The present study aimed to screen several differentially expressed genes (DEGs) and differentially expressed microRNAs (miRNAs) for two types of mesenchymal stem cell (MSC) differentiation. Bone morphogenetic protein 6 (BMP-6) and dexamethasone were used to induce MSCs towards osteoblastic differentiation or adipocytic differentiation. The t-test in the Bioconductor bioinformatics software tool was used to screen DEGs and differentially expressed miRNAs in the two samples. Subsequent gene ontology (GO) and pathway analyses on the DEGs were performed using the GO and Kyoto Encyclopedia of Genes and Genomes databases, respectively; potential target genes for the screened miRNAs were predicted using the TargetScan database. In addition, an interaction network between the DEGs and miRNAs was constructed. Numerous DEGs and miRNAs were screened during osteoblastic and adipocytic differentiation of MSCs. Important pathways, such as glutathione metabolism, pathogenic Escherichia coli infection and Parkinson's disease, and GO terms, including cytoskeletal protein binding and phospholipase inhibitor activity, were enriched in the screened DEGs from MSCs undergoing osteogenic differentiation and adipocytic differentiation. miRNAs, including miRNA (miR)-382 and miR-203, and DEGs, including neuronal growth regulator 1 (NEGR1), phosphatidic acid phosphatase 2B (PPAP2B), platelet-derived growth factor receptor alpha (PDGFRA), interleukin 6 signal transducer (IL6ST) and sortilin 1 (SORT1), were demonstrated to be involved in osteoblastic differentiation. In addition, the downregulated miRNAs (including miR-495, miR-376a and miR-543), the upregulated miR-106a, the upregulated DEGs, including enabled homolog (ENAH), polypeptide N-acetylgalactosaminyltransferase 1 and acyl-CoA synthetase long-chain family member 1, and the downregulated repulsive guidance molecule family member B and semaphorin SEMA7A were demonstrated to be involved in adipocytic differentiation. The results of the present study suggested that miRNAs (miR-203 and miR-382) and DEGs (NEGR1, PPAP2B, PDGFRA, IL6ST and SORT1) may serve pivotal functions in the osteoblastic differentiation of MSCs, whereas miR-495, which is also involved in osteoblast differentiation and had four targets, including NEGR1, miR-376a, miR-543 and ENAH may have crucial roles in adipocytic differentiation of MSCs.

Ubiquitin-specific processing enzyme 22 (USP22) plays a direct role in regulating cell cycle, and its overexpression has been reported to be involved in tumor progression. However, little is known about the regulation of USP22 transcription. In this study, we cloned and characterized the human USP22 promoter. Using 5′ RACE (rapid amplification of cDNA ends) analysis, the transcriptional initiation site was identified. Promoter deletion analysis showed that the sequence between −210 and −7 contains the basal promoter for USP22 in human fibroblast and tumor cells. Surprisingly, mutations in a putative Sp1 binding site immediately upstream of the USP22 transcriptional start site (−13 to −7) resulted in a significant induction of promoter activity. Further study revealed that Sp1 binds to this site in human normal fibroblast cells, and treatment with the Sp1 inhibitor mithramycin A led to a marked increase in USP22 transcript levels. Forced expression of exogenous Sp1 repressed the USP22 promoter activity in HeLa cells. In contrast, knockdown of Sp1 enhanced USP22 promoter activity and mRNA levels. These data suggest that Sp1 is a crucial regulator of USP22 transcription.

MicroRNAs are members of the family of non-coding small RNAs that regulate gene expression either by inhibiting mRNA translation or by promoting mRNA degradation at the post-transcriptional level. They play an important role in the differentiation of human bone marrow mesenchymal stem cells (hMSCs) into adipocytes. However, the role of microRNAs in this process remains to be poorly understood. Here, we observed that miR-377-3p expression was markedly decreased during adipogenic differentiation of hMSCs. Overexpression of miR-377-3p decreased adipocyte differentiation and downregulated the expression of adipogenic markers. Meanwhile, bioinformatics-based studies suggested that LIFR is a target of miR-377-3p. Further analysis confirmed that expression of LIFR present markedly increased during adipogenic differentiation of hMSCs. In addition, downregulation expression of LIFR significantly inhibited the process of adipocyte differentiation. To confirm the relation between miR-377-3p and LIFR, luciferase reporter assays were carried out. The results indicated that miR-377-3p bound directly to the 3'-untranslated region of LIFR. These data indicate that miR-377-3p suppressed adipogenesis of hMSCs by targeting LIFR, which provides novel insights into the molecular mechanism of miRNA-mediated cellular differentiation.

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The ubiquitin-specific protease 22 (USP22) is an oncogene and its expression is upregulated in many types of cancer. In the nucleus, USP22 functions as one subunit of the SAGA to regulate gene transcription. However, the genome-wide USP22 binding sites and its direct target genes are yet clear. In this study, we characterized the potential genomic binding sites of UPS22 and GCN5 by ChIP-seq using specific antibodies in HeLa cells. There were 408 overlapping putative target genes bound by both USP22 and GCN5. Motif analysis showed that the sequences bound by USP22 and GCN5 shared two common motifs. Gene ontology (GO) and pathway analysis indicated that the genes targeted by USP22 and GCN5 were involved in different physiological processes and pathways. Further RNA-seq, GO and pathway analyses revealed that knockdown of UPS22 induced differential expression of many genes that participated in diverse physiological processes, such as metabolic process. Integration of ChIP-seq and RNA-seq data revealed that UPS22 bound to the promoters of 56 genes. These findings may provide new insights into the regulation of USP22 on gene expression during the development of cervical cancer.

Abstract. Elevated expression of ubiquitin-specific processing enzyme 22 (USP22) was identified in multiple types of human cancers, and was correlated with tumorigenesis and progression. Despite an increase in the numbers of studies in the physiological function of USP22, little is known regarding the regulation of its expression. The 5' flanking sequence of the USP22 gene was recently characterized. In the present study, USP22 transcription was regulated by p38 mitogen-activated protein kinase (MAPK). Treatment of human cervical carcinoma (HeLa) cells with SB203580, an inhibitor of p38 MAPK, enhanced basal USP22 promoter activity and mRNA abundance. Transfection of MAPK kinase 6 (MKK6), an upstream activator of p38 MAPK, resulted in a 40% decrease in USP22 mRNA, while the dominant negative MKK6 increased the transcription level of the USP22, similar to SB203580. Dual luciferase report assays showed that mutations of the Sp1 binding site ahead of the transcription start site abolished the promoting effect of the USP22 promoter by SB203580. Cisplatin, the activator of p38 MAPK, also suppressed USP22 expression. This suppression was blocked by SB203580. In conclusion, p38 MAPK acts as an upstream negative regulator of USP22 transcription in HeLa cells. IntroductionUbiquitin-specific processing enzyme 22 (USP22) is a novel deubiquitinating enzyme that can cleave ubiquitin (Ub) from Ub-conjugated protein substrates (1). As the subunit of the human SAGA coactivator complex, USP22 is linked to the regulation of gene transcription by deubiquitinating histones H2A and H2B (2,3). In addition, USP22 deubiquitinates intracellular protein, including the shelterin protein telomeric repeat binding factor 1 (4), the histone deacetylase sirtuin 1 (5) and the far upstream element-binding protein 1 fructose-1,6-bisphosphatase 1 (4), and therefore performs an extensive physiological function. A murine study showed that USP22 also regulates embryonic stem cell differentiation (6). In humans, the USP22 gene is located on chromosome 17, consists of 14 exons, and is transcribed and produced broadly across various tissues (7). Of note, elevated levels of USP22 have been identified in numerous types of human cancer, including colorectal (8) lung (9) and breast cancer (10). USP22 has been indicated in tumorigenesis. Deletion of USP22 leads to the accumulation of cells in the G 1 phase of the cell cycle (2). For these reasons, USP22 is a putative cancer stem cell marker. Reducing the rate of USP22 expression may be a suitable target for cancer therapy (11). However, the mechanisms that lead to USP22 transcriptional activation, particularly in the human tumor cells, remain unknown.Previously, USP22 transcription was activated by mitogen stimulation or viral infection in normal T and B lymphocytes (12), suggesting the regulation of USP22 gene expression occurs mainly at the transcriptional level. However, the mechanism in which signal transduction pathways regulate USP22 transcription is unclear. It is well-known that activation of the mito...

Adipogenesis and osteoblastogenesis (adipo-osteoblastogenesis) are closely related processes involving with the phosphorylation of numerous cytoplasmic proteins and key transcription factors.

The ubiquitin‑specific protease 22 (USP22) gene is overexpressed in the majority of types of cancer cells, and has been implicated in tumorigenesis. However, the mechanisms that regulate its expression remain unclear. The results of the present study demonstrated that the expression of USP22 is negatively regulated by trichostatin A (TSA), a classical histone deacetylase inhibitor. Furthermore, TSA was revealed to interfere with the binding of RNA polymerase II to the USP22 promoter, directly suppressing its transcription. In addition, the overexpression of USP22 was observed to attenuate TSA‑induced apoptosis in HeLa cells. To the best of our knowledge, these results provide the first insight into the regulation of the USP22 gene by antitumor drugs and into the mechanisms underlying the anticancer activity of TSA.