| Gastric cancer is one of the most common malignant tumors in the world,and induced by a variety of factors and dynamically regulated by different molecules,and has extremely complex pathological mechanisms.In recent years,research on the molecular mechanism of gastric cancer has continued to advance,but there is still a lack of diagnostic and therapeutic targets.Based on the construction of the molecular co-regulatory network in gastric cancer and the in-depth exploration of key regulatory molecules,we could further elucidate the underlying mechanisms of gastric cancer occurrence and development,and find effective diagnosis and treatment targets,meanwhile develop personalized treatment plans.Studies have shown that long non-coding RNAs(lncRNAs)can participate in the proliferation,apoptosis,migration,and migration of gastric cancer cells through various modes,and play a key role in the occurrence and development of gastric cancer,and are also vital for the early diagnosis and prognostic evaluation of gastric cancer patients.Therefore,through differentially expressed analysis of lncRNAs in gastric cancer and constructing lncRNA-related regulatory network,we could explore the key functional lncRNAs,which provide a new perspective for the gastric cancer pathogenesis research and the development of new diagnosis and treatment programs.In this study,the differentially expressed lncRNA,miRNA and mRNA in gastric cancer samples of TCGA database were analyzed,and the lncRNA/miRNA/mRNA co-regulatory network was constructed by using bioinformatic tools.We also performed functional analysis of the network-related genes,and extracted core lncRNAs through modularization.Then the clinical feature correlation analysis and survival analysis were further carried out for the core lncRNA.In addition,we detected the expression of survival-related lncRNA RP11-7K24.3 in gastric cancer cells and gastric cancer tissues,as well as the targeted regulatory relationship among RP11-7K24.3,miR-17-5p and ZBTB4,so as to clarify its important regulatory role in the occurrence and development of gastric cancer.Part ?: Construction of lncRNA/miRNA/mRNA co-regulatory network and screening for key lncRNAs in gastric cancerBasing on the RNA profiles of 372 gastric cancer samples and 32 non-cancerous samples,along with clinical information,we screened 2221 differentially expressed lncRNAs,168 differentially expressed miRNAs and 3879 differentially expressed mRNAs,then constructed a gastric cancer-related lncRNA/miRNA/mRNA co-regulatory network by utilizing Target Scan,miRDB and DIANA database resources.Meanwhile,the GO and KEGG functional enrichment analysis were performed to reveal potential biological function.Through the modularization of protein-protein interaction network(PPI),76 core lncRNAs were extracted.According to the correlation analysis and survival analysis of core lncRNAs,we found that HULC and RP11-314B1.2 were closely related to multiple clinical features,AC018647.3,MAGI2-AS3,MIR99 AHG,NR2F1-AS1,LINC00106,PVT1,RP5-1074L1.4 and RP11-7K24.3 were related to overall survival,and we also established a prognostic risk model composed of LINC00106 and RP11-999E24.3.At the same time,the expression levels of 11 clinical-related key lncRNAs in gastric cancer cells were detected by q RT-PCR,we found that RP11-7K24.3 was consistently low expressed in various gastric cancer cell lines.RP11-7K24.3 can be used as a specific biomarker for prognostic analysis of gastric cancer,and may also be closely related to the occurrence and development of gastric cancer.It is worth further exploring its potential biological functions and molecular regulation mechanisms.Part ?: RP11-7K24.3 biological function in gastric cancerBy verifying the expression level of RP11-7K24.3 in 55 cases of gastric cancer and adjacent samples,it was found that RP11-7K24.3 was significantly low expressed in gastric cancer tissues,and its low expression status was consistent with the tumor size and distant metastasis of gastric cancer patients.Using nucleoplasmic RNA isolation experiments,it was found that RP11-7K24.3 was mainly located in the cytoplasm.The pc DNA3.1 RP11-7K24.3 was transfected into gastric cancer cells HGC-27 and SGC-7901 to up-regulate RP11-7K24.3.Meanwhile,CCK-8 and Ed U experiments were used to detect cell growth,the results suggest that RP11-7K24.3 overexpressed can significantly inhibit the proliferation of gastric cancer cells.The wound healing assay and transwell cell migration/invasion assays showed that RP11-7K24.3overexpressed can inhibit the migration and invasion abilities of gastric cancer cells.Through detecting the expression of E-cadherin and N-cadherin by western blot,we also found that RP11-7K24.3 overexpressed can inhibit the EMT process in gastric cancer cells.While RP11-7K24.3 could promote apoptosis detected by flow cytometry assay.In addition,the results of tumor xenograft assay showed that RP11-7K24.3 overexpressed can reduce the growth rate and tumor volume of gastric cancer in vivo.Both in vivo and in vitro experiments illustrated that RP11-7K24.3plays a certain protective role in the occurrence and development of gastric cancer.Part ?: Research for molecular mechanism of RP11-7K24.3 in gastric cancerLuciferase reporter gene assay and RNA immunoprecipitation assay confirmed the binding relationship between miR-17-5p and RP11-7K24.3.It was detected by q RT-PCR that miR-17-5p was highly expressed in gastric cancer cells,and RP11-7K24.3 overexpressed could inhibit the expression level of miR-17-5p.We utilized miR-17-5p mimics or miR-17-5p inhibitor to transfect gastric cancer cells HGC-27 and SGC-7901 for up-or down-regulate miR-17-5p.Through CCK-8 and Ed U assays,we found that miR-17-5p promoted the proliferation of gastric cancer cells.According to the results of wound healing assay and transwell assay,it was found that miR-17-5p promoted the migration and invasion of gastric cancer cells.Meanwhile the results of western blot showed that miR-17-5p could promote the EMT process.Otherwise,miR-17-5p inhibited the apoptosis of gastric cancer cells detected by flow cytometry assay.Furthermore,co-transfection of miR-17-5p mimics and pc DNA3.1 RP11-7K24.3 could reverse the promoting effect of miR-17-5p on the proliferation,migration and invasion of gastric cancer cells and the inhibitory effect on apoptosis.We also validated the binding relationship between miR-17-5p and ZBTB4 by luciferase reporter gene assay and RNA immunoprecipitation assay.ZBTB4 was downregulated in gastric cell lines detected by q RT-PCR and western blot,and miR-17-5p reduced the expression of ZBTB4,while RP11-7K24.3 increased it,which suggested that RP11-7K24.3 could inhibit the silencing effect of miR-17-5p on ZBTB4 by competitively binding miR-17-5p.In addition,through CCK-8 assay,Ed U assay,wound healing assay,transwell assay,western blot and flow cytometry assay,it was found that ZBTB4 overexpressed could inhibit the proliferation,migration,and invasion of gastric cancer cells,also regulate EMT process,while promote the apoptosis of gastric cancer cells.However,co-transfection of miR-17-5p mimics reversed the regulatory function of ZBTB4.We also detected the regulatory effect of RP11-7K24.3 on the transcription factors Sp1,Sp3 and Sp4 through miR-17-5p/ZBTB4.In short,we speculated that the RP11-7K24.3/miR-17-5p/ZBTB4 axis may be involved in the proliferation,migration,invasion and apoptosis of gastric cancer cells,and affect the pathogenesis of gastric cancer. |
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