Genome Wide Differential Expression Profiles in Nevus Sebaceous Uncovered Low Expression of CDKN2AIP and Construction of a ceRNA Network.

Background: Nevus sebaceous (NS) is a benign hamartoma of the skin, characterized by hyperplasia of the epidermis, in addition to immature hair follicles. The exact mechanisms of folliculo-sebaceous-apocrine defects and adnexal tumorigenesis are unknown in NS, but benign and malignant neoplasms are often due to a complex etiology in NS. Long noncoding RNAs (lncRNAs) have been implicated in various important biological processes and regulate inflammatory diseases and tumors. However, the role of lncRNAs in nevus sebaceous is unclear. Objective: To identify NS-associated mRNA and lncRNA profiles and predict their potential roles in the development of the folliculo-sebaceous-apocrine unit and adnexal tumorigenesis. Methods: RNA-seq was used to identify NS-associated genes and lncRNAs. Analysis software Illumina NovaSeq 6000 was used to analyze the sequences, and real-time PCR and Western blot were used to validate the differentially expressed genes. Competing endogenous RNAs (ceRNA) networks were constructed by prediction software TargetScan & miRanda. Results: Many mRNAs were significantly differentially expressed between nevus sebaceous and adjacent normal scalp tissues. Among them, 72 were upregulated and 18 were downregulated. KEGG pathway analysis further revealed that 32 functional pathways were associated with the upregulated mRNAs, while only 1 pathway was associated with the downregulated mRNAs. Verification by real-time PCR and Western blot indicated that CDKN2AIP gene was downregulated consistently in NS tissue compared to normal scalp skin. Additionally, 7 upregulated and 10 downregulated significantly differentially expressed lncRNAs were detected between NS and adjacent normal scalp tissues. Three downregulated lncRNAs including AL355607.2, RP5-1024G6.8 and AC007780.1 were predicted to consistently associate with CDKN2AIP expression by competing endogenous RNAs(ceRNA) construction. Conclusion: Both mRNA and lncRNA profiles were altered in NS scalp tissues. We identified a downregulated gene, CDKN2AIP, as a target of differentially expressed mRNA and predicted a ceRNA network of CDKN2AIP with differentially expressed lncRNA.

miR-128 targets the CC chemokine ligand 18 gene (CCL18) in cutaneous malignant melanoma progression.

BACKGROUND: The CC chemokine ligand 18 (CCL18) has a higher expression in some tumors, while the CCL18 level can be a marker of tumor progression and prognosis. We previously reported that the expression of CCL18 gene was dramatically up-regulated in cutaneous malignant melanoma (CMM) and its expression levels were correlated with tumor thickness. OBJECTIVE: To investigate miRNAs which could target the CCL18 gene so as to mediate CMM development and improvement. METHODS: The expression of miR-128 and CCL18 in CMM were measured by qRT-PCR. The interaction of miR-128 with CCL18 3'UTR was verified by Luciferase reporter gene assay. The changes in expression of CCL18 after miR-128 mimic transfection of A375 melanoma cells were determined by both qRT-PCR and Western-bloting. Cell viability was accessed by CCK8-assay. Flow cytometry was employed to detect the incidence of apoptosis. Clonogenic assay was used to detect the ability of colony formation. Cell migration was evaluated by Transwell migration study. The protein levels of epithelial-mesenchymal transition (EMT), such as E-cadherin, N-cadherin and ß-catenin were analyzed by Western-bloting. RESULTS: The expression of miR-128 had negative relevance with CCL18 in CMM. miR-128 could interact with CCL18 3'UTR. Transfected miR-128 mimic significantly reduced CCL18 expression and this impairment of CCL18 gene promoted apoptosis, inhibited migration and colony formation of A375 melanoma cells. Furthermore, the relative expression of N-cadherin was decreased. CONCLUSION: CCL18 is a target gene of miR-128. Overexpression of miR-128 inhibits the oncogenic effect of CCL18.

Up-regulation of long noncoding RNA FALEC predicts poor prognosis and promotes melanoma cell proliferation through epigenetically silencing p21.

Accumulating evidences have suggested that focally amplified lncRNA on chromosome 1 (FALEC) serves as an oncogenic long non-coding RNA (lncRNA) and has been identified to be dysregulated in various tumors. However, the expression, clinical values, and biological function of FALEC in melanoma are still unknown. In this study we detected the expression level of FALEC in tumor tissues and cell lines and measured the prognostic value of FALEC for melanoma patients and the biological effects of FALEC on melanoma cell proliferation, cell cycle, and apoptosis. Our results indicated that FALEC was more highly expressed in melanoma tissues and cell lines than in non-neoplastic nevi tissues and normal cell lines. Moreover, functional assays showed that silenced FALEC suppressed the proliferation of melanoma cells, resulted in cell cycle arrest, and induced apoptosis. Mechanically, we discovered that FALEC boosted melanoma progression via epigenetically repressing p21 through recruiting EZH2 to the promoter of p21. Generally, our results suggested that FALEC acted as an oncogene in melanoma and had the potential to be a prognostic biomarker and therapeutic target for melanoma.

Expression of Rho GTPases family in melanoma cells and its influence on cytoskeleton and migration.

Rho GTPases family members influenced the filopodia, lamellipodia, stress fiber and adhesion plaque of melanoma cells through regulating cytoskeleton recombination. The role of Rho GTPases family in the migration and invasion of melanoma and its molecular mechanism were explored. The morphological difference between three types of melanoma cells (M14, A375 and MV3) and human melanocyte (MC) was observed by the Hoffman microscope. Cells were stained by phalloidin labeled by rhodamine. The differences between 4 types of cells in filopodia, lamellipodia, stress fiber and adhesion plaque (microfilament is the main constituent) were observed under the super-high resolution microscope. The migration ability of 4 types of cells was detected by Transwell migration assay. QPCR was used to detect the mRNA transcription level of Rho GTPases family. WB was adopted to detect the expression of RhoD and DIAPH2 proteins. There were significant differences in filopodia, lamellipodia, stress fiber and adhesion plaque between MC and 3 types of melanoma cells (M14, A375 and MV3). MC did not have stress fiber or adhesion plaque, while M14, A375 and MV3 had stress fiber and adhesion plaque. All 4 types of cells had thin and short filopodia. MV3 had fewer but thicker stress fibers than the latter two. Transwell migration test indicated the followings: M14 and A375 had a similar high migration rate; the migration rate of MV3 was slightly low; MC did not have the ability of transmembrane migration. QPCR results of Rho GTPases family in 4 types of cells showed the change corresponding to immunofluorescence. WB results showed that RhoD was barely expressed in M14, A375 or MV3. DIAPH2, the downstream effector molecule of RhoD, had the corresponding change. Rho GTPases influences the migration and invasion of melanoma cells through regulating filopodia, lamellipodia, stress fiber and adhesion plaque (microfilament is the main constituent).

The inhibitory effects of NKX3.1 on IGF-1R expression and its signalling pathway in human prostatic carcinoma PC3 cells.

NKX3.1, which is a prostate-specific homeobox gene, plays an important role in prostate cancer and usually functions as a tumour suppressor gene. In this study, we investigated the inhibitory effect of NKX3.1 on insulin-like growth factor (IGF)-1R expression and its downstream signalling pathway in PC3 cells. PC3 cells were stably transfected with NKX3.1 expression plasmid (pcDNA3.1-NKX3.1) or vector plasmid (pcDNA3.1+). The IGF-IR mRNA and protein expression levels were assessed in PC3-NKX3.1 transfectants by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The expression and activation of IGF-1/IGF-1R downstream signalling targets were examined by Western blotting and luciferase reporter assay. The cells were subsequently treated with relevant concentrations of IGF-1. The effect of IGF-1 on cell growth was examined by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT) assay and flow cytometry analysis. A significant suppression of IGF-1R mRNA and protein expression was observed after forced expression of NKX3.1 in PC3 cells. Correspondingly, the forced expression of NKX3.1 decreased IGF-1-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (AKT) and activation of the Elk-1 transcription factor and downregulated the expression of the downstream target genes c-fos and cyclin D1. Furthermore, the forced expression of NKX3.1 inhibited IGF-1-induced cell growth. In conclusion, NKX3.1 could downregulate IGF-1R expression and could inhibit IGF-1R-mediated mitogen-activated protein kinase (MAPK)/ERK and AKT signalling pathways, which might partially leads to the inhibition of IGF-1-induced cell growth. This study provides new insights into the molecular mechanisms that NKX3.1 exerts against prostate cancer and ultimately expands the scope of alternative approaches in advanced prostate cancer therapy.

Characterization and functional analysis of the human microRNA let-7a2 promoter in lung cancer A549 cell lines.

Recent studies have revealed that microRNAs have a strong association with cancer in humans. The miRNA let-7 is highly expressed in normal lung tissue, but frequently expressed at reduced levels in lung cancers. Let-7a2 is a member of the let-7 family. So far, little is known about the transcriptional regulation of let-7a2. Our study is focused on the characterization and functional analysis of the promoter of the human miRNA let-7a2 in A549 cell lines. Firstly, 5' rapid amplification of cDNA ends (5' RACE) was carried out and a 2.8 kb fragment in the upstream of let-7a2 gene was then cloned into pGL3-basic vector. Sequence analysis with the MatInspector database revealed that there were putative binding sites for some important transcriptional factors in the promoter region of let-7a2, such as p53, c-Myc, Ras, CEBPα, RORA, RXR, TCF, and GR. Additionally, a series of transfection and luciferase reporter assays were carried out to test let-7a2 promoter activity. RT-PCR and transfection of let-7a target sequence-reporter plasmid were performed to detect transcription levels of the let-7a2 gene in A549 cells treated with 9-cis-RA, all-trans-RA, lithium chloride or dexamethasone. Our results showed that the recombinant pGL3-p7a2 could acts as a promoter. The promoter activity of the 2.8 kb fragment could be downregulated by transfection with CEBPα or treatment with lithium chloride and enhanced by 9-cis-RA or all-trans-RA treatment. Furthermore, the results of RT-PCR analysis and transfection of let-7a target sequence-reporter plasmid showed that 9-cis-RA and all-trans-RA both upregulated let-7a2 expression, while lithium chloride downregulated its expression. Our results suggest that 9-cis-RA, all-trans-RA,lithium chloride and CEBPα might play important regulatory roles in let-7a2 gene expression in A549 cells.