Expression And Mechanism Of Long Non-coding RNA TCF7 In Multiple Myeloma

Objective This study aimed to detect the expression of long non-coding RNA transcription factor 7(lnc-TCF7)as a new biomarker in multiple myeloma(MM)patients;investigate the correlation of lnc-TCF7 with clinical features and prognosis of MM patients;evaluate the effects of lnc-TCF7 knockdown on the proliferation and apoptotic function of MM cells;verify the targeted regulatory relationship between lnc-TCF7 and miR-203,and explore the mechanism of action of lnc-TCF7-MiR-203 axis in MM cells.Methods(1)Bone marrow plasma cells were collected from 86 newly diagnosed MM patients and 30 healthy controls,and the expression of Inc-TCF7 in the two groups were detected by RT-qPCR.(2)Collected and sorted out relevant clinical data of MM patients;used ROC curve to analyze the cut-off value of lnc-TCF7 expression,and performed statistical analysis with various clinical indicators of the patient.(3)Lnc-TCF7 expression in normal bone marrow plasma cells and MM cell lines(OPM2,RPMI8226,NCI-H929,U266)were detected by RT-qPCR;the location of lnc-TCF7 in MM cell lines(RPMI8226,U266)was detected by fluorescence in situ hybridization.(4)The effects of knockdown of lnc-TCF7 on the proliferation and apoptosis of MM cell lines(RPMI8226,U266)were investigated using CCK-8 and flow cytometry,respectively;bioinformatics software predicted the target molecule of lnc-TCF7,observed the change of target molecule expression after knocking down lnc-TCF7 in MM cell lines(RPMI8226,U266);the dual luciferase reporter assay was used to verify whether miR-203 was the target of lnc-TCF7.(5)Knock down lnc-TCF7 expression in MM cell lines(RPMI8226,U266)by transfecting lnc-TCF7 shRNA(Sh-TCF7 group),and used RT-qPCR to detect its effect on the expression of the target molecule miR-203;also observed whether miR-203 Inhibitor(Sh-TCF7&miR Inhibitor group)could rescue miR-203 expression changes caused by knockdown of lnc-TCF7;at the same time,the CCK-8 method and flow cytometry were used to detect changes in the proliferation and apoptotic capacity of the two cell lines.(6)Bioinformatics software predicted the signal regulation pathways mediated by lnc-TCF7 and miR-203;in rescue experiments,RT-qPCR and Western Blot were used to detect the mRNA and protein expression of related molecules in the Jagged1-Notch1 signaling pathway,respectively.Results(1)Compared with the healthy control group,the expression of lnc-TCF7 in bone marrow plasma cells of newly diagnosed MM patients was up-regulated,and the difference was statistically significant.(2)Analysis of clinical data showed that the expression of lnc-TCF7 had a certain correlation with the risk of MM and had a good predictive value;the expression of lnc-TCF7 was correlated with patients’β2 microglobulin levels and ISS staging;the CR rate of the lnc-TCF7 high expression group was lower than that of the lnc-TCF7 low expression group,but the difference was not statistically significant,ORR was similar between the two groups;survival analysis showed that EFS and OS in the high-expression group of lnc-TCF7 were shorter than those in the low-expression group of lnc-TCF7,indicating that it is related to the prognosis of patients.(3)Univariate or multivariate Logistic regression model analysis showed that lnc-TCF7 and other clinical indicators were not related to patients’ CR;univariate or multivariate Cox proportional hazard regression model analysis showed that lnc-TCF7 was not an independent risk factor for EFS,but age,DS staging,and ISS staging were independent risk factors for EFS;analysis of univariate Cox proportional hazard regression model analysis showed that lnc-TCF7 and ISS staging were associated with worse OS,however,the multivariate Cox proportional hazard regression model analysis showed that lnc-TCF7 was not an independent risk factor for OS and ISS stage was an independent risk factor for OS.(4)Compared with normal bone marrow plasma cells,the expression of lnc-TCF7 in MM cell lines(OPM2,RPMI8226,NCI-H929,U266)was significantly increased,the difference was statistically significant;the results of fluorescence in situ hybridization showed that lnc-TCF7 was mainly located in the cytoplasm of MM cell lines(RPMI8226,U266).(5)Compared with the control group,knocking down lnc-TCF7 could inhibit MM cell lines proliferation and promote MM cell lines apoptosis,the difference was statistically significant;detection of the double luciferase reporter gene showed that lnc-TCF7 could bind to miR-203,and knocking down lnc-TCF7 could up-regulate miR-203 expression.(6)Compared with the Sh-TCF7 group,the expression of miR-203 in the Sh-TCF7&miR Inhibitor group decreased without significant change in the expression level of lnc-TCF7;compared with the Sh-TCF7 group,the proliferation ability of MM cell lines in Sh-TCF7&miR Inhibitor group was significantly increased,which weakened the proliferation inhibition effect of knockdown lnc-TCF7 on MM cells.(7)Compared with the Sh-TCF7 group,the expression of Jagged1 and Notch 1 in the MM cell lines of the Sh-TCF7&miR Inhibitor group were both up-regulated by RT-qPCR and Western Blot,the difference was statistically significant.Conclusions(1)Lnc-TCF7 expression is up-regulated in MM patients and MM cell lines,which is related to the deterioration of clinical characteristics and decreased survival rate of patients,and has a good predictive value for the risk of MM disease,so it can be used as one of the ideal biomarkers of MM.(2)Knockdown of lnc-TCF7 plays a role in inhibiting MM cell proliferation and promoting apoptosis by regulating miR-203-mediated Jagged 1-Notch 1 signaling pathway.