Rs6757 in microRNA-3976 binding site of CD147 confers risk of hepatocellular carcinoma in South Chinese population

Abstract

Background: Cluster of differentiation 147 (CD147) overexpression plays a key role in the proliferation, differentiation, invasion, metastasis, and prognosis of hepatocellular carcinoma (HCC). The aim of this study was to explore the relationship between rs6757 and the HCC risk in the South Chinese population, and the functional significance of rs6757 by affecting the efficacy of microRNA-3976 (miR-3976) binding to the CD147 3'-UTR.

Methods: We performed a retrospective case-control study to analyze the association between rs6757 and the risk of HCC. We chose candidate microRNAs with the potential of interacting with rs6757 through a series of silico analyses. A luciferase reporter gene assay was implemented to detect the binding extent of microRNAs to each polymorphic allele of rs6757.

Results: An obvious association between rs6757 and the risk of HCC was detected in C vs. T (OR = 1.826, 95% CI [1.263-2.642]), CC vs. TT (OR = 4.513, 95% CI [1.510-13.489]), dominant genetic model (OR = 1.824, 95% CI [1.120-2.965]), and recessive genetic model (OR = 3.765, 95% CI [1.286-11.020]). Bioinformatics analysis indicated that miR-3976 binding sites containing the rs6757-T allele had lower free energies than those with the C allele, the lower free energies, the higher affinities. Luciferase activity was remarkably decreased by miR-3976 binding to the CD147 3'-UTR bearing rs6757 T allele, which could be reversed by miR-3976 inhibitors. Furthermore, miR-3976 reduced the luciferase expression in a manner of dose-dependent when cotransfected with constructs with the CD147-TT-pSICHECK2.

Conclusions: The research we have done suggests that rs6757 confers the CD147 allele-specific translational suppression by miR-3976, which provides a theoretical basis for antineoplastic therapy targeting CD147.

Keywords: CD147; Hepatocellular carcinoma (HCC); MicroRNA-3976 (miR-3976); Single nucleotide polymorphism (SNP).

Fig. 1

Combination of different alleles of rs6757 to hsa-miR-3976. The T/C variant (rs6757) is located in the seed region of miRNA-3976, which means that the C/T variant (rs6757) has the potential to disrupt the binding of miR-3976 to CD147 3′-UTR

Fig. 2

In silico analysis of the pairing of miR-3976 to the binding site in the 3′-UTR of CD147. The arrow indicates the SNP site in the CD147 3′-UTR in each folding structure. The lower minimal folding energies, the more stable binding; the higher the sum of the ΔΔG value, the more likely to be a functional SNP

Fig. 3

Sequencing results of the inserts in CD147-TT-pSICHECK2 plasmid. Arrows indicate the inserted rs6757 TT genotype fragment

Fig. 4

The Blast of the inserts in CD147-TT-pSICHECK2 plasmid. The sequencing results were analyzed by Blast, the inserted sequence of target gene was 100% consistent with the sequence of CD147 rs6757 TT genotype in the NCBI database

Fig. 5

Sequencing results of the inserts in CD147-CC-pSICHECK2 plasmid. The sequencing result showed that the mutation from nucleotide T to C at the target site (marked in green) of CD147 gene had successfully achieved

Fig. 6

The Blast of the inserts in CD147-CC-pSICHECK2 plasmid. After mutating from nucleotide T to C, the inserted sequence was 99% consistent with the sequence of CD147 in the NCBI database by Blast analysis

Fig. 7

The R/F analysis of a luciferase reporter vector. A Luciferase expression was significantly reduced following transfection with CD147-TT-pSICHECK2 (P < 0.01); the translational suppression of miR-3976 can be reversed by its inhibitor. B There was no significant statistical difference between the groups following transfection with CD147-CC-pSICHECK2. C Luciferase activity was decreased by miR-3976 in dose-dependent manner for the constructs with a TT genotype but not changed for constructs with a CC genotype at the rs6757:T>C polymorphism. Each transfection was carried out in triplicate. * denotes a p value < 0.05; ** denotes a p value < 0.01