生物技术通报 ›› 2021, Vol. 37 ›› Issue (11): 72-80.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0444
• 食用菌生物技术专题(专题主编: 黄晨阳) • 上一篇 下一篇
黄海辰1(), 吴文雅1,2, 戚梦1,2, 薛帆正1, 吴小平1,2, 张君丽3, 傅俊生1,2()
收稿日期:
2021-04-07
出版日期:
2021-11-26
发布日期:
2021-12-03
作者简介:
黄海辰,男,研究方向:食用菌活性物质研究;E-mail: 基金资助:
HUANG Hai-chen1(), WU Wen-ya1,2, QI Meng1,2, XUE Fan-zheng1, WU Xiao-ping1,2, ZHANG Jun-li3, FU Jun-sheng1,2()
Received:
2021-04-07
Published:
2021-11-26
Online:
2021-12-03
摘要:
前期研究表明,蛹虫草活性成分虫草素能有效抑制乳腺癌移植瘤的生长和转移,但其作用机制未清楚。本研究取三阴性乳腺癌移植瘤样品,通过转录组测序技术分析虫草素抗乳腺癌移植瘤的分子作用机制。结果表明,以P<0.05、log2|FC|>1为标准,筛选获得584个差异表达基因,其中上调基因69个,下调基因515个;KEGG分析表明,虫草素对乳腺癌移植瘤的分子调控涉及Hippo signaling pathway、Hedgehog signaling pathway、ECM-receptor interction、TGF-beta signaling pathway等多条癌症相关信号通路;GO分析表明,这些差异基因富集在biological adhesion、growth、metabolic process、locomotion和biological process等16个生物学过程中,提示虫草素对乳腺癌移植瘤生长与转移有极显著的调节作用;进一步对生长和转移过程的基因进行PPI互作分析,提示Hedgehog signaling pathway、Hippo signaling pathway是虫草素抗三阴性乳腺癌的重要潜在作用通路。研究结果提示Hippo-Yap/TAZ信号通路和Hedgehog-Gli信号通路为潜在作用通路,为进一步理解虫草素治疗三阴性乳腺癌的分子机制奠定了基础。
黄海辰, 吴文雅, 戚梦, 薛帆正, 吴小平, 张君丽, 傅俊生. 虫草素抗三阴性乳腺癌的转录组学分析[J]. 生物技术通报, 2021, 37(11): 72-80.
HUANG Hai-chen, WU Wen-ya, QI Meng, XUE Fan-zheng, WU Xiao-ping, ZHANG Jun-li, FU Jun-sheng. RNA-Seq Analysis of Cordycepin Against Triple-negative Breast Cancer[J]. Biotechnology Bulletin, 2021, 37(11): 72-80.
基因Gene | 引物序列Primer sequence(5'-3') |
---|---|
PDK4 | F:5'-GAGGATTACTGACCGCCTCTTTAG-3' R:5'-TTCCGGGAATTGTCCATCAC-3' |
CD24 | F:5'-CCCACGCAGATTTATTCCAG-3' R:5'-GACTTCCAGACGCCATTTG-3' |
MMP7 | F:5'-AGCCAAACTCAAGGAGATGC-3' R:5'-GCCAATCATGATGTCAGCAG-3' |
SHH | F:5'-AGGGCACCATTCTCATCAAC-3' R:5'-GGAGCGGTTAGGGCTACTCT-3' |
GLI2 | F:5'-GCCCTT CCTGAAAAGAAGAC-3' R:5'-CATTGGAGA AACAGGATTGG-3' |
GADPH | F:5'-GAAGGACTCATGACCACAG-3' R:5'-CTTCACCACCTTCTTGATG-3' |
表1 实时荧光定量qPCR的引物序列和片段长度
Table 1 Primers sequences and fragment lengths for qPCR
基因Gene | 引物序列Primer sequence(5'-3') |
---|---|
PDK4 | F:5'-GAGGATTACTGACCGCCTCTTTAG-3' R:5'-TTCCGGGAATTGTCCATCAC-3' |
CD24 | F:5'-CCCACGCAGATTTATTCCAG-3' R:5'-GACTTCCAGACGCCATTTG-3' |
MMP7 | F:5'-AGCCAAACTCAAGGAGATGC-3' R:5'-GCCAATCATGATGTCAGCAG-3' |
SHH | F:5'-AGGGCACCATTCTCATCAAC-3' R:5'-GGAGCGGTTAGGGCTACTCT-3' |
GLI2 | F:5'-GCCCTT CCTGAAAAGAAGAC-3' R:5'-CATTGGAGA AACAGGATTGG-3' |
GADPH | F:5'-GAAGGACTCATGACCACAG-3' R:5'-CTTCACCACCTTCTTGATG-3' |
样品 Sample | 浓度 Concentration/(ng·μL-1) | 体积 Volume /μL | 总量 Total /μg | RIN值 RIN value | 文库类型 Library type | 结论 Conclusion |
---|---|---|---|---|---|---|
C0R1 | 295 | 35 | 10.33 | 6.8 | RNAseq | A |
C0R2 | 528 | 35 | 18.46 | 7.9 | RNAseq | A |
C0R3 | 761 | 35 | 26.67 | 9.3 | RNAseq | A |
C0N1 | 652 | 76 | 49.55 | 8.3 | RNAseq | A |
C0N2 | 695 | 76 | 52.78 | 9.3 | RNAseq | A |
C0N3 | 602 | 74 | 44.51 | 9.3 | RNAseq | A |
表2 RNA测序质量评估
Table 2 RNA sequencing quality assessment
样品 Sample | 浓度 Concentration/(ng·μL-1) | 体积 Volume /μL | 总量 Total /μg | RIN值 RIN value | 文库类型 Library type | 结论 Conclusion |
---|---|---|---|---|---|---|
C0R1 | 295 | 35 | 10.33 | 6.8 | RNAseq | A |
C0R2 | 528 | 35 | 18.46 | 7.9 | RNAseq | A |
C0R3 | 761 | 35 | 26.67 | 9.3 | RNAseq | A |
C0N1 | 652 | 76 | 49.55 | 8.3 | RNAseq | A |
C0N2 | 695 | 76 | 52.78 | 9.3 | RNAseq | A |
C0N3 | 602 | 74 | 44.51 | 9.3 | RNAseq | A |
图2 样品reads分类图 CON1:对照组1;CON2:对照组2;CON3:对照组3;COR1:实验组1;COR2:实验组2;COR3:实验组3;Adaptor:含有接头序列的reads数占总reads数的比例;High rate:含未知碱基的reads数占reads总数的比例;Low quality:从原始序列数据中去除杂质获得的数据与总读数之比;High quality:总读数
Fig.2 Classification chart for sample reads CON1:Control group 1. CON2:Control group 2. CON3:Control group 3. COR1:Cordycepin 1. COR2:Cordycepin 2. COR3:Cordycepin 3. Adaptor:The ratio of the number of reads containing the adaptor sequence to the total number of reads. High rate:The ratio of the number of reads containing unknown bases to the total number of reads. Low quality:The ratio of the data obtained by removing impurities from the original sequence data to the total number of reads. High quality:Total readings
样品名 Sample | 总reads数量 Number of total reads | 比对上rRNA的 reads 数及占总数的比例 Mapped reads(ratio in total) | 未比对上rRNA的 reads 数及占总数的比例 Unmapped reads(ratio in total) |
---|---|---|---|
CON1 | 53195320 | 410412(0.77%) | 52784908(99.23%) |
CON2 | 69510386 | 493598(0.71%) | 69016788(99.29%) |
CON3 | 65958874 | 550350(0.83%) | 65408524(99.17%) |
COR1 | 90497008 | 812122(0.90%) | 89684886(99.10%) |
COR2 | 78992736 | 387314(0.49%) | 78605422(99.51%) |
COR3 | 56738684 | 310270(0.55%) | 56428414(99.45%) |
表3 HQ clean data与rRNA的比对统计表
Table 3 HQ clean data and rRNA alignment statistics
样品名 Sample | 总reads数量 Number of total reads | 比对上rRNA的 reads 数及占总数的比例 Mapped reads(ratio in total) | 未比对上rRNA的 reads 数及占总数的比例 Unmapped reads(ratio in total) |
---|---|---|---|
CON1 | 53195320 | 410412(0.77%) | 52784908(99.23%) |
CON2 | 69510386 | 493598(0.71%) | 69016788(99.29%) |
CON3 | 65958874 | 550350(0.83%) | 65408524(99.17%) |
COR1 | 90497008 | 812122(0.90%) | 89684886(99.10%) |
COR2 | 78992736 | 387314(0.49%) | 78605422(99.51%) |
COR3 | 56738684 | 310270(0.55%) | 56428414(99.45%) |
样品名 Sample | 总reads数量 Total reads | 未比对上参考基因组的 reads 数及占总数的比例 Unmapped reads(ratio in total) | 唯一比对上参考基因组的 reads 数及占总数的比例 Unique mapped reads(ratio in total) | 多处比对上参考基因组的 reads 数及占总数的比例 Multiple mapped reads(ratio in total) | 比对上参考基因组的 reads占总数的比例 Mapping ratio |
---|---|---|---|---|---|
CON1 | 52784908 | 19192339(36.36%) | 33372603(63.22%) | 219966(0.42%) | 63.64% |
CON2 | 69016788 | 24438545(35.41%) | 44287405(64.17%) | 290838(0.42%) | 64.59% |
CON3 | 65408524 | 14470073(22.12%) | 50589691(77.34%) | 348760(0.53%) | 77.88% |
COR1 | 89684886 | 16369614(18.25%) | 72826044(81.20%) | 489228(0.55%) | 81.75% |
COR2 | 78605422 | 19831015(25.23%) | 58342213(74.22%) | 432194(0.55%) | 74.77% |
COR3 | 56428414 | 18462152(32.72%) | 37706418(66.82%) | 259844(0.46%) | 67.28% |
表4 比对核糖体后得到的unmapped reads与参考基因组的比对统计表
Table 4 Comparison of unmapped reads and reference genomes obtained after ribosomal alignment
样品名 Sample | 总reads数量 Total reads | 未比对上参考基因组的 reads 数及占总数的比例 Unmapped reads(ratio in total) | 唯一比对上参考基因组的 reads 数及占总数的比例 Unique mapped reads(ratio in total) | 多处比对上参考基因组的 reads 数及占总数的比例 Multiple mapped reads(ratio in total) | 比对上参考基因组的 reads占总数的比例 Mapping ratio |
---|---|---|---|---|---|
CON1 | 52784908 | 19192339(36.36%) | 33372603(63.22%) | 219966(0.42%) | 63.64% |
CON2 | 69016788 | 24438545(35.41%) | 44287405(64.17%) | 290838(0.42%) | 64.59% |
CON3 | 65408524 | 14470073(22.12%) | 50589691(77.34%) | 348760(0.53%) | 77.88% |
COR1 | 89684886 | 16369614(18.25%) | 72826044(81.20%) | 489228(0.55%) | 81.75% |
COR2 | 78605422 | 19831015(25.23%) | 58342213(74.22%) | 432194(0.55%) | 74.77% |
COR3 | 56428414 | 18462152(32.72%) | 37706418(66.82%) | 259844(0.46%) | 67.28% |
各分组名称 Group name | 检测到的已知基因数及比率 Number of known genes(ratio) | 检测到的新基因数目 Number of newly detected genes | 所有基因数目 Number of all genes |
---|---|---|---|
COR | 15 730(80.23%) | 1 405 | 17 135 |
CON | 15 658(79.86%) | 1 394 | 17 052 |
表5 各组检测基因数目统计
Table 5 Statistics of the number of detected genes in each group
各分组名称 Group name | 检测到的已知基因数及比率 Number of known genes(ratio) | 检测到的新基因数目 Number of newly detected genes | 所有基因数目 Number of all genes |
---|---|---|---|
COR | 15 730(80.23%) | 1 405 | 17 135 |
CON | 15 658(79.86%) | 1 394 | 17 052 |
图3 分组间差异统计 A:样品间差异基因统计柱状图;B:CON vs COR差异基因火山图
Fig.3 Inter-group difference statistics A:Inter-sample differential gene statistics histogram. B:CON vs COR differential gene volcano map
检测项目 Project | MMP7 | PDK4 | CD24 | LIFR | GLI2 |
---|---|---|---|---|---|
RNA-seq | -2.55 | -2.09 | -2.37 | -1.35 | -2.34 |
qRT-PCR/GAPDH | -2.05 | -1.25 | -2.31 | -2.07 | -2.87 |
表6 5个随机基因的RNA-seq与qPCR表达量对比
Table 6 Comparison between RNA-seq and qPCR expression levels of 5 random genes
检测项目 Project | MMP7 | PDK4 | CD24 | LIFR | GLI2 |
---|---|---|---|---|---|
RNA-seq | -2.55 | -2.09 | -2.37 | -1.35 | -2.34 |
qRT-PCR/GAPDH | -2.05 | -1.25 | -2.31 | -2.07 | -2.87 |
条目 Term | 基因数量 Number of genes | 基因占比 Percentage/% | 基因 Gene |
---|---|---|---|
生长 Growth | 21 | 2.142 | GLI2, ULK2, HNF4A, COBL, SEMA5A, WNT5A, IGFBP2, IGFBP5, PROX1, STRA6, DUOX2, NKD1, NLGN4X, DRAXIN, SHH, ISLR2, KLK6, HOPX, PSAPL1, SOX2, SELENOP |
黏附 Biological adhesion | 31 | 2.683 | ADAM28, GLI2, HNF4A, LHB, MDFI, CLIC5, WNT5A, IGFBP2, IGFBP5, CNR1, NDP, HOXD10, HAVCR2, NKX21, MMP7, STRA6, LGR5, DUOX2, ACE, DNALI1, HPGD, SHH, SPINT1, PDE3A, ADGRG2, PSAPL1, CADM1, TMEM119, CCIN, PAX5, SELENOP |
运动 Locomotion | 28 | 1.374 | GLI2, TESC, HNF4A, FLT1, FA2H, SEMA5A, WNT5A, IGFBP2, IGFBP5, PROX1, NDP, HAVCR2, LGR5, VASH2, CD109, ACE, TFF1, SHH, CTHRC1, SPINT1, ATOH8, TP53I11, SOX2, TMEM119, GNG2, TNFRSF18, SULF2, CD24 |
表7 生长与转移差异基因
Table 7 Growth and metastasis difference gene
条目 Term | 基因数量 Number of genes | 基因占比 Percentage/% | 基因 Gene |
---|---|---|---|
生长 Growth | 21 | 2.142 | GLI2, ULK2, HNF4A, COBL, SEMA5A, WNT5A, IGFBP2, IGFBP5, PROX1, STRA6, DUOX2, NKD1, NLGN4X, DRAXIN, SHH, ISLR2, KLK6, HOPX, PSAPL1, SOX2, SELENOP |
黏附 Biological adhesion | 31 | 2.683 | ADAM28, GLI2, HNF4A, LHB, MDFI, CLIC5, WNT5A, IGFBP2, IGFBP5, CNR1, NDP, HOXD10, HAVCR2, NKX21, MMP7, STRA6, LGR5, DUOX2, ACE, DNALI1, HPGD, SHH, SPINT1, PDE3A, ADGRG2, PSAPL1, CADM1, TMEM119, CCIN, PAX5, SELENOP |
运动 Locomotion | 28 | 1.374 | GLI2, TESC, HNF4A, FLT1, FA2H, SEMA5A, WNT5A, IGFBP2, IGFBP5, PROX1, NDP, HAVCR2, LGR5, VASH2, CD109, ACE, TFF1, SHH, CTHRC1, SPINT1, ATOH8, TP53I11, SOX2, TMEM119, GNG2, TNFRSF18, SULF2, CD24 |
序号 Index | 名称 Name | P值 P-value | 调整后P值 Adjusted P-value | 综合得分 Combined score |
---|---|---|---|---|
1 | Hedgehog signaling pathway | 0.0006293 | 0.02289 | 13.42 |
2 | Basal cell carcinoma | 0.0008323 | 0.02289 | 13.28 |
3 | Hippo signaling pathway | 0.001736 | 0.03183 | 10.73 |
4 | Focal adhesion | 0.004714 | 0.05655 | 9.89 |
5 | T cell receptor signaling pathway | 0.005141 | 0.05655 | 9.33 |
6 | Pathways in cancer | 0.01061 | 0.09472 | 8.98 |
7 | Cell adhesion molecules(CAMs) | 0.01206 | 0.09472 | 6.99 |
8 | Fc epsilon RI signaling pathway | 0.02185 | 0.1247 | 6.78 |
表8 基于Enrichr信号通路富集分析
Table 8 Enichr-based signal path enrichment analysis
序号 Index | 名称 Name | P值 P-value | 调整后P值 Adjusted P-value | 综合得分 Combined score |
---|---|---|---|---|
1 | Hedgehog signaling pathway | 0.0006293 | 0.02289 | 13.42 |
2 | Basal cell carcinoma | 0.0008323 | 0.02289 | 13.28 |
3 | Hippo signaling pathway | 0.001736 | 0.03183 | 10.73 |
4 | Focal adhesion | 0.004714 | 0.05655 | 9.89 |
5 | T cell receptor signaling pathway | 0.005141 | 0.05655 | 9.33 |
6 | Pathways in cancer | 0.01061 | 0.09472 | 8.98 |
7 | Cell adhesion molecules(CAMs) | 0.01206 | 0.09472 | 6.99 |
8 | Fc epsilon RI signaling pathway | 0.02185 | 0.1247 | 6.78 |
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