| Objective: Gastric cancer is one of the most common malignant tumors in the world,with the fourth incidence and the fifth cancer-related mortality in the world.Asia has a high incidence of gastric cancer in the world,and China ranks the fifth in incidence and the sixth in mortality among 183 countries in the world.Moreover,due to the large population,the number of cases and deaths of gastric cancer in China accounts for 42.6%and 45.0% of the global incidence and mortality of gastric cancer respectively.The onset of gastric cancer is insidious,and there are usually no obvious symptoms in the early stage.Its pathogenesis is complex and affected by many factors,such as genetic factors,infection factors(including Helicobacter pylori,Epstein-Barr virus infection,etc.),dietary habits and hobbies(smoking,drinking history),and mental factors.Therefore,it is of great significance to discover new biomarkers that can accurately predict or reflect individual cancer risk and judge poor prognosis of gastric cancer for patients to implement timely and individualized precise diagnosis and treatment.Previous studies have suggested that tumors are caused by mutations in protein-coding genes.In recent years,with the further development of gene research,it has been found that long non-coding RNA(lnc RNA)can be abnormally expressed in various cancers by regulating gene expression.lnc RNA has the potential to play a role in epigenetic changes as oncogenes and/or tumor suppressors.The abnormal expression of lnc RNA is also closely related to gastric cancer,which affects the occurrence,development and prognosis of gastric cancer by participating in the biological behaviors such as proliferation,invasion and migration of gastric cancer cells.In-depth and systematic understanding and analysis of the differential expression of related lnc RNAs in gastric cancer,study the biological functions of key lnc RNAs,and construct their related molecular regulatory networks can not only provide new markers for early detection,risk prediction and prognosis judgment of gastric cancer,but also find new targets for the treatment of gastric cancer.It is a new way to study the molecular mechanism of gastric cancer.It has been confirmed that lnc RNAs can act as competitive endogenous RNAs(ce RNAs)and bind to mi RNAs(micro RNA)sites to regulate the expression of target genes.The role of mi RNAs in disease and tumor biology is well known.In our study,we predicted that LINC02408 targeted mi R-1226-3p,which has been shown to act as a tumor suppressor in various malignant tumors.For example,mi R-1226-3p suppresses an aggressive phenotype in triple-negative breast cancer and reduces drug resistance in synovial sarcoma.In non-small cell lung cancer cells,mi R-1226-3p has also been reported to inhibit tumor growth and metastasis.In addition,we also predicted the interaction between mi R-1226-3p and APPL1.APPL1 is a DBF2-related kinase with tumor-promoting effects.Studies have shown that APPL1 regulates cell development by regulating the function of downstream effectors.APPL1 can promote tumor metastasis,and it is also regulated by mi RNA such as mi R-302 and mi R-372-3p.The high level expression of APPL1 indicates poor prognosis of gastric cancer and lung cancer.APPL1 can inhibit cell apoptosis,promote tumor cell growth,and enhance tumor cell migration and activity.Although the respective roles of LINC02408,mi R-1226-3p and APPL1 have been found in different malignant tumors,the role of LINC02408 in gastric cancer and the interaction between them has never been studied.In this study,we investigated whether LINC02408 affects the proliferation and metastasis of tumor cells by regulating the mi R-1226-3p /APPL1 axis,in order to provide relevant theoretical basis for the treatment of gastric cancer.In this study,by using bioinformatics analysis technology,the differentially expressed lnc RNAs in gastric cancer samples in TCGA database were analyzed,and a new key lnc RNA LINC02408 was screened.The correlation analysis of clinical characteristics and survival analysis of LINC02408 verified its role in promoting the progression of gastric cancer.Then,the biological function of LINC02408 in the occurrence and development of gastric cancer was further explored by in vivo and in vitro experiments,and the key molecules in the regulation mechanism of LINC02408 in the occurrence and development of gastric cancer were further screened,and the targeted regulatory relationship between LINC02408/mi R-1226-3p/APPL1 was explored.The effect of LINC02408/mi R-1226-3p/APPL1 axis on the biological function of gastric cancer was analyzed,so as to clarify the important regulatory role and mechanism of LINC02408 as a ce RNA in the occurrence and development of gastric cancer.Methods and results:Part I Screening of the key lnc RNA in gastric cancer and construct the lnc RNA/mi RNA/m RNA ce RNA regulatory networkTCGA database was used to load the transcriptome sequencing data and corresponding clinical characteristics of 375 gastric cancer samples and 32 paracancerous tissue samples,and the characteristic lnc RNAs expression profile of gastric cancer tissues was obtained by differential analysis.DIANA,mi RDB,Target Scan and other database resources were integrated.The lnc RNA/mi RNA/m RNA molecular regulatory network of gastric cancer was constructed,and the most key differentially expressed lnc RNA LINC02408 was selected as the target of this study.Further survival analysis of LINC02408 showed that LINC02408 was related to overall survival.It is speculated that LINC02408 may not only be a potential specific marker for the prognosis of gastric cancer,but also be closely related to the occurrence and development of gastric cancer.It is worthy of further research and exploration on its biological function and molecular regulation mechanism.Part II The biological function of LINC02408 in gastric cancer in vitro and in vivoThe expression level of LINC02408 was detected by q PCR in 44 cases of gastric cancer and adjacent tissues,and it was found that LINC02408 was highly expressed in gastric cancer tissues,and its high expression status was closely related to the degree of tumor differentiation,depth of invasion and lymphatic metastasis.The expression levels of LINC02408 in gastric cancer cell lines SGC-7901、BGC-823、MKN45、AGS、HGC-27、MGC-803 and normal gastric mucosa GES-1 cells were detected.LINC02408 was highly expressed in a variety of gastric cancer cells.LINC02408 was found to be mainly localized in the cytoplasm using a nucleocytoplasmic separation assay.The LINC02408 interfering lentiviral vector p LKO.1-sh LINC02408(3 sites)were successfully constructed and transfected into gastric cancer cells MKN45 and MGC-803 to interfere(silence)the expression of LINC02408.The results showed that it could significantly inhibit the proliferation of gastric cancer cells.Flow cytometry showed that silencing LINC02408 expression could promote the apoptosis of gastric cancer cells.Transwell assay showed that silencing LINC02408 expression can inhibit the migration and invasion of gastric cancer cells,and LINC02408 has a significant promoting effect on the metastasis of gastric cancer cells.Using subcutaneous tumor formation experiment in nude mice,it was found that silencing LINC02408 expression could reduce the growth rate of gastric cancer and slow down the increase of tumor volume in vivo.Both in vitro and in vivo experiments confirmed that LINC02408 promote the proliferation and metastasis of gastric cancer.Part III Study on the mechanism of LINC02408 regulating the occurrence and development of gastric cancerAfter silencing the expression of LINC02408,the expression level of mi R-1226-3p was detected in the two gastric cancer cell lines,and it was found that mi R-1226-3p was significantly overexpressed.In the above tumor formation experiments in nude mice,q PCR detected that mi R-1226-3p was significantly down-regulated in mouse gastric cancer cells,and the expression level of mi R-1226-3p was significantly increased after silencing the expression of LINC02408.Luciferase reporter gene assay confirmed the binding relationship between mi R-1226-3p and LINC02408.CCK-8 assay showed that mi R-1226-3p could inhibit the proliferation of gastric cancer cells,and flow cytometry showed that mi R-1226-3p could promote the apoptosis of gastric cancer cells.After co-transfection of mi R-1226-3p inhibitor and vector of silencing LINC02408 expression in gastric cancer cells MKN45,the inhibitory effect of mi R-1226-3p on proliferation and the promotion effect on apoptosis of gastric cancer cells could be reversed.After silencing the expression of LINC02408,APPL1 expression was detected in the two gastric cancer cell lines,and it was found that APPL1 was significantly down-regulated.In the above tumor formation experiments in nude mice,APPL1 was significantly overexpressed in mouse gastric cancer cells as detected by q PCR,and the expression level of APPL1 was significantly reduced after silencing the expression of LINC02408.Luciferase reporter gene assay was used to confirm the binding relationship between mi R-1226-3p and APPL1.APPL1 was highly expressed in gastric cancer MKN45 cells as detected by q PCR and Western-Blot.Over-expression of mi R-1226-3p could inhibit APPL1 expression,while silencing LINC02408 expression could inhibit APPL1 expression.These results indicated that LINC02408 could inhibit the silencing effect of mi R-1226-3p on APPL1 by competitively binding to mi R-1226-3p.In addition,CCK-8 assay showed that APPL1 over-expression could promote the proliferation of gastric cancer cells.The expression levels of EMT-related proteins N-cadherin,Vimentin and E-cadherin were detected in gastric cancer cells co-transfected with sh LINC02408,mi R-1226-3p mimics,mi R-1226-3p inhibitor,oe APPL1 or negative control,respectively.The results showed that silencing LINC02408 promoted E-cadherin expression and inhibited N-cadherin and Vimentin expression,while inhibition of mi R-1226-3p reversed the effect of silencing LINC02408.In addition,over-expression of mi R-1226-3p significantly promoted E-cadherin expression,while inhibited N-cadherin and Vimentin expression.Furthermore,these changes were reversed by over-expression of APPL1.This functional recovery experiment indicated that LINC02408/mi R-1226-3p/APPL1 axis could regulate gastric cancer metastasis by affecting EMT-related proteins in human gastric cancer cells.Conclusions:LINC02408 can act as a ce RNA to competitively bind mi R-1226-3p to regulate APPL1 in human gastric cancer cells.LINC02408 regulates the proliferation and metastasis of gastric cancer cells by targeting the mi R-1226-3p/APPL1 axis. |
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