MiRNA-HNF1α Regulatory Axis In The Inflammatory Reaction Of Hepatocytes

【Background and Objective】Liver injury is the common pathological basis of various liver diseases. It has beenrecognized that inflammatory reaction is a crucial factor of liver damage and chronic liverdiseases. Hepatocyte is one of the most important cellular components of the liver, whichis involved in the inflammatory reaction and repair process of both acute and chronic liverinjury. Moreover, there are many inflammatory cytokine receptors (such as interleukinreceptor, TNF-α receptor and TGF-β receptor) in the surface of hepatocytes, suggesting theimportant roles hepatocytes play in the liver inflammatory reaction.Hepatocyte nuclear factors (HNFs) are transcription factors enriched in the liver.Hepatocyte nuclear factor-1α (HNF1α) is one of the key members of HNFs. It playsimportant roles in the differentiation, maturation and function maintenance of hepatocytesas well as the development of the liver. A previous study showed that HNF1α wasinvolved in the inflammatory reaction and repair process in acute liver inflammation bypromoting the expression of C-reactive protein (CRP). Moreover, interleukin-6(IL-6), animportant inflammatory cytokine, can inhibit the expression and transcriptional activity ofHNF1α, indicating that HNF1α may be involved in the regulation of liver inflammationreaction and the occurrence and development of liver fibrosis.Widely existed in C. elegans, Drosophila and human tissue cells, microRNA (miRNA)is a single-stranded non-coding RNA containing21-25nucleotides. miRNAs can inhibittranslation of target mRNAs by binding to the3′untranslated region (3′UTR) of thesemRNAs. Many studies have shown that miRNAs are implicated in inflammation. Theyexert inflammation-promoting or-inhibition effect by targeting important proteins ofinflammation-related pathways such as NF-κB and IL6-STAT3pathway. miR-21, anwell-known inflammation-associated miRNA, has been reported to regulate HNF4α whichis an important member of HNFs. Our previous study has shown that HNF4α wasdownregulated during the development of liver fibrosis, and upregulation of HNF4αinhibited the progression of liver fibrosis. These studies suggest that inflammation-relatedmiRNA may be associated with the role of HNF1α in the inflammatory reaction activatedin liver fibrosis. The present study aims to investigate the expression of HNF1α in the inflammation ofhepatocytes and its influence on the inflammation of hepatocytes, as well as the role ofmiRNAs in the downregulation of HNF1α. It may help to make clear the role ofmiRNAs-HNF1α regulatory axis in the inflammation of hepatocytes, thus developing anew strategy for the prevention and treatment of chronic liver disease.【Methods】1. Expression of HNF1α in hepatocytes inflammatory reaction(1)Expression of HNF1α and inflammatory cytokines in ratprimary hepatocytes during in vitro culturePrimary hepatocytes were isolated from normal SD rats and cultured in vitro. Proteinand mRNA were isolated from hepatocytes at day1,3,5,7. Real-time PCR was used todetect the expression of TNF-α and IL-6. Western Blot was used to detect the expressionof HNF1α.(2) Expression of HNF1α and inflammatory cytokines inDMN-treated ratsMale SD rats were injected intraperitoneally with a dose of100μl/100g of1%DMNsolution three times a week for2weeks. Primary hepatocytes were isolated from the liverof the rats. Real-time PCR and Western Blot were used to detect expression of HNF1α andinflammatory cytokines.2. Construction of recombinant adenovirus vectors expressingsiRNA against HNF1αThe construction of the adenoviral vector containing the siRNA targeting HNF1α(AdshHNF1α) and the control adenovirus (AdshNC) was performed as previouslydescribed. Real-time PCR and Western Blot were used to detect mRNA and proteinexpression of HNF1α in primary hepatocytes infected with adenovirus. 3. Effect of HNF1α downregulation on hepatocyteinflammation reaction(1) Effect of HNF1α downregulation on the expression ofinflammatory cytokines in vitroPrimary hepatocytes were isolated from normal SD rats and cultured in vitro.Twenty-four hours after isolation, hepatocytes were infected with AdshHNF1α. Real-timePCR was used to detect the expression of TNF-α and IL-6mRNA. Western Blot was usedto detect the expression of HNF1α.(2) Effect of HNF1α downregulation on the expression ofinflammatory cytokines in vivoSD rats were injected with a single dose of2×109pfu AdshHNF1α via tail vein. Twoweeks later, rats were sacrificed for immunohistological analysis for HNF1α, IL-6andTNF-α.4. miRNA-HNF1α regulatory axis regulates hepatocyteinflammatory reaction(1) Inflammation-related miRNAs impact the expression ofHNF1αWe screened inflammation-related miRNAs which contain binding sites in the3’ UTRof HNF1α using software. Primary hepatocytes isolated from SD rats were transfected withmimics of screened miRNAs miR-21, miR-31and miR-146a. Real-time PCR and Westernblot were used to detect the expression of HNF1α. (2) Identification of HNF1α as a target gene ofinflammation-related miRNAsWe constructed a luciferase reporter plasmid containing HNF1α3’UTR. Cells wereco-transfected with miR-21mimic or miR-146a mimic and luciferase reporter plasmid.Dual reporter gene assay kit was used to detect the activity of reporter vector.(3) The impact of inflammatory cytokines on the expression ofinflammation-related miRNAs and HNF1αPrimary hepatocytes were isolated and cultured with mediums containing50ng/ml IL-6or20ng/ml TNF-α for48h. Western blot and real-time PCR were used to detect theexpression of HNF1α, miR-21and miR-146a.(4) The correlation between HNF1α, miR-21and miR-146a inDMN-or BDL-treated ratsRat DMN or BDL model were established as described previously. Two weeks later,rats were sacrificed for detections of HNF1α、miR-21and miR-146a. Correlation analysiswas performed to investigate their correlation.5. Statistical analysisThe arithmetic mean and standard deviation (s.d.) were calculated for the data, andstatistically evaluated using2-tailed unpaired t-test. P <0.05was considered statisticallysignificant.【Results】1. HNF1α was decreased in the inflammatory reaction ofhepatocytesInflammatory cytokines such as IL-6and TNF-α were gradually increased accompaniedby a decrease of HNF1α in primary hepatocyte cultured in vitro. The same changes weredetected in primary hepatocytes isolated from DMN model rats.2. Depression of HNF1α promoted the inflammatory reactionof hepatocytes in vitro and in vivo The downregulation of HNF1α and the upregulation of IL-6and TNF-α were detected inprimary hepatocytes isolated from SD rats and infected with AdshHNF1α. SD rats wereinjected with AdshHNF1α via tail vein. Immunohistochemical results showed that theexpression of HNF1α in the liver was significantly reduced, while Real-time PCR showedthat the expression of IL-6and TNF-α were increased.3. The role of miRNA-HNF1α regulatory axis in theinflammatory reaction of hepatocytesPrimary hepatocytes were isolated and transfected with mimics of miR-21, miR-31andmiR-146a. Western blot and Real-time PCR showed that both mRNA and protein level ofHNF1α were significantly depressed by miR-21and miR-146a mimic but not miR-31mimic. The further reporter assays showed that HNF1α was the target gene of miR-21andmiR-146a.Downregulation of HNF1α protein and upregulation of miR-21and miR-146a weredetected in primary hepatocytes treated with IL-6or TNF-α. Two weeks after theestablishments of DMN or BDL models, the rats were sacrificed for the detections ofHNF1α, miR-21and miR-146a. The correlation analysis showed that both miR-21andmiR-146a levels were negatively correlated with HNF1α level.【Conclusion】1. HNF1α was decreased in the inflammatory reaction of hepatocytes.2. Depression of HNF1α promoted inflammatory reaction of hepatocytes both in vitroand in vivo.3. HNF1α is a direct target of miR-21and miR-146a.4. miRNA-HNF1α regulatory axis was involved in the regulation of inflammatoryreaction in hepatocytes.