Expression and functions of long non-coding RNA actin filament-associated protein 1-antisense RNA1 in oral squamous cell carcinoma

doi:10.7518/hxkq.2019.06.005

Abstract

Abstract:

Objective To analyze the expression and clinical significance of long non-coding RNA (lncRNA) actin filament-associated protein 1-antisense RNA1 (AFAP1-AS1) in oral squamous cell carcinoma (OSCC) and its effect on the biobehavior of OSCC cells.Methods Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of lncRNA AFAP1-AS1 in the tumor tissue and matching adjacent normal tissue of OSCC patients (n=55), SCC25 cells, and normal oral keratinocyte lines (NOK) cells. The correlation between AFAP1-AS1 expression and the clinicopathological characteristics of OSCC patients was analyzed. The relationship between AFAP1-AS1 and prognosis was also studied with the Kaplan-Meier method. AFAP1-AS1 siRNA was transfected into the SCC25 cells. Cell counting kit-8 (CCK-8) and Transwell were used to detect cell proliferation, invasion, and migration. The expression of the invasion-associated protein, AFAP1, and Rho GTPase family members, was detected by Western blot. In addition, the immunofluorescence of the cytoskeletal actin filament was observed.Results The expression of AFAP1-AS1 was higher in the OSCC tissues than in the NOK cells, and the relative expression of AFAP1-AS1 was higher in the SCC25 cells than in the NOK cells (P<0.001). AFAP1-AS1 expression was associated with the degree of differentiation, TNM stage, lymphatic metastasis, and poor prognosis of OSCC (P<0.05). Patients with a high expression of AFAP1-AS1 had lower survival rates than those with a low expression of AFAP1-AS1 (P<0.05). After transfected by AFAP1-AS1 siRNA, the expression of AFAP1-AS1 was downregulated. The inhibition of AFAP1-AS1 expression consequently suppressed the proliferation, invasion, and migration of SCC25. Moreover, AFAP1-AS1 siRNA upregulated the expression levels of AFAP1, RhoA, Rac2, Rab10, RhoGDI, and Pfn1 but downregulated the expression of RhoC. Immunofluorescence showed that AFAP1-AS1 also reduced the formation of stress filaments in the cytoskeleton and affected the integrity of the actin filament.Conclusion The expression of AFAP1-AS1 was high in the OSCC tissues and SCC25 cells and is associated with the development and prognosis of OSCC. The knockdown of AFAP1-AS1 might inhibit the proliferation and invasion of OSCC cells by regulating the integrity of the actin filament.

Key words: oral squamous cell carcinoma, long non-coding RNA, actin filament-associated protein 1-antisense RNA1, prognosis, proliferation, invasion

CLC Number:

R739.8

Geng Yudong,Wang Shubin,Lu Taiqing,Teng Wei. Expression and functions of long non-coding RNA actin filament-associated protein 1-antisense RNA1 in oral squamous cell carcinoma[J]. West China Journal of Stomatology, 2019, 37(6): 594-601.

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基因名称	序列（5′-3′）
GAPDH	上游：AGGCAACTAGGATGGTGTGG
	下游：TTGATTTTGGAGGGATCTCG
AFAP1-AS1	上游：ATCTTCGTTAGCACTGAGACTGG
	下游：AGAAAGAAGGTGGGAGTTAGGTG
si-AFAP1-AS1-1	CAUUAUUUUGCUAAUUCAACA
si-AFAP1-AS1-2	CGUUAUAUCUGAAAAAAAUCU
si-AFAP1-AS1-3	GAAGUUAGUACAUUAAACAAA
si-NC	CUGAAACAUACAGCAAUAAAG

临床病理特征		n	AFAP1-AS1低表达组	AFAP1-AS1高表达组	χ²值	P值
年龄/岁	≤60	31	18	13	2.289	0.130
	>60	24	9	15
性别	男	36	16	20	0.900	0.343
	女	19	11	8
肿瘤大小/cm	≤3	41	22	19	1.345	0.246
	>3	14	5	9
肿瘤部位	舌	24	14	10	1.582	0.663
	牙龈	15	6	9
	腭	4	2	2
	颊黏膜	12	5	7
分化程度	低分化	44	25	19	5.256	0.022
	中、高分化	11	2	9
TNM分期	Ⅰ、Ⅱ期	35	21	14	4.583	0.032
	Ⅲ、Ⅳ期	20	6	14
淋巴结转移	无	42	24	18	4.610	0.032
	有	13	3	10

组别	AFAP1-AS1的表达量	P值
空白对照组	1	-
si-NC	0.98±0.09	>0.05
si-AFAP1-AS1-1	0.58±0.06	<0.05
si-AFAP1-AS1-2	0.39±0.05	<0.05
si-AFAP1-AS1-3	0.72±0.06	<0.05