FOXO3 Inhibits the Cisplatin Resistance and Progression of Melanoma Cells by Promoting CDKN1C Transcription.

BACKGROUND: Forkhead box O3 (FOXO3) and cyclin dependent kinase inhibitor 1 C Gene (CDKN1C) have been shown to be involved in the melanoma process, but their roles in the cisplatin (DDP) resistance of melanoma remain unclear. METHODS: The mRNA levels of CDKN1C and FOXO3 were measured using quantitative real-time PCR. The protein levels of CDKN1C, FOXO3 and mitochondrial oxidative phosphorylation (mtOXPHOS)-related markers were determinant by western blot analysis. The DDP resistance, proliferation, and apoptosis of melanoma cells were assessed by cell counting kit 8 assay, colony formation assay and flow cytometry. Glucose consumption, lactate production and ATP level were detected to assess glycolysis. The regulation of FOXO3 on CDKN1C was confirmed by ChIP assay and dual-luciferase reporter assay. In vivo experiments were performed to evaluate the effect of FOXO3 on DDP sensitivity in melanoma tumor tissues. RESULTS: CDKN1C and FOXO3 were downregulated in chemoresistant melanoma tissues, and their low expression levels were related to the poor prognosis of melanoma patients. Overexpression of CDKN1C and FOXO3 repressed DDP resistance, proliferation, and glycolysis, while promoted apoptosis and mtOXPHOS in DDP-resistant melanoma cells. Further analysis suggested that FOXO3 could bind to CDKN1C promoter region to enhance its transcription. Besides, CDKN1C knockdown reversed the regulation of FOXO3 on melanoma cell DDP resistance and progression. Moreover, FOXO3 overexpression enhanced the DDP sensitivity of melanoma tumor tissues in vivo. CONCLUSION: FOXO3 promoted the transcription of CDKN1C, thereby inhibiting the DDP resistance and progression of melanoma cells.

A Comprehensive Study to Investigate the Tumor-Suppressive Role of Radix Bupleuri on Gastric Cancer with Network Pharmacology and Molecular Docking.

Background: Gastric cancer (GC) is a common fatal malignancy. The aim of this study was to explore and validate the tumor-suppressive role and mechanism of Radix Bupleuri in GC. Methods: The active constituents of Radix Bupleuri were screened using TCMSP database. SwissTargetPrediction database was used to predict potential target genes of the compounds. GeneCards, TTD, DisGeNET, OMIM, and PharmGKB databases were used to search for GC-related targets. STRING database and Cytoscape 3.10 software were used for protein-protein interaction network construction and screening of core targets. DAVID database was used for GO and KEGG analyses. Core targets were validated using molecular docking. Cell proliferation and apoptosis were detected using CCK-8 and flow cytometry after GC cells were treated with isorhamnetin. The mRNA and protein expression levels of genes were detected using qRT PCR and Western blot. The metastasis potential of GC cells was evaluated in a nude mouse model. Results: A total of 371 potential targets were retrieved by searching the intersection of Radix Bupleuri and GC targets. Petunidin, 3',4',5',3,5,6,7-Heptamethoxyflavone, quercetin, kaempferol, and isorhamnetin were identified as the main bioactive compounds in Radix Bupleuri. SRC, HSP90AA1, AKT1, and EGFR, were core targets through which Radix Bupleuri suppressed GC. The tumor-suppressive effect of Radix Bupleuri on GC was mediated by multiple pathways, including PI3K-AKT, cAMP, and TNF signaling. The key compounds of Radix Bupleuri had good binding affinity with the core target. Isorhamnetin, a key component of Radix Bupleuri, could inhibit proliferation and metastasis, and induces apoptosis of GC cells. In addition, isorhamnetin could also reduce the mRNA expression of core targets, and the activation of PI3K/AKT pathway. Conclusion: This study identified potential targets and pathways of Radix Bupleuri against GC through network pharmacology and molecular docking, providing new insights into the pharmacological mechanisms of Radix Bupleuri in GC treatment.

Circular RNA hsa_circ_0026344 suppresses gastric cancer cell proliferation, migration and invasion via the miR-590-5p/PDCD4 axis.

OBJECTIVES: Circular RNA (CircRNA) is a class of non-coding RNA transcripts, with multiple pathophysiological functions. Instead, the mechanism and function of circRNA in gastric cancer (GC) are not fully deciphered. METHODS: CircRNA_0026344 (circ_0026344), microRNA (miR)-590-5p and programmed cell death 4 (PDCD4) mRNA expression levels in GC tissues and cells were probed by quantitative real-time PCR. Cell viability, migration and aggressiveness were examined by cell counting kit-8 and transwell assays. Additionally, the interplay among circ_0026344, miR-590-5p and PDCD4 was verified with bioinformatics and dual-luciferase reporter gene assay. Western blot was conducted to probe PDCD4 protein expression. KEY FINDINGS: Circ_0026344 expression was underexpressed in GC tissues and cells, which was associated with clinicopathological characteristics such as tumour size, tumor-node-metastasis stage and lymph node metastasis. Circ_0026344 overexpression restrained the malignant biological behaviours of GC cells, while circ_0026344 knockdown functioned oppositely. Circ_0026344 could act as a competing endogenous RNA of miR-590-5p to negatively modulate its expression, and this miRNA could mitigate the impact of circ_0026344 on GC cells. In addition, PDCD4 was identified as the downstream target of miR-590-5p, and PDCD4 expression was positively modulated by circ_0026344. CONCLUSIONS: Circ_0026344 up-regulates PDCD4 expression via sponging miR-590-5p, thus inhibiting the progression of GC. This study further expounds the underlying molecular mechanism in the GC progression.

Long non-coding RNA GAS5 accelerates oxidative stress in melanoma cells by rescuing EZH2-mediated CDKN1C downregulation.

BACKGROUND: The significance of long non-coding RNAs (lncRNAs) in mediating oxidative stress of cancers has been implicated recently. This study proposed a potential therapeutic target lncRNA growth arrest-specific transcript 5 (GAS5) for melanoma, due to its crucial role in oxidative stress and apoptosis of melanoma cells by regulating the enhancer of zeste homolog 2 (EZH2)-mediated CDKN1C expression. METHODS: The lncRNA GAS5 expression pattern was examined in melanoma tissues and cells. The correlation of lncRNA GAS5, EZH2, and CDKN1C with survival rate of melanoma patients was analyzed. In melanoma cell lines, lncRNA GAS5 expression was overexpressed or knocked down to clarify its effects on cell viability, apoptosis, and oxidative stress. The interaction between lncRNA GAS5 and EZH2 was examined by RIP and RNA pull-down assays followed by verification of the target relationship between EZH2 and CDKN1C. RESULTS: High expression of EZH2 and poor expression of lncRNA GAS5 and CDKN1C was observed in melanoma tissues and found to be correlated with the reduction in survival expectancy of melanoma patients. Overexpression of lncRNA GAS5 or CDKN1C or EZH2 knockdown could inhibit cell viability but enhance melanoma cell apoptosis and oxidative stress. Importantly, lncRNA GAS5 attenuated EZH2 expression by recruiting E2F4 to the EZH2 promoter region and knockdown of EZH2 upregulated CDKN1C expression by inhibiting the H3K27me3. CONCLUSION: The evidence provided by our study highlighted the involvement of lncRNA GAS5 in the translational suppression of EZH2 as well as the upregulation of CDKN1C, resulting in the promotion of melanoma cell apoptosis and oxidative stress.

Silencing of microRNA-517a induces oxidative stress injury in melanoma cells via inactivation of the JNK signaling pathway by upregulating CDKN1C.

BACKGROUND: Melanoma is notoriously resistant to current treatments, and less than 25% of metastatic melanoma cases respond to existing therapies. Growing evidence has shown that microRNAs (miRNAs) play a vital role in the prognosis of melanoma. MiR-517a has been implicated in many types of cancer; however, its expressional features and potential biological functions in melanoma remain unclear. The present study aimed to investigate the possible effects of miR-517a on oxidative stress (OS) in melanoma cells. METHODS: miR-517a expression in melanoma was determined using RT-qPCR. After treatment with different concentrations of H2O2, cell viability was determined in order to identify the most appropriate H2O2 concentration. Through loss and gain of function experiments, the interactions between miR-517a, the cyclin dependent kinase inhibitor 1C (CDKN1C) and the c-Jun NH2-terminal kinase (JNK) signaling pathway, as well as their roles in OS of melanoma cells were identified. Moreover, the expression of Cleaved Caspase-3, extent of ERK1/2 phosphorylation, Bax/Bcl-2 ratio, levels of T-AOC, ROS and MDA, and SOD activity were also tested. Finally, melanoma cell viability and apoptosis were detected. RESULTS: MiR-517a was upregulated, while CDKN1C was downregulated in melanoma tissues and cells. MiR-517a targets CDKN1C and consequently reduced its expression. Inhibition of miR-517a was shown to increase Cleaved Caspase-3 expression, Bax/Bcl-2 ratio, levels of ROS and MDA, as well as cell apoptosis but decrease extent of ERK1/2 phosphorylation, T-AOC levels, SOD activity, along with cell proliferation and mitochondrial membrane potential. CONCLUSIONS: Overall, silencing miR-517a results in upregulated CDKN1C expression, and inhibited JNK signaling pathway activation, consequently promoting OS in melanoma cells.

TNNT1, negatively regulated by miR-873, promotes the progression of colorectal cancer.

BACKGROUND: Troponin T1 (TNNT1) is a subunit of troponin that has been linked to neuromuscular disorder. Recently, it was reported that TNNT1 facilitates the proliferation of breast cancer cells. Interestingly, Cancer Genome Atlas data indicate that its overexpression is associated with an unfavorable prognosis of colorectal cancer (CRC) patients. The present study aimed to explore the expression, function and mechanism of dysregulation of TNNT1 in CRC. METHODS: Immunohistochemical staining and a real-time polymerase chain reaction were used to compare the expression level of TNNT1 in CRC tissues and adjacent tissues. Western blotting was used to detect the expression of TNNT1 in cell lines. Kaplan-Meier analysis and a chi-squared test were applied to evaluate the potential of TNNT1 to function as a cancer biomarker. RNA interference was used to inhibit TNNT1 expression in CRC cells, followed by detection of cell proliferation, apoptosis, migration and invasion. A luciferase reporter gene assay was used to determine the regulatory relationship between miR-873 and TNNT1. RESULTS: In the present study, we found that TNNT1 was significantly up-regulated in CRC samples and cell lines. The up-regulation of TNNT1 was also associated with several clinicopathologic features, and its high expression was correlated with an unfavorable prognosis of the patients. Knockdown of TNNT1 markedly arrested proliferation, migration and invasion, whereas it also promoted apoptosis. TNNT1 was identified as a target gene of miR-873, and there was a negative correlation among CRC samples. CONCLUSIONS: In conclusion, we have demonstrated that TNNT1, regulated by miR-873, is an oncogene of CRC associated with patient prognosis.