△S2 SF1a-PRLR对人类乳腺癌细胞转录组的影响

Analysis of the effect of △S2 SF1a-PRLR on the transcriptome of human breast cancer cells MCF-7

目的探讨SF1a-PRLR和ΔS2 SF1a-PRLR对人类乳腺癌细胞转录组的影响,分析两者在乳腺癌细胞中的基因表达差异。方法构建携带ΔS2 SF1a-PRLR与SF1a-PRLR的慢病毒载体,将稳转细胞总RNA分离纯化,然后进行RNA测序,整理数据并进行生物信息学统计和分析。将人乳腺癌MCF-7细胞分为空白对照组(con组)(空载体)、过表达SF1a-PRLR基因组(过表达SF1a-PRLR基因)、过表达ΔS2 SF1a-PRLR基因组(过表达ΔS2SF1a-PRLR基因)。结果 G8-3(将ΔS2 SF1a简称为G8)、Con-1、1a-3(将SF1a简称为1a)与G8-1的相似度均偏低,G8-3与1a-1的相似度最高。主成分分析(PCA)结果显示,G8-1、Con-2为离群样本,G8-3、G8-2、1a-3、1a-2、1a-1为相似性高的样本。高通量测序结果显示,过表达SF1a-PRLR基因组、过表达ΔS2 SF1a-PRLR基因组和con组的转录组表达情况存在差异。con组和过表达SF1a-PRLR基因组的差异基因进行京都基因和基因组数据库(KEGG)通路富集分析,发现涉及调节肿瘤生长和维持肿瘤细胞多能性的多条信号通路获得富集,如丝裂原激活蛋白激酶(MAPK)、肿瘤坏死因子(TNF)、转化生长因子-β(TGF-β)、磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(PKB,又称AKT)、环腺苷一磷酸(cAMP)、Hedgehog、Hippo信号通路。对con组和过表达ΔS2 SF1a-PRLR基因组的差异基因进行KEGG通路富集分析,发现涉及调节肿瘤生长的多条信号通路获得富集,如MAPK、TNF、TGF-β、PI3K/AKT、ErbB、Hedgehog、Hippo信号通路。通过可变剪切分析发现,3组样本中主要发现了6种不同的剪切模式。单核苷酸多态性(SNP)分析结果显示,过表达SF1a-PRLR基因组的MCF-7细胞中有42 885个SNP位点,过表达ΔS2 SF1a-PRLR基因组MCF-7细胞中有37 305个SNP位点,con组的MCF-7细胞中有31 583个SNP位点。结论过表达SF1a-PRLR基因和过表达ΔS2 SF1a-PRLR基因能够影响MCF-7乳腺癌细胞的多种基因的表达,且可通过MAPK、TGF-β、PI3K/AKT、Hedgehog等信号通路影响乳腺癌的生长。

Objective To investigate the effect of SF1a-PRLR and ΔS2 SF1a-PRLR on the transcriptome of human breast cancer cells, and analyze the differences of gene expression in breast cancer cells. Method Lentivirus vectors carrying SF1a-PRLR or ΔS2 SF1a-PRLR were established, and then the total RNA was extracted and purified for RNA sequencing, of which the data were collected to perform bioinformatical statistical analysis. MCF-7 cell can be divided into blank control group (con group)(empty carrier), over express SF1a- PRLR genome group (over espress SF1a- PRLR gene), and over express ΔS2 SF1a-PRLR genome group (over express ΔS2 SF1a-PRLR gene). Result G8-3 (G8 is short for ΔS2 SF1a), Con-1, 1a-3 (1a short for SF1a) had relatively lower similarity with G8-1, while the similarity between G8- 3 and 1a-1 were the highest. Principal component analysis (PCA) revealed that, G8-1 and Con-2 were outliers, however, G8-3, G8-2, 1a-3, 1a-2, and 1a-1 were samples of high similarity. High throughput sequencing showed that, the transcriptome expression in SF1a-PRLR,ΔS2 SF1a-PRLR and blank control group exhibited significant differences. KEGG pathway enrichment analysis was performed on differential gene expressing between the blank control group and the SF1a- PRLR over expressing group, it was found that a variety of signaling pathways involved in the regulation of tumor growth and maintenance of tumor cell pluripotency were enriched, such as mitogen-activated protein kinase (MAPK), tumor necrosis factor (TNF), transforming growth factor-β(TGF-β), phosphatidylinositol- 3- kinase (PI3K)/ protein kinase B (AKT), cyclic adenosine monophosphate (cAMP), Hedgehog, and Hippo signaling pathways. Besides, the KEGG pathway enrichment analysis performed on differential gene expressing between the blank control group and the ΔS2 SF1a- PRLR over expressing group demonstrated a number of signaling pathways involved in the regulation of tumor growth were found to be enriched, such as MAPK, TNF, TGF-β, PI3K/AKT, ErbB, Hedgehog, and Hippo. By alternative splicing, 6 different splicing modes were identified in the three groups of samples. Single nucleotide polymorphism (SNP) analysis indicated that, there were 42 885 SNP sites in the MCF-7 cells of over expressed SF1a-PRLR genome, and 37 305 in that of ΔS2 SF1a-PRLR, and 31 583 in that of blank control group. Conclusion The over expression of SF1a- PRLR gene and ΔS2 SF1a-PRLR gene could affect various gene expression of MCF-7 breast cancer cells, and can exert influential effect on the growth of breast cancer through MAPK, TGF-β, PI3K/AKT, and Hedgehog and other signaling pathways.