Effect And Mechanism Of LncRNA GAS5 On Inhibition Of Pituitary Tumor Growth By MiR-27a-5p/CYLD

Pituitary adenoma is a common intracranial tumor,accounting for about fifteen percent of intracranial tumors.Aggressive pituitary tumors often cause severe clinical syndromes such as visual impairment,hormonal abnormalities,or metabolic syndrome.Clinical treatment of pituitary tumors remains a great challenge because of the lack of effective therapeutic targets.Therefore,in recent years,researchers have been exploring the mechanisms of pituitary tumor development at the molecular level and searching for potential anti-tumor targets to open up new avenues for clinical treatment of pituitary tumors.Long non-coding RNA(lncRNA)is a class of non-coding RNA molecules more than two hundred nucleotides in length,whose abnormal expression is closely related to cell proliferation,apoptosis and tumor growth.Studies have shown that long-stranded non-coding RNA growth arrest-special transcript 5(GAS5)is expressed in a variety of tumor tissues and plays dual oncogenic or oncogenic functions in different tumors.However,the expression of lncRNA GAS5 in pituitary tumors and its mechanism of action are still unclear.Therefore,this thesis focuses on the effect of lncRNA GAS5 on pituitary tumor growth and its related mechanisms.1.Research Methodology(1)Tumor samples and cell line selection.Sixty-three patients with pituitary adenoma,rat normal pituitary gland cells and rat pituitary adenoma cell lines GH1,GH3,RC-4B/C and MMQ cells were included in the study,and lncRNA GAS5 expression levels of pituitary tumor tissues and cell lines were detected by fluorescence real-time quantitative PCR(RT-qPCR).(2)Analysis of lncRNA GAS5 overexpression on pituitary tumor cell activity.The lncRNA GAS5 overexpression plasmid pcDNA3.1 GAS was constructed,and the constructed plasmid was transfected with pituitary tumor cell lines,and the proliferation,clone formation and apoptosis of the transfected cells were detected by CCK-8 method,clone formation assay and flow cytometry.(3)lncRNA GAS5 targeting miR-27a-5p analysis.The miRNAs that bind to lncRNA GAS5 were predicted according to Starbase software,miR-27a-5p was screened,RNA binding protein immunoprecipitation technique and dual luciferase reporter analysis system were applied to verify lncRNA GAS5/miR-27a-5p binding,and RT-qPCR was performed to determine the effect of lncRNA GAS5 expression in pituitary tumor cells on miR-27a-5p expression in pituitary tumor cells.(4)Analysis of the effect of miR-27a-5p expression changes on downstream CYLD(Cylindromatosis)expression.CYLD was screened using the TargetScan tool to predict miR-27a-5p target genes.miR-27a-5p mRNA and protein expression level changes were detected after transfection of pituitary tumor cells with miR-27a-5p analogs or suppressors,and the dual luciferase reporter analysis system was used to verify miR-27a-5p binding to CYLD.(5)Analysis of the activity of lncRNA GAS5 cotransfected with miR-27a-5p/CYLD in pituitary tumor cells.Cells were transfected with pcDNA3.1-GAS5,pcDNA3.1-GAS5+miR-27a-5p mimic or pcDNA3.1-GAS5+sh-CYLD expression plasmids,and the expression levels of CYLD protein in each group of cells were detected by Western blot,CCK-8 method,clone formation assay and flow cytometry.The proliferation,clone formation and apoptosis of the transfected cells were detected by Western(6)In vivo experiments to analyze the growth of overexpressed lncRNA GAS5 pituitary tumors.BALB/c nude rats were selected,and lncRNA GAS5 overexpressing GH3 cells were then transfected with miR-27a-5p mimics or sh-CYLD expression vectors,respectively.Rats were injected subcutaneously with each group of plasmid-transfected cells to observe the growth of transplanted tumors,and immunohistochemical staining was performed to detect the expression of CYLD in each group of transplanted tumors.(7)Enzyme-linked immunosorbent assay(ELISA)was performed to detect TGF-β1 in pituitary tumor tissue homogenates,which is an important immunosuppressive factor promoting tumor growth.The homogenates were prepared from each group of transplanted tumor tissues in(6)above,and the TGF-β1 levels in each tissue homogenate were measured according to the kit instructions.2.Research results(1)LncRNA GAS5 expression was down-regulated in human invasive pituitary adenoma tissues and rat pituitary tumor cell lines.RT-qPCR results showed that the expression level of LncRNA GAS5 was significantly decreased in human invasive pituitary adenoma tissues compared with non-invasive pituitary adenoma tissues.It was also seen that the expression levels of LncRNA GAS5 were lower in rat pituitary adenoma cell lines GH1,GH3,RC-4B/C and MMQ cells than in normal pituitary gland cells.(2)LncRNA GAS5 overexpression induces apoptosis in pituitary tumor cells.GH3 and MMQ cells overexpressing LncRNA GAS5 showed reduced cell colony formation,decreased cell viability,and increased apoptosis.Flow cytometry results showed increased cell cycle arrest in pituitary tumor cells overexpressing LncRNA GAS5.(3)LncRNA GAS5 regulates pituitary tumor cell activity via miR-27a-5p/CYLD.①Bioinformatics analysis and RNA-binding protein immunoprecipitation techniques and dual luciferase reporter assays showed that LncRNA GAS5 could bind to a novel target miRNA,miR-27a-5p,and down-regulate miR-27a-5p expression.②Bioinformatics analysis and dual luciferase reporter assay showed that miR-27a-5p could bind to CYLD and down-regulate CYLD mRNA and protein expression.③Expression plasmids were cotransfected with salivary tumor cells,and the results showed that miR-27a-5p overexpression and knockdown of CYLD attenuated the effects of reduced colony formation,elevated apoptosis and increased cell cycle arrest in salivary tumor cells caused by LncRNA GAS5 overexpression.(4)LncRNA GAS5 inhibits the growth of transplanted tumors in nude mouse pituitary tumor cells.①LncRNA GAS5 overexpression significantly inhibited the growth of transplanted tumors in GH3 cells.miR-27a-5p overexpression or knockdown of CYLD both attenuated the inhibitory effect of LncRNA GAS5 overexpression on the growth of transplanted tumors.②LncRNA GAS5 overexpression resulted in increased CYLD expression in GH3 cell transplants.miR-27a-5p overexpression or knockdown of CYLD attenuated the effect of LncRNA GAS5 overexpression to increase CYLD.(5)LncRNA GAS5 inhibits TGF-β1 secretion from pituitary tumor cells.The TGF-β1 level in the tissue homogenate of transplanted GH3 cells with LncRNA GAS5 overexpression was significantly lower than that of the control group.miR-27a-5p overexpression or knockdown of CYLD both attenuated the inhibitory effect of LncRNA GAS5 on TGF-β1 secretion.3.Research conclusionsIn summary,LncRNA GAS5 is lowly expressed in human pituitary tumor tissues,and overexpression of LncRNA GAS5 inhibits pituitary tumor cell colony formation,promotes pituitary tumor cell apoptosis,inhibits pituitary tumor cell transplantation tumor growth in vivo,and downregulates TGF-β1 production.LncRNA GAS5 targets binding to miR-27a-5p,inhibits miR-27a-5p expression and upregulates CYLD expression.miR-27a-5p,on the other hand,can downregulate CYLD expression by binding to CYLD.miR-27a-5p overexpression or knockdown of CYLD attenuates the effect of LncRNA GAS5 overexpression.The above study revealed that LncRNA GAS5 may inhibit pituitary tumor growth through the miR-27a-5p/CYLD axis and may also indirectly affect the tumor immune microenvironment by inhibiting TGF-β1 production.However,how it affects the tumor immune microenvironment through TGF-β1 still needs to be further explored in depth.The expression and role of LncRNA GAS5 in pituitary tumors hold promise as a new target for clinical pituitary tumor therapy.