Long non‑coding RNA MIR4435‑2HG promotes the progression of head and neck squamous cell carcinoma by regulating the miR‑383‑5p/RBM3 axis

Abstract

Recent studies have shown that long non‑coding RNAs (lncRNAs) are strongly related to the progression of various types of cancer. The lncRNA MIR4435‑2 host gene (MIR4435‑2HG) has been recently recognized as a tumor‑related lncRNA that is upregulated in several tumors. However, its possible functions in head and neck squamous cell carcinoma (HNSCC) remain unclear. In tShe present study, we observed that MIR4435‑2HG expression was markedly upregulated in HNSCC tissues based on a Gene Expression Profiling Interactive Analysis dataset. This result was further confirmed in HNSCC tissues and cell lines using quantitative real‑time polymerase chain reaction. In addition, the high expression level of MIR4435‑2HG was significantly associated with poor disease‑free survival and overall survival in all HNSCC cases and was associated with advanced tumor‑metastasis‑node stage and poor prognosis. In vitro and in vivo assays demonstrated that MIR4435‑2HG knockdown suppressed HNSCC cell proliferation and invasion, epithelial‑mesenchymal transition (EMT), and tumor growth as determined by Cell Counting Kit‑8, Transwell assays and western blotting. Furthermore, MIR4435‑2HG affected HNSCC cell proliferation and migration and EMT by modulating the microRNA miR‑383‑5p to positively regulate the protein expression level of RNA‑binding motif protein 3 (RBM3). In conclusion, we provide a detailed analysis of the roles of MIR4435‑2HG in HNSCC and identified the MIR4435‑2HG/miR‑383‑5p/RBM3 axis as a potential therapeutic target for HNSCC treatment.

Keywords: MIR4435‑2HG; head and neck squamous cell carcinoma; cell proliferation; cell migration; epithelial–mesenchymal transition; miR‑383‑5p/RBM3.

Figure 1.

MIR4435-2HG expression is upregulated in HNSCC tissues. (A) The expression level of MIR4435-2HG in 519 HNSCC tissues and 44 normal tissues was analyzed using a GEPIA database. (B and C) Prognostic significance of MIR4435-2HG in HNSCC tissues obtained from the GEPIA database. (D) The expression level of MIR4435-2HG in different clinical stages of HNSCC from the GEPIA database. (E) The expression level of MIR4435-2HG in 18 HNSCC tissue samples and adjacent normal tissues was determined using qPCR. (F) The expression level of MIR4435-2HG in HNSCC tissues from patients with different clinical stages was determined. *P<0.05 and **P<0.01. MIR4435-2HG, lncRNA MIR4435-2 host gene; HNSCC, head and neck squamous cell carcinoma.

Figure 2.

MIR4435-2HG knockdown suppresses HNSCC cell proliferation, migration, and invasion in vitro. (A) CAL27 and SCC25 cells were infected with MIR4435-2HG shRNA (shMIR4435-2HG) or control shRNA (shNC), respectively, and qPCR was used to determine the expression level of MIR4435-2HG. (B) Cell viability was determined using the CKK-8 assay. (C) Cell proliferation was determined using the colony formation assay. (D) Cell migration was determined using the Transwell migration assay. (E) Cell invasion was determined using the Transwell invasion assay. (F and G) The protein expression levels of E-cadherin (E-cad) and vimentin (Vim) were determined using western blot analysis. *P<0.05 and **P<0.01. MIR4435-2HG, lncRNA MIR4435-2 host gene; HNSCC, head and neck squamous cell carcinoma.

Figure 3.

MIR4435-2HG knockdown inhibits HNSCC tumor growth in vivo. The in vivo xenograft assay was performed using MIR4435-2HG-knockdown CAL27 cells or control cells. (A and B) Tumor growth (volume in A and weight in B) was determined in the indicated groups. (C) Representative images of the xenograft tumors. (D) Representative image of hematoxylin and eosin (H&E) staining and immunohistochemical staining with Ki67, E-cadherin (E-cad) and vimentin (Vim) antibodies. (E) The protein expression levels of E-cad and Vim were determined using western blot analysis. *P<0.05 and **P<0.01. MIR4435-2HG, lncRNA MIR4435-2 host gene; HNSCC, head and neck squamous cell carcinoma.

Figure 4.

MIR4435-2HG serves as a miR-383-5p sponge and inhibits its expression in HNSCC cells. (A) The binding site of miR-383-5p was predicted using the miRDB program. (B) The luciferase activity of HNSCC cells transfected with miR-383-5p mimic and MIR4435-2HG-WT (or MIR4435-2HG-MT) was determined. (C) RNA immunoprecipitation (RIP) assay was performed using cell lysates, normal IgG, or anti-Ago2 antibodies. The relative expression levels of MIR4435-2HG and miR-383-5p were determined using qPCR. (D) The expression level of miR-383-5p in shMIR4435-2HG (or shNC)-transfected CAL27 and SCC25 cells was determined using qPCR. **P<0.01. MIR4435-2HG, lncRNA MIR4435-2 host gene; HNSCC, head and neck squamous cell carcinoma.

Figure 5.

RBM3 is a target of miR-383-5p. (A) The 3′-UTR (untranslated region) of RBM3 is potentially targeted by miR-383-5p, as predicted using TargetScan. (B) The relative luciferase activity of HNSCC cells after co-transfection with wild-type (WT) or mutant (MT) RBM3 3′-UTR reporter genes and miR-383-5p mimics or control mimics. (C) The mRNA expression level of RBM3 in HNSCC cells transfected with miR-383-5p mimics or control mimics was analyzed using qPCR. (D and E) The protein expression level of RBM3 in HNSCC cells transfected with miR-383-5p mimics or control mimics was measured using western blot analysis. **P<0.01. RBM3, RNA-binding motif protein 3; HNSCC, head and neck squamous cell carcinoma.

Figure 6.

miR-383-5p abolishes the function of MIR4435-2HG in HNSCC cells by regulating RBM3 expression. (A) The protein expression level of RBM3 was determined in HNSCC cells transfected with MIR4435-2HG, miR-383-5p mimics alone, or co-transfected with MIR4435-2HG and miR-383-5p mimics. (B) Cell proliferation, (C) cell colony formation, (D) cell migration, and (E) cell invasion were detected in the different groups. (F) The protein expression levels of E-cad (E-cadherin) and Vim (vimentin) were measured using western blotting. *P<0.05 and **P<0.01. MIR4435-2HG, lncRNA MIR4435-2 host gene; RBM3, RNA-binding motif protein 3; HNSCC, head and neck squamous cell carcinoma.