| Background and Objective: Colorectal cancer(CRC)is one of the most common malignancies worldwide.According to the global annual cancer report released in 2020,the colorectal cancer incidence rate ranks third,and the cancer-related mortality rate ranks second,posing a serious threat to human health.With the development of molecular biology,the pathogenesis of colorectal cancer has been partially explained,the popularization of early cancer screening and the application of comprehensive treatment such as surgery,radiotherapy and chemotherapy,targeted and immunotherapy,making the diagnosis and efficacy of colorectal cancer have been significantly improved.However,most colorectal cancer patients in China are already advanced,and they are prone to progression within a short term,leading to treatment failure.Therefore,intensively exploring the molecular mechanism of colorectal cancer occurrence and development will contribute to seeking new biomarkers,therapeutic targets and the improvement of clinical treatment strategies,which will finally improve the quality of life and prolong the survival time of patients.Long non-codingRNAs(lncRNAs)are a class ofRNAs longer than 200 nucleotides,non-encoding or encoding a small amount of short peptides,and widely found in various eukaryotes.Current studies have confirmed that lncRNAs can participate in the regulation of almost all pathophysiological processes of the body life cycle,especially as oncogenes or tumor suppressor gene involved in the regulation of tumor malignant phenotype.We mainly focused on the field of lncRNAs and tumor malignant phenotype,and found the lncRNA GATA2-AS1 related to colon cancer progression by analyzing the TCGA database and the GEO database in the previous study.However,the role and specific mechanism of GATA2-AS1 in colon cancer progression are still unclear.Therefore,this project aims to clarify the role and specific molecular mechanism of GATA2-AS1 in colon cancer progression by analyzing public databases and combining in vitro and in vivo experiments.Methods:1.The GATA2-AS1 expression in colon cancer was explored in TCGA and GEO datasets through bioinformatics analysis.2.The GATA2-AS1 expression in colon cancer cells were detected by q RT-PCR.Colony formation experiments,EDU experiments,flow cytometry,Td T-mediated d UTP nick-end labeling(TUNEL),Transwell experiments,cell spheroidization experiments and Western blot to confirm the functions of GATA2-AS1 in colon cancer cells.3.Nucleoplasmic separation assay and FISH(Fluorescence in situ hybridization)clarified the substructure localization of GATA2-AS1 in colon cancer cells,and the downstream genes of GATA2-AS1 were confirmed by bioinformatics analysis,Western blot,luciferase assay,RIP,RNA pull-down,RNA stability and Ch IP-PCR.4.Rescue experiments and animal experiments were used to confirm the role of GATA2-AS1 in the malignant biological behavior of colon cancer.Results:1.GATA2-AS1 was highly expressed in colon cancer,and knockdown of GATA2-AS1 inhibited the malignant phenotype of colon cancer cells.The results of bioinformatics analysis suggested that GATA2-AS1 was highly expressed in colon cancer tissues.The results of q RT-PCR indicated that GATA2-AS1 expression was elevated in colon cancer cells.Colony formation assay,EDU assay,flow cytometry,TUNEL test,Transwell assay,Epithelial mesenchymal transition(EMT)marker detection,and cell spheroidization assay,confirmed that knockdown of GATA2-AS1 can inhibit clone formation ability,proliferation,invasion,EMT,and cancer stem cell stemness in colon cancer cells,and promote colon cancer cell apoptosis.2.The expression of GATA2 was positively correlated with GATA2-AS1 expression in colon cancer and they formed a positive feedback regulatory loop.The results of nucleoplasmic separation assay and FISH confirmed that GATA2-AS1 was mainly localized in the cytoplasm.TCGA database showed that GATA2 was highly expressed in colon cancer and positively correlated with GATA2-AS1 expression and poor prognosis;The result of q RT-PCR showed that GATA2 was elevated in colon cancer cells;The result of q RT-PCR showed that the expression level of GATA2 was downregulated in colon cancer cells when GATA2-AS1 was knocked down.Actinomycin D experiment,RNA pull-down and RIP experiment confirmed that GATA2-AS1 promoted the mRNA stability of GATA2 to upregulated its mRNA and protein expression levels by recruiting DEADbox helicase 3X-linked(DDX3X).q RT-PCR,RIP,dual luciferase experiments and Ch IP-PCR experiments confirmed that GATA2 was the upstream transcription factor of GATA2-AS1 and promote the transcription and expression of GATA2-AS1,thus forming a positive feedback loop.3.GATA2-AS1 promotes the malignant biological behavior of colon cancer cells by upregulation of GATA2.Rescue experiments confirmed that overexpression of GATA2 effectively reversed the inhibited effect of proliferation ability,invasion,EMT,tumor cell stemness and apoptosis induction of GATA2-AS1 silenced colon cancer cells on colon cancer cells.4.Establishing xenograft tumor model further verified that knockdown of GATA2-AS1 inhibited the growth and proliferation of colon cancer in vivo.The results of subcutaneous tumor transplantation experiments in nude mice showed that the growth rate and weight of the GATA2-AS1-silenced group were less than those of the control group.Immunohistochemistry and Western blot indicated that the expression levels of Ki-67,PCNA,N-cadherin,Nanog,and OCT4 were lower in the GATA2-AS1-silenced group,and E-cadherin expression was higher than that of the control group.Conclusion:1.GATA2-AS1 is highly expressed in colon cancer tissues and cells and knocking down GATA2-AS1 can inhibit the proliferation,invasion and stemness maintenance of colon cancer cells,and promote their apoptosis.2.GATA2-AS1 recruits DDX3 X to enhance the stability of GATA2 mRNA and the expression of GATA2 mRNA and protein.GATA2 can promote the transcription and expression of GATA2-AS1 as a transcription factor,forming a positive feedback regulatory loop between them.3.GATA2-AS1 facilitated the malignant phenotype of colon cancer via upregulating the expression of GATA2. |
PDF Full Text Request