| ObjectiveAlternative splicing is associated with human diseases, and it could also play an important role in the process of L-type PML-RARαfusion gene of acute promyelocytic leukemia(APL). This study was designed to discuss the relation between the three isoforms of PML-RARαfusion gene and the prognosis of patients with APL, and then to verify the conclusion of clinical trials through experiments with NB4 cells, which right possessed the three isoforms. Moreover, test in which exogenous gene was transferred into cells was also performed to approve that different isoforms of L-type PML-RARαfusion gene may affect the sensibility of cells treated with ATRA. To discuss the molecular mechanism of this process, subcellular localization and differentiation experiments were conducted. In addition, in order to explore the way to improve the cells'sensitivity to ATRA and to excavate new ideas of increasing alleviation rate of APL, more tests were performed to prove whether the ratios of the three different isoforms produced during the differentiation of leukemia cell line were associated with the expression of the splicing factors ASF/SF2 and hnRNP A1 in the protein level.Methods1. To detect the relative expression of isoforms of PML-RARαfusion gene in APL by means of RT-PCRPML-RARαfusion gene of Death group (9 cases) and the first complete remission (CR1) group (66 cases) were detected by RT-PCR. The optical density of each band, which presents respectively E5(+)E6(+)(no deletion), E5(-)E6(+)(deleting exon5) and E5(-)E6(-) (deleting exon5 and exon6), was recorded by UVP analysis system and its proportion was then calculated. The relative expression of alternative splice isoforms, initial WBC and age were given multi-factor analysis with prognosis.2. To detect the relative expression of isoforms of PML-RARαfusion gene in NB4 cells by means of FQ-RT-PCRWe prepared the positive standard substances and probes of three isoforms of PML-RARαfusion gene and ABL house-keeping gene, and constructed the FQ-RT-PCR amplification system. To observe the sensitivity of three isoforms to ATRA, NB4 cells were treated with 1μM ATRA and the expression of the isoforms were detected by means of FQ-RT-PCR at different intervals. To judge the differentiation of NB4 cells during the ATRA treatment, morphocytology, FCM and NBT reduction were performed.3. To constructe the different isoforms of PML-RARαfusion gene eukaryotic expression vectorAfter the open reading frames of three isoforms of PML-RARαfusion gene were amplified, the fragments were subcloned into pEGFP-N3 vectors. The identified recombinant plasmids were then transfected transiently into 293T cells, and the expression of green fluorescent protein (GFP) were observed by confocal laser scanning microscope at 48 hours.4. To constructe recombinant Lentiviral Vector with pCDH1-MCS1-EF1-copGFPRecombinant lentiviral vectors pCDH1-E5(+)E6(+),pCDH1-E5(-)E6(+),pCDH1-E5(-)E6(-) were constructed and were co-transfected into 293T cells with packaging plasmids (pPACKH1-GAG, pPACKH1-REV and pVSV-G) with the optimum proportion. After condensing and measuring the titers, we use the lentiviral system to infecte U937 cells. Positive colonies were screened by means of limited dilution.5. To detect the relative expression of single isoform in cloned cells by means of FQ-RT-PCRThe cloned U937 cells were induced by 1μM ATRA, and some tests including morphocytology and FCM were used to confirm the standard of differentiation. The expression of single isoforms was detected by means of FQ-RT-PCR in different intervals in order to verify the different sensitivity to ATRA of isoforms.6. Study of the relation between the expression of splicing factor ASF/SF2,hnRNPA1 and the alternative splicing L-type PML-RARαTwo groups were designed due to the expression of isoforms E5(-)E6(-) in APL. Total proteins extracted from the bone marrow of the two groups and NB4, K562, U973, 293T cells were detected the expression of splicing factor ASF/SF2 and hnRNPA1 by western blot. The rations of expression were used to analyze the correlation.Results1. Study of the relation between the relative expression of isoforms of L-type PML-RARαand the prognosis of patients with APL The expression of E5(-)E6(-), E5(-)E6(+) and E5(+)E6(+) in Death group is obviously different from that in CR1group (P=0.005, P=0.000 and P=0.000). Initial WBC and age were not correlated with prognosis significantly between two groups (P=0.219 and P=0.086). Logistic regression analysis showed that the expression of E5(-)E6(-) was the most relevant with prognosis, and it was a negative correlation (B=-13.490, P=0.001).2. The relative expression of three isoforms of PML-RARαfusion gene in NB4 cellsThe morphology of granulocytic nuclear can be observed during the differentiation of NB4 cell. At the same time, the NBT reduction rate and the CD11b positive rate both increased gradually. Results from FQ-PT-PCR showed that the expression of isoforms E6(+) and E5(-)E6(-) both declined gradually. Compared with control group, the expression level at various intervals were significant difference. And the descend of isoform E6(+) was found early. The descending level of the expression was significant difference at 24,48,72,96 hours (P <0.05).3. Study of subcellular localizationThe eukaryotic expression constructs named pEGFP-N3-E5(+)E6(+), pEGFP-N3-E5(-)E6(+) and pEGFP-N3-E5(-)E6(-) were identified by restriction enzyme disgestion and sequencing analysis. The results from subcellular localization showed that pEGFP-N3-E5(-)E6(-) fusion proteins attached by GFP were concentrated in cytoplasma while both in the other two, GFP dispersively distributed in the cell nucleus and cytoplasm.4. Construction of recombinant lentiviral vector with pCDH1-MCS1-EF1-copGFPTo identified the lentiviral system, restriction enzyme disgestion, sequencing analysis, testing the viruses titers , PCR amplification and western blot were performed.The results showed that recombinant lentiviral vector pCDH1-E5(+)E6(+),pCDH1-E5(-)E6(+),pCDH1-E5(-)E6(-) were constructed successfully.5.The relative expression of single isoform in cloned cells treated with ATRAThe result showed: The expression of isoforms in the groups of pCDH1-E5(+)E6(+), pCDH1-E5(-)E6(+) and pCDH1-E5(-)E6(-)both declined with the increase of time. Compared with control group, the expressing level at various intervals were significant difference (P<0.05). The descending rate of isoforms in different groups was remarkably different, and the groups of pCDH1-E5(+)E6(+), pCDH1-E5(-)E6(+) and pCDH1-E5(-)E6(-) were in turn. The descending level of isoforms in group pCDH1-E5(-)E6(-) was significant difference compared with that in the group of pCDH1-E5(-)E6(+) and pCDH1-E5(+)E6(+) in 24, 48, 72, 96 hours (P<0.05). And it was significant difference between the group of pCDH1-E5(-)E6(+) and pCDH1-E5(+)E6(+) in 24, 48 hours (P<0.05), howerer, it was no significant difference in 72, 96 hours (P>0.05).6. Study of the relation between the expression of splicing factor ASF/SF2,hnRNPA1 and the alternative splicing of L-type PML-RARαfusion geneThe western blot results showed that the splicing factors differentially expressed in various cell lines including NB4, K562, U973 and 293T cells. Significant differences in the expression of splicing factor ASF/SF2 and hnRNPA1were observed between the group of CR1 and Death (P<0.05).Conclusion1. The increasing of E5(-)E6(-) is related with the poor prognosis for patients with APL.2. Compared with the isoform E6(+), the isoform E5(-)E6(-) was lower sensitivity to ATRA treatment.3. The isoform E5(-)E6(-) located in the cytoplasm resulting from lossing of nuclear localization signal, which maybe one of mechanisms underlying the insensitivity to ATRA treatment.4. The three recombinant lentiviral systems, which expressed respectively the isoforms of E5(-)E6(-), E5(-)E6(+) and E5(+)E6(+), were constructed successfully. It once again was proved that the isoform E5(-)E6(-) was lower sensitivity to ATRA treatment than the isoforms E5(-)E6(+) and E5(+)E6(+) at the level of single isoform.5. There is a positive association between the expressing ratio of three alternative splicing isoforms of L-type PML-RARαand the expression of splicing factor ASF/SF2, hnRNPA1. |
