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Objective:To determine the effects of silencing glucose regulated protein ( GRP94 ) on the proliferation and apoptosis of breast carcinoma MCF7 cells. Methods:Chemically synthesized siRNA targeting GRP94 gene and transfected into MCF7 cells used by Liopfectamine RNAIMAX. The mRNA and protein expression levels of GRP94,cyclinD1,Bax and Bcl-2 were detected by Real-time PCR and Western blot. CCK8 assay was used to detect the effect of specific GRP94 siRNA on cell proliferation and the effect on cell cycle and apoptosis were analyzed by flow cytometry and Hoechst 33258 staining. Results:Compared with the siRNA-NC cells, the expression of GRP94 was significantly down-regulated in MCF7 cells. Knockdown of GRP94 in MCF7 cells decreased cell proliferation and promoted cell apoptosis. The expression of cyclinD1and Bcl-2levels were significantly reduced, and Bax level was increased in siRNA-GRP94 MCF7 cells. Conclusion: The siRNA-mediated GRP94 silence significantly inhibits MCF7 cell proliferation,promoted cell apoptosis by down-regulating cyclin D1,Bcl-2 expression and up-regulating the Bax expression in MCF7 cells.
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Aim To investigate ALEX1 gene expres-sion in cervical cancer tissues and adjacent non-can-cerous tissues, and to explore the ALEX1 genetic influ-ence on cell proliferation,cycle and apoptosis of human cervical cancer cell line HeLa. Methods ALEX1 protein expression in cervical cancers and in non-can-cerous cervical tissues was evaluated using immunohis-tochemical method. A small interference RNA targeting ALEX1 gene was transfected into HeLa cells′, and the effect of ALEX1 interference on HeLa cells′ cycle and apoptosis was analysed by flow cytometry. The effect of ALEX1 interference on HeLa cells′ proliferation and sensitivity to resveratrol was analysed by CCK-8 assay. Results ALEX1 protein expression was significantly increased in cervical cancer tissues compared with non-cancerous tissues. HeLa cells′proliferation was inhibi-ted compared with control group and blank group. He-La cells′ sensitivity to resveratrol was enhanced com-pared with control group blank group. Conclution SiRNA silencing of ALEX1 gene could significantly in-hibit HeLa cells′ proliferation and enhance resveratrol ability of inhibiting HeLa cells′proliferation.
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Objective:To investigate the effects of ALEX1 overexpression on cell proliferation and apoptosis of human breast cancer cell line MCF-7.Methods: MCF-7 cells were infected recombinant lentivirus LV5-ALEX1 and the negative control lentivirus LV5-NC,respectively.After 72 h, the expression of ALEX1 was detected by Real-Time PCR and Western blot.CCK8 assay were performed to observe the proliferation ability after 24 h, 48 h, 72 h, 96 h.The effect of overexpression ALEX1 on apoptosis was determined by flow cytometry.The level of Bax,BCL-2 and active caspase3 was detected by Western blot.Results:The mRNA level of ALEX1 markedly increased after 72 h(165.81±12.14 vs 52.29±2.32,P<0.01).In CCK8 assay,the results revealed that the cell pro-liferation was inhibited compared with control group in 48 h,72 h,96 h( P<0.05).The results revealed that overexpression of ALEX1 enhanced MCF-7 apoptosis(20.55%±2.50 % vs 3.60%±1.614%,P<0.05).The results by Western blot showed that the protein levels of Bax and active caspase were increased in LV5-ALEX1 group compared with LV5-NC group.However,the protein levels of BCL-2 was decreased in LV5-ALEX1 group compared with LV5-NC group.Conclusion:Overexpression of ALEX1 significantly reduced MCF-7 cancer cells proliferation and induced MCF-7 cells apoptosis.
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Aim To investigate the effect of small inter-ference RNA-mediated silcencing of the Bmi-1 gene on cell invasion and metastasis in human nasopharyngeal carcinoma cell line CNE-1 . Methods Chemically syn-thesized siRNA targeting the Bmi-1 gene was transfect-ed into CNE-1 cells, which had high invasive and me-tastatic potential. The expression of Bmi-1 mRNA and protein were detected by quantative Real-time PCR and Western blot, respectively. The effects of Bmi-1 knockdown on CNE-1 cells migration and invasion were analysied by Transwell migration assay and Matrigel in-vasion assay. Results Transfected with Bmi-1 siRNA significantly down-regulated the expression of Bmi-1 mRNA and protein as compared with the control group. CNE-1 cells transfected with Bmi-1 siRNA had lower levels of invasion and migration capacity than cells in the control group. Conclusion SiRNA-media-ted silencing of the Bmi-1 gene could significantly in-hibit cell migration and invasion in human nasopharyn-geal carcinoma cell line CNE-1 .
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Molecular medicine and cancer research center in Chongqing Medical University adhered to academic honesty and credit education ,established original laboratory records system , regularly carried out seminar and improved paper submission program thus to reject academic miscon-ducts from the source,guarantee the authenticity of the data and improve the academic moral level of postgraduates.
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In this paper,the recent advances in both biomedical sciences and higher medical education reform were reviewed and analyzed.Furthermore,we proposed and reconstructed the teaching system of biochemistry and molecular biology course in our university,including its teaching content,teaching methods,teacher team,teaching management,etc.The preliminary practice of this system has obtained significant positive effects on teaching quality and student performance.
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In order to strengthen the graduate management in scientific research platform and to ensure the quality of graduate training,the ideological and moral education was invigorated through establishing virtual party branch,the behavior was regulated through establishing and amplifying the daily management system,the student interests were protected through establishing financial management system and the cultivation quality was guaranteed through perfecting the academic management system.Satisfactory results were achieved in molecular medicine and cancer research center in Chongqing Medical University.
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X-box binding protein1 (XBP1) is an important transcription factor, which participates in many signal transduction procession.To investigate the biological function of XBP1, yeast two-hybrid system to screen proteins interacting with XBP1 in hepatocytes was performed. The XBP1 coding sequence was amplified by polymerase chain reaction (PCR) method, and was cloned in pGEM-T vector. After the target region was sequenced, it was subcloned into the bait plasmid pGBKT7, then was transformed into yeast AH109(a type). After the expression of bait plasmid pGBKT7-XBP1 in AH109 yeast strains were proved by Western blot. The transformed yeast AH109 was mated with yeast Y187(α type) containing hepatocyte cDNA library plasmids pACT2 in 2 ×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medilium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After sequencing and verifying ORF of positive colonies,7 different kinds of proteins were obtained. In order to further testify the interaction between the screened proteins and XBP1, one of positive colonies MT1E was cloned. The interaction between MT1E and XBP1 in vitro/in vivo were examined successfully with GST pulldown and coimmunoprecipitations. It was shown that MT1E would be a new regulatory protein of XBP1. These screened proteins by yeast two-hybird system were closely correlated with liver fundamental metabolism,protein synthesis and transport,cell proliferation and apoptosis. The results mentioned above contributed to reveal the XBP 1 biological function, and brought some new clues for further exploration of the expressing and regulating mechanism of XBP1.
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Through setting up the elective course Protein Abnormity and Diseases for the undergraduates,we combine basic courses and clinic courses to enhance the students' enthusiasm in biochemistry.And at the same time modern molecule biology knowledge is organically combined with clinic lessons.
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Objective To learn the effect on carcinoma xenograft in nude mice by inhibiting human papillomavirus 16(HPV16) E6 gene expression in CaSki cell. Methods The recombinant plasmids expressing HPV16 E6 small interference RNA (siRNA) were transfected into CaSki cell. The cells expressing recombinant plasmid was screened out with G418. The expression of E6 mRNA was determined by RT-PCR. The cells were inoculated into BALB/c nude mice subcutaneouly and the growth of the xenograft carcinoma was observed. After the pGensil-CH2 recombinant was injected into the carcinoma, the growth of carcinoma and pathological changes of carcinoma were observed. Results The CaSki cell expressing E6 siRNA was obtained, and HPV16 E6 mRNA expression in CaSki cell was down-regulated. The oncogenicity of the CaSki cell expressing E6 siRNA was degraded, the inhibition rate was up to 71.4% as compared with that of control group. The growth of tumor in nude mice was inhibited after the E6 siRNA plasmids were injected into the nude mice. The volume and weight of the tumor treated by siRNA were smaller than that of control group significantly. More necrotic area and less cell division phase were observed under light microscope in the E6 siRNA treated tumor. Conclusion The oncogenicity of the CaSki cell was degraded after silencing HPV16 E6 gene in CaSki cell by E6 siRNA.
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Aim To construct a eukaryotic expression vector of human MCHR2 and to analyze the stable expression and signal transduction pathways of MCHR2 in CHO cells. Methods The full-length MCHR2 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and inserted into eukaryotic expres-sion vector pcDNA3.1(+), resulted in the recombinant expression vector pcDNA3.1-MCHR2. The recombinant plasmid was confirmed by the restriction enzymatic digestion and DNA sequencing analysis, and then transfected into CHO cells by lipofectamine. A stably-transfected cell line was obtained by the dominant G418 selection, and the expression of the MCHR2 gene on transcription and translation levels were identified by RT-PCR and Western blot. Signal transduction pathways mediated by MCHR2 were analyzed by measurement of intracellular cAMP and calcium. Results The eukaryotic expression vector pcDNA3.1-MCHR2 was successfully constructed and the MCHR2 gene was stably transfected into CHO cells. A stably-transfected cell line was established and the MCHR2 gene was efficiently transcribed and translated. MCH stimulation had no effect on the production of cAMP, however, it could induce a clear and transient increase of intracellular calcium, suggesting that MCHR2 was only coupled to Gq protein. Conclusion The stable expression of MCHR2 and analysis of its signal transduction pathways in CHO cell line provided a solid experimental foundation for further studies on the function of the MCHR2 gene in vitro.