Amino acid networks (AANs) are undirected networks consisting of amino acid residues and their interactions in three-dimensional protein structures. The analysis of AANs provides novel insight into protein science, and several common amino acid network properties have revealed diverse classes of proteins. In this review, we first summarize methods for the construction and characterization of AANs. We then compare software tools for the construction and analysis of AANs. Finally, we review the application of AANs for understanding protein structure and function, including the identification of functional residues, the prediction of protein folding, analyzing protein stability and protein-protein interactions, and for understanding communication within and between proteins.

Transforming growth factor beta 1 (TGF-β1) is the most potent inhibitor of myogenic differentiation (MyoD) of rhabdomyosarcoma (RMS); however, the underlying mechanisms of this inhibition remain unclear. In this study, we identified novel TGF-β1-related microRNAs (miRNAs); among these, miR-450b-5p is significantly regulated by TGF-β1. We provide evidence that TGF-β1 exerts it function by suppressing miR-450b-5p. Both in cultured cells and tumor implants, miR-450b-5p significantly arrested the growth of RMS and promoted its MyoD. Utilizing a bioinformatics approach, we identified miR-450b-5p target mRNAs. Among these candidates, only the expression of ecto-NOX disulfide-thiol exchanger 2 (ENOX2) and paired box 9 (PAX9) was augmented by miR-450b-5p knockdown examined by western blot; the engineered inhibition antagonized TGF-β1-mediated differentiation inhibition. Furthermore, we found that the Smad3 and Smad4 pathways, but not Smad2, are the principal mediator of TGF-β1 suppression of miR-450b-5p. Taken together, these results suggest that disrupting the TGF-β1 suppression of miR-450b-5p, or knockdown of ENOX2 and PAX9, are effective approaches in inducing RMS MyoD.

The origin of rhabdomyosarcoma (RMS) remains controversial. However, specific microRNAs (miRNAs) are downregulated in RMS and it is possible that re-expression of these miRNAs may lead to differentiation. Transforming growth factor-β1 (TGF-β1) is known to block differentiation of RMS. We therefore analyzed miRNA microarrays of RMS cell lines with or without TGF-β1 knockdown and identified a novel anti-oncogene miR-411-5p. Re-expression of miR-411-5p inhibited RMS cell proliferation in vitro and tumorigenicity in vivo. Using a luciferase reporting system and sequence analysis, the potential target of miR-411-5p was identified as sprouty homolog 4 (SPRY4), which inhibits protein kinase Cα-mediated activation of mitogen-activated protein kinases (MAPKs), especially p38MAPK phosphorylation. These results revealed an inverse correlation between TGF-β1/SPRY4 and miR-411-5p levels. SPRY4 small interfering RNA and miR-411-5p both activated p38MAPK phosphorylation and also promoted apoptosis and myogenic differentiation, indicated by increased caspase-3, myosin heavy chain, and myosin expression. SPRY4 and miR-411 mRNA levels correlated with TGF-β1 expression levels in RMS tissues, which was confirmed by immunohistochemical staining for TGF-β1, SPRY4, and phosphorylated p38MAPK proteins. Overall, these results indicate that miR-411-5p acts as an RMS differentiation-inducing miRNA prompting p38MAPK activation via directly downregulating SPRY4. These results establish an autoregulatory loop between TGF-β1/miR-411-5p/SPRY4 and MAPK in RMS, which governs the switch between proliferation and differentiation.

Transforming growth factor-β1 is considered a key contributor to the progression of breast cancer. MicroRNAs are important factors in the development and progression of many malignancies. In the present study, upon studies of breast cancer cell lines and tissues, we showed that microRNA -196a-3p is decreased by transforming growth factor-β1 in breast cancer cells and associated with breast cancer progression. We identified neuropilin-2 as a target gene of microRNA -196a-3p and showed that it is regulated by transforming growth factor-β1. Moreover, transforming growth factor-β1-mediated inhibition of microRNA -196a-3p and activation of neuropilin-2were required for transforming growth factor-β1-induced migration and invasion of breast cancer cells. In addition, neuropilin-2 expression was suppressed in breast tumors, particularly in triple-negative breast cancers. Collectively, our findings strongly indicate that microRNA -196a-3p is a predictive biomarker of breast cancer metastasis and patient survival and a potential therapeutic target in metastatic breast cancer.

Overexpression of transforming growth factor-b1 (TGF-b1) and its downstream molecules in the rhabdomyosarcoma (RMS) RD cell line has been reported previously, but the regulatory role of TGFb1 on RMS has not been studied extensively. In the present study, we showed that expression of TGF-b1 and its downstream molecules type II TGF-b receptor (TbRII) and Smad4 was significantly higher in RMS than in normal skeletal muscle, and there was a significant relationship between TGF-b1 expression and histological grade. Gene silencing with TGF-b1 short-hairpin RNA (shRNA)-expressing vectors significantly decreased the growth of RD cells, which was confirmed by caspase-3 (in vitro) and TUNEL (in vivo) assays. Moreover, a proportion of treated rhabdomyosarcoma (RD) cells changed to a round shape from the normal fusiform or polygonal shape and expressed myofilaments. Myogenin is one of the myogenic differentiation genes (MyoD) family of myogenic regulators, and was obviously higher in TGF-b1-shRNA-treated tumors than it in control at the mRNA and protein level. Immunohistochemical staining with myogenic differentiation markers such as myosin and desmin in subcutaneous RMS tissue showed that TGF-b1 shRNA increased staining for myosin. These results provide new insight into the biological function of TGF-b1 in malignant tumors, and imply that the TGF-b1 signal pathway is a potential therapeutic target for drugs that induce differentiation of RMS.