Retraction Note: Antisense oligonucleotides targeting midkine induced apoptosis and increased chemosensitivity in hepatocellular carcinoma cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Retraction Note: Antisense oligonucleotides targeting midkine inhibit tumor growth in an in situ human hepatocellular carcinoma model.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Inhibitory effect of midkine-binding peptide on tumor proliferation and migration.

BACKGROUND: To investigate the inhibitory effect of midkine-binding peptides on human umbilical vein endothelial cells (HUVECs) proliferation and angiogenesis of xenograft tumor. METHODS: The midkine-binding peptides were panned by Ph.D.-7(™) Phage Display Peptide Library Kit, and the specific binding activities of positive clones to target protein were examined by phage ELISA. The effect of midkine-binding peptides on proliferation of HUVECs was confirmed by MTT test. The xenograft tumor model was formed in BALB/c mice with the murine hepatocarcinoma cells H22 (H22). Microvessel density (MVD) was analyzed by immunohistochemistry of factor VIII staining. RESULTS: Midkine-binding peptides have the inhibitory effects on tumor angiogenesis, a proliferation assay using human umbilical vein endothelial cells (HUVECs) indicated that particular midkine-binding peptides significantly inhibited the proliferation of the HUVECs. Midkine-binding peptides were also observed to efficiently suppress angiogenesis induced by murine hepatocarcinoma H22 cells in BALB/c nude mice. CONCLUSION: The midkine-binding peptides can inhibit solid tumor growth by retarding the formation of new blood vessels. The results indicate that midkine-binding peptides may represent potent anti-angiogenesis agents in vivo.

Development of hybrid-type modified chitosan derivative nanoparticles for the intracellular delivery of midkine-siRNA in hepatocellular carcinoma cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Most of the patients with HCC lose the surgical opportunity at the time of diagnosis. Some novel therapeutic modalities, like gene therapy, are promising for the treatment of HCC. However, the success of gene therapy depends on two aspects: efficient gene materials and gene delivery vectors. The present study was to develop new chitosan-based nanoparticles for a midkine-siRNA (anti-HCC gene drug) delivery. METHODS: The novel gene delivery vector (MixNCH) was synthesized by hybrid-type modification of chitosan with 2-chloroethylamine hydrochloride and N, N-dimethyl-2-chloroethylamine hydrochloride. The chemical structure of MixNCH was characterized by FT-IR and 1HNMR. The cytotoxicity of MixNCH was determined by MTS assay. The gene condensation ability and size, zeta potential and morphology of MixNCH/MK-siRNA nanoparticles were measured. The in vitro transfection and gene knockdown efficiency of midkine by MixNCH/MK-siRNA nanoparticles was detected by qRT-PCR and Western blotting. Gene knockdown effect at the molecule level on the proliferation of HepG2 in vitro was determined by MTS assay. RESULTS: MixNCH was successfully acquired by aminoalkylation modification of chitosan. The MixNCH could condense MK-siRNA well above the weight ratio of 3. The average size of MixNCH/MK-siRNA nanoparticles was 100-200 nm, and the surface charge was about +5 mV. Morphologically, MixNCH/MK-siRNA nanoparticles were in regular spherical shape with no aggregation. Regarding to the in vitro transfection of nanoparticles, the MixNCH/MK-siRNA nanoparticles reduced MK mRNA level to 14.03%+/-4.03%, which were comparable to Biotrans (8.94%+/-3.77%). MixNCH/MK-siRNA effectively inhibited the proliferation of HepG2 in vitro. CONCLUSION: MixNCH/MK-siRNA nanoparticles could be effective for the treatment of hepatocellular carcinoma.

Upregulation of miR-1280 expression in non-small cell lung cancer tissues.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a prolific and high-mortality disease with few effective treatments. Although the detection and surgical techniques for NSCLC continue to advance, the survival rate for the patients with NSCLC remains poor. Enhanced predictive biomarkers such as microRNAs (miRNAs) are needed at the time of diagnosis to better tailor therapies for patients. This study focused on the expression of miR-1280 in NSCLC tissues and distal normal tissues in order to explore the association between miR-1280 expression and NSCLC. METHODS: A total of 72 newly diagnosed primary NSCLC patients were enrolled in this study. Quantitative real-time polymerase chain reaction (PCR) was performed to identify the expression level of miR-1280 in the NSCLC tissues and distal normal tissues of these patients. RESULTS: The miR-1280 expression was significantly higher in the NSCLC tissues (0.084 ± 0.099) than distal normal tissues (0.014 ± 0.015, P = 0.009). In 54 patients (75%), the miR-1280 expression in the NSCLC tissues was upregulated (2-ΔΔct > 2), and no case showed a downregulation of miR-1280 expression. CONCLUSIONS: The expression level of miR-1280 could be regarded as a biomarker for NSCLC.

[Development of EV71, CA16 and other enterovirus vrial real-time qualitative PCR diagnostic kit].

OBJECTIVE: A novel multiplex real-time RT-PCR kit was developed to detect EV71, CoxA16 and other human enteroviruses simultaneously with an internal amplification control to avoids false negatives, which used for hand, foot and mouth disease in the clinical diagnosis and epidemiological surveillance. METHODS: Design specific primers and probes of EV71, CA16, other intestinal virus and internal amplification control, improve the extraction method of virus nucleic acid. Optimization the detection system of real-time quantitative PCR. Research the products of the accuracy, stability, precision, amplification efficiency and detection of linear range. RESULTS: The primers and probes had high spicificity. The Viral RNA extraction effect of this Kit is as same as that of QIAamp Viral RNA mini Kit (QIAGEN company), but less reagent cost. The optimal concentrations of primers and probes are 0.2 micromol/L for all the upstream and downstream primers, 0.06 micromol/L for probes of other human enteroviruse, 0.08 micromol/L for probes of EV71 and CA16 respectively. The kit has good stability, accuracy and precision. The amplification efficiencies of EV71, CoxA16 and other human enteroviruses are 106% ,101% and 105% and the detection of linear range is from 10(9) copies/microl-10(2) copies/microl. CONCLUSION: The novel multiplex real-time RT-PCR kit for detecting EV71, CoxA16 and other human enteroviruses simultaneously with an internal amplification control has good stability, accuracy, precision and amplification efficiencies. So it has great value in clinical application.

[Preparation and characterization the polyclonal antibody of the nonstructural protein of human highly pathogenic H5N1 avian influenza viruses].

OBJECTIVE: Of this study was to prepare high sensitivity and high specificity of highly pathogenic H5N1 subtype avian influenza virus NS1 protein antibody and a preliminary assessment of its potency. METHODS: Construct pET-28a (+) recombinant vector containing the H5N1 subtype of avian influenza virus NS1 sequences of E. coli BL21 (DE3), induced expression of NS1 protein, NS1 recombinant protein was obtained by Ni-NTA column purified by affinity chromatography, and SDS-PAGE and Western Blot analysis. Purified protein antigen to immunize New Zealand white rabbits, obtained rabbit anti-NS1 serum, affinity-purified polyclonal antibodies. Using ELISA and Western Blot analysis of purified antibody titer and specificity. RESULTS: NS1 fusion protein was highly expressed in a purity of greater than 90%, with the fusion protein was used to immunize New Zealand white rabbits anti-NS1 polyclonal antibody titer of 1:80 000, and specific recognition of the H5N1 subtype of avian influenza virus NS1 protein. CONCLUSIONS: NS1 polyclonal antibodies to NS1 recombinant protein purified antigen, with better potency and specificity, and to prepare the conditions for the development of the H5N1 subtype of avian influenza virus detection kit.

Large scale preparation of midkine antisense oligonucleotides nanoliposomes by a cross-flow injection technique combined with ultrafiltration and high-pressure extrusion procedures.

The midkine antisense oligonucleotide (MK-ASODN, 5'-CCC CGG GCC GCC CTT CTT CA-3') nanoliposomes have been identified to suppress hepatocellular carcinoma (HCC) growth effectively, and have a great potential to be an effective target drug for HCC. In this study, a facile and reproducible method for large-scale preparation of MK-ASODN nanoliposomes followed by lyophilization has been developed successfully. Meanwhile, the MK-ASODN nanoliposomes characteristics, storage stability and their antitumor efficiency were studied. The mean particle size of MK-ASODN nanoliposomes were 229.43±15.11 nm, and the zeta potential were 29.7±1.1 mV. High entrapment efficiency values were achieved around 90%. Transmission electron microscopy images revealed spherical shaped nanoliposomes. Nanoliposomes allowed sustained MK-ASODN release for as long as 14 days. During 180 days of storage, freeze-dried nanoliposomes showed no significant change in the mean size, zeta potential, entrapment efficiency and drug release ratio. Regarding their antitumor efficiency, the in vitro proliferation of human liver cancer cells were significantly inhibited by the MK-ASODN nanoliposomes. Furthermore, the MK-ASOND nanoliposomes also significantly inhibited the growth of HCC in the mouse model. In summary, the results confirmed that this large-scale preparation of MK-ASOND nanoliposomes was facile and reproducible, and potentially, could speed up the application process of our MK-ASOND nanoliposomes for HCC therapy.

Novel functional proteins interact with midkine in hepatic cancer cells.

BACKGROUND: Midkine is a heparin-binding growth factor that promotes the proliferation, survival, migration and differentiation of various target cells. Midkine plays an important role in tumorigenesis and tumor progression, and is overexpressed in many human malignant tumors. Patients with high tumor midkine expression frequently have a worse prognosis than those with low expression. The present study was designed to investigate the interaction network of midkine in hepatic cancer cells, and to elucidate its role in hepatocellular carcinoma. METHODS: DNA encoding full-length midkine was cloned into pDBLeu vector to serve as bait in yeast two-hybrid screening to identify interacting proteins. Candidate proteins were examined on SC-Leu-Trp-His+3-AT (20 mmol/L) plates and assayed for X-gal activity, then sequenced and classified according to the GenBank. Finally, identified proteins were expressed by the in vitro expression system pCMVTnT, and protein interactions were confirmed by co-immunoprecipitation. RESULTS: Using the yeast two-hybrid system, we found 6 proteins that interacted with midkine: NK-kappa-B inhibitor alpha (I-κ-B-alpha), Dvl-binding protein naked cuticle 2, granulin, latent active TGF-beta binding protein 3, latent active TGF-beta binding protein 4, and phospholipid scramblase 1. In vitro co-immunoprecipitation demonstrated that all identified proteins directly interacted with midkine. CONCLUSION: The identification of midkine-interacting proteins in hepatic cancer cells indicates that midkine is a multifunctional factor that may participate in cell migration, differentiation, and proliferation, and is also associated with the multicellular response feedback during the development of hepatocellular carcinoma.

Progranulin expression in breast cancer with different intrinsic subtypes.

Progranulin is a newly discovered 88-kDa glycoprotein originally purified from the highly tumorigenic mouse teratoma-derived cell line PC. We found that high progranulin expression was associated with higher breast carcinoma angiogenesis, reflected by increased vascular endothelial growth factor expression and higher microvessel density. However, no immunohistochemical evidence currently exists to correlate progranulin expression with clinicopathological features in different intrinsic subtypes of breast carcinoma biopsies. The aim of this study was to investigate the progranulin expression profiles in the intrinsic subtypes of breast carcinomas and their relevance to histopathological and clinicopathological features. Tissue blocks containing 264 cases of breast carcinomas from 2006 to 2009 were classified as different intrinsic subtypes. Tissues of four intrinsic subtypes were immunostained for progranulin, vascular endothelial growth factor and CD105. Their relevance to histopathological and clinicopathological features was also analyzed. Twenty tissue samples from breast fibroadenomas were included in this study. Progranulin expression showed no significant differences in different intrinsic subtypes, although an increasing tendency could be found in the triple-negative breast cancer (TNBC) subgroup (χ(2)=5.00, df=3, p=0.17). However, differences were significant when pathologically node metastasis-positive (pN(+)) TNBC were excluded (χ(2)=17.84, df=3, p<0.01). Some clinicopathological parameters, including CK5/6 (χ(2)=0.08, df=3, p=0.78), E-cadherin (χ(2)=0.71, df=3, p=0.40) and P53 (χ(2)=0.05, df=3, p=0.83), displayed no correlation with activity of progranulin in pathologically node metastasis-negative (pN(-)) TNBC. It was noted that the EGFR expression level of the pN(-) TNBC subtype was significantly higher in cases with strong progranulin expression than in cases with weak progranulin expression (χ(2)=11.26, df=1, p<0.01). A significantly higher expression level of progranulin in pN(-) TNBC suggests that progranulin is a promising new target for pN(-) TNBC treatment. Strong expression of progranulin correlates with positive EGFR expression in the pN(-) TNBC subtype. The close relationship between EGFR and progranulin/VEGF/CD105 expression may partly play a role in high angiogenesis levels in the pN(-) TNBC subtype.

Clinicopathologic and prognostic implications of progranulin in breast carcinoma.

BACKGROUND: Progranulin is a newly discovered 88-kDa glycoprotein originally purified from the highly tumorigenic mouse teratoma-derived cell line PC. Its expression is closely correlated with the development and metastasis of several cancers. However, no immunohistochemical evidence currently exists to correlate progranulin expression with clinicopathologic features in breast carcinoma biopsies, and the role of progranulin as a new marker of metastatic risk and prognosis in breast cancer has not yet been studied. The aim of this study was to investigate the clinicopathologic and prognostic implications of progranulin expression in breast carcinoma and its correlation with tumor angiogenesis. METHODS: Progranulin expression was determined immunohistochemically in 183 surgical specimens from patients with breast cancer and 20 tissue samples from breast fibroadenomas. The tumor angiogenesis-related biomarker, vascular endothelial growth factor was assayed and microvessel density was assessed by counting vascular endothelial cells in tumor tissues labeled with endoglin antibody. The relationship between progranulin expression and the clinicopathologic data were analyzed. RESULTS: Progranulin proteins were overexpressed in breast cancer. The level of progranulin expression was significantly correlated with tumor size (P = 0.004), lymph node metastasis (P < 0.001) and TNM staging (P < 0.001). High progranulin expression was associated with higher tumor angiogenesis, reflected by increased vascular endothelial growth factor expression (P < 0.001) and higher microvessel density (P = 0.002). CONCLUSION: Progranulin may be a valuable marker for assessing the metastasis and prognosis of breast cancer, and could provide the basis for new combination regimens with antiangiogenic activity.

Preparation and preliminary characterization of rabbit monoclonal antibodies against human midkine.

We prepared rabbit monoclonal antibodies that target human midkine (MK). The MK gene was amplified by PCR from the plasmid pEGFP-MK and subcloned into the prokaryotic expression vector pGEX-1λT to generate an N-terminally glutathione S-transferase (GST)-tagged fusion protein construct. Expression of the GST-MK fusion protein was achieved by IPTG induction in Escherichia coli cells. The expressed protein was purified using the GST system. After verifying purification, the fusion protein was used to immunize rabbits to prepare monoclonal antibodies against human MK by the rabbit hybridoma technique. The hybridomas generated were screened by an enzyme-link immunoassay (ELISA) for specificity, which was further characterized by Western blotting and ELISA. SDS-PAGE analysis showed that the purified protein corresponds to the calculated molecular weight. The GST-MK fusion protein was prepared. At least one hybridoma cell line secreting anti-MK MAb was obtained. Western blotting analysis confirmed the identity of the MAb. The titer of the MAbs measured by an indirect ELISA was 1:64,000. The affinity constant, which was measured by a non-competitive ELISA, was found to be 3.0 × 10(9) M(-1). Western blotting and immunohistochemistry analysis showed that the produced MAbs bind to the MK protein in cancerous tissues. The GST-MK fusion protein was successfully expressed and purified. The MAbs against MK were subsequently prepared, which should further aid research and the application of MK MAbs in clinical settings.

Expression of midkine and endoglin in breast carcinomas with different immunohistochemical profiles.

The aim of this study was to investigate the midkine and endoglin expression in breast carcinomas with five different immunohistochemical profiles and their relevance to histopathologic and clinicopathologic features. We analyzed 161 archival tissues immunohistologically. The level of midkine expression in breast cancer significantly correlated with lymph node metastasis (p = 0.001) and TNM staging (p = 0.003). High microvessel density (MVD) was associated with higher midkine reactivity group (p = 0.036). Although the basal-like subtype had higher midkine expression level and MVD, no significant difference with the other breast cancer subtypes was found. In conclusion, midkine was a promising target for tumor prognosis in clinical diagnosis and treatment. This study found no significant differences in tumor angiogenesis in different molecular subtypes of breast cancer.

Allele polymorphisms of interleukin-10 and hepatitis B, C virus infection.

BACKGROUND: Interleukin 10 (IL-10) is an important cytokine with anti-inflammatory, anti-immune and anti-fibrotic functions. This study aimed at evaluating the relationship between allele polymorphisms in the IL-10 promoter region and hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. METHODS: The odds ratios (ORs) of IL-10 allele distributions in patients with HBV or HCV infection were analyzed against healthy controls. All the relevant studies in PubMed were identified, and poor qualified studies were excluded. The meta-analysis software REVMAN 4.2 was applied for investigating heterogeneity among individual studies and summarizing all the studies. The publication bias was also evaluated. RESULTS: This study demonstrated a significant association between the IL-10-592 A/C polymorphism and HBV infection in the Asian population under the overall effect size of allele A versus C. In our subgroup meta-analysis, we found a significant association of IL-10-592 A/C polymorphism to HCV infection susceptibility in Asian populations, although sensitivity analysis showed that the combined result was not associated with the worldwide population. Other IL-10 allele polymorphisms were not associated with HBV or HCV infection. CONCLUSION: IL-10-592 A/C allele might be a risk factor for HBV or HCV in Asians but not in Europeans.

Promotion of self-renewal of embryonic stem cells by midkine.

AIM: To characterize the expression and function of midkine (MK) in an in vitro embryonic stem cell (ESC) culture system. METHODS: To investigate the potential roles of MK, the expression of MK in ESCs was evaluated by RT-PCR and immunocytochemistry. The effects of MK on the self-renewal of ESCs were measured using alkaline phosphatase assays, immunocytochemistry, RT-PCR and colony-forming assays. The mechanism of the growth-promoting effect of MK in mESCs was assessed by cell cycle analysis and Western blot analysis. RESULTS: MK is expressed in mouse embryonic stem cells (mESCs), human embryonic stem cells (hESCs) and mouse embryonic fibroblasts (MEFs). MK promotes proliferation and self-renewal of mESCs both in feeder and feeder free culture systems. It also promotes self-renewal and proliferation of hESCs. Further study showed that MK promotes the growth of mESCs by inhibiting apoptosis while accelerating the progression toward the S phase, and enhances mESC self-renewal through PI3K/Akt signaling pathway. CONCLUSION: MK plays profound roles in ESCs. MK/PTPzeta signaling pathway is a novel pathway in the signal network maintaining pluripotency of ESCs. The results extend our knowledge on pluripotency control of ESCs and the relationship between ESCs and cancers.

Peroxisome proliferator-activated receptor-gamma 34C>G polymorphism and colorectal cancer risk: a meta-analysis.

AIM: To investigate the association between peroxisome proliferator-activated receptor-gamma (PPAR-gamma) gene polymorphism 34 C>G and colorectal cancer (CRC), a meta-analysis review was performed in this report. METHODS: A systematic literature search and selection of eligible relevant studies were carried out. Nine independent studies with a total number of 4533 cases and 6483 controls were included in the meta-analysis on the association between polymorphism 34 C>G and CRC. RESULTS: There was no evidence for the association between PPAR-gamma 34 C>G and CRC if all of the subjects in the nine studies were included. However, CG + GG showed a marginally significant difference from CC (OR = 0.84, 95% CI: 0.69-1.01, P = 0.07) in random-effect model. Stratified meta-analysis indicated that PPAR-gamma 34 C>G was associated with colon cancer (OR = 0.8, 95% CI: 0.65-0.99, P = 0.04) in random-effect model, and the G allele decreased colon cancer risk. No significant association was observed between PPAR-gamma 34 C>G and rectal cancer. CONCLUSION: PPAR-gamma 34 C>G is associated with colon cancer risk, but not associated with CRC and rectal cancer risk.

Histone deacetylase inhibitor promotes differentiation of embryonic stem cells into neural cells in adherent monoculture.

BACKGROUND: Embryonic stem (ES) cells poss unlimited self-renewal capacity and the ability to differentiate into cell of all three germ layers in vitro. Induced differentiation of ES cells to neural lineage cells has great potential in basic study of neurogenesis and regeneration therapy of neurodegenerative diseases. Histone deacetylase (HDAC) inhibitors enhance histone acetylation so that globularly activate gene expression and may initiate multilineage differentiation. In this study, we aimed to develop a method to induce the differentiation of ES cells to neural cells combining HDAC inhibition and neural cell selection. METHODS: In this study, we used HDAC inhibitor sodium butyrate (NaB) to induce the differentiation of mouse embryonic stem cells to neural cells through monolayer culture. After differentiation initiation by histone deacetylase inhibitor sodium butyrate, neural cells were induced and selected with a serum free culture system. RESULTS: Homogeneous neurons without glial cells demonstrated by molecular marker expression were differentiated with the method. The resultant neurons were excitable. CONCLUSION: The method combined differentiation induction effect of HDAC inhibitors and selective culture system to derive neural cells from ES cells, and implied the involvement of epigenetic regulation in neural differentiation.

5-Azacytidine facilitates osteogenic gene expression and differentiation of mesenchymal stem cells by alteration in DNA methylation.

Mesenchymal stem cells (MSCs) are considered to be one of the most promising therapeutic cell sources as they encompass a plasticity of multiple cell lineages. The challenge in using these cells lies in developing well-defined protocols for directing cellular differentiation to generate a desired lineage. In this study, we investigated the effect of 5-azacytidine, a DNA demethylating agent, on osteogenic differentiation of MSCs. The cells were exposed to 5-azacytidine in culture medium for 24 h prior to osteogenic induction. Osteogenic differentiation was determined by several the appearance of a number of osteogenesis characteristics, including gene expression, ALP activity, and calcium mineralization. Pretreatment of MSCs with 5-azacytidine significantly facilitated osteogenic differentiation and was accompanied by hypomethylation of genomic DNA and increased osteogenic gene expression. Taking dlx5 as a representative, methylation alterations of the "CpG island shore" in the promoter caused by 5-azacytidine appeared to contribute to osteogenic differentiation.

In vitro and in vivo suppression of hepatocellular carcinoma growth by midkine-antisense oligonucleotide-loaded nanoparticles.

AIM: To synthesize antisense oligonucleotides (ASODNs) of midkine (MK), package the ASODNs with nanoparticles, and to inhibit hepatocellular carcinoma (HCC) growth using these nanoparticles. METHODS: HepG2 cell proliferation was analyzed in vitro using the 3-(4,5-dimethythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt assay. The in vivo activity of nanoparticles delivering the MK-ASODNs was analyzed by histopathological and immunohistochemical staining and quantitative real time polymerase chain reaction (PCR). RESULTS: The in vitro proliferation of HepG2 cells was significantly inhibited by the nanoparticles packaged with MK-ASODNs (NANO-ASODNs). Furthermore, the NANO-ASODNs significantly inhibited the growth of HCC in the mouse model. CONCLUSION: NANO-ASODNs can significantly suppress the growth of HCC in vitro and in vivo.