| BackgroundHematological malignancy including leukemia, malignant lymphoma and multiple myeloma, the incidence of the former two types cancer is currently ranked in the top ten human malignant tumors.Where the mortality rate of leukemia in the malignancy, accounting for 6 (male) and 8 (female); children and adults under the age of 35, the No.1 ranking. And the incidence increased year by year, it is a serious threat to human health and life, leukemia include Acute leukemia (AL) and chronic leukemia (CL), in china the incidence of AL is more than CL (about 5.5:1), of which the most is acute myeloid leukemia AML (1.62/100000)in china. When human get leukemia,the acute leukemia blast cells and immature abnormal cells (leukemia cells) will be full of the bone marrow and they proliferate rapidly, and they inhibit normal hematopoiesis and extensively infiltrative the liver, spleen, lymph nodes and other organs. Manifested as anemia, bleeding, infection and signs of infiltration.In recent years, with the strengthening of supportive care, the application of multi-drug joint programs, high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT) in the promotion, AML patients with chemotherapy, complete remission rate (CR) up to 75%, but there are still 20%～30% AML failed to obtain through formal remission after chemotherapy, known as refractory. Even though about 50% patients relapse after CR, if relapse, patients with poor prognosis. It is now generally considered that the basic reasons of leukemia refractory and relapsed are:①the first reason is leukemia cells resist apoptosis pathway;②the second reason is leukemia cells with high expression of multidrug resistance;③The third reason is most leukemia cells stay in the Go quiescent state and they are not sensitive to chemotherapy.Due to leukemia cells are not sensitive and resistant to drugs, in clinical treatment the use of high-dose chemotherapy, a considerable part of the patients died of toxic effects of chemotherapy rather than the progress of leukemia.Therefore, the most failure fundamental reasons for the treatment AML are refractory and relapsed, It is aslo a problem for treatment AML.Eps8 (EGF receptor pathway substrate NO.8) epidermal growth factor receptor pathway substrate 8, Fazioli et al found it expreesed in NIH3T3 murine fibroblasts and proposed the concept in 1993. Eps8 widely distribute in different forms of cells, including stromal cells, epithelial cells, there is also a small amount of hematopoietic stem cells. And main branch in the cytoplasm, nuclear membrane and the membrane surrounding, it is a receptor-type tyrosine kinase receptor (RTKs), in addition to it is phosphorylated by EGFR completely apart, but also phosphorylated and activated by other RTKs. Eps8 with the EGFR binding, making cells to be the cancer, DNA synthesis and increased capabilities, in addition to Eps8 is a scaffold protein, Eps8 with F-actin and Cortractin combination cytoskeletal reorganization, membrane folds occur, and promote cell migration.The structure of Eps8 includes phosphotyrosine binding domain (phosphotyrosine binding protein, PTB), SH3 and the SAM-PNT zone area (sterile alpha-pointed).Many studies have shown that Eps8 is highly expressed in the solid tumors, and through various mechanisms to promote tumor cell migration and rapid growth, and progression of tumor metastasis,it is a bad prognostic indicator.For example, in colon cancer, Eps8 Facilitates celluar growth and motility of colon cancer cell by incressing the expression and activity of Focal Adhesion Kniase (FAK). Oral squamous cell carcinoma cells with high expression of Eps8,and by regulating the activity of Racl increased cell invasion and migration capacity. Pancreatic ductal carcinoma, the Eps8 mRNA expression was significantly increased and positively correlated with tumor metastasis, and Eps8 play an important role in maintaining cell cytoskeleton structure and cell-cell junctions. Besides, Eps8 is involved in but not essential,for cell migration, overexpression makes cells to malignant cell transformation.Eps8 decreases chemosensitivity and affects survival of cervical patients. Mithramycin (MTM) inhibits human epithelial carcinoma cell proliferation and migration involving downregulation of Eps8 expression.However, what it the Eps8 express in Hematologic Neoplasms cell lines? there are no people to study, therefore, It is very important to explore the Eps8 express in hematological malignant cell lines and hematological malignancy and its relationship with prognosis.ObjectionSelecting Hela cell as a positive control, using RT-PCR to detect Eps8 mRNA expression of seven kinds of hematological malignant cell lines about KGla,K562,HL-60,ARH-77,8226,Raji,Jurkat, and to determine the Eps8 mRNA expressions in hematological malignant cell lines.Builded real-time fluorescence quantitative PCR method for detect Eps8 genes, further used of RQ-PCR (Relative Quantification-PCR) and immunoblotting (Western Blot) to dectect the expression of Eps8 in the 7 kinds of hematological malignant cell lines.Drugs interfered KGla cell, using placenta Blue resist staining live cells to get the Inhibition rate of anti-KGla.RQ-PCR and Western Blot assayed with different concentrations of the drug effected the expression of Eps8 KGla cells.MethodsCulturing Hela,KG1a,K562,HL-60,ARH-77,8226,Raji,Jurkat cells, using TRIzol extracted total cellular RNA. searched for Eps8 gene in NCBI, designed primers, usedβ-actin as internal reference, used RT-PCR to detect the expression of Eps8 in the 7 kinds of cell lines, Hela cells as a positive control group, healthy human peripheral blood mononuclear cells (PBMNCs) as the control group.Established RQ-PCR method, selected randomly one cDNA template samples, diluted at 100 10-1 10-2 10-3 10-4 concentration gradient, using SYBER GreenⅠtested the objective gene and reference gene amplification efficiency, to make a straight line graph about the log value of dilution gradient andΔCT (ΔCT= CTEps8-CTGAPDH), the slope is less than 0.1 can be considered the two genes have the same amplification efficiency. Using SYBR Green I RQ-PCR and Western Blot to detected the Eps8 expression in the cell lines and normal bone marrow mononuclear cells (BMMNCs), and analysis the difference between them.Using fluorescent probe PE labeled KGla, K562 cells, flow cytometry tested the expression of CD34 positive cells.Used anthracycline antitumor drugs daunorubicin (DNR) to kill KG1a, (1)DNR concentration was 0.05μg/ml,0.1μg/ml,0.4μg/ml,0.8μg/ml, after 24 hours we used placenta Blue resist staining live cells to count the inhibition rate DNR to anti-KG1a.Using RQ-PCR and Western Blot assayed the Eps8 expressionof the 0.05μg/ml,0.1μg/ml,0.4μg/ml groups. (2)non-adding Drug group as control group, experimental group of drug concentrations were 0.1μg/ml,0.2μg/ml,0.4μg/ml, respectively, after dosing measured 24h,48h,72h to get the DNR inhibition rate of anti-KG1a and Using RQ-PCR and Western Blot assayed 0.1μg/ml,0.4μg/ml group Eps8 expression. Applicating 2-ΔΔCT to processe the quantitative PCR data, calculated of the average CT value of each sample,ΔCT values (ΔCt=CtEps8-CtGAPDH).Calculated of 2-ΔΔCT(ΔΔCt=ΔCt purpose of the sample -ΔCt internal reference sample), the purpose of the digital value used to represent a multiple of the value relative to the reference. Weatern Blot protein bands using ImageJ software analysised, protein expression was Eps8 optical density/β-actin optical density (ODEps8/ODβ-actin).Statistical analysis, applied SPSS13.0 statistical software to deal with the data, measurement data use Mean±deviation state(x±s). compared differences among the groups use One-Way-ANONA. when homogeneity, using LSD test to compare multiple between groups, Heterogeneity of variance, used Welch test,and used Dunnett T3 test to compare multiple between groups; P<0.05 indicated significant difference.ResultsSelected one sample of total RNA,by 1.5% agarose gel electrophoresis, showing 28s and 18s and 5s three roads with a clear, Proved the quality of the RNA, no DNA contamination the final RT-PCR products by 1.5% agarose gel electrophoresis, observed in ultraviolet light can be seen with the same fragment size of 165bp (Eps8), and 510bp (β-actin) of electrophoretic bands.Gene amplification efficiency of the two genes, the results from the slope of a straight line y=-0.065x+1.743,absolute value was 0.065 (slope of absolute value <0.1), indicated that the amplification efficiency of the two genes are similar.The results could be used 2-ΔΔCT relative quantitative analysis method. RQ-PCR tested method is established.There were significant differences in the cells population means (F=2082.917, P<0.001), KG1a is 3177.48-fold relative to BMMNCs (P<0.001), ARH-77 is 377.40-fold relative to BMMNCs (P=0.002),8226 is 1/10 relative to BMMNCs (P= 0.025), Raji is 3/100 relative to BMMNCs (P=0.001). The remaining three kinds of malignant blood cells relative to BMMNCs have no significant difference (K562, Jurkat, HL-60) (P=0.831, P=0.960, P=0.969). Western Blot tested results also show that the Eps8 expression in KG1a,and ARH-77 cell lines, and the other cells; and BMMNCs had no positive bands. Experiment was repeated 5 times independently and got the same results.The CD34 positive rates of KGla, K562 cells respectively were positive (98.3±1.5)%, (1.3±0.6)%.The concentrations of DNR respectively were 0.05μg/ml,0.1μg/ml,0.4μg/ml, 0.8μg/ml to kill KG1a after 24 hours, the inhibition rates were respectively (12.36±4.07)%, (26.83±3.68)%, (60.38±3.57)%, (81.44±3.38)%.there was significant difference that different concentrations of DNR on the inhibition of anti-KG1a (F= 317.11, P<0.001), and when the concentrations increased the inhibition of anti-KG1a increased (P<0.05). The difference of expression of Eps8 mRNA between the groups was significant by different concentration drug (F=1069.725, P<0.001), Comparison with the no drug group (0μg/ml), the role of drug concentration 0.05μg/ml group Eps8 mRNA expression is about 1/5 (P=0.023); drug concentration O.1μg/ml group expression is about 1/20 (P= 0.011); the role of drug concentration was 0.4μg/ml group expression is about 1/100 (P=0.004).Western Blot results showed that different concentration groups, Eps8 expression was significantly different (F= 99.802, P<0.001), compared with the control group, the Eps8 protein downregulated in the concentration of 0.05g/ml, 0.1g/ml,0.4μg/ml group (P<0.001, P<0.001, P<0.001),0.05μg/ml group was lower than 0.1μg/ml group (P=0.044). 0.4μg/ml was group lower than 0.1μg/ml group (P<0.001). and when the concentration was 0.4μg/ml Eps8 expression was significantly decreased. In short, with the drug concentration increased, the inhibition of anti-KG1a 24h after by DNR 24h raised, The expression of Eps8 mRNA and protein were corresponding lower. The concentrations of DNR respectively were 0.1μg/ml,0.2μg/ml,0.4μg/ml to kill KG1a after 24h,48h,72h the inhibition rate of anti-KGla respectively were (26.91±2.70)%, (45.65±0.54)%, (74.79±1.37)%, (43.15±1.78)%, (61.29±1.97)%, (85.70±1.45)%, (61.28±1.35)%, (88.59±0.76)%, (95.76±0.95)%. Different DNR concentration and different time for the inhibition rate of anti-KG1a, the expression of Eps8 mRNA and protein had exists interaction (F=42.33, P<0.001; F=25.916, P<0.001; F=12.768, P=0.000). Under the same concentration Eps8 decreased with the time (P<0.05), the same reaction time Eps8 decreased with the drug concentration (P<0.05). In short, with the DNR concentration and the time, the inhibition rate of anti-KG1a decreased, Eps8 expression decreased.ConclusionEstablished the method of SYBR GreenⅠRQ-PCR to assay the Eps8 mRNA expression.2-ΔΔCT analytical method to analyze gene expression in different cells is a very good relative method.Compared with normal BMMNCs, Eps8 mRNA expression in KG1a and ARH-77 were highly expressed,8226 and Raji cell lines were low expression, the difference about Eps8 mRNA expression between the others selected cell lines was not statistically significant.Eps8 protein expressed in KG1a and ARH-77, and the other cell lines showed no expression and BMMNCs, Eps8 were high expression in same hematopoietic stem cells cell lines, Eps8 plays a role in early differentiation in some malignant tumor cells.With the drug concentration increased and treatment time prolonged, the expression of Eps8 decreased, Maybe DNR inhibits KG1a cell proliferation involving downregulation of Eps8 expression. |
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