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MiR-122 modulates type I interferon expression through blocking suppressor of cytokine signaling 1
The International Journal of Biochemistry & Cell Biology 45 (2013) 858–865
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MiR-122 modulates type I interferon expression through blocking suppressor of cytokine signaling 1 Aimei Li a,b,1 , Wuqi Song a,b,c,1 , Jun Qian a,b,1 , Yujun Li a,b,c , Junming He a,b , Qingmeng Zhang a,b , Wenhui Li a,b , Aixia Zhai a,b , Wenping Kao a,b , Yunlong Hu a,b , Hui Li a,b , Jing Wu a,b , Hong Ling a,b , Zhaohua Zhong a,b,∗∗ , Fengmin Zhang a,b,c,∗ a
Department of Microbiology, Harbin Medical University, Harbin 150081, China Heilongjiang Key Laboratory of Immunity and Infection, Harbin 150081, China c Bio-pharmaceutical Key Laboratory in Harbin Medical University, Ministry of Education of China, Harbin 150081, China b
a r t i c l e
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Article history: Received 20 October 2012 Received in revised form 9 January 2013 Accepted 14 January 2013 Available online 22 January 2013 Keywords: MiRNA MiR-122 Suppressor of cytokine signaling 1 Type I interferon Hepatocytes
a b s t r a c t MiR-122 is a liver-specific miRNA. Recent studies demonstrated that the interferon (IFN) therapy efficacy is poor in the hepatitis C virus (HCV)-infected patients with lower miR-122 abundance in the livers. The hepatocarcinoma patients also have low miR-122 levels in their livers. We previously found that the IFN expression was reduced when miR-122 was knocked down in human oligodendrocytes. The mechanism is unclear. In this study, the miR-122-abundant cell Huh7 was used to explore the regulatory mechanism of miR-122 on type I IFN expression. We found that miR-122 significantly increased the type I IFN expression in Huh7 cells, while knocking down miR-122 decreased the type I IFN expression. By screening potential miR-122 targets among the negative regulators in IFN signaling pathways, we found that there were putative miR-122 targets in the suppressor of cytokine signaling 1 (SOCS1) mRNA. Over-expressing miR-122 decreased the SOCS1 expression by 50.55% in Huh7 cells, while knocking down miR-122 increased SOCS1 expression by 62.56%. Using a green fluorescence protein (EGFP) fused SOCS1expressing plasmid, the SOCS1-EGFP fluorescence intensity and protein were lower in miR-122 mimictreated cells than those in mock-miRNA-treated cells, while miR-122 knockdown significantly increased the SOCS1-EGFP fluorescence intensity and protein expression. Mutations in the nt359–nt375 region abandoned the impact of miR-122 on SOCS1-EGFP expression. Taken together, SOCS1 is a target of miR122. MiR-122 can regulate the type I IFN expression through modulating the SOCS1 expression. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Abbreviations: IFN, interferon; HCV, hepatitis C virus; SOCS1, suppressor of cytokine signaling 1; EGFP, enhanced green fluorescence protein; HCC, hepatocellular carcinoma; mfe, minimum free energy; DMEM, Dulbecco’s modified eagle medium; FBS, fetal bovine serum; AMO-122, anti-miR-122 oligonucleotide; PCR, polymerase chain reaction; PMSF, protease inhibitor phenylmethylsulfonyl fluoride; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; qRT-PCR, quantitative RT-PCR; SEM, standard error of the mean; ADAM, a disintegrin and metalloprotease. ∗ Corresponding author at: No. 194, Xuefu Road, Harbin 150081, China. Tel.: +86 451 8666 9576; fax: +86 451 8666 9576. ∗∗ Corresponding author at: Department of Microbiology, Harbin Medical University, Harbin 150081, China. Tel.: +86 451 8668 5122; fax: +86 451 8668 5122. E-mail addresses: [email protected] (A. Li), [email protected] (W. Song), [email protected] (J. Qian), Li [email protected] (Y. Li), [email protected] (J. He), [email protected] (Q. Zhang), [email protected] (W. Li), [email protected] (A. Zhai), hm [email protected] (W. Kao), [email protected] (Y. Hu), [email protected] (H. Li), wujing [email protected] (J. Wu), profl[email protected] (H. Ling), [email protected] (Z. Zhong), [email protected] (F. Zhang). 1 These authors contributed equally to this work. 1357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2013.01.008
MiR-122 is the most abundant miRNA in hepatocytes, constituting 70% of the total miRNA population and is extensively involved in the physiological and pathological processes of hepatocytes (Chang et al., 2004; Lewis and Jopling, 2010; Girard et al., 2008). MiR-122 can up-regulate hepatitis C virus (HCV) replication by targeting the 5 un-translational region of the HCV genome in Huh7 cells (Jopling et al., 2005). The miR-122 abundance in hepatocellular carcinoma (HCC) tissues is significantly lower than that in the healthy livers, and the decrease of the miR-122 abundance is closely correlated to the treatment outcome and prognosis of HCC (Coulouarn et al., 2009). A recent study revealed that the HCV patients with low miR122 levels responded poorly to IFN therapy (Sarasin-Filipowicz et al., 2009). These studies suggest that miR-122 plays an important role in HCV infection, hepatocarcinogenesis and IFN treatment. We previously found that the induction of type I IFN was significantly reduced when the miR-122 expression was suppressed in human oligodendrocytes (Qian et al., 2010). However, the mechanism is
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unclear. Therefore, in this study, miR-122-abundant cell Huh7 was used to explore the regulatory mechanism of miR-122 on type I IFN expression. We demonstrate that miR-122 negatively modulates SOCS1 expression and then induce the type I IFN expression. Our data suggest that miR-122 modulates host’s innate immunity through blocking SOCS1 and may play important role in the viral infection and carcinogenesis in the liver. 2. Materials and methods
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(sense); 5 -AGCAGCTCGAAGAGGCAGTC-3 (antisense). The primers of GAPDH, IFN-␣, IFN-, and miR-122 were described previously (Qian et al., 2010). 2.6. ELISA The production of IFN-␣ and IFN- in culture supernatant was measured using the VeriKine Human IFN-␣ and IFN- ELISA kits (PBL interferon source, Piscataway, NJ) according to the manufacturer’s instructions.
2.1. MiRNA target prediction 2.7. Site-directed mutagenesis The potential targets of miR-122 in the SOCS1 mRNA were predicted by RNAhybrid 2.2 (bibiserv.techfak.unibielefeld.de/rnahybrid) based on the complementary sequences and minimum free energy (mfe) (Alves et al., 2009; Rajewsky, 2006). 2.2. Cell culture Hepatoma cell lines Huh7 was cultured in Dulbecco’s modified eagle medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Israel) and 100 units/ml of penicillin and 100 g/ml of streptomycin in humidified incubator at 37 ◦ C with 5% CO2 . 2.3. Nucleotides, plasmids, and IFN inducers The sense and antisense miR-122 mimic (5 -TGGAGTGTGACAATGGTGTTTG-3 and 5 -CAAACACCATTGTCACACTCCA-3 ), the 2 -O-methylated, anti-miR-122 oligonucleotide (AMO-122) (5 CAAACACCAUUGUCACACUCCA-3 ), and the mock-miRNA were synthesized by GenePharma (Shanghai, China). The plasmid pSOCS1-EGFP, which expresses the SOCS1 and enhanced green fluorescence protein (EGFP) fusion protein, was obtained from GeneChem (Shanghai, China). Human recombinant IFN-␣ and IFN- were purchased from Pestka Biomedical Laboratories (Piscataway, NJ). To induce type I IFN expression, the cells cultured in antibiotics-free medium were stimulated with IFN-␣ and IFN- (100 units/ml), respectively. 2.4. DNA and RNA transfections Huh7 cells in antibiotics-free DMEM with 10% FBS were seeded in 24-well plates and cultured at 37 ◦ C with 5% CO2 for 18–24 h. Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was used to transfect the 40 pmol of miR-122 mimic, AMO-122, and mock-miRNA into the cells, respectively. For fluorescence and protein assay, the cells were also co-transfected with 0.8 g pSOCS1-EGFP and harvested at 32 h or 48 h post-transfection according to the experiments. For mRNA assay, the cells were harvested at 24 h post-transfection. For ELISA assay, the culture supernatant was collected at 48 h posttransfection. 2.5. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) Total RNA was isolated from the cells using TRIzol reagents (Invitrogen) according to the manufacturer’s instructions, and then incubated with specific reverse primers (for miRNAs) or oligo-d(T) (for mRNAs). Quantitative PCR was performed using 2 l of the synthesized cDNA and SYBR PrimeScript Ex Taq I (TaKaRa, Otsu, Shiga, Japan) in a LightCycler 2.0 (Roche, Basel, Switzerland). Fold variations between RNA samples were calculated after normalizing to the U6 RNA or the GAPDH mRNA. The SOCS1 primer sequences were 5 -GGAACTGCTTTTTCGCCCTTA-3
Overlapping polymerase chain reaction (PCR) was used to mutate the two putative miR-122 targets in the SOCS1. The primers included primer pair A (A1: 5 -CCGCTCGAGATGGTAGCACACAACCAGGTGGCAGCCGACAAT-3 , A2: 5 -CACGGTACCGTAATCTGGAAGGGGAAGGAGCTCAGGTAGT-3 ; A1 underlined: Xho I restriction site; A2 underlined: Kpn I restriction site), primer pair B (B1: 5 -CGCGTGCTAGTGCGTTATTGGACGCCTGCGGATTCTACT-3 , B2: 5 -TCCAATAACGCACTAGCACGCGTGATGCGCCGGTAAT3 ), primer pair C (C1: 5 -GCGATACGCATTTCCGTACGTTTAGATCGCACGCCGATTACCGG-3 , C2: 5 -ATCTAAACGTACGGAAATGCGTATCGCCGGGGGCCGGGGCCGGGACCGCGG-3 ). The pSOCS1-EGFP DNA, served as the template, was amplified with the primers A1 and B2, A2 and B1, respectively. The PCR products were purified and mixed to amplify with primers A1 and A2. Use the same protocol with primer pairs A and C. The amplified DNA was purified and digested with Xho I and Kpn I, and ligated into pEGFP/Xho I + Kpn I. The resultant plasmids contained the mutations in the nt359–nt375 and nt309–nt331 of SOCS1 sequence, respectively. The mutations in these plasmids were verified by DNA sequencing. 2.8. Quantitation of SOCS1-EGFP expression SOCS1-EGFP expression in the pSOCS1-EGFP-transfected cells was analyzed at 16–48 h post-transfection using a fluorescence microscopy (Axiovert 200, Carl Zeiss, Gottingen, Germany) and a fluorescent spectrometer NanoDrop 3300 (Thermo, Rockford, IL). For fluorescence spectrometry, Hoechst 33342 (Invitrogen) was added to the culture medium at 37 ◦ C with 5% CO2 for 30 min to stain the nucleus 2 h before harvest. The proteins were extracted with the Pierce RIPA Buffer (Thermo, Rockford, IL) along with a protease inhibitor phenylmethylsulfonyl fluoride (PMSF) cocktail (Beyotime, Beijing, China), and 2 l of the protein extract was detected with NanoDrop 3300. The SOCS1-EGFP fluorescence intensity was normalized to the Hoechst 33342 fluorescence intensity to eliminate variation in the cell populations of each sample. 2.9. Western blotting The proteins were extracted by Pierce RIPA Buffer (Thermo) with PMSF cocktail, and separated by 12% SDS-polyacrylamide gel electrophoresis. A polyclonal SOCS1 antibody (sc-9021, Santa Cruz Biotechnology, Santa Cruz, CA), and the monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (TA08, Zhongshan Golden Bridge, Beijing, China) were used for immunoblotting detection. The blots were stained with a SuperSignal kit (Pierce, Rockford, IL) and photographed by a charge-coupled camera LAS4000 (Fujifilm, Tokyo, Japan). 2.10. Data analysis All values were presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed using SigmaStat 3.0 (Systat Software, Richmond, CA). The Student’s t test was used to
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Fig. 1. Response of IFN-␣ and IFN- expression to miR-122 over-expression or knockdown. Huh7 cells at 70% confluency were transfected with miR-122 mimic, AMO-122, and mock-miRNA, respectively. The total RNA was extracted at 24 h post-transfection. The culture supernatant was collected at 48 h post-transfection. (A) The miR-122 abundance. (B) The IFN-␣ mRNA expression. (C) The IFN- mRNA expression. (D) The IFN-␣ protein expression. (E) The IFN- protein expression. (F) Pearson correlation analysis for the miR-122 expression and IFN-␣ levels. r = 0.7572, P < 0.001. (G) Pearson correlation analysis for miR-122 expression and IFN- levels. r = 0.7446, P < 0.001. The mock-miRNA was used as a normal control for the miR-122 mimic and AMO-122. Values are presented as the mean ± SEM (n = 3). * refers to P < 0.05, ** refers to P < 0.01 and *** refers to P < 0.001.
evaluate the differences between two groups. A P value of <0.05 was considered statistically significant. All experiments were repeated at least three times. 3. Results 3.1. Type I IFN expression is consistent with miR-122 abundances in hepatocytes To evaluate whether miR-122 impacts the type I IFN expression in hepatocytes, we over-expressed miR-122 by transfection of miR-122 mimic or knocked down miR-122 expression by transfection of AMO-122 in Huh7 cells. The miR-122 levels in miR-122 mimic treated cells were 9.51-fold higher than that in mock-miRNA treated cells, while in AMO-122 treated cells, the miR-122 levels were significantly suppressed (P < 0.001) (Fig. 1A). qRT-PCR and ELISA showed that miR-122 over-expression resulted in a significant increase in the IFN-␣/ expression. Conversely, miR-122 knockdown resulted in a suppression of the IFN-␣/ expression (Fig. 1B–E). To further evaluate the relationship between miR-122 and type I IFN expression, Huh7 cells were transfected with various doses of miR-122 mimic or AMO-122 and the IFN-␣ and IFN- mRNA
expression was measured by qRT-PCR. Pearson’s correlation analysis showed a direct correlation between the IFN-␣ and IFN- levels and miR-122 over-expression (two-tailed Pearson’s test, r = 0.7572 for IFN-␣ and 0.7446 for IFN-, respectively; P < 0.0001) (Fig. 1F and G). To further confirm the involvement of miR-122 on the induction of IFN, we knocked down the miR-122 expression in Huh7 cells and then treated the cells with exogenous IFN-␣/. As shown in Fig. 2, IFN stimulation (100 units/ml) could not effectively induce the IFN expression, suggesting that the miR-122 knockdown could counteract the stimulation of IFN inducers to the cellular IFN expression. These results indicate that miR-122 may be an important intracellular regulator of IFN synthesis after stimulation by IFN treatment. Therefore, we suspected that certain negative regulator in the IFN signaling was activated or de-repressed by knocking down miR-122 and inhibited further the IFN expression in hepatocytes. 3.2. Prediction of miR-122 targets in the SOCS1 mRNA To verify our hypothesis, we used the RNAhybrid, a web-based RNA analysis tool, to screen potential miR-122 targets among the negative regulators of the IFN signaling pathway. Interestingly, we
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Fig. 2. The IFN mRNA expression in Huh7 cells treated with AMO-122 and recombinant IFN. Huh7 cells were transfected with AMO-122 for 24 h, and then stimulated with exogenous IFN-␣ or IFN- (100 units/ml) for 4 h. (A) The IFN-␣ mRNA expression. (B) The IFN- mRNA expression. The mock-miRNA was used as a normal control for the miR-122 mimic and AMO-122. Values are presented as the mean ± SEM (n = 3). * refers to P < 0.05, ** refers to P < 0.01 and *** refers to P < 0.001.
found that there were two putative miR-122 targets in the SOCS1 mRNA (Fig. 3). The target sequences were perfectly matched to the seed sequence of miR-122. Among them, one was located in the extended SH2 subdomain-coding region (nt359–nt375), and the mfe was −27.8 kcal/mol, while the other resided in the kinase inhibitory region-coding region (nt309–nt331), and the mfe was −24.3 kcal/mol (Fig. 3). 3.3. MiR-122 suppresses the SOCS1 protein expression To verify the effect of miR-122 on the SOCS1 expression, the Huh7 cells were transfected with miR-122 mimic, AMO-122, and mock-miRNA, respectively. qRT-PCR showed that there was no significant difference among the SOCS1 mRNA expression in the cells treated with miR-122 mimic, AMO-122, and mock-miRNA, respectively (Fig. 4A). However, Western blotting showed that the SOCS1 protein expression was significantly suppressed in the miR-122
mimic-treated cells (49.45% ± 8.44%) (P < 0.01) and up-regulated in the AMO-122-treated cells (162.56% ± 19.47%) (P < 0.05), compared to the mock-miRNA-treated cells (Fig. 4B). These findings demonstrate that the miR-122 mimic may inhibit, but AMO-122 may increase, the SOCS1 protein expression in hepatocytes posttransciptionally. To validate the inhibition of miR-122 on the SOCS1 expression in hepatocytes post-transciptionally, we introduced miR-122 mimic, AMO-122, and mock-miRNA separately into Huh7 cells. The pSOCS1-EGFP was also co-transfected with the above miRNAs. The SOCS1-EGFP fusion protein expression was detected by counting SOCS1-EGFP-positive cells using a fluorescence microscope and measuring SOCS1-EGFP fluorescence intensity using a NanoDrop 3300. The SOCS1-EGFP levels determined at 4 h-intervals from 16 h to 48 h post-transfection showed that the differentiated SOCS1-EGFP expression occurred at 28 h and 32 h among the various groups. As shown in Fig. 4, AMO-122 significantly increased the SOCS1-EGFP expression, whereas miR-122 mimic reduced the SOCS1-EGFP expression at 28 h and 32 h post-transfection. By counting SOCS1-EGFP-positive cells, the SOCS1-EGFP expression was 89% higher in the Huh7 cells co-transfected with AMO-122, and 41.12% lower in the Huh7 cells co-transfected with miR-122 mimic than those co-transfected with mock-miRNA (P < 0.05) (Fig. 4D). Moreover, the SOCS1-EGFP fluorescence intensities were consistent with that of SOCS1-EGFP-positive cell counts (Fig. 4E). Western blotting showed that the SOCS1-EGFP expression was also consistent with the SOCS1-EGFP fluorescence quantitation (Fig. 4F). These results confirm that miR-122 specifically suppresses the SOCS1 expression in a post-transcriptional manner. 3.4. MiR-122 targets the nt359–nt375 region of SOCS1 mRNA
Fig. 3. Prediction of miR-122 targets in the SOCS1 mRNA. (A) The structure of SOCS1 mRNA. The locations of the putative miR-122 targets are marked by arrows. (B) The complementary sequences between the miR-122 and SOCS1 mRNA. “nt” stands for nucleotide, the number counts from the first nucleotide at the 5 end of the SOCS1 mRNA (GenBank accession: NM 003745).
To verify the putative targets, two mutated pSOCS1-EGFP plasmids were generated by over-lapping PCR: (1) in the pSOCS1-EGFP 359 (mut-359), six nucleotides between nt359 and nt375 were substituted, including C361U, C364U, C367U, C371U, C373A, and C374U; (2) in the pSOCS1-EGFP 309 (mut-309), seven nucleotides between nt309 and nt331 were substituted, including C310U, C316U, C322U, A325G, C328U, C329A, and U331A (Fig. 5A and B). As these mutations occurred in the open reading frame, all mutated codons were synonymous with its prototype to avoid changing the translational products. Therefore, the mutations significantly reduced the match quality of the putative targets to miR-122 but did not change the amino acid sequence of the translational products. Using the same protocol above, miR-122 mimic, AMO-122, and mock-miRNA were separately co-transfected into Huh7 cells with
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Fig. 4. Identification of SOCS1 as a target of miR-122. For (A) and (B) The total RNAs and proteins of Huh7 cells transfected with miR-122 mimic, AMO-122, and mockmiRNA, respectively, were extracted at 24 h (mRNA) and 48 h (protein) post-transfection. (A) The SOCS1 mRNA level (n = 4). (B) The SOCS1 protein level (n = 6). For (C)–(F) MiR-122 mimic, AMO-122 or mock-miRNA were co-transfected into Huh7 cells with pEGFP-SOCS1, respectively. The proteins were then extracted after 28 h and 32 h post-transfection. Hoechst 33342 was added into the culture medium to stain the nuclei half hour before protein extraction. The SOCS1-EGFP expression in these cells was measured by fluorescence microscope and NanoDrop 3300. The SOCS1-EGFP protein was detected by Western blotting. (C) The SOCS1-EGFP expression observed by fluorescence microscope. (D) The SOCS1-EGFP-positive cells counted and normalized to the number of nuclei in each view. The relative counts of the SOCS1-EGFP-positive cells of miR-122 mimic-treated group or AMO-122-treated group were calculated by normalizing to the mock-miRNA-treated groups (n = 6). (E) The SOCS1-EGFP fluorescence intensity measured by NanoDrop 3300 (n = 5). The SOCS1-EGFP intensity was normalized to the Hoechst 33342 intensity in each sample. The relative EGFP intensities of the miR-122-treated or AMO-122-treated groups were calculated by normalizing to the mock-miRNA-treated groups. (F) The SOCS1-EGFP protein expression (molecular weight: 51 kDa) detected by Western blotting (n = 6). The mock-miRNA was used as a normal control for the miR-122 mimic and AMO-122. Values are presented as the mean ± SEM. * refers to P < 0.05, ** refers to P < 0.01 and *** refers to P < 0.001.
mut-359 or mut-309. The SOCS1-EGFP expression was measured at 32 h post-transfection with a fluorescence microscope and a NanoDrop 3300, and the SOCS1 protein expression was measured by Western blotting. Among the cells transfected with mut-309, the proportion of SOCS1-EGFP-positive cells was approximately 40.26% lower in the miR-122 mimic-treated cells and 35.39% higher in the AMO-122-treated cells than those in the mock-miRNAtreated cells (P < 0.05) (Fig. 5D). The SOCS1-EGFP fluorescence
intensity and SOCS1 protein expression results were consistent with that of SOCS1-EGFP-positive cell counts (Fig. 5C, E, and G). However, the SOCS1-EGFP-positive cell counts, the SOCS1-EGFP fluorescence intensity, and the SOCS1 protein expression in the cells co-transfected with miRNAs and mut-359 were identical to each other (P > 0.05) (Fig. 5C–F). These results indicate that the mutations in the nt309–nt331 region did not affect the interaction between the SOCS1-EGFP and miR-122, while the mutations
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Fig. 5. Verification of miR-122 targets in the SOCS1 mRNA. For (A) and (B) The mutated nucleotides in the putative targets of SOCS1 and the sequencing data of the mutated pSOCS1-EGFP. Two mutated pSOCS1-EGFP were generated by over-lapping PCR to minimize the match of miR-122 with SOCS1 mRNA in the region of nt359–nt375 or nt309–nt331, respectively. The red boxes are the mutated regions. (A) mut-359-SOCS1-EGFP. (B) mut-309-SOCS1-EGFP. For (C)–(G) MiR-122 mimic, AMO-122, and mockmiRNA were co-transfected into Huh7 cells with mut-359-SOCS1-EGFP and mut-309-SOCS1-EGFP plasmid, respectively. Hoechst 33342 was added into the culture medium to stain the nuclei half hour before the detection. (C) SOCS1-EGFP expression in the treated cells was observed by fluorescence microscope at 32 h post-transfection. (D) SOCS1-EGFP-positive cell counts and (E) SOCS1-EGFP fluorescence intensity were measured as described in Fig. 4. The SOCS1-EGFP protein expression detected by Western blotting after transfecting the mut-359-SOCS1-EGFP (F) and mut-309-SOCS1-EGFP plasmid (G), respectively. The mock-miRNA was used as a normal control for the miR-122 mimic and AMO-122. Values are presented as the mean ± SEM (n = 4). * refers to P < 0.05, ** refers to P < 0.01 and *** refers to P < 0.001.
in the nt359–nt375 region greatly reduced the effect of miR-122 on SOCS1-EGFP expression. These data confirm that miR-122 targeted the nt359–nt375 region of SOCS1 mRNA and regulated the SOCS1 protein expression. 4. Discussion As a liver-specific miRNA, miR-122 is extensively involved in the physiological and pathological processes of the hepatocytes
(Lewis and Jopling, 2010; Girard et al., 2008). MiR-122 can significantly enhance HCV replication in Huh7 cells (Jopling et al., 2005). The miR-122 abundance is significantly reduced in hepatoma tissues, and a rise in levels of the miR-122 is beneficial to improve the treatment outcome and prognosis of HCC (Coulouarn et al., 2009). A recent study revealed that there is an association between miR-122 levels and the response to IFN therapy, the HCV patients with low miR-122 levels responded poorly to IFN therapy (SarasinFilipowicz et al., 2009). We previously found that knocking down
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the miR-122 expression restricted the induction of type I IFN in human oligodendrocytes (Qian et al., 2010). These findings suggest that miR-122 plays an important role in hepatitis virus infection, hepatocarcinogenesis, and IFN treatment. However, the mechanisms of these effects remain unclear. In this study, we found a direct correlation between the miR122 abundance and the IFN-␣ or IFN- expression in hepatocytes. By screening the negative regulators of the IFN signaling pathway, we found that miR-122 could regulate the IFN expression through targeting the SOCS1 mRNA at the nt359–nt375 region. Our data demonstrate that miR-122 can regulate the innate immunity and impact the IFN therapy against hepatitis virus infections and hepatocarcinogenesis. Previous studies have showed that miR-122 can also target several other genes in hepatocytes (Chang et al., 2004; Gramantieri et al., 2007; Fan et al., 2011; Lin et al., 2008; Tsai et al., 2009; Bai et al., 2009). For example, there is miR-122-binding site in the cationic amino acid transporter mRNA (Chang et al., 2004). MiR-122 can specifically target the cyclin G1 mRNA and regulate for the cyclin G1 expression (Gramantieri et al., 2007). MiR-122 also targets N-myc downstream-regulated gene 3 (Fan et al., 2011), Bcl-w (Lin et al., 2008), a disintegrin and metalloprotease 17 (ADAM17) (Tsai et al., 2009), an ADAM10, serum response factor, and insulin-like growth factor 1 receptor (Bai et al., 2009). These findings suggest that miR122 can modulate various biological functions of the hepatocytes by targeting multiple genes. Based on the intricate relationship between miR-122 and IFN induction (Figs. 1 and 2), we reasoned that miR-122 might modulate the IFN expression by targeting on a negative regulator in the IFN signaling pathways, e.g., JAK/STAT signaling pathway. The binding of type I IFN to its receptor results in the activation of the associated JAKs, which in turn regulate the phosphorylation of STATs (Darnell et al., 1994). Though STAT1 and STAT2 are the most important mediators for type I IFN response, other STATs, including STAT3 and STAT5, can also be activated by type I IFN (Brierley and Fish, 2002; Aaronson and Horvath, 2002; Meinke et al., 1996). The activation of STAT4 and STAT6 by IFN seems to be restricted to certain cell types, such as endothelial cells or Daudi cells (Torpey et al., 2004; Fasler-Kan et al., 1998). The activated STATs induced by type I IFN form homodimers or heterodimers, including STAT1–STAT1, STAT3–STAT3, STAT4–STAT4, STAT5–STAT5 and STAT6–STAT6 homodimers, as well as STAT1–STAT2, STAT1–STAT3, STAT1–STAT4, STAT1–STAT5, STAT2–STAT3 and STAT5–STAT6 heterodimers (Aaronson and Horvath, 2002; Parmar and Platanias, 2003). Thus, the functional diversity of type I IFN regulated pathways mediates multiple biologic responses. It is widely acknowledged that there are three families of proteins which negatively regulate the JAK/STAT signaling pathway: the phosphotyrosine phosphatases, the SOCS and the protein inhibitor of activated STAT (Valentino and Pierre, 2006). Among them, the SOCS family is thought to be closely associated with IFN expression and the SOCS1 is the most effective suppressor in the IFN signaling (Fenner et al., 2006). By predicting with RNAhybrid 2.2, we found that there were two putative miR-122 targets in the SOCS1 mRNA (Fig. 3). Thus, we hypothesized that miR-122 might modulate the IFN expression in hepatocytes by targeting SOCS1 expression. To verify our hypothesis, we further ectopic expression and knockdown of miR-122, the results demonstrate that miR-122 effectively suppresses the SOCS1 expression (Fig. 4). To understand the interaction between miR-122 and SOCS1 mRNA, site-directed mutagenesis strategy was employed to identify the miR-122 target sequence in SOCS1 mRNA. These mutations, which were carefully selected to avoid changing the reading frame, minimized the complementary sequences to miR-122 at
nt359–nt375 and nt309–nt331 region of the SOCS1 mRNA. The mutations at nt309–nt331 did not affect the inhibitory effect of miR-122 mimic on SOCS1-EGFP expression, while the mutations at nt359–nt375 significantly reduced the effect of miR-122 mimic on SOCS1-EGFP expression (Fig. 5). Although the nt359–nt375 and nt309–nt331 sequences in SOCS1 mRNA both perfectly match with the miR-122 seed sequence, miR-122 forms a large stem-loop in the nt309–nt331 region and probably impedes the binding of miR-122 with the nt309–nt331 region of SOCS1 mRNA. Our results indicate that miR-122 specifically inhibited the SOCS1 expression through targeting the nt359–nt375 region of the SOCS1 mRNA. The type I IFN is recognized as key component of the innate immune response and plays a vital role for hepatocytes to resist viral infection (Liu et al., 2011). Accordingly, type I IFN is currently used as a therapeutic agent, with the most noteworthy example, to combat HCV infection (Liu et al., 2011). However, previous investigations demonstrated that the effectiveness of IFN therapy against HCV infection was less than 50% (Pfeffer et al., 2009), and the patients with higher miR-122 abundance had a better outcome (Sarasin-Filipowicz et al., 2009). The SOCS1 expression was significantly higher in the HCV-infected patients, particularly those who were not responsive to subsequent IFN therapy (Imanaka et al., 2005). These data suggest that there is a connection between the miR-122 abundance and the efficiency of the IFN therapy against HCV infection. Our findings offer a molecular interpretation of the unresponsive cases of IFN therapy against HCV infection. Though our data support that higher miR-122 abundance is beneficial to the IFN therapy against hepatitis viruses, it is paradoxical in certain situations. For example, miR-122 can target the 5 un-translated region of the HCV genome and up-regulate the HCV replication (Jopling et al., 2005). Low levels of miR-122 may be helpful to restrict the HCV replication in hepatocytes, but it may also limit the innate antiviral mechanism of the hepatocytes. Other studies also revealed that the miR-122 abundances were usually lower in the livers of HCC patients than that of healthy individuals (Coulouarn et al., 2009; Zhang et al., 2010). The loss of miR-122 resulted in an increase of cell migration and invasion and that restoration of miR-122 reversed this phenotype (Ma et al., 2010). Several molecular clues have identified to elucidate the role of miR-122 in HCC pathogenesis, e.g., miR-122 can specifically target the cyclin G1 mRNA and regulate for its expression (Gramantieri et al., 2007). MiR-122 can directly repress Bcl-w mRNA, an anti-apoptotic gene (Lin et al., 2008). MiR-122 can inhibit the intra-hepatic metastasis of HCC by modulating ADAM17, a key component in angiogenesis (Tsai et al., 2009). MiR-122 can also inhibit ADAM10, serum response factor and insulin-like growth factor 1 receptor that promote tumorigenesis (Bai et al., 2009). Inducing type I IFN expression is an essential component for the host restriction of tumor growth (Gresser and Belardelli, 2002). Increasing evidence suggests that IFN signaling could effectively promote cellular apoptosis and thus restrict tumor growth (Takaoka et al., 2003; Herzer et al., 2009). Our data suggest that the reduction of miR-122 in HCC leads to the de-repression of SOCS1 and inactivation of type I IFN expression and this obstructs the therapeutic effect of IFN on HCC. Thus, our findings provide another molecular clue to explain the role played by miR-122 in the pathology of hepatocytes by modulating various gene expressions, and a strategy that utilizes the induction of miR-122 in HCC might contribute to effectiveness of therapy. In conclusion, we demonstrate that SOCS1 is a target of miR122. MiR-122 can regulate the type I IFN expression through modulating the SOCS1 expression. These data suggest a novel function and a therapeutic application of miR-122 in viral infection and carcinogenesis.
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Competing interests The authors declare that they have no competing interests. Acknowledgments We are grateful to Prof. Yong-Chuan Wong, Department of Anatomy, Faculty of Medicine, the University of Hong Kong, Hong Kong SAR, China, and James Ogutu, Department of Microbiology, Harbin Medical University, China, for their critical reading the manuscript. This work was supported by grants from the National Natural Science Foundation of China (NSFC) (J1103609, J1210062 and 81202296), Natural Science Foundation of Heilongjiang Province (QC2012C058), Heilongjiang Postdoctoral Grant (LBH-Z11095) and the postdoctoral granted financial support from China Postdoctoral Science Foundation (20110491115). References Aaronson DS, Horvath CM. A road map for those who don’t know JAK-STAT. Science 2002;296:1653–5. Alves Jr L, Niemeier S, Hauenschild A, Rehmsmeier M, Merkle T. Comprehensive prediction of novel microRNA targets in Arabidopsis thaliana. Nucleic Acids Research 2009;37:4010–21. Bai S, Nasser MW, Wang B, Hsu SH, Datta J, Kutay H, et al. MicroRNA-122 inhibits tumorigenic properties of hepatocellular carcinoma cells and sensitizes these cells to sorafenib. Journal of Biological Chemistry 2009;284:32015–27. Brierley MM, Fish EN. Review IFN-alpha/beta receptor interactions to biologic outcomes: understanding the circuitry. Journal of Interferon and Cytokine Research 2002;22:835–45. Chang J, Nicolas E, Marks D, Sander C, Lerro A, Buendia MA, et al. miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1. RNA Biology 2004;1:106–13. Coulouarn C, Factor VM, Andersen JB, Durkin ME, Thorgeirsson SS. Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties. Oncogene 2009;28:3526–36. Darnell Jr JE, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994;264:1415–21. Fan CG, Wang CM, Tian C, Wang Y, Li L, Sun WS, et al. miR-122 inhibits viral replication and cell proliferation in hepatitis B virus-related hepatocellular carcinoma and targets NDRG3. Oncology Reports 2011;26:1281–6. Fasler-Kan E, Pansky A, Wiederkehr M, Battegay M, Heim MH. Interferon-alpha activates signal transducers and activators of transcription 5 and 6 in Daudi cells. European Journal of Biochemistry 1998;254:514–9. Fenner JE, Starr R, Cornish AL, Zhang JG, Metcalf D, Schreiber RD, et al. Suppressor of cytokine signaling 1 regulates the immune response to infection by a unique inhibition of type I interferon activity. Nature Immunology 2006;7:33–9. Girard, Jacquemin M, Munnich E, Lyonnet A, Henrion-Caude S. A. miR-122, a paradigm for the role of microRNAs in the liver. Journal of Hepatology 2008;48:648–56.
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Gramantieri L, Ferracin M, Fornari F, Veronese A, Sabbioni S, Liu CG, et al. Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma. Cancer Research 2007;67:6092–9. Gresser I, Belardelli F. Endogenous type I interferons as a defense against tumors. Cytokine and Growth Factor Reviews 2002;13:111–8. Herzer K, Hofmann TG, Teufel A, Schimanski CC, Moehler M, Kanzler S, et al. IFN-alpha-induced apoptosis in hepatocellular carcinoma involves promyelocytic leukemia protein and TRAIL independently of p53. Cancer Research 2009;69:855–62. Imanaka K, Tamura S, Fukui K, Ito N, Kiso S, Imai Y, et al. Enhanced expression of suppressor of cytokine signalling-1 in the liver of chronic hepatitis C: possible involvement in resistance to interferon therapy. Journal of Viral Hepatitis 2005;12:130–8. Jopling CL, Yi M, Lancaster AM, Lemon SM, Sarnow P. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science 2005;309: 1577–81. Lewis AP, Jopling CL. Regulation and biological function of the liver-specific miR-122. Biochemical Society Transactions 2010;38:1553–7. Lin CJ, Gong HY, Tseng HC, Wang WL, Wu JL. miR-122 targets an anti-apoptotic gene, Bcl-w, in human hepatocellular carcinoma cell lines. Biochemical and Biophysical Research Communications 2008;375:315–20. Liu JP, Wang X YL, Li JL, Ho WZ. Cyclosporin A inhibits hepatitis C virus replication and restores interferon-alpha expression in hepatocytes. Transplant Infectious Disease 2011;13:24–32. Ma L, Liu J, Shen J, Liu L, Wu J, Li W, et al. Expression of miR-122 mediated by adenoviral vector induces apoptosis and cell cycle arrest of cancer cells. Cancer Biology and Therapy 2010;9:554–61. Meinke A, Barahmand-Pour F, Wohrl S, Stoiber D, Decker T. Activation of different Stat5 isoforms contributes to cell-type-restricted signaling in response to interferons. Molecular and Cellular Biology 1996;16: 6937–44. Parmar S, Platanias LC. Interferons: mechanisms of action and clinical applications. Current Opinion in Oncology 2003;15:431–9. Pfeffer LM, Madey MA, Riely CA, Fleckenstein JF. The induction of type I interferon production in hepatitis C-infected patients. Journal of Interferon and Cytokine Research 2009;29:299–306. Qian J, Zhai A, Kao W, Li Y, Song W, Fu Y, et al. Modulation of miR-122 on persistently Borna disease virus infected human oligodendroglial cells. Antiviral Research 2010;87:249–56. Rajewsky N. microRNA target predictions in animals. Nature Genetics 2006;38(Suppl.):S8–13. Sarasin-Filipowicz M, Krol J, Markiewicz I, Heim MH, Filipowicz W. Decreased levels of microRNA miR-122 in individuals with hepatitis C responding poorly to interferon therapy. Nature Medicine 2009;15:31–3. Takaoka A, Hayakawa S, Yanai H, Stoiber D, Negishi H, Kikuchi H, et al. Integration of interferon-alpha/beta signalling to p53 responses in tumour suppression and antiviral defence. Nature 2003;424:516–23. Torpey N, Maher SE, Bothwell AL, Pober JS. Interferon alpha but not interleukin 12 activates STAT4 signaling in human vascular endothelial cells. Journal of Biological Chemistry 2004;279:26789–96. Tsai WC, Hsu PW, Lai TC, Chau GY, Lin CW, Chen CM, et al. MicroRNA-122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma. Hepatology 2009;49:1571–82. Valentino L, Pierre J. JAK/STAT signal transduction: regulators and implication in hematological malignancies. Biochemical Pharmacology 2006;71: 713–21. Zhang G, Wang Q, Xu R. Therapeutics based on microRNA: a new approach for liver cancer. Current Genomics 2010;11:311–25.
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The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
MiR-122 modulates type I interferon expression through blocking suppressor of cytokine signaling 1 Aimei Li a,b,1 , Wuqi Song a,b,c,1 , Jun Qian a,b,1 , Yujun Li a,b,c , Junming He a,b , Qingmeng Zhang a,b , Wenhui Li a,b , Aixia Zhai a,b , Wenping Kao a,b , Yunlong Hu a,b , Hui Li a,b , Jing Wu a,b , Hong Ling a,b , Zhaohua Zhong a,b,∗∗ , Fengmin Zhang a,b,c,∗ a
Department of Microbiology, Harbin Medical University, Harbin 150081, China Heilongjiang Key Laboratory of Immunity and Infection, Harbin 150081, China c Bio-pharmaceutical Key Laboratory in Harbin Medical University, Ministry of Education of China, Harbin 150081, China b
a r t i c l e
i n f o
Article history: Received 20 October 2012 Received in revised form 9 January 2013 Accepted 14 January 2013 Available online 22 January 2013 Keywords: MiRNA MiR-122 Suppressor of cytokine signaling 1 Type I interferon Hepatocytes
a b s t r a c t MiR-122 is a liver-specific miRNA. Recent studies demonstrated that the interferon (IFN) therapy efficacy is poor in the hepatitis C virus (HCV)-infected patients with lower miR-122 abundance in the livers. The hepatocarcinoma patients also have low miR-122 levels in their livers. We previously found that the IFN expression was reduced when miR-122 was knocked down in human oligodendrocytes. The mechanism is unclear. In this study, the miR-122-abundant cell Huh7 was used to explore the regulatory mechanism of miR-122 on type I IFN expression. We found that miR-122 significantly increased the type I IFN expression in Huh7 cells, while knocking down miR-122 decreased the type I IFN expression. By screening potential miR-122 targets among the negative regulators in IFN signaling pathways, we found that there were putative miR-122 targets in the suppressor of cytokine signaling 1 (SOCS1) mRNA. Over-expressing miR-122 decreased the SOCS1 expression by 50.55% in Huh7 cells, while knocking down miR-122 increased SOCS1 expression by 62.56%. Using a green fluorescence protein (EGFP) fused SOCS1expressing plasmid, the SOCS1-EGFP fluorescence intensity and protein were lower in miR-122 mimictreated cells than those in mock-miRNA-treated cells, while miR-122 knockdown significantly increased the SOCS1-EGFP fluorescence intensity and protein expression. Mutations in the nt359–nt375 region abandoned the impact of miR-122 on SOCS1-EGFP expression. Taken together, SOCS1 is a target of miR122. MiR-122 can regulate the type I IFN expression through modulating the SOCS1 expression. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Abbreviations: IFN, interferon; HCV, hepatitis C virus; SOCS1, suppressor of cytokine signaling 1; EGFP, enhanced green fluorescence protein; HCC, hepatocellular carcinoma; mfe, minimum free energy; DMEM, Dulbecco’s modified eagle medium; FBS, fetal bovine serum; AMO-122, anti-miR-122 oligonucleotide; PCR, polymerase chain reaction; PMSF, protease inhibitor phenylmethylsulfonyl fluoride; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; qRT-PCR, quantitative RT-PCR; SEM, standard error of the mean; ADAM, a disintegrin and metalloprotease. ∗ Corresponding author at: No. 194, Xuefu Road, Harbin 150081, China. Tel.: +86 451 8666 9576; fax: +86 451 8666 9576. ∗∗ Corresponding author at: Department of Microbiology, Harbin Medical University, Harbin 150081, China. Tel.: +86 451 8668 5122; fax: +86 451 8668 5122. E-mail addresses: [email protected] (A. Li), [email protected] (W. Song), [email protected] (J. Qian), Li [email protected] (Y. Li), [email protected] (J. He), [email protected] (Q. Zhang), [email protected] (W. Li), [email protected] (A. Zhai), hm [email protected] (W. Kao), [email protected] (Y. Hu), [email protected] (H. Li), wujing [email protected] (J. Wu), profl[email protected] (H. Ling), [email protected] (Z. Zhong), [email protected] (F. Zhang). 1 These authors contributed equally to this work. 1357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2013.01.008
MiR-122 is the most abundant miRNA in hepatocytes, constituting 70% of the total miRNA population and is extensively involved in the physiological and pathological processes of hepatocytes (Chang et al., 2004; Lewis and Jopling, 2010; Girard et al., 2008). MiR-122 can up-regulate hepatitis C virus (HCV) replication by targeting the 5 un-translational region of the HCV genome in Huh7 cells (Jopling et al., 2005). The miR-122 abundance in hepatocellular carcinoma (HCC) tissues is significantly lower than that in the healthy livers, and the decrease of the miR-122 abundance is closely correlated to the treatment outcome and prognosis of HCC (Coulouarn et al., 2009). A recent study revealed that the HCV patients with low miR122 levels responded poorly to IFN therapy (Sarasin-Filipowicz et al., 2009). These studies suggest that miR-122 plays an important role in HCV infection, hepatocarcinogenesis and IFN treatment. We previously found that the induction of type I IFN was significantly reduced when the miR-122 expression was suppressed in human oligodendrocytes (Qian et al., 2010). However, the mechanism is
A. Li et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 858–865
unclear. Therefore, in this study, miR-122-abundant cell Huh7 was used to explore the regulatory mechanism of miR-122 on type I IFN expression. We demonstrate that miR-122 negatively modulates SOCS1 expression and then induce the type I IFN expression. Our data suggest that miR-122 modulates host’s innate immunity through blocking SOCS1 and may play important role in the viral infection and carcinogenesis in the liver. 2. Materials and methods
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(sense); 5 -AGCAGCTCGAAGAGGCAGTC-3 (antisense). The primers of GAPDH, IFN-␣, IFN-, and miR-122 were described previously (Qian et al., 2010). 2.6. ELISA The production of IFN-␣ and IFN- in culture supernatant was measured using the VeriKine Human IFN-␣ and IFN- ELISA kits (PBL interferon source, Piscataway, NJ) according to the manufacturer’s instructions.
2.1. MiRNA target prediction 2.7. Site-directed mutagenesis The potential targets of miR-122 in the SOCS1 mRNA were predicted by RNAhybrid 2.2 (bibiserv.techfak.unibielefeld.de/rnahybrid) based on the complementary sequences and minimum free energy (mfe) (Alves et al., 2009; Rajewsky, 2006). 2.2. Cell culture Hepatoma cell lines Huh7 was cultured in Dulbecco’s modified eagle medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Israel) and 100 units/ml of penicillin and 100 g/ml of streptomycin in humidified incubator at 37 ◦ C with 5% CO2 . 2.3. Nucleotides, plasmids, and IFN inducers The sense and antisense miR-122 mimic (5 -TGGAGTGTGACAATGGTGTTTG-3 and 5 -CAAACACCATTGTCACACTCCA-3 ), the 2 -O-methylated, anti-miR-122 oligonucleotide (AMO-122) (5 CAAACACCAUUGUCACACUCCA-3 ), and the mock-miRNA were synthesized by GenePharma (Shanghai, China). The plasmid pSOCS1-EGFP, which expresses the SOCS1 and enhanced green fluorescence protein (EGFP) fusion protein, was obtained from GeneChem (Shanghai, China). Human recombinant IFN-␣ and IFN- were purchased from Pestka Biomedical Laboratories (Piscataway, NJ). To induce type I IFN expression, the cells cultured in antibiotics-free medium were stimulated with IFN-␣ and IFN- (100 units/ml), respectively. 2.4. DNA and RNA transfections Huh7 cells in antibiotics-free DMEM with 10% FBS were seeded in 24-well plates and cultured at 37 ◦ C with 5% CO2 for 18–24 h. Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was used to transfect the 40 pmol of miR-122 mimic, AMO-122, and mock-miRNA into the cells, respectively. For fluorescence and protein assay, the cells were also co-transfected with 0.8 g pSOCS1-EGFP and harvested at 32 h or 48 h post-transfection according to the experiments. For mRNA assay, the cells were harvested at 24 h post-transfection. For ELISA assay, the culture supernatant was collected at 48 h posttransfection. 2.5. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) Total RNA was isolated from the cells using TRIzol reagents (Invitrogen) according to the manufacturer’s instructions, and then incubated with specific reverse primers (for miRNAs) or oligo-d(T) (for mRNAs). Quantitative PCR was performed using 2 l of the synthesized cDNA and SYBR PrimeScript Ex Taq I (TaKaRa, Otsu, Shiga, Japan) in a LightCycler 2.0 (Roche, Basel, Switzerland). Fold variations between RNA samples were calculated after normalizing to the U6 RNA or the GAPDH mRNA. The SOCS1 primer sequences were 5 -GGAACTGCTTTTTCGCCCTTA-3
Overlapping polymerase chain reaction (PCR) was used to mutate the two putative miR-122 targets in the SOCS1. The primers included primer pair A (A1: 5 -CCGCTCGAGATGGTAGCACACAACCAGGTGGCAGCCGACAAT-3 , A2: 5 -CACGGTACCGTAATCTGGAAGGGGAAGGAGCTCAGGTAGT-3 ; A1 underlined: Xho I restriction site; A2 underlined: Kpn I restriction site), primer pair B (B1: 5 -CGCGTGCTAGTGCGTTATTGGACGCCTGCGGATTCTACT-3 , B2: 5 -TCCAATAACGCACTAGCACGCGTGATGCGCCGGTAAT3 ), primer pair C (C1: 5 -GCGATACGCATTTCCGTACGTTTAGATCGCACGCCGATTACCGG-3 , C2: 5 -ATCTAAACGTACGGAAATGCGTATCGCCGGGGGCCGGGGCCGGGACCGCGG-3 ). The pSOCS1-EGFP DNA, served as the template, was amplified with the primers A1 and B2, A2 and B1, respectively. The PCR products were purified and mixed to amplify with primers A1 and A2. Use the same protocol with primer pairs A and C. The amplified DNA was purified and digested with Xho I and Kpn I, and ligated into pEGFP/Xho I + Kpn I. The resultant plasmids contained the mutations in the nt359–nt375 and nt309–nt331 of SOCS1 sequence, respectively. The mutations in these plasmids were verified by DNA sequencing. 2.8. Quantitation of SOCS1-EGFP expression SOCS1-EGFP expression in the pSOCS1-EGFP-transfected cells was analyzed at 16–48 h post-transfection using a fluorescence microscopy (Axiovert 200, Carl Zeiss, Gottingen, Germany) and a fluorescent spectrometer NanoDrop 3300 (Thermo, Rockford, IL). For fluorescence spectrometry, Hoechst 33342 (Invitrogen) was added to the culture medium at 37 ◦ C with 5% CO2 for 30 min to stain the nucleus 2 h before harvest. The proteins were extracted with the Pierce RIPA Buffer (Thermo, Rockford, IL) along with a protease inhibitor phenylmethylsulfonyl fluoride (PMSF) cocktail (Beyotime, Beijing, China), and 2 l of the protein extract was detected with NanoDrop 3300. The SOCS1-EGFP fluorescence intensity was normalized to the Hoechst 33342 fluorescence intensity to eliminate variation in the cell populations of each sample. 2.9. Western blotting The proteins were extracted by Pierce RIPA Buffer (Thermo) with PMSF cocktail, and separated by 12% SDS-polyacrylamide gel electrophoresis. A polyclonal SOCS1 antibody (sc-9021, Santa Cruz Biotechnology, Santa Cruz, CA), and the monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (TA08, Zhongshan Golden Bridge, Beijing, China) were used for immunoblotting detection. The blots were stained with a SuperSignal kit (Pierce, Rockford, IL) and photographed by a charge-coupled camera LAS4000 (Fujifilm, Tokyo, Japan). 2.10. Data analysis All values were presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed using SigmaStat 3.0 (Systat Software, Richmond, CA). The Student’s t test was used to
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Fig. 1. Response of IFN-␣ and IFN- expression to miR-122 over-expression or knockdown. Huh7 cells at 70% confluency were transfected with miR-122 mimic, AMO-122, and mock-miRNA, respectively. The total RNA was extracted at 24 h post-transfection. The culture supernatant was collected at 48 h post-transfection. (A) The miR-122 abundance. (B) The IFN-␣ mRNA expression. (C) The IFN- mRNA expression. (D) The IFN-␣ protein expression. (E) The IFN- protein expression. (F) Pearson correlation analysis for the miR-122 expression and IFN-␣ levels. r = 0.7572, P < 0.001. (G) Pearson correlation analysis for miR-122 expression and IFN- levels. r = 0.7446, P < 0.001. The mock-miRNA was used as a normal control for the miR-122 mimic and AMO-122. Values are presented as the mean ± SEM (n = 3). * refers to P < 0.05, ** refers to P < 0.01 and *** refers to P < 0.001.
evaluate the differences between two groups. A P value of <0.05 was considered statistically significant. All experiments were repeated at least three times. 3. Results 3.1. Type I IFN expression is consistent with miR-122 abundances in hepatocytes To evaluate whether miR-122 impacts the type I IFN expression in hepatocytes, we over-expressed miR-122 by transfection of miR-122 mimic or knocked down miR-122 expression by transfection of AMO-122 in Huh7 cells. The miR-122 levels in miR-122 mimic treated cells were 9.51-fold higher than that in mock-miRNA treated cells, while in AMO-122 treated cells, the miR-122 levels were significantly suppressed (P < 0.001) (Fig. 1A). qRT-PCR and ELISA showed that miR-122 over-expression resulted in a significant increase in the IFN-␣/ expression. Conversely, miR-122 knockdown resulted in a suppression of the IFN-␣/ expression (Fig. 1B–E). To further evaluate the relationship between miR-122 and type I IFN expression, Huh7 cells were transfected with various doses of miR-122 mimic or AMO-122 and the IFN-␣ and IFN- mRNA
expression was measured by qRT-PCR. Pearson’s correlation analysis showed a direct correlation between the IFN-␣ and IFN- levels and miR-122 over-expression (two-tailed Pearson’s test, r = 0.7572 for IFN-␣ and 0.7446 for IFN-, respectively; P < 0.0001) (Fig. 1F and G). To further confirm the involvement of miR-122 on the induction of IFN, we knocked down the miR-122 expression in Huh7 cells and then treated the cells with exogenous IFN-␣/. As shown in Fig. 2, IFN stimulation (100 units/ml) could not effectively induce the IFN expression, suggesting that the miR-122 knockdown could counteract the stimulation of IFN inducers to the cellular IFN expression. These results indicate that miR-122 may be an important intracellular regulator of IFN synthesis after stimulation by IFN treatment. Therefore, we suspected that certain negative regulator in the IFN signaling was activated or de-repressed by knocking down miR-122 and inhibited further the IFN expression in hepatocytes. 3.2. Prediction of miR-122 targets in the SOCS1 mRNA To verify our hypothesis, we used the RNAhybrid, a web-based RNA analysis tool, to screen potential miR-122 targets among the negative regulators of the IFN signaling pathway. Interestingly, we
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Fig. 2. The IFN mRNA expression in Huh7 cells treated with AMO-122 and recombinant IFN. Huh7 cells were transfected with AMO-122 for 24 h, and then stimulated with exogenous IFN-␣ or IFN- (100 units/ml) for 4 h. (A) The IFN-␣ mRNA expression. (B) The IFN- mRNA expression. The mock-miRNA was used as a normal control for the miR-122 mimic and AMO-122. Values are presented as the mean ± SEM (n = 3). * refers to P < 0.05, ** refers to P < 0.01 and *** refers to P < 0.001.
found that there were two putative miR-122 targets in the SOCS1 mRNA (Fig. 3). The target sequences were perfectly matched to the seed sequence of miR-122. Among them, one was located in the extended SH2 subdomain-coding region (nt359–nt375), and the mfe was −27.8 kcal/mol, while the other resided in the kinase inhibitory region-coding region (nt309–nt331), and the mfe was −24.3 kcal/mol (Fig. 3). 3.3. MiR-122 suppresses the SOCS1 protein expression To verify the effect of miR-122 on the SOCS1 expression, the Huh7 cells were transfected with miR-122 mimic, AMO-122, and mock-miRNA, respectively. qRT-PCR showed that there was no significant difference among the SOCS1 mRNA expression in the cells treated with miR-122 mimic, AMO-122, and mock-miRNA, respectively (Fig. 4A). However, Western blotting showed that the SOCS1 protein expression was significantly suppressed in the miR-122
mimic-treated cells (49.45% ± 8.44%) (P < 0.01) and up-regulated in the AMO-122-treated cells (162.56% ± 19.47%) (P < 0.05), compared to the mock-miRNA-treated cells (Fig. 4B). These findings demonstrate that the miR-122 mimic may inhibit, but AMO-122 may increase, the SOCS1 protein expression in hepatocytes posttransciptionally. To validate the inhibition of miR-122 on the SOCS1 expression in hepatocytes post-transciptionally, we introduced miR-122 mimic, AMO-122, and mock-miRNA separately into Huh7 cells. The pSOCS1-EGFP was also co-transfected with the above miRNAs. The SOCS1-EGFP fusion protein expression was detected by counting SOCS1-EGFP-positive cells using a fluorescence microscope and measuring SOCS1-EGFP fluorescence intensity using a NanoDrop 3300. The SOCS1-EGFP levels determined at 4 h-intervals from 16 h to 48 h post-transfection showed that the differentiated SOCS1-EGFP expression occurred at 28 h and 32 h among the various groups. As shown in Fig. 4, AMO-122 significantly increased the SOCS1-EGFP expression, whereas miR-122 mimic reduced the SOCS1-EGFP expression at 28 h and 32 h post-transfection. By counting SOCS1-EGFP-positive cells, the SOCS1-EGFP expression was 89% higher in the Huh7 cells co-transfected with AMO-122, and 41.12% lower in the Huh7 cells co-transfected with miR-122 mimic than those co-transfected with mock-miRNA (P < 0.05) (Fig. 4D). Moreover, the SOCS1-EGFP fluorescence intensities were consistent with that of SOCS1-EGFP-positive cell counts (Fig. 4E). Western blotting showed that the SOCS1-EGFP expression was also consistent with the SOCS1-EGFP fluorescence quantitation (Fig. 4F). These results confirm that miR-122 specifically suppresses the SOCS1 expression in a post-transcriptional manner. 3.4. MiR-122 targets the nt359–nt375 region of SOCS1 mRNA
Fig. 3. Prediction of miR-122 targets in the SOCS1 mRNA. (A) The structure of SOCS1 mRNA. The locations of the putative miR-122 targets are marked by arrows. (B) The complementary sequences between the miR-122 and SOCS1 mRNA. “nt” stands for nucleotide, the number counts from the first nucleotide at the 5 end of the SOCS1 mRNA (GenBank accession: NM 003745).
To verify the putative targets, two mutated pSOCS1-EGFP plasmids were generated by over-lapping PCR: (1) in the pSOCS1-EGFP 359 (mut-359), six nucleotides between nt359 and nt375 were substituted, including C361U, C364U, C367U, C371U, C373A, and C374U; (2) in the pSOCS1-EGFP 309 (mut-309), seven nucleotides between nt309 and nt331 were substituted, including C310U, C316U, C322U, A325G, C328U, C329A, and U331A (Fig. 5A and B). As these mutations occurred in the open reading frame, all mutated codons were synonymous with its prototype to avoid changing the translational products. Therefore, the mutations significantly reduced the match quality of the putative targets to miR-122 but did not change the amino acid sequence of the translational products. Using the same protocol above, miR-122 mimic, AMO-122, and mock-miRNA were separately co-transfected into Huh7 cells with
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Fig. 4. Identification of SOCS1 as a target of miR-122. For (A) and (B) The total RNAs and proteins of Huh7 cells transfected with miR-122 mimic, AMO-122, and mockmiRNA, respectively, were extracted at 24 h (mRNA) and 48 h (protein) post-transfection. (A) The SOCS1 mRNA level (n = 4). (B) The SOCS1 protein level (n = 6). For (C)–(F) MiR-122 mimic, AMO-122 or mock-miRNA were co-transfected into Huh7 cells with pEGFP-SOCS1, respectively. The proteins were then extracted after 28 h and 32 h post-transfection. Hoechst 33342 was added into the culture medium to stain the nuclei half hour before protein extraction. The SOCS1-EGFP expression in these cells was measured by fluorescence microscope and NanoDrop 3300. The SOCS1-EGFP protein was detected by Western blotting. (C) The SOCS1-EGFP expression observed by fluorescence microscope. (D) The SOCS1-EGFP-positive cells counted and normalized to the number of nuclei in each view. The relative counts of the SOCS1-EGFP-positive cells of miR-122 mimic-treated group or AMO-122-treated group were calculated by normalizing to the mock-miRNA-treated groups (n = 6). (E) The SOCS1-EGFP fluorescence intensity measured by NanoDrop 3300 (n = 5). The SOCS1-EGFP intensity was normalized to the Hoechst 33342 intensity in each sample. The relative EGFP intensities of the miR-122-treated or AMO-122-treated groups were calculated by normalizing to the mock-miRNA-treated groups. (F) The SOCS1-EGFP protein expression (molecular weight: 51 kDa) detected by Western blotting (n = 6). The mock-miRNA was used as a normal control for the miR-122 mimic and AMO-122. Values are presented as the mean ± SEM. * refers to P < 0.05, ** refers to P < 0.01 and *** refers to P < 0.001.
mut-359 or mut-309. The SOCS1-EGFP expression was measured at 32 h post-transfection with a fluorescence microscope and a NanoDrop 3300, and the SOCS1 protein expression was measured by Western blotting. Among the cells transfected with mut-309, the proportion of SOCS1-EGFP-positive cells was approximately 40.26% lower in the miR-122 mimic-treated cells and 35.39% higher in the AMO-122-treated cells than those in the mock-miRNAtreated cells (P < 0.05) (Fig. 5D). The SOCS1-EGFP fluorescence
intensity and SOCS1 protein expression results were consistent with that of SOCS1-EGFP-positive cell counts (Fig. 5C, E, and G). However, the SOCS1-EGFP-positive cell counts, the SOCS1-EGFP fluorescence intensity, and the SOCS1 protein expression in the cells co-transfected with miRNAs and mut-359 were identical to each other (P > 0.05) (Fig. 5C–F). These results indicate that the mutations in the nt309–nt331 region did not affect the interaction between the SOCS1-EGFP and miR-122, while the mutations
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Fig. 5. Verification of miR-122 targets in the SOCS1 mRNA. For (A) and (B) The mutated nucleotides in the putative targets of SOCS1 and the sequencing data of the mutated pSOCS1-EGFP. Two mutated pSOCS1-EGFP were generated by over-lapping PCR to minimize the match of miR-122 with SOCS1 mRNA in the region of nt359–nt375 or nt309–nt331, respectively. The red boxes are the mutated regions. (A) mut-359-SOCS1-EGFP. (B) mut-309-SOCS1-EGFP. For (C)–(G) MiR-122 mimic, AMO-122, and mockmiRNA were co-transfected into Huh7 cells with mut-359-SOCS1-EGFP and mut-309-SOCS1-EGFP plasmid, respectively. Hoechst 33342 was added into the culture medium to stain the nuclei half hour before the detection. (C) SOCS1-EGFP expression in the treated cells was observed by fluorescence microscope at 32 h post-transfection. (D) SOCS1-EGFP-positive cell counts and (E) SOCS1-EGFP fluorescence intensity were measured as described in Fig. 4. The SOCS1-EGFP protein expression detected by Western blotting after transfecting the mut-359-SOCS1-EGFP (F) and mut-309-SOCS1-EGFP plasmid (G), respectively. The mock-miRNA was used as a normal control for the miR-122 mimic and AMO-122. Values are presented as the mean ± SEM (n = 4). * refers to P < 0.05, ** refers to P < 0.01 and *** refers to P < 0.001.
in the nt359–nt375 region greatly reduced the effect of miR-122 on SOCS1-EGFP expression. These data confirm that miR-122 targeted the nt359–nt375 region of SOCS1 mRNA and regulated the SOCS1 protein expression. 4. Discussion As a liver-specific miRNA, miR-122 is extensively involved in the physiological and pathological processes of the hepatocytes
(Lewis and Jopling, 2010; Girard et al., 2008). MiR-122 can significantly enhance HCV replication in Huh7 cells (Jopling et al., 2005). The miR-122 abundance is significantly reduced in hepatoma tissues, and a rise in levels of the miR-122 is beneficial to improve the treatment outcome and prognosis of HCC (Coulouarn et al., 2009). A recent study revealed that there is an association between miR-122 levels and the response to IFN therapy, the HCV patients with low miR-122 levels responded poorly to IFN therapy (SarasinFilipowicz et al., 2009). We previously found that knocking down
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the miR-122 expression restricted the induction of type I IFN in human oligodendrocytes (Qian et al., 2010). These findings suggest that miR-122 plays an important role in hepatitis virus infection, hepatocarcinogenesis, and IFN treatment. However, the mechanisms of these effects remain unclear. In this study, we found a direct correlation between the miR122 abundance and the IFN-␣ or IFN- expression in hepatocytes. By screening the negative regulators of the IFN signaling pathway, we found that miR-122 could regulate the IFN expression through targeting the SOCS1 mRNA at the nt359–nt375 region. Our data demonstrate that miR-122 can regulate the innate immunity and impact the IFN therapy against hepatitis virus infections and hepatocarcinogenesis. Previous studies have showed that miR-122 can also target several other genes in hepatocytes (Chang et al., 2004; Gramantieri et al., 2007; Fan et al., 2011; Lin et al., 2008; Tsai et al., 2009; Bai et al., 2009). For example, there is miR-122-binding site in the cationic amino acid transporter mRNA (Chang et al., 2004). MiR-122 can specifically target the cyclin G1 mRNA and regulate for the cyclin G1 expression (Gramantieri et al., 2007). MiR-122 also targets N-myc downstream-regulated gene 3 (Fan et al., 2011), Bcl-w (Lin et al., 2008), a disintegrin and metalloprotease 17 (ADAM17) (Tsai et al., 2009), an ADAM10, serum response factor, and insulin-like growth factor 1 receptor (Bai et al., 2009). These findings suggest that miR122 can modulate various biological functions of the hepatocytes by targeting multiple genes. Based on the intricate relationship between miR-122 and IFN induction (Figs. 1 and 2), we reasoned that miR-122 might modulate the IFN expression by targeting on a negative regulator in the IFN signaling pathways, e.g., JAK/STAT signaling pathway. The binding of type I IFN to its receptor results in the activation of the associated JAKs, which in turn regulate the phosphorylation of STATs (Darnell et al., 1994). Though STAT1 and STAT2 are the most important mediators for type I IFN response, other STATs, including STAT3 and STAT5, can also be activated by type I IFN (Brierley and Fish, 2002; Aaronson and Horvath, 2002; Meinke et al., 1996). The activation of STAT4 and STAT6 by IFN seems to be restricted to certain cell types, such as endothelial cells or Daudi cells (Torpey et al., 2004; Fasler-Kan et al., 1998). The activated STATs induced by type I IFN form homodimers or heterodimers, including STAT1–STAT1, STAT3–STAT3, STAT4–STAT4, STAT5–STAT5 and STAT6–STAT6 homodimers, as well as STAT1–STAT2, STAT1–STAT3, STAT1–STAT4, STAT1–STAT5, STAT2–STAT3 and STAT5–STAT6 heterodimers (Aaronson and Horvath, 2002; Parmar and Platanias, 2003). Thus, the functional diversity of type I IFN regulated pathways mediates multiple biologic responses. It is widely acknowledged that there are three families of proteins which negatively regulate the JAK/STAT signaling pathway: the phosphotyrosine phosphatases, the SOCS and the protein inhibitor of activated STAT (Valentino and Pierre, 2006). Among them, the SOCS family is thought to be closely associated with IFN expression and the SOCS1 is the most effective suppressor in the IFN signaling (Fenner et al., 2006). By predicting with RNAhybrid 2.2, we found that there were two putative miR-122 targets in the SOCS1 mRNA (Fig. 3). Thus, we hypothesized that miR-122 might modulate the IFN expression in hepatocytes by targeting SOCS1 expression. To verify our hypothesis, we further ectopic expression and knockdown of miR-122, the results demonstrate that miR-122 effectively suppresses the SOCS1 expression (Fig. 4). To understand the interaction between miR-122 and SOCS1 mRNA, site-directed mutagenesis strategy was employed to identify the miR-122 target sequence in SOCS1 mRNA. These mutations, which were carefully selected to avoid changing the reading frame, minimized the complementary sequences to miR-122 at
nt359–nt375 and nt309–nt331 region of the SOCS1 mRNA. The mutations at nt309–nt331 did not affect the inhibitory effect of miR-122 mimic on SOCS1-EGFP expression, while the mutations at nt359–nt375 significantly reduced the effect of miR-122 mimic on SOCS1-EGFP expression (Fig. 5). Although the nt359–nt375 and nt309–nt331 sequences in SOCS1 mRNA both perfectly match with the miR-122 seed sequence, miR-122 forms a large stem-loop in the nt309–nt331 region and probably impedes the binding of miR-122 with the nt309–nt331 region of SOCS1 mRNA. Our results indicate that miR-122 specifically inhibited the SOCS1 expression through targeting the nt359–nt375 region of the SOCS1 mRNA. The type I IFN is recognized as key component of the innate immune response and plays a vital role for hepatocytes to resist viral infection (Liu et al., 2011). Accordingly, type I IFN is currently used as a therapeutic agent, with the most noteworthy example, to combat HCV infection (Liu et al., 2011). However, previous investigations demonstrated that the effectiveness of IFN therapy against HCV infection was less than 50% (Pfeffer et al., 2009), and the patients with higher miR-122 abundance had a better outcome (Sarasin-Filipowicz et al., 2009). The SOCS1 expression was significantly higher in the HCV-infected patients, particularly those who were not responsive to subsequent IFN therapy (Imanaka et al., 2005). These data suggest that there is a connection between the miR-122 abundance and the efficiency of the IFN therapy against HCV infection. Our findings offer a molecular interpretation of the unresponsive cases of IFN therapy against HCV infection. Though our data support that higher miR-122 abundance is beneficial to the IFN therapy against hepatitis viruses, it is paradoxical in certain situations. For example, miR-122 can target the 5 un-translated region of the HCV genome and up-regulate the HCV replication (Jopling et al., 2005). Low levels of miR-122 may be helpful to restrict the HCV replication in hepatocytes, but it may also limit the innate antiviral mechanism of the hepatocytes. Other studies also revealed that the miR-122 abundances were usually lower in the livers of HCC patients than that of healthy individuals (Coulouarn et al., 2009; Zhang et al., 2010). The loss of miR-122 resulted in an increase of cell migration and invasion and that restoration of miR-122 reversed this phenotype (Ma et al., 2010). Several molecular clues have identified to elucidate the role of miR-122 in HCC pathogenesis, e.g., miR-122 can specifically target the cyclin G1 mRNA and regulate for its expression (Gramantieri et al., 2007). MiR-122 can directly repress Bcl-w mRNA, an anti-apoptotic gene (Lin et al., 2008). MiR-122 can inhibit the intra-hepatic metastasis of HCC by modulating ADAM17, a key component in angiogenesis (Tsai et al., 2009). MiR-122 can also inhibit ADAM10, serum response factor and insulin-like growth factor 1 receptor that promote tumorigenesis (Bai et al., 2009). Inducing type I IFN expression is an essential component for the host restriction of tumor growth (Gresser and Belardelli, 2002). Increasing evidence suggests that IFN signaling could effectively promote cellular apoptosis and thus restrict tumor growth (Takaoka et al., 2003; Herzer et al., 2009). Our data suggest that the reduction of miR-122 in HCC leads to the de-repression of SOCS1 and inactivation of type I IFN expression and this obstructs the therapeutic effect of IFN on HCC. Thus, our findings provide another molecular clue to explain the role played by miR-122 in the pathology of hepatocytes by modulating various gene expressions, and a strategy that utilizes the induction of miR-122 in HCC might contribute to effectiveness of therapy. In conclusion, we demonstrate that SOCS1 is a target of miR122. MiR-122 can regulate the type I IFN expression through modulating the SOCS1 expression. These data suggest a novel function and a therapeutic application of miR-122 in viral infection and carcinogenesis.
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