| Background:In 2020,the World Health Organization released its latest global cancer report,which showed that in the past year,China ranked first in the world in the number of new cancer cases and deaths.Among them,gastric cancer has become one of the top cancers in morbidity and mortality in China.Recent studies have improved the early diagnosis and treatment of gastric cancer.However,the incidence of gastric cancer is relatively insidious,clinical symptoms are not obvious,and routine physical examination is extremely difficult to detect.As a result,a considerable number of gastric cancer patients are in the advanced stage when diagnosed,so the situation of prevention and treatment of gastric cancer is still grim.As early as 40 years ago,the concept of cancer stem cells was proposed.This theory believes that tumor growth is similar to the growth and renewal of normal tissues and is starting by a small number of stem cells with specific functions.In the past ten years,with the continuous deepening of research on cancer stem cells,cancer stem cells are considered to be a type of tumor cell subgroup responsible for tumor initiation,tumor chemotherapy and radiotherapy resistance,tumor aggressive growth,metastasis,and tumor recurrence it plays an important role in tumor development,recurrence and metastasis.However,many tumor stem cell markers also exist on normal stem cells,which is a disadvantage for cancer stem cells as therapeutic targets.Therefore,it is very important to explore the molecular mechanism of tumor stem cell self-renewal regulation.In tumor cells,in addition to the abnormal expression of genes encoding proteins,scientific research in recent years has found that the abnormal regulation of long-chain non-coding RNAs(lncRNAs)also plays an important role.Although the research on lncRNA is advancing rapidly,the function of most lncRNA is still unclear,and the reports of lncRNA related to gastric cancer metastasis are even rarer.Methods:In this study,the lncRNA HNF1A-AS1,which is significantly related to the stemness of gastric tumor cells,was selected from the existing long non-coding RNAs in the research group,and the ALDH enzyme activity detection experiment and the tumor cell microsphere formation experiment were used in gastric tumor cell lines.Validated in MKN-45 and BGC-823.Real-time quantitative PCR(RT-qPCR)was used to detect the expression of HNF1A-AS1 in spheroid cells and non-spheroid cells in the tumor microsphere formation experiment,and further analyze the correlation between HNF1 A-AS1 expression and the stemness of gastric tumor cells.The effect of lncRNA HNF1A-AS 1 on the stemness of gastric cancer cells was researched by the tumor microsphere formation and counting experiment and the monoclonal formation experiment under in vitro conditions.Bioinformatics analysis was used to predict the miRNAs that lncRNA hnF1A-AS1 might bind,and then the binding effect between the two was verified by luciferase assay.The signaling pathways and molecules of HNF1A-AS1 related to tumor cell stemness maintenance were predicted by literature retrieval and bioinformatics analysis,and the regulatory effect of miRNA on target genes was verified by luciferase assay and Western-blot.Fluorescence immunositu hybridization experiments and western-blot experiments were used to further confirm the ceRNA regulatory network of HNF1A-AS1-miRNA-target gene and the regulation effect of downstream molecular mechanism on the stemness of gastric cancer cells.Results:1.Through ALDH enzyme activity detection experiments,it is found that compared with other long-chain non-coding RNA indicators in the research group,lncRNA HNF1A-AS1 has a significant promoting effect on the stemness of gastric cancer cells.2.Tumor microspheres formation experiment was used to establish an in vitro amplification model of tumor stem cells.Then the expression of HNF1A-AS1 in tumor stem cells and non-tumor stem cells was analyzed by RT-qPCR assay.The results showed that the expression of HNF1A-AS1 in tumor stem cells was significantly higher than that in non-tumor stem cells.3.Over-expressing lncRNA HNF1A-AS1 in MKN-45 and BGC-823 cell lines,the activity of ALDH enzyme of tumor cells is enhanced,the number of tumor cell microspheres is increased,and the number of single clones is increased.4.Luciferase experiment confirmed that miR-150-5p could be adsorbed by lncRNA HNF1A-AS1.5.Western blot experiments and fluorescence immunositu hybridization experiments confirmed that lncRNA HNF1A-AS1 could positively regulate the expression ofβ-catenin and promote the activation of Wnt signaling pathway.6.Luciferase experiment and western-blot experiment verify that miR-150-5p has a regulatory effect on the target gene β-catenin.Use the bioinformatics website to predict the miRNA that lncRNA HNF1A-AS1 may bind,and verify the binding effect between the two through luciferase experiment.Conclusions:The expression of HNF1A-AS1 in gastric cancer stem cells is higher than that of non-tumor stem cells.HNF1A-AS1 can promote the stemness of gastric cancer cells and positively regulate the self-renewal of gastric cancer cells.HNF1A-AS1,as ancompeting endogenous RNA RNA(ceRNA),up-regulates the expression ofβ-catenin by adsorbing miR-150-5p,and promotes its nuclear entry to activate the Wnt/β-catenin pathway,thereby promoting the stemness of tumor cells. |
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