Circ-CDYL Affects The Survival Of Multiple Myeloma Cells Through MiR-1180 Targeting YAP

Objective: Multiple myeloma accounts for about 10% of hematological malignancies.It is the second most common hematological malignant tumor.It is a form of malignant clonal hyperplasia of plasma cells.It is still an incurable disease.Although with the autologous hematopoietic stem cell transplantation,the application of new drugs and the immunotherapy,the efficacy and prognosis of patients with multiple myeloma have improved significantly,and the 5-year overall survival has been significantly extended,but most patients eventually still relapse in the end.There are many factors that affect the progression and prognosis of multiple myeloma.Chromosomal abnormalities,activation of oncogene mutations,and changes in the bone marrow microenvironment all play key roles in the pathogenesis,progression and prognosis of multiple myeloma.However,there is no ideal molecular index for judging the prognosis of the disease,and exploring the factors that affect the prognosis is the current research hotspot.circRNA,as a new type of non-coding RNA,is a covalently closed endogenous non-coding RNA.It has neither 5 ’and 3’ end polarities nor PolyA tail,so it is structurally stable and not easily degraded by endonucleases.CircRNA has tissue-and disease-specific expression,and plays an important regulatory function in the development of cancer.In-depth research on circRNA is of great significance to reveal the development of tumors.circRNA has a variety of biological functions,including miRNA "sponge" adsorption of miRNA,translation of proteins,regulation of RNA binding proteins,regulation of parental gene expression and so on.At present,the most frequently reported functions of circRNA are its competitive binding to miRNA,inhibiting the binding of miRNA to downstream target genes,regulating the expression of target genes,affecting cell function and biological behavior,and playing an important role in canceration and invasive progress.Through database analysis and literature review,three circRNAs related to cancer or their parental genes related to cancer were selected,including circ-CDYL,circ-HIPK3,and circ-ACC1.The pre-experiment used qRT-PCR to detect multiple myeloma samples The expression of these three circRNAs in the control sample was found to be stable and highly expressed in multiple myelomacompared to the control group.Therefore,circ-CDYL was selected as the target gene for subsequent research.circ-CDYL is located on chromosome 6 chr6: 4891946-4892613 and is derived from head and tail splicing of the exon of its parent gene CDYL.There are reports in the literature that circ-CDYL is involved in regulating the occurrence and development of tumors.For example,circ-CDYL has an important driving role in the development of hepatocellular carcinoma and is related to the disease process in mantle cell lymphoma.However,the molecular function and biological role of circ-CDYL in MM have not been reported.In this study,the differential expression of circ-CDYL between newly diagnosed MM patients and normal people were determined through the detection of clinical samples,and the possibility and rationality of circ-CDYL for judging the prognosis of MM was explored in combination with clinical data and follow-up analysis methods of patients.According to its structural characteristics and localization in MM cells,its biological function in MM was speculated,and it was verified through cell experiments and animal experiments,and its possible mechanism of action was initially explored.Methods: 1.Through bioinformatics methods,we selected 3 circRNAs associated with tumors or their parental genes associated with tumors,and determined circ-CDYL as the target gene for research by Realtime PCR pre-experiment.Increasing the collection of samples,the relative expression of circ-CDYL was detected by Realtime PCR in 72 MM samples and 13 normal control samples.and the possibility and rationality of circ-CDYL as a potential prognostic biomarker for MM were explored by clinical data analysis and follow-up analysis.2.Lentiviral packaging was used to construct a stable circ-CDYL knockdown cell model.Cell proliferation and cell viability were measured by EdU and CCK-8 methods,and flow cytometry detected the apoptosis of tumor cells.3.Nucleus and plasma were separated.The nucleus and cytosolic RNA were extracted separately,and the location of circ-CDYL in the cells was determined by Realtime PCR experiments.4.The bioinformatics method predicted miRNAs that may be targeted by circ-CDYL.Realtime PCR experiments detected miRNA expression levels,determined miR-1180 as downstream miRNA,and detected the relative expression of miR-1180 in clinical samples,and analyzed the correlation between miR-1180 with circ-CDYL.The targeting effect of miR-1180 and circ-CDYL were further verified by circRNA pull-downexperiment and dual luciferase reporter gene system.5.Bioinformatics methods predicted that the YAP gene may be the downstream target gene of miR-1180,and verified by the double luciferase reporter gene.6.Realtime PCR experiments were used to detect mRNA expression of YAP after overexpression and knockdown of miR-1180.7.In stable silence circ-CDYL cells,after transient transfection of miR-1180 inhibitors,Realtime PCR and Western blot were used to detect the expression of mRNA and protein of YAP,further proving that miR-1180 targets YAP.8.In order to prove the circ-CDYL,miR-1180 and YAP regulatory network,we transfected miR-1180 inhibitors in stable silence circ-CDYL cells to restore the expression of YAP,and then detected cell proliferation and invasion ability by EdU method and transwell experiment.9.In vivo experiments,establish a nude mouse xenograft model,and test the effect of silence circ-CDYL on tumorigenicity of multiple myeloma in vivo.HE staining was used to observe the proliferation of tumor cells.Realtime PCR was used to detect the mi R-1180 and YAP in tumor tissues.The expression of YAP and Ki-67 were detected by immunohistochemistry to evaluate the proliferation and invasion ability of tumor cells.Results: 1.Compared with normal control samples,circ-CDYL was significantly overexpressed in myeloma cells of newly diagnosed multiple myeloma patients.2.We grouped patients according to the median of circ-CDYL relative expression,and analyzed the correlation between circ-CDYL expression and clinical risk factors of myeloma in combination with clinical data.It showed that: circ-CDYL expression was closely related to ISS and DS stage,and the patients with high expression of circ-CDYL were more likely to have stage III of ISS and DS,and they were more likely to be associated with hypercalcemia,and the survival time of patients with high expression of circ-CDYL were shorter than the patients with low expression of circ-CDYL.3.In vitro cell experiments showed that compared with the control group,silencing circ-CDYL significantly reduced the DNA synthesis rate of MM cells,reduced the activity of MM cells,and promoted the apoptosis of MM cells.4.Realtime PCR experiments proved that circ-CDYL was mainly localized in MM cytoplasm.5.CircRNA pulled down,dual luciferase reporter gene and Realtime PCR experiments showed that miR-1180 was the downstream target gene of circ-CDYL.6.The dual luciferase reporter gene and Realtime PCR experiments verified YAP association with miR-1180.7.Realtime PCR and Western Blot experiments verifiedthat in silenced circ-CDYL cells,YAP mRNA and protein levels were down-regulated,and miR-1180 knockdown effectively prevented this inhibition.Thus,the circ-CDYL,miR-1180 and YAP regulatory networks were identified.8.After the expression of YAP was restored in silenced circ-CDYL cells,both the cell’s proliferation ability and invasion ability were enhanced.9.In vivo experimental results: Subcutaneous tumor formation experiments in nude mice confirmed that multiple myeloma tumor cell growth was slowed down,tumor volume and weight were significantly reduced,and miR-1180 was up-regulated in tumor tissue cells while YAP expression was suppressed after circ-CDYL was silenced.Down-regulation and HE staining showed that MM cells had reduced proliferation and invasion.At the same time,circ-CDYL / miR-1180 / YAP regulatory axis also existed in vivo.Conclusion: 1.circ-CDYL is significantly overexpressed in myeloma cells of newly diagnosed multiple myeloma patients,and patients with overexpressed circ-CDYL had a poor prognosis,and the expression of circ-CDYL could jugde the prognosis of MM.2.Circ-CDYL targeted mi R-1180,which was negatively correlated with circ-CDYL expression.3.MiR-1180 targeted the 3′-UTR region of YAP to inhibit YAP expression.4.circ-CDYL up-regulated YAP expression in MM cells through targeted binding to miR-1180,and promoted MM cell proliferation and invasion.5.Animal experiments showed that the circ-CDYL / miR-1180 / YAP regulatory network existed in vivo.