| Objective:1. To study the potential biological function of lncRNA HEIG and validate its carcinogenic activity in vivo.2. To screen lncRNA HEIG target genes, validate the regulatory effect of HEIG on its target genes via the corresponding signaling pathways, and further explore whether HEIG participate in the molecular mechanisms of cell apoptosis, invasion and migration in gastric cancer.Methods:1. Large scale expression profiling of lncRNA and mRNA in samples of gastric cancer tissues and their matched adjacent normal tissue was performed with lncRNA microarray technology and validated by qPCR. Differentially expressed lncRNAs were subjected to bioinformatic analysis for target gene prediction, and findings were integrated with differentially expressed mRNA data. GO and pathway analysis were performed to determine associated functions and pathway of the predicted targets.2. After HEIG overexpression and RNA interference, the MTS assay, colony formation assay, flow cytometry, TUNEL assay, Transwell migration and invasion assays were applied to evaluate the effects of HEIG on cell biological behaviors. Subsequently, xenograft-transplanted NOD/SCID mouse tumor model of human gastric cancer growth was used to reveal the carcinogenic potential of HEIG in vivo.3. Microarray screening for HEIG target genes was performed by human apoptosis qPCR array and human tumor metastasis qPCR array. Subsequently, the corresponding signaling pathway for it target genes were analyzed. QPCR and western blot assay were performed to validate the regulatory effect of HEIG on its target genes and corresponding signaling pathway.Results:1. The lncRNA and mRNA expression profiling data showed that 2,621 lncRNAs and 3,121 mRNA were differentially expressed (fold change≥2.0) in gastric cancer samples relative to their matched counterparts. The qPCR results demonstrated that the expression trends of 8 differentially expressed lncRNAs and 8 differentially expressed mRNAs, which were randomly selected, were consistent with the microarray data. In addition, the lncRNA-mRNA correlation network was constructed to include differentially expressed lncRNAs and their target genes. Pathway analysis showed that lncRNA target genes were significantly enriched in 7 different pathways, including p53 pathway, apoptosis pathway and Jak-STAT signaling pathway, etc.2. The qPCR technology demonstrated that a new lncRNA HEIG which screened via lncRNA microarray high expressed in gastric cancer tissues and 4 gastric cancer cell lines compared with normal controls. HEIG could promote cell proliferation, enhance the colony formation, inhibit apoptosis and promote cell invasion and migration in SGC-7901 and HGC-27 cells. Animal experiments of human gastric cancer xenograft-transplanted NOD/SCID mouse models revealed that HEIG had the carcinogenic potential in vivo. 3. The qPCR microarray screening for HEIG target genes showed that 7 target genes were involved in cell apoptosis and 3 target genes were implicated in tumor metastasis of gastric cancer. Among these target genes, PI3K, PTEN, AKT, Bcl-2 and Bcl-XL were found to involve in PI3K-AKT signaling pathway. We validated the regulatory effect of HEIG on its target genes by qPCR in SGC-7901 and HGC-27 cells. When lncRNA HEIG and PTEN were simultaneously overexpressed, mRNA and protein expression levels of caspase-3, caspase-6, Bcl-2, Bcl-XL, MMP2, MMP9 and phosphorylation levels of AKT were all reversed.Conclusion:Overall, the results of the present study suggest that HEIG has the carcinogenic potential in gastric cancer, and it inhibits the apoptosis and promotes the invasion and migration in gastric cancer cells through regulating the PI3K-AKT signaling pathway. |
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