Many miRNAs affect invasion and migration of cancer cells through directly regulating the inactivation of mRNA or the expressions of downstream effector molecules [15,16]

Many miRNAs affect invasion and migration of cancer cells through directly regulating the inactivation of mRNA or the expressions of downstream effector molecules [15,16]. function of FAT4 in CRC cells, thus playing a carcinogenic role by targeting FAT4 in the CRC cells. adipose tissues [8]. It was reported that expression of FAT4 is low-expressed in gastric cancer [9], endometrial cancer [10] and hepatocellular carcinoma [11]. A previous study found that overexpression of FAT4 promotes cell cycle, proliferation, invasion and migration of certain cancers and inhibits tumor cell apoptosis [12]. However, the role and mechanism of FAT4 in CRC are less reported. MicroRNAs (miRNAs) are non-coding RNAs that affect the stability of messenger RNA (mRNA) as negative regulators of protein translation, and regulate many signaling pathways and cellular processes to participate in intercellular communication [13,14]. Many miRNAs affect invasion and migration of cancer cells through directly regulating the inactivation of mRNA or the expressions of downstream effector molecules [15,16]. As FAT4 and miRNAs could affect the proliferation and migration of tumor cells, the current study aimed to determine the specific miRNA regulating FAT4 expression in CRC. In this research, we explored the role and underlying mechanism of FAT4 in proliferation, migration and invasion of CRC cells, hoping to provide theoretical basis for CRC treatment. Materials and methods Patient samples Fifty patients who were diagnosed with CRC from 2018 to 2019 in Guilin Peoples Hospital were selected as the research subjects. The CRC tissues and paired adjacent tissues from these patients were then collected. All the tissue samples were fixed by formalin and paraffin-embedded. The current study was approved by the Ethics Committee of Guilin Peoples Hospital Ethics Committee (approval number: SH20185665). The written informed consents were signed by all patients. Cell culture Human normal colon cell CCD-18Co and CRC cell line (LS174T, LOVO, HT29, HCT116 and SW-620) were purchased from American Type Culture Collection (ATCC, Manassas, Virginia, U.S.A.) and these cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS; Gibco, U.S.A.) at Walrycin B 37C with 5% CO2 in a humidified incubator. Cell transfection The cells were transfected with FAT4 siRNA and pc-DNA3.1-FAT4 plasmid (Shanghai Sangon Biotech, Shanghai, China). The primers were as follows: SiNC, 5-GCGCGATAGCGCGAATATA-3; pcNC RPD3L1 sense 5-UUCUCCGAACGUGUCACGUTT-3, and pcNC antisense 5-ACGUGACACGUUCGGAGAATT-3; Scramble, 5-TTCTCCGAACGTGTCACGT-3; miR-106b-5p mimics, 5-TAAAGTGCTGACAGTGCAGAT-3; miR-106b-5p inhibitor, 5-ATCTGCACTGTCAGCACTTTA-3. The cell Walrycin B transfection Walrycin B was performed using the Lipofectamine 2000 Kit (Invitrogen, Carlsbad, CA). The cells were cultured in an incubator with 5% CO2 at 37C for 4 days and prepared for further experiment. Grouping To Walrycin B investigate the function of FAT4 in CRC, the cells were divided into control group (untreated cells), siNC (cells transfected with siNC), pcNC group (cells transfected with pcNC), siFAT4 (cells treated with FAT4 siRNA), and pcFAT4 group (cells treated with pc-DNA3.1-FAT4 plasmid). Moreover, to further explore the effects of miR-106b-5p and FAT4 on the CRC cells, the cells were divided into Scramble+pcNC (cells transfected with scramble and pcNC), siNC group (cell were transfected with scramble and siNC), mimics+pcNC (cells transfected with miR-106b-5p mimic and pcNC), inhibitor+siNC group (cells transfected with miR-106b-5p inhibitor and siNC), Scramble+pcFAT4 (cells transfected with scramble and pc-DNA3.1-FAT4 plasmid), siFAT4 (cells transfected with scramble and FAT4 siRNA), mimics+pcFAT4 (cells transfected with miR-106b-5p mimic and pc-DNA3.1-FAT4 plasmid), and inhibitor+siFAT4 group (cells transfected with miR-106b-5p inhibitor and FAT4 siRNA). The quantitative real-time PCR analysis Total RNAs were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.). First-strand DNA was synthesized from 2 g of total RNAs for the detection of expressions of.