Expression And Molecular Mechanism Of Long Non-coding RNA HULC In Gastric Cancer

[Background] Gastric cancer (GC) is the second leading cause of cancer-related mortality globally. There are～750,000new cases diagnosed annually around the world and5-year overall survival rates are <25%.Early diagnosis and treatment is an important means to improve survival.With the progress in understanding the pathophysiological mechanisms and treatment for GC in recent years, a deeper understanding of the molecular and genetic networks that control the initiation and progression of GC is imperative.Long non-coding RNAs (lncRNAs) are a class of newfound non-coding RNAs,such as HULC, H19have been suggested to have a functional role in tumorigenesis and tumor progression.However, investigations of HULC in GC are scarce. This study tries to detect the expression level of HULC in GC and the effects of HULC on gastric cancer biological function, to reveal the function and molecular mechanisms of HULC in GC preliminary.[Objective] In the present study, we examined the role of HULC in GC and the potential mechanisms involved by a retrospective analysis of58GC patients, and by carrying outin vitro experiments to clarify the contribution of HULC to various aspects of the malignant phenotype of human GC,which provides us with a new biomarker in GC and perhaps a potential target for GC prevention, diagnosis and therapeutic treatment.[Methods]1. Fifty eight surgical samples of GC were collected, the expression levels of HULC were detected by qRT-PCR, the relationship between the expression of HULC and the clinicopathological factors of GC was analyzed; the expression of HULC levels in GC cell lineMKN28,MKN45,SGC7901,BGC823,AGS and human gastric epithelial mucosa cell line GES-1were detected by qRT-PCR.2. By lentivirus construction, stably transfected gastric cancer cell lines of HULC-downregulated and HULC-upregulated were established respectively, the effect of HULC on cell proliferation,apoptosis and invasion in vitro were detected by CCK-8assays, transwell assays,wound healing assays and flow cytometry.3. Changes in cell morphology were observed under inverted microscope before and after the HULC expression was changed; the changes on the protein level and mRNA level of EMT associated gene (E-Cadherin and Vimentin) in HULC-downregulated were detected by qRT-PCR and Western blot, respectively.4. Cells were treated with3-MA (an inhibitor of autophagic sequestration blocker),we also determined a change in the ratio of LC3-Ⅱ/LC3-Ⅰ using wstem blot, and apoptosis was detected using flow cytometry.[Results]1. lncRNA HULC is upregulated in GC tissues in comparison with the adjacent normal gastric tissues. Of note, its level was significantly associated with lymph node metastasis, distant metastasis. In addition, a receiver operating characteristic (ROC) curve was constructed to evaluate the diagnostic values and HULC could be used as a marker of GC. Compared with human gastric epithelial mucosa cell line GES-1, expression of HULC in BGC-823,SGC-7901and AGS cells increased significantly,while no significant difference were found in MKN28or MKN45cells.2. Stably transfected cell lines of HULC-upregulated and HULC-downregulated were established successfully, CCK-8assays showed that abnormally expressed HULC promotes the proliferation of GC cells.3. Wound healing assays and transwell assays confirmed that HULC positively regulates GC cell migration and invasion and deletion of HULC reverses EMT, indicating that HULC could act as a possible regulator of EMT. Under an inverted microscope, silencing of HULC in SGC-7901cells induced round spheroids with no or few protrusions. Western blot and qRT-PCR results showed that the downregulation of HULC levels in SGC7901cells was associated with upregulated E-cadherin and downregulated Vimentin both on the protein and mRNA levels.4. After using autophagy inhibitor3-MA, the flow cytometry assays and western blot showed upregulated HULC inhibits cell apoptosis by activating autophagy.[Conclusion] HULC,a cancer-related lncRNA,contributed to the malignant phenotype of GC cells through its regulation of diverse cellular processes, including proliferation, apoptosis, migration and invasion and its potential effect on autophagy and EMT.