[Mechanism of piR-hsa-26925 in regulating invasion and metastasis of lung adenocarcinoma via METTL3-mediated m6A methylation modification]

Abstract

Objective: To investigate the mechanism by which PIWI interacting RNA piR-hsa-26925 regulates the invasion and metastasis of lung adenocarcinoma through Methyltransferase-like 3 (METTL3)-mediated m6A methylation modification. Methods: The expression levels of piR-hsa-26925 were detected in lung adenocarcinoma cell lines (H1650, H1299, H1975, and A549) and normal lung epithelial cells (BEAS-2B) using real-time fluorescent quantitative PCR (qRT-PCR). Lung adenocarcinoma cells were transfected using transient RNA transfection technology, divided into a piR-hsa-26925 knockdown group in the A549 lung adenocarcinoma cell line and a negative control (NC-1) group; the lung adenocarcinoma H1299 cell line piR-hsa-26925 overexpression group and negative control (NC-2) group. qRT-PCR was used to detect the relative expression levels of piR-hsa-26925 to validate transfection success. Cell migration and invasion capabilities were assessed using cell scratch and Transwell assays; Western blot analysis was performed to detect the expression levels of E-cadherin, vimentin, and METTL3 proteins in different groups of cells; m6A spot blot analysis was conducted to assess differences in m6A methylation levels following piR-hsa-26925 overexpression and knockdown; the interaction between piR-hsa-26925 and METTL3 was detected using RNA pull-down experiments and RNA immunoprecipitation (RIP) experiments; fluorescence in situ hybridization (FISH) experiments was used to investigate the cellular localization of piR-hsa-26925 and METTL3 in A549 cells. RNA sequencing and MeRIP sequencing analyses to were combined identify downstream targets; m6A-RIP-qRT-PCR was used to validate the effects of piR-hsa-26925 knockdown on m6A methylation levels of downstream targets, and the regulatory roles of piR-hsa-26925 and METTL3 on the downstream molecule erythropoietin-producing hepatocellular kinase receptor B2 (EPHB2). We then applied transient RNA transfection technology to A549 cell lines, dividing them into METTL3 knockdown, METTL3 overexpression, and negative control (METTL3-NC) groups, and used Western blotting to detect EPHB2 expression levels. In the piR-hsa-26925 knockdown group, the METTL3 activity inhibitor STM2457 was added to observe whether it could reverse the downregulation of lung adenocarcinoma cell migration and invasion caused by piR-hsa-26925 knockdown. In the piR-hsa-26925 overexpression group, EPHB2 was further overexpressed to determine whether it could reverse the upregulation of lung adenocarcinoma cell migration and invasion caused by piR-hsa-26925 overexpression. All experimental data were repeated three times. Results: The level of piR-hsa-26925 expression was significantly higher in human lung adenocarcinoma cell lines than in normal lung epithelial cells (all P<0.05). Compared with the piR-hsa-26925-NC-1 group, the piR-hsa-26925 knockdown group showed decreased E-cadherin expression, increased vimentin expression, and increased lung adenocarcinoma cell wound healing area, migration, and invasion (all P<0.05). Compared with the piR-hsa-26925-NC-2 group, the piR-hsa-26925 knockdown group showed increased expression of E-cadherin, decreased expression of vimentin, and reduced wound healing area, cell migration, and invasiveness of lung adenocarcinoma cells (all P<0.05). FISH results showed that piR-hsa-26925 colocalized with METTL3 protein in the cytoplasm of A549 cells. m6A spot hybridization experiment results showed that piR-hsa-26925 was negatively correlated with m6A methylation levels. RNA pull-down and RIP experiments confirmed an interaction between piR-hsa-26925 and METTL3, but Western blot analysis showed that METTL3 expression levels were lower in both the piR-hsa-26925 knockdown and overexpression groups compared to the control group (all P<0.05). Downstream molecules EPHB2 were identified through bioinformatics analysis; Western blot analysis results showed that EPHB2 expression levels were higher in the piR-hsa-26925 knockdown group than in the piR-hsa-26925-NC-1 group, and lower in the piR-hsa-26925 overexpression group than in the piR-hsa-26925-NC-2 group (all P<0.05). In the A549 cell line, the EPHB2 expression level in the METTL3 knockdown group was lower than that in the METTL3-NC group, while the EPHB2 expression level in the METTL3 overexpression group was higher than that in the METTL3-NC group (all P<0.05). m6A-RIP-qRT-PCR results showed that the m6A modification level of EPHB2 in the piR-hsa-26925 knockdown group was higher than that in the piR-hsa-26925-NC-1 group. After treatment with the METTL3 activity inhibitor STM2457, the m6A modification levels of EPHB2 decreased (all P<0.05). Transwell results showed that the numbers of migrating and invading cells increased after treatment with STM2457 in piR-hsa-26925 knockdown group and the numbers of migrating and invading cells decreased after overexpression of EPHB2 in piR-hsa-26925 overexpression group (all P<0.05). Conclusions: piR-hsa-26925 is significantly upregulated in lung adenocarcinoma tissues and cells and functions as an oncogene to enhance the migration and invasion abilities of lung adenocarcinoma cells, potentially playing a critical role in their invasion and metastasis. piR-hsa-26925 binds to METTL3 and modulates the m6A methylation level of the downstream molecule EPHB2 by regulating METTL3 activity, thereby inhibiting EPHB2 expression and promoting the malignant progression of lung adenocarcinoma.