Molecular Mechanism Research Of Histone Deacetylase Inhibitor Mediated Epigenetic Regulation Of Tumor Related Genes Expression And Anti-cancer Effect

Object:Histone deacetylase inhibitors (HDACi) represent a novel class of targeted drugs which alter the acetylation status of several cellular proteins. These agents, modulating both chromatin structure through histone acetylation, and the activity of several non-histone substrates, are able to determine changes in gene transcription and to induce a plethora of biological effects ranging from cell apoptosis induction, to cell-cycle arrest, differentiation or modulation of immune responses. HDACi displayed impressive anticancer activity against solid and hematological malignancies with high efficiency and low toxicity in preclinical models; preferential effect on cancer cells and significant therapeutic advantage over traditional chemotherapy agents. Two HADC inhibitors, suberoylanilide hydroxamic acid (SAHA or Vorinostat) and FK-228(Romidepsin) have been approved by the FDA for the treatment of cutaneous T-cell leukemia and more HDACis (MS275, LBH589, PXD101etc.) are under clinical trials. To date, the precise mechanisms of HDAC inhibitors in cancer therapy have not yet been fully elucidated. Improved understanding of the molecular mechanisms by which HDACi elicit anti-tumor activity would undoubtedly assist their clinical development as anticancer agents. This research will provide the experimental basis for elucidation the mechanism by which HDAC inhibitor regulates miRNA expression, and ultimately provide new research targets and therapeutic strategies for cancer treatment.Methods:Part1:1. We found that SAHA enhanced the susceptibility of HCC cells to NK lysis evaluated with MTT assay and enhanced the expression levels of MICA/B molecules using flow cytometric analysis.2. Real-time quantitative PCR was employed to detect the effect of SAHA on the expression of miRNAs (miR-20a, miR-93, miR-106b) targeting MICA/B, which showing down-regulation of the expression of these miRNAs in S AHA-treated HCC cells in a dose-dependent manner. A dose-and time-dependent decrease in the levels of miR-17, miR-18a, miR-19a, pri-miR-17-92and MCM-7was also verified with Real-time quantitative PCR. Then we recognized that these miRNAs are transcribed as part of the host gene and share the same promoter with their host genes.3. The miRNA (miR-20a, miR-93, miR-106b) mimics or control RNA were transiently transfected into HepG2or H7402cells and FACS were employed to confirm that all of the three miRNAs significantly down-regulated MICA surface expression.4. We verified the HDAC inhibition activity of HDACis by Western Blotting using anti-AcH3or anti-AcH4antibodies and examined the histone acetylation status around the promoter region of MICA, miR-17-92or MCM7with Ch1P.5. We next analyzed the sequences of miR-17-92cluster genome to predict the exact location of its promoter with Promoter2.0Prediction Serve, then the human miR-17-92promoter region was amplified by PCR and cloned in the luciferase reporter pGL3-basic vector to generated miR-17-92promoter luciferase reporter construct.6. The binding elements of transcription factor in the promoter regions of miR-17-92were predicted by JASPAR database and TFSEARCH.7. To overexpress GATA-2, we generated pEGFP-N1-GATA-2construct. Western blotting assay was used to monitor GATA-2expression. The effect of GATA-2overexpression on the expression of miR-20a in HCC cells were tested with qPCR.8. Western blot was used to detect the activation of STAT3with anti-p-STAT3antibodies and the effect of SAHA on STAT3activation. IL-6was used as STAT3activator. Real-time quantitative PCR were employed to detect the effect of IL-6on the expression of miR-20a and pri-miR-17-92. Using Ch1P, we verified p-STAT3could bind to miR-17-92promoter, with the reduction of p-STAT3enrichment in miR-17-92promoter after SAHA treatment, which mediated the inhibition of miR-17-92gene cluster by SAHA. Part2:1. We examined the growth inhibition activity of ZYJ-34c in leukemia cells (K562, HL-60and primary AML cells) with WST-1assay.2. PI and Annexin V/PI staining were employed to detect cell cycle and apoptosis after HDACi treatment. Western blotting and semi-quantitative RT-PCR were used to detect the expression of apoptosis-regulating genes and various cell cycle regulators.3. We verified the HDAC inhibition activity of HDACis by Western Blotting using anti-AcH3or anti-AcH4antibodies. We examined the histone acetylation status around the promoter region of p21WAF1with Ch1P.4. To silence the expression of p21, we generated p21-specific short hairpin RNA (shRNA) pTZU6+1construct. The vector was transient transferred into K562cells by electroporation. Then, SAHA or ZYJ-34c was added to the cells. FACS analysis (ModFit LT) of the cell cycle was performed after24hours of SAHA or ZYJ-34c induction.Results:Part1:SAHA enhance NK sensitivity in hepatocellular carcinoma cells through epigenetic regulation of miR-17-92clusters and MICA1. SAHA treatment significantly increased the susceptibility of the two HCC cell lines to cytolysis by NK cellsSAHA treatment increased the expression of MICA/B on hepatoma cells, and further promoted the interaction between NKG2D and its ligands, MICA and MICB, thus enhanced the susceptibility of HCC cells to cytotoxicity of NK cells.2. Expression of miRNAs targeting MICA/B is down-regulated in SAHA-treated HCC cells in a dose-dependent mannerSAHA treatment resulted in a two-to four-fold dose-dependent decrease in the expression of miR-20a and miR-106b in both HCC cells. miR-93also showed a modest decrease in HepG2cells. Since these miRNAs have been implicated in tumorigenesis, and their transcription seems to be affected by SAHA’s epigenetic regulation, we sought to further explore the molecular mechanisms.3. Expression of miRNAs (miR-17, miR18a, miR-19a) in C13ORF25BOX and MCM7(the host gene of miR-106b-93-25cluster) are down-regulated in SAHA-treated HCC cells in a dose-dependent mannerWhen HCC cells were treated with SAHA, dose-and time-dependent decrease in the levels of miR-17, miR-18a, miR-19a, pri-miR-17-92and MCM7was observed. All these results demonstrated that these miRNAs are transcribed as part of the host gene and share the same promoter with the host genes.4. miR-20a, miR-93and miR-106b specifically downregulate MICA expression in HCC cell linesMiRNA-20a,-93,-106b mimics or control RNA were transiently transfected into HepG2or H7402cells, which had high expression of MICA, and demonstrated that all of the three miRNAs significantly down-regulated MICA surface expression.5. SAHA induces the accumulation of acetylated histones in chromatin associated with the MICA gene, but decreases the binding of acetyl-histone H4to the MCM7promotersWe verified the HDAC inhibition activity of SAHA by Western Blotting using antibody to AcH3or AcH4. The enhancement of acetylation of histones H3associated with the MICA promoter is involved in the MICA upregulation induced by SAHA. SAHA decreased histone H4acetylation at MCM7promoters, but did not affected the histone acetylation levels of miR-17-92cluster promoter.6. In SAHA treated HCC cells, GATA-2was not involved in the transcriptional repression of miR-17-92cluster.Although the protein levels of GATA-2was decreased after SAHA treatment, over-expression of GATA-2in HCC cells had no effect on the expression of miR-17-92.7. SAHA downregulated miR-17-92cluster via abolishing tyrosine phosphorylation of STAT3SAHA was able to dephosphorylate STAT3tyrosine phosphorylation in both HCC cells. After stimulation with IL-6, the expression of miR-20a and pri-miR-17-92increased significantly in both HCC cell lines. Using ChIP, we verified p-STAT3could bind to miR-17-92promoter, with the reduction of p-STAT3enrichment in miR-17-92promoter after SAHA treatment.8. miR-17-92promoter luciferase reporter vector construction, expression of recombinant plasmids in mammalian cells was enhanced by SAHAThe human miR-17-92promoter region was amplified by PCR and then cloned into the luciferase reporter pGL3-basic vector. HepG2cells transfected with pEGFP-Nl (G418selection was conducted after the transfection) express more GFP after SAHA treatment. The fluorescence intensity of SAHA treated group was higher than that of control group.Part2:Superior activity of a new histone deacetylase inhibitor (ZYJ-34c) in inhibiting growth of human leukemia cells via induction p21WAF1expression and cell cycle arrest1. ZYJ-34c possesses more potent antiproliferative activity than SAHA We use K562, HL-60cells and primary AML cells to demonstrate ZYJ-34c exerts a more potent inhibitory effect of hematological malignancies than SAHA.2. ZYJ-34c does not cause more significant cell apoptosis than SAHAThere were no significant differences in apoptosis or apoptosis-regulating genes induction between SAHA and ZYJ-34c treatment. The superior growth inhibition by ZYJ-34c is not caused by apoptosis.3. ZYJ-34c induces Gl phase arrest more significantly than SAHACompared to SAHA, ZYJ-34c demonstrated stronger G1phase arrest effects on K562and HL-60cells.4. ZYJ-34c induces the expression of p21WAF1mRNA and p21WAF1protein more significantly than SAHA We examined the effect of SAHA and ZYJ-34c on the expression of various cell cycle regulators (p21, p27, p57, c-myc, p53, bcr-abl) and demonstrated that the mechanism of superior cell cycle arrest of ZYJ-34c was mediated by a greater ability to upregulate p21WAF1and downregulate bcr-abl.5. ZYJ-34c induces accumulation of acetylated histones in chromatin associated with the p21WAF1gene more significantly than SAHAZYJ-34c had a more potent ability to induce histones acetylation compared with SAHA. Using ChIP assay we verified the significant p21upregulation induced by ZYJ-34c was mainly a result of enhanced acetylation of histones H3and H4associated with the p21WAF1promoter.6. G1cell cycle arrest induced by ZYJ-34c or SAHA is p21-dependentTo silence the expression of p21, we generated p21specific short hairpin RNA (shRNA) pTZU6+l construct. Silencing of p21WAF1abrogated SAHA-induced G1arrest, while ZYJ-34c-induced cell cycle arrest was also attenuated.7. SAHA selectivity lowered Cyclin A in leukemias cells and this may contribute to the mechanism of more sensitive of hematologic tumors to HDACi than that of solid tumors.Cyclin A influences the sensitivity of tumors to HDAC inhibitors. SAHA selectivity lowered Cyclin A in leukemias cells (K562cells), which with high expression of Cyclin A, while the expression of Cyclin A in solid tumor (H7402cells) was low.Conclusion:Natural killer cells (NK cells) play an important role in immune surveillance against tumor. Immunomodulatory effects of HDACi, which can promote tumor cells to express MICA (the ligand of NK activation receptor NKG2D), regulate the expression of NK lysis-related miRNAs, and enhance tumor’s sensitivity to NK cytotoxicity, provides a new way to treat liver cancer. In this study, we first report that the enhancement of acetylation of histones associated with the MICA promoter is involved in the MICA upregulation induced by S AHA.Histone acetylation modification and post-transcriptional regulation mediated by microRNA are both important in epigenetic research, closely related to the development of tumors. Generally, the acetylation of histones marks active, transcriptionally competent regions, whereas hypoacetylated histones are found in transcriptionally inactive euchromatic or heterochromatic regions. However, there is considerable evidence that these models are oversimplified. Regulation of gene expression by acetylation is more highly selective than would be expected by a simple and unregulated physical disruption of histone-DNA structure, and also likely involves chromatin-associated nonhistone proteins. The discovery of miRNAs opens up a new field for the therapeutic targets of cancer, whereas little is known about the regulation mechanism of miRNA itself. This study explores the anti-tumor mechanism of the histone deacetylase inhibitor SAHA, which inhibits miR-17-92cluster via abolishing tyrosine phosphorylation of STAT3, under the conditions of high acetylated histone, loose chromatin, transcriptionally activate genomic status induced by itself. Our study indicates that non-coding genes including miRNAs are also under the epigenetic regulation.In addition, we screened a series of novel HDAC inhibitor based on the HDAC inhibition and growth inhibition in multiple tumor cell lines, made a study of the antitumor activity of ZYJ-34c, which was found to have promising antitumor potency. Using a variety of cell biology and molecular biology techniques, we explored its roles in inhibiting proliferation, arresting cell cycle and inducing apoptosis in human leukemia cells. We interpretated how ZYJ-34c-induced histone acetylation regulate the expression of relevant target protein p21WAF1. Finally we knocked down the expression of p21WAF1and demonstrated G1cell cycle arrest induced by ZYJ-34c was p21-dependent. This study constituted the theoretical foundation for development and application of ZYJ-34c as anticancer drugs. This study also provided a meaningful experiment reference for development of novel histone deacetylase inhibitors.