The Role And Molecular Mechanism Of Long Noncoding RNA LINC00675 In Gastric Cancer Progression

Background:Gastric cancer?GC?is the most common malignancy worldwide.In the United States,26,370 new cases and 10,730 deaths occurred in 2016.A worse situation appears in China,where the incidence and mortality for GC both rank second among total cancers.Although the incidence of GC has decreased in western countries,the overall 5-year relative survival rate remains approximately 20%in most areas of the world;for stage IV disease,this rate is less than 5%.Surgical resection for early GC has a curable effect,but most patients with GC have advanced-stage disease when diagnosed.Therefore,it is urgent to explore novel biomarkers and therapeutic targets for the diagnosis and treatment of GC.Long noncoding RNAs?lncRNAs?are a class of noncoding RNAs>200 nucleotides in length that do not encode proteins.The ENCyclopedia of DNA Elements?ENCODE?project has indicated that more than 80%of the human genome transcribes RNA products and has biological effects,but less than 2%of the total genome encodes proteins,suggesting that abundant noncoding RNAs have potential bio-functions.In recent years,increasing evidence has shown the role of IncRNAs in cancer development,such as HOTAIR,H19,and Xist.In GC?the IncRNA HOXA11-AS functions as a scaffold to recruit EZH2 and LSD1,resulting in a complex-integrated phenotype affecting cell growth,migration,invasion,and apoptosis.Several IncRNAs in serum or tissue were considered as biomarkers for the diagnosis and prognosis of GC.Until now,many IncRNAs have not been functionally characterized,and the mechanism underlying their biological effects on the development of GC remains largely unknown.The present study aimed to identify a novel lncRNA with aberrant expression in gastric cancer?GC?progression,and investigate its role and molecular mechanism in GC progression.Methods:1.The IncRNAs with aberrant expressions in GC tissues were screened using lncRNA microarray.2.Candidate lncRNAs were selected and identified using qRT-PCR experiment,and finally a novel IncRNA,termed LINC00675?gastric cancer-associated suppressor of Vimentin?,was locked to investigate.3.LINC00675 expression was analyzed in large-scale samples of GC and adjacent normal tissues using ISH and qRT-PCR,and the relationship between LINC00675 expression and clinical characterization was assessed.4.GC cells with stable expression of LINC00675 were constucted using lentivirus containing pcDNA3.1 vector and shRNA sequence.5.The role of LINC00675 in regulating GC cells was evaluated using CCK-8,colony formation,Trans-well,nude mouse models with subcutaneous injection or caudal vein injection or peritoneal cavity injection.6.pcDNA3.1-LINC00675-MS2 and GFP-MS2 vectors were constructed and transfected into GC cells,and the complex interacting with LINC00675 were extracted using RIP experiment.7.The complex were subject to isolation using SDS-PAGE,and stained using silver,and finally the component was analyzed using mass spectrum.8.The protein interating with LINC00675 was identified using Western blot experiment.9.The complex interating with the protein was extracted using RIP experiment based on an antibody of the protein,and the enrichment of LINC00675 in the complex was detected using qRT-PCR experiment.10.The expression and phosphorylation level of the binding protein if regulated by LINC00675 were detected using Western blot experiment.11.The gene expression profile in LINC00675-overexpressing GC cells was analyzed using genome-wide transcript microarray.Results:1.The aberrant expression profile of lncRNAs in GC tissues was screened using lncRNA microarray.The candidate lncRNA LINC00675 was selected,and the relationship between its expression and clinical characterization was evaluated.?1?LncRNA microarray experiment revealed 154 lncRNAs with aberrant expressions,among which 59 lncRNAs were overexpressed,and 95 lncRNAs were downregulated.In the downregulated lncRNAs,the expression of LOC400573 was decreased by 5 fold in GC tissues compared with adjacent normal tissues?ranked the third?.Currently,the expression and role in GC had not been explored.LOC400573 was called LINC00675.?2?LINC00675 expression was found to be significantly decreased in GC tissues compared with adjacent normal tissues using qRT-PCR and ISH experiments.ROC curve analysis showed that LINC00675 expression had an AUC of 0.7256 in distinguishing GC tissues from adjacent normal tissues,with sensitivity of 78.05%and specificity of 72.5%.GC patients with decreased expression of LINC00675 had more poor survival rates than those with increased expression of LINC00675.?3?Clinical data analysis showed that LINC00675 expression was correlated with tumor size,TNM stage,lyphnode metastasis,and distal metastasis.2.Study on the role of LINC00675 in vitro.?1?GC cells?MKN-45 and SGC-7901?with stably inhibitory or enforced expression of LINC00675 were respectively constructed.?2?CCK-8 and colony formation experiments showed that LINC00675-overexpressing MKN-45 and SGC-7901 cells had decreased abilities of cell proliferation and colony formation,whereas LINC00675-knockdown MKN-45 and SGC-7901 cells had increased abilities of cell proliferation and colony formation.?3?Flow cytometry experiment showed that the number of LINC00675-overexpressing GC cells in G1 phase was significantly increased,and in S phase was significantly decreased,whereas the number of LINC00675-knockdown GC cells in G1 phase was significantly decreased,and in S phase was significantly increased,suggesting that LINC00675 suppressed the G1/S transition.?4?Trans-well experiments showed that LINC00675-overexpressing MKN-45 and SGC-7901 cells had decreased abilities of cell migration and invasion,whereas LINC00675-knockdown MKN-45 and SGC-7901 cells had increased abilities of cell migration and invasion.?5?Subcutaneous injection mouse model experiments showed that LINC00675 upregulation resulted in significantly decreased size and weight of formative tumors,and the expression of Ki67 was decreased in the formative tumors.However,LINC00675 downregulation caused significantly increased size and weight of formative tumors,and the expression of Ki67 was increased in the formative tumors.?6?Caudal vein injection mouse model experiments showed that LINC00675-upregulation GC cells had a significantly subdued ability of lung metastasis,whereas LINC00675-knockdown GC cells had a significantly enhanced ability of lung metastasis.Intraperitoneal injection mouse model experiments showed that LINC00675-upregulation GC cells had a significantly subdued ability of liver metastasis,whereas LINC00675-knockdown GC cells had a significantly enhanced ability of liver metastasis.3.The mechanism by which LINC00675 suppresses GC cells proliferation,migration and invasion.?1?The vectors of pcDNA3.1-LINC00675-MS2 and GFP-MS2 were successfully constructed,and co-transfected into SGC-7901 cells.QRT-PCR experiment showed that the expression of LINC00675 was significantly increased in the transfected SGC-7901 cells,and Western blot experiments revealed that the expression of GFP was significantly increased the transfected SGC-7901 cells.?2?LINC00675-interacting complex were isolated using RIP experiments.SDS-PAGE and silver staining revealed that significantly aberrant expressions of proteins existed in GFP group compared with IgG group.The specific band was cut and underwent mass spectrum experiment,and the results showed that Vimentin protein,whose molecular weight was 57 kDa,was included in the complex.?3?Western blot experiment confirmed that Vimentin protein existed in LINC00675-interating complex.By using Vimentin antibody,RIP and qRT-PCR experiments showed that LINC00675 was largely enriched in the isolated complex.These data suggested that LINC00675 interacted with Vimentin.?4?Western blot experiment showed that Vimentin had a high phosphorylation level on Ser83 in LINC00675-overexpressing SGC-7901 cells compared with parallel control SGC-7901 cells,whereas a low phosphorylation level on Ser83 was found in LINC00675-knockdown SGC-7901 cells compared with parallel control SGC-7901 cells.However,the expression and phosphorylation level on Ser39 of Vimentin had not significantly changed.?5?Genome-wide transcript microarray showed that multiple signaling pathways were activated,including p53,and some were inactiviated,including TGF-beta,in LINC00675-overexpressing SGC-7901 cells.Furthermore,the expressions of multiple genes were decreased in "BENPPRATH_ES_WITH_H3K27ME3" and "ZWANG_TRANS IMENTLY_UP_BY₁ST_EGF_PULSE_ONLY",and the expressions of multiple genes were increased in "MANALO_HYPOXIA_DN" and "GO_RIBONUCLEOPR OTEIN_COMPLEX”.Conclusion:1.We first found that LINC00675 expression was significantly decreased in GC tissues,and was associated with tumor size,TNM stage,lymphnode metastasis,distal metastasis,and patients' survival rates.2.LINC00675 suppresses GC cells proliferation,migration and invasion.3.LINC00675 interacts with Vimentin and enhances its phosphorylation on Ser83,resulting in the collapse of Vimentin IF.4.LINC00675 overexpression causes decreased expressions of multiple oncogenes and increased expressions of multiple tumor suppressors,and regulates several cancer-related signaling pathway.In conclusion,LINC00675 interacts with Vimentin and enhances its phosphorylation on Ser83 to suppress GC progression.