Effect of miR-126 on intracranial aneurysms and its predictive value for rupture of aneurysms.

OBJECTIVE: This study aimed to investigate the effect of miR-126 on intracranial aneurysm (IA) and its predictive value for aneurysm rupture. PATIENTS AND METHODS: Altogether 102 patients (patient group) with IA diagnosed in the Jinhua Municipal Central Hospital from July 2016 to April 2018, and 80 healthy people (normal group) who underwent physical examination during the same period were collected. QRT-PCR was used to detect the expression of miR-126 in serum, analyze the expression of miR-126 in IA, and explore the predictive value on IA rupture. Potential target genes of miR-126 were analyzed by target gene prediction website, and David was used to analyze the enrichment of miR-126 target gene GO and KEGG. RESULTS: The expression of miR-126 in serum of patient group was significantly higher than that of normal group (p < 0.05), ROC curve area was 0.966. The high expressions of miR-126 were directly related to the possibility of large lesions (p < 0.05). Multivariate analysis showed that lesion size and miR-126 expression were independent risk factors for rupture of IA patients. ROC curve showed that lesion size and miR-126 expression area under the curve were 0.707 and 0.827. Altogether 520 potential target sites were found by Venn diagram of Targetscan, miRDB, and Starbase online miR-126 prediction website. GO enrichment and KEGG analysis by David online software found that miR-126 target genes were mainly enriched in 169 biological processes, such as nucleus, transcription, DNA-templated, transcription factor activity, sequence-specific DNA binding, protein binding, and phosphatidylinositol phosphorylation. KEGG analysis found that miR-126 target genes were significantly enriched in MAPK signaling pathway, pathways in cancer, ErbB signaling pathway, MicroRNAs in cancer, and Thyroid hormone signaling pathway. CONCLUSIONS: MiR-126 can be used as a potential diagnostic and predictive indicator for IA occurrence and IA rupture.

Electronic structure and unusual exchange splitting in the spin-density-wave state of the BaFe2As2 parent compound of iron-based superconductors.

The magnetic properties in the parent compounds are often intimately related to the microscopic mechanism of superconductivity. Here we report the first direct measurements on the electronic structure of a parent compound of the newly discovered iron-based superconductor, BaFe2As2, which provides a foundation for further studies. We show that the energy of the spin density wave in BaFe2As2 is mainly lowered through exotic exchange splitting of the band structure.

Reduced expression of small GTPases and hypermethylation of the folate binding protein gene in cisplatin-resistant cells.

Reduced accumulation of cisplatin is the most consistent feature seen in cisplatin-resistant (CP-r) cells that are cross-resistant to other cytotoxic compounds, such as methotrexate. In this report, defective uptake of a broad range of compounds, including [(14)C]-carboplatin, [(3)H]MTX, [(3)H]folic acid (FA), [(125)I]epidermal growth factor, (59)Fe, [(3)H]glucose, and [(3)H]proline, as well as (73)As(5+) and (73)As(3+), was detected in CP-r human hepatoma and epidermal carcinoma cells that we have previously shown are defective in fluid-phase endocytosis. Downregulation of several small GTPases, such as rab5, rac1, and rhoA, which regulate endocytosis, was found in CP-r cells. However, expression of an early endosomal protein and clathrin heavy chain was not changed, suggesting that the defective endocytic pathway is clathrin independent. Reduced expression of the cell surface protein, folate-binding protein (FBP), which is a carrier for the uptake of MTX, was also observed in the CP-r cells by confocal immunofluorescence microscopy and Real-Time PCR. Reactivation of the silenced FBP gene in the CP-r cells by a DNA demethylation agent, 2-deoxy-5-aza-cytidine (DAC) demonstrates that hypermethylation occurred in the CP-r cells. The uptake of [(14)C]carboplatin, [(3)H]FA, and [(3)H]MTX increased in an early stage CP-r cell line (KB-CP1) after treatment with DAC. Both a defective endocytic pathway and DNA hypermethylation resulting in the downregulation of small regulatory GTPases and cell surface receptors contribute to the reduced accumulation of a broad range of compounds in CP-r cells.

Reduced endocytosis and altered lysosome function in cisplatin-resistant cell lines.

We isolated human KB adenocarcinoma cisplatin-resistant (CP-r) cell lines with multidrug-resistance phenotypes because of reduced accumulation of cisplatin and other cytotoxic compounds such as methotrexate and heavy metals. The uptake of horseradish peroxidase (HRPO) and Texas Red dextran was decreased several-fold in KB-CP-r cells, indicating a general defect in fluid-phase endocytosis. In contrast, although EGF receptors were decreased in amount, the kinetics of EGF uptake, a marker of receptor-mediated endocytosis, was similar in sensitive and resistant cells. However, 40-60% of the (125)I-EGF released into the medium after uptake into lysosomes of KB-CP-r cells was TCA precipitable as compared to only 10% released by sensitive cells. These results indicate inefficient degradation of internalised (125)I-EGF in the lysosomes of KB-CP-r cells, consistent with slower processing of cathepsin L, a lysosomal cysteine protease. Treatment of KB cells by bafilomycin A(1), a known inhibitor of the vacuolar proton pump, mimicked the phenotype seen in KB-CP-r cells with reduced uptake of HRPO, (125)I-EGF, (14)C-carboplatin, and release of TCA precipitable (125)I-EGF. KB-CP-r cells also had less acidic lysosomes. KB-CP-r cells were crossresistant to Pseudomonas exotoxin, and Pseudomonas exotoxin-resistant KB cells were crossresistant to cisplatin. Since cells with endosomal acidification defects are known to be resistant to Pseudomonas exotoxin and blocking of endosomal acidification mimics the CP-r phenotype, we conclude that defective endosomal acidification may contribute to acquired cisplatin resistance.

Decreased accumulation of [14C]carboplatin in human cisplatin-resistant cells results from reduced energy-dependent uptake.

We have isolated cisplatin-resistant human liver carcinoma (7404-CP20) cells with reduced accumulation of cisplatin and other drugs (methotrexate, arsenate, and arsenite) to which these cells are cross-resistant. To determine whether the reduction of drug accumulation in cisplatin-resistant cells results from impaired uptake or from active efflux, [(14)C]carboplatin was used for kinetic analysis of drug uptake and efflux. We demonstrate here that the uptake of [(14)C]carboplatin in 7404 parental cells is time, temperature, and energy dependent, and that the rate of uptake is reduced in 7404-CP20 cells. Efflux of [(14)C]carboplatin in cisplatin-resistant cells was comparable to efflux in the parental cisplatin-sensitive cells. There was little effect of temperature (between 37 degrees C and 4 degrees C) on efflux in cisplatin-resistant cells. Immunoblotting with specific antibodies directed to MRP1 and MRP2 (cMOAT) also showed that expression of these two ABC transporter genes was considerably reduced in 7404-CP20 cells and another cisplatin-resistant cell line KB-CP20, in contradistinction to previous studies suggesting that MRP might be responsible for cisplatin efflux. To rule out a generalized defect in uptake of small molecules, fluorescence-activated cell sorter (FACS) analysis of rhodamine 123 uptake showed that there was no difference between cisplatin-sensitive and -resistant cells. The presence of a pleiotropic defect in uptake of [(14)C]carboplatin, [(3)H]methotrexate, [(73)As]arsenate, and [(73)As]arsenite in cisplatin-resistant cells, in association with reduced expression of related cell surface proteins as demonstrated in our previous work, suggests a novel mechanism for acquisition of resistance to cisplatin associated with reduced activity of many different specific uptake systems.

Characterisation of high-level cisplatin-resistant cell lines established from a human hepatoma cell line and human KB adenocarcinoma cells: cross-resistance and protein changes.

Human liver carcinoma cells (BEL-7404) and human KB adenocarcinoma cells were selected by stepwise increases in cisplatin. Drug sensitivity assays indicated that the IC50 value for 7404-CP7.5 cells was 49 micrograms ml-1 cisplatin, 111-fold higher than for the parental hepatoma cells. The IC50 value for KB-CP10 cells was 38 micrograms ml-1 cisplatin, which is 1152-fold higher than for the parental KB cells. The 7404-CP7.5 cells were cross-resistant to methotrexate (39 x), 5-fluorouracil (23 x) and 6-mercaptopurine (13 x), but were sensitive to drugs which are known substrates for the multidrug transporter (P-glycoprotein), including colchicine, vinblastine and actinomycin D. Similar cross-resistance patterns were observed for KB-CP10 cells. No evidence of DNA amplification or expression of the MDR1 gene was found. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed increases in 52 kDa protein(s) in both the soluble cytosolic and crude membrane fractions in 7404-CP(r) cells and in KB-CP(r) cells. The amount of 52 kDa protein was proportional to the degree of resistance of the 7404-CP(r) cells to cisplatin. Two-dimensional gel analysis demonstrated that two polypeptides of molecular mass 52 and 50 kDa were overexpressed in the membrane fractions in both 7404-CP20 and KB-CP20 cells. Using amino acid microsequencing and Western blotting, major 52 kDa protein was identified as the mitochondrial heat shock protein hsp60. Two-dimensional gels of [35S]methionine-labelled polypeptides showed many other changes, including reduction in soluble proteins of approximately 57 kDa molecular weight in KB-CP20 cells, and of 35 kDa in both 7404-CP20 and KB-CP20 cells. These results suggest that alterations of certain proteins occur commonly in cisplatin-resistant cells, particularly proteins of molecular weight 52 and 50 kDa.

[Expression of human multidrug resistance gene (mdr1) cDNA in murine ES cells and in chimeric mice].

Human multidrug resistance gene (mdr1) was introduced into mouse embryonic stem cells (ES-5 line) by calcium phosphate mediated transfection, and transfected ES-5 cells were then selected by stepwise increase in colchicine concentration (30, 50, 100, and 200 ng/ml respectively). Finally, we obtained 4 clones that could be stably grown in culture medium with colchicine at 200 ng/ml and designated as ES-mdr1 clones A, B, C, and D. Southern blot analysis of DNA from ES-mdr1 A and D cells digested by Hind III and hybridized with mdr1 cDNA 5 A probe was shown in Fig. 3. Characteristic 4.8 and 2.4 kb fragments of mdr1 gene were found as expected and their amplification under increased concentration of colchicine in culture medium was also evident from the figure. Slot blot and Northern analysis of total RNA and poly A+ RNA extracted from ES-mdr1 cells were shown in Fig. 4 and 5, demonstrating that ES-mdr1 cells could express mdr1 mRNA. Indirect immunofluorescence analysis with antibodies against p170 glycoprotein indicated that p170 protein translated from mdr1 mRNA was present at the surface of ES-mdr1 cells (Plate I, Fig. 2). The biological characteristics of ES-mdr1 cells cultured in medium containing 200 ng/ml colchicine were investigated. The cells maintained their undifferentiated morphology and grew in nests (Plate I, Fig. 1), like the parental ES-5 cells. When ES-mdr1 cells were cultured in suspension in vitro, these cells were still capable of producing simple and cystic embryoid bodies. ES-mdr1 cells injected subcutaneously into 129 mice formed tumor-like outgrowths giving a great variety of cell types (Plate I, Fig. 4). These results indicated that the integration and expression of human mdr1 gene and selection against colchicine did not affect the pluripotency of ES-mdr1 cells both in vitro and in vivo. However, ES-mdr1 cells, unlike their parental ES-5 cells, could no longer be induced to differentiate by either RA or HMBA (Plate I, Figs. 3a, 3b), indicating that the human mdr1 gene transfected ES cells had changed their competence of inducible response to differentiation in vitro. The details and possible significance of such change require further studies. From the above preliminary data, we are of the opinion that ES-mdr1 cells may serve as a model to study the mode of action of p170 glycoprotein at cellular level and to screen possible means to counteract the action of mdr1 gene.(ABSTRACT TRUNCATED AT 400 WORDS)

[Purification of 31/32 kDa proteins of adult Schistosoma japonicum and studies on their protective immunity].

Schistosoma japonicum adult worm 31/32 kDa proteins (Sj 31/32) were separated on polyacrylamide slab gels and purified by electrophoretic elution. These purified proteins were used to immunize mice in order to observe their protective immunity against challenge. The results of SDS-PAGE,EITB and ELISA indicated that the 31/32 kDa proteins separated and purified by means of these methods were pure and active. It was assumed that Sj 31/32 proteins could reduce worm burden and inhibit the fecundity of schistosome and formation of egg granuloma. The results suggested that 31/32 kDa S. japonicum proteins might be an important component of a multivalent vaccine against schistosomiasis japonica (Figs. 1-4).

Double minute chromosomes carrying the human multidrug resistance 1 and 2 genes are generated from the dimerization of submicroscopic circular DNAs in colchicine-selected KB carcinoma cells.

This study characterizes amplified structures carrying the human multidrug resistance (MDR) genes in colchicine-selected multidrug resistant KB cell lines and strongly supports a model of gene amplification in which small circular extrachromosomal DNA elements generated from contiguous chromosomal DNA regions multimerize to form cytologically detectable double minute chromosomes (DMs). The human MDR1 gene encodes the 170-kDa P-glycoprotein, which is a plasma membrane pump for many structurally unrelated chemotherapeutic drugs. MDR1 and its homolog, MDR2, undergo amplification when KB cells are subjected to stepwise selection in increasing concentrations of colchicine. The structure of the amplification unit at each step of drug selection was characterized using both high-voltage gel electrophoresis and pulsed-field gel electrophoresis (PFGE) techniques. An 890-kb submicroscopic extrachromosomal circular DNA element carrying the MDR1 and MDR2 genes was detected in cell line KB-ChR-8-5-11, the earliest step in drug selection in which conventional Southern/hybridization analyses detected MDR gene amplification. When KB-ChR-8-5-11 was subjected to stepwise increases in colchicine, this circular DNA element dimerized as detected by PFGE with and without digestion with Not 1, which linearizes the 890-kb amplicon. This dimerization process, which also occurred at the next step of colchicine selection, resulted in the formation of cytologically detectable DMs revealed by analysis of Giemsa-stained metaphase spreads.

Human hepatocellular carcinoma cell lines exhibit multidrug resistance unrelated to MRD1 gene expression.

Multidrug resistance of human cancer cells may result from expression of P-glycoprotein, the product of the MRD1 gene, acting as an energy-dependent drug efflux pump. However, direct evidence that expression of the MDR1 gene contributes to the multidrug resistance of human liver carcinomas has not been established. In this study, we tested five cell lines derived from human hepatocellular carcinomas for sensitivity to a variety of drugs used widely as anticancer agents; these included vinblastine, doxorubicin, actinomycin D, mitomycin C, 5-fluorouracil, 6-mercaptopurine, melphalan, methotrexate, cis-platinum and etoposide (VP-16). All five hepatoma cell lines were resistant at different levels to these chemicals compared to human KB cells. Although it has been demonstrated that resistance to vinblastine, colchicine, doxorubicin and actinomycin D in human multidrug-resistant cells is associated with overexpression of P-glycoprotein, very little expression of P-glycoprotein was found in these human hepatoma cells. Neither verapamil nor quinidine, inhibitors of the drug efflux pump, were able to overcome multidrug resistance in hepatoma cells. These results indicate that the multidrug resistance phenotype in human hepatocellular carcinoma cells cannot be attributed to expression of the MDR1 gene, but that novel mechanisms may account for the resistance of these cancer cells.

In situ hybridization analysis of acquisition and loss of the human multidrug-resistance gene.

The extent of multidrug-resistance of human KB carcinoma cell lines has been shown to be proportional to the level of expression of the MDR1 gene. Using an in situ hybridization analysis with 35S-labeled RNA probes, we have found that there is some heterogeneity in expression of the MDR1 gene from cell to cell, but that the average level of expression is proportional to the resistance of the cell line. In the absence of selective pressure, a colchicine-selected multidrug-resistant population with a highly amplified MDR1 gene loses its resistance in parallel with the loss of the amplified gene. Loss of resistance also parallels a decrease in MDR1 RNA expression in the whole cell population. Loss of MDR1 expression in this population is highly heterogeneous, with small clusters of cells maintaining expression even after the population as a whole has become relatively sensitive. This heterogenous loss of expression of the MDR1 gene is consistent with random segregation of amplified DNA segments in the selected cells. The analysis of MDR1 RNA expression by in situ hybridization which is validated by this study should be useful in the study of normal human tissue and tumor samples expressing the MDR1 gene.

Intrinsic drug resistance in human kidney cancer is associated with expression of a human multidrug-resistance gene.

The cloning of the cDNA for the mdr1 gene, whose expression is associated with the development of multidrug-resistance in cultured cells, has made it possible to explore the mechanism of multidrug resistance in human tumors. We have found that normal human kidney, six of eight adenocarcinomas of the kidney, and four cell lines derived from kidney adenocarcinomas express high levels of mdr1 mRNA. Two criteria suggest that primary multidrug resistance in human adenocarcinomas of the kidney results, at least in part, from expression of the mdr1 gene: (1) mdr1 mRNA levels are elevated in four unselected kidney adenocarcinoma cell lines that show a multidrug-resistant phenotype; and (2) multidrug resistance in these kidney cancer cell lines is reversed by verapamil and quinidine, agents known to reverse mdr1-associated drug resistance in cell lines selected for multidrug resistance in vitro. These results suggest that appropriate pharmacological intervention to reverse multidrug resistance might make adenocarcinomas of the kidney more sensitive to chemotherapy with agents such as Adriamycin (Adria Laboratories, Columbus, OH) and the vinca alkaloids.

Multidrug resistance of DNA-mediated transformants is linked to transfer of the human mdr1 gene.

Mouse NIH 3T3 cells were transformed to multidrug resistance with high-molecular-weight DNA from multidrug-resistant human KB carcinoma cells. The patterns of cross resistance to colchicine, vinblastine, and doxorubicin hydrochloride (Adriamycin; Adria Laboratories Inc.) of the human donor cell line and mouse recipients were similar. The multidrug-resistant human donor cell line contains amplified sequences of the mdr1 gene which are expressed at high levels. Both primary and secondary NIH 3T3 transformants contained and expressed these amplified human mdr1 sequences. Amplification and expression of the human mdr1 sequences and amplification of cotransferred human Alu sequences in the mouse cells correlated with the degree of multidrug resistance. These data suggest that the mdr1 gene is likely to be responsible for multidrug resistance in cultured cells.

Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine, adriamycin, or vinblastine show changes in expression of specific proteins.

We have established four cell lines derived from the human KB carcinoma cell line which express high-level multiple drug resistance. One of these lines was selected for resistance to colchicine, one was selected for resistance to colchicine in the presence of the tumor promoter, mezerein, one for resistance to vinblastine, and one for resistance to adriamycin. All of these cell lines are cross-resistant to the other selective agents. The development of multidrug resistance in these cultured human carcinoma cells is associated with a limited number of specific protein alterations revealed by high resolution two-dimensional gel electrophoresis and Western blot analysis. These protein alterations in multidrug-resistant lines include the decreased prevalence of members of a family of proteins of molecular mass 70,000 to 80,000 daltons, pI 4.8-5.0, the increased synthesis of a protein of molecular mass 21,000 daltons, pI 5.0, in the colchicine-resistant cell lines only, and the increased expression of a 170,000-dalton protein in membrane preparations from all of the resistant cells. The loss of the 70,000- to 80,000-dalton proteins in the multidrug-resistant lines, which can also be demonstrated by immunoprecipitation of these proteins with specific antisera, is associated with a loss of translatable mRNA for these proteins. These studies suggest that only a limited number of protein changes occur in multidrug-resistant cell lines.

Isolation of human mdr DNA sequences amplified in multidrug-resistant KB carcinoma cells.

The ability of tumor cells to develop simultaneous resistance to structurally different cytotoxic drugs constitutes a major problem in cancer chemotherapy. It was previously demonstrated that multidrug-resistant Chinese hamster cell lines contain an amplified, transcriptionally active DNA sequence designated mdr. This report presents evidence that multidrug-resistant sublines of human KB carcinoma cells, selected for resistance to either colchicine, vinblastine, or Adriamycin (doxorubicin), display amplification of two different DNA sequences homologous to the hamster mdr gene. Segments of the human mdr DNA sequences, designated mdr1 and mdr2, have been cloned. mdr1 sequences were amplified in all of the highly drug-resistant sublines and were expressed as a poly(A)+ RNA species of 4.5 kilobases that was detected in the resistant cells but not in the parental cell line. No expression of mdr2 sequences was detected. mdr2 sequences were coamplified with mdr1 in some of the multidrug-resistant sublines and, in two independently derived cell lines, underwent very similar rearrangements. The data suggest that the mdr1 gene is involved in multidrug resistance in human cells.

Human multidrug-resistant cell lines: increased mdr1 expression can precede gene amplification.

The development of simultaneous resistance to multiple structurally unrelated drugs is a major impediment to cancer chemotherapy. Multidrug resistance in human KB carcinoma cells selected in colchicine, vinblastine, or Adriamycin is associated with amplification of specific DNA sequences (the multidrug resistance locus, mdr1). During colchicine selection resistance is initially accompanied by elevated expression of a 4.5-kilobase mdr1 messenger RNA (mRNA) without amplification of the corresponding genomic sequences. During selection for increased levels of resistance, expression of this mRNA is increased simultaneously with amplification of mdr1 DNA. Increased expression and amplification of mdr1 sequences were also found in multidrug-resistant sublines of human leukemia and ovarian carcinoma cells. These results suggest that increased expression of mdr1 mRNA is a common mechanism for multidrug resistance in human cells. Activation of the mdr1 gene by mutations or epigenetic changes may precede its amplification during the development of resistance.

Protein p53 and inducer-mediated erythroleukemia cell commitment to terminal cell division.

Inducer-mediated murine erythroleukemia cell (MELC) differentiation provides a model for examining factors determining terminal cell differentiation. The nuclear protein, p53, has been implicated as a potential determinant of cell cycle progression and cell differentiation. In this study p53 content and synthesis, during inducer-mediated MELC differentiation, has been examined with monoclonal antibodies to p53. A decrease in p53 synthesis and content was demonstrated during induced differentiation. As determined by cell cycle fractionation, the decrease in p53 is manifest at all stages of the cell cycle. Hemin, which induces globin mRNA accumulation but not terminal cell division, fails to decrease p53 content. A MELC variant resistant to inducer-mediated commitment to terminal cell division also fails to decrease p53 levels in response to inducers. These experiments suggest that p53 is implicated in MELC cell proliferation and that an induced decrease in p53 may be responsible for G1 phase prolongation and terminal G1 arrest.