The Connection Of Mi RNA-21 And NK/T Cell Lymphoma Cells Growth Controlling And Clinical Characteristics

Objective: Through analyzing the differences among contents of mi RNA-21 in peripheral blood of NK/T cell lymphoma patients, normal person and SNK-6 cell line, we detected the connection of mi RNA-21 and NK/T cell lymphoma cells’ clinical characteristics and prognosis. Furthermore, we studied the relationship and mechanisms between mi RNA-21 and SNK-6 cell in growth and regulation, in order to provide theoretical basis and research direction for the strategy of pathogenesis, treatment of NK/T cell lymphoma.Methods: 1 Subject: peripheral blood of NK/T cell lymphoma were from patients, who were confirmed as NK/T cell lymphoma via pathology in Hebei No. 4 hospital and easily collected integrated clinical materials, were gathered among 2013 to 2015. Normal peripheral blood was collected from healthy volunteers. SNK-6 cell line, a human NK/T cell lymphoma cell line, was a generously gift from Prof. Zhang mingzhi in the NO. 1 hospital of Zhenzhou University. 2 Cultivating and passaging of human NK/T cell lymphoma cell line: the culture medium containing 10 % volume fraction of human AB blood type plasma, 1000 u/ml IL-2 and RPMI 1640 incubate medium, and putted in 37℃, saturated humidity and 5% CO2 environment. The first passaging was in 48 hours.All experiments have been done among the ogarithmic phase. 3 Extracting of mi RNA gene: Applying mi RNA extracting kit to refine the peripheral blood of NK/T cell lymphoma patients and healthy volunteers, and SNK-6 cell line. Applying total RNA extracting kit to extract total RNA from SNK-6 cell line, and reverse transcription it to c DNA. Then, stored c DNA into-20 ℃ refrigerator. 4 Realtime-PCR was the method we use to test the expression level ofmi RNA-21. 5 Transient transfection is the majority way of silence mi RNA-21 in SNK-6 cell line. Randomly separate SNK-6 cell line into untreated group, si RNA blocking group and mi RNA-21 blocking group. Setting up si RNA- sequence homology of Endo FectionTM complex control to except the cytotoxic effection of Endo FectionTM. Blank group was cultivated normally, control group transfected si RNAoriginal sequence, and experiment group was transfected si RNA-21. After incubating 48 hours in equal conditions, we each extracted three groups of cells to detect the expressing level of mi RNA-21 applying RT-PCR to confirm the success of transfection 5.1 Using CCK-8 method to observe the growing trend of experiment grops after successfully transfection. Using three groups of transfected cells, after cultivating 48 hours. Then, Adjust the density of cell to 1*105/ml and seeded cells into 96-well plates. Each groups have 5 repeated plates. 48 hours Later, after treating with 10μl CCK-8, all plates was incubated in 37℃, saturated humidity and 5% CO2 environment. At last, we tested OD in each groups at 30min、1h、2h、3h、4h、5h、6h、7h on 450 nm ELIASA. 5.2 Realtime-PCR was applied on testng the changes of m RNA expression in PTEN, PI3 K and AKT. After 48 hours transfection, total RNA kit was used to extract total RNA from SNK-6 cell line, RT-PCR method was applied again to exam m RNA expressing of PTEN、PI3K、AKT. 6 Using the expression level of mi RNA-21 of each patient to connect with their mainly clinical characteristics 7 We inputted all the database into SPSS21.0 statistical software to analyze in statistics. P< 0.05 shows the meaning in statistics. According to the normal distribution, we observed mi RNA-21 gene in the peripheral blood of NK/T cell lymphoma patients and normal volunteers. To artistically analyze them,we adopted t test or non parametric test. Applying χ2 test and multiple factors analysis for expression of mi RNA-21 and clinical characteristics of NK/T cell lymphoma.Results: 1 In the samples of peripheral blood, NK/T cell lymphoma patients showed statistic difference(P=0.000), comparing to control groups. Samples of peripheral blood in NK/T cell lymphoma patients shows higher expression level than these in healthy volunteers. This phenomena showed a highexpression of mi RNA-21 in human peripheral blood of NK/T cell lymphoma patients. 2 In SNK-6 cell lines, a human NK/T cell lymphoma cell lines, the expression level showed a statistic difference(P=0.001). In SNK-6 cell lines, the expression level of mi RNA-21 is high. 3 After blocking of mi RNA-21 in SNK-6 cell lines, CCK-8 data illustrate low survive of cells, their OD exist differences(P=0.001、P=0.003), which proved that mi RNA-21 expression motivate tumor proliferation. 4 On blocking mi RNA-21’s SNK-6 cell line, RT-PCR revealed a highlevel expression of m RNA in PTEN, as well as a low-level expression of PI3 K and AKT, which indicate that mi RNA-21 could motivate the activity of PI3K/AKT pathway. 5 Relevance between mi RNA-21 and clinical characteristics of NK/T cell lymphoma: the expression level of mi RNA-21 is connected with phases, marrow infiltration, Ki-67, β2-MG,albumin expression level. Precisely speaking, high level expression of mi RNA-21, late phase of disease, high probability of marrow infiltration, high possibility of Ki-67 positive, high level of β2-MG, low expression of albumin.Conclusions: 1 Data shows that in our country, an exceptional highly expression of mi RNA-21 in patients’ peripheral blood and SNK-6 cell line, which might connect with nosogenesis of NK/T cell lymphoma. 2 The proliferation and apoptosis of NK/T cell lymphoma cells wouldrelative to mi RNA-21. 3 mi RNA-21 affects PI3K/AKT pathway and participates in NK/T cell lymphoma’s development via controlling target gene- PTEN. 4 There are connection between mi RNA-21 and NK/T cell lymphoma in separating disease stages, marrow infiltration, positive rate of Ki-67, β2-MG and albumin, and et al.