-125b高表达后MGC-803细胞的增殖降低、凋亡率增加、裂解caspase-3与裂解PARP表达增加(P0.01);mi R-125b与MCL1基因的3′UTR(2 613~2 620)结合,抑制MCL1的m RNA及蛋白质表达(P0.01);沉默MCL1基因表达后MGC-803细胞的增殖降低、凋亡率增加、裂解caspase-3与裂解PARP表达增加(P0.01).从而得出结论,mi R-125b在胃癌组织中低表达,其表达与胃癌组织分化程度、TNM分期、淋巴结转移及患者预后密切相关;mi R-125b靶向抑制MCL1基因表达,活化caspase-3信号通路,抑制MGC-803细胞增殖.     

Downregulation of miR-33b promotes non-small cell lung cancer cell growth through reprogramming glucose metabolism miR-33b regulates non-small cell lung cancer cell growth

Glucose metabolism is a common target for cancer regulation and microRNAs (miRNAs) are important regulators of this process. Here we aim to investigate a tumor-suppressing miRNA, miR-33b, in regulating the glucose metabolism of non-small cell lung cancer (NSCLC). In our study, quantitative real-time polymerase chain reaction (qRT-PCR) showed that miR-33b was downregulated in NSCLC tissues and cell lines, which was correlated with increased cell proliferation and colony formation. Overexpression of miR-33b through miR-33b mimics transfection suppressed NSCLC proliferation, colony formation, and induced cell-cycle arrest and apoptosis. Meanwhile, miR-33b overexpression inhibited glucose metabolism in NSCLC cells. Luciferase reporter assay confirmed that miR-33b directly binds to the 3′-untranslated region of lactate dehydrogenase A (LDHA). qRT-PCR and Western blot analysis showed that miR-33b downregulated the expression of LDHA. Moreover, introducing LDHA mRNA into cells over-expressing miR-33b attenuated the inhibitory effect of miR-33b on the growth and glucose metabolism in NSCLC cells. Taken together, these results confirm that miR-33b is an anti-oncogenic miRNA, which inhibits NSCLC cell growth by targeting LDHA through reprogramming glucose metabolism.     

miR-29c targets TNFAIP3, inhibits cell proliferation and induces apoptosis in hepatitis B virus-related hepatocellular carcinoma

Recent studies have revealed that microRNA-29c (miR-29c) is involved in a variety of biological processes including carcinogenesis. Here, we report that miR-29c was significantly downregulated in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) cell lines as well as in clinical tissues compared with their corresponding controls. Tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a key regulator in inflammation and immunity, was found to be inversely correlated with miR-29c levels and was identified as a target of miR-29c. Overexpression of miR-29c in HepG2.2.15 cells effectively suppressed TNFAIP3 expression and HBV DNA replication as well as inhibited cell proliferation and induced apoptosis. We conclude that miR-29c may play an important role as a tumor suppressive microRNA in the development and progression of HBV-related HCC by targeting TNFAIP3. Thus miR-29c and TNFAIP3 represent key diagnostic markers and potential therapeutic targets for the prevention and treatment of HBV infection.     

The competing endogenous circular RNA ADAMTS14 suppressed hepatocellular carcinoma progression through regulating microRNA-572/regulator of calcineurin 1

Emerging evidence have discovered that circular RNAs (circRNAs) may serve as diagnostic or tumor promising biomarkers. This study aimed to investigate how circular RNA ADAMTS14 (circADAMTS14) regulates microRNA-572/ regulator of calcineurin 1(miR-572/ RCAN1) in hepatocellular carcinoma (HCC). The expression profiles of circRNA/microRNA (mRNA) between HCC tissues and paired adjacent tissues were analyzed via microarray analysis. The expressions of circADAMTS14, miR-572, and RCAN1 were measured by real-time polymerase chain reaction (PCR). The protein expression level of RCAN1 in HCC cells was detected by western blot. The viability and apoptosis levels of HCC cell lines were measured by the cell counting Kit-8 (CCK-8) assay and fluorescence-activated cell sorter. The invasiveness and migration of cells were detected based on the transwell and wound-healing assay, respectively. The dual-luciferase reporter assays were used to reveal circADAMTS14 and RCAN1 as a potential target of miR-572, which was predicted by TargetScan and miRBase. The effect of circADAMTS14 on HCC cells was demonstrated by tumor formation in nude mice in vivo. CircADAMTS14 and RCAN1 were lowly expressed in HCC clinical specimens and cell lines using microarrays and qRT-PCR, but miR-572 inversely. Our study further verified the direct interaction between circADAMTS14 and RCAN1 with miR-572 via the dual-luciferase reporter gene assay. Overexpressed circADAMTS14 and RCAN1 induced apoptosis of HCC cells and inhibited cell proliferation and invasion. But overexpressed miR-572 could decrease apoptosis of HCC cells and promote proliferation and invasion. In vivo, circADAMTS14 inhibited the tumor growth, correlated positively with the protein expression levels of RCAN1. Our results demonstrated that circADAMTS14 might suppress HCC progression through regulating miR-572/ RCAN1 as the competing endogenous RNA.     

MicroRNA-33b regulates hepatocellular carcinoma cell proliferation,apoptosis, and mobility via targeting Fli-1-mediated Notch1 pathway

MicroRNAs (miRNAs) have been confirmed to play pivotal roles in hepatocellular carcinoma (HCC) carcinogenesis. However, the underlying function of microRNA-33b (miR-33b) in HCC remains unclear. Here, we found that miR-33b level was significantly reduced in both HCC tissues and tumor cell lines. Further, luciferase reporter assay and western blot analysis confirmed that Friend leukemia virus integration 1 (Fli-1) was a direct target of miR-33b. Overexpression of miR-33b dramatically suppressed HCC tumor cell proliferation and cell mobility, but facilitated tumor cell apoptosis in vitro. Besides, restoration of Fli-1 partially attenuated miR-33b-mediated inhibition of cell growth and metastasis via activating Notch1 signaling and its downstream effectors. Our findings demonstrate the important role of miR-33b/Fli-1 axis in HCC progression and provide novel therapeutic candidates for HCC clinical treatment.     

MiR-125b regulates the proliferation and metastasis of triple negative breast cancer cells via the Wnt/β-catenin pathway and EMT

Background/aim: MiR-125b plays an important role in breast cancer. The current study was to explore the expression and function of miR-125b in triple negative breast cancer cells. Materials and methods: The expression of miR-125b in human TNBC samples and cell lines were examined by qRT-PCR. MTT, scratch assays and transwell assays were utilized to observe the proliferation, migration and invasion ability. MiR-125b’s target gene and downstream signaling pathways were investigated by Luciferase Reporter Assays, qRT-PCR, immunofluorescence assays and western bolt. Results: MiR-125b was highly expressed in human TNBC tissues and cell lines. Inhibiting miR-125b expression suppressed the proliferation, cell migration and invasion. The three-prime untranslated region (3´-UTR) of adenomatous polyposis coli (APC) mRNA contains miR-125b binding sites, and inhibiting miR-125b expression suppressed the activity of the intracellular Wnt/β-catenin pathways and EMT. Conclusion: Inhibiting miR-125b regulates the Wnt/β-catenin pathway and EMT to suppress the proliferation and migration of MDA-MB-468 TNBC cells.     

Loss of exosomal miR-148b from cancer-associated fibroblasts promotes endometrial cancer cell invasion and cancer metastasis

Cancer-associated fibroblasts (CAFs) play crucial roles in tumor progression, given the dependence of cancer cells on stromal support. Therefore, understanding how CAFs communicate with endometrial cancer cell in tumor environment is important for endometrial cancer therapy. Exosomes, which contain proteins and noncoding RNA, are identified as an important mediator of cell–cell communication. However, the function of exosomes in endometrial cancer metastasis remains poorly understood. In the current study we found that CAF-derived exosomes significantly promoted endometrial cancer cell invasion comparing to those from normal fibroblasts (NFs). We identified a significant decrease of miR-148b in CAFs and CAFs-derived exosomes. By exogenously transfect microRNAs, we demonstrated that miR-148b could be transferred from CAFs to endometrial cancer cell through exosomes. In vitro and in vivo studies further revealed that miR-148b functioned as a tumor suppressor by directly binding to its downstream target gene, DNMT1 to suppress endometrial cancer metastasis. In endometrial cancer DNMT1 presented a potential role in enhancing cancer cell metastasis by inducing epithelial–mesenchymal transition (EMT). Therefore, downregulated miR-148b induced EMT of endometrial cancer cell as a result of relieving the suppression of DNMT1. Taken together, these results suggest that CAFs-mediated endometrial cancer progression is partially related to the loss of miR-148b in the exosomes of CAFs and promoting the transfer of stromal cell-derived miR-148b might be a potential treatment to prevent endometrial cancer progression.     

MicroRNA-125b Confers the Resistance of Breast Cancer Cells to Paclitaxel through Suppression of Pro-apoptotic Bcl-2 Antagonist Killer 1 (Bak1) Expression

Paclitaxel (Taxol) is an effective chemotherapeutic agent for treatment of cancer patients. Despite impressive initial clinical responses, the majority of patients eventually develop some degree of resistance to Taxol-based therapy. The mechanisms underlying cancer cells resistance to Taxol are not fully understood. MicroRNA (miRNA) has emerged to play important roles in tumorigenesis and drug resistance. However, the interaction between the development of Taxol resistance and miRNA has not been previously explored. In this study we utilized a miRNA array to compare the differentially expressed miRNAs in Taxol-resistant and their Taxol-sensitive parental cells. We verified that miR-125b, miR-221, miR-222, and miR-923 were up-regulated in Taxol-resistant cancer cells by real-time PCR. We further investigated the role and mechanisms of miR-125b in Taxol resistance. We found that miR-125b was up-regulated in Taxol-resistant cells, causing a marked inhibition of Taxol-induced cytotoxicity and apoptosis and a subsequent increase in the resistance to Taxol in cancer cells. Moreover, we demonstrated that the pro-apoptotic Bcl-2 antagonist killer 1 (Bak1) is a direct target of miR-125b. Down-regulation of Bak1 suppressed Taxol-induced apoptosis and led to an increased resistance to Taxol. Restoring Bak1 expression by either miR-125b inhibitor or re-expression of Bak1 in miR-125b-overexpressing cells recovered Taxol sensitivity, overcoming miR-125-mediated Taxol resistance. Taken together, our data strongly support a central role for miR-125b in conferring Taxol resistance through the suppression of Bak1 expression. This finding has important implications in the development of targeted therapeutics for overcoming Taxol resistance in a number of different tumor histologies.