| Summary: | 博士 === 國立陽明大學 === 藥理學研究所 === 106 === Primary liver cancer (majorly hepatocellular carcinoma, HCC) is the second leading cause of cancer death worldwide. Recent studies indicate that microRNAs play an important role in the development and progression of HCC. MicroRNAs are a class of small non-coding single-stranded RNAs that regulate post-transcriptional gene expression through mRNA degradation and translational repression of mRNA in mammalian cells. Glycine N-methyltransferase (GNMT) is a tumor suppressor for HCC. It is abundant in the liver and has multiple functions. It regulates the pool of methyl groups by controlling the ratio of S-adenosylmethionine to S-adenosylhomocysteine through the one-carbon metabolism. GNMT is also diminished in both human HCC cell lines and tumor tissues. Both male and female GNMT-/- mice develop chronic hepatitis and HCC spontaneously. Global DNA hypomethylation was seen in the genome of GNMT-/- mouse and there were changes to gene expression profiling during HCC progression. However, the mechanisms of GNMT down-regulation and microRNAs profiling are not fully understood in HCC. We hypothesize that used the microRNAs prediction database to search for possible oncogenic microRNAs (onco-miRs) targeting at the CDS and 3’UTR of human GNMT mRNA, and whether there is specific novel microRNAs signature of GNMT-/- mouse identified from genome-wide small RNA sequencing analysis serves as a biomarker for liver cancer diagnosis. We tested the microRNA-guided regulation of GNMT in HCC. The nucleotides 601-609 in the CDS of GNMT mRNA served as a miR-224 response element by miRWalk and miRTar prediction software. The 3’UTR dual-luciferase reporter system showed that miR-224 directly targeted the CDS of GNMT mRNA. Use of lentivirus for ectopic miR-224 and GNMT expression in human hepatoma cells indicated that miR-224 counteracted the effects of GNMT on the reduction of cell proliferation and tumor growth in vitro and in vivo. We noticed a significant inverse correlation between miR-224 and GNMT expression in HBV-related HCC specimens and HBx transgenic mice liver tissue. In addition, we also observed a reduction in the expression of GNMT and a significant inverse correlation between miR-224 and GNMT expression in CCl4-induced inflammation response and AFB1-induced liver cancer of mice. Moreover, AAV-GNMT reduced CCl4-induced miR-224 expression and fibrosis formation in mouse liver. Emerging evidence indicates that miR-224 regulates GNMT expression in liver cancer, while miR-224/GNMT may act as a bridge between inflammation and tumorigenesis. In addition, small RNA deep sequencing analysis was performed to evaluate the non-coding RNAs expression profiles from 4 months old and HCC formation in both genders of GNMT-/- mice. We found distributions of the 24 up- and 12 down-regulated miRNAs in both genders of GNMT-/- mice HCC tissues. The miRdeep2.0 software predicted novel miRNAs from the unannotated region of sequencing data. Alignments between known/novel mouse miRNAs and human miRNAs respective BLAST hits found their homologous sites by miRbase and UCSC database. We found 4 human microRNAs hsa-miR-3960, -4532, -4492 and -4508 from 3 novel mouse microRNAs. TCGA-liver cancer database revealed 11 up-regulated microRNAs, 7 down-regulated microRNAs and there was no available data on 5 novel microRNAs (hsa-miR-4791, 3960, -4532, -4492 and -4508). These include up-regulation of hsa-miR-146b-5p, -132-3p, -425-5p, -140-3p, -181a-5p, -182-5p, -183-5p, -96-5p, -21-5p- 34a-5p, -10b-5p, and down-regulation of hsa-miR-451a, 144-3p, 101-3p, 99a-5p, 125b-5p, 145-3p, 122-5p in the TCGA-liver cancer database. Furthermore, we assessed the potential for early HCC detection of these five newly identified miRNAs (hsa-miR-4791, 3960, -4532, -4492 and -4508) in 60 paired HCC specimens from TLCN cohort and serum of liver cirrhosis patients (n = 51) and HCC patients (n = 55) from KUMH by RT-qPCR. The tumor tissue and serum levels of these five miRNAs were significantly increased, whereas the serum levels in patients with liver cirrhosis of miR-3960 and miR-4508 were markedly increased. Notes on serum miR-panel optimization of logistic regression revealed the four combined miRNAs (hsa-miR-4791,-4532, -4508, and -4492) and the two combined miRNAs (hsa-miR-3960 and -4508) for HCC and liver cirrhosis (LC) diagnosis. The areas under the ROC (receiver operating characteristic) curve (AUC) for the HCC miR-panel and LC miR-panel were 0.95 and 0.84, respectively. The high expression of these five combined miRNAs was significantly correlated with higher recurrence rates. Finally, the five miRNAs inhibitors enhanced the rate of cell death in hepatoma cell lines. Interestingly, the five miRNAs mimics did not enhanced the rate of living cells in hepatoma cell lines. In the present study, anoikis resistance is an essential prerequisite for tumor metastasis. The hsa-miR-4532 and -4508 elevated wounding-healing behaviors, transwell migration and transwell invasion in the high metastatic efficiency of Mahlavu cell line and low metastatic efficiency of Huh6 cell line. Furthermore, treatment with lower doses of the miR-4492/-4532/-4508 inhibitors mixture repressed the migration and invasion of hepatoma cells in a synergistic manner. Taken together, these results strongly suggested that the miR-4492, -4532, -4508 may mediate the progression of liver cancer through the regulation of metastatic pathways. This study provides insight into understanding the regulatory mechanisms of GNMT and the discovery of novel microRNAs in liver cancer. This suggests a new therapeutic strategy of liver cancer by manipulating the level of GNMT using miR-224 inhibitors. Alternatively, silent mutants of GNMT cDNA can resist miR-224's attack, whereas the anti-tumor cell growth and anti-metastatic properties of five novel microRNAs inhibitors can be used in gene therapy. The serum miRNA panels may have the potential to be used clinically as an auxiliary tool for liver cirrhosis and HCC diagnosis and HCC prognosis, which may lead to more personalized therapy.
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