Effect of gap junctions on anti-proliferation effect of miR-124 and its mechanism / 中国药理学通报

was observed by patch clamp. Results Cx32 or Cx26 expression and GJ function were induced by doxycycline (Dox, the promotor for PBI plasmid) in transfected Hela cells. MiR-124 reduced the proliferation of Hela cells, dox incubation alone did not affect Hela cell growth, and also had no effect on anti-tumor effect of miR-124 when combined with miR-124 transfection. Compare with U 87shRNA-Cx43 , the Cx43 expression and GJ function significantly decreased in U87shRNA-Cx43. Similar to the effect on Hela cells, MiR-124 also reduced U87 cell growth. Reducing Cx43 expression did not influence U87 cell proliferation, but attenuated the growth-inhibition effect of miR-124 when combined with miR-124 transfection. Under the microscope, the transfer of fluorescence-labelled miR-124 from "donor" cell to adjacent " non-injection " cell was observed. Conclusions The role of GJ on anti-tumor effect of miR-124 possesses connexin heterogeneity. Compare with Cx26 or Cx32, GJ composed of Cx43 has more obvious effect, which may be related to the maximum permeability of junction channel to miR-124.

Construction of lentiviral vector-mediated Cx32 stably over-expressed Huh7 cell line and its effect on cell proliferation / 中国药理学通报

Aim To construct a lentiviral vector for stable delivery of the connexin32 (Cx32) gene in human hepatocellular carcinoma cell line Huh7,and also to detect its effect on cell proliferation.Methods Human Cx32 gene sequence was obtained by whole gene synthesis and amplified by PCR,and was then inserted into LV5-GFP lentiviral vector to construct the recombinant plasmid LV5-GFP-hCx32.Following identified by restriction endonuclease digestion and DNA sequencing,this plasmid together with lentiviral package plasmid was transfected into 293T cells to produce the lentiviral particles,and the viral titer was assessed under fluorescent microscope.Targeted Huh7 cells were infected with the lentivirus (LV5-hCx32 and LV5-NC),and the infected cells after selection with puromycin were amplified and cultured.The expression and localization of Cx32 were detected by real-time RT-PCR,Western blot and immunofluorescence assay,respectively.The gap junction (GJ) function between adjacent cells was measured by dye transfer assay.The Huh7 cell proliferation capacity was determined by MTT and colony formation assays.Results The results of double enzyme digestion and DNA sequencing proved that the recombinant lentiviral vector LV5-GFP-hCx32 was successfully constructed.After packing in 293T cells,the recombinant lentivirus LV5-GFP-hCx32 with virus drops to 3 × 1011 TU · L-1 was obtained.Huh7 cells transiently infected with the lentivirus LV5-GFP-hCx32 remarkably over-expressed Cx32 at both mRNA and protein levels.Moreover,Cx32 expression was also significantly up-regulated in stably transfected Huh7 cells,and the presence of enhanced functional GJ in intact cells could be detected due to an increased amount of Cx32 protein along the plasma membrane at cell-cell contacts.Compared to LV5-NC group,the proliferation ability in Huh7 cells with recombinant Cx32 declined (P ＜ 0.05).Conclusions The lentiviral vector over-expressing Cx32 gene is successfully constructed,and can stably transfect Huh7 cells to yield sustained over-expression of exogenous Cx32 gene,thus eventually inhibits cell proliferation.

X-linked Charcot-Marie-Tooth Disease Type 1 Harboring Unusual Electrophysiological Features

Charcot-Marie-Tooth X type 1 (CMTX1) is caused by mutations in the connexin 32 gene (Cx32) on the X chromosome. Electrophysiologically, CMTX1 is usually associated with intermediate slowing of conduction velocities and severe impairments in male patients. In addition, patients with CMTX1 rarely present conduction block and temporal dispersion, which are characteristic findings in acquired demyelinating neuropathy. We report herein, for the first time in Korea, two patients with Cx32 mutations who exhibited unusual electrophysiological findings.

Expression of Cx43 and CX32 in the epileptogenic lesions of patients with refractory temporal epilepsy / 中国综合临床

Objective In order to investigate the role of Gap Junction (GJ) in the generation and development of epilepsy,we examined the expression of connexin43 (Cx43)and Cx32 in the epileptogenic lesions and surrounding brain tissues of the patients with refractory temporal epilepsy.Methods Thirty intractable epilepsy patients were performed the resection of epileptogenic lesions under the ECC monitor.The expression of Cx43 and Cx32 in the epileptogenic lesions and the surrounding brain tissues of the patients were examined by immunohistochemical staining(the two step method).The data were statistically analyzed.Results The results from the immunohistochemistry staining showed that Cx43 and Cx32 were expressed in the epileptogenic lesions and the surrounding brain tissues.The expression of Cx43 in the epileptogenic lesions was increased obviously in comparison with the surrounding brain tissues ( U =4.066,P ＜ 0.001 ).There was no significant difference in the expression of Cx32 between the epileptogenic lesions and the surrounding brain tissues ( U =1.866,P ＞ 0.05 )Conclusion The expression of Cx43 in the epileptogenic focus is increased in comparison with that in the surrounding brain tissues,indicating that GJ plays an important role in the generation and development of epilepsy.There is no significant difference in the expression of Cx32 between the epileptogenic lesions and the surrounding brain tissues,which may be related to the apoptosis of the neurons after multiple seizure attacks.

Expressions of Connexin32 and Connexin43 in Hippocampus and Effect of Carbenoxolone on Their Expression in Epileptic Immature Rats Induced by Lithium-Pilocarpine / 实用儿科临床杂志

Objective To observe the behavior changes and connexin 32(CX32),connexin 43(CX43)expressions in hippocampus and the effect of carbenoxolone on their expression in epileptic immature rats induced by lithium-pilocarpine.Methods Seventy-two SD immature rats of 21 d were randomly divided into control group(n=24),lithium-pilocarpine kindled group(n=24)and carbenoxolone treated group(n=24),each group by 24 h,3 d,7 d and 30 d were subdivided into 4 groups(n=6).Immuno-histochemisty was used to observe the expressions of CX32 and CX43 in hippocampus areas of immature rats,and to observe their behavior changes.Results The scores of the severe elileptiform seizures(Racine Ⅳ/Ⅴlevel)in lithium-pilocarpine group were significantly higher than those in carbenoxolone treated group;The latency in carbenoxolone treated group was prolonged significantly(P

X-linked Charcot-Marie-Tooth Patient with a Novel Cys168Arg Missense Mutation in the Connexin32 Gene

X-linked Charcot-Marie-Tooth (CMTX) disease is a clinically heterogeneous hereditary motor and sensory neuropathy. The X-linked inheritance showed an absence of male-to-male transmission and a more severe disease phenotype in affected males compared to that in affected female. A missense mutation, Cys168Arg, was found in connexin 32 gene (Cx32/GJB1) from a patient with CMTX neuropathy. The familial history of this patient also suggested that the disease is X-linked CMT. Thus, we report a CMTX family having the novel Cys168Arg mutation in the Cx32 gene.

Mutation of Cx32 gene,clinical and electrophysiological features in patients with Charcot-Marie-Tooth disease / 临床神经病学杂志

Objective To investigate the mutation of Cx32 gene,clinical and electrophysiological features of Chinese patients with Charcot-Marie-Tooth(CMT) disease.Methods 24 CMT probands were selected for Cx32 mutation screening after the exclusion of the CMT1A 1.5 Mb duplication and male-to-male transmission.The motor and sensory nerve conduction studies were performed in all probands and most of their affected family members to establish the clinical CMT1,CMT2 or CMT intermediate diagnosis.The presence of mutations in the coding region of Cx32 was detected by single-strand conformation polymorphism analysis combined with direct sequencing.Results It was found 7 different point mutations in the coding region of Cx32 in 1 sporadic CMT1 patient and 6 X-linked inherited families,including 4 families with CMT1 diagnosis and 2 families with CMT intermediate diagnosis.There were 20 male CMTX patients,6 female CMTX patients and 12 asymptomatic female carriers among 38 family members bearing Cx32 mutation.All of the 26 patients were mildly to moderately affected clinically.Conclusions Seven different Cx32 point mutations were detected and the percentage of Chinese CMT families with Cx32 mutation is about 10% in our study.The inheritance model of CMT secondary to Cx32 mutation could be X-linked dominant,X-linked recessive or sporadic.Male patients are usually more severly affected than females with slower nerve conduction velocities.Cx32 mutation screening should be firstly performed in those CMT families without male-to-male transmission and CMT1A duplication.

Expression of CDK 4, Connexin 32, and PCNA in N-diethylnitrosamine-Induced Lesions of Rat Liver

Cellular altered foci (CAF), hyperplastic nodules (HN) including regenerating and adenomatous nodules, and hepatocellular carcinomas (HCC) were induced in Sprague-Dawley rat liver by prolonged administration of N-diethylnitrosamine (DEN, 200 ppm). Immunohistochemical expression of connexin 32 (Cx 32), cyclin-dependent kinase 4 (CDK 4), and proliferating cell nuclear antigen (PCNA) was assessed for the evaluation of preneoplastic potential of CAF. Regardless the duration of DEN administration, basophilic cell foci were the most frequently observed lesion in both CAF and cellular expanding hyperplastic nodules. Eosinophilic cell foci, however, were concomitantly increased with adenomatous nodules in later experimental groups. Cx 32 showed perimembranous spot-like expression. Its number was 7.25 2.10 per cell in normal hepatocytes. CAF and adenomatous nodules showed markedly decreased Cx 32 spots. Moreover, its reduction was more prominent in HCC. PCNA-labelled hepatocytes were scattered in the most CAF, showing no significant difference between each CAF. PCNA labelling index (LI) in adenomatous nodule and HCC was markedly increased. CDK 4 was localized in the cytoplasm and/or nucleus of hepatocytes. Eosinophilic cell foci revealed more nuclear expression of CDK 4 than other types of CAF, of which expression incidence was comparable to that of adenomatous nodule. Nuclear CDK 4 expression in HN and HCC was increased, although significant difference between regenerating nodule and adenomatous nodule was not seen. In conclusion, the incidence of CDK 4 was concomitantly increased with PCNA LI, however, reciprocally decreased with Cx 32 in accordance with the advance of DEN-induced HCC in rat. Phenotypically altered foci manifested as CAF are early valuable preneoplastic marker lesion for evaluation. In addition, basophilic cell foci can be considered a discernible marker of cellular expansion within nodules. However, eosinophilic cell foci might be an indeterminate marker for the advance of DEN-induced HCC in rat.