SPI1 involvement in malignant melanoma pathogenesis by regulation of HK2 through the AKT1/mTOR pathway.

Spi-1 proto-oncogene (SPI1) plays a vital role in carcinogenesis. Our work aimed to investigate the potential regulatory mechanism of SPI1 in melanoma. The mRNA and protein levels were measured via qRT-PCR and Western blotting. Cell viability was assessed by CCK-8 assay. The target relationship between SPI1 and hexokinase 2 (HK2) was determined using dual-luciferase reporter detection. ChIP was conducted to confirm the targeted relationship between SPI1 and the HK2 promoter. Immunohistochemistry analysis was conducted to measure the positive cell number of SPI1 and HK2 in melanoma tissues. The cell migration abilities were determined using a wound healing assay. Glucose consumption, pyruvate dehydrogenase activity, lactate production and ATP levels were measured to assess glycolysis. SPI1 transcription in melanoma cells and tissues was dramatically higher than that in adjacent normal tissues and epidermal melanocyte HEMa-LP, respectively. Knockdown of SPI1 restrained cell viability, metastasis and glycolysis in melanoma cells. SPI1 directly targeted HK2, and knockdown of SPI1 repressed HK2 expression. Overexpression of HK2 weakened the inhibitory effects of SPI1 knockdown on the viability, metastasis and glycolysis of melanoma cells. The serine-threonine kinase 1 (AKT1)/mammalian target of rapamycin (mTOR) axis is involved in melanoma progression. SPI1 knockdown restrained melanoma cell proliferation, metastasis and glycolysis by regulating the AKT1/mTOR pathway.

lncRNA RNCR2 facilitates cell proliferation and epithelial-mesenchymal transition in melanoma through HK2-mediated Warburg effect via targeting miR-495-3p.

Melanoma is a potentially lethal skin cancer with a high death rate. LncRNAs were reported to be implicated in melanoma progression. However, the function and mechanisms of lncRNA RNCR2 in melanoma are little known. In this study, RNCR2, miR-495-3p, and HK2 expression levels were measured in melanoma tissue specimens and cell lines by qPCR. EdU and CCK-8 assays were performed to assess cell proliferation. Enolase activity, ATP level, lactate production, and glucose consumption measurement kits were used to evaluate the glycolysis of tumor cells. Immunofluorescence and western blot were used to detect the expression of epithelial-mesenchymal transition (EMT) and glycolysis-related proteins. Luciferase reporter assay was applied to confirm the target relationships. The role of RNCR2 in tumorigenesis was examined using murine xenograft models. LncRNA RNCR2 was upregulated in melanoma tissues and cell lines. Cell function detection showed that RNCR2 knockdown remarkably inhibited cell proliferation and EMT via glycolysis, as well as reduced the growth of a tumor. Mechanically, RNCR2 was confirmed to bind to miR-495-3p and positively regulated HK2 expression level, and the miR-495-3p level was negatively correlated with RNCR2 or HK2 in melanoma tissues. Further, miR-495-3p downregulation or HK2 upregulation partially reversed RNCR2 knockdown-induced inhibition of melanoma cell growth, EMT, and glycolysis. Collectively, RNCR2 might be an oncogenic lncRNA to promote tumor cell glycolysis and accelerate tumor growth via the miR-495-3p/HK2 axis, providing a promising treatment target for melanoma.

CREB1 regulates KPNA2 by inhibiting mir-495-3p transcription to control melanoma progression : The role of the CREB1/miR-495-3p/KPNA2 axis in melanoma progression.

BACKGROUND: Melanoma is a common type of skin cancer, and its incidence is increasing gradually. Exploring melanoma pathogenesis helps to find new treatments. OBJECTIVE: We aimed to explore the potential molecular mechanisms by which CREB1 regulates melanoma. METHODS: TransmiR and ALGGEN were used to predict targets of CREB1 in the promoter of miR-495-3p or miR-495-3p and KPNA2, and a dual-luciferase reporter assay was performed to detect binding of CREB1 to these promoters. In addition, binding of CREB1 to the miR-495-3p promoter was confirmed by a ChIP assay. qRT‒PCR was carried out to detect mRNA levels of miR-495-3p, CREB1 and KPNA2. An EdU assay was conducted to detect cell viability. Transwell assays and flow cytometry were performed to assess cell migration and invasion and apoptosis, respectively. Moreover, factors associated with overall survival were analysed by using the Cox proportional hazards model. RESULTS: Our results show miR-495-3p to be significantly decreased in melanoma. Additionally, miR-495-3p overexpression inhibited melanoma cell viability. CREB1 targeted miR-495-3p, and CREB1 overexpression enhanced melanoma cell viability by inhibiting miR-495-3p transcription. Moreover, miR-495-3p targeted KPNA2, and CREB1 regulated KPNA2 by inhibiting miR-495-3p transcription to enhance melanoma cell viability. CONCLUSION: CREB1 regulates KPNA2 by inhibiting miR-495-3p transcription to control melanoma progression. Our results indicate the molecular mechanism by which the CREB1/miR-495-3p/KPNA2 axis regulates melanoma progression.

Influence of intense pulsed light on the secretion of TGF-beta1 in cultured human fibroblasts and intervention of JNK inhibitor.

OBJECTIVE: To determine the influence of intense pulsed light (IPL) on the secretion of TGF-beta1 in cultured human fibroblasts and the intervention of JNK inhibitor. METHODS: The callan foreskin fibroblasts were cultured and divided into 2 groups. In the IPL treatment group, cells were irradiated with IPL with fluences of 0 (negative control), 10, 18, 27, 36, and 36 J/cm2*2 (irradiated with IPL with fluences of 36 J/cm2 twice). In the IPL+inhibitor group, cells were irradiated with IPL with fluences of 36 J/cm2 after incubation with the inhibitor SP600125 for 2 h. TGF-beta1 in the culture supernatant was evaluated 48 h after the irradiation using enzyme-linked immunosorbent assay. RESULTS: Compared with the negative control, TGF-beta1 in the culture supernatant decreased at the IPL irradiation of 10, 18, 27, and 36 J/cm2, whereas TGF-beta1 increased at the IPL irradiation of 36 J/cm2*2. In the IPL+inhibitor group, the concentration of TGF-beta1 in the culture supernatant decreased compared with the controls (P<0.05). CONCLUSION: IPL can suppress the secretion of TGF-beta1 at the lower fluence and promote the secretion at a higher fluence. JNK inhibitor may play an inhibitive role when IPL regulates the TGF-beta1 secretion in cultured human fibroblasts. IPL may stimulate TGF-beta1 secretion of the fibroblast cells in human skin via JNK signal pathway.