Correlation between the expression level of nucleostemin gene in marrow cells of the patients with acute leukemia and its clinical types,therapy efficacy

AIM: To investigate nucleostemin gene expression in bone marrow of acute leukemia and its clinical significance.METHODS: The expression of nucleostemin in 67 acute leukemia patients was detected by fluorescent quantitative polymerase chain reaction (FQ-PCR). The correlation between the expression level of nucleostemin gene and clinical significance was analyzed.RESULTS: Significantly higher expression levels of nucleostemin gene were detected in the initially-treated acute leukemia patients than those in normal control group and complete remission (CR) group by FQ-PCR (P<0.01). The expression level of nucleostemin gene in the cells from ALL was significantly lower than that of the cells in ANLL (P<0.05). No significant difference of nucleostemin expression in various differentiation stages (M2, M3, M4, M5) of ANLL was found (P>0.05). No significant association was observed between nucleostemin expression levels and age, sex, hepatauxe, splenomegaly, WBC count of acute leukemia patients by logistic analysis. The patients with positive expression of nucleostemin had significantly lower complete remission rate than those with negative expression (51.3% vs 83.3%, P<0.05). The nucleostemin expression level was significantly reduced during complete remission. Long-term follow-up of nucleostemin expression level showed that continuous or significant increase in nucleostemin expression in acute leukemia patients predicts refractoriness and impending relapse.CONCLUSION: Expression level of nucleostemin in acute leukemia patients is obviously higher than that in normal control. Nucleostemin can be a marker for evaluating therapy efficacy and monitoring minimal residual diseases (MRD) in leukemias.     

Clinical significance of detection of p16 gene methylation in early diagnosis of lung cancer

AIM: To investigate the aberrant methylation in the promoter of p16 in plasma, sputum, bronchoalveolar lavage fluid (BALF), pleural effusion and biopsy specimens from suspected lung cancer patients and to evaluate the clinical significance in the early diagnosis of lung cancer.METHODS: Using methylation specific PCR (MSP) for the detection of promoter methylation of p16 gene in plasma, sputum, BALF, pleural effusion and biopsy specimens from suspected lung cancer patients.RESULTS: Of the 67 cases of suspected lung cancer patients, 42 were proved by pathology. The positive percentages of p16 gene promoter methylation of the lung cancer patients are as follows: 52.4%（22/42）in plasma, 47.6%（20/42）in sputum, 59.5%（25/42）in BALF, 71.4%（10/14）in pleural effusion and 61.9%（26/42）in biopsy specimens, respectively；while promoter methylation in p16 gene was found only one in plasma and one in pleural effusion in 25 patients with various benign lesions (P<0.05). The positive expression of p16 gene promoter methylation in lung cancer patients was irrelevant to histological type, clinical stage, pathological grade, lymph node metastasis and distant metastasis of the lung carcinomas (P>0.05).CONCLUSION: Detection of the aberrant methylation in the promoter of p16 gene in plasma, sputum, BALF, pleural effusion and biopsy specimens from lung cancer patients by MSP method is a kind of rising technology with development potential for lung cancer early diagnosis.     

Methylation of p15INK4B gene and its mechanism in patients with myelodysplastic syndrome and leukemia

AIM: To explore the relationship between hypermethylation of p15INK4B gene and the pathogenesis of hematopoietic malignances. METHODS: The expression and methylation of p15INK4B gene and the expression of DNA methyltransferase genes (DNMTs) in bone marrow cells from 54 cases with hematopoietic malignances were detected by RT-PCR, Western blot, and methylation-specific PCR. RESULTS: The p15INK4B gene was methylated more often in high-risk myelodysplastic syndrome (MDS) patients, patients at blast phase of chronic myeloid leukemia (CML-BP) and acute leukemia patients than that in low-risk MDS patients (P<0.01). The expression levels of DNMT_3A and DNMT_3B in acute leukemia patients, high-risk MDS patients, and CML-BP patients were higher than that in low-risk MDS patients (P<0.05). CONCLUSION: The hypermethylation of p15INK4B gene may be one of the most common genetic event in pathogenesis of acute leukemia, high-risk MDS, and blast phase of chronic myeloid leukemia. Furthermore, DNMT_3A and DNMT_3B are substantially over-expressed in the bone marrow cells of these patients.     

Study on hyper methylation of p15INK4B gene in multiple myeloma

AIM: To study the characteristics and regulations of p15 (MST INK4B) methylation in multiple myeloma (MM). METHODS: Method of methylation-specific PCR was applied in 23 cases of MM about the methylation rate of p15 gene. RESULTS: Methyaltion rate in MM was 73.5%(17/23). The PCR product was a fragment of 148 bp. In four stageⅠcases of MM with plasma cell-typed bone marrow profile, p15^INK4B gene was non-methylated; In one case of stag ⅡMM and one case of stage Ⅲ MM with mature plasma typed bone marrow profile, p15^INK4B gene was no-methylated, too, while in many cases of stageⅠ、stage Ⅱand stage Ⅲ with naive plasma cell in bone marrow profile which were plasma cell-typed or mixed typed, p15^INK4B gene methylation was frequently detected. The methylation rates for stageⅠ、stage Ⅱand stage Ⅲ MM were respectively 55%(5/9),100%(7/7) and 71.4%(5/7). CONCLUSION: p15 gene methylation was a possible pathogenic factor,and might be related to the progression and prognosis of the disease.     

Effect of DNA methylation of miR-30a-5p promoter region on hepatic injury induced by homocysteine

AIM: To explore the role of DNA methylation of microRNA-30a-5p(miR-30a-5p) promoter region in hepatic injury. METHODS: Four-week-old normal mice and cystathionine β-synthase (CBS) single gene knockout mice were used and divided into normal (CBS^+/+, n=12) group and single gene knockout (CBS^+/-, n=12) group, and the mice were fed with high methionine diet for 8 weeks. HL-7702 hepatic cells were routinely cultured in vitro and divided into control group, homocysteine (Hcy) group and Hcy+5-azacytidne (AZC) group. Serum Hcy, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured by automatic biochemical analyzer. The levels of ALT and AST in the cells culture medium were determined by the microplate method. Hepatic injury in the mice were observed with HE staining. Cell viability staining was used to measure the viability of hepatocytes. RT-qPCR was used to detect the expression of miR-30a-5p in the liver tissues and hepatocytes. The correlation between the expression of miR-30a-5p and serum ALT and AST levels was analyzed by Pearson correlation analysis. DNA methylation level of miR-30a-5p promoter region in the liver tissues and hepatocytes was detected by nested landing methylation-specific PCR (nMS-PCR). RESULTS: Compared with the CBS^+/+ mice, the serum levels of Hcy, ALT and AST in the CBS^+/- mice were significantly increased (P < 0.05). HE staining showed the hepatocyte swelling and nuclear fragmentation and dissolution. The expression level of miR-30a-5p in the liver tissues was decreased (P < 0.01). Besides, the expression level of miR-30a-5p in the mice was negatively correlated with serum ALT and AST levels (r²=0.4557, P=0.0003, r²=0.4626, P=0.0003), and the DNA methylation of miR-30a-5p promoter region was increased (P < 0.01). In the HL-7702 cells, compared with control group,the ALT and AST levels were increased in Hcy group (P < 0.05, P < 0.01), and the cell viability was remarkablely decreased. DNA methylation of miR-30a-5p promoter region was increased (P < 0.01), which decreased after treated the cells with AZC (P < 0.05), while the expression level of miR-30a-5p in the cells was increased (P < 0.05). CONCLUSION: Hypermethylation of miR-30a-5p promoter region may play an important role in hepatic injury.     

The impact of scavenger receptor class A typeⅠ/Ⅱ on lipid metabolism in mice

AIM: To investigate the impact of scavenger receptor class A type Ⅰ/Ⅱ(SR-A Ⅰ/Ⅱ) on the lipid metabolism in SR-A Ⅰ/Ⅱ gene deficient mice. METHODS: A probe of 660 bp fragment of SR-A Ⅰ/Ⅱ cDNA digested with PstⅠ and XhoⅠ from plasmid 122 was used to identify whether SR-A Ⅰ/Ⅱ had been knocked out in the tail DNA of the mutant(SR-/-) and control(SR+/+) mice by the method of Southern-blot analysis. The serum levels of triglycerides(TG), cholesterol(CH), low density lipoprotein(LDL), high density lipoprotein(HDL), apolipoprotein(Apo) A and ApoB of the mice fed with normal food and higher lipid food respectively were tested by biochemical method. RESULTS: The serum levels of LDL and body weights of group with SR-A Ⅰ/Ⅱ gene knocked out were higher than that of control group(P<0.05). Although the disorders of serum levels of LDL and body weights deteriorated after high lipid diet in both groups, the disorders in group of SR-/- were worse than those in control group(P<0.01). CONCLUSION: The results demonstrated that SR-A Ⅰ/Ⅱ were involved in the regulation of lipid metabolism, and SR-A Ⅰ/Ⅱ deficiency is harmful for removing LDL from blood circulation.     

Application of multiple gene methylations in plasma for diagnosis of lung cancer

AIM: To determine the aberrant methylation status in the gene promoter regions of CDH13, RASSF1A, DLEC1, SEPT9and RUNX3by detecting the plasma specimens and the value of their combined detection for diagnosis of lung cancers. METHODS: Nest methylation specific PCR (nMSP) was used to detect the promoter methylation status of the 5 genes in the plasma from 106 normal controls， lung cancer tissues, lung benign tissues and the plasma from 106 patients with lung cancers. Multiple displacement amplification (MDA) was used to amplify modified genomic DNA to solve the problem of insufficient of plasma DNA template. RESULTS: The positive rates of promoter methylation of CDH13, RASSF1A, DLEC1, SEPT9and RUNX3in the lung cancer tissues were 51.9%, 44.3%, 54.7%, 36.8%, 24.5%, respectively, and those in the plasma were 46.2%, 41.5%, 50.9%, 31.1%, 19.8%, respectively. The results of the Kappa consistency check showed that the lung cancer tissues and the plasma had obviously coherence in the methylation status of the 5 gene promoter regions. Combination of DLEC1, CDH13, RASSF1A, and SEPT9 had a higher diagnostic efficiency than the others, as their ACC value was 0.8208 and youden index was 0.6415 (with the sensitivity of 81.13% and the specificity of 83.02%). CONCLUSION: Combination detection of promoter methylation of lung cancer-related genes in the plasma is expected to apply to the early diagnosis of lung cancer.     

Changes of DNA methylation at CpG sites in promoter region of TNF-α gene in DENV2-infected peripheral blood mononuclear cells

AIM：To examine DNA methylation at CpG sites in the promoter region of tumor necrosis factor-alpha (TNF-α) gene in dengue virus type 2 (DENV2)-infected peripheral blood mononuclear cells (PBMC).
METHODS：DNA methylation in the promoter region of TNF-α gene was measured by bisulfite sequencing PCR.
RESULTS：The promoter region of TNF-α gene was from -294 bp to +58 bp, including 11 CpG sites. The PCR products identified by aga-rose gel electrophoresis were consistent with the theoretical size. Two sites were methylated at 0 h and 6 h and 6 sites were methylated at 12 h in TNF-α gene promoter region in DENV2-infected PBMC. The average methylation rates were 103%, 121% and 255% at 0 h, 6 h and 12 h, respectively. Significant differences between 0 h and 12 h and between 6 h and 12 h were observed.
CONCLUSION：The DNA methylation in the promoter region of TNF-α gene is increased in DENV2-infected PBMCs.     

Correlation between hypermethylation and expression of Syk gene in colorectal cancer cell lines

AIM:To explore the relationship of methylation status and expression of spleen tyrosine kinase (Syk) gene in colorectal cancer cell lines. METHODS:Bisulfite sodium modification sequencing, methylation specific PCR and Western blotting were used to detect the methylation status and expression of Syk gene in 23 colorectal cancer (CRC) cell lines. Luciferase assay was applied to measure the activity of promoter with or without methylation in CpG islands. Meanwhile, methylation status and expression level were compared in Syk(-) HCT-15 cell line before or after 5-Aza-CdR administration. RESULTS:(1) Among 23 cell lines, loss expression of Syk gene in 9 cell lines due to hypermethylation in promoter, and 14 expression with unmethylation status were observed. The total methylation rate was 39.2%. (2) Microsatellite instability was found in 7 of 9 cell lines with promoter hypermethylation, 4 of 14 were wihtout hypermethylation. The difference between methylation and unmethylation group was significant (P<0.05). (3) 5-Aza-CdR restored methylated-Syk gene promoter activity. Compared to methylated-promoter, luciferase activities increased to 4.5 and 4.7 folds with Syk full size promoter and unmethylated-promoter, respectively. (4) 5-Aza-CdR restored methylated-Syk gene expression and the effect had time-course dependence. CONCLUSION:Hypermethylation of CpG islands in Syk gene promoter silences Syk expression in CRC cell lines, and 5-Aza-CdR restores Syk expression by demethylation.     

IL-1B-511 single nucleotide polymorphism and gastric atrophy in high prevalence regions of gastric cancer in China

AIM: To study the relationship between IL-1B-511 single nucleotide polymorphism,H.pylori infection,and gastric atrophy in high prevalent (Shanxi) region in China.METHODS: Five hundred healthy volunteers from Shanxi Province of China were recruited in this study,which were divided into five subgroups according to age,namely age 20-29,30-39,40-49,50-59 and >60 years,respectively.Genomic DNA was extracted from peripheral blood.IL-1B-511 single nucleotide polymorphism was analyzed by PCR-RFLP.Serum pepsinogen was used as a biomarker of gastric atrophy.Serum pepsinogen Ⅰ (PGⅠ),pepsinogen Ⅱ (PGⅡ) and anti-H.pylori IgG were determined by an ELISA assay.RESULTS: The mean serum PGⅠ concentration and ratio of PGⅠ /PGⅡ decreased gradually with increasing age,and were lower in subjects with IL-1B-511 T/T genotype and H.pylori infection than those without H.pylori infection in each subgroup,respectively (All P<0.05).Multiple linear regression showed that IL-1B-511 T/T genotype,age and Hp infection were significantly associated with gastric atrophy (P<0.05,P<0.05 and P<0.01,respectively).CONCLUSION: Gastric atrophy is closely correlated with IL-1B-511 T/T genotype,age,H.pylori infection in high prevalence region of gastric cancer in China.The risk of gastric atrophy is significantly increased for the IL-1B-511 T/T genotype with Hp infection.     

Relationship between apoptosis,proliferation and expression,mutation of related genes in breast cancer

AIM:To study the relationship between apoptosis, proliferation and expression,mutation of related genes in breast cancer.METHODS:Methods of TUNEL, immunohistochemical S-P and PCR-SSCP were used respectively to study apoptotic index (AI), mitotic index(MI), expression of Bcl-2,p53,c-erbB-2,PCNA,Ki67,TopoⅡ and mutation of p53 in 54 cases of breast cancer.RESULTS:AI and MI were 9.40±3.78 and 5.96±2.36, respectively. There was a significant direct correlation between them(r=0.46.P＜0.01).High expression of Bcl-2,PCNA,Ki67,TopoⅡ coincided with high AI,MI(P＜0.01). High expression of p53,c-erbB-2 and mutation of p53 coincided with high MI(P＜0.01). Type of p53 mutation coincided with AI(P＜0.05).CONCLUSION:Disturbance of gene control between apoptosis and proliferation is related with expression,mutation of related genes in breast cancer.     

Histone acetylation and methylation modification of topoisomerase Ⅱα promoter regulatory factor Sp1 in patients with chronic benzene poisoning

AIM: To investigate the histone modification changes of topoisomerase Ⅱα(TOPOⅡα) promoter regulatory factor Sp1 in the patients with chronic benzene poisoning.METHODS: The bone marrow samples were collected from 25 chronic benzene poisoning cases and 25 controls. The chromatin immunoprecipitation assay was carried out to study the possible mechanism of TOPOⅡα promoter regulatory factor Sp1 expression changes. The mRNA expression of Sp1 was detected by RT-PCR.RESULTS: Compared with the controls, the histone H4 acetylation and histone H3 acetylation of Sp1 in the chronic benzene poisoning patients significantly decreased(P<0.01), and histone H3K9 methylation level of Sp1 increased(P<0.01), but the histone H3K4 methylation level of SP1 was not obviously changed(P>0.05). The mRNA expression of Sp1 in the chronic benzene poisoning patients was significantly lower than that in the controls(P<0.05).CONCLUSION: In chronic benzene poisoning patients, the histone acetylation and methylation modification changes of TOPO Ⅱα promoter regulatory factor Sp1 accompanied with the changes of mRNA level are observed. Histone H4 and H3 acetylation and H3K9 methylation modification of Sp1 may play an important role in the benzene,s hematopoietic toxicities.     

Relationship between ERα gene polymorphisms and endometriosis

AIM: To evaluate the effect of ERα gene polymorphisms in the pathogenesis of endometriosis. METHODS: The distribution of ERα gene PvuⅡ and XbaⅠ polymorphisms in 60 specimens of endometriosis and 56 specimens of normal endometrium was determined by RFLP-PCR and DNA sequencing. RESULTS: The distribution of PvuⅡ genotypes (PP, Pp and pp) and XbaⅠ genotypes (XX, Xx and xx) of the ERα gene was not significantly different between endometriosis and normal endometrium. The six genotype combinations were analyzed, and the distribution of these genotype combinations did not reveal the statistical difference between the cases and controls. The distribution of PvuⅡ genotypes and XbaⅠ genotypes of the ERα gene was not significantly different between the patients with stage I/II endometriosis and those with stage III/IV endometriosis. CONCLUSION: The finding suggests that there is no significant correlation between ERα gene polymorphisms and the pathogenesis of endometriosis.