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Objective To construct the miRNA‐21 eukaryotic overexpression vector pmR‐21 and to explore its regulation effect on the expression of c‐myc gene in HepG2 .2 .15 cells .Methods The miRNA‐21 precursor gene fragment pre‐miRNA‐21 was amplified by PCR ,then connected to the pmR‐mCherry plasmid vector after double enzyme digestion ,the accuracy of the recombi‐nant vector was verified by double enzyme digestion and sequencing ;then the recombinant vector was transfected into HepG2 .2 .15 cells ,the fluorescent protein expression was observed under the fluorescence microscopy at 24 h and the transfection efficiency was detected by flow cytometry ;the expression of miRNA‐21 was evaluated by real‐time quantitative PCR;at 72 h after transfection ,the expression levels of c‐myc gene were detected by RT‐PCR and Western blot ;CCK‐8 was used to detect the cell proliferation in each group .Results The double enzyme digestion and Western blot verified that the target gene fragment was inserted into the pmR‐mCherry vector;at 24 h after transfection ,intracellular strong fluorescence was seen ,the transfection efficiency was higher than 50% ;miRNA‐21 expression level of the pmR‐21 recombinant vector group was significantly increased;c‐myc gene expression was increased in the pmR‐21 recombinant vector group at 72 h after transfection ,the cell proliferation in the pmR‐21 recombinant group was faster than that in the control group(P<0 .05) .Conclusion The pmR‐21 eukaryotic overexpression vector is successfully con‐structed ,this recombinant vector can express miRNA‐21 stably ;miRNA‐21 can up‐regulate c‐myc gene expression ,c‐myc gene is one of miR‐21′s targets for playing a cancer‐promoting action .
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Objective To investigate the clinical significance of c-myc、Bcl-2 protein expression, DNA ploidy and their relationship in breast cancer. MethodsWT5”BZ The expression of c-myc、Bcl-2 protein in 146 breast tumor tissues was examined by using immunohistochemical methods(S-P)and DNA ploidy in 72 cases of breast carcinoma by flow cytometry. ResultsKG1 Nuclear expression of c-myc protein was detected in 9 6% of tumors, and it was related to the DNA aneuploidy and prognosis. Cytoplasmic expression of c-myc protein was present in 91 8% of the carcinomas. Moderate to strong c-myc protein expression in cytoplasmic was associated with the positive status of ER. Bcl-2 protein was positive in 78 8% of cancers. Overexpression of Bcl-2 protein was associated with ER(+) status and the lack of axillary lymph node metastasis. DNA aneuploidy was found in 51 4% of 72 cancers, it was related to nuclear expression of c-myc protein and prognosis.WT5”HZConclusion Nuclear expression of c-myc protein and DNA ploidy are important prognostic factors.
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AIM:To study the induction of apoptosis by c-myc antisense oligonucleotide in osteosarcoma cell(MG-63).METHODS:The designed c-myc antisense oligonucleotide fragment was transfected into human osteosarcoma MG-63 cells.The cell growth and apoptosis were measured by the methods of MTT,FCM,HE staining and transmission electron microscopy.RESULTS:The results showed that the proliferation of human osteosarcoma MG-63 cells was inhibited and apoptotic rate was 37.92% when treated with c-myc antisense oligonucleotide at the does of 10.0 ?mol/L for 48 h.c-myc antisense oligonucleotide(10.0 ?mol/L) also inhibited the expression of c-myc protein.CONCLUSION:c-myc antisense oligonucleotide is able to induce apoptosis in human osteosarcoma MG-63 cells.
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AIM: To evaluate the inhibitory effect of galactose (Gal)-polyethyleneimine (PEI)-c-myc antisense oligodeoxynucleotide (ASODN) complex on proliferation of human hepatocellular carcinoma cells. METHODS: Human hepatocellular carcinoma cell line Bel-7402 was treated with Gal-PEI-ASODN complex. Cell proliferation was tested by trypan blue dye at different time points and with various concentrations of ASODN treatment. Cell morphology was observed under inverted microscope, cell hypodiploid percentage was analyzed by flow cytometry and cell ultrastructure was observed through electron microscopy. RESULTS: Compared with ASODN group (20 ?mol/L) from (0 h) to 96 h, Gal-PEI-ASODN complex (with ASODN 0.75 ?mol/L) significantly suppressed Bel-7402 cells proliferation, the ASODN concentration within Gal-PEI-ASODN complex and time course acquired were significantly lower and shorter, respectively. Incubated with pure ASODN at different concentrations for 72 hours, cell proliferation was inhibited and IC_(50) was 20.9 ?mol/L; while mediated with galactose receptor for 48 hours, ASODN significantly inhibited cell proliferation and IC_(50) was only 0.294 ?mol/L, the inhibitory efficacy of ASODN enhanced 70.9 folds. While Bel-7402 cells were incubated with Gal-PEI-ASODN complex for 48 hours, cell hypodiploid percentage was much higher than ASODN groups and cell apoptosis was seen under electron microscopy. CONCLUSIONS: Galactose receptor mediated ASODN delivery may significantly increase proliferation inhibition efficacy on Bel-7402 cells. [
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AIM: To investigate the transcription regulation of the promoter of human telomerase reverse transcriptase (hTERT) by transcription factors c-myc and [STBX]mad1. METHODS: The various plasmids including wild type hTERT (Tw) or mutant type hTERT (Td) which both harboring luciferase gene, the expression plasmids of c-myc and [STBX]mad1, and their control vectors were constructed. The plasmids were co-trans fected into bladder cancer cell lines T24, EJ and control cells COS-7 or fibrocytes by DOTAP liposome in various combining manner, respectively. The reporter gene luciferase activities in various groups were measured 48 h after transfection. RESULTS: The luciferase activities in T24 and EJ cells treated with Tw were much higher than that in COS-7 and fibrocytes cells treated with Tw, as well as higher than that in T24 and EJ cells treated with Td, respectively. In bladder cancer T24 and EJ cells, transcription factor c-myc and [STBX]mad1 positively and negatively regulated Tw expression in a dose-dependent manner. However, the effects of c-myc and [STBX]mad1 on Td were completely opposite to Tw. Combined with c-myc and [STBX]mad1, down-regulation of Tw expression was observed. CONCLUSION: c-myc and [STBX]mad1 regulates the transcriptional activity of hTERT promoter in bladder cancer cells, and the effects might highly depend on the conservative E-box sequence CACGTG.