LINC00839/miR-144-3p/WTAP (WT1 Associated protein) axis is involved in regulating hepatocellular carcinoma progression

Figures & data

Figure 1. Expression of LINC00839 and its association with HCC prognosis. a) Bioinformatics analysis showed LINC00839 was upregulated in HCC tumor samples from StarBase v3.0. b) High expression level of LINC00839 was confirmed by qRT-PCR in 60 HCC samples and the adjacent normal tissues. c) Expression of LINC00839 was examined in different HCC cell lines (Huh6, Huh7, SK-hep1, HepG2, and PLC5) and normal hepatocyte cell line (LO2). d) High expression of LINC00839 was associated with a poor overall survival according to Kaplan-Meier Plotter with 371 HCC patients (left) and our database with 60 HCC patients (right). The data were shown as the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Figure 2. The effect of LINC00839 silencing on cell phenotypes. a) The knockdown efficiency of si-LINC00839^1# and si-LINC00839^2# by qRT-PCR. b) LINC00839 silencing inhibited cell proliferation in both HepG2 and PLC5 cells. c) LINC00839 silencing inhibited cell colony formation. d-e) Si-LINC00839 impaired cell migration and invasion ability. f) Si-LINC00839 transfection promoted cell apoptosis. The data were shown as the mean ± SD of three independent experiments. **, P < 0.01; ***, P < 0.001

Figure 3. LINC00839 serves as miR-144-3p sponge. a) The predicted binding site of LINC003839 and miR-141-3p. b) LINC00839 silencing upregulated the expression of miR-144-3p in HCC cells. c) Dual-luciferase assay using reporter with wild-type binding site (Luci-LINC00839-WT), mutated reporter (Luci-LINC00839-mut) or empty reporter (Luci-NC) in the presence of MiR-144-3p mimic or miR-NC. d) MiR-144-3p was downregulated in HCC tissues from the database. e) The expression of miR-144-3p was quantified by qRT-PCR in 60 HCC samples and the adjacent normal tissues. f) There was a negative correlation between LINC00839 and miR-144-3p expression in 60 HCC samples. The data were shown as mean ± SD of three independent experiments. **, P < 0.01; ***, P < 0.001

Table 1. Correlation of linc00839 expression with clinicopathologic features of  hepatocellular carcinoma

	LINC00839
Patients	High (n = 30)	Low (n = 30)	P value
Age (years)			0.1965
<58	17	12
≥58	13	18
Gender			0.4475
Male	27	25
Female	3	5
HBV infection			0.176
Positive	22	17
Negative	8	13
Liver cirrhosis history			0.3006
Yes	18	14
No	12	16
Tumor size			0.0201
≤5 cm	11	20
>5 cm	19	10
AFP			0.152
≤200	6	11
>200	24	19
Lymph node metastasis			0.0149
No	15	24
Yes	15	6
Clinical satge			0.0195
I+ II	9	18
III+IV	21	12
Tumor differentiation			0.0384
Well+moderate	12	20
Poor	18	10

Figure 4. The effect of miR0144-3p on cell phenotypes. a) miR-144-3p knockdown efficiency of miR-144-3p inhibitor. b) miR-144-3p inhibitor partially rescued the inhibition effect on cell proliferation of si-LINC00839 in HCC cells. c) miR-144-3p inhibitor partially recovered colony formation ability after LINC00839 silencing. d-e) miR-144-3p inhibitor partially rescued the inhibition effect of si-LINC00839 on migration and invasion ability. f) miR-144-3p inhibitor prevented the apoptotic effect of LINC00839 silencing. The data were shown as mean ± SD of three independent experiments. **, P < 0.01; ***, P < 0.001

Figure 5. WTAP is a downstream target of miR-144-3p. a) The predicted binding sites between WTAP and miR-144-3p. b) Dual-luciferase assay using reporter with wild-type binding site (Luci-WTAP-WT), mutated reporter (Luci-WTAP-mut) or empty reporter (Luci-NC) in the presence of MiR-144-3p mimic or miR-NC. c) The expression level of WTAP in HCC tissues from the database. d) A negative correlation between WTAP and miR-144-3p expression in 60 HCC samples. e) miR-144-3p mimic upregulated miR-144-3p level. f) miR-144-3p mimic downregulated WTAP mRNA level by qRT-PCR. g) Mi-144-3p mimic downregulated the protein level of WTAP. The data were shown as mean ± SD of three independent experiments. **, P < 0.01; ***, P < 0.001

Figure 6. The effect of WTAP overexpression on cell phenotypes. a) The overexpression efficiency after transfecting with pc-WTAP expression plasmid. b) WTAP overexpression rescued the inhibition effect of si-LINC00839 on cell proliferation. c) WTAP overexpression enhanced cell colony formation after LINC00839 silencing. d-e) WTAP overexpression rescued the inhibition effect of si-LINC00839 on the migration and invasion ability of HCC cells. f) WTAP overexpression prevented cell apoptosis induced LINC00839 silencing. The data were shown as the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001