The Functional And Mechanistic Study Of UCA1/miR-203/ZEB2 Axis On Invasion And Metastasis Of Gastric Cancer

Background:Gastric cancer is malignant digestive tract tumor with mortality rate ranking second in the world,it is also a common malignant tumor in the Chinese population.In recent years,although people have made many improvements in the treatment of surgery,chemotherapy,radiotherapy and so on,the average five-year survival rate of patients with gastric cancer in the world is still below 10%.Among them,tumor recurrence and metastasis is the major obstacle to the treatment of gastric cancer.Researchers have focused on the molecular mechanisms of gastric cancer,including gene mutations,gene copy number variation,gene cleavage,gene fusion,m RNA,mi RNA,epigenetics.However,the detailed molecular mechanism of the development and metastasis of gastric cancer remains unclear.Recent studies have revealed that long non-coding RNA(lnc RNA)plays an important regulatory role in the development and progression of tumors.With the elucidation of the structure and function of lnc RNA,the researchers found that lnc RNAs participate in the regulation of various biological functions of gastric cancer cells in various mechanisms,and it is expected to become a new candidate target for the diagnosis and treatment of gastric cancer.Objective:In this study,lnc RNA UCA1 was selected as a candidate molecule for exploring the biological role in the development of gastric cancer and its molecular regulatory network,aiming to understand the contribution of lnc RNA in the development of gastric cancer.Methods: 1.Limma package in R lauguage was used to analyze the lnc RNA chip expression matrix of gastric cancer tissue samples in the GEO database,the gastric cancer tissue sequencing data in The Cancer Genome Atlas(TCGA)database was downloaded,and the differential expression of UCA1 was analyzed using the edge R package;The expression level of UCA1 in 60 pairs of gastric cancer tissues was detected by q PCR method,and the correlation between the expression level of UCA1 and clinicopathological parameters of gastric cancer was analyzed;The expression level of UCA1 in gastric cancer cell lines was also detected by q PCR;RNA-seq was performed on gastric cancer cell line BGC-823 after knocking down UCA1,the functional context of differentially expressed genes associated with UCA1 was assessed by querying the Gene Ontology(GO)method.2.Stable cell line with overexpression or interference of UCA1 was established by lentivirus vectors,scratch and Transwell chamber assay were employed to measure the effect of UCA1 on migration and invasion of gastric cancer cell lines in vitro,the effect of UCA1 on migration in gastric cancer cell lines was measured by mouse tail vein injection model in vivo,HE staining was used for histological evaluation,q PCR and Western Blot were used to detect the expression of migration-related molecules.3.Nuclear and cytoplasmic RNA isolation was performed to study the localization of UCA1,bioinformatics method to construct a regulatory network involving UCA1,to find the target molecule affected by UCA1 sponging mi RNA,Luciferase assay and RNA pull down experiments were used to demonstrate the physical binding between UCA1 and mi R-203,the luciferase experiment demonstrated that mi R-203 inhibits ZEB2 by binding to the 3 ?UTR of ZEB2,the luciferase assay was used to demonstrated that mi R-203 inhibits ZEB2 by binding to the 3 ?UTR of ZEB2,RNA Binding Protein Immunoprecipitation(RIP)was used to verify whether mi R-203 inhibits ZEB2 expression dependent on Ago-2;Rescue experiments were conducted to investigate whether UCA1 regulate ZEB2 expression dependent on sponging mi R-203.Results:1.We analyzed the differential expression results of lnc RNA microarray obtained by our group and the two independent lnc RNA expression profiles in the GEO dataset and discovered that the expression level of lnc RNA UCA1 has significantly increased and ranked first.The abnormal expression level of UCA1 observed from the microarray data was further verified in patients with gastric cancer from TCGA database.2.We analyzed the expression level of UCA1 in 60 pairs fresh gastric cancer tissues and found that the expression level of UCA1 in gastric cancer tissues was significantly higher than that in adjacent tissues(p<0.001),and the average expression level of UCA1 in gastric cancer tissues was 18 times higher than that in adjacent tissues.Analyzing the correlation between the expression level of UCA1 and the clinicopathological parameters of gastric cancer,we found that the expression level of UCA1 was significantly correlated with lymph node metastasis(p=0.038)and age(p=0.027).3.We used the RNA-Seq method to compare differences in gene expression after interfering with UCA1 in gastric cancer cell line BGC-823,the functional context of differentially expressed genes associated with UCA1 was assessed by querying the Gene Ontology(GO)method,the result demonstrated that the changes of cell signaling pathways were mainly concentrated on adhesion,adhesion junction,anchoring junction after interfering with UCA1.4.We demonstrated that overexpression of UCA1 induced an epithelial-mesenchymal transition(EMT)phenotype in the gastric cancer cell line BGC-823.We interrogated some EMT markers in BGC-823 and SGC-7901 by Western Blot,and reasoned that epithelial marker were down-regulated and mesenchymal marker were up-regulated when UCA1 was overexpressed.5.Scratch and Transwell chamber assay demonstrated that the overexpression of UCA1 promoted the ability of the migration and invasion of gastric cancer cell lines SGC-7901 and BGC-823;Knockdown of UCA1 expression inhibited the ability of the migration and invasion of gastric cancer cell lines SGC-7901 and BGC-823.6.The tail vein injection model of nude mice proved that overexpression of UCA1 in gastric cancer cell line SGC-7901 can significantly promote metastasis to liver.On the contrary,knockdown of UCA1 expression in gastric cancer cell line BGC-823 can significantly inhibit metastasis to liver.7.q RT-PCR performed after nuclear and cytoplasmic RNA isolation in gastric cancer cell concluded that UCA1 was mainly localized in the cell cytoplasm.By constructing the UCA1-related sponge network,combinded with the RNA-seq results after interference with UCA1 in the gastric cancer cell line BGC-823,we identified ZEB2 as a candidate target affected by UCA1,knocking down UCA1 could inhibit UCA1 expression level both at the transcriptional level and protein level,while overexpression of UCA1 could increase the expression level of ZEB2.Through sponge regulation network,we have found that UCA1 regulated ZEB2 by sponge mi R-203,thereby abolishing the inhibitory effect of mi R-203 on ZEB2.8.Luciferase assay and RNA pull down assay showed that UCA1 and mi R-203 are physically bound,Luciferase assay showed that mi R-203 inhibits ZEB2 expression by binding to ZEB2 3 ?UTR,RNA Binding Protein Immunoprecipitation(RIP)experiments revealed that mi R-203 can inhibit ZEB2 expression dependent on Ago-2.9.Through rescue experiments,it was found that transfection of mi R-203 mimics in gastric cancer cell lines SGC-7901 and BGC-823 stably overexpressing UCA1 can down-regulate the expression level of ZEB2,and after transfection of mi R-203 inhibitor in gastric cancer cell lines SGC-7901 and BGC-823 stably interfering with UCA1,the expression level of ZEB2 was restored,these results suggest that the regulation of ZEB2 by UCA1 is dependent on the adsorption of mi R-203.Conclusion: 1.UCA1 is highly expressed in gastric cancer tissues,and the expression level of UCA1 is significantly correlated with lymph node metastasis of gastric cancer,which speculates that UCA1 may become a biomarker for the diagnosis and detection of disease progression in gastric cancer.2.Highly expressed UCA1 can promote the invasion and metastasis of gastric cancer cells,combined with the GO analysis based on RNA-seq data after knocking down UCA1 in the gastric cancer cell line BGC-823,it suggests that UCA1 is expected to become a therapeutic target for invasion and metastasis of gastric cancer.3.UCA1 acts as a ce RNA to regulate ZEB2 by sponging mi R-203,the proposition of UCA1/mi R-203/ZEB2 axis is helpful for deeply understanding the mechanism of invasion and metastasis of gastric cancer as well as establishing a theoretical basis for the development of novel drugs.Background:Amplification of chromosome 20q13.33 region is closely related to the occurrence and development of gastric cancer.linc00659 is located in the 20q13.33 region and is involved in the growth of colon cancer cells,but it is unclear whether it is related to the occurrence and progression of gastric cancer.Objective: Differentially expressed linc00659 on chromosome 20q13.33 segment has a strong relationship with survival time of gastric cancer patients,and it has have a profound effect on invasion and migration of gastric cancer cells,providing possible new diagnostic markers and potential therapeutic targets for the diagnosis and treatment of gastric cancer.Methods:1.Downloading TCGA expression data of gastric cancer,To use the edgeR package to analyze the differential expression of lnc RNA on chromosome segment 20q13.33.According to the expression level of linc00659,gastric cancer patients were divided into high expression group of linc00659 and low expression of linc00659.The correlation between the expression of linc00659 and the survival time of gastric cancer patients was analyzed.The correlation between the expression of linc00659 and the clinical stage of gastric cancer was analyzed.2.The expression levels of linc00659 in 60 gastric cancer tissues and adjacent normal tissues were analyzed by q RT-PCR,and the correlation between linc00659 and lymph node metastasis of gastric cancer was also analyzed.The expression of linc00659 in gastric cancer cell lines was detected by q RT-PCR.3.The changes in cellular pathways between the high linc00659 expression group and the low linc00659 expression were analyzed by GSEA enrichment to speculate that linc00659 is involved in the cell biological function of gastric carcinogenesis.4.The effect of inhibition of linc00659 expression on the migration and invasion of gastric cancer cells by scratch assay and Transwell chamber assay after stably interferencing with the expression of linc00659 in gastric cancer cell lines AGS and MKN-74.Results:1.After analysis,the 20q13.33 chromosome segment contains 18 lnc RNAs,of which 4 lnc RNA RTEL1-TNFRSF6 B,linc00659,SLCO4A1-AS1,ZBTB46-AS1 are differentially expressed,and linc00659 is associated with survival time of gastric cancer patients.By analyzing the clinical pathological information of gastric cancer in the TCGA database,it was found that the expression level of linc00659 was significantly correlated with the clinical staging of the tumor.2.The q RT-PCR method was used to detect the expression level of linc00659 in 60 pairs of fresh gastric cancer tissues and adjacent tissues.It was found that the expression level of linc00659 in cancer tissues was significantly higher than that in adjacent tissues,and when lymph nodes were present in patient tissue,the expression level of linc00659 in cancer tissues was significantly higher in the metastatic gastric cancer case than that in adjacent tissues.3.Compared with the gastric mucosal immortalized cell GES-1,linc00659 was up-regulated in gastric cancer cell lines.4.Based on bioinformatics analysis,it is speculated that linc00659 may be mainly involved in cell adhesion-related pathways.5.Scratch and Transwell chamber assay showed that invasion and migration ability of gastric cancer cell lines AGS and MKN-74 was significantly inhibited after linc00659 was knocked down.Conclusion: linc00659 is highly expressed in both gastric cancer and gastric cancer cell lines,suggesting that linc00659 may play an oncogene role in the development of gastric cancer.The high expression of linc00659 is positively correlated with lymph node metastasis and clinical stage in patients with gastric cancer,and is associated with poor prognosis.linc00659 may be involved in the migration and invasion of gastric cancer cells.