The Effect Of LncRNA NKX3-1 On The Proliferation And Invasiveness Of Glioma Cells Through Fem1b/SPDEF Pathway

Objectives:Find the differentially expressed LncRNA in glioma cells by bioinformatics,and select the target differentially expressed LncRNA for next research;Analyze the effect of differentially expressed LncRNA on glioma in vivo and in vitro,and further study the regulation mechanism of differentially expressed LncRNA on downstream pathway in glioma.Methods:(1)Firstly,the glioma related data were downloaded from TCGA database,and analyzed by bioinformatics to find the differentially expressed LncRNAs.Through survival analysis,the impact of all differentially expressed LncRNAs on the prognosis of glioma was analyzed,and the LncRNA NKX3-1 that have the most obvious impact on the prognosis was selected for further experiment.Verify the analysis results of TCGA database through external database.(2)The expression of LncRNA NKX3-1 in clinical glioma samples was detected by q RT PCR,and the subcellular localization of LncRNA NKX3-1 was determined by fluorescence in situ hybridization(FISH).CCK-8,flow cytometry,cell scratch and Transwell analysis were used to detect glioma cells’ proliferation,apoptosis and invasion.Nude mice were implanted subcutaneously to verify the effect of differential expression of LncRNA NKX3-1 on tumor growth.(3)The downstream target of LncRNA NKX3-1 was predicted by targetscan database.Luciferase assay was used to study the effect of LncRNA NKX3-1 overexpression on downstream targets.The effect of LncRNA NKX3-1 on the protein expression level in glioma cells was detected by Western blot,Transwell and cell scratch analysis,and the overexpression of the protein with increased expression level was detected.(1)The differential expression profile of LncRNA in glioma was obtained by analyzing the glioma data sets in TCGA database.LncRNA NKX3-1 was significantly increased in glioma tissues,and the differential expression was statistically significant(P < 0.05).Survival analysis showed that the high expression of LncRNA NKX3-1 had an impact on the prognosis of glioma patients,and the difference had statistical significance(P < 0.01).(2)LncRNA NKX3-1 was mainly expressed in glioma nuclei by fluorescence in situ hybridization(FISH).Using CCK-8,flow cytometry,cell scratch and Transwell analysis found that the proliferation,invasion and immigration of glioma cells increased significantly(P < 0.05)and the ability of apoptosis decreased significantly(P < 0.05)in the high expression group of LncRNA NKX3-1.In nude mice,the tumor volume and weight of LncRNA NKX3-1 high expression group increased significantly(P < 0.05).(3)Targetscan database predicted that the downstream target of LncRNA NKX3-1 was Fem1 b.Double luciferase experiment showed that LncRNA NKX3-1 significantly increased the luciferase activity(P <0.05)of Fem1 b 3 ’-utr-wt reporter gene and SPDEF protein level increasd significantly(P < 0.05),and the protein level of Fem1 b decreased significantly(P < 0.05).The invasion and immigration of glioma cells increased significantly in LncRNA NKX3-1 high expression group and SPDEF group(P < 0.05).Conclusions:(1)Survival analysis of differentially expressed genes showed that patients with high expression of LncRNA NKX3-1 in glioma had a worse prognosis.(2)The expression of LncRNA NKX3-1 in glioma cells was higher than that in adjacent tissues,and it was mainly expressed in the nucleus of glioma cells.The high expression of LncRNA NKX3-1 can improve the proliferation of glioma cells and inhibit their apoptosis.The high expression of LncRNA NKX3-1 in nude mice can promote the growth of subcutaneous tumors.(3)The expression of LncRNA NKX3-1 can increase the proteinResult:level of SPDEF and reduce the protein level of Fem1 b.The high expression of LncRNA NKX3-1 in glioma may affect the level of SPDEF protein by down regulating Fem1 b,so as to affect the proliferation,metastasis and invasiveness of glioma cells.