| Objective:Objective:In this experiment we tested the RNA induced silencing complex(RISC)related proteins AGO2 m RNA and TNRC6 A m RNA expression levels in bone marrow mononuclear cells collected from patients with acute myeloid leukemia(AML) excluding acute promyelocytic leukemia(APL)/M3). We compared the differences of the relative expression level of AGO2 m RNA and TNRC6 A m RNA in different group and analyzed if there was a possible link existing between AGO2 m RNA and TNRC6 A m RNA expression levels and different clinical index of AML patients. Moreover we further detected AGO2 m RNA expression levels in leukemia cell lines(THP1, KG1 a, K562). In this study, we aimed to determine the possible roles of AGO2 and TNRC6 A in the development of AML and the possible relations between RNA induced gene regulations and the occurrence,development,treatment of AML,and to provideclue for exploring new therapeutic targets of AML. Methods: 1. The bone marrow specimens were collected from 92 cases of acute myeloid leukemia(excluding M3) patients and 20 cases of normal. Among them, 82 cases were from untreated AML patients and 10 cases were collected after the first course of chemotherapyfrom the above mentioned cases. The expression levels of AGO2 m RNA and TNRC6 A m RNA in bone marrow mononuclear cells were detected by real time PCR(RT-PCR)and the relative expression quantity of AGO2 m RNA and TNRC6 A m RNA were calculated by the method of 2???ct.The patients were grouped by gender,age,diagnostic classification,risk classification, remission situation after treatment and other clinical features and then the AGO2 m RNA and TNRC6 A m RNA relative expression quantity differences were compared amongdifferent group.2. Three kinds of leukemia cell lines THP1, KG1 a and K562 were cultured in vitro and the total RNA werecollected when the cells were in good growth condition. Peripheral blood mononuclear cells from healthy adult wereused as control.AGO2 m RNA expression levels in leukemia cell lines and normal human peripheral blood mononuclear cells were detected by RT-PCR technique. 2-??ct method was used to calculate the relative expression quantity of AGO2 m RNA. The AGO2 m RNA expression differences were compared between leukemia cell lines and normal peripheral blood mononuclear cells. Results:1. AGO2 m RNA and TNRC6 A m RNA expression in the patients with AML:(1) Both AGO2 m RNA and TNRC6 A m RNA expression in patients with AML were significantly higher than those in normal control group;(2) Bboth AGO2 m RNA and TNRC6 A m RNA expression in the high-risk patients group were higher thanthose in low-risk patients group.(3) Both AGO2 m RNA and TNRC6 A m RNA expression in high white blood cells patients group(white blood cells was greater than 100 * 10 ^ 9/L) were higher than those in non high white blood cells patients group(white blood cells was less than 100 * 10 ^ 9/L);(4) Both AGO2 m RNA and TNRC6 A m RNA expression in the bad chromosome karyotype group were higher than those in good chromosome karyotype group,and AGO2 m RNA expression in poor chromosome karyotype group was higher than that in middle chromosome karyotype group;(5)In groups based on prognostic genes, both AGO2 m RNA and TNRC6 A m RNA expression were higher in FLT3-ITD positive group than in FLT3-ITD negative group. TNRC6 A m RNA expression is higher in NMP1 positive or(and) CEBPA positive group than in negativeprognostic genes group(6) Both AGO2 m RNA and TNRC6 A m RNA expression in patients after the first time chemotherapy significantly decreased after the first course of chemotherapy;(7) Both AGO2 m RNA and TNRC6 A m RNA expression in non-remission group were higher than those in complete remission group and partial response group;(8)All the patients were grouped by diagnosis and each two groups were compared. The results showed that AGO2 m RNA expression in M6 patients was higher than that in M1 patients and TNRC6 A m RNA expression in M1 patients was higher than in M2patients;The above comparative differences between groups were statistically significant(p < 0.05). There was no significant difference between differentgender and age group(P > 0.05).2. The correlation of AGO2 m RNA and TNRC6 A m RNA expression: There was no correlationof the expression of these two genes in normal control group(Person correlation coefficient r = 0.0275, P = 0.936);A positive correlation existed in the expression of these two genes in AML group(Person correlation coefficient r = 0.381, P = 0.009)3. AGO2 m RNA expression in THP1,KG1 a and K562 leukemia cell line: the expression of AGO2 m RNA in the three leukemia cell lines were all higher than that in normal mononuclear cells and the expression in K562 cells was highest. The expression in THP1 cells was lowest,all of the differences were statistically significant(P ? 0.05). Conclusion:1. Both AGO2 m RNA and TNRC6 A m RNA showed higher expression in AML patients compared with healthy control group and the expression of AGO2 m RNA in the three kinds of leukemia cell lines were all higher than that in normal peripheral blood mononuclear cells. It is highly suggested that there may be a close relationship between AGO2, the core protein of RNA induced silencing complex(RISC) and AML. The high expression of AGO2 m RNA and TNRC6 A m RNA in the pre-chemotherapy group, the high risk group, the high white blood cells group, bad chromosome karyotype group, adverse prognosis molecular biology group further illustrates elevated expression of AGO2 protein and TNRC6 A protein may be associated with the development and poor prognosis of AML and predicts RNA induced gene regulation may play important roles in the occurrence of leukemia. AGO2 m RNAand TNRC6 A m RNA showed high expression in the patients who did not acquire complete remission after chemotherapy, which suggested AGO2 and TNRC6 A may be associated with the drug resistance of AML patients.The expression of AGO2 m RNA in M6 patients was significantly higher among different FAB type suggestedthat AGO2 may play a unique role in the occurrence and development of M6.TNRC6 Am RNA showed high expression both in good prognostic gene group and poor prognostic gene group indicated that it may have an undiscovered role in the molecular changes of AML.2. There has no correlation of the expression of AGO2 m RNA and TNRC6 A m RNA in healthy control group but has a positive correlation in AML patients group, indicating the presence of synergy of AGO2 protein and TNRC6 A protein in the development of leukemia. In addition, both of the two proteins are the core components of RISC, further indicating an important role of RNA-induced gene regulation in the occurrence and development of leukemia. |
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