Xing Cai

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.

Secreted Monocytic miR-150 Enhances Targeted Endothelial Cell Migration

MicroRNAs (miRNAs) are a class of noncoding RNAs that regulate target gene expression at the posttranscriptional level. Here, we report that secreted miRNAs can serve as signaling molecules mediating intercellular communication. In human blood cells and cultured THP-1 cells, miR-150 was selectively packaged into microvesicles (MVs) and actively secreted. THP-1-derived MVs can enter and deliver miR-150 into human HMEC-1 cells, and elevated exogenous miR-150 effectively reduced c-Myb expression and enhanced cell migration in HMEC-1 cells. In vivo studies confirmed that intravenous injection of THP-1 MVs significantly increased the level of miR-150 in mouse blood vessels. MVs isolated from the plasma of patients with atherosclerosis contained higher levels of miR-150, and they more effectively promoted HMEC-1 cell migration than MVs from healthy donors. These results demonstrate that cells can secrete miRNAs and deliver them into recipient cells where the exogenous miRNAs can regulate target gene expression and recipient cell function.

Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA

Our previous studies have demonstrated that stable microRNAs (miRNAs) in mammalian serum and plasma are actively secreted from tissues and cells and can serve as a novel class of biomarkers for diseases, and act as signaling molecules in intercellular communication. Here, we report the surprising finding that exogenous plant miRNAs are present in the sera and tissues of various animals and that these exogenous plant miRNAs are primarily acquired orally, through food intake. MIR168a is abundant in rice and is one of the most highly enriched exogenous plant miRNAs in the sera of Chinese subjects. Functional studies in vitro and in vivo demonstrated that MIR168a could bind to the human/mouse low-density lipoprotein receptor adapter protein 1 (LDLRAP1) mRNA, inhibit LDLRAP1 expression in liver, and consequently decrease LDL removal from mouse plasma. These findings demonstrate that exogenous plant miRNAs in food can regulate the expression of target genes in mammals.

Role of miR-143 targeting KRAS in colorectal tumorigenesis.

Dysregulated expression of microRNAs (miRNAs) is associated with a variety of diseases, including colorectal cancer. By comparing more than 200 miRNAs in 13 pairs of matched colorectal cancer and normal adjacent tissue samples through qRT-PCR and microarray analysis, we found a widespread disruption of miRNA expression during colorectal tumorigenesis. In particular, among a panel of presumed targets generated by in silico analysis that may interact with these aberrantly expressed miRNAs, KRAS oncogene has been further experimentally validated as the target of miR-143. First, an inverse correlation between KRAS protein and miR-143 in vivo was found. Second, KRAS expression in Lovo cells was significantly abolished by treatment with miR-143 mimic, whereas miR-143 inhibitor increased KRAS protein level. Third, luciferase reporter assay confirmed that miR-143 directly recognize the 3′-untranslated region of KRAS transcripts. Four, Lovo cells treated with miR-143 inhibitor showed a stimulated cell proliferation, whereas miR-143 overexpression had an opposite effect. Finally, inhibition of KRAS expression by miR-143 inhibits constitutive phosphorylation of ERK1/2. Taken together, the present study provides the first evidences that miR-143 is significant in suppressing colorectal cancer cell growth through inhibition of KRAS translation.

Microvesicle-mediated Transfer of MicroRNA-150 from Monocytes to Endothelial Cells Promotes Angiogenesis

Background: The mechanism of secreted miRNA promoting angiogenesis is still unclear. Results: Secreted miR-150 from monocyte induce endothelial cell tube formation in vitro and angiogenesis in vivo, and down-regulation of miR-150 inhibits angiogenesis caused by diabetes, cancer, and atherosclerosis. Conclusion: Monocyte-derived miR-150 can induce angiogenesis via targeting endothelial cells. Significance: Our study illustrates the new role of a secreted miRNA in angiogenesis. Recent studies by our group and others show that microRNAs can be actively secreted into the extracellular environment through microvesicles (MVs) and function as secretory signaling molecules that influence the recipient cell phenotypes. Here we investigate the role of monocyte-secreted miR-150 in promoting the capillary tube formation of endothelial cells and in enhancing angiogenesis. In vitro capillary tube formation and in vivo angiogenesis assays showed that monocyte-derived MVs have strong pro-angiogenic activities. By depleting miR-150 from monocytic MVs and increasing miR-150 in MVs derived from cells that normally contain low levels of miR-150, we further demonstrated that the miR-150 content accounted for the pro-angiogenic activity of monocytic MVs in these assays. Using tumor-implanted mice and ob/ob mice as models, we revealed that miR-150 secretion, which is increased for diseases such as cancers and diabetes, significantly promotes angiogenesis. The delivery of anti-miR-150 antisense oligonucleotides into tumor-implanted mice and ob/ob mice via MVs, however, strongly reduced angiogenesis in both types of mice. Our results collectively demonstrate that secretion of miR-150 via MVs can promote angiogenesis in vitro and in vivo, and we also present a novel microRNA-based therapeutic approach for disease treatment.

Tumor-secreted miR-214 induces regulatory T cells: a major link between immune evasion and tumor growth.

An increased population of CD4+CD25highFoxp3+ regulatory T cells (Tregs) in the tumor-associated microenvironment plays an important role in cancer immune evasion. However, the underlying mechanism remains unclear. Here we observed an increased secretion of miR-214 in various types of human cancers and mouse tumor models. Tumor-secreted miR-214 was sufficiently delivered into recipient T cells by microvesicles (MVs). In targeted mouse peripheral CD4+ T cells, tumor-derived miR-214 efficiently downregulated phosphatase and tensin homolog (PTEN) and promoted Treg expansion. The miR-214-induced Tregs secreted higher levels of IL-10 and promoted tumor growth in nude mice. Furthermore, in vivo studies indicated that Treg expansion mediated by cancer cell-secreted miR-214 resulted in enhanced immune suppression and tumor implantation/growth in mice. The MV delivery of anti-miR-214 antisense oligonucleotides (ASOs) into mice implanted with tumors blocked Treg expansion and tumor growth. Our study reveals a novel mechanism through which cancer cell actively manipulates immune response via promoting Treg expansion.

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.

The promotion of type 1 T helper cell responses to cationic polymers in vivo via toll-like receptor-4 mediated IL-12 secretion

Cationic polymers with nucleic acid drug delivery ability are widely used in experimental and clinical studies. However, their interactions with the immune systems are rarely studied. In the present study, cationic polymers including PEI, polylysine, cationic dextran and cationic gelatin exhibited strong stimulation on Th1 response which was characterized by the induction of the proliferation of CD4(+) T cells and the secretion of Th1 related cytokines. Experiments performed on macrophages demonstrated that cationic polymers specifically stimulated the macrophage to secrete IL-12 which is one of the main Th1-inducing cytokines. The result that MyD88 inhibitor remarkably reduced the IL-12 expression induced by cationic polymers suggested that this stimulation was mainly mediated by toll-like receptor (TLR) pathway. Additionally, cationic polymers could strongly inhibit LPS-induced TNF-alpha secretion in macrophages. This result implied that cationic polymers may interact with macrophages through TLR-4 which is the receptor of LPS. The following test of inhibiting IL-12 expression stimulated by cationic polymers using TLR-4 antibody proved that the stimulation was mainly mediated by TLR-4. Data in the present study demonstrated that the stimulation ability of cationic polymer was related with its cationic degree and neutralizing cationic polymer with anionic polymer completely abrogated the stimulation effect. The molecular weight of the polymers also influenced their stimulation ability, larger molecular means stronger stimulation ability. In conclusion, the present study revealed that cationic polymers could promote Th1 responses in vivo via TLR-4 mediated IL-12 secretion and the molecular weight and cationic degree of the polymers determined the stimulation ability.

A microarray-based approach identifies ADP ribosylation factor-like protein 2 as a target of microRNA-16.

microRNAs (miRNAs) are generally thought to negatively regulate the expression of their target genes by mRNA degradation or by translation repression. Here we show an efficient way to identify miRNA target genes by screening alterations in global mRNA levels following changes in miRNA levels. In this study, we used mRNA microarrays to measure global mRNA expression in three cell lines with increased or decreased levels of miR-16 and performed bioinformatics analysis based on multiple target prediction algorithms. For further investigation among the predicted miR-16 target genes, we selected genes that show an expression pattern opposite to that of miR-16. One of the candidate target genes that may interact with miR-16, ADP-ribosylation factor-like protein 2 (ARL2), was further investigated. First, ARL2 was deduced to be an ideal miR-16 target by computational predictions. Second, ARL2 mRNA and protein levels were significantly abolished by treatment with miR-16 precursors, whereas a miR-16 inhibitor increased ARL2 mRNA and protein levels. Third, a luciferase reporter assay confirmed that miR-16 directly recognizes the 3′-untranslated region (3′-UTR) of ARL2. Finally, we showed that miR-16 could regulate proliferation and induce a significant G0/G1 cell cycle arrest, which was due at least in part, to the down-regulation of ARL2. In summary, the present study suggests that integrating global mRNA profiling and bioinformatics tools may provide the basis for further investigation of the potential targets of a given miRNA. These results also illustrate a novel function of miR-16 targeting ARL2 in modulating proliferation and cell cycle progression.

Localized Delivery of Antisense Oligonucleotides by Cationic Hydrogel Suppresses TNF-α Expression and Endotoxin-Induced Osteolysis

ABSTRACTPurposeTo investigate the possibility of using localized nucleic drug delivery methods for the treatment of osteolysis-related bone disease.MethodsA bio-degradable cationic hydrogel composed of gelatin and chitosan was used to deliver an antisense oligonucleotide (ASO) targeting murine TNF-α for the treatment of endotoxin-induced osteolysis.ResultsASO combined with this hydrogel was released when it was digested by adhering cells. The released ASO was efficiently delivered into contacted cells and tissues in vitro and in vivo. When tested in animal models of edotoxin-induced bone resorption, ASO delivered by such means effectively suppressed the expression of TNF-α and subsequently the osteoclastogenesis in vivo. Osteolysis in the edotoxin-induced bone resorption animal models was blocked by the treatment.ConclusionThis is a successful attempt to apply localized gene delivery method to treat inflammatory diseases in vivo.