MiR-199a-5p regulates rat liver regeneration and hepatocyte proliferation by targeting TNF-α TNFR1/TRADD/CASPASE8/CASPASE3 signalling pathway

Figures & data

Table 1. Sequences of miR-199a mimics/inhibitors and related controls.

MiRNA	Sequences (5′→3′)
MiR-199a mimic sense	CCCAGUGUUCAGACUACCUGUUC
MiR-199a mimic antisense	GAACAGGUAGUCUGAACACUGGG
MiR-199a inhibitor	GAACAGGUAGUCUGAACACUGGG
NC mimic sense	UUUGUACUACACAAAAGUACUG
NC mimic antisense	CAGUACUUUUGUGUAGUACAAA
NC inhibitor	CAGUACUUUUGUGUAGUACAAA

Table 2. The sequence of TNF-α siRNAs.

SiRNA	Target sequence	Sequence
TNF-α-01	CCAATGGCATGGATCTCAA	5′-CCAAUGGCAUGGAUCUCAAdTdT-3′
		3′-dTdTGGUUACCGUACCUAGAGUU-5′
TNF-α-02	GCAGATGGGCTGTACCTTA	5′-GCAGAUGGGCUGUACCUUAdTdT-3′
		3′-dTdTCGUCUACCCGACAUGGAAU-5′
TNF-α-03	GCCGATTTGCCACTTCATA	5′-GCCGAUUUGCCACUUCAUAdTdT-3′
		3′-dTdTCGGCUAAACGGUGAAGUAU-5′

Table 3. Primers used in reverse transcription and quantitative real-time PCR.

MiRNA and genes	Pimers sequences (5′→3′)
MiR-199a RT	GTCGTATCCAGTGCAGGGTCCGAGGTATT CGCACTGGATACGACGAACAG
MiR-199a FP	CCCAGTGTTCAGACTACCTG
MiR-199a RP	GTGCAGGGTCCGAGGT
U6 FP	CTCGCTTCGGCAGCACA
U6 RP	AACGCTTCACGAATTTGCGT
TNF-α FP	TACTCCCAGGTTCTCTTCAAGG
TNF-α RP	AGGCTGACTTTCTCCTGGTATG
CASPASE3 FP	GAGCTGGACTGCGGTATTGAG
CASPASE3 RP	AACCATGACCCGTCCCTTGA
CASPASE8 FP	ATATCCCAGATGAGGCAGAC
CASPASE8 RP	GGGTTGTCTTTATTGCTCACATCAT
TRADD FP	AAGAAGAAAGTGGCAATCTACAAGG
TRADD RP	TTCTGGGCTAAGATGTAATTCAAAC
TNFR1 FP	GGTACTGCCGTGCTGTTGC
TNFR1 RP	GCTGAAGGCTGGGATAGAGG
BCL2 FP	CGACCTCTGTTTGATTTCTCCTG
BCL2 RP	CTTTTCATATTTGTTTGGGGCA
CCND1 FP	AAAATGCCAGAGGCGGATGA
CCND1 RP	GAAAGTGCGTTGTGCGGTAG
CCNA2 FP	CTTTTAGTGCCGCTGTCTCTTT
CCNA2 RP	GCCCGCATACTGTTAGTGATGT
β-actin FP	ACATCCGTAAAGACCTCTATGCCAACA
β-actin RP	GTGCTAGGAGCCAGGGCAGTAATCT

Figure 1. MiR-199a is upregulated in liver tissue at 12–30 h after PH compared to those at SO group and rat hepatocyte BRL-3A. (A) qRT-PCR assay was used to test miR-199a level at 12–30 h after PH and SO. (B) Relative miR-199a levels were tested in rat hepatocyte BRL-3A by qRT-PCR assay. All data are represented as the mean ± SD, *p < .05, **p < .01.

Figure 2. Down-regulate expression of miR-199a inhibits hepatocyte proliferation a. (A, B) qRT-PCR analysis was used to test transfection efficiency of miR-199a mimics (miR-199a-m)/inhibitors (miR-199a-i), or NCs (NC-m, NC-i). (C) CCK-8 kit was used for Cell viability assay. (D) Flow cytometry was used to detect the effect of miR-199a on the cell cycle. (E) Edu (red) assay was used to detect the proliferation of miR-199a in hepatocyte. (F) The percentage of Edu-positive cells was quantified. All data are represented as the mean ± SD, *p < .05, **p < .01.

Figure 3. Down-regulate expression of miR-199a promotes hepatocyte apoptosis. (A) Apoptosis analysis of miR-199a in BRL-3A were tested by flow cytometry after treatment with miR-199a inhibitors or NC; (B) Quantification of the cell apoptosis rate in each group. All data are represented as the mean ± SD, *p < .05.

Figure 4. MiR-199a negatively correlates with TNF-α expression at protein level both in vitro and in vivo. (A) qRT-PCR analysis for TNF-α level in liver tissue at 12–30 h after PH (compared with those at SO group). (B) Western blot analysis for TNF-α level in liver tissue at 12–30 h after PH (compared with those at SO group). (C) qRT-PCR analysis for TNF-α expression in BRL-3A after transfection with miR-199a inhibitor (miR-199a-i) or NC-i. (D) Western blot analysis for TNF-α expression in BRL-3A transfected with miR-199a inhibitor (miR-199a-i) or NC-i. β-actin/U6 were used as loading control. All data are represented as the mean ± SD, *p < .05, **p < .01.

Figure 5. TNF-α is a direct target of miR-199a. (A) The luciferase report analysis for the relative fluorescence level in BRL-3A cells after co-transfection of TNF-α wild-type and mutant 3′UTR with miR-199a mimic/inhibitor. (B) The miR-199a binding site in the 3′UTR of TNF-α and the corresponding mutation site. All data are represented as the mean ± SD, *p < .05.

Figure 6. SiRNA- TNF-α reverses miR-199a inhibition-inhibited cell growth in vitro. (A,B) TNF-α level were measured in BRL-3A after treatment with siRNA- TNF-α (sequence-1,-2 or -3) by qRT-PCR and Western blot analysis. (C) Cell viability assay. (D) Edu (red) cell proliferation assay. (E) The percentage of Edu-positive cells was quantified. The results demonstrated that siRNA- TNF-α reversed the inhibitive effect of miR-199a-i on hepatocyte proliferation. All data are represented as the mean ± SD, *p < .05, **p < .01.

Figure 7. MiR-199a regulates proliferation in rat hepatocyte via TNF-α/TNFR1/TRADD/CASPASE8/CAPASE3 signalling pathway. (A) qRT-PCR analysis for TNF-α, TNFR1, TRADD, CASPASE8, and CAPASE3, BCL2, CCND1 and CCNA2 in rat hepatocyte BRL-3A after co-transfection with siR- TNF-α, miR-199a inhibitor (miR-199a-i) or its negative control (NC-i). (B) Western blot analysis for TNF-α, TNFR1, TRADD, CASPASE8, and CAPASE3, BCL2, CCND1, and CCNA2 in rat hepatocyte BRL-3A after co-transfection with siR-TNF-α, miR-199a inhibitor (miR-199a-i) or its negative control (NC-i). All data are represented as the mean ± SD.*p < .05, **p < .01.