星形细胞上调基因-1下游分子miR-885-5p裸鼠肝癌转移实验
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摘要
目的研究星形细胞上调基因-1(metadherin,MTDH)调控的下游分子mi R-885-5p在调控肝癌转移中的作用。方法收集10名癌症患者的癌组织和癌旁样品,检测MTDH和mi R-885-5p表达量。分别构建mi R-885-5p过表达慢病毒质粒和随机序列的阴性对照质粒,带有荧光标签;慢病毒包装后侵染人类肝癌细胞MHCC-97H,使用流式细胞仪分选出稳定细胞系;分别以稳定表达mi R-885-5p的MHCC-97H细胞和稳定表达随机序列的MHCC-97H阴性对照细胞对裸鼠进行腹腔及肝注射,4周后使用活体荧光成像系统检测荧光素酶信号,观察肝癌细胞在裸鼠体内成瘤的动态变化过程。结果 MTDH在肝癌组织样品的表达量较癌旁样品提高2~5倍,mi R-885-5p在肝癌组织样品的表达量较癌旁样品降低40%~90%。注射表达mi R-885-5p的肝癌细胞的动物中,荧光面积和荧光强度较对照组均明显降低(50%)。结论在肝癌组织中MTDH高表达,并且可以负调控mi R-885-5p。动物实验证明,mi R-885-5p对肝癌转移起到抑制作用。
Objective To investigate the function of miR-885-5p mediated by Metadherin(MTDH) in liver cancer metastasis in vivo. Methods MTDH and miR-885-5p expression levels were detected in cancer tissues and paraneoplastic samples of 10 cancer patients. miR-885-5p over-expressed lentiviral plasmid and scrambled negative control plasmid were constructed and infected into human liver cancer cell MHCC-97 H and the stable cell lines were selected by flow cytometry. Then MHCC-97 H cell of stably expressed mi R-885-5p and negative control plasmid were injected into nude mice. After 4 weeks, Luciferase signal was detected by in vivo fluorescence imaging system, and dynamic changes of tumors in nude mice were observed. Results The expression of MTDH in liver cancer tissue sample was 2-5 times higher than that in paraneoplastic sample, while the expression of miR-885-5p in liver cancer sample was 40%- 90% lower than that in paraneoplastic sample. The fluorescence area and intensity decreased 50% after injecting hepatoma carcinoma cell expressed by miR-885-5p in experimental group when compared with control group.Conclusion MTDH shows high-expression in liver cancer samples and it can mediate mi R-885-5p. This experiment indicates that miR-885-5p plays an inhibitory role in liver cancer metastasis in vivo.
Objective To investigate the function of miR-885-5p mediated by Metadherin(MTDH) in liver cancer metastasis in vivo. Methods MTDH and miR-885-5p expression levels were detected in cancer tissues and paraneoplastic samples of 10 cancer patients. miR-885-5p over-expressed lentiviral plasmid and scrambled negative control plasmid were constructed and infected into human liver cancer cell MHCC-97 H and the stable cell lines were selected by flow cytometry. Then MHCC-97 H cell of stably expressed mi R-885-5p and negative control plasmid were injected into nude mice. After 4 weeks, Luciferase signal was detected by in vivo fluorescence imaging system, and dynamic changes of tumors in nude mice were observed. Results The expression of MTDH in liver cancer tissue sample was 2-5 times higher than that in paraneoplastic sample, while the expression of miR-885-5p in liver cancer sample was 40%- 90% lower than that in paraneoplastic sample. The fluorescence area and intensity decreased 50% after injecting hepatoma carcinoma cell expressed by miR-885-5p in experimental group when compared with control group.Conclusion MTDH shows high-expression in liver cancer samples and it can mediate mi R-885-5p. This experiment indicates that miR-885-5p plays an inhibitory role in liver cancer metastasis in vivo.
引文
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2 Srivastava J,Siddiq A,Emdad L,et al.Astrocyte elevated gene-1promotes hepatocarcinogenesis:novel insights from a mouse model[J].Hepatology,2012,56(5):1782-1791.
3 Srivastava J,Siddiq A,Gredler R,et al.Astrocyte elevated gene-1and c-Myc cooperate to promote hepatocarcinogenesis in mice[J].Hepatology,2015,61(3):915-929.
4 Croce CM.Causes and Consequences of micro RNA dysregulation in cancer[J].Nat Rev Genet,2009,10(10):704-714.
5 Valverde BJ.Liver Cancer:Causes,Diagnosis and Treatment[M].New York:Nova Science Publishers,2011:201-205.
6 Makarova-Rusher OV,Medina-Echeverz J,Duffy AG,et al.The yin and yang of evasion and immune activation in HCC[J].J Hepatol,2015,62(6):1420-1429.
7 Scorsetti M,Comito T,Cozzi L,et al.The challenge of inoperable hepatocellular carcinoma(HCC):results of a single-institutional experience on stereotactic body radiation therapy(SBRT)[J].J Cancer Res Clin Oncol,2015,141(7):1301-1309.
8 Dwek M,Schumacher U,Brooks SA.Metastasis Research Protocols[M].2nd ed.New York:Humana Press,2012:10-21.
9 Martin TA,Jiang WG.Tight Junctions in Cancer Metastasis[M].New York:Springer,2013:53-71.
10 Jiang T,Zhu A,Zhu Y,et al.Clinical implications of AEG-1 in liver metastasis of colorectal cancer[J].Med Oncol,2012,29(4):2858-2863.
11 Li C,Liu J,Lu R,et al.AEG-1 overexpression:a novel indicator for peritoneal dissemination and lymph node metastasis in epithelial ovarian cancers[J].Int J Gynecol Cancer,2011,21(4):602-608.
12 Qian BJ,Yan F,Li N,et al.MTDH/AEG-1-based DNA vaccine suppresses lung metastasis and enhances chemosensitivity to doxorubicin in breast cancer[J].Cancer Immunol Immunother,2011,60(6):883-893.
13 Zheng J,Li C,Wu X,et al.Huaier polysaccharides suppresses hepatocarcinoma MHCC97-H cell metastasis via inactivation of EMT and AEG-1 pathway[J].Int J Biol Macromol,2014,64:106-110.
14 Li C,Wu X,Zhang H,et al.A huaier polysaccharide reduced metastasis of human hepatocellular carcinoma SMMC-7721 cells via modulating AUF-1 signaling pathway[J].Tumour Biol,2015,36(8):6285-6293.
15 Nicoloso MS,Spizzo R,Shimizu M,et al.Micro RNAs--the micro steering wheel of tumour metastases[J].Nat Rev Cancer,2009,9(4):293-302.
16 Hayes EL,Lewis-Wambi JS.Mechanisms of endocrine resistance in breast cancer:an overview of the proposed roles of noncoding RNA[J].Breast Cancer Res,2015,17:40.
17 Zhang H,Hao Y,Yang J,et al.Genome-wide functional screening of mi R-23b as a pleiotropic modulator suppressing cancer metastasis[J].Nat Commun,2011,2:554.
18 Wang S,Lv C,Jin H,et al.A common genetic variation in the promoter of mi R-107 is associated with gastric adenocarcinoma susceptibility and survival[J].Mutat Res,2014,769:35-41.