MicroRNA-21对食管鳞状上皮癌细胞TE-1生物学特性的影响

目的:食管鳞状上皮癌TE-1细胞中microRNA-21(miR-21)高表达,本研究探讨miR-21对TE-1细胞生物学特性的影响。方法:转染miRZip-21慢病毒,持久抑制TE-1细胞中高表达的miR-21,将由此获得的稳定细胞系命名为anti-miR-21细胞系,实时定量PCR方法验证,以TE-1细胞作为对照。细胞增殖实验检测anti-miR-21细胞增殖能力,Transwell侵袭实验观察细胞侵袭能力,Transwell迁移实验及划痕实验观察细胞迁移能力,克隆形成实验及细胞毒性实验分别观察放射线及药物敏感性变化。结果:Anti-miR-21细胞的相对增殖率较对照组TE-1细胞低,差异具有统计学意义(P<0.05)。Transwell侵袭实验及迁移实验显示穿过小室的anti-miR-21细胞较TE-1细胞分别减少51.97%及64.31%,差异具有统计学意义(P<0.05)。划痕实验显示anti-miR-21细胞较TE-1细胞的迁移率降低,差异具有统计学意义(P<0.01)。克隆形成实验分析显示anti-miR-21细胞的D0值(2.73Gy vs 3.60Gy)和Dq值(1.25Gy vs 2.75Gy)均低于对照组,差异具有统计学意义(P<0.05)。细胞毒性实验显示anti-miR-21细胞在顺铂及氟尿嘧啶各个梯度浓度下细胞存活率均降低,差异具有统计学意义(P<0.05)。结论:MiR-21影响食管鳞状上皮癌TE-1细胞的生物学特性。抑制miR-21表达后,降低了TE-1细胞的增殖、侵袭及迁移能力,同时增加了其对放射及化学药物的敏感性。MiR-21可能成为食管鳞癌治疗的潜在靶点。     

Targeting the microRNA-21/AP1 axis by 5-fluorouracil and pirarubicin in human hepatocellular carcinoma

MicroRNAs function as oncomiRs and tumor suppressors in diverse cancers. However, the utility of specific microRNAs in predicting the clinical benefit of chemotherapy has not been well-established. Here, we investigated the correlation between microRNA-21 expression and hepatic arterial infusion chemotherapy with 5-fluorouracil and pirarubicin (HAIC) for hepatocellular carcinoma (HCC). We found that HCC patients with low microRNA-21 levels in tumors tended to have a longer time to recurrence and disease-free survival. We demonstrated that microRNA-21 suppression in combination with 5-fluorouracil and pirarubicin treatment inhibited tumor growth in subcutaneous xenograft mice models. Mechanistically, the AP-1 and microRNA-21-mediated axis was verified to be a therapeutic target of cytotoxic drugs and deregulation of this axis led to an enhanced cell growth in HCC. Taken together, our findings demonstrate that microRNA-21 is a chemotherapy responsive microRNA and can serve as a prognostic biomarker for HCC patients undergoing HAIC. Targeting microRNA-21 enhances the effect of chemotherapeutic drugs, thereby suggesting that microRNA-21 suppression in combination with HAIC may be a novel approach for HCC treatment.     

miR-874 functions as a tumor suppressor by inhibiting angiogenesis through STAT3/VEGF-A pathway in gastric cancer

MicroRNAs are endogenously expressed, small non-coding RNAs that regulate gene expression by targeting mRNAs for translational repression or degradation. Our previous studies indicated that miR-874 played a suppressive role in gastric cancer (GC) development and progression. However, the role of miR-874 in tumor angiogenesis and the mechanisms underlying its function in GC remained to be clarified. Here, gain- and loss-of-function assays demonstrated that miR-874 inhibited the tumor angiogenesis of GC cells in vitro and in vivo. Through reporter gene and western blot assays, STAT3 was shown to be a direct target of miR-874. Overexpression of STAT3 rescued the loss of tumor angiogenesis caused by miR-874. Conversely, the STAT3-shRNA attenuated the increased tumor angiogenesis caused by the miR-874-inhibitor. Furthermore, the levels of miR-874 were inversely correlated with those of STAT3 protein in GC tissues. Taken together, these findings indicate that down-regulation of miR-874 contributes to tumor angiogenesis through STAT3 in GC, highlighting the potential of miR-874 as a target for human GC therapy.     

miR-1236 down-regulates alpha-fetoprotein,thus causing PTEN accumulation,which inhibits the PI3K/Akt pathway and malignant phenotype in hepatoma cells

Alpha fetoprotein (AFP) is a clinical biomarker of hepatocellular carcinoma (HCC). Here, we found that miR-1236 is down-regulated, whereas AFP is highly expressed in HCC tissues and cells. We demonstrated that miR-1236 directly targets the 3′UTR of AFP and down-regulates its expression. Also, miR-1236 inhibited and AFP stimulated proliferation, migration, invasion and vasculogenic mimicry (VM) of HCC. In agreement, AFP over-expression counteracted the inhibitory effect of miR-1236. We demonstrated that AFP promoted the ubiquitination of PTEN, thus decreasing PTEN levels, while miR-1236 inhibited the PI3K/Akt pathway.     

Overexpression of microRNA-95-3p suppresses brain metastasis of lung adenocarcinoma through downregulation of cyclin D1

Despite great efforts to improve survival rates, the prognosis of lung cancer patients is still very poor, mainly due to high invasiveness. We developed brain metastatic PC14PE6/LvBr4 cells through intracardiac injection of lung adenocarcinoma PC14PE6 cells. Western blot and RT-qPCR analyses revealed that PC14PE6/LvBr4 cells had mesenchymal characteristics and higher invasiveness than PC14PE6 cells. We found that cyclin D1 was upregulated, miR-95-3p was inversely downregulated, and pri-miR-95 and its host gene, ABLIM2, were consistently decreased in PC14PE6/LvBr4 cells. MiR-95-3p suppressed cyclin D1 expression through direct binding to the 3′ UTR of cyclin D1 mRNA and suppressed invasiveness, proliferation, and clonogenicity of PC14PE6/LvBr4 cells. Ectopic cyclin D1 reversed miR-95-3p-mediated inhibition of invasiveness and clonogenicity, demonstrating cyclin D1 downregulation is involved in function of miR-95-3p. Using bioluminescence imaging, we found that miR-95-3p suppressed orthotopic tumorigenicity and brain metastasis in vivo and increased overall survival and brain metastasis-free survival. Consistent with in vitro metastatic cells, the levels of miR-95-3p, pri-miR-95, and ABLIM2 mRNA were decreased in brain metastatic tissues compared with lung cancer tissues and higher cyclin D1 expression was involved in poor prognosis. Taken together, our results demonstrate that miR-95- 3p is a potential therapeutic target for brain metastasis of lung adenocarcinoma cells.     

Oxidized low-density lipoprotein is a common risk factor for cardiovascular diseases and gastroenterological cancers via epigenomical regulation of microRNA-210

Hyperlipidemia, including the oxidized low-density lipoprotein (oxLDL) accumulation, is a risk and highly associated with the development of cancers and cardiovascular diseases. microRNA-210 (miR-210), a hypoxia-responsive microRNA regulated by HIF-1α, has been implicated in cancer and cardiovascular disease formation. Furthermore, Bioinformatics analysis revealed that the promoter of the miR-210 gene contains CpG-rich regions. It is unclear whether miR-210 expression could be epigenetically regulated in these disease progresses. The study aimed to explore the relationships between lipid and miR-210 in the context of cardiovascular disease and gastrointestinal cancer. We demonstrated oxLDL can decrease methylation in the miR-210 promoter to up-regulate miR-210. HIF-1α can bind to miR-210 promoter, but this HIF-1α binding site can be blocked by methylation. We showed that subjects of carotid atherosclerosis, stroke patients and cancer patients had hypomethylation in the miR-210 promoter, especially the HIF-1α binding site. Furthermore, miR-210 can directly inhibit sprouty-related EVH1 domain 2 (SPRED2) expressions, and SPRED2 reduces cell migration via ERK/c-Fos/MMPs pathways. Increased miR-210 and reduced SPRED2 levels were found in aorta of mice under high-fat diet and tumor tissues, which implied that miR-210 can be an underlying mechanism to explain oxLDL as a common risk factor for cardiovascular disease and gastrointestinal cancer.     

Intravesical administration of exogenous microRNA-145 as a therapy for mouse orthotopic human bladder cancer xenograft

We previously reported that the level of microRNA (miR)-145 is attenuated in human bladder cancer cells. In this current study, we investigated whether intravesical administration of miR-145 could be a potential therapeutic strategy for controlling bladder cancer by using an orthotopic human bladder cancer xenograft model. Following transfection of 253J B-V cells with miR-145, the effects of the ectopic expression of miR-145 were examined by performing MTT, Western blotting analysis, Hoechst33342 staining, and wound healing assay in vitro. Also, a mouse orthotopic human bladder cancer model was established by inoculating 253J B-V cells into the bladder wall of mice. The anti-cancer effects of intravesical injections of miR-145 into these mice were then assessed. Transfection of 253J B-V cells with miR-145 induced apoptosis and suppression of cell migration in vitro. Western blotting showed that the levels of c-Myc, socs7, FSCN1, E-cadherin, β-catenin, and catenin δ-1 were decreased and that the PI3K/Akt and Erk1/2 signaling pathways were increased in compensatory fashion. In vivo, mice treated with miR-145 showed 76% inhibition of tumor growth, with a significant prolongation of animal survival (p = 0.0183 vs. control). Western blotting showed that both apoptosis and cell motility-related genes were significantly decreased as seen in vitro. Furthermore, PI3k/Akt and Erk1/2 signaling pathways, which were activated in a compensatory manner in vitro, were decreased in vivo. Intravesical administration of exogenous miR-145 was thus concluded to be a valid therapy for bladder cancer in this human bladder cancer xenograft model.     

Androgen receptor (AR) suppresses miRNA-145 to promote renal cell carcinoma (RCC) progression independent of VHL status

Mutational inactivation of the VHL tumor suppressor plays key roles in the development of renal cell carcinoma (RCC), and mutated VHL-mediated VEGF induction has become the main target for the current RCC therapy. Here we identified a signal pathway of VEGF induction by androgen receptor (AR)/miRNA-145 as a new target to suppress RCC progression. Mechanism dissection revealed that AR might function through binding to the androgen receptor element (ARE) located on the promoter region of miRNA-145 to suppress p53''s ability to induce expression of miRNA-145 that normally suppresses expression of HIF2α/VEGF/MMP9/CCND1. Suppressing AR with AR-shRNA or introducing exogenous miRNA-145 mimic can attenuate RCC progression independent of VHL status. MiR-145 mimic in preclinical RCC orthotopic xenograft mouse model revealed its efficacy in suppression of RCC progression. These results together identified signals by AR-suppressed miRNA-145 as a key player in the RCC progression via regulating HIF2α/VEGF/MMP9/CCND1 expression levels. Blockade of the newly identified signal by AR inhibition or miRNA-145 mimics has promising therapeutic benefit to suppress RCC progression.     

Comprehensive profiling of novel microRNA-9 targets and a tumor suppressor role of microRNA-9 via targeting IGF2BP1 in hepatocellular carcinoma

MicroRNA-9 (miR-9) dysregulation is implicated in a variety of human malignancies including hepatocellular carcinoma (HCC), but its role remains contradictory. In this study, we explored the expression and methylation status of miR-9 in HCC samples, as well as the tumor-related functions of miR-9 in vitro. Bioinformatics analysis, array-based RNA expression profile, and literature retrieval were used to identify miR-9 targets in HCC. The potential downstream candidates were then validated by luciferase reporter assay, real-time quantitative PCR, and western blot or enzyme linked immunosorbent assay (ELISA). The expression status and clinicopathologic significances of miR-9 target genes in clinical samples were further explored. The results showed that miR-9 was frequently downregulated in primary HCC. Its silencing was largely contributed by a high frequency (42.5%) of mir-9-1 hypermethylation, which was correlated with bigger tumor size (P = 0.0234). In vitro functional studies revealed that miR-9 restoration retarded HCC cell proliferation and migration. IL-6, AP3B1, TC10, ONECUT2, IGF2BP1, MYO1D, and ANXA2 were confirmed to be miR-9 targets in HCC. Among them, ONECUT2, IGF2BP1, and ANXA2 were confirmed to be aberrantly upregulated in HCC. Moreover, upregulation of ONECUT2, IGF2BP1, and IL-6 were significantly associated with poor post-surgery prognosis (P = 0.0458, P = 0.0037 and P = 0.0461, respectively). Mechanically, miR-9 plays a tumor suppressive role partially through a functional miR-9/IGF2BP1/AKT&ERK axis. Our study suggests that miR-9 functions as a tumor suppressor in HCC progression by inhibiting a series of target genes, including the newly validated miR-9/IGF2BP1/AKT&ERK axis, thus providing potential therapeutic targets and novel prognostic biomarkers for HCC patients.