Long non-coding RNA HOXA-AS3 promotes cell proliferation of oral squamous cell carcinoma through sponging microRNA miR-218-5p

Abstract

Increasing evidence demonstrated long non-coding RNAs (lncRNAs) play important roles in the occurrence and development of oral squamous cell carcinoma (OSCC). This study aimed to explore the role and molecular mechanism of lncRNA HOXA-AS3 in the progression of OSCC. Here, we found that the expression of lncRNA HOXA-AS3 was upregulated in OSCC tissues and cell lines compared with the para-cancerous tissues and normal human oral keratinocyte (NHOK), respectively. Inhibition of HOXA-AS3 significantly inhibited the proliferation and colony formation of OSCC cells. Further, the luciferase reporter assay showed that HOXA-AS3 was directly bound to miR-218-5p. Moreover, the expression of miR-218-5p was negatively regulated by HOXA-AS3, and miR-218-5p could inhibit the expression of collagen type I alpha1 (COL1A1) and lysophosphatidylcholine acyltransferase 1 (LPCAT1). In addition, silencing miR-218-5p reversed the inhibitory effect of HOXA-AS3 knockdown on the proliferative potential of OSCC cells. In summary, our study illustrated that HOXA-AS3 promoted cancer cell proliferation in OSCC, possibly by sponging miR-218-5p for the first time, which provides a new target or a potential diagnostic biomarker for OSCC.

Keywords: HOXA-AS3; LncRNA; Oral squamous cell carcinoma; miR-218-5p; proliferation.

Figure 1.

HOXA-AS3 expression is upregulated in OSCC tissues and cell lines. (a) The expression of HOXA-AS3 in OSCC tissues and para-cancerous tissues was measured using qRT-PCR. (b) Expression levels of HOXA-AS3 in NHOK and OSCC cell lines (TSCCA, CAL-27, SCC-9, and Tca8113) were detected via qRT-PCR. (c) The Kaplan-Meier survival curve indicated that the prognosis of patients in HOXA-AS3 high-expression group was significantly worse than that of patients in HOXA-AS3 low-expression group. *P < 0.05, **P < 0.01

Figure 2.

HOXA-AS3 knockdown inhibited OSCC cell proliferation and colony formation in vitro and in vivo. (a) Transfection efficacy of sh-HOXA-AS3 in SCC-9 and CAL-27 cells. (b, c) Cell Counting Kit-8 assay showed that HOXA-AS3 knockdown inhibited cell proliferation in SCC-9 and CAL-27 cells. (d) Colony formation assay showed that HOXA-AS3 knockdown significantly reduced the number of colonies. *P < 0.05, **P < 0.01

Figure 3.

HOXA-AS3 directly interacted with miR-218-5p. (a) Predicted binding of human miR-218-5p with the wild-type 3ʹUTR region of HOXA-AS3 mRNA and a mutated 3ʹUTR of HOXA-AS3. (b, c) Luciferase reporter gene assay verified that HOXA-AS3 could directly bind to miR-218-5p in SCC-9 and CAL-27 cells. (d, e) SCC-9 and CAL-27 cells were transfected with miR-218-5p mimics or control, followed by the measurement of HOXA-AS3 mRNA enrichment with anti-Ago2 by qRT-PCR, and anti-IgG used as control. *P < 0.05, **P < 0.01

Figure 4.

MiR-218-5p was downregulated in OSCC tissues and cells and inversely correlated with HOXA-AS3 expression. (a) The expression of miR-218-5p in OSCC cell lines and NHOK was detected by qRT-PCR. (b) The expression of miR-218-5p in OSCC tissues and para-cancerous tissues was detected by qRT-PCR. (c) HOXA-AS3 and miR-218-5p expression level was negatively correlated in OSCC tissues (r = −0.759, P < 0.01, n = 38). (d) qRT-PCR was used to measure the expression level of miR-218-5p after HOXA-AS3 knockdown in OSCC cell lines. *P < 0.05, **P < 0.01

Figure 5.

Overexpression of miR-218-5p inhibited the proliferation of OSCC cells. (a) Transfection efficacy of miR-218-5p mimics in SCC-9 and CAL-27 cells. (b, c) Cell Counting Kit-8 assay showed that overexpression of miR-218-5p inhibited cell proliferation in OSCC cells. (d) Colony formation assay showed that overexpression of miR-218-5p significantly reduced the number of colonies. *P < 0.05, **P < 0.01

Figure 6.

MiR-218-5p inhibited COL1A1 and LPCAT1 in OSCC cells. (a) The poteintial targeted mRNAs of miR-218-5p were predicted by PicTar, miRanda and TargetScan online databases. (b) Volcano plot showing the DEGs identified from GSE138206. X axis represents log transformed P value, and Y axis indicates the mean expression differences of genes between OSCC samples and normal samples. (c) qRT-PCR was used to measure the expression of COL1A1 after miR-218-5p overexpression in OSCC-9 cells. (d) qRT-PCR was used to measure the expression of LPCAT1 after miR-218-5p overexpression in OSCC cells. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 7.

LncRNA HOXA-AS3 promoted OSCC development through regulating miR-218-5p. (a, b) The expression level of HOXA-AS3 in cells co-transfected with sh-HOXA-AS3 and anti-miR-218-5p was detected by qRT-PCR. (c-f) The inhibited proliferation of SCC-9 and CAL-27 cells by HOXA-AS3 knockdown was reversed by anti-miR-218-5p. *P < 0.05, **P < 0.01