Altered Expressions of NF1 and NF1-Related microRNAs as Biomarkers in the Diagnosis of Undifferentiated Pleomorphic Sarcoma

Abstract

Objective: Undifferentiated pleomorphic sarcoma (UPS) is a highly malignant, aggressive, and pleomorphic subtype of soft tissue sarcoma in adults. However, UPS is difficult to be diagnosed due to the lack of specific morphological and immunophenotypic features. Here, we aimed to identify new biomarkers for the diagnosis of UPS. Methods: The mRNA and protein expression of neurofibromin 1 (NF1) in 68 pairs of UPS and adjacent normal tissues were detected by qRT-PCR and immunohistochemistry, and the correlation between the NF1 protein expression and clinicopathological characteristics was analyzed. Then, differentially expressed microRNAs (DE miRNAs) were identified between the UPS tumor tissue and matched adjacent normal tissue using Hisep sequencing, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG). The DE miRNAs of the regulating NF1 gene were also identified using the TargetScan and miRanda databases and validated by qRT-PCR. Results: Compared with the adjacent normal tissue, both mRNA and protein expressions of NF1 in the UPS tumor tissue were significantly decreased, and the positive rate of NF1 protein was associated with the tumor size, metastasis, and recurrence. A total of 125 known DE miRNAs were identified from the screened miRNAs based on | log₂(Fold Change) ≥5 and p-value < 0.05 (A total of 82 upregulated and 43 downregulated DE miRNAs in the UPS tissue). Target genes regulated by the DE miRNAs were enriched in pathways of metabolisms, RNA degradation, PI3K-Akt, and Rap1 pathway. In total, 11 miRNAs which were predicted to regulate the NF1 gene were screened. After verification, the relative expressions of hsa-miR-199a-3p and hsa-miR-34a-5p were increased and decreased in the UPS tumor tissue compared with those in the adjacent normal tissue, respectively. Conclusion: NF1 and NF1-related microRNAs including hsa-miR-199a-3p and hsa-miR-34a-5p may be novel biomarkers in the diagnosis of undifferentiated pleomorphic sarcoma (UPS).

Keywords: NF1; biomarkers; microRNA; soft tissue sarcomas; undifferentiated pleomorphic sarcoma.

FIGURE 1

Expression of NF1 in the UPS sarcoma tissue and adjacent normal tissue. (A): NF1 mRNA expression in the UPS sarcoma tissue and adjacent normal tissue by qRT-PCR, *: vs. adjacent normal tissue, p < 0.05; (B) Representative images of the protein expression of NF1 in the UPS sarcoma tissue and adjacent normal tissue by immunohistochemistry (200×) (a): High protein expression of NF1 in the adjacent normal tissue; (b): Low or negative protein expression of NF1 in the adjacent normal tissue; (c): High protein expression of NF1 in the UPS sarcoma tissue; (d): Low or negative protein expression of NF1 in the UPS sarcoma tissue.

FIGURE 2

Protein expression of p-Akt, p-mTOR, and p-S6 in the UPS sarcoma tissue and adjacent normal tissue. (A): Representative images of the protein expression of p-Akt, p-mTOR, and p-S6 in the UPS sarcoma tissue and adjacent normal tissue by immunohistochemistry (200×); (B): The comparison of the ratios of the positive area to the total area of p-Akt, p-mTOR, and p-S6 between the UPS sarcoma tissue and adjacent normal tissue. *: vs. adjacent normal tissue, p < 0.05.

FIGURE 3

Scatter plot indicating the differentially expressed miRNAs between the adjacent normal tissue and UPS tissue. The principles of identifying the differentially expressed miRNAs were based on | log₂(Fold Change) | ≥2 and p-value <0.05. Each dot represented one miRNA. The red dots represented upregulated miRNAs, the green dots represented downregulated miRNAs, and the blue dots represented equally-expressed miRNAs.

FIGURE 4

GO and KEGG analysis of genes targeted by the DE miRNAs. (A–C) Top four GO biological processes (BPs), cellular components (CCs), and molecular functions (MFs) enriched in target genes regulated by all the DE miRNAs. (D) KEGG enrichment analysis of signal pathways.

FIGURE 5

Validation by qRT-PCR of the three miRNAs regulating the NF1 gene. (A–C) were the expression and comparison of miR-127-3p, miR-199a-3p, and miR-34a-5p in 68 pairs of the UPS tumor tissue and adjacent normal tissue. U6 was used as the reference gene. *: vs. adjacent normal tissue, p < 0.05.

FIGURE 6

Conclusion image of NF1 and NF1-related miRNAs involved in signal pathways and occurrence of UPS. Note: Blue arrows: downregulated expression; Red arrows: upregulated expression; and Dotted line: Need to be validated.