| BackgroundAcute lymphoblastic leukemia (ALL) and non-Hodgkin’s lymphoma (NHL) are malignant tumors that originate in lymphocytes and occur most frequently in children. With the improvement of diagnosis and treatment level in recent years, long-term disease-free survival rate of ALL has been up to 70%, especially for low-risk ALL, the rate even up to 90%. On the other hand, the prognosis of NHL has a great relationship with its type and staging. At present, among Children’s NHL Collaborative Treatment Group,5-year disease-free survival rate has reached 60-80%. Burkitt lymphoma (BL) is a kind of invasive malignant tumor, which is belong to NHL. BL has a poor prognosis. Intensive chemotherapy may improve the prognosis. But because of drug-resistance, relapse occurs in some patients and eventually patients suffer from treatment failure and death.All cases of BL and parts of ALL cases have at least a kind of chromosomal abnormality:t (8; 14), t (2; 8) or t (8; 22). And their common feature is that the c-myc gene at chromosome 8q24 is involved in these translocations resulting in gene over-expression. The clonal abnormalities are closely related with the immunophenotyping, histological type, clinical manifestations, treatment response and prognosis. It has been shown that the protein product of c-myc gene, c-Myc, carries out multiple functions, such as DNA replication, cell growth, proliferation and differentiation. And uncontrolled over-expression of c-myc gene plays a pivotal role in the accurance and development of the tumor. t (8; 14) is an adverse prognostic factor and the over-expression of c-myc gene is mainly responsible for its phenotypic character. The clinical manifestations, such as early extramedullary infiltration, poor sensitivity of the usual chemotherapy, poor prognosis, and rapid disease progression, contribute to the primary drug resistance of tumor.Given the key role of over-expression of c-myc gene in many cancers, it is assumed that the inhibition of its expression could make their phenotypes lost. RNA interference (RNAi), also known as post-transcriptional gene silencing (PTGS), is an evolutionary method that effects gene post-transcriptional regulation and expression. It can specificly inhibit gene expression and has been widely used in functional genomics study and gene therapy. Consequently, RNAi, having highly specific sequence and inhibiting target gene expression accordingly, has developed as a powerful tool for reverse genetics and showed broad prospects of therapeutic application.Malignant tumors that originate in lymphocytes are the most common malignancy in childhood, and are also major diseases causing death. Although the current intensive chemotherapy and hematopoietic stem cell transplantation allow some patients to be long-term disease-free survival or even cure, there are still some patients relapse because of drug-resistance, so it is critical for researchers to work in order to further improve the therapeutic effects.In this study, the lentiviral vector-based short-interfering RNA (siRNA) targeting c-myc gene was constructed by RNAi technique and transfected into Jiyoye cell lines. By observing c-myc mRNA and protein expression levels, the most efficient of interference sequence, pLVX-c-myc-3, was chosen out, further transfected into Jiyoye cells lines and assessed the impact of down-expression of c-myc on the tumor cell proliferation, cell cycle and apoptosis so as to provide the experimental evidence for novel therapeutic approaches targeting c-myc gene for leukemia and malignant lymphoma.ObjectiveTo construct the lentiviral vector-based siRNA targeting c-myc gene by RNAi technique, and explore the suppression of c-myc gene expression in Jiyoye cells, so as to assess the impact of down-expression of c-myc on the tumor cell proliferation, cell cycle and apoptosis, and therefore provide the experimental evidence for novel therapeutic approaches targeting c-myc gene for leukemia and malignant lymphoma in vitro.MethodsThree short hairpin RNA (shRNA) interference sequences targeting c-myc gene (c-myc-1, c-myc-2 and c-myc-3), and one negative control sequence (c-myc-neg) were designed and synthesis. The sequence of them:c-myc-1: 5’-TCCGTACAGCCCTATTTCA-3’; c-myc-2:5’-TGGAGATGATGACCGAGTT-3’; c-myc-3:5’-GAGCAAGAAGATGAGGAAG-3’respectively. The negative control is c-myc-neg:5’-CGGCGCATAAGAAGCATAT-3’.They were annealed and cloned into pLVX vector to produce the recombinant plasmid, and then transfected into Jiyoye cells by lentivirus. After transfection and having been cultured for 72h, transfection rates in every group were detected by flow cytometry (FCM), c-myc mRNA and protein level were detected by real-time PCR and Western blot, the cell proliferation activities were assayed by MTT, and cell cycle and apoptosis were analyzed by flow cytometry with PI single staining and Annexin V/PI double staining respectively.Results1. Identification of recombinant vector by PCRpLVX-c-myc was determined by PCR, every lane has target band that is consistent with the expected size. But no fragments appeared in the control lane (pEGFP). It showed that the recombinant expression vector of pLVX-c-myc was successfully constructed.2. Comparison of transfection rates in different groups by flow cytometry72h after transfection, positive cells were detected by flow cytometry in every group. Positive cell rate of c-myc-neg group was (54.3±4.2)%, mean fluorescence intensity was 5.98±0.41; Positive cell rate of c-myc-1 group was (51.6±3.8)%, mean fluorescence intensity was 6.12±0.63; Positive cell rate of c-myc-2 group was (53.7±3.3)%, mean fluorescence intensity was 6.04±0.37; Positive cell rate of c-myc-3 group was (52.8±2.9)%, mean fluorescence intensity was 6.01±0.52. Positive cell rates of the cells in every group had no significant difference (p>0.05), and mean fluorescence intensity of the cells had also no significant differences(p>0.05). It show that the transfection efficiency of each group was not significantly different.3. Analysis of the expression of c-myc by real-time PCR72h after transfection, real-time PCR was used to detect the expression of c-myc in every group. GAPDH was as internal reference gene. The ratio of the value of the gene under test divided by the value of internal reference in the same sample was the relative content of test gene. The c-myc mRNA expression level of the groups that transfected with c-myc-1, c-myc-2 and c-myc-3 respectively were all lower than that of the negative control group transfected with c-myc-neg (p<0.05), and among which the c-myc mRNA expression level of c-myc-3 was the lowest (p<0.05). There was no difference between untransfected control group and transfected with c-myc-neg group (p>0.05). It indicated that the c-myc-3 could specifically inhibit c-myc gene expression most.4. Analysis of c-Myc protein expression by Western blot72h after transfection, Western blot was used to detect the expression of c-Myc protein in every group. The c-Myc protein expression level of the groups that transfected with c-myc-1, c-myc-2 and c-myc-3 respectively were all lower than that of the negative control group transfected with c-myc-neg (p<0.05), and among which the c-Myc protein expression level of c-myc-3 was the lowest (p<0.05).5. The impact of pLVX-c-myc-3 on the proliferation of Jiyoye cellDuring 168h after transfection, the growth curve showed that the cells transfected with c-myc-3 all grew slowly, and the differences were statistically significant (p<0.05).72h after being tranfected with c-myc-3, the cell proliferation activities were assayed by MTT. Cell inhibition ratio of the experimental groups was higher than the negative control group and blank control group, and the cell proliferation activities of the experimental groups significantly decreased, the differences were statistically significant (p<0.05). Cell inhibition ratio of the negative control group was higher than that of the blank control group (p<0.05), and its reason might be the cytotoxicity after transfection.6. The impact of pLVX-c-myc-3 on cell cycleFlow cytometry with PI single staining was used to analyze the cell cycle, G0/G1 phase cells in experimental group were (72.4±4.6)%, and G0/G1 phase cells in negative control group and blank control group are (81.5±4.7)% and (79.3±2.8)% respectively. The cells in experimental group were lower than that of the negative control group and blank control group (p<0.05). G2/M phase cells in experimental group were (19.8±4.4)%, and G2/M phase cells in negative control group and blank control group were (11.6±4.2)% and (12.2±1.8)% respectively. The cells in experimental group were higher than that of the negative control group and blank control group (p<0.05). There was no difference between the S phase cells in experimental group and that of the negative control group and blank control group (p> 0.05).7. The impact of pLVX-c-myc-3 on cell apoptosis72h after transfection with pLVX-c-myc-3, flow cytometry with Annexin V/PI double staining was used to detect the cell apoptosis. The proportion of apoptotic cells in the experimental group was (25.6±4.2)%, and that in negative control group and blank control group was (15.1±4.2)% and (12.7±1.8)% respectively. The proportion of early-term apoptotic cells in the experimental group was higher than that of negative control group and blank control group (p<0.05). The proportion of late-term apoptostic cells cells in the experimental group was (11.5±4.7)%, and that in negative control group and blank control group was (9.3±4.7) % and (8.4±2.8)% respectively. The proportion of late-term apoptostic cells in the experimental group was higher than that of negative control group and blank control group (p<0.05). The results showed that compared to blank control groups, the proportion of apoptotic cells in the negative control group increased, but the difference was not statistically significant (p>0.05). It suggested siRNA targeting c-myc gene could induce Jiyoye cells apoptosis.Conclusions1. The fourth-generation lentivirus was used as vector, and its advantages are low immunogenicity and non-carcinogenicity which are different from other viral vectors. The recent researches on gene therapy of lentiviral vector have been carried out a series of systematic work. Given that the experiments of the lentiviral vector-based HIV-1 in vivo and in vitro have not yet been found an example of host immune response, its biological safety is guaranteed.2. Three pairs of c-myc specific siRNA and a pair of negtive siRNA were designed and synthesized, and real-time PCR and Western blotting were used to detect the expression level of c-myc mRNA and protein after Jiyoye cells were transfected by pLVX-c-myc respectively. It was confirmed that c-myc specific siRNA can down regulate the c-myc gene mRNA and protein expression in Jiyoye cells. The most efficient interference sequence was chosen out, which further provide an experimental basis for c-myc gene silencing in targeting therapy of leukemia and lymphoma.3. siRNA interference targeting c-myc may change the cell cycle of Jiyoye cell, and block the cell in G2/M phase.4. siRNA targeting c-myc inhibited tumor cell proliferation and induced cell apoptosis. Its mechanism may be related to the block of tumor cells in G2/M phase and induction apoptosis of tumor cells. |
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