Effect of miR-153-targeted PRDM2 on invasion and migration abilities of bladder cancer cells by JAK/STAT signaling pathway / 中国病理生理杂志

AIM:To investigate the effect of microRNA(miR)-153-targeted positive regulatory domain zinc finger protein 2(PRDM2 )on invasion and migration abilities of bladder cancer cells through JAK /STAT signaling path-way.METHODS:The expression levels of miR-153 in bladder cancer tissues were detected by qPCR.The expression of PRDM2 in normal tissues and bladder cancer tissues was detected by immunohistochemistry.The effect of miR-153 on the transcriptional activity of PRDM2 was examined by dual-luciferase reporter assay system.The effect of miR-153 over-ex-pression on the invasion ability of bladder cancer RT 4 cells was detected by Transwell assay ,and the cell invasion ability was analyzed by scratch test.The protein levels of PRDM2,p-JAK2 and p-STAT3 were determined by Western blot.RE-SULTS:The expression of miR-153 was significantly lower in the bladder cancer tissues than that in normal tissues(P<0.05).PRDM2 was highly expressed in the bladder cancer tissue(P<0.05).The results of dual-luciferase reporter assay system showed that miR-153 regulated the expression of PRDM2.miR-153 over-expression significantly decreased the invasion and migration abilities of bladder cancer RT 4 cells.miR-153 over-expression also down-regulated the protein levels of p-JAK2 and p-STAT3(P<0.05).CONCLUSION:miR-153 targets PRDM2,and regulates the invasion and migra-tion abilities of bladder cancer cells by JAK/STAT signaling pathway.

Expression of miR-133b in prostate cancer and its effect on the proliferation of tumor cells / 临床检验杂志

Objective To investigate the expression level of miR-133b in cancer tissues of patients with prostate cancer and its effect on the proliferation of prostate cancer cells.Methods The total RNAs in resected prostate cancer tissues and adjacent tissues from 30 patients with prostate cancer were extracted and reversely transcripted into cDNA,and then the expression levels of miR-133b were detected by real-time quantitative PCR.The correlations between the expression levels of miR-133b and the patients' clinicopathological features were analyzed.The expression of positive regulatory domain I-binding factor 16 (PRDM16) and proliferation of PC-3 cells transfected with miR-133b mimics by LipofectamineTM 3000 were determined by real-time quantitative PCR and the CCK8 method,respectively.Results The expression levels of miR-133b in prostate cancer tissues [(16.85 ± 0.94) × 10-4] was significantly lower than that in adjacent tissues [(22.95 ± 1.567) × 10-4,t =3.335,P ＜ 0.01].The expression levels of PRDM16 in PC-3 cells transfected with miR-133b mimics were significantly lower than that in the control group (0.371 ±0.031 vs 1.000 ±0.022,t =12.53,P ＜ 0.01).The proliferation ability of PC3 cells transfected with miR-133b mimics for 72 hours was significantly lower than that in the control group (t =6.811,P ＜ 0.01).Similarly,the proliferation ability of PC-3 cells transfected with PRDM16 inhibitor for 72 hours was also significantly lower than that in the control group (t =9.048,P ＜0.01).Conclusion The expression levels of miR-133b in prostate cancer tissues are significantly down-regulated,which regulate the proliferation of prostate cancer cells possibly through PRDM16.

Switch of Regulatory Domains of P-protein and T-protein from E. coli / 中国生物化学与分子生物学报

Chorismic acid is a mid-metabolite that plays a central role in the metablism process distributing in the bacterium, epiphyte and plants. It is a common precursor substance of the all aromatic amino acids that can turn into phenylalanine and tyrosine catalyzed by bi-functional enzyme chorismate mutase (CM)-prephenate dehydratase (PDT) and chorismate mutase-prephenate dehydrogenase (PDH) respectively. CMp-PDT with its regulate domain Rp were called P-protein and CMt-PDH with its regulate domain Rt were called T-protein. P-protein and T-protein from E. coli. have a similar structure, both of which contained three domains: CMp, PDT, Rp in P-protein and CMt, PDH, Rt in T-protein. P-protein and T-protein are regulated by their effectors phenylalanine and tyrosine respectively through binding to their Rp and Rt domains. Rp and Rt domains were switched between P-protein and T-protein by cloning of chimeric proteins. The results showed that regulatory effects were switched along the switch of R domains and the switch of the regulatory domains lead to the switch of effectors. It means that the combination of the regulatory domain and the effector is specific and the regulating of the regulatory domain to the enzyme activity is non-specific. This property of R domains may make them possible molecular elements in the study of molecular machines.

Effects of Deletions in the Regulatory Domain on the Stability and Enzymatic Characteristics of Tyrosine Hydroxylase

BACKGROUND: Various vectors have been developed and tried for the delivery of tyrosine hydroxylase (TH) in order to supplement dopamine, which is severely deficient in Parkinson's disease, however, none of the protocols tried have yielded fruitful results that can be applied directly to humans. One of the problems revealed from previous trials was a short duration of expression of the delivered gene, that is, tyrosine hydroxylase. METHODS: To extend the stability and to improve the enzymatic characteristics of the protein, part of the regulatory domain was deleted via PCR technique. The cDNA for regulatory domain-deleted THs (dTH) were sub-cloned into a retroviral vector and the resulting recom-binant retrovirus was used to infect NIH-3T3. After selection, expression levels of TH were determined by Western blot analysis and the enzymatic characteristics were examined. RESULTS: The deletion increased steady state expression level of TH protein by 7-fold for d19TH (TH with amino acids #2-19 are deleted) and 3-fold for d31TH (TH with amino acids #2-31 are deleted. The elevated expression level of d19TH is likely due to the enhanced stability of the protein as determined by a treatment of cycloheximide. The activity of d19TH was also increased approximately by 3-fold but no increase of the L-dopa production was observed. However, the production of L-dopa was dramatically increased when GTP cyclohydrolase I (GTPCH I) was co-transfected suggesting that the activity of d19TH is dependent on the presence of cofactor. d19TH seem to be free of feedback inhibition at low concentration of dopamine (10 nM~1 nM) but more sensitive to the inhibition at high concentration of dopamine (10 mM). CONCLUSIONS: The deletion of 18 amino acids on the regulatory domain increases the stability of the protein, reduces the activity, and frees it from the feedback inhibi-tion by the end product.

Effects of Cofactor Tetrahydrobiopterin and Deletions on the Regulatory Domain of Tyrosine Hydroxylase on the Expression of Tyrosine Hydroxylase

BACKGROUND: For the treatment of Parkinson's disease, dopamine-producing cells or genes involved in producing dopamine or supporting neurons have been tested to replace conventional chemical therapies. Of these, tyrosine hydroxylase (TH) was the most widely used gene for the therapy. Trials using TH via various vectors yielded behavioral improvements in animal models but the effectiveness did not last long enough. As one of the approaches for solving this problem, the regulation of expression of the protein and mRNA of TH was studied. METHODS: Two approaches for a higher and/or more stable expression of TH were pursued. First, the effect of cofactor tetrahydrobiopterin (BH4) on the expression level of TH and second, the effect of deletion which enables TH protein to escape from protease attack, were examined. RESULTS: Cells producing BH4 showed an approximately 10-fold higher TH expression than cells expressing TH alone. When the in vitro modified TH was expressed in NIH-3T3, mutant THs showed elevated protein (17.5 ~68.6 fold) and mRNA (1.8 ~4.6 fold) expression levels at a steady state. CONCLUSIONS: Results suggest that an addition of BH4 has a more positive effect on mRNA expression levels than protein. However, the deletions seem to have a tremen-dous effect on the translation and/or protein stability, but a small effect on the mRNA level. (J Korean Neurol Assoc 19(5):514~519, 2001)