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. 2015 Aug;6(8):575-588.
doi: 10.1007/s13238-015-0142-8. Epub 2015 Apr 28.

Two less common human microRNAs miR-875 and miR-3144 target a conserved site of E6 oncogene in most high-risk human papillomavirus subtypes

Lin Lin #  1 , Qingqing Cai #  1   2 , Xiaoyan Zhang  3 , Hongwei Zhang  1 , Yang Zhong  4   5 , Congjian Xu  1   6   2   7 , Yanyun Li  1
Affiliations

Two less common human microRNAs miR-875 and miR-3144 target a conserved site of E6 oncogene in most high-risk human papillomavirus subtypes

Lin Lin et al. Protein Cell. 2015 Aug.

Abstract

Human papillomaviruses (HPVs) including high-risk (HR) and low-risk (LR) subtypes have distinguishable variation on both genotypes and phenotypes. The co-infection of multiple HR-HPVs, headed by HPV16, is common in cervical cancer in female. Recently accumulating reports have focused on the interaction between virus and host, particularly the role of human microRNAs (miRNAs) in anti-viral defense by targeting viral genome. Here, we found a well-conserved target site of miRNAs in the genomes of most HR-HPVs, not LR-HPVs, by scanning all potential target sites of human miRNAs on 24 HPVs of unambiguous subtypes of risk. The site is targeted by two less common human miRNAs, miR-875 and miR-3144, and is located in E6 oncogene open reading frame (ORF) and overlap with the first alternative splice exon of viral early transcripts. In validation tests, miR-875 and miR-3144 were identified to suppress the target reporter activity markedly and inhibit the expression of both synthetically exogenous E6 and endogenous E6 oncogene. High level of two miRNAs can inhibit cell growth and promote apoptosis in HPV16-positive cervical cancer cells. This study provides a promising common target of miRNAs for most HR-HPVs and highlights the effects of two low expressed human miRNAs on tumour suppression.

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Figures

Figure 1
Figure 1
The sequence alignments for 15 HR-HPV subtypes (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82) and 9 LR-HPV subtypes (HPV6, 11, 40, 42, 43, 44, 54, 61, 81). Yellow highlights the targets of miR-875, red highlights the targets of miR-3144, orange show their overlapping districts. Blue shows the degree of conservation in each Nucleotide site. The more conserved it is, the darker blue it shows. Black columns at the bottom show the consensus of conservation in each Nucleotide site
Figure 2
Figure 2
MiR-875 and miR-3144 inhibit luciferase reporter expression. (A) Analysis of the relative luciferase activity in C33A cells co-transfected with miR-875 vector and target (209–231) reporter, or empty vector and target (209–231) reporter, or miR-875 vector and mutated target (∆209–231) reporter. (B) Analysis of the relative luciferase activity in C33A cells co-transfected with miR-3144 vector and target (207–232) reporter, or empty vector and target (207–232) reporter, or miR-3144 vector and mutated target (∆207–232) reporter. P-values at Student’s t-test were *P < 0.05
Figure 3
Figure 3
MiR-875 and miR-3144 decrease the expression of exogenous E6. The relative expression of E6 mRNA obtained by qRT-PCR in C33A cells after co-transfection with synthetically E6 construct along with miRNAs vectors or empty vector. P-values at Student’s t-test were *P < 0.05
Figure 4
Figure 4
Transfection with miR-875 and miR-3144 interferes the expression of endogenous E6. (A) The expression levels of miR-875 or miR-3144 by qRT-PCR in SiHa cells transfected with miR-875 or miR-3144 expression vectors, SiHa cells transfected with empty vectors (Vector), or untreated SiHa cells (Mock). (B and C) RT-PCR analysis of E6 mRNA relative expression in SiHa cells transfected with miR-875 or miR-3144 expression vectors, SiHa cells transfected with empty vectors (Vector), or untreated SiHa cells (Mock). P-values at Student’s t-test were *P < 0.05, ***P < 0.001
Figure 5
Figure 5
High-level of miR-875 and miR-3144 inhibit cell growth. Time kinetics of cell growth after 120 h of monitoring every 4 h with the xCELLigence RTCA DP System in SiHa cells transfected with miR-875 (A) or miR-3144 (B) vectors, or with empty vectors (Vector), or untreated SiHa cells (Mock). P-values at Student’s t-test were *P < 0.05, ***P < 0.001
Figure 6
Figure 6
High-level of miR-875 and miR-3144 promoted cell apoptosis. (A) Annexin-V/PI analysis in SiHa cells transfected with miR-875 or miR-3144 vector, or with empty vectors (Vector), or untreated SiHa cells (Mock). (B) Early apoptosis, late apoptosis and the sum total of apoptosis in different group cells. P-values at Student’s t-test were *P < 0.05, **P < 0.01
Figure 7
Figure 7
High-level of miR-875 and miR-3144 induced apoptotic morphological changes. (A) Apoptotic morphological changes (white arrows) with Hoechst staining (blue) in fluorescence microscope (200 × times) in SiHa cells transfected with miR-875 or miR-3144 vector, or with empty vectors (Vector), or untreated SiHa cells (Mock). (B) Histograms for quantification of the cell death (%) in different groups. P-values at Student’s t-test were ***P < 0.001
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