Establishing artificial gene connections through RNA displacement-assembly-controlled CRISPR/Cas9 function.

Construction of synthetic circuits that can reprogram genetic networks and signal pathways is a long-term goal for manipulation of biosystems. However, it is still highly challenging to build artificial genetic communications among endogenous RNA species due to their sequence independence and structural diversities. Here we report an RNA-based synthetic circuit that can establish regulatory linkages between expression of endogenous genes in both Escherichiacoli and mammalian cells. This design employs a displacement-assembly approach to modulate the activity of guide RNA for function control of CRISPR/Cas9. Our experiments demonstrate the great effectiveness of this RNA circuit for building artificial connections between expression of originally unrelated genes. Both exogenous and naturally occurring RNAs, including small/microRNAs and long mRNAs, are capable of controlling expression of another endogenous gene through this approach. Moreover, an artificial signal pathway inside mammalian cells is also successfully established to control cell apoptosis through our designed synthetic circuit. This study provides a general strategy for constructing synthetic RNA circuits, which can introduce artificial connections into the genetic networks of mammalian cells and alter the cellular phenotypes.

[Expression of ROBO3 and Its Effect on Cell Proliferation and Apoptosis in Pediatric Patients with Acute Myeloid Leukemia].

OBJECTIVE: To investigate the expression of ROBO3 in pediatric AML patients and explore its function on cell proliferation and apoptosis. METHODS: The expression of ROBO3 in pediatric AML patients at different treatment stage was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The relationship between the expression of ROBO3 and clinic pathological characteristics in newly diagnosed pediatric AML patients was analyzed. Moreover, the effects of ROBO3 on the proliferation and apoptosis of AML cell lines HL-60 and THP-1 were estimated by using CCK-8 and flow cytometry after transfection with ROBO3 siRNA. RESULTS: It was found that ROBO3 expression was significantly increased in most of newly diagnosed pediatric AML patients, especially in non-M3 subtype, younger patients (<10 years old), and high risk group, compared to corresponding controls. Furthermore, the expression level of ROBO3 was sharply decreased in patients who achieved complete remission. Targeting ROBO3 significantly inhibited AML cell proliferation, as well as increased apoptosis by ROBO3 siRNA transfection in vitro. CONCLUSION: ROBO3 is differentially expressed within distinct subtypes of the pediatric AML patients, which suggested that ROBO3 may be a potential biomarker and a new therapeutic target for pediatric AML.

[Bmi-1 as A Molecular Marker for Prognosis of Pediatric ALL].

OBJECTIVE: To study whether the Bmi-1 gene can be a biomarker for analysis of clinical risk stratification and prognosis of ALL patients. METHODS: The expression level of Bmi-1 gene in bone marrow samples from 127 cases of newly diagnosed ALL was detected by qRT-PCR, at the same time the expression level of Bmi-1 protein in bone marrow samples from above-mentioned cases was detected by Western blot. The collected samples were divided into 3 groups: high, intermediate and low risk according to clinical risk stratfication, the relationship between Bmi-1 expression and risk grade of ALL patients was analyzed; at the same time the collected samples were divided into 2 groups: prednisone good response (PGR) and prednisone poor respouse (PPR) according to the sensitivity of prednison test, and the sensitivily to prednisone in 2 groups was compared; moreover, the collected samples were divided into 2 groups: high level and low level according to median of Bmi-1 level, and the relation of Bmi-1 level with prognosis of patients was analyzed by using the Kaplan-Meier method. RESULTS: The expression level of Bmi-1 in low risk group was lowest, while that in high risk group was highest, however that in intermediat risk group was between the low and high risk groups, statistical analysis showed significant difference (P＜0.05). The expression level of Bmi-1 in PPR group was significantly higher than that in PGR group (P＜0.001). The Kaplan-Meier analysis showed that the RFS rate in Bmi-1 high expression group was significantly lower than that in Bmi-1 low expression group (73.0% vs 90.6%) (P＜0.001). CONCLUSION: The Bmi-1 can be used as a molecular marker for the analysis of chinical risk and prognosis of pediatric ALL.

[Down-Regulation of MiR-125b Reverses Drug Resistance of Doxorubicin-Resistant Leukemia Cells].

OBJECTIVE: To investigate whether the down-regulation of miR-125b can reverse the drug-resistence of doxorubicine-resistant leukemia cell lines or not, so as to explore a new method for treatment of drug-resistant leukemia patients. METHODS: The expression levels of miR125b in doxorubicine drug-sensitive and doxorubicine drug-resistant leukemia cell lines.HL-60, K562 and HL-60/Dox, the K562/Dox were detected by using RT-qPCR; the up-regulation or inhibition of miR-1256 expression in HL-60/Dox were performed by electroporation transfection, then the viability of cells treated with doxorubicine of different concentration was detected by CCK-8 method, the proliferation inhibition curve of cells was drawed, and the IC50 was calculated. RESULTS: The miR-125b expression was obviously up-regulated in drug-resistant cell lines HL-60/DOX and K562/DOX, as compared with HL-60 and K562 cell lines. The miR-125b expression level in HL-60/DOX and K562/DOX cells was 15 times and 5 times higher than that in HL-60 and K562 cells, respectively. The up-regulating or inhibiting expression of miR-125b in HL-60/DOX cells found that the proliferation inhibition rate in cells transfected with miR-125b mimic significantly decreased, compared with control group (P<0.01), while the proliferation inhibition rate in cells transfected with miR-125b inhibitor significantly increased, compared with control group(P<0.01). CONCLUSION: The miR-125b expression in HL-60/Dox and K562/Dox cells has been up-regulated, down-regulation of miR-125b expression can reverse the drug resistance of leukemia cells to doxorubicine.