Trichostatin A suppresses up-regulation of histone deacetylase 4 and reverses differential expressions of miRNAs in the spinal cord of rats with chronic constrictive injury / 南方医科大学学报

OBJECTIVE@#To explore the analgesic mechanism of intrathecal trichostatin A (TSA) injection in a rat model of neuropathic pain induced by chronic constrictive injury (CCI).@*METHODS@#Male SD rats were randomized into sham operation+ DMSO group (group S), CCI +DMSO group (group C), CCI +10 μg TSA group (group T), and in the latter two groups, rat models of neuropathic pain were established induced by CCI. The rats were given intrathecal injections of 10 μL 5% DMSO or 10 μg TSA (in 5% DMSO) once a day on days 7 to 9 after CCI or sham operation. The rats were euthanized after behavioral tests on day 10, and the lumbar segment of the spinal cord was sampled to determine the expression of histone deacetylase 4 (HDAC4) protein and mRNA and detect the differentially expressed miRNAs using a miRNA chip. MiR-190b-5p and miR-142-3p were selected for validation of the results using RT-qPCR.@*RESULTS@#Compared with those in group S, the rats in group C showed significantly decreased paw withdrawal mechanical threshold (PWMT) from day 3 to day 10 after CCI ( < 0.05); intrathecal injection of TSA significantly reversed the reduction of PWMT following CCI ( < 0.05). Positive HDAC4 expression was detected mainly in the cytoplasm of the neurons in the gray matter of the spinal cord, and was obviously up-regulated after CCI ( < 0.05). Intrathecal injection of TSA significantly suppressed CCI-induced up-regulation of HDAC4 at 10 days after the operation ( < 0.05). Compared with the miRNA profile in group S, miRNA profiling identified 83 differentially expressed miRNAs in group C (fold change ≥2 or ≤0.5, < 0.05); TSA treatment reversed the expressions of 58 of the differentially expressed miRNAs following CCI, including 41 miRNAs that were decreased after CCI but up-regulated following TSA treatment. The results of real-time PCR validated the changes in the expressions of miR-190b-5p and miR-142-3p.@*CONCLUSIONS@#TSA suppresses CCI-induced up-regulation of HDAC4 and reverses differential expressions of miRNAs in the spinal cord of rats, which may contribute to the analgesic effect of TSA on neuropathic pain.

Expression of miR-140 in human gastric cancer and its effect on function of SGC-7901 cells / 中国病理生理杂志

AIM:To explore the expression level of microRNA-140 ( miR-140 ) in human gastric cancer and normal gastric tissues, and the regulatory effect of miR-140 expression on the function of SGC-7901 cells.METHODS:The expression levels of miR-140 in human gastric cancer and normal gastric tissues were detected by real-time PCR.miR-140 mimics ( miR-140 up-regulated expression) and miR-140 inhibitors ( miR-140 down-regulated expression) were trans-fected into human gastric cancer SGC-7901 cells by liposome method.At the same time, the untransfected control group ( control group) and miRNA nonsense sequence transfection group ( NC group) were set up .The expression of miR-140 in the cells after transfection was detected by real-time PCR.The cell viability and growth inhibition rate with DDP were meas-ured by MTT assay.The cell cycle and apoptotic rate of SGC-7901 cells were analyzed by flow cytometry.The invasion a-bility of SGC-7901 cells was measured by Transwell assay.The protein expression of histone deacetylase 4(HDAC4) in the cells was determined by Western blot.RESULTS:The expression level of miR-140 in human gastric cancer tissues was significantly lower than that in normal gastric tissues (P<0.05).Compared with control group and NC group, the viability and invasion ability of the SGC-7901 cells were decreased, the cell cycle was arrested, the cell growth inhibition rate and apoptotic rate with DDP treatment were increased, and the protein expression of HDAC4 was down-regulated ( P<0.05) in miR-140 mimics group.However, in miR-140 inhibitors group, the viability and invasion ability of the SGC-7901 cells were increased, the cell cycle was promoted, the cell growth inhibition rate and apoptotic rate with DDP treatment were de-creased, and the protein expression of HDAC4 was up-regulated ( P<0.05 ) .CONCLUSION:The expression level of miR-140 in the gastric cancer tissues is low.miR-140 serves as a tumor suppressor to regulate the viability, apoptosis and invasion ability of gastric cancer cells, and to play a role by down-regulating HDAC4 protein.miR-140 may serve as a new target for diagnosis and treatment of gastric cancer.

Effect of microRNA-548ah targeting histone deacetylase-4 on the replication and expression of hepatitis B vi rus / 中华传染病杂志

Objective To investigate the effect of microRNA (miRNA )‐548ah targeting histone deacetylase‐4 (HDAC4) on the replication and expression of hepatitis B virus (HBV) .Methods HepG2 , 2 ,15 cells were transfected by mimics and inhibitors . The expressions of miRNA‐548ah and HDAC4 before and after transfection were detected by fluorescent quantitative polymerase chain reaction (PCR) . The expression of HDAC4 protein in HepG2 ,2 ,15 cells was detected by Western blotting .The target gene of miRNA‐548ah was analyzed by bioinformatics methods .3′UTR dual‐luciferase expression vector containing candidate HDAC4 target genes was built to test the luciferase activity .The levels of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) in the supernatant of cultured HepG2 ,2 ,15 cells were detected by enzyme‐linked immunosorbent assay (ELISA) .The level of HBV DNA in the supernatant of HepG2 ,2 ,15 cells was detected by fluorescent quantitative PCR .The t‐test was used for comparison between two groups ,SNK‐q tests were used for multiple groups comparisons .Results The expressions of miRNA‐548ah in HepG2 ,2 ,15 and HepG2 cells were 5 .74 ± 0 .02 and 2 .96 ± 0 .40 , respectively (t= 11 .89 ,P< 0 .01) ,and the expressions of HDAC4 mRNA were 9 .38 ± 0 .39 and 18 .13 ±0 .34 ,respectively (t = 29 .39 , P < 0 .01) . The expression of miRNA‐548ah in HepG2 ,2 ,15 cells was inhibited by transfection of miRNA‐548ah inhibitors (1 .01 ± 0 .13 ,t= 15 .48 , P< 0 .01) .Compared with control group ,the levels of HBsAg ([6 .45 ± 0 .46 ] IU/mL vs [2 .60 ± 0 .20 ] IU/mL , t = 7 .48 , P <0 .01) ,HBeAg ([5 .49 ± 0 .27] NCU/mL vs [4 .15 ± 0 .34 ] NCU/mL , t = 3 .10 , P < 0 .05 ) and HBV DNA ([3 .93 ± 0 .06] lg copy/mL vs[2 .04 ± 0 .07] lg copy/mL ,t = 18 .89 , P< 0 .01) in the supernatant of cultured HepG2 ,2 ,15 cells significantly decreased in inhibitor group . The expression of HDAC4 in HepG2 ,2 ,15 cells significantly decreased after transfection of miRNA‐548ah mimics (2 .98 ± 0 .94) ,but significantly increased after transfection of miRNA‐548ah inhibitors (23 .77 ± 6 .74 ) , with statistical significance (F= 9 .34 , P< 0 .01) .The expression of HDAC4 protein was also significantly inhibited after transfection of miRNA‐548ah mimics (0 .53 ± 0 .14 vs 0 .23 ± 0 .02 , t = 3 .58 , P = 0 .02) .The activity of luciferase was significantly inhibited by transfection of miRNA‐548ah mimics (7 .62 ± 0 .45 vs 6 .65 ±0 .27 ,t = 3 .18 , P = 0 .03 ) .Conclusion miRNA‐548ah may promote the replication and expression of HBV through the regulation of target gene HDAC4 .