Jiangning Chen

Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases

Dysregulated expression of microRNAs (miRNAs) in various tissues has been associated with a variety of diseases, including cancers. Here we demonstrate that miRNAs are present in the serum and plasma of humans and other animals such as mice, rats, bovine fetuses, calves, and horses. The levels of miRNAs in serum are stable, reproducible, and consistent among individuals of the same species. Employing Solexa, we sequenced all serum miRNAs of healthy Chinese subjects and found over 100 and 91 serum miRNAs in male and female subjects, respectively. We also identified specific expression patterns of serum miRNAs for lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases. Two non-small cell lung cancer-specific serum miRNAs obtained by Solexa were further validated in an independent trial of 75 healthy donors and 152 cancer patients, using quantitative reverse transcription polymerase chain reaction assays. Through these analyses, we conclude that serum miRNAs can serve as potential biomarkers for the detection of various cancers and other diseases.

Preparation and drug release behaviors of nimodipine-loaded poly(caprolactone) poly(ethylene oxide) polylactide amphiphilic copolymer nanoparticles

Amphiphilic block copolymers, poly(caprolactone)-poly(ethylene glycol)-poly(lactide) (PCELA), were synthesized by ring opening polymerization of caprolactone and lactide initiated with the hydroxyl groups of poly(ethylene glycol) (PEG). These copolymers could form micelle-like nanoparticles due to their amphiphilic characteristic. From the observation of transmission electron microscopy (TEM), the nanoparticles exhibited a regular spherical shape with core-shell structure. The critical micelle concentrations (CMC) of these nanoparticles in water were decreased as molecular weight of PEG decreased. The particle sizes obtained by dynamic light scattering of these nanoparticles were in the range of 100-200 nm, and increased as the hydrophobic property of the nanoparticles increased. Nimodipine as a model drug was loaded in these nanoparticles to investigate the drug release behavior. It was found that the chemical composition of the nanoparticles was a key factor in controlling nanoparticle size, nanoparticle yields, drug-entrapment efficiency, and drug release behavior. When the PEG content is about 2% (wt), the release profile of PCELA nanoparticles appeared to follow zero-order kinetics.

Immobilization of chitosan onto poly-L-lactic acid film surface by plasma graft polymerization to control the morphology of fibroblast and liver cells.

Surface functionalization of biodegradable poly-L-lactic acid (PLLA) was achieved by plasma coupling reaction of chitosan. The structure of modified PLLA surfaces was characterized by contact angle measurements and X-ray photoelectron spectroscopy. Two cell lines, L929 (mouse fibroblasts) and L02 (human hepatocytes), were cultured on the modified PLLA surface. It was found that cells cultured on this film could hardly spread and tend to become round, and the film was demonstrated to be a poorly adhering substrate. However, cells grown on this substrate can proliferate at almost the same speed as cultured on a glass surface. These results suggest that the new substrate can be used to control the morphology of cells, and has potential applications in tissue engineering. It may be helpful in understanding the mechanism of the switch between cell phases of growth and differentiation, which is necessary for the design of tissue regeneration biomaterials.

MicroRNA-31 activates the RAS pathway and functions as an oncogenic MicroRNA in human colorectal cancer by repressing RAS p21 GTPase activating protein 1 (RASA1).

Background: MicroRNAs are important for colorectal cancer signal transduction. Results: miR-31 stimulates colorectal cancer cell proliferation and tumorigenesis by directly targeting RASA1. Conclusion: miR-31 activates the RAS pathway and functions as an oncogenic microRNA in human colorectal cancer. Significance: Learning how miRNAs participate in tumor signaling is crucial for understanding tumor signal transduction and cancer therapy. MicroRNAs (miRNAs) are known to play a vital role in colorectal cancer. We found a widespread disruption in miRNA expression during colorectal tumorigenesis using microarray and quantitative RT-PCR analysis; of the 161 miRNAs altered in colorectal cancer compared with normal adjacent tissue samples, miR-31 was the most significantly dysregulated. We identified candidate targets of miR-31 using bioinformatics approaches and validated RAS p21 GTPase activating protein 1 (RASA1) as a direct target. First, we found an inverse correlation between miR-31 and RASA1 protein levels in vivo. Second, in vitro evidence demonstrated that RASA1 expression was significantly decreased by treatment with pre-miR-31-LV, whereas anti-miR-31-LV treatment increased RASA1 protein levels. Third, a luciferase reporter assay confirmed that miR-31 directly recognizes a specific location within the 3′-untranslated region of RASA1 transcripts. Furthermore, the biological consequences of miR-31 targeting RASA1 were examined by the cell proliferation assay in vitro and by the immunodeficient mouse xenograft tumor model in vivo. Taken together, our results demonstrate for the first time that miR-31 plays a significant role in activating the RAS signaling pathway through the inhibition of RASA1 translation, thereby improving colorectal cancer cell growth and stimulating tumorigenesis.

A pH/Enzyme-responsive tumor-specific delivery system for doxorubicin

To overcome the cardiotoxicity of doxorubicin, a self-assembled pH/enzyme-responsive system was developed. Cationic gelatin combined polyGC-DOX intercalation tightly to form compact nanoscale complexes (CPX1) which then combined by a pH-sensitive pegylated alginate to form CPX2. CPX2 could be digested and release DOX under the co-digestion of gelatinase (GA) and Dnase I when pH < 6.9. More importantly, tumor homogenate supernatant (THS) could effectively release DOX from CPX2 while the plasma and liver homogenate supernatant (LHS) could not, which was confirmed by an in vivo test. The results indicated that this formulation had the tumor-specific drug-release effect. This effect resulted in an increased drug concentration in tumor tissue and decreased content in heart and liver. The changed bio-distribution of DOX delivered by CPX2 greatly enhanced the anti-cancer activity and reduced the cardiotoxicity of the drug. The anti-cancer efficiency of DOX delivered by CPX2 is more than 2 times of the free doxorubicin, and the mortality caused by the high-dose DOX was completely prevented by CPX2. All results suggested that this easy-manufactured, cost-effective nanocomplex holds great promise to be developed into a formulation of doxorubicin and the other anthracyclines with high anti-cancer activity and low cardiotoxicity.

miR-141 Regulates colonic leukocytic trafficking by targeting CXCL12β during murine colitis and human Crohn's disease

Objective Emerging evidence suggests that microRNA (miRNA)-mediated gene regulation influences a variety of autoimmune disease processes, including Crohns disease (CD), but the biological function of miRNAs in CD remains unclear. We examine miRNA level in colon tissues and study the potential functions of miRNAs that regulate pathological genes during the inflammation process. Design miRNA levels were assayed in the inflamed colon of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced and IL-10 knockout (KO) chronic colitis mice and CD patients by microarray or qRT-PCR. The influence of differently expressed miR-141 on its putative target genes, CXCL12β, and leukocyte migration was investigated in colonic epithelia cells, colitis models and CD patients. The role of miR-141 was further studied in the experimental colitis mice by intracolonic administration of miR-141 precursors or inhibitors. Results An inverse correlation between miR-141 and CXCL12β/total-CXCL12 was observed predominantly in the epithelial cells of the inflamed colons from colitic mice and CD patients. Further study demonstrated that miR-141 directly regulated CXCL12β expression and CXCL12β-mediated leukocyte migration. Upregulation or downregulation of miR-141 in the TNBS-induced or IL-10 KO colitic colon regulated leukocyte infiltration and alleviated or aggravated experimental colitis, respectively. Additionally, colonic overexpression of CXCL12β abolished the therapeutic effect of miR-141 in TNBS-induced colitis. Conclusions This study showed that the pathway of miR-141 targeting CXCL12β is a possible mechanism underlying inflammatory cell trafficking during colonic inflammation process. Inhibiting colonic CXCL12β expression and blocking colonic immune cell recruitment by using miRNA precursors represents a promising approach that may be valuable for CD treatment.

In Vitro Evidence Suggests That miR-133a-mediated Regulation of Uncoupling Protein 2 (UCP2) Is an Indispensable Step in Myogenic Differentiation

UCP2 and UCP3, two novel uncoupling proteins, are important regulators of energy expenditure and thermogenesis in various organisms. The striking disparity between UCP2 mRNA and protein levels in muscle tissues prompted initial speculation that microRNAs are implicated in the regulatory pathway of UCP2. We found, for the first time, that the repression of UCP2 expression in cardiac and skeletal muscle resulted from its targeting by a muscle-specific microRNA, miR-133a. Moreover, our findings illustrate a novel function of UCP2 as a brake for muscle development. We also show that MyoD can remove the braking role of UCP2 via direct up-regulation of miR-133a during myogenic differentiation. Taken together, our current work delineates a novel regulatory network employing MyoD, microRNA, and uncoupling proteins to fine-tune the balance between muscle differentiation and proliferation during myogenesis.

MicroRNA‐101 suppresses liver fibrosis by targeting the TGFβ signalling pathway

Transforming growth factor‐β (TGFβ) is crucial for liver fibrogenesis and the blunting of TGFβ signalling in hepatic stellate cells (HSCs) or hepatocytes can effectively inhibit liver fibrosis. microRNAs (miRNAs) have emerged as key regulators in modulating TGFβ signalling and liver fibrogenesis. However, the regulation of TGFβ receptor I (TβRI) production by miRNA remains poorly understood. Here we demonstrate that the miR‐101 family members act as suppressors of TGFβ signalling by targeting TβRI and its transcriptional activator Kruppel‐like factor 6 (KLF6) during liver fibrogenesis. Using a mouse model of carbon tetrachloride (CCl4)‐induced liver fibrosis, we conducted a time‐course experiment and observed significant down‐regulation of miR‐101 in the fibrotic liver as well as in the activated HSCs and injured hepatocytes in the process of liver fibrosis. Meanwhile, up‐regulation of TβRI/KLF6 was observed in the fibrotic liver. Subsequent investigations validated that TβRI and KLF6 were direct targets of miR‐101. Lentivirus‐mediated ectopic expression of miR‐101 in liver greatly reduced CCl4‐induced liver fibrosis, whereas intravenous administration of antisense miR‐101 oligonucleotides aggravated hepatic fibrogenesis. Mechanistic studies revealed that miR‐101 inhibited profibrogenic TGFβ signalling by suppressing TβRI expression in both HSCs and hepatocytes. Additionally, miR‐101 promoted the reversal of activated HSCs to a quiescent state, as indicated by suppression of proliferation and migration, loss of activation markers and gain of quiescent HSC‐specific markers. In hepatocytes, miR‐101 attenuated profibrogenic TGFβ signalling and suppressed the consequent up‐regulation of profibrogenic cytokines, as well as TGFβ‐induced hepatocyte apoptosis and the inhibition of cell proliferation. The pleiotropic roles of miR‐101 in hepatic fibrogenesis suggest that it could be a potential therapeutic target for liver fibrosis. Copyright

Targeted delivery of oligonucleotides into tumor-associated macrophages for cancer immunotherapy

Tumor-associated macrophages (TAMs) have been proven to be a driving force in the initiation, proliferation, metastasis and angiogenesis of various tumors. Specifically, alterations in the functions of TAMs exhibited inhibitory effects on tumor growth. However, there is currently no research being conducted on the targeting delivery of drugs into TAMs for cell-specific tumor immunotherapy. In the present study, we developed a TAMs targeted delivery system that is triggered by the acidic microenvironment in the tumor to release a TAMs-recognizing nano-complex loaded with oligonucleotides. By using this system, we demonstrated a significant anti-tumor effect of an oligonucleotide combination of CpG oligonucleotide, anti-IL-10 and anti-IL-10 receptor oligonucleotides. These nucleic acid drugs delivered by the delivery system accumulated in the TAMs of an allograft hepatoma murine model by intravenous injection, suppressed the pro-tumor functions and stimulated the anti-tumor activities of TAMs. More importantly, the nucleic acid drug-based immune-regulation was restricted to the tumor microenvironment and did not cause an upregulation of serum inflammatory cytokines. Our present study provides an effective therapeutic strategy for regulating cell-specific functions using nucleic acid drugs.

The Autoregulatory Feedback Loop of MicroRNA-21/Programmed Cell Death Protein 4/Activation Protein-1 (MiR-21/PDCD4/AP-1) as a Driving Force for Hepatic Fibrosis Development

Background: MicroRNA-21 is important in hepatic fibrosis development, but the mechanism is unclear. Results: MicroRNA-21 is predominantly up-regulated in activated hepatic stellate cells and could form a double negative feedback loop that links fibrogenic machinery. Conclusion: The microRNA-21-mediated loop is a main driving force for hepatic fibrosis progression. Significance: It suggests a mechanism for how microRNA-21 contributes to hepatic fibrosis. Sustained activation of hepatic stellate cells (HSCs) leads to hepatic fibrosis, which is characterized by excessive collagen production, and for which there is no available drug clinically. Despite tremendous progress, the cellular activities underlying HSC activation, especially the driving force in the perpetuation stage, are only partially understood. Recently, microRNA-21 (miR-21) has been found to be prevalently up-regulated during fibrogenesis in different tissues, although its detailed role needs to be further elucidated. In the present study, miR-21 expression was examined in human cirrhotic liver samples and in murine fibrotic livers induced by thioacetamide or carbon tetrachloride. A dramatic miR-21 increase was noted in activated HSCs. We further found that miR-21 maintained itself at constant high levels by using a microRNA-21/programmed cell death protein 4/activation protein-1 (miR-21/PDCD4/AP-1) feedback loop. Disrupting this loop with miR-21 antagomir or AP-1 inhibitors significantly suppressed fibrogenic activities in HSCs and ameliorated liver fibrosis. In contrast, reinforcing this loop with small interfering RNA (siRNA) against PDCD4 promoted fibrogenesis in HSCs. Further analysis indicated that the up-regulated miR-21 promoted the central transforming growth factor-β (TGF-β) signaling pathway underlying HSC activation. In summary, we suggest that the miR-21/PDCD4/AP-1 autoregulatory loop is one of the main driving forces for hepatic fibrosis progression. Targeting this aberrantly activated feedback loop may provide a new therapeutic strategy and facilitate drug discovery against hepatic fibrosis.