RBM10 regulates alternative splicing of lncRNA Neat1 to inhibit the invasion and metastasis of NSCLC

Abstract

Background: Non-small cell lung cancer (NSCLC) accounts for more than 85% of the total cases with lung cancer. NSCLC is characterized by easy metastasis, which often spreads to bones, brains and livers. RNA-binding motif protein 10 (RBM10) is an alternative splicing (AS) regulator frequently mutated in NSCLC. We found that there were multiple peak binding sites between RBM10 and long non-coding RNA nuclear enriched abundant transcript 1 (LncRNA Neat1) by crosslinking-immunprecipitation and high-throughput sequencing (Clip-Seq). LncRNA Neat1 plays an indispensable role in promoting cancer in a variety of tumors and produces two splicing variants: Neat1_1 and Neat1_2. This study aims to explore the mechanism of RBM10 and LncRNA Neat1 in invasion and metastasis of NSCLC.

Methods: Through histological and cytological experiments, we assessed the expression level of RBM10 protein expression. The interaction between RBM10 and Neat1 was evaluated via Clip-Seq and RNA immunoprecipitation assay. The effect of RBM10 on Neat1 and its splicing variants was identified by RT-qPCR. The effect of RBM10 and Neat1 on invasive and metastasis phenotypes of NSCLC was analyzed using transwell invasion assay and scratch test. Additionally, downstream signaling pathway of RBM10 were identified by immunofluorescence and western blot.

Results: RBM10 exhibited low levels of expression in NSCLC tissues and cells. RBM10 inhibited the invasion and metastasis of NSCLC and recruited Neat1 and Neat1_2. Overexpression of RBM10 simultaneously inhibited Neat1 and Neat1_2, and promoted the expression of Neat1_1. On the other hand, silencing RBM10 promoted Neat1 and Neat1_2, and inhibited the expression of Neat1_1. From this, we concluded that RBM10 regulated AS of Neat1, and the tumor-promoting effect of Neat1 was mainly attributed to Neat1_2. RBM10 had a negative correlation with Neat1_2. In addition, RBM10 upregulated the expression of PTEN and downregulated the phosphorylation of PI3K/AKT/mTOR through Neat1_2, which ultimately inhibited the invasion and metastasis of NSCLC.

Conclusion: The RBM10 regulated AS of Neat1 to cause the imbalance of Neat1_1 and Neat1_2, and RBM10 suppressed the activation of the PTEN/PI3K/AKT/mTOR signal by downregulating Neat1_2, finally affected the invasion and metastasis of NSCLC.

Keywords: Alternative splicing; Invasion; Metastasis; NSCLC; RBM10; lncRNA Neat1.

Fig. 1

RBM10 expression is low in NSCLC tissue and cells. A Immunohistochemical detection of RBM10 protein expression in NSCLC and paracancerous tissues (200 ×, 400 ×). LA, lung adenocarcinoma tissue; SCC, squamous cell carcinoma tissue. B Western blot of RBM10 protein expression in representative tissue samples from NSCLC (T) and non-tumor specimens (N). C RBM10 protein expression in A549, H1299, and BEAS-2B cells. *p < 0.05, **p < 0.01, ***p < 0.001. Data are means ± standard deviation (SD)

Fig. 2

RBM10 inhibits invasion and metastasis of A549 and H1299 cells. A A549 and H1299 cells were transfected with lentiviruses expressing GV358-RBM10 (oe-RBM10), GV248-RBM10 (sh-RNA), and negative control (oe-NC and sh-NC). Using Western blot, total protein extracts were analyzed for RBM10 expression; negative control served as control respectively. B The effect of RBM10 on cell invasion was measured using the transwell invasion assay (200 ×). C The effect of RBM10 on cell metastasis was measured using the scratch test (40 ×). D The effect of RBM10 on the expression of N-cadherin, vimentin, and E-cadherin was measured using Western blotting analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are means ± SD

Fig. 3

RBM10 promotes PTEN expression and inhibits the phosphorylation of PI3K/AKT/mTOR signaling pathway. A PTEN protein expression in oe-RBM10 and sh-RBM10 NSCLC cells by Western blot analysis, normalized to GAPDH. B Immunofluorescence qualitative analysis of PTEN protein expression (100 ×). The blue fluorescence represents the nucleus, the red fluorescence represents the PTEN protein, and the red fluorescence is mainly located in the cytoplasm. C The phosphorylation of PI3K, AKT, and mTOR in oe-RBM10 and sh-RBM10 NSCLC cells by Western blotting analysis, normalized to GAPDH. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are means ± SD

Fig. 4

RBM10 regulates Neat1 AS. A Clip-seq analysis revealed the top 10 LncRNA binding sites in the RBM10 binding peak according to maxHeight. B RIP assay to verify the binding between RBM10 and Neat1. C Neat1 expression in A549 cells after RBM10 overexpressing and silencing by RT-qPCR, normalized to β-actin. D Structure of NEAT1_1 and Neat1_2 and binding site of RBM10 to Neat1. E RIP assay to verify the binding between RBM10 and Neat1_2. F Neat1_2 expression in A549 cells after RBM10 overexpressing and silencing by RT-qPCR, normalized to β-actin. G Proportion of Neat_2 in total Neat1 after RBM10 overexpressing and silencing. H. Neat1_1 expression in A549 cells after RBM10 overexpressing and silencing. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are means ± SD

Fig. 5

The expression of Neat1_2 increased in NSCLC cells and tissues, and RBM10 is clinically correlated with Neat1_2. A Neat1_2 expression in NSCLC cell lines and HBE cells by RT-qPCR, normalized to β-actin. B Expression of Neat1 in NSCLC and paracancerous tissues as determined using RT-qPCR. LA, lung adenocarcinoma tissue (n = 14); SCC, squamous cell carcinoma tissue (n = 13). C Pearson correlation of RBM10 and Neat1_2 expression in LA and SCC. **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are means ± SD

Fig. 6

RBM10 inhibits the invasive and metastasis potentials of NSCLC cells via Neat1_2. A Expression of Neat1_2 in response to Neat1 silencing using different siRNAs as determined by RT-qPCR, normalized to β-actin. B Cell invasion was measured using the transwell assay (200 ×). C Cell metastasis was measured using the scratch test (40 ×). D EMT related proteins were measured using Western blotting analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are means ± SD

Fig. 7

RBM10 regulated PTEN/PI3K/AKT/mTOR signaling pathway via Neat1_2 in NSCLC cells. A PTEN protein expression after silencing Neat1 in RBM10-silenced A549 cells by Western blot analysis, normalized to GAPDH. B PTEN protein expression after silencing Neat1 in RBM10-silenced A549 cells by cell immunofluorescence assay. C The phosphorylation of PI3K, AKT, and mTOR after silencing Neat1 in RBM10-silenced A549 cells by western blot analysis, normalized to GAPDH. *p < 0.05, **p < 0.01, ***p < 0.001. Data are means ± SD

Fig. 8

Mechanism of RBM10 and Neat1 in invasion and metastasis of NSCLC.RBM10 regulates AS of Neat1, leading to changes in the expression level of Neat1_2 and Neat1_1. RBM10 decreases Neat1_2 to inhibit the invasion and metastasis of NSCLC via PTEN/PI3K/AKT/mTOR signaling