Study On The Function And Mechanism Of LncRNA Involved In Helicobacter Pylori Infection

Gastric cancer （GC） is one of the most frequently diagnosed gastrointestinal neoplasms in the world, and the third cause of cancer deaths worldwide. Gastric cancer usually lead to the high mortality, and the 5 year survival rate is only about 20%. Its poor prognosis leads to serious threat to human health. Recent findings implicate lncRNAs as key players of cellular differentiation, cell lineage choice, organogenesis and tissue homeostasis. The object of this study is to screen ectopic expression of lncRNAs in Helicobacter pylori （HP） related gastric carcinoma, providing molecular mechanism for HP related GC diagnosis and therapy.Previously, microarray analysis was used to screen ectopic expression of lncRNAs based on HP infected gastric epithelial （GES-1） cells, HP positive chronic atrophic gastritis and gastric cancer clinical tissue samples. We found 124 upregulated lncRNAs and 81 downregulated lncRNAs which fold changes were more than 10 times in HP infected GES-1 cells for 72 hours compared to normal GES-1 cells using Agilent Arraystar lncRNA chip. And 259 upregulated lncRNAs and 221 downregulated lncRNAs in both gastric carcinoma samples and gastritis samples compared to normal tissues were found in microarray data. We detected 18 lncRNAs those were differentially expressed in both two chips. Then we finally tested and verified aberrant expressions of NR₀38975, NR₀03605, LncHP, PVT1, LINC00260 with Real-time PCR in both HP related inflammatory cell model and GC tissues. Results showed that expression trends of NR038975, LncHP, PVT1 are conformed to those in chip. According to the above verification results and the expression level, expression abundance, bioinformatics analysis, we finally choose LncHP, PVT1 and LINC00260 as further analysis lncRNAs.In order to explore the fuction of LncHP, PVT1 and LINC00260. We further investigated effect of LncHP, PVT1 and LINC00260 on proliferation and migration of GES-1, SGC-7901, MGC-803 and AGS cells with MTS assay and wound healing assay. When knocking down the expression of LncHP, PVT1 or LINC00260, cell migration of gastric cancer cells was significantly inhibited. And inflammatory cytokines such as TNFα, IL-1β and IL-8 were significantly downregulated in HP infected gastric cancer cells when expression of LncHP, PVT1 or LINC00260 were inhibited, which indicates that LncHP, PVT1 and LINC00260 may participate in inflammatory signal pathways.Then we further demonstrated that both LncHP and LINC00260 had polyA tail structure using RT-PCR reverse transcribed with random primers or oligo（dT） primers. FISH assay and Cytoplasm/nucleus separation experiment showed that LncHP mainly distributed in the nucleus of normal gastric epithelial cells and gastric cancer cells. Surprisingly, we found that the expression of LncHP in cytoplasm was increased and expression in nucleus was decreased in GES-1 and AGS cells after HP infection. Results indicated that LncHP might play a key role in gastric cancer caused by HP. Using RACE method, we demonstrated LncHP is a 1164 full-length transcript, and it might has two spliced variants.Taken together, we first systemically studied the aberrent lncRNAs expression in HP infected GC both in cells and tissues. The function and ectopic expression of LncHP, PVT1, LINC00260 were in-depth study. Results showed that LncHP and PVT1 were significantly upregulated in both inflammatory model and GC tissues. LINC00260 is significantly downregulated in inflammatory cell model. Further results showed that LncHP, PVT1 and LINC00260 promoted the migration of gastric cancer cells and might play regulatory roles in cell inflammatory response. LncHP was located in nucleus, and might transferred to cell cytoplasm in HP infected GC cells.Therefore, this study found that LncHP was a lncRNA related to the gastric carcinoma caused by HP infection, and might be new molecular targets for the diagnosis and treatment of gastric cancer.