LncRNA NEAT1 Acts as an miR-148b-3p Sponge to Regulate ROCK1 Inhibition of Retinoblastoma Growth

Figures & data

Table 1 Relationship Between LncRNA NEAT1 and Pathological Data

Factor		LncRNA NEAT1		χ2	P value
		High Expression (n=25)	Low Expression (n=25)
Gender				0.081	0.777
	Male (n=27)	13 (52.00)	14 (56.00)
	Female (n=23)	12 (48.00)	11 (44.00)
Age				1.282	0.258
	≥5 years old (n=24)	10 (40.00)	14 (56.00)
	<5 years old (n=26)	15 (60.00)	11 (44.00)
Affected eye area				2.000	0.157
	Left (n=25)	10 (40.00)	15 (60.00)
	Right (n=25)	15 (60.00)	10 (40.00)
Tumor thickness				5.333	0.021
	≥10mm (n=20)	14 (56.00)	6 (24.00)
	<10mm (n=30)	11 (44.00)	19 (76.00)
TNM staging				8.333	0.004
	I+II (n=20)	5 (20.00)	15 (60.00)
	III+IV (n=30)	20 (80.00)	10 (40.00)
Differentiation				8.117	0.004
	Medium and high differentiation (n=28)	9 (36.00)	19 (76.00)
	Low differentiation (n=22)	16 (64.00)	6 (24.00)

Table 2 Cox Regression Analysis

Factor	Univariate Cox			Multivariate Cox
	P value	HR value	95% CI	P value	HR value	95% CI
Sex	0.777	0.901	0.440~1.848
Age	0.509	1.274	0.621~2.614
Affected eye area	0.693	1.155	0.564~2.369
Tumor thickness	0.917	1.039	0.500~2.159
TNM staging	0.010	2.918	1.292~6.586	0.273	1.661	0.670~4.118
Differentiation	0.011	2.572	1.245~5.311	0.021	2.380	1.139~4.975
LncRNA NEAT1	0.007	0.358	0.170~0.755	0.013	0.382	0.179~0.817

Figure 1 LncRNA NEAT1 in retinoblastoma. (A) LncRNA NEAT1 in patient tissue. (B) LncRNA NEAT1 in serum of patients and normal people. (C) Correlation analysis of LncRNA NEAT1 in serum and tissue. (D) ROC curve of LncRNA NEAT1 in serum for diagnosis of retinoblastoma. (E) The 5-year survival rate of high and low expression group. *** indicates that P<0.001.

Figure 2 Role of LncRNA NEAT1 in retinoblastoma. (A) LncRNA NEAT1 in retinoblastoma. (B) Expression of three designed and synthesized si-NEAT1. (C) Relative expression of LncRNA NEAT1 in cells transfected with si-NEAT1#2. (D) Cell proliferation after transfection of si-NEAT1#2. (E) Change of cell penetrating membrane number after si-NEAT1#2. (F) Apoptosis rate after si-NEAT1#2. (G) Volume changes after tumor allograft. (H) Tumor quality of nude mice after tumor allografting and execution. (I) Expression of LncRNA NEAT1, miR-148b-3p, ROCK1 in tumor tissues of nude mice. * indicates that P<0.05, ** indicates that P<0.01.

Figure 3 LncRNA NEAT1 as a sponge of miR-148b-3p. (A) miR-148b-3p in retinoblastoma chip. (B) Binding sites for LncRNA NEAT1 and miR-148b-3p. (C) Double luciferase report confirmed the relationship between LncRNA NEAT1 and miR-148b-3p. (D) RIP experiment confirmed that LncRNA NEAT1 and miR-148b-3p can be precipitated by Ago2 antibody. (E) Relative expression of miR-148b-3p after transfection of retinoblastoma cells. (F) Proliferation of retinoblastoma cells after transfection. (G) Invasion of retinoblastoma cells after transfection. (H). Apoptosis of retinoblastoma cells after transfection. * indicates that P<0.05, ** indicates that P<0.01, *** indicates that P<0.001.

Figure 4 miR-148b-3p targets ROCK1. (A) Three online prediction of potential target genes of miR-148b-3p. (B) miR-148b-3p targeting ROCK1 binding site. (C) Double luciferase activity detection. (D) Relative expression of ROCK1 in cells transfected with miR-148b-3p with different expressions. (E) Relative expression of ROCK1 protein in cells transfected with miR-148b-3p with different expressions. (F) Relative expression of ROCK1 in cells co-transfected with miR-148b-3p-inhibit and si-ROCK1. (G) Relative expression of ROCK1 protein in cells co-transfected with miR-148b-3p-inhibit and si-ROCK1. (H) Cell proliferation after co-transfection of miR-148b-3p-inhibit and si-ROCK1. (I) Cell invasion after co-transfection of miR-148b-3p-inhibit and si-ROCK1. (J) Apoptosis after co-transfection of miR-148b-3p-inhibit and si-ROCK1. ** indicates that P<0.01.

Figure 5 LncRNA NEAT1 acts as miR-148b-3p sponge to regulate ROCK1 inhibiting cell growth. (A) Cell proliferation after co-transfection. (B) Cell invasion after co-transfection. (C) Apoptosis after co-transfection. ** indicates that P<0.01.