4B). strong class=”kwd-title” Keywords: Scirrhous gastric cancer, Peritoneal dissemination, Methyltransferase-like (METTL) gene, Short hairpin RNA, Molecular targeting therapy against cancer metastasis 1.?Introduction Adaptation of cells to various environmental and physiological conditions is essential for cell survival and development. In a phenomenon called cell reprogramming, cells quickly change their transcriptomic and metabolic dynamics to adapt to extracellular conditions [1,2]. Cell reprogramming is achieved in part by chemical modifications such as methylation, acetylation, and phosphorylation [3,4]. Chemical modifications of DNA, RNA, and proteins substantially affect the stability, activity, and functions of cell components [5]. In particular, methylation on cellular components mediated by over 200 enzymes, is attentive topic for post-transcriptional and post-translational regulation [6]. Furthermore, the aberrant regulation of chemical modification leads to the onset of diverse diseases, including cancers [7]. Human scirrhous gastric cancers are a poorly differentiated and aggressive type of gastric cancer that frequently show peritoneal dissemination, leading to poor prognosis [8,9]. For patients harboring peritoneal dissemination, effective therapeutics have not yet been established because the molecular mechanisms of peritoneal dissemination of the cancer are not fully understood. To elucidate the molecular mechanisms underlying peritoneal dissemination, we previously established patient-derived metastatic cell lines in human scirrhous gastric cancers [10]. By analyzing metastatic cell models, we discovered the expression alteration of methyltransferase-like (METTL) genes. The METTL gene family consists of 27 members, some of which are known as representative writers of N6-methyladenosine (m6A), which is the most abundant chemical modification in an RNA molecule [11]. It is known that dysregulation of METTL3 induces abnormal m6A abundance, which plays important roles in the progression of various cancers [12]. However, the functions of most METTL family genes, including cancer metastasis, remain unclear. So far, the biological functions of METTL9 have been exhibited only in autosomal recessive deafness 22 by genetic analysis [13]. Here we aim to investigate the functions of METTL9 genes in the peritoneal dissemination of human scirrhous gastric cancer using our metastatic cell model and clinical samples (gastric cancer tissues) from patients. As a result, we discovered that elevated METTL9 expression can function to positively promote peritoneal dissemination in scirrhous gastric cancers. 2.?Material and methods 2.1. Cells HSC-58?cells (parental cells) were originally established from a patient with scirrhous gastric cancer [10]. 58As9 cells (metastatic cells) were successfully isolated by repeating orthotopic implantation of HSC-58 in nude mice, as described previously [10]. These cell Bis-PEG4-acid lines were maintained in PRMI-1640 medium (Sigma-Aldrich Japan, Tokyo, Japan) containing 10% fetal bovine serum (FBS) at 37?C in a 5% CO2 incubator. 2.2. Short hairpin RNA (shRNA) expression vector For the METTL9 knockdown, Bis-PEG4-acid an SGEP vector provided by Dr. Johannes Zuber was used [14]. We used the splashRNA algorithm [15] to select target sequences for METTL9 according to the instructions. As a negative Bis-PEG4-acid control, shRNA against a Renilla luciferase was used. After the introduction of the SGEP-based vectors into 58As9 cells (metastatic Bis-PEG4-acid cells), the cells were cultured in the presence of puromycin at 4?g/ml for 1 week. Further, the largest portion of GFP-positive cells ( 10%) was isolated by an SH800 Cell Sorter (SONY, Tokyo, Japan). The target sequences of the two shRNAs for human METTL9 were as follows: shMETTL9#1: 5-TTAAGTATAAAAAATATCTTCC-3. shMETTL9#2: 5-TTAAATTCCTACATGATATTTA-3. Information on the sequences of shRNAs targeting the human METTL9 gene is summarized in Supplementary Table 1. 2.3. Antibodies We purchased anti-human METTL9 antibody (15120-1-AP) from Proteintech Japan (Tokyo, Japan) and anti–actin antibody (A5441) from Sigma-Aldrich Japan (Tokyo, Japan). 2.4. Reverse transcription quantitative PCR (RT-qPCR) To isolate total RNAs, ISOGEN reagent (Nippon Gene, Tokyo, Japan) was used according to the manufacturer’s instructions. Normal human stomach tissue RNAs were Bis-PEG4-acid purchased from TaKaRa Bio (Shiga, Japan). iScript RT Supermix for RT-qPCR (Bio-Rad Laboratories, Hercules, CA, USA) was employed for the synthesis of first-strand cDNA. qPCR was carried out with the StepOne Real-time PCR System (ThermoFisher Scientific). The relative expression level was calculated by the ddCq method using the level of the reference gene, succinate dehydrogenase complex subunit A (SDHA). The sequences of the primer sets used in this study were as follows: SDHA forward, 5-TGGGAACAAGAGGGCATCTG-3 SDHA reverse, 5-CCACCACTGCATCAAATTCATG-3 METTL9 forward, 5-CTGTGATCAGCCCCTGACTT-3 METTL9 reverse, 5- TGATGGTTTCTCCCACTTGCC-3 Rabbit polyclonal to ADCYAP1R1 Information on the sequences of specific primers to determine human METTL9 gene expression is summarized in Supplementary Table 2. 2.5. Immunoblotting Total cell lysates were isolated using RIPA buffer (ThermoFisher Scientific).