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MiR-301a-5p/SCIN promotes gastric cancer progression via regulating STAT3 and NF-κB signaling.

ABSTRACT: Objective: Gastric cancer (GC) is a type of highly malignant cancer. Although the diagnostic and therapeutic methods are innovating, the outcome of GC patients is still poor. Therefore, our research was carried out to explore potential molecular mechanism in the diagnosis of GC. Materials and methods: Bioinformatics analyses were used to obtain microRNA and target mRNA of interest. The expression level of miR-301a-5p and Scinderin (SCIN) mRNA were detected by quantitative real-time PCR (qRT-PCR). Western blot assay was used to investigate SCIN protein level. Cell Counting Kit-8 assay (CCK-8) and colony formation assay were used to investigate cell proliferation ability. Transwell assay was employed to examine cell motility. The interaction between miR-301a-5p and SCIN mRNA was verified by dual-luciferase reporter assay. Results: The qRT-PCR analysis revealed that the expression of miR-301a-5p was higher in gastric cancer tissues than para-cancer tissues (P<0.05). Cox regression analysis showed upregulated miR-301a-5p was associated with larger tumor size (P=0.036) and more advanced TNM stage (P=0.048). The Kaplan-Meier analysis showed a correlation between increased miR-301a-5p expression and shorter overall survival (OS)(P=0.018). By using bioinformatic analysis, SCIN was predicted as one of the targets of miR-301a-5p. Overexpressing miR-301a-5p promoted proliferation and motility of GC cells while knockdown of SCIN exhibited the same performance. Further, we verified the alteration of miR-301a-5p and SCIN expression level could affect the epithelial-mesenchymal transition (EMT) progression on GC cells via STAT3 and NF-κB signaling. Conclusion: Highly expressed miR-301a-5p was associated with aggressiveness of GC. Upregulation of miR-301a-5p promoted malignant phenotype of GC by targeting SCIN. The present results indicated miR-301a-5p might be a promising molecule in the prognosis of GC.

SUBMITTER: Huang Y

PROVIDER: S-EPMC8364655 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:MicroRNA‑301a (miRNA/miR‑301a) and nuclear factor (NF)‑κB signaling play important roles in tumor invasion, migration and progression. However, the role of miRNA‑301a‑3p in human gastric cancer (GC), and specifically in the activation of NF‑κB signaling, remains unclear. The aim of the present study was to investigate miRNA‑301a‑3p expression in GC progression and the molecular mechanisms as regards the regulation of NF‑κB signaling. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was used to detect miRNA‑301a‑3p expression in GC and paired normal tissues. The association between the expression of miRNA‑301a‑3p and patient pathological parameters and the prognosis of GC was statistically analyzed using an in situ hybridization (ISH) assay. An MTS assay and a Transwell assay were performed to evaluate the effects of miRNA‑301a‑3p on the proliferation, invasion and migration of GC cells. RT‑qPCR and western blot analysis were used to analyze the association between miRNA‑301a‑3p and nuclear factor‑κB repressing factor (NKRF) expression and the corresponding downstream NF‑κB signaling molecules. A luciferase assay was used to verify the target effect of miRNA‑301a‑3p and NKRF. It was found that miRNA‑301a‑3p expression was significantly higher in 30 cases of primary GC compared with matched normal tissues. Additionally, the ISH assay indicated that the high expression of miRNA‑301a‑3p in GC was associated with tumor invasion depth, lymph node metastasis, lymph node invasion and tumor metastasis stage. Patients whose tumors had a higher miRNA‑301a‑3p expression level exhibited a poorer prognosis. The in vitro assay indicated that miRNA‑301a‑3p affected the proliferative and invasive ability of GC cells by targeting the expression of NKRF, which then affected NF‑κB signaling. Therefore, it was hypothesize that miRNA‑301a‑3p promotes GC progression and affects the prognosis of patients with GC by targeting NKRF, which in turn, directly influences NF‑κB activation.

Project description:Osteoarthritis (OA) is the most common age-related joint disease, characterized by chronic inflammation, progressive articular cartilage destruction and subchondral osteosclerosis. More and more evidence showed that microRNAs (miRNAs) play a key role in various diseases, but the specific mechanism of miRNAs in OA is not clear. The purpose of this study was to investigate the expression level and role of miR-181a-5p in OA and its related mechanism. Here we identified the key gene DEAD-box RNA helicase 3X (DDX3X) in the OA dataset by bioinformatics analysis. At the same time, miRNAs targeting DDX3X were screened, and miR-181a-5p was selected as the next research object. Then we used different concentrations of interleukin-1 beta (IL-1β)-induced in vitro model of arthritis, and found that IL-1β can stimulate cells to release nitric oxide. The expression levels of miR-181a-5p and DDX3X in mouse chondrocyte cell line ATDC5 induced by IL-1β at a concentration of 10ug/mL were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). IL-1β induced a decrease in the expression of miR-181a-5p and an increase in the expression of DDX3X in ATDC5 cells. mimic miR-181a-5p or inhibitor miR-181a-5p were transfected into ATDC5 cells, and the levels of inflammatory mediators in the cells were detected by enzyme-linked immunosorbent assay, and the results showed that miR-181a-5p could reduce the release of tumor necrosis factor-α, IL-1β, IL-6 and inducible nitric oxide nitric oxide synthase in a cellular model of arthritis. Luciferase reporter assays confirmed that the miR-181a-5p binding site was in the DDX3X gene 3'-untranslated region (3'-UTR), and DDX3X was negatively regulated by miR-181a-5p. Rescue assays confirmed that miR-181a-5p reduced the expression of DDX3X by targeting the 3'-UTR region of DDX3X, thereby reducing the release of inflammatory factors. Finally, in this paper, western blot was used to detect the mechanism of miR-181a-5p regulating OA. The results showed that interfering with the expression of miR-181a-5p could up-regulate the expression of DDX3X protein, increase the expression of nuclear factor- kappaB (NF-κB) related proteins, and reduce the inflammatory response of OA, thereby increasing the secretion of the matrix proteinases MMP-3 and MMP-13. Taken together, the results of the study suggested that miR-181a-5p may be a promising therapeutic target for the treatment of human OA.

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MiR-301a-5p/SCIN promotes gastric cancer progression via regulating STAT3 and NF-κB signaling.

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