mRNA and long non-coding RNA expression profiles of rotator cuff tear patients reveal inflammatory features in long head of biceps tendon

Abstract

Background: This study aimed to identify the differentially expressed mRNAs and lncRNAs in inflammatory long head of biceps tendon (LHBT) of rotator cuff tear (RCT) patients and further explore the function and potential targets of differentially expressed lncRNAs in biceps tendon pathology.

Methods: Human gene expression microarray was made between 3 inflammatory LHBT samples and 3 normal LHBT samples from RCT patients. GO analysis and KEGG pathway analysis were performed to annotate the function of differentially expressed mRNAs. The real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was admitted to verify their expression. LncRNA-mRNA co-expression network, cis-acting element, trans-acting element and transcription factor (TF) regulation analysis were constructed to predict the potential molecular regulatory mechanisms and targets for LHB tendinitis.

Results: 103 differentially expressed lncRNAs and mRNAs, of which 75 were up-regulated and 28 were down-regulated, were detected to be differentially expressed in LHBT. The expressions of 4 most differentially expressed lncRNAs (A2MP1, LOC100996671, COL6A4P, lnc-LRCH1-5) were confirmed by qRT-PCR. GO functional analysis indicated that related lncRNAs and mRNAs were involved in the biological processes of regulation of innate immune response, neutrophil chemotaxis, interleukin-1 cell response and others. KEGG pathway analysis indicated that related lncRNAs and mRNAs were involved in MAPK signaling pathway, NF-kappa B signaling pathway, cAMP signaling pathway and others. TF regulation analysis revealed that COL6A4P2, A2MP1 and LOC100996671 target NFKB2.

Conclusions: LlncRNA-COL6A4P2, A2MP1 and LOC100996671 may regulate the inflammation of LHBT in RCT patients through NFKB2/NF-kappa B signaling pathway, and preliminarily revealed the pathological molecular mechanism of tendinitis of LHBT.

Keywords: Inflammation; Long head of biceps tendon; Long non-coding RNA; Rotator cuff.

Fig. 1

Macroscopic and microscopic view of inflamed and non-inflamed tendon samples. long head of the biceps tendon (LHBT) samples with (A, B) and without (C, D) tendinitis are shown. A, C Intraoperative arthroscopic view. B, D Tendon samples before processing in the laboratory.

Fig. 2

Scatter plot (A), Volcano plots (B) and heat map (C) showing expression profiles of long non-coding RNAs (lncRNAs) and mRNAs in inflammatory and normal long head of biceps tendon (LHBT). Three plots are based on the expression values of all lncRNAs and mRNAs detected by microarray. These maps showing significantly changed lncRNAs and mRNAs with fold change ≥ 2.0 respectively (P < 0.05; false discovery rate < 0.05).

Fig. 3

qRT-PCR validation. qRT-PCR verification of 4 candidate lncRNAs in 3 pairs of inflammatory and normal LHBT tissue. Expression of inflammatory vs. normal samples was analyzed using qRT-PCR, and summarized as mean average ± standard error (SE). P < 0.05 was considered statistically significant.

Fig. 4

Gene Ontology (GO) annotations of up-and down regulated lncRNAs and mRNAs (fold change > 2; p < 0.05) with top 10 enrichment scores of biological processes. This bar chart shows the go items of top 10 in x-axis. Y axis called enrichment score is − log10 (p value), and the higher the bar graph height, the smaller the corresponding p value. Different color distributions correspond to cellular component (green), biological process (red) and molecular function (blue).

Fig. 5

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis scores of up-and down regulated lncRNAs and mRNAs with top 30. This bubble chart shows that the x-axis called gene ratio represents the enrichment degree and the y-axis represents the enrichment pathway. The larger the circle dot is, the more genes fall into the pathway. The greener the color is, and the higher the significance of enrichment is.

Fig. 6

The lncRNA-mRNA co-expression network with significant values of Pearson correlation coefficients (p < 0.05). The rhombuses denote lncRNAs and the ellipses denote mRNAs (green: downregulated genes; red: upregulated genes). An edge represents a co-expression relationship between mRNA and a lncRNA in the development of LHB tendinitis. Data were analyzed and constructed by Cytoscape software. This co-expression network suggests an inter-regulation of lncRNAs and mRNAs in LHB tendinitis.

Fig. 7

Networks analysis of putative interactions between 18 selected lncRNAs and trans-regulated mRNAs related to the tendinopathy of LHBT. Circular (red) and triangular (green) nodes represent lncRNAs and protein-coding genes, respectively.

Fig. 8

Predicted top 20 related transcription factors (TFs) of three differentially expressed lncRNAs including COL6A4P2 (A), A2MP1 (B), LOC100996671 (C). The x-axis represents the enrichment score, and the larger the bubble, the more differential coding genes it contains. The bubble color changes according to purple-blue-green-red. The smaller the enrichment p value, the greater the significance.