Molecular Mechanism Of LINC00473 In The Cell Proliferation Of Pituitary Adenoma

Objective:To screen out the differential expression of lncRNA profiles in invasive pituitary adenomas and non-invasive pituitary adenomas,and further explore the effect of the most significantly up-regulated lncRNA on the cell proliferation of pituitary adenoma and their molecular mechanisms.Methods:1.The differential expression of LncRNAs between in four cases of invasive pituitary adenoma samples(IPA)and three cases of non-invasive pituitary adenoma samples(NIPA)was explored by RNA-sequencing at first.Then,the Pearson correlation coefficient was used for the quality control of the sequencing data.To visualized of the differential expression LncRNAs,the hierarchical clustering analysis map of differentially expression LncRNA was construct based on the FPKM value of all LncRNAs in each sample.The highest expression of LncRNA in invasive pituitary adenomas wae screened out,and plotted the GO function and KEGG pathway by R package.In a word,the potential biological function are preliminary predicted based on bioinformatics analysis.Further verification of the expression level of LINC00473 in invasive pituitary adenoma tissue and non-invasive pituitary adenoma tissue by Q-PCR.2.Construction the over-expression plasmid vector and target silencing siRNA for LINC00473 at first,liposomes were used to transfection of plasmids and siRNA into pituitary adenoma cell lines AtT2 and GT1-1.The efficiency of over-expression and knock down of LINC00473 in two groups were detected by Q-PCR.3.The effect of over-expression and knock down LINC00473 on the proliferation ability of pituitary adenoma AtT20?GT1-1 cells was detected by CCK-8 method and EdU fluorescence staining.The effect of over-expression and knock down LINC00473 on the cell cycle of pituitary adenoma AtT20?GT1-1 cells was detected by flow cytometry.4.Western blot was used to detect over-expression and knock down LINC00473 on cell cycle related proteins CDK2 and Cyclin D1 in pituitary adenoma AtT20?GTl-1 cells.Results:1.Pearson correlation coefficient chart proved that the repeatability of samples between IPA group and NIPA group is good and the datas were reliable.According to the result of hierarchical clustering analysis,the highest expression level in invasive pituitary adenoma was selected as LINC00473.It was verified by Q-PCR that the expression level of LINC00473 in IPA samples was higher than that in NIPA samples,which was consistent with the trend of profiles results.GO enrichment analysis and KEGG signal pathway enrichment analysis suggest that LINC00473 may play a role in regulating cell proliferation and cell cycle.2.The results of CCK-8 and EdU cell proliferation experiment confirmed that over-expression of LINC00473 could promote the cell proliferation ability of pituitary adenoma AtT20?GT1-1 cells,while knocking down LINC00473 could inhibit the cell proliferation ability of pituitary adenoma AtT20?GT1-1 cells.3.The results of flow cytometry showed that over-expression of LINC00473 could decrease the proportion of pituitary adenoma AtT20?GT1-1 cells in G0/G1 phase ratio and increase the proportion of G2/M phase ratio,while knocking down LINC00473 could increase the proportion of pituitary adenoma AtT20?GT1-1 cells in G0/G1 period and decrease the proportion of pituitary adenoma cells in G2/M phase ratio.4.Western blot confirmed that over-expression of LINC00473 could up-regulate the expression of cell cycle related proteins CDK2 and Cyclin D1;knocking down LINC00473 can inhibit the expression of cell cycle related proteins CDK2 and Cyclin D1.Conclusion:LINC00473 is closely related to the occurrence and development of invasive pituitary adenomas;LINC00473 can promote the proliferation of pituitary adenomas,and its mechanism is related to the regulation of cell cycle-related proteins and thereby changing the cell cycle proportion.