The microRNAs (miRNAs) are an extensive class of small noncoding RNAs, 18 to 25 nucleotides in length, with the role of regulating gene expression through translational repression by base-pairing with partially complementary mRNAs.1) Currently, more than 3000 miRNAs have been annotated from a wide range of organisms including plants, worms, flies, mammals, and viruses. 2,3) The expression of miRNAs is regulated developmentally and spatially, and is involved in cell differentiation, proliferation, and death. 2)Recently several reports have indicated the involvement of miRNAs in human cancer and further that miRNAs might be used in future therapeutic and diagnostic applications.4) Especially, the expression level of let-7 miRNA was reduced in human lung cancer, 5) and it was shown to be the anticancer miRNA that represses RAS and/or c-MYC expression at the translational level.6) On the other hand, it has been established that genetic aberration of RAS and over-expression of c-myc are frequently involved in colon cancer. 7-9) Such findings led us to speculate that let-7 may also be reduced in its expression in colon cancer cells.In the current study, we focused on the expression of let-7 miRNA in colon cancer and its expression profile. Our functional analysis indicates that let-7 may function as one of the growth suppressive miRNAs in human colon cancer cells. MATERIALS AND METHODS Patients and Tissue PreparationAll human tissue samples were specimens from patients who had undergone biopsy for histological diagnosis of colon cancer at Saiseikai Ibaraki hospital, Ibaraki, Osaka, between 2000 and 2003. Informed consent in writing was obtained from each patient. The tumor and its adjacent normal tissues were also obtained. Each specimen was divided into 2 parts. One of them was subjected to Western blot analysis, and the other was used for extraction of RNA and DNA.Cell Culture and Cell Viability Human malignant cell lines SW480, DLD-1, and COLO101 (colon cancer); HL60 (myeloid leukemia); U937 (monocytic leukemia); Jurkat (Tcell leukemia); PC3 and LNCaP (prostate cancer); SH-SY5Y (neuroblastoma); HeLa (cervical cancer); and HuH7 (hepatic cancer) were grown in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated FBS (Sigma, St. Louis, MO, U.S.A.) and 2 mM L-glutamine under an atmosphere of 95% air and 5% CO 2 at 37°C. The number of viable cells was determined by the trypan-blue dye exclusion test. Plating efficiencies (the average number of colonies per 60-mm dish divided by the number of cells seeded per dish (ϫ100) were determined 14 d following inoculation of 1000 cells. The ability of the cells to grow as colonies in or on the soft agar was assessed. Briefly, 10 3 individual trypsinized cells were suspended in 5 ml RPMI 1640 medium/10% FBS/0.35% liquid Bacto-agar (Difco Laboratories. Inc.) at 40°C, immediately poured onto a 4-ml basal layer of 0.5% Bactoagar/RPMI 1640 medium/10% FBS in a 60-mm dish, and incubated at 37.5°C. The dishes were examined for the presence of colonies growing in the 0.35% agar ...

Abstract. MicroRNAs (miRNAs) are endogenously expressed RNAs 18-24 nucleotides in length that regulate gene expression through translational repression by binding to a target mRNA. It is thought that the expression level of miRNAs, which act like antioncogenes, is frequently reduced in cancers because of chromosome deletion and epigenetic changes. Since the processing of miRNAs has been characterized to be enzymatic in nature, the expression levels of miRNAs are closely associated with the activity and levels of such enzymes. Here, we found that miRNA 143 and 145 expression levels were extremely reduced in colon cancer cells and commonly in the other kinds of cancer cells tested. The transfection of each precursor miRNA into the cells demonstrated a significant growth inhibition in human colon cancer DLD-1 and SW480 cells, and ERK5 was determined to be the target gene of miRNA 143. Since the presence of genomic loci of the miRNAs was confirmed by PCR in the cell lines and the precursor miRNAs exhibited a growth inhibitory effect in DLD-1 and SW480 cells, the early processes such as transcription and enzymatic modification from primary miRNAs to precursor miRNAs seemed to be commonly disturbed. These findings indicate that the miRNAs 143 and 145 could become good tumor markers and provide an important clue in the study of the mechanism of oncogenesis involving miRNAs.

Objective: Downregulation of specific microRNAs (miRNAs) occurs in human tumors, which suggests a function for miRNAs in tumor suppression. We investigated the role of the miRNAs miR-143 and miR-145 in gastric cancers. Methods: The expression levels of miR-143 and miR-145 in the samples from 43 patients with gastric cancer were determined by real-time PCR using TaqMan assay. The growth inhibitory effect was estimated by the transfection of human gastric cancer cells with the miRNA. Results: The expression levels of miR-143 and -145 were decreased in most human gastric cancers examined, as previously reported to occur in colon tumors. The transfection of human gastric MKN-1 cells with miR-145 resulted in a greater growth inhibitory effect than that with miR-143, results which were contrary to those in colon cancers. In MKN-1 cells, an additive effect on growth inhibition was shown by the combined transfection with miR-143 and miR-145; further, higher sensitivity to 5-fluorouracil was also observed following the transfection with miR-143 or miR-145. The possible candidate target messenger RNAs of miR-145 were identified to be insulin receptor substrate-1 and β-actin. Conclusion: Taken together, these findings suggest that miR-143 and miR-145 act as anti-oncomirs common to gastrointestinal tumors.

Recently, it has been found that inappropriate expression of microRNAs (miRNAs) is strongly associated with carcinogenesis. In this study, we demonstrated that the expression of miRNAs (miRs) -143 and -145, the levels of which were previously shown to be reduced in colon cancers and various kinds of established cancer cell lines, was also decreased in most of the B-cell malignancies examined, including chronic lymphocytic leukemias (

Mangosteen, Garcinia mangostana Linn, is a tree found in South East Asia, and its pericarps have been used as traditional medicine. Phytochemical studies have shown that they contain a variety of secondary metabolites, such as oxygenated and prenylated xanthones. Recent studies revealed that these xanthones exhibited a variety of biological activities containing anti-inflammatory, anti-bacterial, and anti-cancer effects. We previously investigated the anti-proliferative effects of four prenylated xanthones from the pericarps; α-mangostin, β-mangostin, γ-mangostin, and methoxy-β-mangostin in various human cancer cells. These xanthones are different in the number of hydroxyl and methoxy groups. Except for methoxy-β-mangostin, the other three xanthones strongly inhibited cell growth at low concentrations from 5 to 20 μM in human colon cancer DLD-1 cells. Our recent study focused on the mechanism of α-mangostin-induced growth inhibition in DLD-1 cells. It was shown that the anti-proliferative effects of the xanthones were associated with cell-cycle arrest by affecting the expression of cyclins, cdc2, and p27; G1 arrest by α-mangostin and β-mangostin, and S arrest by γ-mangostin. α-Mangostin found to induce apoptosis through the activation of intrinsic pathway following the down-regulation of signaling cascades involving MAP kinases and the serine/threonine kinase Akt. Synergistic effects by the combined treatment of α-mangostin and anti-cancer drug 5-FU was to be noted. α-Mangostin was found to have a cancer preventive effect in rat carcinogenesis bioassay and the extract from pericarps, which contains mainly α-mangostin and γ-mangostin, exhibited an enhancement of NK cell activity in a mouse model. These findings could provide a relevant basis for the development of xanthones as an agent for cancer prevention and the combination therapy with anti-cancer drugs.