Involvement of aberrant miR-139/Jun feedback loop in human gastric cancer.

Accumulating evidence indicates that some miRNAs could form feedback loops with their targets to fine-tune tissue homeostasis, while disruption of these loops constitutes an essential step towards human tumorigenesis. In this study, we report the identification of a novel negative feedback loop formed between miR-139 and its oncogenic target Jun. In this loop, miR-139 could inhibit Jun expression by targeting a conserved site on its 3'-UTR, whereas Jun could induce miR-139 expression in a dose dependent manner through a distant upstream regulatory element. Interestingly, aberration in this loop was found in human gastric cancer, where miR-139 was down-regulated and inversely correlated with Jun expression. Further functional analysis showed that restored expression of miR-139 in gastric cancer cells significantly induces apoptosis, and inhibits cell migration and proliferation as well as tumour growth through targeting Jun. Thus, our data strongly suggests a role of aberrant miR-139/Jun negative feedback loop in the development of human gastric cancer and miR-139 as a potential therapeutic target for gastric cancer. Given that miR-139 and Jun are deregulated in many cancers, our findings here might have broader implication in other types of human cancers.

[Change of cardiac myocyte nuclear inositol 1,3,4,5-tetrakisphosphate receptor binding properties in rat with myocardial ischemic reperfusion].

OBJECTIVE: To observe the alteration of cardiac myocyte nuclear inositol 1,3,4,5-tetrakisphosphate receptor (IP(4)R) binding properties in rat subjected to myocardial ischemic reperfusion in order to further make it clear whether this change is involved in the molecule mechanism of cell apoptosis of rat with myocardial ischemic reperfusion. METHODS: Extracting of cardiac myocyte nucleus was accomplished by saccharose density gradient centrifugation method, the binding properties of nuclear IP(4)R in different conditions were detected by radioligand binding assay. Apoptosis index of myocardial cell was determined by using TUNEL assay. RESULTS: (1) Myocardial cell apoptosis index in rat heart underwent 30 min regional ischemia and 3 h reperfusion increased distinctly compared with that in control group (P < 0.01). (2) There were two IP(4) binding sites located to the nuclear envelope. (3) In ischemic reperfusion injury (IRI) group, Bmax from high affinity binding site of nuclear IP(4)R significantly increased compared with that in sham-operated group, whereas Bmax from low affinity binding site didn't change. Kd values of both sites were all significantly decreased by 63% and 55%, respectively. (4) Phosphorylation of nuclear IP(4)R by PKC increased markedly its binding ability both in IRI and control group (P < 0.05), which was more apparent in IRI group. (5) In sham-operated group, the binding ability of nuclear IP(4)R increased with increasing free calcium concentrations in cytoplasm, and the binding properties of IP(4)R in IRI group were also increased in the condition of calcium overloading. CONCLUSION: The increasing of binding properties of nuclear IP(4)R from ischemic reperfusion heart may be one of important mechanism involved in myocardial cell apoptosis, furthermore resulting in myocardial IRI.

[Characteristics in the feature of 3H-Ryanodine binding to cardiomyocyte nuclei in reperfusion injury in rat].

OBJECTIVE: To investigate the changes in binding features of (3)H -Ryanodine cardiac myocytic nuclei in reperfusion injury in the rat, and the effects of phosphorylation regulation on the binding characteristics. METHODS: Healthy male Wistar rats were randomly divided into ischemia/reperfusion injury (IRI) group and sham-operation group. IRI model was reproduced by ligating the left main coronary artery for 30 minutes followed by reperfusion of the heart for 3 hours, while in the sham-operation group the animals underwent a thoracotomy only for 3.5 hours. Cardiac myocytic nuclei were isolated by sucrose density gradient centrifugation. The maximum binding capacity (Bmax) and the dissociating ratio (Kd) of Ryanodine receptors (RyRs) were measured by radioligand binding analysis as well as Scatchard plot. RESULTS: There was a high affinity of RyRs to bind with (3)H-Ryanodine on the nuclear wall of rat cardiac myocytic nucleus. Compared with that of the sham-operation group, Bmax of RyRs of IRI cardiac myocytic nuclei was decreased by 29% (P<0.01), but there was no difference in Kd between two groups(P>0.05). With phosphorylation by activating the endogenous protein kinase C(PKC) with phorbol myristate acetate (PMA)+phosphatidyl serine(PS), Bmax of both sham and IRI groups were increased markedly (P<0.01), but in the latter group it was less increased (P<0.01). With phosphorylation by Ca(2+)-calmodulin (CaM), Bmax was decreased in both sham-operation and IRI groups (both P<0.05), but was less decreased in the latter(P<0.01). However, both PMA+PS and Ca(2+)-CaM did not change the Kd of nuclear RyR in either group (both P>0.05). CONCLUSION: After IRI, Bmax of (3)H -Ryanodine of cardiac myocytic nuclei is decreased and the impact of PMA+PS and Ca(2+)-CaM on the Bmax is impaired, but the affinity of (3)H -Ryanodine to cardiac myocytic nuclei is not altered under above circumstances.

Alteration of binding sites for [3H]P1075 and [3H]glibenclamide in renovascular hypertensive rat aorta.

AIM: The alterations of the binding sites for ATP-sensitive K+ channel (K(ATP)) openers and blockers in aortic strips were investigated in hypertensive rats. METHODS: Radioligand binding techniques were used to compare the specific binding properties of [3H]P1075 and [3H]glibenclamide (Gli) in normotensive (NWR) and reno-vascular hypertensive rat (RVHR) aortic strips. RESULTS: The KD values of [3H]P1075 binding were increased by 1.5-fold, while the Bmax values were unchanged in RVHR. The IC50 values of P1075 and pinacidil (Pin) for displacing the [3H]P1075 binding in RVHR were increased by 1.8- and 1.7-fold, respectively. The kinetic processes of association and dissociation of [3H]P1075 binding were slower in RVHR. Glibenclamide pretreatment slowed down the kinetic processes of the association and dissociation of [3H]P1075 binding in NWR, but failed to alter the kinetic processes of [3H]P1075 binding in RVHR. The IC50 values of Gli for displacing the [3H]Gli binding at high-affinity sites were increased by 3-fold, while those at low-affinity sites remained to be unchanged in RVHR. The kinetic processes of association of [3H]Gli binding were decreased and those of the dissociation were accelerated in RVHR. The treatment with Pin slowed down the association kinetic processes but accelerated the process of the dissociation of [3H]Gli binding in NWR, but did not alter the kinetics of [3H]Gli binding in RVHR. CONCLUSION: The affinity of binding sites for [3H]P1075 and of high-affinity binding sites for [3H]Gli are decreased, and the negative allosteric interactions between the two binding sites are impaired in RVHR aorta.

Replication and gene expression of mutant hepatitis B virus in a transgenic mouse containing the complete viral genome with mutant s gene.

AIM: To establish the transgenic mouse line harbouring complete hepatitis B virus (HBV) genome with mutant s gene (adr subtype). METHODS: Transgenic mice were generated by microinjecting HBV genome into fertilized eggs. Integration, expression, replication of HBV gene and histological changes in transgenic mice were estimated by genomic DNA PCR, serum DNA PCR, Southern blot, ELISA, HE staining, immunohistochemistry and transmission electron microscopy. Transgenic mice with HBsAg positive in serum were bred and analyzed. RESULTS: A total of 288 eggs survived from microinjections were transplanted into the oviducts of 13 pseudopregnant mice and 49 pups were produced. Twenty-six mice were identified to have the integrated HBV gene. Serum HBsAg and HBeAg were detected in 2 of 43 mice. HBsAg and HBcAg in cytoplasm or nuclei of hepatocytes were detected in 10 mice. Founders with HBsAg in serum were named lineages G145R-15 and G145R-18. Of the 16 F1 offsprings generated by G145R-15 founder, 12 were positive for HBV genome with PCR, 10 were positive for HBsAg and HBcAg with immunohistochemistry and 7 were positive for HBsAg and HBeAg with ELISA. Only 1 of 8 F1 offsprings generated by G145R-18 founder was survived and it was detected positive for HBV genome, HBsAg, HBcAg and HBeAg. Both of the two lineages had some pathological characteristics of mild chronic hepatitis B in the liver, such as swelling of hepatocytes and focal hepatocellular necrosis and parenchymal lymphomononuclear cell infiltrate. CONCLUSION: Transgenic mice harbouring HBV with mutant s gene can be generated. The HBV genes are integrated in the transgenic mice genome and can be expressed, replicated, packaged and excreted. HBV DNA can be stably transmitted in the transgenic mice.

Antigenic and immunogenic changes due to mutation of s gene of HBV.

AIM: To investigate the change of immunological characteristics of HBsAg caused by the mutation at codon 145 of HBsAg using DNA-based immunization. METHODS: Plasmids expressing mutant and wild type envelope antigens were transfected into human hepatocellular carcinoma cells via electrotransformation. The antigenicity of HBsAg was studied with EIA and immunocytochemical staining. Then plasmids were used to immunize 5 C57BL/6 mice. Sera of mice were detected for anti-HBs and anti-preS2 with ELISA. RESULTS: The mutant HBsAg could be detected by native antibody in EIA and immunocytochemical study. But the A((450 nm)) value of the mutant HBsAg in the supernatant was apparently lower than that of the wild-type. Both mutant and native HBsAg expression plasmid could stimulate a strong humoral immune response to HBsAg and preS2 antigen in mice. Protective antibodies against HBsAg elicited by the native HBsAg occurred earlier than that elicited by the mutant HBsAg about one to two weeks. The occurrence of protective antibodies against preS2 antigen was one to two weeks earlier than that of anti-HBs. CONCLUSION: The amino acid substitution causes changes of the antigenicity and immunogenicity of HBsAg, but mutant HBsAg can still induce a protective humoral immune response in mice.

[Induction of specific humoral immune response in mice by recombinant mutant HBV s gene vaccine].

AIM: To construct the recombinant eukaryotic expression vector pCMV-S2. S + 145R(PR) containing mutant HBV s gene and detect the specific humoral immune response to the PR in mice. METHODS: The PR was constructed by positional cloning of restriction endonuclease, and then it was transfected into human hepatocellular carcinoma cell line Hep G2 through electrotransformation. The antigenicity of PR was examined by EIA, ELISA and immunocytochemical staining. The PR and empty vector pcDNA3.0 were then used respectively to immunize intramuscularly 5 C57BL/6 mice, dosage being 100 pg purified plasmid each mouse. The titers of serum anti-HBs and anti-HBs2 antibodies were detected by ELISA. RESULTS: In vitro experiment showed that mutant HBsAg could bind to anti-HBs antibody. The PR could induce anti-HBs and anti-HBs2 antibody production in immunized mice. But the appearance time of serum anti-HBs2 antibody one or two weeks earlier than that of serum anti-HBs antibody. CONCLUSION: The expression product of PR had a good antigenicity, which can induce specific hu-moral immune response in C57BL/6 mice.