| Orbital lymphoma refers to the malignant lymphoma occurring in the eyelid,conjunctiva and orbit.According to the pathological features,it can be divided into two categories:Hodgkin's lymphomas and non-Hodgkin's lymphomas.According to the cell origin,it can be divided into B-cell lymphoma,T-cell lymphoma and NK cell lymphoma.Different subtypes of lymphoma have different biological behavior and prognosis.Orbital diffuse large B cell lymphoma(DLBCL)is intermediate-high malignant lymphoma with the highest incidence in orbit,and mainly occurrs in adults,with high degree of malignancy and poor prognosis.At present,the combination therapy,surgical resection of lesions combined with postoperative radiotherapy and chemotherapy is the main treatment for orbital DLBCL.However,these methods have disadvantages such as serious side effect,high relapse rate.With the deep understanding of the mechanism of tumor occurrence and development and the rapid progress of biotechnology,tumor therapy has changed from the era of traditional cytotoxicity drug therapy to the era of targeted therapy.With the application of targeted drugs,the treatment of lymphoma has made a breakthrough.Target therapy is effective with little adverse reactions,but the current targeted drugs are still not effective for about 40%of dlbcl patients.Thus,it needs to find new targets and develop new better drugs and treatments for this disease.LncRNAs is non-coding RNA with a length of more than 200 nucleotides,having important biological functions and can widely participate in physiological and pathological processes of body.It can participate in many important regulatory processes,such as gene imprinting,chromatin remodeling,chromatin modification,chromosome silencing,transcriptional regulation and translation regulation.More and more evidence shows that LncRNAs is closely related to cell differentiation,proliferation and many human diseases.LncRNAs has become a hot point in tumor research in recent years.Peng w et al found in diffuse large B-cell lymphoma,the expression of HULC in tumor tissues was up-regulated,and kaplan-meier analysis and COX regression analysis showed that the expression level of HULC was positively correlated with the poor prognosis of patients.However,the roles and molecular mechanisms of HULC in DLBCL have not yet been clarified.MicroRNA(miRNA)is a short single chain endogenous non-coding RNA with a length of about 22nt.It can regulate cell differentiation,proliferation and apoptosis by binding with targeted miRNA to inhibit target miRNA translation or direct degradation.According to the study presented by Shu YJ et al,miR-29c-5p regulated the MAPK pathway by inhibiting expression of CPEB4,which functioned as tumor suppressor in gallbladder carcinoma.Recent studies have found that LncRNA can regulate gene expression by binding miRNA.Bioinformatics studies have shown that HULC contains responsive elements of miR-29c-5p.Therefore,it is speculated that HULC may regulate the MAPK/ERK signaling pathway by sponging miR-29c-5p,and play a crucial role in occurrence and development of orbital DLBCL.This study intends to reveal a new regulation pathway,HULC-miR-29c-5p axis in orbital DLBCL,and analyze its roles in occurence and development of orbital DLBCL.At present,there are few studies on the LncRNA in the occurrence and development of orbital DLBCL.This study will help us to better understand the pathogenesis of orbital DLBCL and provide a new therapeutic target based on IncRNAs for the treatment of orbital DLBCL.This study aimed to explore the roles and mechanisms of HULC in orbital DLBCL.Firstly qRT-PCR was used to detect HULC and miR-29c-5p expression in the orbital lymphoma tissues;Sencondly,in vitro studies,miR-29c-5p and HULC overexpression stable cell lines,HULC knockout were constructed using Pfeiffer cell lines,and the effects of HULC knockout and HULC-miR-29c-5p axis on proliferation,invasion,apoptosis and the MAPK/ERK signaling pathway of orbital lymphoma cells were detected;Finally,in vivo studies,the sh-HULC Pfeiffer xenograft models in nude mice were established,and the effect of HULC knockout on the growth of DLBCL xenografts and its mechanism were investigated.Objective:1.To detect the expression of HULC and miR-29c-5p in orbital lymphoma tissues.2.To study the effect of HULC knockout and HULC-miR-29c-5p axis on the cells proliferation,invasion,apoptosis and the MAPK/ERK signaling pathway in DLBCL cells.3.To further study the effect of HULC knockout on growth of xenografts and miR-29c-5p expression,cell proliferation,the MAPK/ERK signaling pathway in DLBCL xenografts.Main content:The first part:The expression of HULC and miR-29c-5p in orbitalDLBCLMethods1.The histological features of HE stained orbital DLBCL tissues were observed by microscope.2.The expression of HULC and miR-29c-5p in orbital lymphoma tissues was determined by qRT-PCR.Results1.HE staining showed that tumor cells were diffusely distributed,large-sizedlymphoid cells,cell shape was round,nucleus with basophilic-staining is large in size,and mitotic figure was often seen.2.Compared with normal adjacent tissues and reactive lymphoproliferative tissues,expression of HULC was significantly increased in orbital DLBCL tissues,and expression of miR-29c-5p was significantly decreased.The second part:The effect of HULC on the biological function of DLBCL cells and its molecular mechanismMethods1.The plenti-sh-HULC,plenti-HULC and plenti-miR-29c-5p lentiviral vectors were constructed.The lentiviral vectors were packaged to gain lentiviruses expressing sh-HULC,HULC or miR-29c-5p and lentiviruses were titrated.Then Pfeiffer cells were infected with lentiviruses,and the stable cell lines were obtained through screening.2.CCK-8,Transwell,flow cytometry was used to detect proliferation,invasion and apoptosis in Pfeiffer cells.3.Western blot was used to measure the expression of the MAPK/ERK signaling pathway related to proteins p-MEK1/2 and p-ERK in Pfeiffer cells.4.The expression of HULC and miR-29c-5p in Peripheral blood mononuclear cells(PBMC cells)and Pfeiffer cells was detected by qRT-PCR.5.Bioinformatics predicted the miRNAs binding with HULC.Dual-luciferase reporter gene experiment verified the relationship between HULC and miR-29c-5p.Results1.The stable sh-HULC and Lv-HULC Pfeiffer cell lines were successfully constructed,and the Lv-HULC Pfeiffer cells were infected with Lv-miR-29c-5p to obtain miR-29c-5p+Lv-HULC cells.2.HULC knockout inhibited the cells proliferation and invasion,promoted cell apoptosis,and supressed the activity of the MAPK/ERK signaling pathway in Pfeiffer cells.3.Compared with PBMC cells,expression of HULC was significantly increased in Pfeiffer cells,and expression of miR-29c-5p was significantly decreased.Both bioinformatics prediction and dual-lucdferase reporter assay proved that HULC directly binded to miR-29c-5p and inhibited expression of miR-29c-5p.4.HULC-miR-29c-5p axis regulated the proliferation,invasion and apoptosis in Pfeiffer cells through influencing the activity of the MAPK/ERK signaling pathway.The third part:The effect of HULC on the growth of DLBCL xenografts and its molecular mechanismMethods1.The stable sh-HULC Pfeiffer cells were used to construct the DLBCL nude mouse models,and the effect of HULC downregulation on volume and weight of xenografts were examined.2.The expression of HULC and miR-29c-5p in DLBCL xenografts was detected by qRT-PCR.3.The effect of HULC downregulation on expression of Ki67 in DLBCL xenografts was assessed by Immunohistochemistry.4.The expression of proteins(p-MEK1/2 and p-ERK)associated with the MAPK/ERK signaling pathway was measured by Western blot in DLBCL xenografts.Results1.It was found that HULC knockout inhibited the growth of DLBCL xenografts in nude mice.2.Results from qRT-PCR analysis showed that HULC expression was significantly reduced in DLBCL xenografts with HULC knockout compared with control group,and miR-29c-5p expression was significantly increased in HULC knockout group.3.Immunohistochemical detection exhibited that the positive expression rate of Ki67 in DLBCL xenografts with HULC knockout was significantly lower than that in control group.4.Western blot analysis showed that expression of p-MEKl/2 and p-ERK were significantly decreased in DLBCL xenografts with HULC knockout compared with control group.Conclusion1.Compared with normal adjacent tissues and reactive lymphoproliferative tissues,a significant increase in expression of HULC in orbital DLBCL tissues was observed,whreras an obvious reduction in expression of miR-29c-5p was exhibited.2.HULC expression was significantly upregulated in Pfeiffer cells compared with PBMC cells,while miR-29c-5p expression was significantly decreased.Bioinformatics software analysis and dual-luciferase reporter assay demonstrated that HULC sponged to miR-29c-5p and inhibited expression of miR-29c-5p.HULC-miR-29c-5p axis regulated proliferation,invasion,and apoptosis in Pfeiffer cells by affecting activity of the MAPK/ERK signaling pathway.3.In vivo experiments further showed that HULC knockout enhanced expression of miR-29c-5p and repressed cell proliferation and growth of DLBCL xenografts through inhibiting the MAPK/ERK signaling pathway. |
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