The Gene Polymorphisms And Expression Of Vitamin D Receptor In Children With Acute Leukemia And Effect Of Vitamin D In Leukemia Celllines

Objective To investigate the distribution of vitamin D receptor (VDR) gene FokI, BsmI, ApaI, TaqI site gene polymorphism of children in Guangxi region.To explore the correlation between the VDR gene polymorphism and hereditary susceptibility of leukemic children. We hope it will be useful to the followed study on the relative mechanism of acute leukemia. Methods This study adopted case-control design. 127 children with acute leukemia and 268 healthy medical examination children (as controls) who had unrelated blood relationship in Guangxi region were recruited. DNA was extracted from peripheral blood sample. Polymerase chain reaction-restrictive fragment length polymerphism (PCR-RFLP) analysis technique and DNA sequencing technology were used to detect gene polymorphisms of FokI, BsmI, ApaI, TaqI locus. The Hardy -Weinberg balance of heredity in the two groups was analyzed by binomial distribution test. The difference between the frequency of VDR alleles of the two groups was analyzed by chi square test. Odds ratio (OR) and 95% confidence intervals (CI) were calculated to assess the relative risk. The linkage disequilibrium of VDR gene was analyzed by SHEsis software. Results The result of Hardy-Weinberg balance of heredity showed that there were insignificant difference between the observed value and the expected value of the frequency of FokI, BsmI, ApaI, TaqI alleles in the two groups, P>0.05. It was a declaration that the samples were representation. In the 268 healthy children, we obtained the percentage of genotypes ff, Ff and FF to be 22.02%, 48.13% and 29.85% respectively and the allelic frequencies of 46.08% and 53.92% for f and F allele. The frequency of bb, Bb and BB in the BsmI site were 91.79%, 7.84% and 0.37%. Frequencies of b and B allelic gene were 95.71% and 4.29%. The frequency of aa, Aa and AA in the ApaI site were 52.61%, 36.57% and 10.82% respectively. Frequencies of a and A allelic gene were 70.90% and 29.10%. tt, Tt and TT in the TaqI site were 0, 9.70% and 90.30% respectively. Frequencies of t and T allelic gene were 4.85% and 95.15%. The result displayed that FokI, BsmI, ApaI, TaqI site gene polymorphism of healthy children in Guangxi region was one and all with the other regions in China. In the 127 children with acute leukemia, we obtained the percentage of genotypes ff, Ff and FF to be 35.43%, 43.31% and 21.26% respectively and the allelic frequencies of 57.09% and 42.91% for f and F allele. The frequency of bb, Bb and BB in the BsmI site were 92.13%, 7.87% and 0. Frequencies of b and B allelic gene were 96.06% and 3.94%. The frequency of aa, Aa and AA in the ApaI site were 48.82%, 37.01% and 14.17% respectively. Frequencies of a and A allelic gene were 67.32% and 32.68%. tt, Tt and TT were 0, 11.81% and 88.19%. Frequencies of t and T allelic gene were 5.91% and 94.09%. Allele frequencies of the FokI polymorphism showed a significant association (χ2= 8.671, P<0.05) between the two groop. The genotype ff in the acute leukemic children was higher than that in the healthy children. The result of one way analysis showed that the subjects carrying ff genotype had 2.26 fold elevated risk for developing acute leukemia compared with the FF genotype (OR=2.260, P<0.05). Using the F allele as a reference, a significant risk of developing acute leukemia was found between the presence of f allele (OR=1.556, P<0.05). The genotype and allele frequencies of BsmI, ApaI and TaqI site were not significantly different from those in control group (P>0.05). There had no strongly apparent linkage disequilibrium between the FokI locus, BsmI locus, ApaI locus and TaqI locus. Conclusions The polymorphism frequency and distribution of this VDR gene in Guangxi population of China exhibit its own characteristics. There maybe the genetic susceptibility effect of VDR gene FokI allele in acute leukemia while the gene polymorphisms of BsmI, ApaI, TaqI enzyme digestion locus may not be related to genetic susceptibility of children acute leukemia. The individual with f allele were more easier to take AL than that with F. The conclusion must confirm by further arge sample arge sample.Objective To study the expression of VDR in children of acute leukemia and to approach the significance of VDR in the occurrence of acute leukemia from transcription and translation.To determine whether the expression is related to the classification of acute leukemia. Methods Total RNA was extracted by trizol from bone marrow cells of thirty untreated children with acute lymphoblastic leukemia (ALL), twelve untreated children with acute myeloid leukemia (AML) and thirty cases nontumorous hematologic diseases children(including iron deficiency anemia, hemolytic anemia and idiopathic thrombocytopenic purpura, et al, as controls). The first strand cDNA was acquired by reverse transcription. SYBR Green I Rea1 time fluorescent quantitative PCR (RT FQ-PCR) was used to investigate VDR mRNA expression in the three groups. The specificity of amplification production was measured by melting curves and agarose gel electrophoresis. The VDR mRNA signal was normalized to in accordance with the house-keeping gene GAPDH mRNA signal through the method of 2-△△Ct. Appropriate amount bone marrow cells from nine children with acute leukemia and four cases nontumorous hematologic diseases children were smeared immediately to detect the expression of VDR protein and cell localization by ultrasensitive streptavidin-biotin peroxidase (S-P) method of immunocytochemical staining. Peripheral blood mononuclear cells (PBMCs) were extracted from peripheral blood prepared of thirty untreated children with ALL, ten untreated children with AML and thirty healthy children (as controls). Appropriate amount PBMCs were smeared immediately to detect the expression of VDR protein and cell localization by immunocytochemical staining while all cell albumen extracted from the other PBMCs by cell lysate was used to detect the expression of VDR protein by Western blots analysis. The different expression of VDR mRNA and protein in the three gene types ff, Ff and Ff was compared by one-factor analysis of variance. The relationship of VDR expression and the leukocyte number and the platelet number in children with acute leukemia was analyzed by bivariate linear correlation. Results VDR mRNA was detected in all the acute leukemic children and nontumorous hematologic diseases children. The expression of VDR mRNA in children with ALL (1.06±0.31) and in children with AML (1.13±0.34) was lower significantly compared with the expression in the controls (3.10±0.18) (F=1701.00, P<0.01) while there was no significant change between children with ALL and children with AML (P>0.05). The expression lever of VDR mRNA in forty two cases children with acute leukemia had no associativity (r=0.045, P=0.078) with the leukocyte number and the platelet number (r=0.067, P=0.675) by bivariate linear correlation while there was no significant changed at the expression lever of VDR mRNA in the three gene types ff (1.25±0.47, n=12), Ff (1.12±0.41, n=17) and FF (1.09±0.38, n=13) (F=0.489, P>0.05). The results of immunocytochemistry techniques showed VDR protein was expressed in all marrow mononuclear cell and PBMNCs of each group. VDR protein was mainly localized in cell nucleus. The expression of VDR protein in nine children with AL(30.80±1.56%) was lower than that in four control subject (62.45±3.73%) but statistics analysis was not done because the sample is only a few. The expression of VDR protein in the children with ALL (27.82±1.78%) and in the children with AML (27.10±2.44%) were lower significantly compared with the expression in the control subjects (59.02±3.46%) (F=1164.47, P<0.01) while there was no significant different between the children with ALL and the children with AML (P>0.05). The result of Western blots analysis was accordance with immunocytochemistry techniques. The expression of VDR protein in children with ALL (0.299±0.071) and in children with AML (0.290±0.094) were lower significantly compared with the expression in the control subjects (0.710±0.041) (F=356.434, P<0.0) while there was no significant different between children with ALL and children with AML (P>0.05). The expression lever of VDR protein in forty cases children with acute leukemia had no associativity with the leukocyte number (r=-0.133, P=0.413) and the platelet number (r=0.006, P=0.917) by bivariate linear correlation while there was no significant changed at the expression lever of VDR protein in the three gene types ff (0.286±0.061, n=13), Ff (0.289±0.079, n=17) and FF (0.324±0.089, n=10) ((F=0.853, P>0.05). Conclusions VDR was expressed in all the acute leukemic children and we get such a conclusion that leukemic cells were one of the target cells of VDR. The expression of VDR mRNA and VDR protein in acute leukemia children was significantly lower than that in control subjects. VDR expression might be a effective monitoring index in diagnosing of leukemia. The expression of VDR may be had no associativity with classification, genotype and peripheral blood index. Objective To investigate the possible mechanism of inducing apoptosis of 1,25-Dihydroxyvitamin D3 (1,25(OH)2 D3 ) and to determine the therapeutic potential of 1,25(OH)2D3 in leukemia, we observed the effects of inhibition, differentiation, apoptosis and the expression of VDR gene and protein on human leukemia cell lines 6T-CEM and HL-60 induced by 1,25(OH)2D3 at different concentrations. Methods Leukemia cell lines 6T-CEM and HL-60 in exponential growth phase were treated by 1,25(OH)2 D3 with different concentration of 10-9, 10-8, 10-7 and 10-6 mol/L in vitro. Viable cells were counted by Trypan blue exclusion. Methy thiazoly tetrazolium (MTT) assay was used to evaluate the cell proliferation. The differentiation of HL-60 cells was detected by nitro blue tetrazolium (NBT) reduction test. The cells late apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining assays (TUNEL). Cell morphology was observed by inverted microscope and Wright-Giemsa staining method. The expression of VDR mRNA of each group was analyzed by RT FQ-PCR. The expressions of VDR protein were detectied by immunocytochemistry technique and Western blot analysis. Results MTT assay showed that 1, 25(OH)2D3 could significantly inhibit the proliferation of 6T-CEM and HL-60 cells in a dose-dependent and time-dependent manner. 1,25(OH)2 D3 could remarkably induce HL-60 cells to differentiate towards mature granulocyte while there was not change in 6T-CEM cells. The result of TUNEL indicated 1,25(OH)2D3 with different concentration could enhanced cell apoptosis rate in a dose-dependent manner. The observation of cell morphology showed the cells treated by 1,25(OH)2D3 had morphologic characteristics of apoptosis. VDR mRNA and protein were expressed in the all 6T-CEM and HL-60 cells before and after the action of 1,25(OH)2D3. VDR protein expression was up-regulated after drug action while VDR mRNA expression was no change. Conclusions 1,25(OH)2D3 at a certain concentration range can significantly inhibit the proliferation and induce apoptosis in 6T-CEM and HL-60 cells. 1,25(OH)2D3 can induce HL-60 cells to differentiate towards mature granulocyte. 1, 25(OH)2D3 can increase expression of VDR protein in 6T-CEM and HL-60 cells but not VDR mRNA...