Expression Of PTEN In NK/T Cell Lymphoma And Its Significance

NK/T cell lymphoma (NKTCL) is one type of Non-Hodgkin’s lymphoma(NHL), and the most popular subtype of T cell lymphoma. The morbidity of NKTCLis higher in Latin America and East Asia than European and American countries withunclear incidence reasons. Researchers found that NKTCL usually accompanies EBvirus infection and chronic rhinitis, while some scholars thought that genetic factorsmight play an important role in genesis and development of NKTCL which made thisdisease the research focus of lymphoma in recent years.Phosphatase and tensinhomology deleted on chromosome Ten (PTEN) gene thefirst tumor suppressor gene with phosphatase activity found in tumor researches.Studies found that PTEN could regulate cells’ proliferation, apoptosis, cell cycle andother malignant characteristics through tyrosine phosphorylation, and its mutation,fuctions missing, and abnormal protein expression could eventually lead to cancer.We first detected the expression of PTEN in both in NKTCL tissue and normalcontrol specimens, and then constructed recombinant lentivirus carried PTEN cDNAand shRNA element target PTEN following with infection in NKTCL cell lineSNK-6, and studied the impact to malignant biological characteristics includinggrowth proliferation, apoptosis, cell cycle, and chemotherapy drugs sensitivity.Finally we also detected expression of relative pathway and drug resistance genes forrevealing the machnism of PTEN in NKTCL. Object:1. To detect the expression of PTEN in both in NKTCL tissue and normal controlspecimens, and analysis of the relationship between PTEN expression and clinicfactors.2. To construct recombinant lentivirus carried PTEN cDNA and shRNA elementtarget PTEN following with infection in NKTCL cell line SNK-6, study theimpact to malignant biological characteristics including growth proliferation,apoptosis, cell cycle, and chemotherapy drugs sensitivity,and detect expression ofrelative pathway and drug resistance genes for revealing the machnism of PTENin NKTCL.Main Content:The first chapter: Expression and significance of PTEN in NK/T celllymphomaMethod:1. HE staining (hematoxylin-eosin staining): Paraffin-embedded tissue sections weredewaxed, stained and dehydrated. Then, we studied the cell morphology andhistological features of this NK/T cell lymphoma tumor.2. The expression of PTEN protein in NK/T-cell lymphoma was detected byImmunohistochemical: After dewaxing, retrieval antigen, eliminated endogenousperoxidase activity, primary antibody incubation, secondary antibody incubation,DAB color hematoxylin dye and mounted sections, we studied the expression ofPTEN in NK/T-cell lymphoma tissue.Results:1. HE staining showed the tumor grew with the diffuse and infiltrativecharacteristics, tissue necrosis, surface of evident mucosal ulcerate. Tumor cellsform a single, medium-sized, the cytoplasm stained lightly, and has invasion anddestruction of the vessel wall. 2. Immunohistochemistry showed that the positive expression rate of PTEN in NK/T-cell lymphoma could reach to32.0%(16/50), and significantly lower than the86.7%(26/30) of reactive hyperplasia lymph nodes, the difference hasstatistically significant (P <0.01)。3. According Evaluation (2cycles), lymphoma patients were divided into effectivetreatment groups (CR+PR) and ineffective treatment group (SD+PD). Theresults showed that the positive rate of PTEN expression in NK/T-celllymphoma in effective treatment group was42.1%, and significantly lower thanthe ineffective treatment group (16.1%)(P <0.05).The second chapter: Preparation of recombinant lentiviruslenti-PTEN/Lenti-shPTEN and evaluation of infection efficiency tolymphoma cellsMethod:1. The construction of PTEN expression lentiviral vector: ThepCDH-CMV-MCS-EF1-copGFP plasmid were digested with EcoRI and BamHI,and the PTEN Fragment was digested with EcoRI and BamHI from the PCRproduct with cloning vector as a template，then it was ligated and transformed.2. The construction of PTEN interference expression lentiviral vector andinterference efficiency filters: the fragment of PTEN shRNA was designed andsynthesis, annealed to form a double-stranded fragment. The interferencelentiviral vector of pLL3.7was digested with HpaI and XhoI. The vector wastransformed, certificated. The interference expression lentiviral vector of PTENconstructed into pLL-shPTEN1, pLL-shPTEN2and pLL-shPTEN3. Real timePCRwas used to screen out the high-interference pLL-shPTEN.3. Package recombinant lentiviruses Lenti-con, Lenti-PTEN and Lenti-shPTEN, bypWPI（pLL3.7）/vsvg/δ8.0system.72h later，the virus were collected.Concentrate and purify viruses, then determine viral titer.4. Evaluate infection efficiency of recombinant lentiviruses to different lymphomacells，and assess the virus-sensitivity of lymphoma cell lines. Results:1. pCDH-PTEN was constructed successfully and was certificated by thesequencing and comparison.2. Three recombinant lentiviruses successfully constructed, and thehigh-interference pLL-shPTEN was certificated. pLL-shRNA2was used topackage the combinate the lentivirus.3. Recombinant lentiviruses Lenti-con, Lenti-PTEN and lenti-shPTEN waspackaged successfully, and the purified virus titer was up to109IFU/ml.4. SNK-6and YTS can be infected effectively by these lentivirus, and the infectionrate can reach to60%.The third chapter: Study of lenti-PTEN and lenti-shPTEN functionand biologic effect in SNK-6cellsMethod:1. Analyze the mRNA and protein of PTEN both in SNK-6and NK cells.2. Infect SNK-6and NK cells by recombinant lentivirus, and detect expression ofPTEN.3. Abserve morphological change and detect growth, proliferation and apoptosis ofSNK-6after infection recombinant lentivirus by using CCK-8and flowcytometry.4. Detect drug sensitivity of DDP in SNK-6after infection recombinant lentivirusby using CCK-8.5. Analyze the protein of PI3K/AKT pathway and drug resistance genes after infectionrecombinant lentivirus by using westernblot.Results:1. Realtime PCR and western blot analysis showed that expression of PTEN inSNK-6cells was lower than normal NK cells significantly (p<0.05), andexpression of PTEN could up-and down-regulated by recombinant lentiviruslenti-PTEN and lenti-shPTEN. 2. SNK-6cells had a obvious pathological change after infection withlenti-PTEN,and more apotosis cells were detected, and growth of cells wereinhibited, and compared with other groups, cells showed a higher drug sensitivitysignificantly (p<0.05).3. PTEN could inhibit the expression of PI3K, p-Akt, mTOR and ABCC4genesprotein without MDR-1.Conclusion:1. Expression of PTEN gene in NK/T cell lymphoma tissue and cell lines wasclosely associated with its genesis and development, and possibly be used inclinic as a potential molecular markers.2. Recombinant lentivirus lenti-PTEN and lenti-shPTEN were succeededconstructed and could infect SNK-6cell line and regulate expression of PTENgene effectively.3. Expression of PTEN gene could inhibit the growth, proliferation of SNK-6cells,and enhance the drug sensitivity of DDP in SNK-6cells. PI3K/Akt pathway andmTOR, ABCC4might involve in these biologic change in SNK-6.