| Background:Pituitary adenoma(PA)is one of the most common intracranial primary tumors,accounting for about 16%of intracranial tumors,with a population prevalence of 78-116 per 100,000 people.At present,for the treatment of PA,in addition to pure prolactinomas that can be treated with drugs to obtain better curative effects,other types of tumors still need to be treated by surgical resection.Although PA is a benign tumor,there are still up to 35%of tumors that invasively grow to the surrounding structures(sellar diaphragm,suprasellar,cavernous sinus,sphenoid sinus,slope),protruding into the brain tissue,ventricle,surrounding the internal carotid artery and cranial nerves,and eroding bone and dura mater,surgical resection is extremely difficult,the incidence of various serious complications during and after the operation is high,the tumor is difficult to be completely resected,and the recurrence rate is high,this type of PA is called invasive pituitary adenoma(IPA),which is a key factor restricting the improvement of PA diagnosis and treatment.There is an urgent need to find a safe and effective method for the treatment of IPA.Therefore,clarifying the pathogenesis of IPA,and then discovering possible molecular markers and therapeutic targets,is essential to comprehensively improve the level of diagnosis and treatment of PA.The pathogenesis of PA is currently believed to be related to factors such as gene mutations,gene expression disorders,and epigenetic changes that lead to cell cycle disorders and uncontrolled cell proliferation.With the development of gene sequencing analysis technology,more and more studies have found that long non-coding RNA(lncRNA)plays a key role in the occurrence and development of many tumors,including PA.However,due to the large number of lncRNAs,our understanding of them is only the tip of the iceberg.In order to further explore the relationship between lncRNA and PA,especially the progress of IPA,we performed whole transcriptome sequencing on IPA and Non-invasive pituitary adenoma(NIPA)tissues and screened out differentially expressed lncRNAs and microRNAs(miRNA),messenger RNA(mRNA),and then determine the target lncRNA and conduct further verification and research on it.Object:Through the whole transcriptome sequencing analysis of IPA and NIPA tissues,the differential expression profiles of lncRNA,miRNA,and mRNA were screened,and the most up-regulated lncRNA LINC00473 in IPA was obtained,and its related competing endogenous RNA(ceRNA)regulatory network(lncRNA/miRNA/mRNA)was constructed.Further verify the gene sequencing results in isolated PA tissues,PA cell lines and PA xenograft models,and explore the impact of LINC00473-related ceRNA regulatory network on PA proliferation and its molecular mechanisms.Methods:1.Perform whole transcriptome sequencing on IPA and NIPA tissue samples,and obtain differentially expressed lncRNA,miRNA and mRNA by analyzing the sequencing results.Among them,the most up-regulated lncRNA in IPA is LINC00473.Through bioinformatics analysis combined with previous research conclusions,a ceRNA regulatory network related to LINC00473 was constructed.Furthermore,the expression levels of LINC00473 and target miRNA and mRNA were verified by qRT-PCR in IPA and NIPA tissue specimens,and the correlation between their expression levels and PA invasiveness was clarified.2.Cultivate PA cell lines AtT-20 and GT1-1 for PA cell related research.Construct a vector that overexpresses LINC00473 and KMT5A,and a targeting siRNA that knocks down LINC00473 and KMT5A,and then transfect PA cells;Construct miR-502-3p mimic and miR-502-3p inhibitor and transfect PA cells.The efficiency of overexpression and knockdown of LINC00473,miR-502-3p and KMT5A were detected by qRT-PCR.3.CCK-8 and EdU fluorescent staining were used to detect the effects of different experimental conditions on the proliferation of PA cells,and flow cytometry was used to detect the effects of different experimental conditions on the cell cycle of PA cells.Western blot was used to detect the effects of different experimental conditions on the expression of cyclin D1,Cyclin-Dependent Kinases 2(CDK2)and KMT5A in PA cells.4.To study the effects of the following experimental conditions on PA cell proliferation,cell cycle and the expression of cyclin D1,CDK2 and KMT5A:Overexpression and knockdown of LINC00473;Overexpression and inhibition of miR-502-3p and overexpression of miR-502-3p while overexpression of LINC00473;Overexpression and knockdown of KMT5A and knockdown of KMT5A while inhibition of miR-502-3p at the same time.5.The dual luciferase reporter gene experiment verified that there are functional binding sites between LINC00473 and miR-502-3p,and between miR-502-3p and KMT5A.6.PA xenograft model verified the effects of overexpression and knockdown of LINC00473 on tumor volume,proliferation capacity,and expression levels of miR-502-3p,KMT5A,cyclin D1 and CDK2.Results:1.Through the whole transcriptome sequencing analysis of 4 cases of IPA and 3 cases of NIPA tissues,the differentially expressed lncRNA,miRNA and mRNA between IPA and NIPA were obtained.Among them,LINC00473 is the most up-regulated lncRNA in IPA.Through bioinformatics analysis and previous research conclusions,the LINC00473-related ceRNA regulatory network LINC00473/miR-502-3p/KMT5A was constructed,and qRT-PCR detection confirmed that the expression levels of the three in vitro IPA and NIPA tissues were consistent with the sequencing results,and the Pearson correlation analysis of the expression levels between the three was consistent with the ceRNA regulatory mechanism.2.CCK-8 and EdU cell proliferation experiments confirmed that overexpression of LINC00473 and KMT5A and inhibition of miR-502-3p can promote the proliferation of PA cells,while knockdown of LINC00473 and KMT5A and overexpression of miR-502-3p can inhibit the proliferation of PA cells.The inhibitory effect of overexpression of miR-502-3p can be neutralized by overexpression of LINC00473,and the promotion effect of inhibiting miR-502-3p can be offset by knocking down KMT5 A directly.3.Flow cytometry results show that overexpression of LINC00473 and KMT5A and inhibition of miR-502-3p can reduce the number of cells in G0/G1 phase of the PA cell cycle,while increasing the number of cells in G2/M phase;knockdown of LINC00473 and KMT5A and overexpression The expression of miR-502-3p can increase the number of G0/G1 phase cells in the PA cell cycle,while reducing the number of G2/M phase cells.The effect of overexpression of miR-502-3p can be neutralized by overexpression of LINC00473,and the promotion effect of inhibiting miR-502-3p can be offset by knocking down KMT5A directly.4.Western blot confirmed that overexpression of LINC00473 and KMT5A and inhibition of miR-502-3p can up-regulate the expression of KMT5A,cyclin D1 and CDK2.Knockdown of LINC00473 and KMT5A and overexpression of miR-502-3p can inhibit the expression of KMT5A,cyclin D1 and CDK2 Express.The effect of overexpression of miR-502-3p can be neutralized by overexpression of LINC00473,and the promotion effect of inhibiting miR-502-3p can be offset by knocking down KMT5 A directly.5.Dual luciferase reporter gene experiment confirmed that there are functional binding sites between LINC00473 and miR-502-3P,and between miR-502-3p and KMT5A.6.PA xenograft model showed that overexpression of LINC00473 can significantly increase tumor volume,enhance tumor proliferation,increase the expression of KMT5A,cyclin D1 and CDK2,and decrease the expression of miR-502-3p.While knocking down LINC00473 can significantly reduce tumor volume,the ability of tumor proliferation is weakened,the expression of KMT5A,cyclin D1 and CDK2 is reduced,and the expression of miR-502-3p is increased.Conclusions:1.LINC00473 and KMT5A were significantly up-regulated in IPA,while miR-502-3p was significantly down-regulated.2.The LINC00473/miR-502-3p/KMT5A axis is closely related to the invasiveness of PA.3.LINC00473 can promote the proliferation of PA.This effect is achieved by inhibiting miR-502-3p to promote the expression of KMT5A.4.The mechanism of LINC00473 to promote PA proliferation may be achieved by inhibiting miR-502-3p to promote the expression of KMT5A,thereby promoting the expression of cyclin D1 and CDK2,and accelerating the progress of the cell cycle. |
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