MicroRNA-22 can reduce parathymosin expression in transdifferentiated hepatocytes

Abstract

Pancreatic acinar cells AR42J-B13 can transdifferentiate into hepatocyte-like cells permissive for efficient hepatitis B virus (HBV) replication. Here, we profiled miRNAs differentially expressed in AR42J-B13 cells before and after transdifferentiation to hepatocytes, using chip-based microarray. Significant increase of miRNA expression, including miR-21, miR-22, and miR-122a, was confirmed by stem-loop real-time PCR and Northern blot analyses. In contrast, miR-93, miR-130b, and a number of other miRNAs, were significantly reduced after transdifferentiation. To investigate the potential significance of miR-22 in hepatocytes, we generated cell lines stably expressing miR-22. By 2D-DIGE, LC-MS/MS, and Western blot analyses, we identified several potential target genes of miR-22, including parathymosin. In transdifferentiated hepatocytes, miR-22 can inhibit both mRNA and protein expression of parathymosin, probably through a direct and an indirect mechanism. We tested two computer predicted miR-22 target sites at the 3' UTR of parathymosin, by the 3' UTR reporter gene assay. Treatment with anti-miR-22 resulted in significant elevation of the reporter activity. In addition, we observed an in vivo inverse correlation between miR-22 and parathymosin mRNA in their tissue distribution in a rat model. The phenomenon that miR-22 can reduce parathymosin protein was also observed in human hepatoma cell lines Huh7 and HepG2. So far, we detected no major effect on several transdifferentiation markers when AR42J-B13 cells were transfected with miR-22, or anti-miR-22, or a parathymosin expression vector, with or without dexamethasone treatment. Therefore, miR-22 appears to be neither necessary nor sufficient for transdifferentiation. We discussed the possibility that altered expression of some other microRNAs could induce cell cycle arrest leading to transdifferentiation.

Figure 1. Northern blot and stem-loop real-time PCR analysis of differentially expressed miRNAs before and after transdifferentiation of AR42J-B13 cells.

The differentially expressed miRNAs identified by miRNA microarray were confirmed by miRNA Northern blot (A) and stem-loop real-time PCR analysis (B and C). Total RNA extracted from Dex/OSM treated AR42J-B13 cells (7 Days) and mock controls were used for Northern blot analysis using antisense probes against down-regulated miRNAs (miR-93, miR-106b and miR-130b) and up-regulated miRNAs (miR-21, miR-22, miR-122a and miR-182). The relative fold changes of stem–loop real-time PCR were normalized to AR42J-B13 cells before transdifferentiation and U6 was used as a loading control. (*** P<0.001).

Figure 2. The miRNA expression profile of transdifferentiated AR42J-B13 cells is most closely related to those of hepatoma cell lines by clustering analysis.

The expression levels of miRNAs of various hepatoma cell lines (HepG2, Huh7 and Q7 cells), primary tissues (rat spleen, pancreas and liver), and NIH3T3 cells, were measured by stem-loop real-time PCR analysis. Clustering analysis using Genespring V11.0 software was performed by the normalization of the expression level from each sample relative to that of AR42J-B13 cells. Both up-regulated miRNAs (miR-21, miR-22, miR-122a and miR-182) and down-regulated miRNAs (miR-17-5p, miR-18a, miR-93, miR-106a, miR-106b, miR-130b and miR-375) were chosen as a parameter for comparison.

Figure 3. Ectopic overexpression of miR-22 in AR42J-B13 cells was analyzed by Northern blot (A) and stem-loop real-time PCR analysis (B).

(A) pIRES is the empty vector control. pIRES-miR-22 is the miR-22 expression vector. (B) B13-pIRES is the AR42J-B13 cells stably transfected with the pIRES vector control, while B13-pIRES-miR-22 is the AR42J-B13 cells stably transfected with the miR-22 expression vector. MicroRNA-93 was used as a negative control. (***, p<0.001).

Figure 4. 2D-DIGE analysis identified differentially expressed proteins in miR-22 overexpressing AR42J-B13 cells.

Protein lysates of pooled B13-pIRES or B13-pIRES-miR-22 cells were labeled with Cy3 and Cy5, respectively (Experimental Procedures). Three differentially expressed features, Features A, B and C, were picked up for identification by LC-MS/MS. Proteins with altered expression were indicated by arrows. The candidate proteins identified were listed in the table below the image. Parathymosin protein was down-regulated as assayed by 2D-DIGE (A) and Western blot analysis (C) in AR42J-B13 cells stably transfected with pIRES-miR-22. The miR-22 expression level in transiently transfected AR42J-B13 cells was measured by stem-loop real-time PCR (B). (***, p<0.001).

Figure 5. Reduction of parathymosin mRNA and protein levels were observed in AR42J-B13 cells treated with Dex/OSM or a miR-22 expression vector.

(A) Reduced expression of parathymosin protein in AR42J-B13 cells was induced by Dex/OSM treatment (B13+DM) or by stable expression of miR-22 using Western blot analysis. (B & C) The expression of parathymosin mRNA was reduced to approximately 70% level in Dex/OSM induced AR42J-B13 cells (B), and in the stably transfected AR42J-B13 cells overexpressing miR-22 (C). The mRNA level was measured by real-time PCR analysis (***, p<0.001).

Figure 6. The suppression effect of miR-22 on the expression of parathymosin depends on the predicted miR-22 target sites at the 3′UTR of parathymosin.

(A) RNA Hybrid software predicted two miR-22 binding sites, designated as “1” and “2” in the cartoon, at the 3′UTR of parathymosin with free energy less than −20 kcal/mol. The reporter plasmid was engineered by fusing DsRED with the rat parathymosin 3′UTR. (B) Site-directed mutagenesis at the predicted miR-22 binding sites of the parathymosin 3′UTR were illustrated. Two nucleotide substitutions were introduced into each of the two predicted binding sites. The arrows indicate the mutation sites at the 3′UTR of parathymosin. The sequences at the 3′UTR of parathymosin were changed from 5′-CU-3′ to 5′-GA-3′ in mu1, and from 5′-GG-3′ to 5′-CC-3′ in mu2. (C) The reporter assay was performed by transient cotransfection of AR42J-B13 cells with the plasmid of a DsRED reporter containing various wild type and mutant parathymosin 3′UTR, and plasmid pIRES-miR-22. The mean fluorescence intensity (MFI) of DsRED was measured by FACS analysis gated on DsRED positive cells. The relative MFI was calculated by normalization to the pIRES control. Mutations at both predicted sites have an additive effect on miR-22 mediated silencing (***, p<0.001; **, p<0.005; ns, not significant). (D) The reduction of the DsRED reporter mRNA, containing wild type parathymosin 3′UTR, was abolished by mutations at the predicted binding sites of miR-22. Real-time PCR analysis on DsRED mRNA was performed by using the same reporter assay as described above in Fig. 6C. (*, p<0.5).

Figure 7. Compensatory mutations of miR-22 restored the repression effect of miR-22 on the 3′UTR of a parathymosin reporter containing a mu2 target site.

(A) Compensatory mutation from 5′-CC-3′ to 5′-GG.-3′ at the predicted seed sequences of miR-22. (B) Reporter gene assay was performed by co-transfection into AR42J-B13 cells with the various DsRED reporter plasmids, containing various wild type and mutant parathymosin 3′UTR, and pIRES-miR-22 containing the compensatory mutation. FACS analysis was performed as in Fig. 6. (***, p<0.001; ns, not significant).

Figure 8. LNA-based antagomiR-22 increased parathymosin 3′UTR reporter activity in a hepatoma cell line Q7 by knockdown endogenous miR-22.

(A) The reporter assay was conducted by co-transfection of DsRED-parathymosin 3′UTR with LNA-antagomiR-22 in Q7 cells using Lipofectamine 2000. Reporter activity was significantly increased when 100 pmol of LNA-anti-miR-22 was used. (B) Different concentrations of LNA-antagomiR-22 were used to knockdown the endogenous miR-22 expression level, which was measured by stem-loop real-time PCR. (***, p<0.001) (C) Parathymosin mRNA was increased significantly by stem-loop real-time PCR, when transdifferentiated AR42J-B13 cells were treated with LNA-anti-miR-22. (*, p<0.5).

Figure 9. An inverse correlation between the expression levels of parathymosin mRNA and miR-22 was observed in most rat primary tissues.

Real-time PCR quantified parathymosin mRNA expression profile in various rat primary tissues, including cerebellum, cerebrum, eye, fat, muscle, pancreas, kidney, heart, lung, spleen, thymus and liver. AR42J-B13 cells with or without Dex/OSM induced transdifferentiation were included as references. The relative mRNA level of parathymosin was normalized to that of untreated AR42J-B13 cells (A). The expression of miR-22 in these tissues and cells were measured by stem-loop real-time PCR (B). The binding sites of transcription factors on the miR-22 promoter are predicted by the Transfac software (27) and presented in the schematic illustration (C).

Figure 10. The potential effect of parathymosin or miR22 on the transdifferentiation of AR42J-B13 cells.

(A) Similar levels of HBV e antigen (HBeAg) were detected in the media of B13-1 cells transfected with LNA anti-miR22 vs. a LNA scramble control. (B) Increased secretion of HBV surface antigen (HBsAg) was detected by ELISA in the medium of B13-1 cells transfected with LNA anti-miR22. (C) The reduction of the endogenous level of miR-22 in B13-1 cells after LNA anti-miR-22 treatment, was measured by real time PCR. (D) Treatment of B13-1 cells with LNA anti-miR22 resulted in no significant effect on transdifferentiation markers of alpha1-antitrypsin and albumin by Western blot analysis. (E & F) No significant effect on secreted HBsAg and HBeAg was detected by ELISA in B13-1 cells transfected with a parathymosin (PTMS) expression vector. (G) A time course of the gradual decrease of parathymosin protein in B13-1 cells was conducted by Western blot analysis. B13-1 cells were transfected with a vector control (upper panel) or a parathymosin expression vector (lower panel) prior to Dex/OSM induction. (H) Transdifferentiation markers of albumin and alpha1-antitrypsin were measured by Western blot analysis, using B13-1 cells transfected with a parathymosin expression vector or a control vector pCDNA prior to Dex/OSM induction.

Figure 11. Parathymosin can be targeted by miR-22 in human hepatoma cell lines Huh7 and HepG2.

Increased expression of parathymosin protein can be detected by Western blot when Huh7 and HepG2 cells were treated with LNA anti-miR-22 (Experimental Procedures).