Downregulation of microRNA-122 promotes proliferation, migration, and invasion of human hepatocellular carcinoma cells by activating epithelial–mesenchymal transition

Figures & data

Table 1 The transfection sequences in miR-122, anti-miR-122, miR-NC, and anti-miR-NC groups

Groups	Sequences
miR-122	Sense: 5′-UGGAGUGUGACAAUGGUGUUUG-3′ Antisense: 5′-AACACCAUUGUCACACUCCAUU-3′
Anti-miR-122	5′-CAAACACCAUUGUCACACUCCA-3′
miR-NC	Sense: 5′-UUCUCCGAACGUGUCACGUTT-3′ Antisense: 5′-ACGUGACACGUUCGGAGAATT-3′
Anti-miR-NC	5′-CAGUACUUUUGUGUAGUACAA-3′

Abbreviations: miR-122, microRNA-122; NC, negative control.

Table 2 The sequences of forward primers and reverse primers of miR-122, U6, Wnt1, E-cadherin, vimentin, β-cadherin, N-cadherin, and GAPDH

Genes	Primer sequences
miR-122	F: 5′-TTGAATTCTAACACCTTCGTGGCTACAGAG-3′ R: 5′-TTAGATCTCATTTATCGAGGGAAGGATTG-3′
U6	F: 5′-CTCGCTTCGGCAGCACA-3′ R: 5′-AACGCTTCACGAATTTGCGT-3′
Wnt1	F: 5′-TGGCTGGGTTTCTGCTACG-3′ R: 5′-TCGAGGACGGTCGGACT-3′
E-cadherin	F: 5′-CGGTGGTCAAAGAGCCCTTACT-3′ R: 5′-TGAGGGTTGGTGCAACAACGTCGTTA-3′
Vimentin	F: 5′-TGAGTACCGGAGACAGGTCGAG-3′ R: 5′-TAGCAGCTTCAACGCAAAGTTC-3′
β-Cadherin	F: 5′-ATTGTCCACGCTGGATTTC-3′ R: 5′-GACCCAAATGTCGCAGTCAG-3′
N-cadherin	F: 5′-GTGCCATTAGCCAAGGGAATTCAGC-3′ R: 5′-GCGTTCCTGTTCCACTCATAGGAGG-3′
GAPDH	F: 5′-ACAGTCAGCCGCATCTTCTT-3′ R: 5′-GACAAGCTTCCCGTTCTCAG-3′

Abbreviations: miR-122, microRNA-122; GAPDH, glyceraldehyde phosphate dehydrogenase; F, forward; R, reverse.

Figure 1 miR-122 targeting Wnt1.

Notes: (A) TargetScan predicted that Wnt1 was a target gene of miR-122; (B) dual-luciferase reporter gene detection confirmed that Wnt1 was a target gene of miR-122 in HepG2 and huh7 cells. Compared with the miR-22-mimics + pWnt1-Mut, ^aP<0.001.
Abbreviations: miR-122, microRNA-122; NC, negative control; Wt, wild-type; Mut, mutated.

Figure 2 qRT-PCR detecting miR-122 detection levels after transfection in HepG2 and huh7 cells.

Notes: Compared with the miR-NC group, ^aP<0.05; compared with the anti-miR-NC group, ^bP<0.05.
Abbreviations: qRT-PCR, quantitative real-time polymerase chain reaction; miR-122, microRNA-122; NC, negative control.

Figure 3 qRT-PCR detecting mRNA expressions of Wnt1- and EMT-related genes.

Notes: Compared with the miR-NC group, ^aP<0.05; compared with the anti-miR-NC group, ^bP<0.05.
Abbreviations: qRT-PCR, quantitative real-time polymerase chain reaction; EMT, epithelial–mesenchymal transition; mRNA, messenger RNA; miR-122, microRNA-122; NC, negative control.

Figure 4 Western blot detecting Wnt1 protein and EMT-associated proteins.

Notes: Compared with the miR-NC group, ^aP<0.05; compared with the anti-miR-NC group, ^bP<0.05.
Abbreviations: EMT, epithelial–mesenchymal transition; miR-122, microRNA-122; NC, negative control.

Figure 5 MTT assay detecting the effects of miR-122 on cell proliferation of HepG2 and huh7 cells.

Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; miR-122, microRNA-122; h, hours; NC, negative control; OD, optical density.

Figure 6 Transwell assay detecting migration capacity of HepG2 cells (A: miR-NC group; B: miR-122 group; C: anti-miR-NC group; D: anti-miR-122 group; E: miR-122 + vector group; F: miR-122 + Wnt1 group).

Note: Magnification was ×200.
Abbreviations: miR-122, microRNA-122; NC, negative control.

Figure 7 Transwell assay detecting migration capacity of huh7 cells (A: miR-NC group; B: miR-122 group; C: anti-miR-NC group; D: anti-miR-122 group; E: miR-122 + vector group; F: miR-122 + Wnt1 group).

Note: Magnification was ×200.
Abbreviations: miR-122, microRNA-122; NC, negative control.

Table 3 Wound-healing assay detecting cell migration rate in HepG2 and huh7 cells at different time points

Groups	Migration rate (%)
	HepG2 cells		huh7 cells
	24 hours	48 hours	24 hours	48 hours
miR-NC	17.10±2.01	24.33±3.20	10.11±1.02	29.36±3.00
miR-122	6.21±0.23a	14.10±1.65a	4.09±0.21a	16.70±1.96a
Anti-miR-NC	17.22±3.17	25.01±2.99	9.99±1.13	28.97±5.31
Anti-miR-122	30.96±7.03b	40.44±6.15b	26.87±4.33b	47.65±6.11b
miR-122 + vector	4.96±0.11	13.15±1.33	3.98±0.33	15.43±1.11
miR-122 + Wnt1	17.31±2.96a	24.56±2.27a	11.00±0.98a	30.01±4.11a
F	22.15	26.28	56.92	25.32
P	<0.001	<0.001	<0.001	<0.001

Notes: Compared with the miR-NC group,

a P<0.05; compared with the anti-miR-NC group,

b P<0.05.

Abbreviations: miR-122, microRNA-122; NC, negative control.

Figure 8 Transwell assay detecting invasion capacity of HepG2 cells (A: miR-NC group; B: miR-122 group; C: anti-miR-NC group; D: anti-miR-122 group; E: miR-122 + vector group; F: miR-122 + Wnt1 group).

Note: Magnification was ×200.
Abbreviations: miR-122, microRNA-122; NC, negative control.

Figure 9 Transwell assay detecting invasion capacity of huh7 cells (A: miR-NC group; B: miR-122 group; C: anti-miR-NC group; D: anti-miR-122 group; E: miR-122 + vector group; F: miR-122 + Wnt1 group).

Note: Magnification was ×200.
Abbreviations: miR-122, microRNA-122; NC, negative control.