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

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(P0.05),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(P0.05),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。

Screening and Verification of RNA-seq-based PBCRS for Treating Key Genes of Breast Cancer

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.