| Bachground Leukemia is the world's top ten high incidence of malignant tumors,accounting for 5% of the total cancer incidence above.Leukemia is a malignant proliferative diseases of the hematopoietic system,its main feature is the abnormal proliferation of leukemic cells in the bone marrow or other hematopoietic tissues,thereby infiltrating the body tissues and organs,resulting occurs relevant pathological changes in clinically,in addition to blood cytoplasm and volume always make changes.The incidence and mortality rate of leukemia were the highest below 35 years oid,and the incidence was rising year by year,especially in children and adolescents,the incidence rate living first.Currently,relying solely on chemotherapy could significantly improve long-term disease-free survival rate of childhood ALL,but there were still some patients were not sensitive to chemotherapy,leading to failure or induce relapse.Micro RNA(mernas)is a small RNA with multiple biological functions.Due to its important role in the growth,development,and physiological processes of organisms,it has become the focus of research and has been studied by meteorologists.Especially in the process of the occurrence and development of malignant tumors.Mirnas regulates the signaling pathways associated with several human malignant tumors by acting as a cancer gene involved in tumorigenesis and development,or as an auxiliary gene to inhibit the initiation and progression of tumors.According to reports,more than one-third of human genes are regulated by precise gene targets,involving cell proliferation,differentiation,apoptosis,and a series of physiological processes,including many human malignant tumors.The present study has demonstrated that the expression of miR-195 in a variety of human malignant tissues was reduced,suggesting that it might have the function of tumor suppressor genes in the development of malignant tumors,but its acute lymphocytic leukemia in children expression study was relatively small.Objective To develop new approach for the treatment of childhood ALL and New clinical treatments were established,and the expression of mir-195 and bcl-2 in childhood was studied,as well as the role of mir-195 in inhibiting cell proliferation,apoptosis,and cell cycle control in children with peripheral blood leukemia.Method(1)Bioinformatics software was used to predict and screen mir-195 target genes that regulate cell proliferation and cell cycle.(2)We collected 64 cases of children with acute lymphoblastic leukemia bone marrow samples,using real-time PCR method for detecting the samples miR-195 as well as the expression of Bcl-2 m RNA,while the expression of Bcl-2 protein was detected by western blot analysis.miR-195 and Bcl-2 expression in childhood acute lymphoblastic leukemia patients with clinical and pathological data.(3)To increase the expression amount of miR-195 by transfecting miR-34 a mimic into Jurkat cells.We detected the expression of miR-195 m RNA by realtime PCR.Applying MTT method respectively;Annexin V-FITC/PI double dyeing method;Flow cytometry;Transpell Small Room Law;Cell scratches were used to detect the effects of miR-195 on cell proliferation,apoptosis and cycle.And the ability to invade and migrate cells.(4)The experimental cells were randomly divided into 4 groups,the group is in accordance with the design scheme to be divided,the control method is mainly related to the transfection of miR-mimics,and whether to add inhibitors.According to the scheme of the group includes two kinds of control methods negative and positive groups.The negative group transfection adds a kind of material,namely mock.and expression of various molecules in cells of the observation is the main use of amplification means to carry out the detection and other aspects of the function is mainly achieved through the proliferation experiment;Cell proliferation and the expression of bcl-2 protein was determined by Western blotting method.Results(1)We conducted target gene prediction and screening through bioinformatics software,and the results showed that mir-195 could complement CCND1,bcl-2,bcl-6,CCNE1,CDK6,CDC25 A m RNA3 'UTR region.After transfected mir-195 mimics in the cell line,the changes of six candidate genes were detected by luciferase reporter gene experiment,and the results showed that bcl-2 changed most significantly.Therefore,mir-195 and its target gene bcl-2 were selected as the research objects in this study.(2)The expression of mir-195 was significantly higher in ALL control groups and slow-release groups than in children with primary diagnosis and recurrence(P<0.05).The expression of mir-195 was significantly decreased in the recurrence group,while the expression of mir-195 was significantly decreased in the initial diagnosis and recurrence group.There were also significant differences in the expression of mir-195 in at-risk and medium-risk children,and the expression of mir-195 decreased gradually with the increase of ALL risk.The expression of Bcl-2 m RNA and protein in ALL control group and slow-release group was significantly lower than that in children with primary diagnosis and recurrence,and the difference is statistically significant(P<0.05).The expression of bcl-2 protein in the remission group was significantly decreased,and the expression of bcl-2 was abnormal again.The Bcl-2protein expression in crisis,endanger in high-risk children also obvious difference,at the same time,with the improvement of ALL dangerous degree,the Bcl-2 protein expression was increased gradually trend.(3)The mir-195 has been shown to inhibit cell proliferation and promote apoptosis in jurkat cells,and affect cell growth by blocking cell layers at G1.The mir-195 also inhibits the invasion of Jurkat cells and the ability of Migarin determined by MTT method transfection was detected in 24,48,72,96 h Jurkat cell proliferation inhibition rate,the results showed proliferation inhibition rate at each time point transfection group were higher than with the negative control group,difference has statistical significance(P < 0.05),and the comparison difference between the four time points in the transfection group was also statistically significant(P=0.000),which promots high mir-195 inhibited cell proliferation.Annexin v-fitc /PI double staining for apoptosis detection showed that apoptosis of Jurkat cells in the transfection group increased gradually over time,and the difference is also statistically significant(P<0.05),suggesting that high mir-195 may induce apoptosis of Jurkat cells.Flow cytometry detection effects on cell cycle,according to the results when the transfection miR-195 after the mimic,Jurkat cells of the cell cycle by stagnation in the G1 phase,within 0 ~ 96 h after transfection,longer duration of miR-195 mimic effect,the number of Jurkat cells in G1 phase gradually increased,when the miR-195 mimic at 96 h after the transfection Jurkat cells from the peak of the cells in the G1 phase,prompt miR-195 can make the Jurkat cell growth stagnation in the G1 phase.The Transwell small chamber method showed that mir-195 mimics were transfected for 48 h,and the average number of invasive cells in the field of vision in the transfected group and the control group was significantly decreased,suggesting that mir-195 had inhibitory effect on the invasion of Jurkat cells.Cell scratch the experimental results show that the Jurkat cell transfection 24 h,control group develop the migration distance of hole has been significantly reduced,and miR-195 mimic group culture in cell migration distance is short,transfection scratch area of 48 h control cells has basically been cover closed,scratches the healing rate difference was statistically significant compared with controls,prompt miR-195 inhibits Jurkat cells' ability to migrate.(4)After the mir-195 simulated transfection,the cell proliferation activity of jurkat cells was shown to increase,but this activity was reduced after the addition of the mir-195 inhibitor.The protein expression of bcl-2 in Jurkat cells showed a decrease in the mimetic transfection of mir-195,but a significant increase in the addition of mir-195 inhibitors.Conclusion It is found that mir-195 was involved in the progression of acute lymphoblastic leukemia in children and is related to the prognosis of patients.Its participation may be achieved mainly through the regulation of bcl-2.After the regulation of bcl-2 by mir-195,cell differentiation,proliferation and invasion and metastasis regulated by bcl-2 can be controlled,suggesting that mir-195 may be used as a marker to predict the prognosis of diseases.On the other hand,it also provides a new theoretical basis for finding new targets for targeted therapy. |
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