Validation of the association between PSMA6 -8 C/G polymorphism and type 2 diabetes mellitus in Chinese Dongxiang and Han populations.

OBJECTIVE: The aim of our study was to validate association between -8 C/G variant of PSMA6 gene and T2DM in Chinese Dongxiang and Han populations. METHOD: We genotyped PSMA6 gene -8 C/G polymorphism in the control groups and T2DM groups in two populations from China using PCR-RFLP technique. Phenotypes and biochemical indicators were measured by biochemical technique. RESULT: The frequencies of CG+GG genotype were observably different from CC genotype in the T2DM groups and control groups (for Dongxiang population: OR = 1.341, 95% CI: 1.101-1.632, P = 0.004; for Han population: OR = 1.313, 95% CI: 1.085-1.569, P = 0.006 after adjusting for gender, age, and BMI, respectively). In the Dongxiang population, the FPG, HOMA-IR, SBP and TG levels of CG+GG genotype were markedly higher than those of the CC genotype in control group (all P < 0.05). However, in the Han population, we only found that the FPI level of the CC genotype was significantly higher than that of the CG+GG genotype in control group (P < 0.05). CONCLUSION: Our investigation suggests that -8 C/G variant of PSMA6 gene may be associated with T2DM and diabetes-related metabolic traits in Chinese Dongxiang and Han populations.

Association of P213S polymorphism of the L-selectin gene with type 2 diabetes and insulin resistance in Chinese population.

AIMS: L-selectin belongs to selectin family of adhesion molecule and participates in the generation and development of type 2 diabetes (T2D). In this study, we evaluated the relationship between the P213S polymorphism of L-selectin gene and T2D and insulin resistance in the Chinese population. METHODS: We genotyped P213S polymorphism in 801 patients with T2D and 834 healthy controls in the Chinese population using polymerase chain reaction-ligase detection reaction (PCR-LDR) technique. Plasma glucose, insulin, lipid, blood urea nitrogen, creatinine and uric acid levels were measured by biochemical technique. RESULTS: The frequency of 213PP genotype and P allele of the L-selectin gene in patients with T2D was significantly higher than that in controls (P=0.007; P=0.019, respectively). The relative risk of allele P suffered from T2D was 1.191 times higher than that of allele S. Moreover, the levels of FPG and HOMA-IR of PP and PS genotype carriers were significantly higher than those of SS genotype carriers in the T2D group (P<0.05). CONCLUSION: These findings indicated that the P213S polymorphism of L-selectin gene may contribute to susceptibility to T2D and insulin resistance in the Chinese population, and P allele appears to be a risk factor for T2D.

Cloning and analysis of rat osteoclast inhibitory lectin gene promoter.

Osteoclast inhibitory lectin (OCIL) is a novel regulator of bone remodeling, however, little is known concerning how OCIL is regulated to date. In this study, approximately 4.4 kb of the 5'-flanking sequence of rat OCIL gene was cloned into the promoter-less reporter vector pGL3-basic and transiently transfected into three different cell lines. The differences in the levels of luciferase activity paralleled well with the levels of OCIL mRNA expression in these cells, suggesting that the regulation of rat OCIL gene expression occurs mainly at the transcriptional level. Additional luciferase assays using a series of constructs containing unidirectionally deleted fragments showed that the construct-1819/pGL3 (-1819 to +118) exhibited the highest luciferase activity, suggesting the presence of functional promoter in this region. The region from -4370 to -2805 might contain negative regulatory elements, while the region from -1819 to -1336 might have important positive regulatory elements that enhance OCIL transcription. Sequence analysis of the promoter revealed the absence of both TATA and CAAT boxes. However, in the proximal promoter region (-81 to +118), several potential transcription factor binding sites that may be responsible for the basal transcriptional activity of rat OCIL promoter were observed. The promoter contains several potential Sp1 binding sites, and cotransfection of a shRNA expression plasmid that knockdowns Sp1 significantly reduced OCIL promoter activity and endogenous gene expression and moreover, overexpressing Sp7, a Sp1 family member that also binds to Sp1 binding sequence, increased OCIL promoter activity and gene expression, suggesting a role of Sp1 family proteins in regulation of OCIL transcription.

Parathyroid hormone-related protein regulates osteoclast inhibitory lectin expression via multiple signaling pathways in osteoblast-like cells.

Osteoclast inhibitory lectin (OCIL) is a recently identified inhibitor of osteoclast formation. A variety of osteotropic factors regulate OCIL expression in osteoblastic cells, however, little information is available to date concerning how this gene is controlled. Using real-time RT-PCR, we examined the regulation of OCIL expression by PTHrp and the signaling pathways used. We demonstrated in rat osteoblast-like UMR-106 cells, rat calvarial primary osteoblastic cells, and murine MC3T3-E1 cells, PTHrp(1-34) increased OCIL expression. In UMR-106 cells, the increase began and reached maximum later than RANKL induction and OPG suppression. cAMP/PKA signaling activators PTH(1-31), forskolin and dibutyryl cAMP (db-cAMP), and calcium ionophore A23187 all increased OCIL levels. In contrast, PKC activator phorbol-12-myristate-13-acetate reduced OCIL expression in short term but induced OCIL mRNA in long term. PKA inhibitor KT5720, mitogen-activated protein kinase (MAPK) cascade inhibitor PD98059, calmodulin antagonist W-7, and Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) inhibitor KN-62 all significantly blunted PTHrp-stimulated OCIL expression. Moreover, PD98059 blocked the stimulation of OCIL by FSK or db-cAMP but not that by A23187. In primarily cultured osteoblasts, the PTHrp induction of OCIL was blocked by KT5720, W-7, and PD98059 as well. The data established that PTHrp(1-34) regulates OCIL expression in vitro through cAMP/PKA, Ca(2+)/CaMK II, and MAPK signaling pathways.

An efficient approach for constructing shRNA expression vectors based on short oligonucleotide synthesis.

Traditional strategies for establishing shRNA expression constructs are inefficient, error-prone, or costly. We describe a simple approach that overcomes these drawbacks. Briefly, the sense and antisense strands of the short hairpin RNA coding sequence are segmented into two parts, respectively, at asymmetric sites. The four resulting short oligonucleotides are synthesized. Each oligonucleotide is annealed with its opposite, resulting in a double-stranded fragment with sticky termini at both ends. The two fragments so generated can be easily spliced by simple ligation to reconstitute the full-length short hairpin RNA coding sequence which can then be cloned into an appropriately restricted vector.

SORS: a universal one-round PCR-based method for site-directed mutagenesis.

We have developed a novel protocol for site-directed mutagenesis of double-stranded DNA. The procedure, termed SORS (named because it undergoes the sequential procedure of segmentation-overhang creating PCR-reannealing-splicing) mutagenesis, is exemplified by a substitution, a deletion, and an insertion of nucleotide(s) in target genes. The template DNA is PCR-amplified into two separate segments divided at the prospective mutation site, and each segment is amplified in two parallel PCRs using primers introducing the mutation. The primers are designed to be able to create protruding bases upon pooling, denaturing, and reannealing the two parallel reactions. The protruding bases at the prospective junction of the two segments are mutually complementary; therefore, the two segments can be re-spliced together to generate the mutated gene. Compared to previously published protocols, this procedure is rapid, restriction-independent and ensures higher success rate and lower potential to produce second-site mutations.

[Effect of nylestriol on bone remodeling in ovariectomized rats].

OBJECTIVE: To clarify the effects of nylestriol on microarchitecture and interleukin-6 (IL-6) mRNA expression in tibial bone in ovariectomized rats. METHODS: 30 female rats were randomly allocated into 3 groups: sham, OVX and nylestriol-treated group. Nylestriol-treated group were ovariectomized, then fed with nylestriol for 3 months and the bone mineral density (BMD) was measured in lumbar vertebra by dual energy x-ray absorptiometry. After sacrifice of the animal, bone histomorphometric parameters were measured to study the changes in bone microarchitecture, and RT-PCR was performed to detect the expression of IL-6 mRNA in bone tissue. RESULTS: BMD was significantly reduced, while IL-6 mRNA level elevated in the OVX group compared with the sham group. Static histomorphometric data showed that the trabecular bone volume, mean trabecular plate thickness and density were reduced while the mean trabecular plate space elevated remarkably in the OVX group in comparison with that in the sham group. As for dynamic parameters, trabecular osteoid surface, tetracyclin labeled surface and bone turnover rate were increased while osteoid maturation rate decreased significantly in the OVX group compared with the sham group. BMD, IL-6 mRNA expression and bone histomorphometric parameters were improved in nylestriol-treated rats. CONCLUSION: Nylestriol plays an important role in maintaining bone volume and improving bone microarchitecture by markedly inhibiting bone turnover and bone resorption, which might be to some degree attributed to reduced IL-6 expression.