| Background:As the fourth most common cancer in the world,Gastric Cancer has seriously threatens human health.Once found the patients have developed to advanced stages.Therefore,early diagnosis and treatment of gastric cancer patients are crucial to their prognosis.At present,the diagnosis of gastric cancer mainly depends on the traditional methods such as clinical manifestations,endoscopic,histopathological examinations and imaging examinations.Lacking of suitable molecular biomarkers,most patients have missed the best opportunity for surgery.Therefore,developing suitable molecular biomarkers will inevitably contribute to the early diagnosis and gastric cancer therapy.The Long non-coding RNA(LncRNA)expressed abnormally in cancer cells are expected as promising markers for tumor diagnosis.LncRNAs are non-coding RNAs with a length of more than 200 nucleotides.Currently,tumor-associated LncRNAs have been detected in tissues,blood,urine,and saliva.He Ye et al.in College of Computer Science and Technology of Jilin University has used bioinformatics methods to dig deeply into the transcriptome data and analyzed the three datasets of GSE30727,GSE13195 and GSE33335 for gastric cancer.They found LncRNA LINC00365 and SCGB2A1 is commonly expressed in gastric cancer and has a correlation.At the same time,it is found that LINC00365 is commonly expressed in gastric cancer,is highly significant.The chromosomal location of LINC00365 is 13q12.3.After analyzing the coding genes that are co-expressed with LINC00365,we find the protein SCGB2A1 that can be secreted into the blood and urine.Therefore,the combination of LncRNA and SCGB2A1 may provide new biomarkers for gastric cancer diagnosis and treatment.Objective:This study has detected the expression of LINC00365 and SCGB2A1 in clinical gastric cancer tissues and human gastric cancer cell lines.To further clarify the regulation mechanism of LINC00365 in gastric cancer,we overexpressed LINC00365 in gastric cancer cell lines to examine the effect on SCGB2A1 expression and cell proliferation.Method:Tissue microarrays were performed on 90 patients with confirmed gastric cancer.The expression of LINC00365 was detected by in situ hybridization(ISH).The expression of SCGB2A1 protein was detected by immunohistochemistry(IHC).The cancers of LINC00365 and SCGB2A1 were detected in 30 patients with gastric cancer by RT-qPCR.It was differentially expressed with paracancerous tissues and gastric normal mucosa cell line GES-1,gastric cancer cell lines SGC-7901,MGC-803,and BGC-823.Human gastric cancer BGC-823 and MGC-803 cells were transfected with pc DNA3.1-LINC00365.RT-qPCR and Western Blot were used to detect the effect of overexpression of LINC00365 on the transcription and translation of SCGB2A1.EDU cell proliferation assay and colony formation assay were used to detect the effect of overexpression of LINC00365 on the proliferation of BGC-823 and MGC-803 cells;Western blot was used to detect the expression of p-IκBα and IκBα protein,and immunofluorescence was used to observe expression of p65 in cells;Detection of NFκB transcriptional activity by dual luciferase reporter gene assay;Observation of the effect of overexpression of LINC00365 on NFκB signaling pathway activity in BGC-823 and MGC-803 cells by using laser confocal microscopy and Hoechst 33258 staining was used to observe the change of cell nucleus.The number of apoptotic cells was detected by flow cytometry.The effect of overexpression of LINC00365 on the apoptosis of BGC-823 and MGC-803 cells was observed.Result:(1)The results of ISH and ICH based on tissue microarray showed that LINC00365 was significantly down-regulated in gastric cancer tissues compared with adjacent tissues,and it was positively correlated with SCGB2A1 protein expression.(2)The results of gastric cancer tissues and corresponding paracancerous tissues in 30 patients with gastric cancer confirmed by the diagnosis showed that LINC00365 and SCGB2A1 were lowly expressed in the cancer tissues compared with the adjacent tissues.Human gastric cancer cell line SGC-7901 The expression levels of LINC00365 and SCGB2A1 in cells,BGC-823 cells and MGC-803 cells were lower than that of normal gastric mucosa cell line GES-1 cells.(3)The pcDNA3.1-LINC00365 expression vector was transfected into BGC-823 cells and MGC-803 cells by transfection with pc DNA3.1 vector.The results showed that after overexpression of LINC00365,the transcription and protein levels of SCGB2A1 were upregulated,cell proliferation rate decreased,p-IκB protein expression decreased,p65 subunit nuclear protein expression decreased.(4)Hoechst 33258 staining showed no chromatin condensation and no significant changes in the number of apoptotic cells,indicating that overexpression of LINC00365 had no significant effect on the apoptosis of BGC-823 cells and MGC-803 cells.Conclusion:(1)LINC00365 was positively correlated expressed with SCGB2A1 in clinical specimens of gastric cancer patients.(2)LINC00365 plays an important role in the proliferation of gastric cancer cells by regulation the expression of SCGB2A1 and NFκB signaling pathway. |
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