LncRNA TTN-AS1 promotes the progression of oral squamous cell carcinoma via miR-411-3p/NFAT5 axis

Abstract

Background: Oral squamous cell carcinoma (OSCC) is a common kind of squamous cell carcinoma of the head and neck, which is a threat to public health. Long noncoding RNAs (lncRNAs) are associated with the development of various diseases, including cancers. LncRNA titin antisense RNA 1 (TTN-AS1) is known as a crucial regulatory factor in several cancers. Nevertheless, the specific functions of TTN-AS1 in OSCC remains obscure.

Methods: The expression of TTN-AS1 in OSCC samples or cells was analyzed through qRT-PCR. Colony formation assay, EdU assay, flow cytometry assay, TUNEL assay and wound healing assay were conducted to estimate the functions of TTN-AS1 in OSCC cells. RIP and luciferase reporter assays were utilized to detect the interaction between TTN-AS1 and miR-411-3p as well as between miR-411-3p and NFAT5.

Results: TTN-AS1 expression was stronger in OSCC cells. Knockdown of TTN-AS1 effectively restrained cell proliferation and migration but had inductive role in apoptosis. Moreover, TTN-AS1 could function as the miR-411-3p sponge in OSCC and miR-411-3p exerted the inhibitory functions on OSCC cell growth. In addition, NFAT5 was proven as the target of miR-411-3p. Rescue assay indicated that overexpressing NFAT5 could reverse the inhibitory function of TTN-AS1 depletion on cell growth.

Conclusion: lncRNA TTN-AS1 contributed to the progression of OSCC via miR-411-3p/NFAT5 axis.

Keywords: NFAT5; Oral squamous cell carcinoma; TTN-AS1; miR-411-3p.

Fig. 1

Knockdown of TTN-AS1 restrains the proliferation and migration of OSCC cells. a The expression of TTN-AS1 was tested through qRT-PCR in OSCC cells. b The interference efficiency of TTN-AS1 was detected by qRT-PCR in SCC-4 and SCC-9 cells. c, d Cell proliferation ability was measured by colony formation and EdU experiments when TTN-AS1 was inhibited. e, f Cell apoptosis was evaluated through flow cytometry and TUNEL experiments after silencing TTN-AS1. g Wound healing assays were utilized to estimate cell migration when TTN-AS1 was subjected to knockdown. *P < 0.05; **P < 0.01

Fig. 2

TTN-AS1 acts as the miR-411-3p sponge in OSCC. a, b The cellular location of TTN-AS1 was identified in SCC-4 and SCC-9 through Subcellular fractionation and FISH. c StarBsae website was utilized to predict the possible miRNAs that could bind with TTN-AS1. d MiR-411-3p expression was detected through qRT-PCR in OSCC cells. e The binding site of TTN-AS1 in miR-411-3p. f RIP assay was utilized to evaluate the relationship between miR-411-3p and TTN-AS1. g The efficiency of miR-411-3p overexpression was tested through qRT-PCR. h Luciferase reporter assays were conducted to verify the correlation of miR-411-3p and TTN-AS1. *P < 0.05; **P < 0.01

Fig. 3

Upregulation of miR-411-3p represses cell proliferation and migration in OSCC. a, b Cell proliferation was estimated through colony formation and EdU experiments when miR-411-3p was overexpressed. c, d Flow cytometry and TUNEL experiments were implemented to measure cell apoptosis after overexpressing miR-411-3p. e Wound healing assays were adopted to test cell migration ability when miR-411-3p was subjected to upregulation. **P < 0.01

Fig. 4

NFAT5 is a target gene of miR-411-3p in OSCC. a 28 mRNAs which had the binding site with miR-411-3p were predicted by starBase. b The qRT-PCR analysis was utilized to screen out the mRNAs which could be inhibited by NFAT5 depletion and miR-411-3p overexpression. c The expressions of TLL2, MGAT4A, RAB21 and NFAT5 in SCC-4 and SCC-9 cells through qRT-PCR. d The binding site of NFAT5 and miR-411-3p. e RIP assay was adopted to test the relationship between TTN-AS1, miR-411-3p and NFAT5. f The interference efficiency of miR-411-3p was tested by qRT-PCR analysis. g The expression of NFAT5 was detected when NFAT5 and miR-411-3p was silenced. h The interference efficiency of NFAT5 was tested by qRT-PCR analysis. i, j Cell proliferation was evaluated through colony formation and EdU experiments when NFAT5 was knocked down. k, l Cell apoptosis was measured through flow cytometry and TUNEL experiments after inhibiting NFAT5. m Wound healing assays were carried out for estimating cell migration after NFAT5 was subjected to inhibition. **P < 0.01

Fig. 5

TTN-AS1 promotes OSCC progression via miR-411-3p/NFAT5 axis. a The qRT-PCR analysis was utilized to examine the overexpression efficiency of NFAT5 in SCC-4 and SCC-9 cells. b, c Cell proliferation capability in SCC-4 and SCC-9 cells was measured by colony formation and EdU assay in different groups. d, e Cell apoptosis was tested through flow cytometry and TUNEL assays in different groups. f Wound healing assays were implemented to detect the cell migration ability in different groups. **P < 0.01

Fig. 6

TTN-AS1 promoted OSCC cell growth in vivo. a Tumors removed from the mice injected with sh-NC-transfected cells or sh-TTN-AS1#1-transfected cells. b, c Volume and weight in different groups were measured. d IHC staining of tumor tissues collected from different groups with anti-Ki-67 and anti-PCNA. **P < 0.01