FABP-5基因沉默对肝癌Bel-7402细胞生物学特性及人参川穹嗪敏感性的影响
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摘要
目的探讨通过siRNA干扰技术沉默表皮脂肪酸结合蛋白-5(FABP-5)基因表达对人肝癌Bel-7402细胞生物学特性及人参川穹嗪敏感性的影响。方法以人肝癌Bel-7402细胞为研究对象,根据FABP-5mRNA编码序列设计并合成干扰siRNA序列,瞬时转染Bel-7402细胞。将人肝癌Bel-7402细胞分为FABP-5siRNA组、阴性对照组、空白对照组。用RT-PCR和Western blot法分别从mRNA水平和蛋白水平检测FABP-5基因的表达;CCK8法测定细胞体外增殖能力;流式细胞技术检测各组细胞周期和凋亡的变化情况;细胞侵袭小室法检测各组细胞的体外侵袭力;MTT法观察FABP-5基因沉默后Bel-7402细胞对人参川穹嗪的敏感性。结果 RT-PCR和Western blot显示,FABP-5 siRNA组较阴性对照组和空白对照组的FABP-5 mRNA和蛋白表达都明显下降;FABP-5 siRNA组细胞的生长速度明显减慢,细胞周期阻滞在G0/G1期,S期细胞数减少;与阴性对照组和空白对照组相比,FABP-5 siRNA组细胞的凋亡率明显升高(P<0.01),增殖、侵袭能力显著下降(P<0.05);FABP-5 siRNA组细胞对人参川穹嗪的敏感性显著增强(P<0.05)。结论特异性沉默FABP-5基因表达能够降低肝癌细胞的侵袭能力,抑制细胞的增殖,将细胞周期阻滞于G_0/G_1期,使其凋亡显著增加。
Objective To investigate the si RNA silencing epidermal fatty acid binding protein-5(FABP-5) gene expression in human hepatocellular carcinoma Bel-7402 cells and the sensitivity of Ginseng ligustrazine. Methods The interference si RNA sequence was designed and synthesized according to the FABP-5 sequence of m RNA, and then transiently transfected into human hepatoma cell line Bel-7402 cells. The cells were divided into 3 groups: FABP-5 si RNA group, negative control group and blank control group. RT-PCR and Western blot method were used to detect the expression of FABP-5 gene at m RNA and protein level. Cell proliferation in vitro was determined by CCK8 method. Flow cytometry was applied to examine periodic change of the cells of each group and cell apoptosis. Cell invasion was detected with transwell inserts. Each experiment was repeated 3 times. The sensitivity of Ginseng ligustrazine to Bel-7402 cells was observed with MTT assay after FABP-5 gene silencing. Results The results from RT-PCR and Western blot showed that the m RNA of FABP-5 or protein in FABP-5 si RNA group was significantly lower than that in negative control group and blank control group. The growth rate of cells in si RNA group was significantly decreased, cell cycle was arrested in G0/G1 phase, and S phase cells decreased. As compared to the negative control group or blank control group, the apoptosis of cells in FABP-5 si RNA group was significantly increased(P < 0.01) and proliferation or invasion ability decreased significantly(P < 0.05). The sensitivity of Ginseng ligustrazine in FABP-5 si RNA group cells increased significantly(P < 0.05). Conclusion The expression of FABP-5 gene reduces the invasion ability of hepatocellular carcinoma cells, inhibits the proliferation of cells, blocks the cell cycle in G0/G1 phase, and increases the apoptosis.
Objective To investigate the si RNA silencing epidermal fatty acid binding protein-5(FABP-5) gene expression in human hepatocellular carcinoma Bel-7402 cells and the sensitivity of Ginseng ligustrazine. Methods The interference si RNA sequence was designed and synthesized according to the FABP-5 sequence of m RNA, and then transiently transfected into human hepatoma cell line Bel-7402 cells. The cells were divided into 3 groups: FABP-5 si RNA group, negative control group and blank control group. RT-PCR and Western blot method were used to detect the expression of FABP-5 gene at m RNA and protein level. Cell proliferation in vitro was determined by CCK8 method. Flow cytometry was applied to examine periodic change of the cells of each group and cell apoptosis. Cell invasion was detected with transwell inserts. Each experiment was repeated 3 times. The sensitivity of Ginseng ligustrazine to Bel-7402 cells was observed with MTT assay after FABP-5 gene silencing. Results The results from RT-PCR and Western blot showed that the m RNA of FABP-5 or protein in FABP-5 si RNA group was significantly lower than that in negative control group and blank control group. The growth rate of cells in si RNA group was significantly decreased, cell cycle was arrested in G0/G1 phase, and S phase cells decreased. As compared to the negative control group or blank control group, the apoptosis of cells in FABP-5 si RNA group was significantly increased(P < 0.01) and proliferation or invasion ability decreased significantly(P < 0.05). The sensitivity of Ginseng ligustrazine in FABP-5 si RNA group cells increased significantly(P < 0.05). Conclusion The expression of FABP-5 gene reduces the invasion ability of hepatocellular carcinoma cells, inhibits the proliferation of cells, blocks the cell cycle in G0/G1 phase, and increases the apoptosis.
引文
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[9]Shi JL,Cao J,Su JJ,et al.Expression of epidermal fatty acid-binding protein in cross-species hepatocellular carcinoma.Chin J Hepatol,2012,20(4):270-274.(in Chinese)史俊林,曹骥,苏建家,等.表皮型脂肪酸结合蛋白在跨种属肝癌组织中的表达.中华肝脏病杂志,2012,20(4):270-274.
[10]El-Serag HB.Hepatocellular carcinoma.N Engl J Med,2011,365(12):1118-1127.
[11]Vivarelli M,Risaliti A.Liver transplantation for hepatocellular carcinoma on cirrhosis:strategies to avoid tumor recurrence.World J Gastroenterol,2011,17(43):4741-4746.
[12]Lu XX,Cao J,Li Y,et al.Expression of Cdc25a in cross-species hepatocellular carcinoma tissues and its significance.Chin J Pathophysiology,2014,30(5):820-824.(in Chinese)卢晓旭,曹骥,李瑗,等.Cdc25a在跨种属肝癌组织中的表达及其意义.中国病理生理杂志,2014,30(5):820-824.
[13]Xie Y,Dai JG.Study on mechanism of TCM treatment of primary liver cancer.J Changchun Univ Traditional Chin Med,2011,27(5):738-740.(in Chinese)谢益,戴建国.原发性肝癌的中医药治疗机制研究.长春中医药大学学报,2011,27(5):738-740.
[14]Chen HY,Lang QB,Yue XQ,et al.Initial analysis on affects and factors of therapeutic prescription with Chinese drugs upon post-operation recurrence of hepatocarcinoma.Jilin J Traditional Chin Med,2011,31(7):636-638.(in Chinese)陈红云,郎庆波,岳小强,等.中药治疗方案对肝癌术后复发的影响因素初步分析.吉林中医药,2011,31(7):636-638.
[15]Jemal A,Bray F,Center MM,et al.Global cancer statistics.CA Cancer J Clin,2011,61(2):69-90.
[16]Jeong CY,Hah YS,Cho BI,et al.Fatty acid-binding protein 5promotes cell proliferation and invasion in human intrahepatic cholangiocarcinoma.Oncol Rep,2012,28(4):1283-1292.
[17]Fang LY,Wong TY,Chiang WF,et al.Fatty-acid-binding protein 5promotes cell proliferation and invasion in oral squamous cell carcinoma.J Oral Pathol Med,2010,39(4):342-348.
[18]Shi JL,Cao J,Su JJ,et al.Clinical significance of upregulated expression of epidermal fatty acid binding protein in hepatocellular carcinoma tissues from different species.World Chin J Digestology,2013,21(11):963-969.(in Chinese)史俊林,曹骥,苏建家,等.表皮型脂肪酸结合蛋白在不同种属肝癌组织中的高表达及其临床意义.世界华人消化杂志,2013,21(11):963-969.
[19]Li J,Xu LZ.Protective effect of chuanhunzine injection on acute alcoholic liver injury in mice.J Huaihai Med,2015,33(3):250-252.(in Chinese)李甲,徐凌忠.川穹嗪注射液对小鼠急性酒精性肝损伤的保护作用观察.淮海医药,2015,33(3):250-252.
[20]Storch J,Thumser AE.Tissue-specific functions in the fatty acid-binding protein family.J Biol Chem,2010,285(43):32679-32683.
[21]Diao JS,Zhang X,Guo SZ.Progression of epidermal fatty acid binding protein.J Int Pathol Clin Med,2009,29(1):50-53.(in Chinese)刁建升,张曦,郭树忠.表皮型脂肪酸结合蛋白的研究进展.国际病理科学与临床杂志,2009,29(1):50-53.
[22]Armstrong EH,Goswami D,Griffin PR,et al.Structural basis for ligand regulation of the fatty acid-binding protein 5,peroxisome proliferator-activated receptorβ/δ(FABP5-PPARβ/δ)signaling pathway.J Biol Chem,2014,289(21):14941-14954.
[23]Zhou LL.Effect of silencing FABP-5 gene by RNA interference on the growth of Hep G2 human hepatoma and xenografts in nude mice.Nanning:Guangxi Medical University,2015.(in Chinese)周玲丽.RNA干扰技术沉默FABP-5基因对人肝癌Hep G2细胞和裸鼠移植瘤生长的影响.南宁:广西医科大学,2015.
[24]Feng ZQ,Zuo GW,Shi QQ,et al.Mechanism of ginsenoside Rh2inhibiting Hep G2 cells migration.Chin J Immunol,2015,31(1):61-65.(in Chinese)冯子强,左国伟,石庆强,等.人参皂苷Rh2抑制肝癌Hep G2细胞迁移的实验研究.中国免疫学杂志,2015,31(1):61-65.
[25]Fan J,Zhang L,Wang X.Effect of ginsenoside Rk3 on apoptosis of Hep G2.Acta Univ Med Anhui,2016,51(11):1604-1607,1608.(in Chinese)范洁,张磊,王啸.人参皂苷Rk3对肝癌Hep G2细胞凋亡的影响.安徽医科大学学报,2016,51(11):1604-1607,1608.
[26]Feng QF,Bi L,Yan XJ,et al.Effect of tetramethypyrazine on apoptosis of Hep G2 cells and expression of associated protein.J Nanjing Univ Traditional Chin Med,2015,31(3):246-249.(in Chinese)冯全服,毕蕾,颜晓静,等.川芎嗪调控肝癌Hep G2细胞凋亡及对相关蛋白表达的影响.南京中医药大学学报,2015,31(3):246-249.
[2]Yang Z,Zhou L,Wu LM,et al.Overexpression of long non-coding RNA HOTAIR predicts tumor recurrence in hepatocellular carcinoma patients following liver transplantation.Ann Surg Oncol,2011,18(5):1243-1250.
[3]Bi L,Yan XJ,Yang Y,et al.Optimization of effective component formula of salviae miltiorrhizae radix et rhizoma and ginseng radix et rhizoma on hepatoma carcinoma using orthogonal design.Chin J Exp Traditional Med Formulae,2015,21(13):82-86.(in Chinese)毕蕾,颜晓静,杨烨,等.正交设计优选丹参-人参组分抗肝癌配伍研究.中国实验方剂学杂志,2015,21(13):82-86.
[4]Thun MJ,De Lancey JO,Center MM,et al.The global burden of cancer:priorities for prevention.Carcinogenesis,2010,31(1):100-110.
[5]Liang HJ,Wei W,Kang XN,et al.Differentially expressed proteins in the precancerous stage of rat hepatocarcinogenesis induced by diethylnitrosamine.Chin J Hepatol,2009,17(9):669-674.(in Chinese)梁宏洁,韦薇,康晓楠,等.二乙基亚硝胺诱发大鼠肝癌过程中癌前病变阶段差异表达蛋白质的筛选.中华肝脏病杂志,2009,17(9):669-674.
[6]Zhou LL,Cao J,Li W,et al.Effect of RNA interference targeting FABP-5 on cell proliferation,apoptosis and invasion in human hepatocellular carcinoma cell line Hep G2.World Chin J Digestology,2015,23(2):179-188.(in Chinese)周玲丽,曹骥,李薇,等.沉默FABP-5基因对人肝癌Hep G2细胞的影响.世界华人消化杂志,2015,23(2):179-188.
[7]Liu Q,Wang SF,Xu H,et al.Expressions and significances of CRABPII and E-FABP in non-small cell lung cancer.Chin J Lung Cancer,2013,16(1):12-19.(in Chinese)刘倩,王世凤,徐缓,等.CRABPII和E-FABP在非小细胞肺癌中的表达及其意义.中国肺癌杂志,2013,16(1):12-19.
[8]Thumser AE,Moore JB,Plant NJ.Fatty acid binding proteins:tissue-specific functions in health and disease.Curr Opin Clin Nutr Metab Care,2014,17(2):124-129.
[9]Shi JL,Cao J,Su JJ,et al.Expression of epidermal fatty acid-binding protein in cross-species hepatocellular carcinoma.Chin J Hepatol,2012,20(4):270-274.(in Chinese)史俊林,曹骥,苏建家,等.表皮型脂肪酸结合蛋白在跨种属肝癌组织中的表达.中华肝脏病杂志,2012,20(4):270-274.
[10]El-Serag HB.Hepatocellular carcinoma.N Engl J Med,2011,365(12):1118-1127.
[11]Vivarelli M,Risaliti A.Liver transplantation for hepatocellular carcinoma on cirrhosis:strategies to avoid tumor recurrence.World J Gastroenterol,2011,17(43):4741-4746.
[12]Lu XX,Cao J,Li Y,et al.Expression of Cdc25a in cross-species hepatocellular carcinoma tissues and its significance.Chin J Pathophysiology,2014,30(5):820-824.(in Chinese)卢晓旭,曹骥,李瑗,等.Cdc25a在跨种属肝癌组织中的表达及其意义.中国病理生理杂志,2014,30(5):820-824.
[13]Xie Y,Dai JG.Study on mechanism of TCM treatment of primary liver cancer.J Changchun Univ Traditional Chin Med,2011,27(5):738-740.(in Chinese)谢益,戴建国.原发性肝癌的中医药治疗机制研究.长春中医药大学学报,2011,27(5):738-740.
[14]Chen HY,Lang QB,Yue XQ,et al.Initial analysis on affects and factors of therapeutic prescription with Chinese drugs upon post-operation recurrence of hepatocarcinoma.Jilin J Traditional Chin Med,2011,31(7):636-638.(in Chinese)陈红云,郎庆波,岳小强,等.中药治疗方案对肝癌术后复发的影响因素初步分析.吉林中医药,2011,31(7):636-638.
[15]Jemal A,Bray F,Center MM,et al.Global cancer statistics.CA Cancer J Clin,2011,61(2):69-90.
[16]Jeong CY,Hah YS,Cho BI,et al.Fatty acid-binding protein 5promotes cell proliferation and invasion in human intrahepatic cholangiocarcinoma.Oncol Rep,2012,28(4):1283-1292.
[17]Fang LY,Wong TY,Chiang WF,et al.Fatty-acid-binding protein 5promotes cell proliferation and invasion in oral squamous cell carcinoma.J Oral Pathol Med,2010,39(4):342-348.
[18]Shi JL,Cao J,Su JJ,et al.Clinical significance of upregulated expression of epidermal fatty acid binding protein in hepatocellular carcinoma tissues from different species.World Chin J Digestology,2013,21(11):963-969.(in Chinese)史俊林,曹骥,苏建家,等.表皮型脂肪酸结合蛋白在不同种属肝癌组织中的高表达及其临床意义.世界华人消化杂志,2013,21(11):963-969.
[19]Li J,Xu LZ.Protective effect of chuanhunzine injection on acute alcoholic liver injury in mice.J Huaihai Med,2015,33(3):250-252.(in Chinese)李甲,徐凌忠.川穹嗪注射液对小鼠急性酒精性肝损伤的保护作用观察.淮海医药,2015,33(3):250-252.
[20]Storch J,Thumser AE.Tissue-specific functions in the fatty acid-binding protein family.J Biol Chem,2010,285(43):32679-32683.
[21]Diao JS,Zhang X,Guo SZ.Progression of epidermal fatty acid binding protein.J Int Pathol Clin Med,2009,29(1):50-53.(in Chinese)刁建升,张曦,郭树忠.表皮型脂肪酸结合蛋白的研究进展.国际病理科学与临床杂志,2009,29(1):50-53.
[22]Armstrong EH,Goswami D,Griffin PR,et al.Structural basis for ligand regulation of the fatty acid-binding protein 5,peroxisome proliferator-activated receptorβ/δ(FABP5-PPARβ/δ)signaling pathway.J Biol Chem,2014,289(21):14941-14954.
[23]Zhou LL.Effect of silencing FABP-5 gene by RNA interference on the growth of Hep G2 human hepatoma and xenografts in nude mice.Nanning:Guangxi Medical University,2015.(in Chinese)周玲丽.RNA干扰技术沉默FABP-5基因对人肝癌Hep G2细胞和裸鼠移植瘤生长的影响.南宁:广西医科大学,2015.
[24]Feng ZQ,Zuo GW,Shi QQ,et al.Mechanism of ginsenoside Rh2inhibiting Hep G2 cells migration.Chin J Immunol,2015,31(1):61-65.(in Chinese)冯子强,左国伟,石庆强,等.人参皂苷Rh2抑制肝癌Hep G2细胞迁移的实验研究.中国免疫学杂志,2015,31(1):61-65.
[25]Fan J,Zhang L,Wang X.Effect of ginsenoside Rk3 on apoptosis of Hep G2.Acta Univ Med Anhui,2016,51(11):1604-1607,1608.(in Chinese)范洁,张磊,王啸.人参皂苷Rk3对肝癌Hep G2细胞凋亡的影响.安徽医科大学学报,2016,51(11):1604-1607,1608.
[26]Feng QF,Bi L,Yan XJ,et al.Effect of tetramethypyrazine on apoptosis of Hep G2 cells and expression of associated protein.J Nanjing Univ Traditional Chin Med,2015,31(3):246-249.(in Chinese)冯全服,毕蕾,颜晓静,等.川芎嗪调控肝癌Hep G2细胞凋亡及对相关蛋白表达的影响.南京中医药大学学报,2015,31(3):246-249.