| BackgroundNon-hodgkin’s lymphoma (NHL) is a malignant tumor in immune system that originated in lymph node and lymphatic tissue. It always occurs associated with the immune cell malignant transformation in lymphocyte proliferation and differentiation during the process of the immune response. NHL can be divided into B-cell and T-cell and the B-cell accounts for most part, including diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Bukitt lymphoma (BL), etc. At present, combined chemotherapy CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) is the main treatment method of NHL, but with the widely application of the chemotherapy drugs, chemoresistance has been an important reasean for NHL treatment failure. The mechanism of tumor multi-drug resistance (MDR) is complex, involving a variety of singnal pathways, in which mutation in the level of genetics of the key genes can result in tumor cell resistance phenomenon. The mechanism may be:increased drug efflux, apoptosis regulating disorders, change of drug target, enhancing DNA damage repair, pharmacokinetic changes, etc. Thus, research on these drug-resistance related genes, proteins and signaling pathways has become a focus both at home and abroad.Adriamycin (ADR) is a classic kind of anthracycline-based antibiotics, that belongs to the cell cycle non-specific drug, which can be embedded into the DNA base pairs, and closely bind to the DNA, stopping the process of RNA transcription, inhibiting RNA synthesis, as well as preventing the DNA replication. ADR antineoplastic spectrum is wide, curative effect is high, which is an important drug in NHL combined chemotherapy CHOP. However, the heart related toxic reaction and chemical resistance severely limit its therapeutic effect in NHL, which become an important cause of treatment failure of NHL. Burkitt lymphoma is a highly invasive lymphima, the current clinical treatment effect of which not ideal. The Burkitt lymphoma cell line established in vitro-Raji, has been widely applied to the study of human B cell lymphoma. By establishing an ADR reduced durg resistant cell line in vitro, and find the expressing difference of genes and proteins between the resistant strain and sensitive strain, in order to find new target genes associated with NHL resistance, which will provide strong support in further knowing the NHL resistance mechanisms and exploring effective multi-drug resistance reversal agent and eventually overcomming the NHL clinical drug resistance problems.Prohibitin (PHB) is a kind of protein that is highly conservative on evolution, and widely exists in living cells. PHB gene families can be located in the mitochondria, nucleus and the cell membrane, and their different positions on cell determined different biological functions. PHB1 and PHB2 in human polymerizing and forming high molecular ring complex, mainly located in the mitochondrial membrane, which plays an important role in maintaining the mitochondrial respiratory chain enzyme complexes and their morphology and function stability. At present, it has been confirmed that in acute lymphoblastic leukemia, ovarian cancer, prostate cancer and other human malignant tumors PHB play an important role in the mechanism of MDR by regulating the function of mitochondrial membrane in cell apoptosis and playing a protective role. Our previous study also found that mitochondria PHB expression up-regulated in Raji cells after ADR treatment, and application of P13K/AKT pathway inhibitor can cut the expression of PHB and increased the sensitivity of Raji cells to ADR. Thus we speculated that PHB may be a target gene closely related with the occurrence of NHL multi-drug resistance.MicroRNAs (miRNAs) are a class of approximately 22-nucleotide endogenous non-coding RNAs, which regulate gene expression at the post transcription level and participate in the regulation of cell proliferation, differentiation, apoptosis and biological functions, thus affect the incidence and development of diseases and become a new potential therapeutic targets and molecular markers. The role of microRNAs in cancer cells MDR has increasingly attracted the human attention both at home and abroad. A number of studies have confirmed that there are a variety of miRNAs existing expression level change in NHL cells as well. They regulated the differentiation, growth and apoptosis of lymphocytes by regulating the expression of target genes and proteins, thus influence the diagnosis and treatment of NHL. MicroRNAs as a breakthrough point to design gene drugs will open up a new way to targeted therapy for NHL and improve the NHL cells resistance. Study found that there were high expression of microRNA-27a (miR-27a) in a variety of tumors, which play a role of proto-oncogene. In addition, high expression of miR-27a is also closely related to the MDR of tumor cells in acute leukemia, ovarian cancer, stomach cancer, esophageal cancer, liver cancer, colon cancer and so on. The change of its expression level still played an important role in resistance of leukemia, stomach cancer and esophagus cancer cells to ADR by changing multi-resistant genes expression level. So we speculated that miR-27a may be involved in NHL MDR mechanism and is another important target gene.At present we had not see report on PHB and miR-27a in MDR of NHL both at home and abroad. In this study, we established the ADR resistance Raji cell line, and detected the expression level difference of PHB and miR-27a between drug-resistant strain and sensitive strain, expounding new molecular targets that was closely related with ADR resistance of NHL from the angle of the mitochondrial protein and microRNAs, and providing new therapeutic targets to reverse the NHL multi-drug resistance.ObjectiveTo establish adriamycin (ADR)-resistant Raji cell line and analyse the expression difference of mitochondria Prohibitin (PHB) and microRNA-27a (miR-27a), as well as discuss there clinical significance.MethodsBuilding the ADR-resistant Raji cell line, observing their morphology and growth characteristics in general and counted by microscope, detecting their inhibited rate by CCK-8, flowcytometry detecting their apoptosis rate; evaluating the expression level of gene PHB mRNA and miR-27a in Raji/A and Raji/S cells via real-time quantitative polymerase chain reaction (RT-PCR); western blot detecting the expression of PHB 1 protein in mitochondria of Raji/A and Raji/S cells.ResultsBuilt the Raji/A cell line; compared with Raji/S cell line, Raji/A cell line appeared difference in general morphology and growth characteristics, proliferation at a slower pace, the resistance ratio was 306.47 times, to vincristine (VCR), homoharringtonine (HHT) appeared cross drug resistance, the resistance ratio were 3.44 times and 2.88 times respectively, to methotrexate (MTX) appeared no cross drug resistance, cultivated a month without ADR remained drug resistance, after ADR treatment, the apoptosis obviously decreased; Expression of miR-27a in Raji/A cells was much higher than in Raji/S cells with (128.40±31.59) times (P<0.05); Expression of gene PHB1 mRNA in Raji/A cells was evidently higher than in Raji/S cells with (7.66±3.27) times (P<0.05); Expression of gene PHB2 mRNA in Raji/A cells was (1.25±0.47) times than in Raji/S cells and the difference was statistically meaningless (P>0.05); Expression of PHB 1 protein in Raji/A cells was obviously higher than that of in Raji/S cells.ConclusionsSuccessfully built the stable experimental model of Raji/A cell line, high expression level of PHB 1 and miR-27a may participated in the resistance mechanism of Raji cells. |
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