| Background and objectiveDiffuse large B cell lymphoma is one of the most type of adult non-Hodgkin lymophma. There is an upward trend in the incidence rate of DLBCL in recent years. The incidence rate accounts for about 30%-40% in western countries, while in China the incidence is higher accounting for about 40-50% of NHL. A large number of studies have shown that diffuse large B cell lymphoma is a high heterogeneous and invasive tumor, mainly in clinical manifestation, immune phenotype, genetic characteristics, response to chemotherapy and prognosis, etc. With the combination of chemotherapy and targeted drugs such as rituximab, the treatment effect of DLBCL patients is better, the survival time was significantly extended, but there are still some patients died of this disease because of refractory, relapse or resistance to chemotherapy. Therefore, it is particularly important to assess the risk factors of DLBCL for us to judge prognosis and adopt corresponding chemotherpy before treatment. Currently, the International Prognostic Index (IPI) is widely used clinically for assessment of the prognosis of patients with DLBCL and is the indicators of risk stratification. The clinical judgment has brought a lot of benefits, but we found that the patients with the same IPI risk group had quite different clinical curative effect and prognosis in the clinical work.Gene expression profiling studies have led the division of DLBCL into two major subtypes based on the putiative cell of orgin: germinal centre B-cell-like (GCB) and activated B-cell-like(ABC) subtypes. Survival is better in the GCB group compared with the ABC group. In recent years, the biological research of GCB-DLBCL developed significantly, however the insight in the biology and the carcinogenesis of ABC-DLBCLremains poorly understood. DLBCL may involve the bone marrow or extensive infiltration in some cases, while some are not. The pathophysiological mechanism is still unclear.Therefore, the identification of specific prognostic biomarkers and investigation of the mechanisn is essential for designing target chemotherapy.CD44 is a cell adhesion molecule and broadly distributed on the surface of normal cells and tumor cells.CD44 was also identified as a receptor for hyaluronan andparticipates in both physiological and pathological processes, including cell adhesion, angiogenesis, inflammation, tumor development, tumor invasion, metastasis, recurrence and chemoresistance. Therefore, CD44 could be used as a promising prognostic target of cancer treatment. Recent studies showed that high CD44 expression is closely linked to poor prognosis in diffuse large B cell lymphoma. Our previous study found that the CD44H and CD44v6 were effective immune phenotypes. The CD44 expression was elevated in activated B-cell-like diffuse large B cell lymphoma compared with that in germinal center B-cell-like group. What is more, CD44H expression level is closely related to the occurrence of bone marrow involvement. Our study also suggested that CD44 intracellular domain(CD44ICD) participates in the regulation of the biological characteristics andtranscription in ABC-DLBCL.Then,we established a stable low expression of CD44 in ABC-DLBCL cell line and found that CD44 knowdownby shRNA can inhibit the proliferation, migration and induce apoptosis of ABC-DLBCL cell. Although the evidences implicated that CD44 gene is associated with tumor development, tumor invasion, and metastasis in ABC-DLBCL cells.In order to further investigate the mechanisn and the related pathway protein.We first established the overexpression lentivirus vector (LEGO-CD44-GFP) using lentivirus vector, transfected them into ABC-DLBCL cell line OCI-ly3, sorting the GFP positive cells with stable expressing CD44 gene. We then study the biological function of overexpression CD44 gene associated with proliferation, invasion and chemoresistancein OCI-ly3 cell.Materials and Methods1. Sesearching the CD44 Gene CDS area (Gene ID:960) from PubMed Gene database and designing the primers according to the enzyme loci (PmeI and AsisI), then we choose the pEASY-T vector to make the TA clone to vertify the full-length CD44 cDNA sequence by sequencing. We use double enzymes (PmeI and AsisI) and Solution I to make CD44 gene into backbone vertor to construct the LEGO-CD44-GFP expression vertor. The lentivirus vectors were transformed into TOP 10. We confirmed the target gene and its sequence by double enzyme (Pmel and AsisI) and direct sequencing.2. Transfections of the packaging cell 293T with lentivirus expression vectors were performed with PEI transfection reagent. After transfecting for 24h,48h and 72h,We collected virus supernatant and centrifuged 2 hours at 20000 rpm at 4℃. The virus was used to infect OCI-ly3 cell,72 hours later, we dectect and sorted the Green Fluorescent Protein (GFP) positive cells by Fluorescence-Activated Cell Sorting (FACS) to obtain the stable expression of CD44 gene OCI-ly3 cells. Then, RT-PCR and Western bloting were used to confirm the CD44 gene expression.3. The proliferation, migration were detected through MTT test and Transwell assay, respectively. Cell apoptosis were examined by flow cytometry.4. MTT assay and Annexin V assay detected by FACS was used to investigate the effect of doxorubicin on the cell growth of ABC-DLBCL cell line.5.The statistical analysis was performed with the statistical software SPSS for windows 13.0, and the significance was defined at P value≤0.05 in our test.Resultsl.The CD44 gene was cloned into LEGO-eGFP vector performing with the technology of digestion by double enzyme (PmeI and AsisI) and sequencing to confirm that the CD44 gene was correctly insected into the lentivirus vertors.2.The expression lentivirus vector or the empty vetor combined with helper-plasmid PMD2G and PSPAX2 were transfected into the packaging cell 293T using PEI transfection reagent. Harvest lentivirus supernatant to transfect the OCI-ly3 cell line. After transfection for 72 hours, we performed the Flow Cytometry to detect the GFP expression cells. The GFP positive cells were 15.6% in LEGO-GFP group and 5.36% in LEGO-CD44-GFP group. Then we detected the CD44 gene expression in the GFP positive cells, the expression efficiency was 0% in LEGO-GFP group and 98% in LEGO-CD44-GFP group. We sorted the GFP positive cells by FACS and cultured the cells for a few days. We measured the CD44 mRNA expression level in OCI-ly3 cells by RT-PCR. Western Blot and the FACS were performed to examine the protein expression of CD44 gene in OCI-ly3 cell lines. We confirmed that the stable over-expressing CD44 gene in diffuse large B cell lymphoma cell line was established.3. MTT showed that the cell proliferation rate of OCI-ly3-CD44-GFP cells was significantly faster than that in OCI-ly3-GFP cells and the OCI-ly3 cells (p<0.001 and P＜0.001).4. Annexin V-APC/PI assay results showed that the apoptosis rates displayed no difference after the over-expression of CD44 gene in OCI-ly3 cell lines (p=0.676).5. The cell migration assay was determined by Transwell, CD44 overexpressed cells showed significantly increased cell migration compared to OCI-ly3-GFP cell line group and the OCI-ly3 cell line group(p=0.031).6. Different concentrations of doxorubicin (0.05uM、0.1uM、0.25um、0.5uM、1uM) treated for 48h, MTT assay was performed to detect the doxorubicin sentivity. The results showed that the overexpression of CD44 gene could elevated the resistence to doxorubicin than OCI-ly3-GFP cell line group and the OCI-ly3 cell line group (p<0.05).7.0.5uM doxorubicin treated for 24h, Annexin V-APC stainig assay was performed to detect the pencertage of appototic cells. The results showed that the overexpression of CD44 gene could reduce the the pencertage of appototic cells treated with doxorubicin than OCI-ly3-GFP cell line group and the OCI-ly3 cell line group (p<0.05).ConclusionThe LEGO-CD44-GFP vector was successfully constructed and stable CD44 gene overexpression cell line was obtained. The CD44 gene may increase the proliferation, migration and chemoresistence to doxorubicin of OCI-ly3 cell line. |
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