Haiying Wan

Glycyrrhetinic acid-modified chitosan/poly(ethylene glycol) nanoparticles for liver-targeted delivery

A liver-targeted drug delivery carrier, composed of chitosan/poly(ethylene glycol)-glycyrrhetinic acid (CTS/PEG-GA) nanoparticles, was prepared by an ionic gelation process, in which glycyrrhetinic acid (GA) acted as the targeting ligand. The formation and characterization of these nanoparticles were confirmed by FT-IR, dynamic light scattering (DLS) and zeta potential measurements. The biodistribution of the nanoparticles was assessed by single-photon emission computed tomography (SPECT), and the cellular uptake was evaluated using human hepatic carcinoma cells (QGY-7703 cells). The anti-neoplastic effect of the doxorubicin.HCl-loaded nanoparticles (DOX-loaded nanoparticles) was also investigated in vitro and in vivo. The results showed that the CTS/PEG-GA nanoparticles were remarkably targeted to the liver, and keep at a high level during the experiment. The accumulation in the liver was 51.3% at 3 h after injection; this was nearly 2.6 times that obtained with the CTS/PEG nanoparticles. The DOX-loaded nanoparticles were greatly cytotoxic to QGY-7703 cells, and the IC(50) (50% inhibitory concentration) for the free doxorubicin.HCl (DOX.HCl) and the DOX-loaded CTS/PEG-GA nanoparticles were 47 and 79 ng/mL, respectively. Moreover, the DOX-loaded CTS/PEG-GA nanoparticles could effectively inhibit tumor growth in H22 cell-bearing mice.

Regulation of the cell cycle gene, BTG2, by miR-21 in human laryngeal carcinoma

MicroRNAs are short regulatory RNAs that negatively modulate gene expression at the post-transcriptional level, and are deeply involved in the pathogenesis of several types of cancers. To investigate whether specific miRNAs and their target genes participate in the molecular pathogenesis of laryngeal carcinoma, oligonucleotide microarrays were used to assess the differential expression profiles of microRNAs and mRNAs in laryngeal carcinoma tissues compared with normal tissues. The oncogenic miRNA, microRNA-21 (miR-21), was found to be upregulated in laryngeal carcinoma tissues. Knockdown of miR-21 by specific antisense oligonucleotides inhibited the proliferation potential of HEp-2 cells, whereas overexpression of miR-21 elevated growth activity of the cells, as detected by the colony formation assay. The cell number reduction caused by miR-21 inhibition was due to the loss of control of the G1-S phase transition, instead of a noticeable increase in apoptosis. Subsequently, a new target gene of miR-21, BTG2, was found to be downregulated in laryngeal carcinoma tissues. BTG2 is known to act as a pan-cell cycle regulator and tumor suppressor. These findings indicate that aberrant expression of miR-21 may contribute to the malignant phenotype of laryngeal carcinoma by maintaining a low level of BTG2. The identification of the oncogenic miR-21 and its target gene, BTG2, in laryngeal carcinoma is potentially valuable for cancer diagnosis and therapy.

MicroRNA-214 Suppresses Growth and Invasiveness of Cervical Cancer Cells by Targeting UDP-N-acetyl-α-d-galactosamine:Polypeptide N-Acetylgalactosaminyltransferase 7

Background: Recent research has uncovered tumor-suppressive and oncogenic potential of miRNAs. Results: miR-214 suppresses the proliferation, migration, and invasiveness of cervical cancer cells by targeting GALNT7. Conclusion: Down-regulation of miR-214 results in overexpressed GALNT7, contributing to tumorigenesis of cervical cancer. Significance: The identification of tumor suppressor miR-214 and its oncogenic target GALNT7 in cervical cancer cells is potentially valuable for cancer diagnosis and therapy. MicroRNAs are a class of small noncoding RNAs that function as key regulators of gene expression at the post-transcriptional level. In this study, we demonstrate that miR-214 is frequently down-regulated in cervical cancer, and its expression reduces the proliferation, migration, and invasiveness of cervical cancer cells, whereas inhibiting its expression results in enhanced proliferation, migration, and invasion. miR-214 binds to the 3′-UTR of UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase 7 (GALNT7), thereby repressing GALNT7 expression. Furthermore, we are the first to show, using quantitative real-time PCR, that GALNT7 is frequently up-regulated in cervical cancer. The knockdown of GALNT7 markedly inhibits cervical cancer cell proliferation, migration, and invasion, whereas ectopic expression of GALNT7 significantly enhances these properties, indicating that GALNT7 might function as an oncogene in cervical cancer. The restoration of GALNT7 expression can counteract the effect of miR-214 on cell proliferation, migration, and invasiveness of cervical cancer cells. Together, these results indicate that miR-214 is a new regulator of GALNT7, and both miR-214 and GALNT7 play important roles in the pathogenesis of cervical cancer.

Glycyrrhetinic acid-modified poly(ethylene glycol)–b-poly(γ-benzyl l-glutamate) micelles for liver targeting therapy

Liver targeted micelles were successfully constructed via self-assembly of glycyrrhetinic acid (GA)-modified poly(ethylene glycol)-b-poly(gamma-benzyl l-glutamate) (GA-PEG-PBLG) block co-polymers, which were fabricated via ring opening polymerization of gamma-benzyl l-glutamate N-carboxyanhydride monomer initiated by GA-modified PEG. The in vivo biodistribution and the in vitro cellular uptake of these micelles were investigated. The results showed that the relative uptake of doxorubicin (DOX)-loaded micelles (DOX/GA-PEG-PBLG) in liver was much higher than in other tissues, and the resulting DOX concentration in liver was about 2.2-fold higher than that from the micelles without modification by GA. Moreover, the cellular uptake study demonstrated that the introduction of GA to the micelles could significantly increase the affinity for human hepatic carcinoma 7703 cells, which induced a 3.7-fold higher endocytosis than unmodified ones. The cytotoxicity of DOX/GA-PEG-PBLG micelles (IC(50) 47 ngml(-1)) was much higher than that of free DOX (IC(50) 90 ngml(-1)). These results indicate that GA-modified micelles have great potential in liver targeting therapy.

miR-346 and miR-138 competitively regulate hTERT in GRSF1- and AGO2-dependent manners, respectively.

miRNAs typically downregulate the expression of target genes by binding to their 3′UTR, and dysregulation of miRNAs may contribute to tumorigenesis. Here, we found that miR-346 and miR-138 competitively bind to a common region in the 3′UTR of hTERT mRNA and have opposite effects on the expression and function of hTERT in human cervical cancer cells. Furthermore, G-rich RNA sequence binding factor 1 (GRSF1) mediates the miR-346-dependent upregulation of hTERT by binding to the miR-346 middle sequence motif (CCGCAU) which forms a “bulge loop” when miR-346 is bound to the hTERT 3′UTR, facilitating the recruitment of hTERT mRNA to ribosomes to promote translation in an AGO2-independent manner. Conversely, miR-138 suppresses hTERT expression in an AGO2-dependent manner. Interestingly, replacement of the miR-138 middle sequence with that of miR-346 results in an upregulation of hTERT expression in a GRSF1-dependent manner. Moreover, miR-346 depends on GRSF1 to upregulate another target gene, activin A receptor, type IIB (ACVR2B), in which miR-346 “CCGCAU” motif is essential. These findings reveal novel mechanisms of miRNA-mediated upregulation of target gene expression and describe the coordinated action of multiple miRNAs to control the fate of a single target mRNA through binding to its 3′UTR.

Contribution of hydrophobic/hydrophilic modification on cationic chains of poly(ε-caprolactone)-graft-poly(dimethylamino ethylmethacrylate) amphiphilic co-polymer in gene delivery.

Nanoparticles (NPs) assembled from amphiphilic polycations have been certified as potential carriers for gene delivery. Structural modification of polycation moieties may be an efficient route to further enhance gene delivery efficiency. In this study two electroneutral monomers with different hydrophobicities, 2-hydroxyethyl methacrylate (HEMA) and 2-hydroxyethyl acrylate (HEA), were incorporated into the cationic poly(dimethylamino ethyl methacrylate) (PDMAEMA) side-chains of amphiphilic poly(ε-caprolactone)-graft-poly(dimethylamino ethylmethacrylate) (PCD) by random co-polymerization, to obtain poly(ε-caprolactone)-graft-poly(dimethylamino ethyl methacrylate-co-2-hydroxyethyl methacrylate) (PCD-HEMA) and poly(ε-caprolactone)-graft-poly(dimethylamino ethyl methacrylate-co-2-hydroxyethyl acrylate) (PCD-HEA). Minimal HEA or HEMA moieties in PDMAEMA do not lead to statistically significant changes in particle size, zeta potential, DNA condensation properties and buffering capacity of the naked NPs. However, the incorporation of HEMA and HEA lead to reductions and increases, respectively, in the surface hydrophilicity of the naked NPs and NPs/DNA complexes, which was confirmed by water contact angle assay. These simple modifications of PDMAEMA with HEA and HEMA moieties significantly affect the gene transfection efficiency on HeLa cells in vitro: PCD-HEMA NP/DNA complexes show a much higher transfection efficiency than PCD NPs/DNA complexes, while PCD-HEA NPs/DNA complexes show a lower transfection efficiency than PCD NP/DNA complexes. Fluorescence activated cell sorter and confocal laser scanning microscope results indicate that the incorporation of hydrophobic HEMA moieties facilitates an enhancement in both cellular uptake and endosomal/lysosomal escape, leading to a higher transfection efficiency. Moreover, the process of endosomal/lysosomal escape confirmed in our research that PCD and its derivatives do not just rely on the proton sponge mechanism, but also on membrane damage due to the polycation chains, especially hydrophobic modified ones. Hence, it is proved that hydrophobic modification of cationic side-chains is a crucial route to improve gene transfection mediated by polycation NPs.

miR-1228 promotes the proliferation and metastasis of hepatoma cells through a p53 forward feedback loop.

Background:The effective mechanisms of microRNAs (miRNAs) functions as oncogenes or tumour suppressors in human hepatocellular carcinoma (HCC) are still obscure. Here, we investigated the function and expression of miR-1228 in HCC.Methods:The role of miR-1228 in HCC was determined by colony formation, transwell, and nude mice xenograft experiments. miR-1228 target gene were identified by EGFP reporter assays, real-time PCR, and western blot analysis. Dual-luciferase reporter assay and real-time PCR analysis are used to examine the regulation of p53.Results:miR-1228 promoted proliferation and metastasis, and facilitated the transition of cell cycle in hepatoma cells. miR-1228 downregulated p53 expression by binding to its 3′UTR. The ectopic expression of p53 abrogated the phenotypes induced by miR-1228. An inverse correlation existed between miR-1228 and p53 expression in hepatoma tissues compared with the adjacent tissues and three hepatoma cell lines. Moreover, we found that p53 suppressed the expression and promoter activity of miR-1228.Conclusions:miR-1228 functions as an oncogene by promoting cell cycle progression and cell mobility and negatively regulates the expression of p53. p53 downregulation in turn leads to an increase in miR-1228 expression, thereby forming a positive feedback loop that contributes to cancerogenesis in HCC.

miR-371-5p down-regulates pre mRNA processing factor 4 homolog B (PRPF4B) and facilitates the G1/S transition in human hepatocellular carcinoma cells.

Increasing evidence has lent support to the notion that miRNAs regulate hepatocellular carcinoma (HCC) cell proliferation by directly targeting cell cycle-related genes. Among these genes, we identified PRPF4B, a CDK-like kinase, as a new target of miR-371-5p. Over-expression of miR-371-5p and knockdown of PRPF4B promotes cell growth by accelerating the G1/S transition in HCC cell lines. Moreover, miR-371-5p promotes tumor growth of QGY-7703 cells in vivo. Conversely, inhibition of miR-371-5p yields an opposing effect. Ectopic expression of PFPF4B abolishes the malignant phenotypes caused by miR-371-5p. Furthermore, contrary to PRPF4B, miR-371 was up-regulated in HCC tissues. Collectively, we highlight the significance of miR-371-5p and PRPF4B in cell cycle progression and hepatocarcinogenesis.

CREB1-driven expression of miR-320a promotes mitophagy by down-regulating VDAC1 expression during serum starvation in cervical cancer cells.

The altered expression of miRNAs in response to stresses contributes to cancer pathogenesis. However, little is known regarding the mechanism by which cellular stresses drive alterations in miRNA expression. Here, we found that serum starvation enhanced mitophagy by downregulating the mitophagy-associated protein voltage-dependent anion channel 1 (VDAC1) and by inducing the expression of miR-320a and the transcription factor cAMP responsive element binding protein 1(CREB1). Furthermore, we cloned the promoter of miR-320a and identified the core promoter of miR-320a in the upstream −16 to −130 region of pre-miR-320a. Moreover, CREB1 was found to bind to the promoter of miR-320a to activate its expression and to induce mitophagy during serum starvation. Collectively, our results reveal a new mechanism underlying serum starvation-induced mitophagy in which serum starvation induces CREB1 expression, in turn activating miR-320a expression, which then down-regulates VDAC1 expression to facilitate mitophagy. These findings may provide new insights into cancer cell survival in response to environmental stresses.