Mechanism Of YY1-induced Long Noncoding RNA TUG1 Promoting Multiple Myeloma Malignant Progression By Targeting YOD1

Objective To detect the relative expression level of lncRNA-TUG1 in the serum of patients with MM,and to explore the clinical value of serum TUG1 in the diagnosis and treatment of MM.To study the effect of TUG1 on the proliferation,apoptosis and migration of MM cells in vitro and in vivo.To study the regulation of TUG1 transcriptional activity and expression level by transcription factors.To explore the specific mechanism of TUG1 in the nucleus of MM cells.Methods Specimens of MM patients confirmed by the department of hematology of Affiliated Hospital of Nantong University were collected.The relative expression level of TUG1 was detected by qRT-PCR,and the methodology was evaluated.The correlation between the expression level of TUG1 and clinicopathological parameters was analyzed,and the diagnostic efficacy was analyzed by ROC curve.TUG1 interference lentivirus was constructed and transfected into MM cells to screen the fragments with high interference efficiency.CCK-8 assay and Transwell assay were used to detect the effect of TUG1 on the proliferation and migration of MM cells.The effects of TUG1 on MM cell cycle and apoptosis were determined by flow cytometry and western blot.The xenograft model of nude mice was established to analyze the effect of TUG1 on tumor tissue growth by growth curve and immunohistochemistry.FISH and nucleo-plasma separation PCR assay confirmed the subcellular localization of TUG 1.Bioinformatics predicted transcription factors binding to the TUG1 promoter region,and qRT-PCR correlation was verified.YY1 interference and over-expression vector were constructed to interfere with MM cells to detect the regulation of TUG1 expression level.The dual luciferase reporter gene verified the regulation of transcriptional activity of TUG1 by YY1.Transcriptome sequencing and RNA pulldown were used to search for TUG1 target protein,and qRT-PCR correlation was verified.Interference and overexpression vectors of YOD1 were constructed and transfected into MM cells.CCK-8 and Transwell assay were used to detect the effect of YOD1 on proliferation and migration of MM cells.The effects of YOD1 on MM cell cycle and apoptosis were determined by flow cytometry and western blot.YOD1 overexpression vector and TUG1 interference vector were co-transfected into MM cells.CCK-8 and Transwell assay were used to detect the effect of YOD1 overexpression on the proliferation and migration of TUG1 interfered MM cells.Flow cytometry and western blot were used to detect the effect of YOD1 overexpression on cell cycle and apoptosis of TUG1 interfered MM cells.Results Using 18S rRNA as internal parameter,the qRT-PCR method for TUG1 detection has good linearity,repeatability,specificity and stability.The expression of TUG1 in bone marrow of newly diagnosed MM patients was higher than healthy controls(P<0.05).The expression of TUG1 in the serum of the newly diagnosed MM patients was higher than healthy controls(P<0.05),which decreased after treatment(P<0.05),and increased in the relapsed patients(P<0.05).The expression of TUG1 in MM cell lines were also higher than normal plasma cells(P<0.05).The correlation analysis between serum TUG1 expression and clinicopathological parameters showed that highly expressed TUG1 was correlated with total protein(P=0.035),ALB(P=0.037),globulin(P=0.035),β2-MG(P=0.037),and bone injury(P=0.007).Univariate and multivariate analyses showed that TUG1 could be used as an independent predictor of DS staging in MM(P<0.05).The ROC curve showed that the AUC of TUG1 was 0.792,the sensitivity and specificity were 65.5%and 94.9%,higher than that of β2-MG and ALB.Cytofunctional experiments confirmed that after transfection of MM cells with TUG1 interfering vector,the proliferation rate of cells was slowed down(P<0.05),and the migration ability was decreased(P<0.05).Cells showed G0/G1 phase arrest,S-phase cells were decreased(P<0.05),and apoptotic cells were increased(P<0.05).The xenograft model of nude mice confirmed that after TUG1 interference,the tumorigenesis capacity of the cells was decreased significantly and the growth rate slowed down.Immunohistochemisty results of tumor tissues showed that proliferation cells decreased and apoptosis cells increased significantly after TUG1 interference.FISH and nucleo-plasma isolation PCR experiment confirmed that TUG1 was mainly located in the MM nucleus.Bioinformatics analysis predicted that the transcription factor YY1 could bind to the promoter region of TUG1.The expression of YY1 was positively correlated with that of TUG1(P<0.05),and it was highly expressed in MM patients(P<0.05).MM cells were transfected with YY1 interference and overexpression vector,and the results confirmed that TUG1 was down-regulated after YY1 interference(P<0.05),and up-regulated after YY1 overexpression(P<0.05).The dual luciferase reporter gene confirmed that YY1 could promote the transcriptional activity of TUG1(P<0.05).Transcriptome sequencing after TUG1 interference,RNA pulldown and correlation analysis showed that YOD1 was a potential target protein of TUG1.Cytofunctional experiments confirmed that after transfection of MM cells with YOD1 interfering vector,the proliferation rate of cells was slowed down(P<0.05),the migration ability was decreased(P<0.05),S-phase cells were decreased(P<0.05),and apoptotic cells were increased(P<0.05).After YOD1 overexpression vector was transfected into MM cells,the cell proliferation rate and migration ability was increased(P<0.05),S-phase cells were significantly increased(P<0.05),and the apoptotic cells were significantly decreased(P<0.05).Functional recovery experiments confirmed that after co-transfection of YOD1 overexpression vector and TUG 1 interference vector,the cell proliferation rate was accelerated(P<0.05),the migration ability was increased(P<0.05),the S-phase cells were significantly increased(P<0.05),and the apoptotic cells were significantly decreased(P<0.05).Conclusion Serum TUG1 expression in newly diagnosed MM patients increased significantly,but decreased significantly after treatment,suggesting that it may be a potential clinical biomarker for MM diagnosis and treatment monitoring.The expression of TUG1 regulated by transcription factor YY1 increased in MM cells,and targeted YOD1 to promote cell proliferation and migration,inhibit cell apoptosis,and further promote the malignant progression of MM.This study suggests that TUG1 may be a novel marker for MM diagnosis and a potential target for MM treatment.