Long Noncoding RNAs Act As Competing Endogenous RNAs In Gastric Carcinogenesis

ObjectiveRecent studies showed that long noncoding RNAs (lncRNAs) contribute to oncogenesis and tumormetastasis. LncRNAs are involved in proliferation, apoptosis, invasion and metastasis of cancer cells.LncRNAs play biological roles by various mechanisms, including transcriptional regulation,post-transcriptional regulation and translational regulation. It is not easy to explore lncRNAs’ biologicalfunctions, because of their various kinds and roles.For the reason that gastric cancer is one of the most frequently occurring cancers in China, the presentstudy focused on this type of cancer. By using multiple biochemistry and molecular biology experimentaltechniques, we hope to demonstrate that affecting gastric carcinogenesis by acting as competingendogenous RNAs (ceRNAs) is one of lncRNAs’ biological roles.Methods1. LncRNAs differentially expressed between gastric cancer tissues and paracancerous tissues werescreened by lncRNA microarray. Then, lncRNA-miRNA-mRNA interactions were predicted by miRcodealgorithm and TarBase database.2. Since lncRNA–FER1L4is the most differentially expressed lncRNA in lncRNA microarrayscreening, to validate this result, its expression level in gastric cancer tissues and paracancerous tissues,human normal gastric epithelial cell line GES-1and human gastric cancer cell lines AGS, MGC-803, andSGC-7901was quantified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR).3. FER1L4and miR-106a-5p interaction was validated by dual luciferase reporter assay.4. The expression levels of FER1L4and PTEN mRNA, the targets of miR-106a-5p, were quantified byqRT-PCR after transfecting miR-106a-5p mimics into GES-1, AGS, MGC-803and SGC-7901cells.5. Small interfering RNA (siRNA) targeting FER1L4was designed and synthesized, then transfectedinto GES-1, AGS, MGC-803and SGC-7901cells. The expression levels of FER1L4, miR-106a-5p andPTEN mRNA were quantified by qRT-PCR, and the expression level of PTEN protein was analysed byWestern blot.6. After knockdown of FER1L4in human normal gastric epithelial cell line and human gastric cancercell lines, the cell cycle distribution and cell proliferation were analysed by flow cytometer and Real-TimeCell Analyzer (RTCA), respectively.Results1. Total53lncRNAs were found differentially expressed between gastric cancer tissues andparacancerous tissues (fold change≥3). A gastric cancer associated-ceRNA (lncRNA-miRNA-mRNA)network was constructed by bioinformatic method.2. The results of qRT-PCR with large samples of gastric cancer tissues and three gastric cancer celllines confirmed that FER1L4was low expressed in gastric cancer tissues and gastric cancer cells.3. Dual luciferase reporter assay indicated that FER1L4and miR-106a-5p was interacting directly. The former was one target of the latter.4. FER1L4and PTEN mRNA were down-regulated in GES-1, AGS, MGC-803and SGC-7901cellsafter transfecting miR-106a-5p mimics.5. siRNA effectively knocked down FER1L4in GES-1, AGS, MGC-803and SGC-7901cells,simultaneously up-regulated miR-106a-5p, and down-regulated PTEN in mRNA and protein levels.6. Mechanism analysis found that the reason of promoting cell proliferation by knockdown FER1L4,was promoting G0/G1to S phase transition.ConclusionsFER1L4, low expressed in gastric cancer tissues and gastric cancer cells, is one target of miR-106a-5p,one of onco-miRNAs. In human normal gastric epithelial cell line GES-1and human gastric cancer celllines AGS, MGC-803, and SGC-7901, FER1L4down-regulation leads to miR-106a-5p up-regulation andPTEN down-regulation simultaneously. Take together, FER1L4may act as ceRNA to regulate theexpression of PTEN, one of the famous tumor suppressor genes.