The Expression Of MiR-23b And Its Potential Role In The Termination Stage Of Liver Regeneration After Partial Hepatectomy In Rats

Following 70% partial hepatectomy (PHx), the remnant liver has a remarkable ability to regenerate to its original mass and function within 7-10 days after surgery in a process called liver regeneration (LR). LR after PHx is generally divided into three distinct phases: initiation, proliferation and termination. Despite multiple studies of LR, many aspects of this phenomenon remain unknown, including the proper temporal regulation of termination.MicroRNAs (miRNAs) are a class of small regulatory RNAs that silence messenger RNAs by binding to their 3'-untranslated regions (UTRs). MiRNAs have been reported to modulate a variety of biological processes, including cellular differentiation and proliferation, metabolism and apoptosis. It is reported that miRNAs (miR-21 and miR-378) play critical roles during the early phases of LR by directly inhibiting the expression of target genes that associate with DNA synthesis. However, the miRNAs that function in the termination stage of LR remain unknown.In a preliminary study using a miRNA microarray analysis, we found that miRNA 23b (miR-23b) was down-regulated in regenerating rat liver tissues 120 hours (h) after 70% PHx compared to the SH group. MiR-23b has been reported to be involved in many cell functions including cell proliferation, migration and differentiation.In view of the above analysis, this study sought to elucidate if and how miR-23b was involved in the termination stage of LR, and this will provide basis to better illustrate the molecular mechanism of the termination liver regeneration after partial hepatectomy. The inquiry will also provide the basis for exploring therapeutic targets for liver cancer.SectionⅠ: The expression of miR-23b and its role in the termination stage of liver regeneration after partial hepatectomy in ratsMicroRNAs (miRNAs) are a class of small regulatory RNAs that silence messenger RNAs by binding to their 3'-untranslated regions (UTRs). MiRNAs have been reported to modulate a variety of biological processes, including cellular differentiation and proliferation, metabolism and apoptosis. It is reported that miRNAs (miR-21 and miR-378) play critical roles during the early phases of LR by directly inhibiting the expression of target genes that associate with DNA synthesis. However, the miRNAs that function in the termination stage of LR remain unknown. MiR-23b has been reported to be involved in many cell functions including cell proliferation, migration and differentiation. In a preliminary study using a miRNA microarray analysis, we found that miRNA23b (miR-23b) was down-regulated in regenerating rat liver tissues 120 hours (h) after 70% PHx compared to the SH group. And a recent study has shown that miR-23b served as a molecular switch in regulating Transforming growth factor-β1 (TGF-β1) signaling by targeting Smads. Considering the important role TGF-β1 plays during the termination stage of LR, the present work sought to elucidate if and how miR-23b was involved in the termination stage of LR. We also report evidences that miR-23b expression is remarkably diminished during the termination of LR, and miR-23b may contribute to the TGF-β1/Smad3 signalling pathway during the termination stage of LR.In the present study we confirmed that miR-23b is down-regulated during the termination stage of LR. Our study also found that down-regulation of miR-23b inhibits cell proliferation, which may be attributed to G2/M arrest by flow cytometry investigation, as shown in figure 2. Therefore, down-regulation of miR-23b may contribute to the termination of LR, which is characterised by reduced proliferation and enhanced cellular apoptosis.To further investigate the specific role of miR-23b during the termination stage of LR, it is very important to identify the target gene of miR-23b. A recent study has shown that miR-23b served as a molecular switch in regulating Transforming growth factor-β1 (TGF-β1) signaling by targeting Smads. Thus, miR-23b plays a critical role in TGF-β1 /Smads signaling pathway. More importantly, it is widely known that TGF-β1 and activinA are natural terminators of LR. Therefore, we speculate miR-23b may take part in the termination stage of LR by targeting smads. Our study has confirmed that Smad3 but not Smad4 or Smad5 is a putative target gene of miR-23b in livers following PHx.There is evidence that Smad3 plays a more significant role in the induction of TGF-β1-induced apoptosis than other Smads downstream of TGF-β1. Over-expression of Smad3 greatly potentiates the induction of apoptosis in response to TGF-β1, while expression of a dominant-negative mutant of Smad3 inhibits TGF-β1 induced apoptosis. Therefore, Smad3 is an important downstream molecular in TGF-β1 pathway. Interestingly, we have found that up-regulation of miR-23b could partially inhibit TGF-β1-induced apoptosis in BRL-3A cells by reducing the expression of Smad3. In addition, TGF-β1, Smad3, phosphorylated Smad3 and apoptotic activity increased and proliferative activity decreased when miR-23b was decreased from 72 to 168 h after PHx. Taken together, down-regulation of miR-23b may contribute to activation of the TGF-β1/Smad3 signalling pathway during the termination stage of LR.In exploring the specific relationship between TGF-β1, miR-23b and Smad3, we considered TGF-β1 as a possible upstream regulator of miR-23b. Firstly, several reports have demonstrated that TGF-β1 can either positively or negatively regulate the expression of miRNAs including miR-192 and miR-24. More interestingly, even in the process of LR, miR-23b down-regulation occurs after 72h PHx, when TGF-β1 expression is at its highest levels. In addition, miR-23b can directly regulate the expression of Smad3 which is a downstream molecular in TGF-β1 signaling. Therefore, we presumed that miR-23b may act as a pitch point in the TGF-β1 signalling pathway. Then we confirmed that TGF-β1 can regulate the expression of miR-23b at the transcriptional level. Our present findings suggest at TGF-β1-miR-23b-smad3 signaling may act as a novel mechanism in the termination stage of LR.In conclusion, miR-23b may contribute to TGF-β1/Smad3 signaling pathway during the termination stage of LR. Our findings reveal a miRNA-mediated regulation pattern during the termination stage of LR.SectionⅡ: The expression of neurogranin and its role in the termination stage of liver regeneration after partial hepatectomy in ratsNeurogranin (Ng), a calmodulin (CaM)-binding protein kinase C (PKC) substrate, regulates the availability of Ca2+/CaM complex and modulates the homeostasis of intracellular calcium in neurons. In recent years, studies have found that Ng exists in the thymus, spleen and other lymphatic organs.In the first experimental part, we use Target scan5.1 to predict the potential target genes of miR - 23b have found that Ng is one of the potential target genes of miR-23b. but our study proved that miR - 23b is not the upstream regulator of neurogranin in the BRL-3A cells. Intereatingly, our prestudy using gene chip to indentify the differentially expressed gene during the termination stage (120,168h after partial hepatectomy) of liver regeneration after partial hepatectomy have found that the gene expression of Ng upregulated. In view of these above factors, we predicted that apoptosis related proteins Ng might be involved in the termination of liver regeneration after partial hepatectomy in rats. Based on the above analysis, this experimental part aimed at the investigation of the expression pattern of Ng during the termination stage of liver regeneration and the exploration of the related molecular mechanisms.Our results showed that: the expression of Ng protein upregulated during the termination stage of liver regeneration which is characterized with a high apoptosis activity; the concentration of NO and T3 increased during the termination stage of liver regeneration; downregulation of Ng in BRL-3A cells will inhibits the growth of BRL-3A cells and partially inhibits TGF-β1 induced BRL-3A cell apoptosis through depressing the mitochondrial apoptosis pathway; exogenous NO donors SNP could upregulate the expression of Ng at the transcriptional level in BRL-3A cells, while there were no obvious change on the expression of Ng when BRL-3A cells were treated with miR-23b or T3.This experimental part suggests that during the termination stage of liver regeneration, the upregulated expression of Ng attributed to the increased concentration of NO and contributed to the termination of liver regeneration by inducing mitochondrial apoptosis pathway.SectionⅢ: The association of miR-23b expression with surgical outcome in Chinese hepatocellular carcinoma patientsHepatocellular carcinoma (HCC) is the fifth most frequent human cancer worldwide, and, the second leading cause of cancer-related death in China. Major HCC risk factors include infection with hepatitis B (HBV) or C viruses (HCV) and cirrhosis associated with chronic inflammation. Hepatectomy is the first choice for HCC patients in China, but the outcome is poor due to high recurrence after surgery. Since most HCCs have dissemination within the liver before resection and are undetectable by imaging techniques, new and efficacious adjuvant therapy strategies are needed, along with new prognostic biomarkers for early detection of at-risk patients. Therefore, it is clinically meaningful that metastasis and prognosis related molecule and its functional mechanism were found. MicroRNAs (miRs) are small (21-25 nucleotide) nonprotein-coding RNAs that posttranscriptionally regulate gene expression. MiRNAs possess oncogenic or tumor suppressor activity in various tumors but little is known about miRNA expression pattern in hepatocellular carcinoma (HCC). The purpose of this part of study was to investigate the relationship between miR-23b expression and prognosis of HCC patients after curative resection.We use quantitative reverse-transcriptase-polymerase-chain-reaction assays to investigate the expression of miR-23b in 115 patients with HCC who had undergone radical resection between 2004 and 2005. We assessed the association of miR-23b with tumor recurrence and patients'survival with HCC. The results showed that: Tumors had reduced levels of miR-23b expression, as compared with paired nontumorous tissues. In comparison with high miR-23b expression, low miR-23b expression in tumor tissue, which was correlated with encapsulation (P=.009) and TNM stage (P<.001) was associated with both tumor recurrence (median time to recurrence: 10.3±1.8 months vs 36.3±2.6 months, P<.001) and patients survival (1-, 3-, 5-year survival rates: 52%, 31%, 29% vs 84%, 63% 57%, P<.001). Multivariate analysis indicated that the expression level of miR-23b in tumor tissue was also an independent prognostic factor for both recurrence (hazard ratio [HR]: 2.483, 95% confidence interval [CI]: 1.563-3.944, P<.001) and survival (HR 2.256, 95%CI 1.377-3.697, P=.001).Our analyses revealed that low expression of miR-23b was associated with multiple malignant characteristics of HCC. MiR-23b expression was low in a subgroup of patients with higher serum AFP level, >5cm in diameter and no complete capsule. In addition, patients with lower miR-23b expression were mostly in TNM stageⅢ, and these patients usually display microvascular invasion, and portal vein tumor thrombus. This suggests that miR-23b may be a tumor suppressor and that miR-23b low expression in hepatocytes may contribute to the development of a more aggressive phenotype of hepatocellular carcinoma. Furthermore, low expression of miR-23b might be associated with worse prognosis. Patients whose tumors had high miR-23b expression had a prolonged TTR and survival as compared with patients whose tumors had high miR-23b expression.In short, this part of study evaluated for the first time the correlation between miR-23b expression, tumor recurrence, and survival in HCC patients. Our data strongly indicates that miR-23b expression in HCC is a potential biomarker for prognosis evaluation.In conclusion, through the investigation of the termination stage of liver regeneration, we have found the potential role of miR-23b and neurogranin during the termination stage of liver regeneration in rat which will further improve the molecular mechanism of liver regeneration. Meantime, by analysing the association of miR-23b expression with surgical outcome in Chinese hepatocellular carcinoma patients, we have confirmed that miR-23b might be one of the prognostic indicators of HCC patients after curative resection which will provide the basis for exploring therapeutic targets for HCC.