| The mistletoe (Viscum album) is a semi-parasitic plant widely spread over Europe and parts of Asia. Aqueous mistletoe extracts have been used as a complementary cancer drug for almost a century. The main biological activity of the extracts has now been attributed to the mistletoe lectins (MLs). The mistletoe contains at least three lectins: I, II, and III. ML-I is known to be the major active constituent of natural mistletoe extracts. ML-I consists of two subunits (A and B) and belongs to the family of ribosome inactivating proteins (RIPs), type II. Subunit A exhibits RNA N-glycosidase activity and selectively cleaves the N-glycosidic bond of the adenine-4324 residue in the conserved GAGA hairpin loop of the eukaryotic 28S rRNA. This effect leads to a catalytical inactivation of ribosomes, and thereby inhibits translation and protein synthesis. The B subunit binds to galactose containing cell surface receptors of glycoproteins and glycolipids, thereby facilitating penetration of the toxic A-chain to the cytosol and promoting subsequent ribosome inactivation. RIPs were originally thought to act exclusively on ribosomes or rRNA. Recently, evidence is accumulating that RIPs are also capable of inactivating many nonribosomal nucleic acid substrates. Although the rRNA in native ribosomes is the preferred substrate for RIPs, naked (deproteinized) rRNA, tRNA, DNA, poly(A) and even a synthetic 35-residue oligoribonucleotide that mimics the single-stranded GAGA loop, can also serve as the substrate for RIP activity. The obvious capacity of RIPs to deadenylate different polynucleotides implies that they can be considered polynucleotide:adenosine glycosidases. Therefore, it is reasonable to hypothesize that other important RNA, such as microRNA, may also be the substrate for RIPs.MicroRNAs (miRNAs) are a recently discovered class of small noncoding RNAs that negatively regulate the expression of genes involved in development, differentiation, proliferation, apoptosis and other crucial biological process. The genes encoding miRNAs is typically transcribed by RNA polymerase II or III as primary miRNAs (pri-miRNAs), which range from a few hundred to thousand of nucleotides (nt) in length. The pri-miRNA of each miRNA has a characteristic stem-loop structure that can be recognized and cleaved by ribonuclease Drosha within the nucleus. The cleavage product, a ~70-nt precursor hairpin (pre-miRNA), is exported from the nucleus by Exportin-5-Ran-GTP. In the cytoplasm, the ribonuclease Dicer further processes the pre-miRNA hairpin into its mature miRNA (~21 nt). The functional strand of the mature miRNA is loaded together with Argonaute (Ago2) proteins into the RNA-induced silencing complex (RISC), where it guides RISC to silence target mRNAs through mRNA cleavage, translational repression or deadenylation, whereas the passenger strand is degraded. In view of the ability of RIPs to remove an adenine in a conserved loop in the 28S rRNA, we hypothesize that some pri-miRNAs or pre-miRNAs would also be the substrate for RIPs (such as mistletoe lectins) due to their characteristic stem-loop structures and the specific mature miRNAs levels would be down-regulated eventually due to the destruction of their precursors.In the prior study, a mistletoe lectin named CM-1 was isolated from leaves of the Chinese mistletoe harvested from poplar between November and February and purified using affinity chromatography and cation exchange chromatography. The further analysis of cDNA sequence indicated A-chains of CM-1 showed 91% of identity to that of European mistletoe lectin-1 (ML-1) at primary structure level and the key active sites residues Tyr76, Tyr115, Glu165, Arg168 and Trp199, essential for the RNA N-glycosidase activity of mistletoe lectin, were not mutated. While the European and Korean mistletoe have been studied intensively, less is known about the Chinese mistletoe as an anti-cancer drug. So we want to research the CM-1. The first aim of this study is to analyze the anti-cancer effects of purified CM-1 on colorectal cancer cell growth. The second aim is to identify the potential substrate miRNAs for CM-1and analyze the mechanisms responsible for the down-regulated effects of the specific miRNAs induced by CM-1. The third aim is to investigate the down-regulated effects of the specific miRNAs on their target genes and downstream signaling pathways. Furthermore, the correlation between the specific miRNAs levels and CM-1 sensitivity has also been investigated in this study.The result revealed that CM-1 showed remarkable anti-neoplastic activity towards colorectal cancer cells both in vitro and in vivo. Besides inhibition of protein synthesis caused by catalyzing an irreversible damage to ribosomes through the RNA glycosidase activity of CM-1, the mistletoe lectin also induced the down-regulation of some specific miRNAs, such as miR-135a and miR-135b, and this effect mainly attributed to the damage on the precursors of these miRNA. Moreover, the target gene APC and the downstream Wnt pathway of miR-135a&b were regulated correspondingly. Furthermore, there existed a direct correlation between miR-135a&b levels and cellular sensitivity towards CM-1, according to the cell proliferation assay carried on some stably transfected cell lines with different miR-135a&b levels. These fundings indicate CM-1 had the ability to down-regulate some specific miRNAs by depurinating and destroying their precursor miRNAs, further regulate their target genes and downstream signaling pathway, and eventually affect the proliferation of some specific cell lines.
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