RBM15 promotes COAD progression by regulating the m6A modification of TMC5

Abstract

Background: Colon adenocarcinoma (COAD) is a frequent digestive system malignancy with high mortality and poor prognosis. Transmembrane Channel-like 5 (TMC5) has been reported to play an oncological role in various cancers. However, the role and mechanism of TMC5 in COAD remain unclear.

Methods: TIMER and UALCAN databases analyzed the expression of TMC5 in COAD. TMC5, RNA-binding motif protein-15 (RBM15), E-cadherin, N-cadherin, Vimentin, Fibronectin, and RAD51 protein levels were determined using western blot. TMC5, RBM15, Ferritin heavy chain 1 (FTH1), and cystine/glutamate antiporter SLC7A11 (also commonly known as xCT) mRNA levels were examined using real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation, apoptosis, migration, and invasion were assessed using 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, and transwell assays. Caspase 3 activity, ROS level, Fe⁺ level, and glycolysis level were detected using commercial kits. Immunofluorescence assay analyzed 53BP1 and γH2AX foci. Role of TMC5 on COAD tumor growth was examined using xenograft tumor model in vivo. After SRAMP database analysis, interaction between RBM15 and TMC5 was verified using methylated RNA immunoprecipitation (MeRIP) and dual-luciferase reporter assay.

Results: TMC5 and RBM15 levels were significantly increased in COAD tissues and cells. Moreover, TMC5 silencing could inhibit COAD cell proliferation, migration, invasion, EMT, glycolysis, and induce apoptosis and ferroptosis in vitro, as well as repress tumor growth in vivo. At the molecular level, RBM15 could sustain RNA stability and TMC5 expression through regulating the m6Amodification.

Conclusion: RBM15 could facilitate COAD cell malignant behaviors at least by regulating the stability of TMC5 mRNA, providing a powerful and hopeful target for COAD treatment.

Keywords: COAD; Ferroptosis; Proliferation; RBM15; TMC5.

Fig. 1

Expression level of TMC5 in COAD. (A) Analysis of mRNA expression of TMC5 in different cancers based on TIMER2.0 database. (B) GEPIA database (http://gepia.cancer-pku.cn/) exhibited the expression level of TMC5 in COAD samples (n = 275) and normal samples (n = 349). (C and D) UALCAN database showed TMC5 mRNA and protein expression based on The Cancer Genome Atlas (TCGA) and Clinical Proteomic Tumor Analysis Consortium (CPTAC) samples. (E) TMC5 protein level was determined in 5 normal tissues and 5 COAD tumor tissues using western blot. (F) TMC5 mRNA level was detected in 69 normal tissues and 69 COAD tumor tissues using RT-qPCR. (G) Immunohistochemical observation of TMC5 expression in normal and COAD tumor tissues. (H) Western blot analysis of TMC5 protein level in NCM460 cell line and COAD cell lines (Caco-2, SW620, and LoVo). ***P < 0.001

Fig. 2

TMC5 downregulation repressed COAD cell development. Caco-2 and LoVo cells were transfected with sh-NC or sh-TMC5. (A) TMC5 protein level was determined in transfected Caco-2 and LoVo cells using western blot. (B) Cell proliferation was assessed using EdU assay. (C) Cell apoptosis was measured using flow cytometry assay. (D) Caspase 3 activity was detected using a commercial kit. (E and F) Cell migration and invasion were examined using Transwell assays. (G) ROS level was examined using a commercial kit. (H) Iron assay kit was carried out to detect Fe⁺ level in transfected Caco-2 and LoVo cells. (I and J) FTH1 and xCT mRNA levels were determined using RT-qPCR. **P < 0.01, ***P < 0.001

Fig. 3

Inhibiting TMC5 impaired COAD cell growth in vivo. sh-NC or sh-TMC5-transfected LoVo cells were subcutaneously injected into mice. (A and B) Tumor volume and tumor weight were examined. (C) IHC staining was used to assess the positive expression of TMC5, ki-67, and FTH1 in xenografted tumor samples. **P < 0.01, ***P < 0.001

Fig. 4

RBM15 modulated m6A enrichment and stability of TMC5 mRNA. (A) The online tool SRAMP was used to predict m6A sites of TMC5. (B) Alterations in the m6A methylation level of TMC5 after inhibition of RBM15 were analyzed by MeRIP-qPCR assay. (C) Influences of RBM15 overexpression or downregulation on TMC5 mRNA stability after Actinomycin D treatment was measured using RT-qPCR in Caco-2 and LoVo cells. (D) RBM15 and TMC5 protein levels were determined in Caco-2 and LoVo cells transfected with vector, RBM15, sh-NC, or sh-RBM15 using western blot. (E) Their interaction was confirmed using dual-luciferase reporter assay in Caco-2 and LoVo cells. (F) UALCAN database presented the expression level of RBM15 in COAD samples (n = 286) and normal samples (n = 41) based on sample types TCGA samples. (G) RT-qPCR analysis of RBM15 mRNA level in 69 normal tissues and 69 COAD tumor tissues. (H) Western blot analysis of RBM15 protein level in 5 normal tissues and 5 COAD tumor tissues. (I) IHC staining analyzed the positive expression rate RBM15 in normal tissues and COAD tumor tissues. (J) GEPIA database exhibited the association between RBM15 and TMC5 expression in COAD samples. (K) Pearson correlation analysis was applied to evaluate the expression correlation between RBM15 and TMC5 in COAD tissues. (L) RBM15 protein level was detected in NCM460 cell line and COAD cell lines (Caco-2 and LoVo) using western blot. **P < 0.01, ***P < 0.001

Fig. 5

RBM15/TMC5 regulated COAD cell malignant behaviors. (A) Western blot analysis of TMC5 protein level in Caco-2 and LoVo cells transfected with vector or TMC5. (B-K) Caco-2 and LoVo cells were transfected with sh-NC + vector, sh-RBM15 + vector, or sh-RBM15 + TMC5. (B) TMC5 protein level was monitored by western blot. (C and D) Cell proliferation and apoptosis were tested using EdU and flow cytometry assays. (E) Caspase 3 activity was examined using a commercial kit. (F and G) Cell migration and invasion were measured using Transwell assays. (H and I) ROS level and Fe⁺ level were determined by special kits. (J and K) FTH1 and xCT mRNA levels were examined using RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001