P-glycoprotein enhances radiation-induced apoptotic cell death through the regulation of miR-16 and Bcl-2 expressions in hepatocellular carcinoma cells

P-glycoprotein (Pgp), an efflux pump, was confirmed the first time to regulate the expressions of miR/gene in cells. Pgp is known to be associated with multidrug resistance. RHepG2 cells, the multidrug resistant subline of human hepatocellular carcinoma HepG2 cells, expressed higher levels of Pgp as well as miR-16, and lower level of Bcl-2 than the parental cells. In addition, RHepG2 cells were more radiation sensitive and showed more pronounced radiation-induced apoptotic cell death than the parental cells. Mechanistic analysis revealed that transfection with mdr1 specific antisense oligos suppressed radiation-induced apoptosis in HepG2 cells. On the other hand, ectopic mdr1 expression enhanced radiation-induced apoptosis in HepG2 cells, SK-HEP-1 cells, MiHa cells, and furthermore, induced miR-16 and suppressed its target gene Bcl-2 in HepG2 cells. Moreover, the enhancement effects of Pgp and miR-16 on radiation-induced apoptosis were counteracted by overexpression of Bcl-2. The Pgp effect on miR-16/Bcl-2 was suppressed by Pgp blocker verapamil indicating the importance of the efflux of Pgp substrates. The present study is the first to reveal the role of Pgp in regulation of miRNA/gene expressions. The findings may provide new perspective in understanding the biological function of Pgp.     

MicroRNA 122对肝癌细胞基因表达谱的影响

为研究microRNA（miR-122）对肝癌细胞Hep3B基因表达谱的影响,并探讨其在肝癌发过程中的可能作用,构建了miR-122稳定高表达的Hep3B细胞,利用基因表达谱芯片技术筛选得到和对照组细胞比较的差异表达基因.研究结果显示,2倍以上变化的差异表达基因有490个,其中上调的有345个,下调的有145个.这些基因中有16个与肿瘤发生相关,其它基因涉及细胞周期、信号转导、细胞凋亡和细胞增殖分化等众多生物学过程.这些结果提示,miR-122可能在肝癌发生的过程中发挥作用,并可能与这些差异表达基因密切相关.另外,还结合生物信息学方法,在下调表达的基因中预测了miR-122可能直接作用的靶基因.本研究初步探讨了miR-122在肝癌细胞中的生物学功能,为进一步研究miR-122在肝癌发生中的作用奠定了基础,同时也为miRNA的生物学功能及其作用机制的研究提供了一些参考.     

The anti-proliferative effects of norcantharidin on human HepG2 cells in cell culture

Many lines of evidence have shown that Chinese medicine contains many chemical compounds with anticancer effects. Therefore, we tested whether the active ingredients of blister beetles have a therapeutic effect on hepatoma. The aim of this study was to investigate the inhibitive effects of norcantharidin which is extracted from blister beetles on human hepatoma cells HepG2 in vitro and its anticancer mechanism.MTT assay, agarose gel electrophoresis and flow cytometry were used to evaluate HepG2 cells proliferation and apoptosis. The role of caspase activities were assayed using caspase apoptosis detection kit. Western blot analysis was used to evaluate the level of Bcl-2/Bax expression. Our results indicate that norcantharidin inhibited HepG2 cell growth in a time- and dose-dependent manner by MTT assay. HepG2 cells treated with norcantharidin showed typical characteristics of apoptosis including the DNA fragmentation. The activities of caspase-3, -9 were up-regulated after norcantharidin treatment. By western blot analysis, we found the level of Bcl-2 were down-regulated, whereas, the level of Bcl-2 Up-regulated.so we suggest that up-regulation of mitochondrial Bax expression and down-regulation of Bcl-2 expression participated in the apoptosis induced by NCTD. These results suggest that norcantharidin triggers apoptosis in hepato cancer cell lines via the activation of the caspses, mitochondrial pathways, and that this agent may be useful for developing new therapeutic regimens for the treatment of colorectal carcinoma.     

MicroRNA-15b enhances hypoxia/reoxygenation-induced apoptosis of cardiomyocytes via a mitochondrial apoptotic pathway

Myocardial ischemia reperfusion (I/R) can induce altered expression of microRNAs (miRNAs). The miRNAs—miR-15a, miR-15b and miR-16 have been shown to play a role in apoptosis, although not in cardiac-related models. We investigated the roles of miR-15b in hypoxia/reoxygenation (H/R)-induced apoptosis of cardiomyocytes. Quantitative real time polymerase chain reaction results showed that the expression of miR-15a and miR-15b were up-regulated in Sprague–Dawley rat hearts subjected to I/R. Expression levels of miR-15b increased more than four fold above basal levels. Similar results were obtained for cardiomyocytes exposed to H/R. Recombinant adenoviral vectors were generated to explore the functional role of miR-15b in cultured cardiomyocytes exposed to H/R. Overexpression of miR-15b enhanced cell apoptosis and the loss of mitochondrial membrane potential, as determined by flow cytometric analysis. Conversely, down-regulated expression was cytoprotective. The effects of miR-15b can by mimicked by Bcl-2 short-interfering RNAs. The inhibition of miR-15b increased expression levels of the Bcl-2 protein without affecting Bcl-2 mRNA levels, suppressed the release of mitochondrial cytochrome c to the cytosol and decreased the activities of caspase-3 and 9. It is possible that miR-15b is the upstream regulator of a mitochondrial signaling pathway for H/R induced apoptosis.     

miR-15b and miR-16 regulate TNF mediated hepatocyte apoptosis via BCL2 in acute liver failure

Acute liver failure (ALF) still has an unacceptable high mortality rate, despite substantial improvements with multidisciplinary care. The precise underlying mechanism of ALF remains to be explored. It has been reported that microRNAs (miRNAs) are novel regulators in a number of liver diseases, but the role of miRNAs in the development of ALF is not fully understood. An ALF murine model was generated by ip injection of D: -GalN/LPS, which was confirmed with histopathology and biochemistry. The hepatic miRNA expression profile in ALF was determined by microarray and verified by qRT-PCR. The functions and signal pathways of the targeted genes of these deregulated miRNAs were predicted, using bioinformatics analysis. The possible underlying mechanism was investigated by exploring the relationship between miRNA modification and hepatocyte apoptosis. There were a total of 95 significantly changed miRNAs in ALF compared to mock-treated (P < 0.01). Among these 95 miRNAs, 20 were up-regulated and 26 were down-regulated at both 5 and 7 h time points. Bioinformatics analysis predicted that some of these 46 miRNAs were involved in apoptosis. Among the up-regulated miRNAs involved in apoptosis, miR-15b and miR-16 showed the highest enrichment and targeted the common anti-apoptotic gene, BCL2. Our in vitro data demonstrated that miR-15b and/or miR-16 regulated BCL2 at the protein level. Inhibition of miR-15b and/or miR-16 reduced hepatic apoptosis and TNF production. These data suggest that miR-15b and miR-16 regulate TNF mediated hepatic apoptosis via BCL2 during ALF, and may shed light on the development of a therapeutic strategy for treatment of ALF.     

miR-135a inhibition protects A549 cells from LPS-induced apoptosis by targeting Bcl-2

Acute lung injury (ALI) is a severe clinical condition with high morbidity and mortality. Apoptosis is a key pathologic feature of ALI, and Bcl-2 plays an important role during the pathogenesis of ALI via the regulation of apoptosis. However, the regulation of Bcl-2 during ALI, particularly through microRNAs, remains unclear. We hypothesize that certain miRNAs may play deleterious or protective roles in ALI via the regulation of Bcl-2. The LPS stimulation of A549 cells was used to mimic ALI in vitro. First, we confirmed that Bcl-2 is involved in LPS-induced apoptosis in A549 cells. Then, bioinformatic analyses and quantitative real-time polymerase chain reaction assays were performed to screen for miRNAs targeting Bcl-2. We observed that miR-135a was markedly increased in LPS-challenged A549 cells. miR-135a inhibition markedly restored Bcl-2 expression and protected A549 cells from LPS-induced apoptosis. Furthermore, bioinformatic analysis and luciferase activity assays were conducted to confirm that miR-135a binds directly to the 3′-untranslated region of Bcl-2 and suppresses its expression. Interestingly, the inhibition of miR-135a did not attenuate apoptosis under LPS-treated conditions when Bcl-2 was knocked down. Therefore, we suggest that miR-135a regulation of LPS-induced apoptosis in A549 cells may depend in part on the regulation of Bcl-2. The miR-135a/Bcl-2 signaling pathway may be a novel therapeutic target for the prevention of ALI.     

Resveratrol chemosensitizes adriamycin-resistant breast cancer cells by modulating miR-122-5p

Breast cancer is one of the major malignancies threatening women's health worldwide, and chemotherapy tolerance has become a severe limitation of clinical treatment. Recent findings have revealed that resveratrol, as a dietary agent with antitumour activity, could prevent cancer progression by regulating microRNAs (miRNAs). Additionally, dysregulated miRNAs have been found to contribute significantly to chemoresistance by an increasing number of studies. In this study, experiments were designed to study the functional role of resveratrol in MCF-7 cells (low-invasive breast cancer) in chemosensitivity to adriamycin and to determine the targeted miRNAs of resveratrol and their key target proteins linked to cell activity. We demonstrated that in resveratrol-induced chemosensitivity, cell cycle and apoptosis were arrested in adriamycin-resistant breast cancer cells after modulation of the critical suppresser, miR-122-5p. Further miRNA modulation with miR-122-5p mimics or miR-122-5p inhibitors indicated a major effect of miR-122-5p on the regulation of key antiapoptotic proteins (B-cell lymphoma 2 [Bcl-2]) and cyclin-dependent kinases (CDK2, CDK4, and CDK6) in drug-resistant breast cancer cells in response to resveratrol. In conclusion, our results indicate that resveratrol acts as a potential inducer to enhance the chemosensitivity of breast cancer and also suggest that miR-122-5p is involved in the pathway of cell-cycle arrest by targeting Bcl-2 and CDKs.     

Involvement of miR-1 in the protective effect of hydrogen sulfide against cardiomyocyte apoptosis induced by ischemia/reperfusion

The protective effect of hydrogen sulfide (H₂S) against myocardial ischemia/reperfusion (IR) injury via anti-apoptotic signaling is well established, but the underlying mechanism remains unclear. Recently, miRNAs have been identified as important mediators of myocardial injury by regulating apoptosis-related genes. It was found in our previous preliminary study that microRNA-1 (miR-1) expression underwent a significant change in IR group compared to H₂S preconditioned group, indicating that miR-1 possessed myocyte-specific properties. In the present study, we intended to see whether miR-1 participated in H₂S protection of cardiomyocytes against IR-induced apoptosis by regulating apoptosis-related genes. Cardiomyocytes of neonatal rats were subjected to hypoxia/reoxygenation (HR) injury with or without H₂S preconditioning, while the myocardium of adult SD rats was subjected to IR with or without H₂S preconditioning. It was found that HR injury increased apoptosis of cardiac myocytes, up-regulated the expression of miR-1, and down-regulated the expression of Bcl-2. H₂S preconditioning attenuated cardiomyocyte apoptosis and LDH release, as well as enhanced cell viability following HR injury. MiR-1 was up-regulated by HR and down-regulated by H₂S preconditioning. In contrast, Bcl-2 was down-regulated by HR and up-regulated by H₂S preconditioning. In addition, Bcl-2 protein was down-regulated by the miR-1 mimic in a dose-dependent manner. H₂S also attenuated IR-induced cardiomyocyte apoptosis in vivo. MiR-1 regulated H₂S protection of cardiomyocytes against IR-induced apoptosis by stimulating Bcl-2. These results implicate miR-1 as an important regulator of H₂S on the IR myocardium.     

In vitro induction of apoptosis by isosclerone from marine-derived fungus Aspergillus fumigatus

In the present study, we investigated the apoptotic effects of isosclerone from marine-derived fungi on human breast cancer MCF-7 cells. Treatment with isosclerone exhibited a characteristic feature of apoptosis including significant cytotoxicity and DNA fragmentation in cancer cells. In addition, The apoptosis induction abilities of the isosclerone was studied by analyzing the expression of caspase-3, -8 and -9, Bcl-2 family, NF-κ-B P50, P65, and IKK proteins. Western blot and RT-PCR analysis have indicated that isosclerone induce cancer cells apoptosis through down-regulated Bcl-2 family and up-regulated caspases, and activating the NF-κ-B signaling pathway. Our data demonstrate that isosclerone specifically binds to crystal structure of apoptosis regulator BCL-2 and pseudo-activated procaspase-3 proteins through down-regulated Bcl-2 family and up-regulated caspases, and activating the NF-κ-B signaling pathway. Our proof-of-principle study should have a positive impact on future drug discovery.     

Genome-wide profiling identified a set of miRNAs that are differentially expressed in glioblastoma stem cells and normal neural stem cells

A major challenge in cancer research field is to define molecular features that distinguish cancer stem cells from normal stem cells. In this study, we compared microRNA (miRNA) expression profiles in human glioblastoma stem cells and normal neural stem cells using combined microarray and deep sequencing analyses. These studies allowed us to identify a set of 10 miRNAs that are considerably up-regulated or down-regulated in glioblastoma stem cells. Among them, 5 miRNAs were further confirmed to have altered expression in three independent lines of glioblastoma stem cells by real-time RT-PCR analysis. Moreover, two of the miRNAs with increased expression in glioblastoma stem cells also exhibited elevated expression in glioblastoma patient tissues examined, while two miRNAs with decreased expression in glioblastoma stem cells displayed reduced expression in tumor tissues. Furthermore, we identified two oncogenes, NRAS and PIM3, as downstream targets of miR-124, one of the down-regulated miRNAs; and a tumor suppressor, CSMD1, as a downstream target of miR-10a and miR-10b, two of the up-regulated miRNAs. In summary, this study led to the identification of a set of miRNAs that are differentially expressed in glioblastoma stem cells and normal neural stem cells. Characterizing the role of these miRNAs in glioblastoma stem cells may lead to the development of miRNA-based therapies that specifically target tumor stem cells, but spare normal stem cells.     

Conjugated linoleic acid supplementation caused reduction of perilipin1 and aberrant lipolysis in epididymal adipose tissue

MicroRNAs (miRNAs) have emerged as powerful regulators of multiple processes linked to human cancer, including cell apoptosis, proliferation and migration, suggesting that the regulation of miRNA function could play a critical role in cancer progression. Recent studies have found that human serum/plasma contains stably expressed miRNAs. If they prove indicative of disease states, miRNAs measured from peripheral blood samples may be a source for routine clinical detection of cancer. Our studies showed that both miR-508-3p and miR-509-3p were down-regulated in renal cancer tissues. The level of miR-508-3p but not miR-509-3p in renal cell carcinoma (RCC) patient plasma demonstrated significant differences from that in control plasma. In addition, the overexpression of miR-508-3p and miR-509-3p suppressed the proliferation of RCC cells (786-0), induced cell apoptosis and inhibited cell migration in vitro. Our data demonstrated that miR-508-3p and miR-509-3p played an important role as tumor suppressor genes during tumor formation and that they may serve as novel diagnostic markers for RCC.     

MiR-181a-5p is involved in the cardiomyocytes apoptosis induced by hypoxia–reoxygenation through regulating SIRT1

ABSTRACT

MiR-181a-5p’s mechanism in hypoxia–reoxygenation (H/R)-induced cardiomyocytes apoptosis has not been clarified. This study verified that SIRT1 was the target of miR-181a-5p. MiR-181a-5p expression was up-regulated or down-regulated in H/R-induced cardiomyocytes, and SIRT1 was transfected into cells alone or in combination with miR-181a-5p. Cell viability, apoptosis, levels of released lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD), as well as the Bcl-2, Bax, and Caspase 3 levels in treated cells were tested. On the one hand, down-regulated miR-181a-5p promoted cell viability, reduced released LDH and MDA, and increased SOD level in H/R-induced cardiomyocytes. On the other hand, miR-181a-5p inhibited apoptosis and elevated Bcl-2 expression while decreasing the expressions of Bax and Caspase 3 in treated cells, but the effects of miR-181a-5p could be rescued by SIRT1. In conclusion, miR-181a-5p involved in H/R-induced cardiomyocytes apoptosis through regulating SIRT1, which might become a novel direction for related diseases.     

miR-222 and miR-29a contribute to the drug-resistance of breast cancer cells

Adriamycin (Adr) and docetaxel (Doc) are two chemotherapeutic agents commonly used in the treatment of breast cancer. However, patients with breast cancer who are treated by the drugs often develop resistance to them and some other drugs. Recently studies have shown that microRNAs (miRNAs, miRs) play an important role in drug-resistance. In present study, miRNA expression profiles of MCF-7/S and its two resistant variant MCF-7/Adr and MCF-7/Doc cells were analyzed using microarray and the results were confirmed by real-time quantitative polymerase chain reaction. Here, 183 differentially expressed miRNAs were identified in the two resistant sublines compared to MCF-7/S. Then, five up-regulated miRNAs (miR-100, miR-29a, miR-196a, miR-222 and miR-30a) in both MCF-7/Adr and MCF-7/Doc were selected to explore their roles in acquisition of drug-resistance using transfection experiment. The results showed that miR-222 and miR-29a mimics and inhibitors had partially changed the drug-resistance of breast cancer cells, which was also confirmed by apoptosis assay. Western blot results suggested that miR-222 and -29a could regulate the expression of PTEN, maybe through which the two miRNAs conferred Adr and Doc resistance in MCF-7 cells. Finally, pathway mapping tools were employed to further analyze signaling pathways affected by the two miRNAs. In summary, this study demonstrates that altered miRNA expression pattern is involved in acquiring resistance to Adr and Doc in breast cancer MCF-7 cells, and that there are some miRNAs who displayed consistent up- or down-regulated expression changes in the two resistant sublines. The most importance is that we identify two miRNAs (miR-222 and miR-29a) involved in drug-resistance, at least in part via targeting PTEN.     

Upregulated microRNA-29a by hepatitis B virus X protein enhances hepatoma cell migration by targeting PTEN in cell culture model

Hepatitis B virus X protein (HBx) plays important roles in the development of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) contribute to cancer development by acting as oncogenes or tumor suppressors. Previously, we reported that HBx was able to promote the migration of hepatoma HepG2 cells. However, the regulation of miRNAs in the development of HBV-related HCC is poorly understood. In the present study, we reported that miR-29a was a novel regulator of migration of hepatoma cells mediated by HBx. Our data showed that the expression of miR-29a was dramatically increased in p21-HBx transgenic mice, HBx-transfected hepatoma HepG2-X (or H7402-X) cells and HepG2.2.15 cells that constitutively replicate HBV. However, our data showed that miR-29a was upregulated in 4 of the 11 clinical HCC samples. We found that the overexpression of miR-29a promoted the migration of HepG2 cells, while a specific miR-29a inhibitor could partially abolish the enhanced migration of HepG2-X cells. Moreover, we identified PTEN was one of the target genes of miR-29a in HepG2 cells. The deletion of the miR-29a-binding site was able to abolish the role of miR-29a in suppression of luciferase activity of the PTEN 3'UTR reporter. Meanwhile, the overexpression of PTEN was able to reverse the promoted migration of HepG2 cells mediated by miR-29a. Moreover, our data showed that the modulation of Akt phosphorylation, a downstream factor of PTEN, was involved in the cell migration enhanced by miR-29a, suggesting that miR-29a is responsible for the cell migration through its target gene PTEN. Thus, we conclude that miR-29a is involved in the regulation of migration of hepatoma cells mediated by HBx through PTEN in cell culture model.     

XB130, a New Adaptor Protein,Regulates Expression of Tumor Suppressive MicroRNAs in Cancer Cells

XB130, a novel adaptor protein, promotes cell growth by controlling expression of many related genes. MicroRNAs (miRNAs), which are frequently mis-expressed in cancer cells, regulate expression of targeted genes. In this present study, we aimed to explore the oncogenic mechanism of XB130 through miRNAs regulation. We analyzed miRNA expression in XB130 short hairpin RNA (shRNA) stably transfected WRO thyroid cancer cells by a miRNA array assay, and 16 miRNAs were up-regulated and 22 miRNAs were down-regulated significantly in these cells, in comparison with non-transfected or negative control shRNA transfected cells. We chose three of the up-regulated miRNAs (miR-33a, miR-149 and miR-193a-3p) and validated them by real-time qRT-PCR. Ectopic overexpression of XB130 suppressed these 3 miRNAs in MRO cells, a cell line with very low expression of XB130. Furthermore, we transfected miR mimics of these 3 miRNAs into WRO cells. They negatively regulated expression of oncogenes (miR-33a: MYC, miR-149: FOSL1, miR-193a-3p: SLC7A5), by targeting their 3′ untranslated region, and reduced cell growth. Our results suggest that XB130 could promote growth of cancer cells by regulating expression of tumor suppressive miRNAs and their targeted genes.     

Identification of miR-7 as an oncogene in renal cell carcinoma

MicroRNA-7 (miR-7) has been described as a tumor suppressor in several human cancers, but the results of a study to identify miRNAs associated with metastatic capability in breast cancer suggested that miR-7 may be characterized as an oncogene. The present study was to determine the expression and function of miR-7 in renal cell carcinoma. Quantitative real-time polymerase chain reaction was used to validate the expressions of miR-7 in 48 paired renal cell carcinomas (RCC) and normal tissues, based on the preliminary sequencing results of miRNAs. Furthermore, the impacts of miR-7 on cell migration, proliferation and apoptosis were analyzed using wound scratch assay, MTT and flow cytometry, respectively. The results demonstrated that miR-7 was up-regulated in RCC compared with normal tissues (p = 0.001). Down-regulation of miR-7 with synthesized inhibitor inhibited cell migration in vitro, suppressed cell proliferation and induced renal cancer cell apoptosis, prompting that miR-7 could be characterized as an oncogene in RCC. The present study was the first to reveal that miR-7 was up-regulated in RCC and it played an important role in RCC by affecting cellular migration, proliferation and apoptosis. Further researches should be conducted to explore the roles and target genes of miR-7 in RCC and other cancers.