基于RNA-seq技术的活血化瘀法治疗乳腺癌关键基因的筛选和验证
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摘要
目的:研究活血化瘀治法(the medicine group of promoting blood circulation and removing stasis,PBCRS)对7,12-二甲基苯蒽[7,12-dimethylbenz(a) anthracene,DMBA]诱发性大鼠乳腺癌的抑制作用,及运用转录组测序技术(RNA sequencing,RNA-seq)和信号网络分析(ingenuity pathway analysis,IPA)软件筛选关键基因和验证。方法:将96只SD大鼠随机分为空白组,DMBA模型组,枸橼酸他莫昔芬(Tamoxifen,TAM)组(1.9 mg·kg~(-1)·d-1),PBCRS高、中、低剂量组(12.96,6.48,3.24 g·kg~(-1)·d-1)。药物干预1周后,除空白组外,采用DMBA诱导大鼠乳腺癌模型(灌服2次,间隔1周,剂量100 mg·kg~(-1))。给药10周后,观察大鼠荷瘤重、荷瘤体积变化,用总RNA提取试剂盒提取总RNA,从DMBA模型组和PBCRS中剂量组各取3个RNA样品进行基因检测,筛选出关键差异基因,用实时荧光定量PCR(Real-time PCR)验证关键基因。结果:与DMBA模型组比较,PBCRS中剂量组荷瘤重和荷瘤体积均明显降低(P<0.05),PBCRS高、低剂量组荷瘤重和荷瘤体积虽有降低,但与DMBA模型组比较差异均无统计学意义;基于RNA-seq技术及IPA分析软件筛选出果糖1,6二磷酸酶1 (fructose-1,6-bisphosphatase1,FBP1),浦肯野细胞蛋白4(purkinje cell protein 4,PCP4),肌红蛋白(myoglobin,MB) 3个关键基因;与DMBA模型组比较,PBCRS高、中剂量组FBP1 mRNA表达均明显升高(P<0.05),PBCRS低剂量组FBP1 mRNA表达升高,但差异无统计学意义,PBCRS高、中、低剂量组PCP4 mRNA的表达增高,但无统计学差异,PBCRS低剂量组MB mRNA的表达虽降低,差异无统计学意义。结论:PBCRS可能通过干预乳腺癌组织中FBP1的表达,抑制乳腺癌的发生。
Objective:To study the inhibitory effect of the medicine group of promoting blood circulation and removing stasis(PBCRS) on breast cancer induced by 7,12-dimethylbenz(a) anthracene(DMBA) in rats,and screen out and verify key genes based on RNA Sequencing(RNA-seq) technology and Ingenuity Pathway Analysis(IPA).Method:Totally 96 Sprague-Dawley(SD) rats were randomly divided into blank group,DMBA model control group,tamoxifen(TAM) group(1.9 mg·kg~(-1)·d-1),high-dose,middle-dose and low-dose PBCRS groups(12.96,6.48,3.24 g·kg~(-1)·d-1).One week after drug intervention,except for the blank group,the DMBA was used to induce the rat model of breast cancer(with an interval of a week,irrigation for two times at the dose of 100 mg·kg~(-1)).After 10 weeks,the changes in tumor weight and tumor volume were observed.The total RNA was extracted by total RNA extraction kit,and three RNA samples were collected from the DMBA model control group and the middle-dose PBCRS group for genetic testing.Based on RNA-seq,key differential genes were screened out and verified by Real-time PCR.Result:Comparing with the DMBA model control group,the tumor volume and tumor weight in middle-dose PBCRS group were decreased significantly(P<0.05).Although the tumor weight and tumor volume in high and low-dose PBCRS groups were decreased,there was no statistically significant difference.Based on RNA-seq technology and IPA analysis software,fructose-1,6-bisphosphatase1(FBP1),purkinje cell protein 4(PCP4),myoglobin(MB) key genes were screened out.Compared with the DMBA model control group,the mRNA expressions of FBP1 in the high and middle-dose PBCRS groups were significantly increased(P<0.05).Although the mRNA expressions of PCP4 in the high,middle and low-dose PBCRS groups were increased,there was no statistical significance.The mRNA expression of MB in the low-dose group was decreased,but there was no statistical significance.Conclusion:PBCRS may inhibit the occurrence of breast cancer by interfering with the expression of FBP1 in breast cancer tissue.
Objective:To study the inhibitory effect of the medicine group of promoting blood circulation and removing stasis(PBCRS) on breast cancer induced by 7,12-dimethylbenz(a) anthracene(DMBA) in rats,and screen out and verify key genes based on RNA Sequencing(RNA-seq) technology and Ingenuity Pathway Analysis(IPA).Method:Totally 96 Sprague-Dawley(SD) rats were randomly divided into blank group,DMBA model control group,tamoxifen(TAM) group(1.9 mg·kg~(-1)·d-1),high-dose,middle-dose and low-dose PBCRS groups(12.96,6.48,3.24 g·kg~(-1)·d-1).One week after drug intervention,except for the blank group,the DMBA was used to induce the rat model of breast cancer(with an interval of a week,irrigation for two times at the dose of 100 mg·kg~(-1)).After 10 weeks,the changes in tumor weight and tumor volume were observed.The total RNA was extracted by total RNA extraction kit,and three RNA samples were collected from the DMBA model control group and the middle-dose PBCRS group for genetic testing.Based on RNA-seq,key differential genes were screened out and verified by Real-time PCR.Result:Comparing with the DMBA model control group,the tumor volume and tumor weight in middle-dose PBCRS group were decreased significantly(P<0.05).Although the tumor weight and tumor volume in high and low-dose PBCRS groups were decreased,there was no statistically significant difference.Based on RNA-seq technology and IPA analysis software,fructose-1,6-bisphosphatase1(FBP1),purkinje cell protein 4(PCP4),myoglobin(MB) key genes were screened out.Compared with the DMBA model control group,the mRNA expressions of FBP1 in the high and middle-dose PBCRS groups were significantly increased(P<0.05).Although the mRNA expressions of PCP4 in the high,middle and low-dose PBCRS groups were increased,there was no statistical significance.The mRNA expression of MB in the low-dose group was decreased,but there was no statistical significance.Conclusion:PBCRS may inhibit the occurrence of breast cancer by interfering with the expression of FBP1 in breast cancer tissue.
引文
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[2]Kolberg H C,Lufter D,Lux M P,et al.Breast cancer2012-new aspects[J].Geburtshilfe Frauenheilkd,2012,72(7):602-615.
[3]吴菲,林国桢,张晋昕.我国恶性肿瘤发病现状及趋势[J].中国肿瘤,2012,21(2):81-85.
[4]刘笑然,李惠平.BRCA1,BRCA2基因突变与乳腺癌风险预测及治疗[J].癌症进展,2015,13(2):114-119.
[5]Roy R,Chun J,Powell S N.BRCA1 and BRCA2:different roles in a common pathway of genome protection[J].Nat Rev Cancer,2011,12(1):68-78.
[6]于春梅,纪凤颖,付登科.女性乳腺癌影响因素病例对照研究[J].中国公共卫生,2009,25(7):772-773.
[7]刘专科.某市2011~2014年乳腺癌发病机制及危险因素分析[J].现代医药卫生,2015,31(1):96-97.
[8]徐雅莉,孙强,单广良,等.中国女性乳腺癌发病相关危险因素:病例对照研究[J].协和医学杂志,2011,2(1):7-14.
[9]宗嘉宝.李仝教授运用中医药治疗乳腺癌经验初探[D].北京:北京中医药大学,2015.
[10]张书娟,王苹.活血化瘀中药防治乳腺癌的研究近况[J].福建中医学院学报,2005,15(S1):42-44.
[11]YUAN S,SONG Y,WANG X,et al.The inhibiting effect of tanshinoneⅡA on the growth of tumor cell lines in vitro[J].West China J Pharm Sci,2003,17(5):327-329.
[12]周瑞芳.PE相关中药黄芪、丹参对人乳腺癌MCF-7、MDA-MB-231细胞增殖凋亡的影响[D].广州:广州中医药大学,2008.
[13]郭朋.基于RNA-seq技术的宫颈鳞癌转录组学研究[D].北京:北京协和医学院,2015.
[14]尚广彬,曾莉萍,赵益,等.二至丸对诱发性乳腺癌组织中VEGF和MMP-9表达的影响[J].中国实验方剂学杂志,2013,19(13):270-273.
[15]范刚启,宋祥龙,王辉,等.活血化瘀治疗癌及癌前病变效应的两重性与血管生成的关系[J].中国中西医结合杂志,2003,23(8):624-626.
[16]吴珊珊,杨宝丽,关慧.三七总皂苷联合三苯氧胺对人乳腺癌细胞的抑制作用及对PI3K-AKT-mTOR信号通路的影响[J].中药药理与临床,2017,33(6):84-89.
[17]宋爱莉,张敬涛,李静蔚,等.莪术油对乳腺癌癌前病变造模大鼠血液流变学及乳房微循环的影响[J].中华中医药学刊,2008,26(3):458-460.
[18]王玉祥,杨洁,杨超,等.疏肝化痰祛瘀方联用紫杉醇对乳腺癌大鼠乳房微循环的影响[J].中国老年学杂志,2016,36(22):5539-5542.
[19]张淑娟,王振涛,韩丽华,等.川芎嗪注射液对心梗后大鼠缺血心肌血管新生及VEGF-mRNA表达的影响[J].中国实验方剂学杂志,2011,17(7):170-173.
[20]王国峰,陆峰,赵霞,等.川芎嗪抗家兔动脉粥样硬化作用[J].中国实验方剂学杂志,2012,18(14):202-205.
[21]赵金明,秦文艳,齐越,等.红花黄色素抗凝血作用及对血小板聚集影响的研究[J].实验动物科学,2009,26(6):30-32.
[22]韩海玲,孙玉芹,宋文刚,等.红花黄色素对内皮细胞增殖及凋亡的保护作用[J].中国实验诊断学,2011,15(1):51-54.
[23]朱业靖.丹参酮对晚期非小细胞肺癌患者凝血指标与临床效果的影响研究[J].中国医药指南,2016,14(6):1-3.
[24]JING J,ZHENG H,WANG J,et al.Growth inhibition and multidrug resistance-reversing effect of tanshinone IA on human breast cancer cell with estrogen receptor negative[J].J Sichuan Univ,2007,38(3):391-395.
[25]刘江亭,李慧芬,崔伟亮,等.酒丹参研究进展[J].山东中医杂志,2018,37(5):432-434.
[26]魏玺,黄焰.DMBA诱导大鼠乳腺癌模型及乳腺癌化学预防实验研究进展[J].现代肿瘤医学,2008,16(3):454-456.
[27]李静蔚,宋爱莉,张敬涛,等.DMBA诱导大鼠乳腺癌癌前病变的组织病理学研究[J].中华中医药学刊,2008,26(3):505-508.
[28]祁艳波,吴嘉慧,刘柏杨,等.血府逐瘀汤对荷瘤小鼠的抑瘤作用[J].现代预防医学,2005,32(5):446-448.
[29]王文杰.红花水煎剂对肿瘤组织病理学改变及血管生成的影响[J].山西中医,2007,23(6):59-60.
[30]Raz T,Kapranov P,Lipson D,et al.Protocol dependence of sequencing-Based gene expression measurements[J].PLo S One,2011,6(5):1-13.
[31]廖钦洪,邹勇,李洪雷,等.基于RNA-seq数据的生姜NAC转录因子家族鉴定及分析[J].中国中药杂志,2018,43(3):493-501.
[32]Harashima S,WANG Y,Horiuchi T,et al.Purkinje cell protein 4 positively regulates neurite outgrowth and neurotransmitter release[J].J Neurosci Res,2011,89(10):1519-1530.
[33]Kanazawa Y,Makino M,Morishima Y,et al.Degradation of PEP-19,a calmodulin-binding protein,by calpain is implicated in neuronal cell death induced by intracellular Ca2+overload[J].Neuroscience,2008,154(2):473-481.
[34]Yoshimura T,Hamada T,Hijioka H,et al.PCP4/PEP19 promotes migration,invasion and adhesion in human breast cancer MCF-7 and T47D cells[J].Oncotarget,2016,7(31):49065-49074.
[35]Hamada T,Souda M,Yoshimura T,et al.Antiapoptotic effects of PCP4/PEP19 in human breast cancer cell lines:a novel oncotarget[J].Oncotarget,2014,5(15):6076-6086.
[36]Souda M,Umekita Y,Abeyama K,et al.Gene expression profiling during rat mammary carcinogenesis induced by 7,12-dimethylbenz[a]anthracene[J].Int JCancer,2009,125(6):1285-1297.
[37]Wittenberg J B,Wittenberg B A.Myoglobin function reassessed[J].J Exp Biol,2003,206(12):2011-2020.
[38]袁瑾,李娜娜,李文花,等.c Tn I、Mb联合应用评价异丙肾上腺素诱导的心肌缺血损伤及凋亡发生[J].中国实验诊断学,2011,15(9):1455-1457.
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