MicroRNA-18a promotes hepatocellular carcinoma proliferation, migration, and invasion by targeting Bcl2L10

Figures & data

Figure 1 Differential expression of miR-18a in HCC tissues and HCC cell lines.

Notes: (A) miR-18a expression was much higher in HCC tissues compared with adjacent non-tumor tissues (P<0.001). (B) miR-18a expression was much higher in HepG2 and SMMC7721 cell lines compared with normal liver cell LO2 (P<0.001). (C) Kaplan–Meier analyses of the correlations between miR-18a expression and overall survival of 123 patients with HCC (log-rank P<0.001). Data were represented as mean ± SD. ***P<0.001.
Abbreviations: HCC, hepatocellular carcinoma; miR-18a, microRNA-18a.

Table 1 Associations of miR-18a with the clinical pathology features of HCC patients

Features	Total	miR-18a expression		P-value
	N=123	High	Low
Gender				0.835
Male	97	47	50
Female	26	12	14
Age, year				0.945
≤50	33	16	17
>50	90	43	47
AFP				0.023*
<400 ng/mL	30	9	21
≥400 ng/mL	93	50	43
TNM stage				<0.001
I–II	62	7	55
III–IV	61	52	9
Tumor size				<0.001
≤5 cm	53	6	47
>5 cm	70	53	17
Intrahepatic vascular invasion				<0.001
Yes	64	45	19
No	59	14	45

Note:

* P<0.05.

Abbreviations: AFP, alpha-fetoprotein; HCC, hepatocellular carcinoma; miR-18a, microRNA-18a.

Table 2 Cox regression analysis for miR-18a in HCC

Variables	Multivariable analysis
	Exp(B)	95% CI	P-value
miR-18a	2.017	1.137–3.578	0.017*
Age, year	1.238	–	0.659
Gender	0.195	–	0.266
Tumor size	2.485	–	0.115
TNM stage	4.396	0.164–0.527	<0.001
AFP	0.434	–	0.510
Intrahepatic vascular invasion	0.294	1.987–9.728	<0.001

Note:

* P<0.05.

Abbreviations: AFP, alpha-fetoprotein; HCC, hepatocellular carcinoma.

Figure 2 Effect of miR-18a on the proliferation of HCC cells.

Notes: (A) miR-18a mimic increased miR-18a level (P<0.01 in the HepG2 cell and P<0.001 in the SMMC7721 cell) and miR-18a inhibitor decreased miR-18a level (P<0.05) in HCC cell lines. (B) Upregulation of miR-18a in HCC cells facilitated cell proliferation while downregulation of miR-18a inhibited cell proliferation. (C) Cell apoptosis rate was detected by flow cytometry after transfection with miR-18a inhibitor or miR-18a mimic. (D) The proportion of cells in each cell cycle phase was detected using flow cytometry after the cells were transfected with miR-18a inhibitor or miR-18a mimic. Data were reported as mean ± SD. *P<0.05, **P<0.01, and ***P<0.001.
Abbreviations: HCC, hepatocellular carcinoma; miR-18a, microRNA-18a; NC, negative control; PI, propidium iodide.

Figure 3 Effect of miR-18a on migration and invasion of HCC cells in vitro.

Notes: (A) The effect of miR-18a on cell migration was determined by a wound scratching assay. Cell migration capacity of miR-18a mimic transfected HCC cells was promoted (P<0.05), whereas cell migration capacity of miR-18a inhibitor transfected HCC cells was inhibited (P<0.05). Scale bars =200 µm; magnification 40×. (B) After transfection of cells with miR-18a, the ability of cells to pass through the basement membrane was enhanced (P<0.001). In contrast, after transfecting cells with miR-18a inhibitor, the ability was inhibited (P<0.001). Scale bars =20 µm; magnification 100×. Data were reported as mean ± SD. ***P<0.001.
Abbreviations: HCC, hepatocellular carcinoma; miR-18a, microRNA-18a; NC, negative control.

Figure 4 Bcl2L10 was a direct target of miR-18a.

Notes: (A) The predicted binding site of miR-18a in the 3′-UTR of Bcl2L10. (B) miR-18a mimic significantly inhibited the luciferase activities of Bcl2L10-3′-UTR-Wt reporter in both cell lines (P<0.001), while it exhibited no inhibitory effects on the luciferase activities of Bcl2L10-3′-UTR-Mut reporter in HCC cells (P>0.05). (C) The mRNA expression of Bcl2L10 was much lower in HCC tissues compared with adjacent non-tumor tissues (P<0.001). (D) Immunochemistry showed that the protein expression of Bcl2L10 was much lower in HCC tissues than that in adjacent non-tumor tissue (P<0.01). Scale bars =20 µm. (E) Western blot analysis revealed that the protein expression of Bcl2L10 was higher in adjacent non-tumor tissues than in HCC tissues (P<0.01). (F) miR-18a expression negatively correlated with Bcl2L10 mRNA expression in HCC tissues (R²=0.405, P<0.01, correlation coefficient =−0.636). Data were reported as mean ± SD. **P<0.01 and ***P<0.001.
Abbreviations: HCC, hepatocellular carcinoma; miR-18a, microRNA-18a; Bcl2L10, Bcl2 like 10; Wt, wild-type; Mut, mutant.

Figure 5 Effect of miR-18a on the expression of Bcl2L10 in vitro.

Notes: (A) Upregulation of miR-18a decreased Bcl2L10 mRNA level, while downregulation of miR-18a increased Bcl2L10 mRNA level in HCC cells (P<0.001). (B) Western blot analysis revealed that the protein expression of Bcl2L10 was decreased in the miR-18a mimic transfected HCC cells (P<0.05) and was increased in the miR-18a inhibitor transfected cells (P<0.05). Data were reported as mean ± SD. *P<0.05 and ***P<0.001.
Abbreviations: HCC, hepatocellular carcinoma; miR-18a, microRNA-18a; Bcl2L10, Bcl2 like 10; NC, negative control.

Figure 6 Overexpression of Bcl2L10 partially reversed the function of miR-18a on HCC cell proliferation in vitro.

Notes: (A) The Bcl2L10 plasmids promoted the mRNA and protein expression of Bcl2L10 in HCC cells (P<0.001). (B) Overexpression of Bcl2L10 partially reversed the effect of miR-18a on HCC cell proliferation (P<0.05). (C) Cell apoptosis rate was detected by flow cytometry after transfection with miR-18a mimic or miR-18a mimic + Bcl2L10 plasmids. (D) The proportion of cells in each cell cycle phase was detected using flow cytometry after cells were transfected with miR-18a mimic or miR-18a mimic + Bcl2L10 plasmids. Data were reported as mean ± SD. *P<0.05 and ***P<0.001.
Abbreviations: HCC, hepatocellular carcinoma; miR-18a, microRNA-18a; Bcl2L10, Bcl2 like 10; PI, propidium iodide.

Figure 7 Overexpression of Bcl2L10 partially reversed the function of miR-18a on HCC cell migration and invasion in vitro.

Notes: (A) Bcl2L10 decreased the migration ability of miR-18a transfected HCC cells in vitro. Scale bars =200 µm; magnification 40×. (B) Bcl2L10 inhibited the invasion capacity of miR-18a transfected HCC cell in vitro. Scale bars =20 µm; magnification 100×. Data were reported as mean ± SD. **P<0.01 and ***P<0.001.
Abbreviations: HCC, hepatocellular carcinoma; miR-18a, microRNA-18a; Bcl2L10, Bcl2 like 10.

AshtariSPourhoseingholiMASharifianAZaliMRHepatocellular carcinoma in Asia: Prevention strategy and planningWorld J Hepatol20157121708171726140091

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