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

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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.

Midkine accumulated in nucleolus of HepG2 cells involved in rRNA transcription.

AIM: To investigate the ultrastructural location of midkine (MK) in nucleolus and function corresponding to its location. METHODS: To investigate the ultrastructural location of MK in nucleolus with immunoelectronic microscopy. To study the role that MK plays in ribosomal biogenesis by real-time PCR. The effect of MK on anti-apoptotic activity of HepG2 cells was studied with FITC-conjugated annexin V and propidium iodide PI double staining through FACS assay. RESULTS: MK mainly localized in the granular component (GC), dense fibrillar component (DFC) and the border between the DFC and fibrillar center (FC). The production of 45S precursor rRNA level was decreased significantly in the presence of MK antisense oligonucleotide in the HepG2 cells. Furthermore, it was found that exogenous MK could protect HepG2 from apoptosis significantly. CONCLUSION: MK was constitutively translocated to the nucleolus of HepG2 cells, where it accumulated and mostly distributed at DFC, GC components and at the region between FC and DFC, MK played an important role in rRNA transcription, ribosome biogenesis, and cell proliferation in HepG2 cells. MK might serve as a molecular target for therapeutic intervention of human carcinomas.

Serum Lipidomics Profiling to Identify Biomarkers for Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide, which ranks top in both incidence and mortality. To broaden our understanding of the lipid metabolic alterations in NSCLC and to identify potential biomarkers for early diagnosis, we performed nontargeted lipidomics analysis in serum from 66 early-stage NSCLC, 40 lung benign disease patients (LBD), and 40 healthy controls (HC) using Ultrahigh Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UHPLC-Q-TOF/MS). The identified biomarker candidates of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) were further externally validated in a cohort including 30 early-stage NSCLC, 30 LBD, and 30 HC by a targeted lipidomic analysis. We observed a significantly altered lipid metabolic profile in early-stage NSCLC and identified panels of PCs and PEs to distinguish NSCLC patients and HC. The levels of PCs and PEs were found to be dysregulated in glycerophospholipid metabolism, which was the top altered pathway in early-stage NSCLC. Receiver operating characteristic (ROC) curve analysis revealed that panels of PCs and PEs exhibited good performance in differentiating early-stage NSCLC and HC. The levels of PE(16:0/16:1), PE(16:0/18:3), PE(16:0/18:2), PE(18:0/16:0), PE(17:0/18:2), PE(18:0/17:1), PE(17:0/18:1), PE(20:5/16:0), PE(18:0/18:1), PE(18:1/20:4), PE(18:0/20:3), PC(15:0/18:1), PC(16:1/20:5), and PC(18:0/20:1) in early-stage NSCLC were significantly increased compared with HC (p<0.05). Overall, our study has thus highlighted the power of using comprehensive lipidomic approaches to identify biomarkers and underlying mechanisms in NSCLC.

Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines.

AIM: To construct an expression plasmid encoding human wild-type midkine (MK) and enhanced green fluorescence protein (EGFP) fusion protein (MK-EGFP), and to analyze the subcellular localization of MK in different carcinoma cell lines. METHODS: Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells, respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected. RESULTS: Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines. CONCLUSION: This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.

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.

[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.

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.

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.

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

AIM: To evaluate the in vivo antitumor effects of antisense oligonucleotides targeting midkine (MK-AS). METHODS: An in situ human hepatocellular carcinoma (HCC) model was established in mice livers orthotopically. The MK-AS and 5- fluorouracil (5-Fu) were administered intravenously. The tumor sizes and plasma alpha-fetoprotein (AFP) were measured by calipers and radiation immunoassay respectively. The morphology of tumors was evaluated by hematoxylin-eosin staining of histological sections. Human MK, p53, Bax, Bcl-2, and caspase-3 protein content were detected by Western blotting. RESULTS: MK-AS significantly inhibited in situ human HCC growth in mice compared with the saline group in a dose-dependent manner. After the treatment with MK-AS or with 5-Fu, the plasma AFP concentration decreased in a dose-dependent manner. Interestingly, MK-AS also clearly downregulated the protein level of Bcl-2, and upregulated p53, Bax, and caspase-3 in the hepatocellular carcinoma tissue. CONCLUSION: These results demonstrated that MK-AS was an effective antitumor antisense oligonucleotide in vivo in mice; its antitumor effect is associated with the increase of pro-apoptotic proteins, such as p53, Bax, and caspase-3, and the decrease of the anti-apoptotic protein, Bcl-2.

Antisense oligonucleotide targeting p53 increased apoptosis of MCF-7 cells induced by ionizing radiation.

AIM: To investigate the effect of antisense compounds (AS) targeting human p53 mRNA on radiosensitivity of MCF-7 cells. METHODS: Western blotting and RTPCR were used to analyze the protein content and mRNA level. Additionally, cell proliferation, cell cycle and cell apoptosis were all analyzed in irradiated or sham-irradiated cells. RESULTS: Among the five antisense compounds (AS), AS3 was identified to efficiently inhibit p53 mRNA level and protein content. Interestingly, AS3 transfer has little effect on cell proliferation in DU-145 cells (mutant p53) after ionizing radiation (IR). In contrast, a marked increase of cell apoptosis and growth inhibition were observed in MCF-7 cells (wild-type p53), suggesting that AS3 can increase radiosensitivity of MCF-7 cells. Additionally, it was also observed that the transfection of AS3 decreased the fraction of G1 phase cells, and increased the proportion of S phase cells compared to untreated cells 24 h after IR in MCF-7 cell lines. CONCLUSION: AS3 transfection increases MCF-7 cell apoptosis induced by 5 Gy-radiation, and this mechanism may be closely associated with abrogation of G1 phase arrest.