LM, and KB, provided the clinical samples and collected clinical information and MR participated in the coordination of the study and helped to draft the manuscript. JV performed the statistical PF-01367338 research buy analysis. All authors
read and approved the final manuscript.”
“Introduction Gastric cancer was one of the major causes of mortality in the world, especially in Asian countries. So far, the pathogenic mechanism underlying gastric carcinogenesis was not fully elucidated. MicroRNAs (miRNAs) were a class of 22-nucleotide noncoding RNAs, which might function as regulators of gene expression [1]. More and more evidences showed that miRNAs might play important roles in various biological processes, including cell proliferation, apoptosis, tumorigenesis and MDR of cancer [2]. So far, the functions of gastric cancer related miRNAs were not clear. MiR-27a might mediate drug resistance of esophageal cancer cells through regulation of MDR1 and apoptosis [3]. However, the role of miR-27a in gastric cancer was not reported yet. To our knowledge, here we have firstly investigated the role of miR-27a in proliferation and multidrug resistance of gastric cancer cells. ARS-1620 purchase Materials and methods Cell culture Human gastric cancer cell line, MKN45, was routinely maintained in DMEM medium (GIBCO, Carlsbad, CA,
USA) supplemented with 10% fetal bovine serum at 37°C in humidified air containing 5% carbon dioxide air atmosphere. MiRNA transfection Cells were plated in plates and cultured for 16 h, and then transfected with the antagomirs of miR-27a or control RNA (Lafayette, CO) as described previously [3]. Real-time
PCR Total RNAs from cells were extracted and cDNA synthesis and amplification were performed as described previously [4]. Primers were designed as: MDR1, forward: 5′-CCCATCATTGCAATAGCAGG-3′, reverse: 5′-TGTTCAAACTTCTGCTCCTGA-3′; cyclinD1, forward: 5′-GGAGCTGCTCCTGGTGAACA-3′, reverse: 5′-TGTTGGGGCTCCTCAG GTTCA-3′; P21, forward: 5′-CCCGTGAGCGATGGAACT-3′, reverse: 5′-CGAGGCACAAGGG TACAAGA-3′; P27, forward: 5′-CAAGTACGAGTGGCAAGAGG-3′, reverse: 5′-GTAGAA GAATCGTCGGTTGC-3′. Comparative real-time PCR PLEK2 was performed in triplicate, including no-template controls. Relative expression was calculated using the comparative Ct method. Cell growth assay Cells were seeded on a 96-well plate at 3 × 104 cells/well. Each sample has four replicates. Viable cells were counted by the MTT assay after 2, 4, 6, and 8 days. Soft agar assay Soft agar assay was performed as described previously [5]. Each assay was performed in triplicate. Tumor growth in nude mice Female Osimertinib datasheet athymic nu/nu mice, 5-6 weeks of age, were used in the experiment. The cells were resuspended in D’Hanks solution, and 5 × 106 cells in 0.2 ml were injected subcutaneously into the right flank of 4-week-age mice. Experimental and control groups had at least 6 mice each. Tumors were measured twice weekly, and the tumor volume was calculated.