The Epigenetic Molecular Mechanism Underlying The Malignancies Of Colorectal Cancer Regulated By Wnt Signaling

【Background】MicroRNAs(miRNAs)are a class of 21-25 nucleotide non-coding RNAs known to post-transcriptionally inhibit or degrade the expression level of target genes by specifically recognizing and binding to their 3’-UTRs.MiRNAs can participate in a wide range of biological processes in cells like differentiation,proliferation,apoptosis and development of tissues and organs.At the same time,they are important regulators in different malignant behaviors of tumor cells,since they are highly conserved,widely functional,and most of them are abnormally expressing and functioning during different carcinogeneses.Among these miRNAs,the widely down-regulated tumor-suppresive miRNAs in cancer cells are considered as important effector of activating oncogenes and promoting tumor progression.As a key pathway in cell development,Wnt/β-catenin signaling is dysregulated in a variety of cancers,including the overwhelming majority of colorectal cancers(CRC).The canonical aberrant Wnt/β-catenin signaling is initiated by the removal of tumor suppressor APC or activation of the proto-oncogene β-catenin and followed by nuclear accumulation of β-catenin and its constitutive binding to TCF4 to activate oncogenic downstream genes,such as c-Myc and CCND1.In addition to this activating regulation,several studies have revealed the repressive roles of TCF4 alone or together with β-catenin on the Wnt signaling targets.In addition to the tumorigenic and development-related genes involved,recent studies have reported the interesting possibility that miRNAs,often functioning as negative posttranscriptional regulators,are also embedded in the Wnt signaling network.Although these phenomena have been observed,the intricate mechanism of Wnt signaling-mediated repression of these anti-growth and anti-tumor miRNAs needs systematic elucidation.Our study will try to fill this knowledge gap.【Objectives】To study the specific mechanism of Wnt-regulated miRNAs and to explore the functions of these miRNAs in CRC malignant behaviors,with the aim of offering novel targets fort the diagnosis and treatment of CRC.【Methods】Part 1: The candidate miRNAs negatively regulated by Wnt signaling were scanned and selected by analyzing a ChIP-seq dataset against TCF4 in HCT116 cells,detecting alterations of miRNA levels after interfering with endogenous TCF4 and carrying out ChIP-qPCR tests in the promoter regions of these miRNAs;The expression level of the candidate miRNA was tested by qPCR after activating or blocking Wnt signaling to confirm the negative regulation of Wnt on it;The enrichment of TCF4-β-catenin in the promoter region of candidate miRNA was detected by ChIP-qPCR assay.Part 2: The expression difference of the objective of study,miR-145,in primary and metastatic CRC was analyzed in GEO datasets and clinical tissue specimen from CRCpatients;The effect of miR-145 on the invasion and metastasis of CRC cells in vitro was detected by Wound-healing and Transwell assays;The effect of miR-145 on the invasion and metastasis of CRC cells in vivo was tested in the liver and lung metastatic tumor model of CRC on nude mice.Part 3: The target gene of miR-145 when it inhibits the CRC cell invasion and metastasis was verified using luciferase reporter assay and Western Blot;If miR-145 suppresses the invasion and metastasis of CRC cells through repressing the target gene was confirmed by “rescue experiment”;The correlation between miR-145 and its target gene was detected in the liver and lung metastatic tumor model of CRC on nude mice and clinical tissue specimen from CRC patients.Part 4: The enrichments of H3K27me3 and its trimethylation enzyme PRC2 complex in the putative promoter region of miR-145(pmiR-145p)were detected by ChIP-qPCR assays and the alterations of miR-145 level were observed after treatment of GSK-J4 to raise the overall level of H3K27me3 in CRC cells and after transfection of siEZH2/siSUZ12 to interfere with the binding of PRC2 complex;Co-IP and Re-ChIP assays were carried out to test if the TCF4-β-catenin and PRC2 complexes can directly bind with each other at the TBE locus;Similarly,the enrichment of H3K27me3 and PRC2 complex in the promoter region were detected by ChIP-qPCR assays and the alterations of expression were detected after CRC cells were treated with GSK-J4 or siEZH2/siSUZ12 to test if the same negative regulation pattern also worked for other candidate miRNAs repressed by Wnt signaling.Part 5: After mimic miR-145 was transfected into CRC cells,the transcriptional activity of Wnt signaling was tested using TOP-Flash/FOP-Flash reporter assay,the expression of pre-miR-145 was detected by qPCR and the enrichment alterations of the negative regulators in pmiR-145 p were tested by ChIP-qPCR assays to confirm if miR-145 can reversely target Wnt signaling to form a double-negative regulation loop;The target genes of miR-145 when it reversely suppresses its negative regulators were verified using RNA Hybrid analysis,Western Blot and luciferase reporter assay;The double-negative regulation loop between miR-145 and its negative regulators wasconfirmed in tumor growth model on nude mice and clinical tissue specimen from CRC patients.【Results】Part 1: After interfering with the endogenous expression of TCF4,miR-145 showed the highest up-regulation and miR-145 had the most significant enrichment of TCF4 in its promoter region,leading us to select miR-145 as the object of study;After Wnt signaling was activated and blocked,the expression of miR-145 was decreased and increased,respectively;A TCF4 binding site(TBE)was found in pmiR-145 p and significant enrichment of TCF4-β-catenin was observed near the TBE locus.Part 2: Analyses from 2 GEO datasets and strictly matched primary and metastatic tumor tissues from 41 CRC patients indicated that the expression of miR-145 is significantly down-regulated in metastatic CRC;Results from Wound-healing and Transwell assays demonstrated that miR-145 inhibits the invasion and metastasis of CRC cells in vitro;The assessments of metastatic tumor models on nude mice revealed that miR-145 inhibits both the liver and lung metastasis of CRC cells in vivo.Part 3: Results from Western Blot and luciferase reporter assay revealed that miR-145 targets LASP1 through direct binding to its 3’-UTR;The “rescue experiments” showed that the rescue of LASP1 expression can partially neutralize the anti-invasion and anti-metastasis effect of miR-145,indicating that miR-145 does inhibit the invasion and metastasis of CRC cells through targeting LASP1;The expressions of miR-145 and LASP1 were reversely correlated in both liver and lung metastatic tumor models on nude mice and clinical tissue specimen from 41 CRC patients.Part 4: ChIP-qPCR assays revealed significant enrichment of H3K27me3 and PRC2 complex at the TBE locus of pmiR-145 p and after treatment of GSK-J4 and siEZH2/siSUZ12,the expression of miR-145 went down and up,respectively;Results from Co-IP and Re-ChIP assays showed that TCF4-β-catenin complex can recruit the PRC2 complex to the TBE locus of pmiR-145 p and thus inhibiting the expression of miR-145 through trimrthylation of H3K27 in this region;In other candidate miRNAsnegatively regulated by Wnt signaling,miR-132/212 had the similar regulation pattern with both TCF4-β-catenin and PRC2 complexes involved in the promoter region.Part 5: After transfection of mimic miR-145 into CRC cells,the transcriptional activity of Wnt signaling was decreased,the expression of pre-miR-145 was stably and continuously up-regulated and the enrichment of TCF4-β-catenin and PRC2 complexes at the TBE locus in pmiR-145 p was decreased,indicating that miR-145 can reversely suppress its negative regulators;Results from Western Blot and luciferase reporter assay revealed that miR-145 targets TCF4 and SUZ12 through direct binding to their 3’-UTRs,thus forming a double-negative regulation loop with its negative regulators;The expressions of miR-145 and its negative regulators/targets were reversely correlated in both tumor growth models on nude mice and clinical tissue specimen from CRC patients.【Conclusions】1.miR-145 inhibits the invasion and metastasis of CRC through directly targeting LASP1;2.TCF4-β-catenin complexes can bind to the TBE in the promoter region of miR-145 and other tumor-suppresive miRNAs,then recruit the histone trimethylase PRC2 to the same locus.When activated by Wnt signaling,PRC2 trimethylates the promoter region and slows the production of miR-145 and other tumor-suppresive miRNAs;3.miR-145 targets TCF4 and SUZ12,thus reversely inhibiting the molecules that suppress it in a double-negative regulation loop.