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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Hepatology. Author manuscript; available in PMC Aug 1, 2012.
Published in final edited form as:
Published online Jun 30, 2011. doi:  10.1002/hep.24421
PMCID: PMC3145019
NIHMSID: NIHMS295892

Genome-wide MicroRNA Downregulation as a Negative Feedback Mechanism in the Early Phases of Liver Regeneration

Abstract

Liver is one of the few organs that have the capacity to regenerate in response to injury. We carried out genome-wide miRNA microarray studies during liver regeneration in rats after 70% PH at early and mid-time points to more thoroughly understand their role. At 3, 12 and 18 hrs post-PH ~ 40% of the miRNAs tested were up-regulated. Conversely, at 24 hrs post-PH, ~ 70% of miRNAs were down-regulated. Further, we established that the genome-wide down-regulation of miRNA expression at 24 hrs was also correlated with decreased expression of genes such as Rnasen, Dgcr8, Dicer, Tarbp2 and Prkra that are associated with miRNA biogenesis. To determine if a potential negative feedback loop between miRNAs and their regulatory genes existed, 11 candidate miRNAs which were predicted to target the above genes were examined and found to be up-regulated at 3 hrs post-PH. Using reporter and functional assays, we determined that expression of these miRNA-processing genes could be regulated by a subset of miRNAs and some miRNAs could target multiple miRNA biogenesis genes simultaneously. We also demonstrated that over-expression of these miRNAs inhibited cell proliferation and modulated the cell cycle in both Huh-7 human hepatoma cells and primary rat hepatocytes. From these observations, we postulated that selective up-regulation of miRNAs in the early-phase after PH was involved in the priming and commitment to liver regeneration, while the subsequent genome-wide down-regulation of miRNAs was required for efficient recovery of liver cell mass.

Conclusion

Our data suggest that miRNA changes are regulated by negative feedback loops between miRNAs and their regulatory genes that may play an important role in the steady-state regulation of liver regeneration.

Keywords: Dicer complex, microarray, non-coding RNA, partial hepatectomy, processing genes

The liver has the remarkable ability to regenerate to its original size after injury. As such, liver regeneration after 70% partial hepatectomy (PH) is a unique model system for the study of in vivo regulation of cell proliferation and gene expression (1). In rat, the entire liver mass can be restored within 7–10 days after PH (2). However, in the first 4–5 hrs after PH, the liver remains refractory to stimulation of growth factors and is believed to be in a so-called “priming” period in which the cells undergoes necessary modifications in preparation for the regenerative process. Priming might be critically related to the liver’s extraordinary ability to accurately restore its original size (3). Cell cycle-related genes such as such as p21, p53, Mdm2 are not expressed until 8 hrs after PH, while the expression of most other genes will remain repressed until 24 hrs, after which many are expressed in a predictable fashion (4). The physiological role of this priming period and its underlying mechanisms remain under investigation. After the priming, DNA synthesis for hepatocytes begins at ~12 hrs and peaks at 24 hrs (5).

It has recently become apparent that microRNAs (miRNAs) might be important players involved in the steady-state regulation of many organ systems. miRNAs are 18–24 bp small noncoding RNAs which can bind to the 3′UTR of mRNAs and regulate their expression and translation. The majority of miRNAs are transcribed by polymerase II (6) and processed by a protein complex including Drosha (RNASEN) and Pasha (DGCR8) (7), among others, to form pre-miRNAs in the nucleus. The 70 bp pre-miRNAs are exported into the cytoplasm and further cleaved by RNA III enzyme Dicer, which interacts with TRBP and PACT to become the mature miRNAs. The TRBP/Dicer complex can further recruit Ago proteins to form RISC, which, when directed to target mRNA by the miRNA, can degrade mRNA and/or inhibit its translation (8).

miRNAs have been implicated in many biological processes, such as tumorigenesis (9), stem cell differentiation (10), and organ development (11). The functions of miRNAs in the physiology and pathology of liver have also been studied. For example, miR-122, which is one of the most abundant miRNAs in adult liver, can regulate hepatic lipid metabolism (1214), control bile acid synthesis (15), and is associated with hepatocellular carcinoma, among other functions (16). Liver-specific conditional Dicer deletion can cause hepatic steatosis, impaired regulation of blood glucose, and promote the hepatocellular carcinoma (17). It is therefore highly likely that miRNAs might also play an important role in the process of liver regeneration. For example, it has been reported that miR-21 is up-regulated during the proliferative phase of liver regeneration, targets Pellino-1, and could provide a negative feedback mechanism to inhibit NF-κB signaling (18).

Under this assumption, we have carried out a study for the expression pattern of miRNAs during liver regeneration using miRNA microarrays. In this study, we discovered a biphasic expression pattern for most of the miRNAs including an early over-expression that coincides with the priming period; and a subsequent reduction that superimposes on the later phases of cell cycle and growth regulated genes in this model. This was most likely mediated by a negative feedback between certain miRNAs and the proteins involved in miRNA maturation and function such as Dicer, Drosha, among others, allowing cell proliferation, and restoration of the liver mass. We therefore conclude that miRNAs play an important role in regulating the homeostasis of cell growth and organ size in liver regeneration after 70% PH.

Materials and Methods

Partial Hepatectomies

Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) ~175 g were subjected to sham surgery or 70% PH as originally described (2). All animal work was approved by the Institutional Animal Care and Use Committee at the University of Minnesota and received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86–23 revised 1985).

Global miRNA Expression Profiling

Genome-wide miRNA changes were studied in both sham and PH samples at the indicated time points by a custom microarray platform (19) as described in Supplemental Material. A minimum of 2–3 replicates were studied in each group. The array data for each of the different time points have been deposited in GEO under accession number GSE28404.

Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR), Immunofluorescence and Western Blot

qRT-PCR, western blots and immunoflurescence were performed following the manufacturer’s instructions. Please refer to Supplemental Material for additional details.

Plasmids, Cloning and anti-miR

Human RNASEN (Drosha), TARBP2 (TRBP) and PRKRA (PACT)-3′UTRs were amplified and cloned into pSGG prom 3′UTR reporter plasmid (SwitchGear Genomics, Menlo Park, CA) by NheI and XhoI. Dicer and DGCR8 3′UTR reporters were purchased from SwitchGear Genomics. Ten individual miRNAs or miRNA clusters were cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) by HindIII/XbaI or NheI/XhoI. The miR-17-92 expression construct was kindly provided by Dr. He Lin (University of California, Berkeley). The miRNAs included in the constructs and primers used in cloning are showed in Supplementary Tables 2 and 3, respectively. Anti-miR-07, anti-miR-424 and anti-miR-let-7a were purchased from Qiagen.

Cell Culture

Human hepatoma Huh-7 cells were cultured in high-glucose DMEM media as previously described (20). The cells were plated at 70% density 24 hrs prior to transfection. Primary rat hepatocytes were obtained from male 225–250 g Sprague Dawley rats via collagenase perfusion as outlined (21). Please refer to Supplemental Material for additional details.

3′UTR Luciferase Reporter Assay

Different 3′UTR reporter constructs were co-transfected with miRNAs constructs (or anti-miRs) and the SV40-RL internal control plasmid (Promega, Madison, WI) by Lipofectamine 2000 into Huh-7 cells. The cells were harvested 24 hrs after transfection, and the luciferase activity determined by the Dual-Glo Luciferase Assay System (Promega) using a Synergy 2 microplate reader (BioTek, Winooski, VT).

Cell Proliferation, Cell Cycle and DNA Synthesis Analyses

A number of different miRNA expression constructs were transfected into Huh-7 cells and the cells harvested after 24 hrs. For cell cycle and cell death studies of both Huh-7 cells and primary hepatocytes, please refer to Supplemental Material.

Results

Biphasic Genome-wide miRNA Changes during Liver Regeneration

We analyzed the hepatic miRNA expression profiles from both sham and 70% hepatectomized rats from 3 to 72 hrs after surgery. Between 300 and 400 miRNAs were expressed at these various time points (Table 1). Comparing sham and PH groups, 208 miRNAs could be detected at all the indicated time points. Based on their expression levels, we grouped these miRNAs into 3 sets and classified them as down-regulated (< 0.8-fold); unchanged (0.8- to 1.2-fold); and up-regulated (> 1.2-fold expression). To more clearly elucidate the pattern of miRNA changes, 181 miRNAs with expression level changes up to 2-fold at all 8 time-points were selected to generate the heatmap and corresponding histogram (Fig. 1A, B). At time points between 3–18 hrs, ~ 50% of miRNAs expression remained unchanged, and 25 to 40% were up-regulated (Table 1). However, at 24 hrs and later we detected a significant reduction in expression levels in up to 70% of the miRNAs (Fig. 1A), with a later trend to normal expression. The distribution of miRNA changes at 3, 24 and 72 hrs showed significant shift in expression levels (Fig.1C).

Fig. 1
Biphasic changes in genome-wide miRNA steady-state levels during liver regeneration by miRNA microarray. (A) Heatmap of miRNA changes between sham and PH samples at the eight different time points post-surgery. The miRNA clustering tree is showed on the ...
Table 1
The miRNA Changes at Different Time Points during Liver Regeneration

Next, we determined the miRNA distribution at the three time points 3, 24 and 72 hrs which showed the greatest change by microarray (Fig. 1D, Table 1). By Venn diagram, only a small subset of miRNAs exhibited the same expression patterns at 3, 24 or 72 hrs post-PH, with 7 up-regulated miRNAs; 21 miRNAs showing no change; and 4 miRNAs that were down-regulated. Taken together, the microarray data suggested that miRNA levels undergo dynamic changes during different stages of liver regeneration after 70% PH and clearly display a biphasic expression pattern, reflecting their key role in regulating the regenerative process (18, 2224).

Besides the mouse and rat miRNAs results described above, we also found that some human miRNAs could also hybridize to the rat liver samples in the microarray study; and determined that the expression changes during the process of liver regeneration displayed similar patterns (Supplementary Table 1).

Confirmation of the Biphasic Expression Pattern of miRNAs during Liver Regeneration

To validate the microarray results, qRT-PCR was performed for 20 miRNAs representing all three expression patterns, i.e., up-regulated, unchanged, and down-regulated. The correlation between microarray and qRT-PCR results was ~ 80% at both 3 and 24 hrs, with the best fit observed in the down-regulated miRNAs (Fig. 2A and 2B, Supplementary Table 2). We also verified the time course of expression of miRNAs, let-7, miR-21, miR-29 and miR-30 at 3, 24 and 72 hrs post-surgery (Fig. 2C). The qRT-PCR data confirmed the microarray results supporting the biphasic genome-wide changes observed in the miRNA expression patterns at the various times post-PH.

Fig. 2
Confirmation of microarray results by qRT-PCR. Changes of selected miRNAs between PH and sham controls at 3 hrs (A) and 24 hrs (B) post-PH by real-time PCR. The miRNAs which showed increased expression by microarray analysis are indicated in black; those ...

Down-regulation of miRNA Processing Genes are Correlated with Repression of miRNAs

We postulated that the regulatory mechanism(s) involved in miRNA processing were responsible for this genome-wide miRNA down-regulation at 24 hrs post-PH (4, 25). To test this hypothesis, we studied the expression patterns of miRNA processing genes Rnasen (Drosha) and Dgcr8 (Pasha), Dicer, Tarbp2 (TRBP) and Prkra (PACT) during LR. Our results indicated that gene expression was not stable in sham controls, suggesting some modulation of gene expression associated with stress of the sham procedure (Supplementary Fig. 1). To obviate the effects from the stress, we normalized the results of treated sample to that of the sham controls, as previously reported (2628). The qRT-PCR results of sham and PH samples revealed the miRNA processing gene transcripts were significantly down-regulated between the 3 to 24 hrs time points (Fig. 3A). We also examined the expression level of Eif2c2 (Ago2) one of the four argonaute proteins well characterized for their critical role in the RISC complex. In contrast to the miRNA processing genes, Ago2 showed a significant increase (40%) at 3 hrs. Because of their critical role in miRNA processing, protein levels of both Dicer and Drosha were studied by western blot (Fig. 3B) and immunofluorescence in 3, 18 and 72 hr samples (Fig. 3C). Expression of both proteins was decreased in PH samples compared with sham and correlated with changes in mRNA levels. There were no detectable differences in immunofluorescence, however, between PH and sham for Dicer at 3 and 72 hrs and for Drosha at 72 hrs (data not shown). These data support the notion that the genome-wide miRNA down-regulation occurring at times later than 3 hrs post-PH is likely due to an early repression of genes responsible for processing miRNAs.

Fig. 3
Changes in the key proteins involved in miRNA processing and function during liver regeneration. (A) mRNA levels of the miRNA processing proteins. RNA from both sham operated and hepatectomized animals from 3, 12, 18, 24 and 72 hrs post-surgery was examined ...

Negative Feedback Mechanism between miRNAs and their Processing Genes in the Regenerating Liver

The above studies indicated that the miRNA processing genes Rnasen, Dgcr8, Dicer, Tarbp2 and Prkra transcripts were down-regulated at 3 and/or 24 hrs in the hepatectomized animals. This occurred concurrently with the genome-wide down-regulation in the majority of miRNAs at 24 hrs post-PH. However, let-7 was up-regulated at 3 hrs (Fig. 2A); and it was previously reported that the let-7 family of miRNAs can target and reduce Dicer expression (29, 30). Therefore, we hypothesized that a negative feedback loop mediated by the up-regulated miRNAs at 3 hrs was a potential mechanism involved in down-regulation of these miRNA processing genes.

To test our hypothesis, the complete 3′UTRs of human RNASEN, DGCR8, DICER, PACT, and TRBP, were inserted after a luciferase reporter cDNA to monitor miRNA activities. Based on TargetScan predictions, we selected 11 candidate miRNAs or miRNA clusters which were also up-regulated at 3 hrs post-PH and can potentially target the 3′UTRs of the 5 miRNA processing genes for further studies (Supplementary Table 4). The targeting sites of these miRNAs on the 3′UTRs of the 5 miRNA processing genes are conserved between human and rat. All the 11 miRNAs or miRNA clusters were cloned into the pcDNA3.1 vector; and constructs of pcDNA3.1-miR and luciferase-3′UTR reporter were co-transfected into human hepatoma Huh-7 cells. Using this luciferase reporter system, with Dicer1 and let-7a as positive controls, we found that expression of all 5 genes could be regulated by a subset of these miRNAs or clusters (Fig. 4A). With Dicer1 as an example, we selected 9 miRNAs including let-7, miR-17-92 cluster, and miR-21, which were over-expressed at 3 hrs and could potentially target Dicer mRNA. We found that over-expression of 7 of these 9 candidate miRNAs could target the Dicer 3′UTR resulting in a significant decrease in luciferase expression, including let-7, consistent with previous reports (29, 30).

Fig. 4
The regulation of the miRNAs processing proteins by miRNAs and miRNA antagonists. (A) Over-expression of miRNAs targeting miRNA processing protein 3′UTRs down-regulate luciferase reporter constructs. Huh-7 human hepatoma cells were co-transfected ...

To confirm the effects of these miRNAs on the processing genes, we also attempted to inhibit them with miRNA antagonists. Among the miRNAs we cloned, only miR-107 and miR-424 are single miRNAs while others are clustered; and let-7a has been reported to function in the regulation of Dicer. Therefore, we selected antagonists for these three miRNAs for further studies. The antagonists were cotransfected with Dicer and TRBP 3′UTR reporter vectors as per Table 2. The empty pcDNA-3.1 vector was used as negative control. Our results indicated that the antagonist was able to reverse the inhibitory effects of endogenous miRNAs, as the luciferase activity was increased about 20% (Fig. 4B). From these studies, we concluded that a negative feedback loop exists between the miRNAs and their processing proteins.

Table 2
miRNAs and Their Validated Target Genes

To validate data from the 3′UTR luciferase reporter assays, the endogenous mRNA levels of Drosha, Pasha, Dicer and TRBP were determined by qRT-PCR and the protein levels of Dicer and Drosha were studied by western blot in Huh-7 cells. The mRNA and/or protein levels of these genes were also decreased with over-expression of the miRNAs that target their 3′UTRs, consistent with the luciferase reporter assay results (Fig. 5 and Supplementary Fig. 3). Interestingly, we found that each of these 5 miRNA processing genes could be regulated by a group of miRNAs; and as an example Dicer could be targeted by 7 of these miRNAs. The similar phenomenon was also observed with Drosha, Pasha, TRBP and PACT (Fig. 4). We also found that 6 of the individual miRNAs could simultaneously target multiple processing genes (Table 2). For example, miR-17-92 cluster could target Drosha, Dicer and PACT, all of which are involved in different stages of miRNA processing.

Fig. 5
Endogenous miRNA processing genes are down-regulated with over-expression of candidate miRNAs in Huh-7 cells. (A) The mRNA levels of four of the processing genes that showed significant changes from the controls were examined after the over-expressing ...

In addition to the 11 miRNAs, many other candidate miRNAs which could potentially target the miRNA processing genes were identified by TargetScan software analysis. We analyzed the expression pattern of the predicted miRNAs that potentially target Drosha, Pasha, Dicer, PACT, and TRBP at 3 hrs post-PH. TargetScan predicted that rat Dicer was targeted by 131 miRNAs, of which 83 could be detected by microarray analysis. Among the miRNA candidates, the majority (55 out of 83, 66%) did not change after PH; 34% (28 out of 83) were up-regulated, and none were down-regulated (Supplementary Table 5). Thus based on these results, Dicer could be down-regulated at 3 hrs post-PH by increased expression of potentially 28 miRNAs targeting its 3′UTR. Similar results were also observed for Drosha, Pasha, TRBP and PACT.

miRNAs Expressed Early in Liver Regeneration Regulate Cell Proliferation and Cell Cycle in Huh-7 Cells

To elucidate the biological relevance of miRNAs that target their own processing genes to mediate a negative feedback mechanism, we used the Huh-7 human hepatoma cell line as an in vitro model. We studied the role of these 10 miRNAs or clusters in cell proliferation after transfecting the Huh-7 cells with each of the pcDNA3.1 miRNA over-expression constructs. We found that over-expression of 8 out of 10 miRNAs, except for the miR-25a and miR-125a clusters reduced the total cell number 10–30% (P < 0.05) (Fig. 6A). This decrease in the total cell number was not the result of cell death, as indicated by propidium iodide staining with flow cytometry (Fig. 6B).

Fig. 6
Cell proliferation and DNA synthesis are regulated by over-expression of candidate miRNAs in Huh-7 cells and primary rat hepatocytes. (A) Cell proliferation (left panel) was determined by CellTiter-Blue assay after 24 hrs of over-expression of the various ...

To elucidate the mechanism by which the miRNAs might regulate cell proliferation we examined if their over-expression arrested cells in specific stages of the cell-cycle in Huh-7 cells. Interestingly, we found that over-expression of 7 out of the 10 miRNA constructs dramatically decreased the cell number in S phase (Fig. 6C, left panel). We also consistently observed minor increases in cell numbers both in the G1 and G2-M phases (Fig. 6C, middle and right panels). The results suggested that these miRNAs in some manner either inhibited DNA synthesis or blocked cell cycle progression at the G1-S-phase check point.

miRNA Mediated Regulation of Cell Proliferation in Primary Rat Hepatocytes

To validate these results in non-transformed hepatocytes, we carried out the miRNA over-expression studies in rat primary hepatocytes induced to proliferate under cell culture conditions. We found that over-expression of several of the miRNAs including let-7a, miR-17-92 cluster, miR-29, miR-30 and miR-424 in rat hepatocytes caused a decrease in the number of viable cells by 10% (Fig. 6D). Interesting, when DNA synthesis was examined in cells over-expressing miRNAs identified as reducing the number of viable cells, a corresponding decrease of 10–20% was observed (Fig. 6E). Taken together the results suggested that these miRNAs play a key role in modulating the proliferative capacity of hepatocytes mediated in part by directly targeting the 3′UTRs of the miRNA processing pathway genes.

Discussion

We have characterized the levels of miRNAs during liver regeneration, and documented a biphasic expression pattern for miRNAs characterized by an early up-regulation and late down-regulation. This biphasic change is most likely caused in part, by a negative feedback mechanism mediated by miRNA processing genes. The early up-regulation of specific miRNAs might be responsible for the priming phase of liver regeneration by inhibiting cell proliferation and DNA synthesis, and their later down-regulation eventually allow liver to grow and regenerate.

Given the important regulatory roles miRNAs played in diverse biological processes, it is very likely that those miRNA also participate actively in coordinating the events of liver regeneration (8). It is of particular interest to note that this early activation of miRNAs coincides with a period that was initially termed the priming period of liver regeneration, i.e., the first 4–5 hrs after PH, in which the hepatocytes are refractory to growth signals. It is tempting to speculate that the up-regulation of miRNAs is a critical mechanism that contributes to the priming period of liver regeneration.

Considering the broad spectrum of down-regulation of miRNAs that were identified in this screen after the initial priming period, i.e., 70% of all miRNAs at 24 hrs, it suggested that miRNA processing was potentially involved in the expression changes. It is possible but unlikely that the transcription of these miRNAs is decreased, since miRNAs are mostly transcribed via RNA polymerase II (Pol II) promoters and a global decrease in RNA Pol II activity during the later phases LR is not observed. Thus, we examined the transcript levels of several miRNA processing genes including Rnasen (Drosha), Dgcr8 (Pasha), Dicer, Tarbp2 (TRBP) and Prkra (PACT); and showed that the mRNA steady-state levels of these genes at 24 hrs was indeed decreased. Prior studies had shown that Dicer was subject to post-translational regulation by miRNAs such as by let-7 (29, 30), suggesting that early up-regulation of miRNAs might, in part, be responsible for down-regulating these miRNA processing genes, which in turn promotes the global decrease of miRNAs observed in the later stages of LR.

Sequence analysis of the 3′UTRs of the miRNA processing genes predicted that many miRNAs could potentially target these genes. These included 11 miRNAs that were significantly up regulated at 3 hrs post-PH, concurrent with the first observed decrease in the miRNA processing genes transcript levels. Using over-expression of a select group of these miRNA and luciferase reporter constructs with the 3′UTRs of the miRNA processing genes in Huh-7 cells, we established that select miRNAs targeted and down-regulated the expression of these genes. This work also extended the target spectrum of the candidate miRNAs.

The early up-regluation of miRNA expression coincides with the priming period after PH, which is characterized by refractory response to growth signals and decrease in DNA synthesis. Some miRNAs have previously been reported to function as tumor related genes, such as let-7 and miR-17-92 (9, 30, 31). We found that over-expression of several miRNAs that target miRNA processing genes, including let-7, miR-17, miR-29, miR-30, and miR-424 decrease cell proliferation and DNA synthesis in HuH-7 cells and primary hepatocytes, and are up-regulated during early liver regeneration. Based on the data, it is likely that they, in fact, contribute to the priming phase of liver regeneration. Finally, the pattern of the miRNA expression in the later phases of LR suggests that their down-regulation is also essential for the termination of replication, consistent with the majority of the hepatocytes completing DNA synthesis followed by cell division by 30 hrs post PH. Genome-wide miRNA down-regulation at 24 hrs may contribute to the S phase peak at 24 hrs post-PH. If it is true, the down-regulation of miRNAs should begin prior to 24 hrs, which is consistent with the micro-array results that the down-regulation begins from 6 hrs post-PH. We examined the expression levels of the 9 miRNAs which can target the miRNA processing genes at 18 hrs by qRT-PCR, and found that most are down-regulated but not as dramatically as at 24 hrs (Supplementary Fig. 2). So we believe the peak of the down-regulation is between 18 hrs and 36 hrs post-PH which may be related to S phase at 24 hrs.

Based on the above findings, our results provide an additional temporal course for miRNA expression between proliferation and return to quiescence in the 70% PH liver (Supplementary Fig. 4) (4). Its biphasic nature appears to define the temporal boundaries between the induction of growth- and cell cycle-regulated gene expression and those activated after the major growth phase has occurred. The major portion of liver mass is reconstituted within 72 to 84 hrs and the entire process is complete within 7–10 days. Several patterns of immediate-early, delayed early, and liver specific genes have been defined during the 10 day period post-PH. The orchestration may be mediated by a negative feedback between up-regulated miRNAs and target mRNAs involved in miRNA maturation and function such as Dicer, Drosha, Pasha, Ago2, PACT, and TRBP, allowing cell proliferation, and restoration of the liver mass.

Overall, this study has documented genome-wide miRNA changes during liver regeneration after 70% PH. We also described a negative feedback loop between miRNAs and their processing genes, which appears to be an efficient mechanism for the homeostatic regulation of miRNAs. The early up-regulation of miRNAs might contribute to the priming period of liver regeneration, while the later normalization of these miRNAs might allow the later accurate cell growth and restoration of liver size. In conclusion, the synchronous model of cell replication of ~ 95% of hepatocytes following 70% liver resection provides a novel model with dynamic flux of the miRNAs affecting their biogenesis and provides a much needed resource for studying both mechanisms controlling their synthesis but also their degradation/loss of which little is known.

Supplementary Material

Supp Fig S1

Supplementary Fig. 1:

Changes in mRNA levels of the miRNA processing proteins. RNA from both sham operated (open bars) and hepatectomized animals from 0, 3, 12, 18, 24 and 72 hrs post-surgery (black bars) was examined by qRT-PCR using 18S rRNA as internal control, and normalized to sham controls at the same time points. Time post-surgery is indicated below and the gene/protein designation above. *, P < 0.05 from sham operated control.

Supp Fig S2

Supplementary Fig. 2:

Changes in expression levels of miRNAs which can target miRNA processing genes at 3, 18, and 24 hrs post-PH by qRT-PCR. The selected miRNAs between PH and sham controls at 3 (white) and 18 (gray) and 24 hrs (black) post-PH were analyzed by real-time PCR. The Y-axis indicates the percent change in PH samples compared to sham controls. U6 was used as the internal control.

Supp Fig S3

Supplementary Fig. 3:

Protein levels of miRNA processing genes are down-regulated with over-expression of candidate miRNAs in Huh-7 cells. Protein expression of Dicer and Drosha were determined by western blot, using tubulin as internal control. Huh-7 cells transfected with the pcDNA 3.1 empty vector were used as control.

Supp Fig S4

Supplementary Fig. 4:

Biphasic expression of miRNAs during rat liver regeneration. The temporal course and representative patterns of gene expression for growth-regulated and cell cycle genes, and those re-expressed after the major growth phase are illustrated for the regenerating rat liver. Hours after PH and patterns of DNA synthesis are indicated for hepatocytes (H) and nonparenchymal cells (NP). The genome-wide biphasic nature of miRNA expression provides additional insight into the complex nature of reestablishing liver mass in this model of liver regeneration after 70% PH. Modified and adapted from ref 4.

Supp Material

Table 1. Human, Mouse and Rat miRNAs Changes during Liver Regeneration

Table 2. The Correlation Between qRT-PCR and Microarray Results

Table 3. Primers Used for qRT-PCR and Cloning

Table 4. Candidate miRNAs Selected for Cloning

Table 5. miRNA Changes Associated with miRNA Biogenesis Genes by Microarray

Acknowledgments

Supported by National Institutes of Health ARRA Grant R01 DK081865-01 (to C. J. S.)

Abbreviations

PH
partial hepatectomy
miRNA
microRNA
Rnasen
ribonuclease III, isoform 2
Dgcr8
DiGeorge syndrome critical region gene 8
Tarbp2
TAR RNA binding protein 2
Prkra
interferon-inducible double stranded RNA-dependent protein kinase activator A
3′UTR
three prime untranslated region
LR
liver regeneration
RISC
RNA induced silencing complex

Footnotes

Potential conflicts of interest: Nothing to report.

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