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Deep sequencing reveals small RNA characterization of invasive micropapillary carcinomas of the breast

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Abstract

Invasive micropapillary carcinoma (IMPC) is an uncommon histological type of breast cancer. IMPC has a special growth pattern and a more aggressive behavior than invasive ductal carcinomas of no special types (IDC-NSTs). microRNAs are a large class of non-coding RNAs involved in the regulation of various biological processes. Here, we analyzed the small RNA transcriptomes of five formalin-fixed paraffin-embedded (FFPE) pure IMPC samples and five FFPE IDC-NSTs samples by means of next-generation sequencing, generating a total of >170,000,000 clean reads. In an unsupervised cluster analysis, differently expressed miRNAs generated a tree with clear distinction between IMPC and IDC-NSTs classes. Paired fresh-frozen and FFPE specimens showed very similar miRNA expression profiles. By means of RT-qPCR, we further investigated miRNA expression in more IMPC (n = 22) and IDC-NSTs (n = 24) FFPE samples and found let-7b, miR-30c, miR-148a, miR-181a, miR-181a*, and miR-181b were significantly differently expressed between the two groups. We also elucidated several features of miRNA in these breast cancer tissues including 5′ variability, miRNA editing, and 3′ untemplated addition. Our findings will lead to further understanding of the invasive potency of IMPC and gain an insight into the diversity and complexity of small RNA molecules in breast cancer tissues.

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Abbreviations

miRNA:

microrna

IMPC:

Invasive micropapillary carcinoma

IDC-NSTs:

Invasive ductal carcinomas of no special types

FFPE tissue:

Formalin-fixed paraffin-embedded tissue

UTR:

Untranslated region

RIN:

RNA integrity numbers

EMT:

Epithelial-to-mesenchymal transition

FEA:

Flat epithelial atypia

DCIS:

Ductal carcinoma in situ

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Acknowledgments

This work was supported by grants from the National Basic Research Program of China (973 Program) [2009CB521700] and the Key Program of Chinese National Natural Science Foundation [30930038], and partially by grants from the Program for Changjiang Scholars and Innovative Research Team in the University of Ministry of Education of China [IRT0743] and the Chinese National Natural Science Foundation [30800355].

Conflict of interest

No conflicts of interest were declared.

Author information

Authors and Affiliations

  1. Department of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital, Tianjin, People’s Republic of China

    Shuai Li, Cuicui Yang, Lili Zhai, Feng Gu, Ronggang Lang, Yu Fan & Li Fu

  2. Key Laboratory of Breast Cancer Prevention and Therapy, Ministry of Education, Tianjin Medical University, Tianjin, People’s Republic of China

    Shuai Li, Cuicui Yang, Lili Zhai, Feng Gu, Ronggang Lang, Yu Fan & Li Fu

  3. Key Laboratory of Cancer Prevention and Therapy, Tianjin, People’s Republic of China

    Shuai Li, Cuicui Yang, Lili Zhai, Feng Gu, Ronggang Lang, Yu Fan & Li Fu

  4. State Key Laboratory of Breast Cancer Research, Huanhuxi Road, 300060, Hexi District, Tianjin, People’s Republic of China

    Shuai Li, Cuicui Yang, Lili Zhai, Feng Gu, Ronggang Lang, Yu Fan & Li Fu

  5. Department of Science & Technology, BGI-Shenzhen, 518083, Shenzhen, People’s Republic of China

    Wenwei Zhang, Jing Yu, Meihua Gong & Xiuqing Zhang

Authors
  1. Shuai Li
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  2. Cuicui Yang
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  3. Lili Zhai
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  8. Yu Fan
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  10. Xiuqing Zhang
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  11. Li Fu
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Corresponding authors

Correspondence to Xiuqing Zhang or Li Fu.

Additional information

Shuai Li and Cuicui Yang contributed equally to this work.

Electronic supplementary material

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10549_2012_2166_MOESM1_ESM.tif

Supplementary Fig. 1 Quality assessment of RNA sample. Representative Agilent 2100 Bioanalyzer electropherograms of one fresh-frozen (left) and one FFPE (right) sample. The fresh-frozen sample had a RIN = 7.9 and the FFPE sample had a RIN = 2.4 (TIFF 3406 kb)

10549_2012_2166_MOESM2_ESM.tif

Supplementary Fig. 2 Concordance of miRNA expression profiling between paired FFPE and fresh-frozen samples by deep sequencing. Pearson’s correlation was used to measure the similarity between paired samples (TIFF 8302 kb)

10549_2012_2166_MOESM3_ESM.tif

Supplementary Fig. 3 Drosha defines more precise 5′ ends than Dicer. Data from miRNAs that generated more than 1000 reads and have exclusively unique genomic mappings are summarized. For each miRNA, the heterogeneity of its 5′ end is calculated as the mean of the absolute distance between the 5′ extremity of mapped read and the 5′ end of miRBase reference sequence. The cutting accuracy was compared by means of the Non-parametric test (TIFF 13904 kb)

10549_2012_2166_MOESM4_ESM.tif

Supplementary Fig. 4 Models of miRNA 5′ ends definition by Drosha and Dicer. Drosha and Dicer define the 5′-ends of 5p and 3p miRNAs, respectively. DGCR8 binds to the stem-ssRNA junction of pri-miRNA and locates Drosha catalytic site ~ 11 bp away. Dicer determines its cleavage sites by measuring a fixed distance (~ 22 bp) from the 3′ overhang of the dsRNA terminus. The 3′ ends of pre-miRNAs can be modified by nucleotidyl transferases or exonucleases leading to floating 3′ overhangs. These unstable reference sites make Dicer produce relatively inaccurate 5′ ends. (TIFF 14030 kb)

Supplementary Table 1. Primer Sequence and Amplification Efficiency (DOC 35 kb)

Supplementary Table 2. Mapping Statistics of small RNAs (DOC 44 kb)

10549_2012_2166_MOESM7_ESM.doc

Supplementary Table 3. Differently Expressed miRNAs between IMPC and IDC-NSTs by FFPE Specimen Deep sequencing Analysis. (DOC 119 kb)

10549_2012_2166_MOESM8_ESM.doc

Supplementary Table 4A. miRNA Editing Forms in IMPC and IDC-NSTs. Supplementary Table 4B. miRNA Editing Frequency of Each Position in IMPC and IDC-NSTs. (DOC 100 kb)

Supplementary Table 5. miRNA 3′ Untemplate Nucleotide Addition in IMPC and IDC-NSTs. (DOC 34 kb)

10549_2012_2166_MOESM10_ESM.doc

Supplementary Table 6. Differentially Expressed Genes between IMPC and IDC-NST and Their Potential Regulating miRNAs. (DOC 138 kb)

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Li, S., Yang, C., Zhai, L. et al. Deep sequencing reveals small RNA characterization of invasive micropapillary carcinomas of the breast. Breast Cancer Res Treat 136, 77–87 (2012). https://doi.org/10.1007/s10549-012-2166-6

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