[Effects of CAAP1 on Proliferation, Migration and Invasion of Hepatoma Cell Line SMMC-7721].

OBJECTIVE: To investigate the effect of caspase activity and apoptosis inhibitor 1 (CAAP1) on the proliferation, migration and invasion of hepatoma cell SMMC-7721. METHODS: pcDNA3/ CAAP1, the overexpression vector of CAAP1, and pSilencer 2.1-U6 neo/shR- CAAP1, the knockdown vector, were constructed and examined. The experiment included 4 groups of SMMC-7721 cells, pcDNA3/ CAAP1 group, pcDNA3 control group, shR- CAAP1 group and pSilencer control group. After the SMMC-7721 cells were cultured, the overexpression vector pcDNA3/ CAAP1 (the pcDNA3/ CAAP1 group), knockdown vector shR- CAAP1 (the shR- CAAP1 group) and their controls (pcDNA3 control group and pSilencer control group) were transfected into SMMC-7721 cells respectively, and the follow-up experiments were carried out 48 h later. The mRNA expression of CAAP1 in each group was examined with qRT-PCR. The protein expression level of CAAP1 and cleaved Caspase-3 were checked with Western blot. The proliferation of cells was examined with CCK-8. The colony formation ability and the motility of cells in each group were assessed with colony formation assay and wound-healing assay, respectively. The migration and invasion of cells were examined with Transwell cell chamber and the apoptosis of cells was examined with flow cytometry. The data of 75 patients with low expression of CAAP1 and 295 patients with high expression of CAAP1 were downloaded from TCGA database and the data of 48 months follow-up were analyzed. Kaplan-Meier survival curve was used to compare the correlation between different levels of CAAP1 expression and overall survival (OS) of hepatocellular carcinoma (HCC) patients. RESULTS: Double enzyme digestion analysis showed that the overexpression vector pcDNA3/ CAAP1 and knockdown vector shR- CAAP1 were constructed successfully. qRT-PCR and Western blot results showed that pcDNA3/ CAAP1 increased the mRNA and protein expression level of CAAP1 in SMMC-7721 cells (in comparison with the pcDNA3 control group, P<0.05), while shR- CAAP1 decreased the mRNA and protein expression of CAAP1 (in comparison with the pSilencer control group, P<0.05). Compared with pcDNA3 control group, the proliferation, colony formation ability, motility, migration and invasion of SMMC-7721 cells in the pcDNA3/ CAAP1 group were increased, while the apoptosis of SMMC-7721 cells was inhibited (all P<0.05). Compared with the pSilencer control group, the proliferation, colony formation ability, motility, migration and invasion ability of SMMC-7721 cells in the shR- CAAP1 group decreased, while the apoptosis increased (all P<0.05). TCGA database analysis showed that HCC patients with low CAAP1 expression had better OS than that of HCC patients with high CAAP1 expression. CONCLUSION: CAAP1 can promote the proliferation, migration and invasion of SMMC-7721 cells while it inhibit their apoptosis.

MicroRNA-185 reduces the expression of hepatitis B virus surface antigen by targeting PRKCH in HepG2 2.2.15 cells.

MicroRNAs (miRNAs) are single-stranded noncoding RNAs with 18 to 25 nucleotides and play critical roles in a wide spectrum of biological processes. We repored that miR-185 inhibited hepatitis B surface antigen (HBsAg) expression and hepatitis B virus (HBV) replication without affecting the proliferation of HepG2 2.2.15 cells, compared with the controls. We identified that protein kinase C eta (PRKCH) is a direct target gene of miR-185 that affects HBV replication and protein expression and that the miR-185 may suppress HBV replication. Our results provide more information for gene therapy in HBV infection. Keywords: miR-185; HBV; HBV surface antigen; viral replication; PRKCH.

Which level is responsible for gluteal pain in lumbar disc hernia?

BACKGROUND: There are many different reasons why patients could be experiencing pain in the gluteal area. Previous studies have shown an association between radicular low back pain (LBP) and gluteal pain (GP). Studies locating the specific level responsible for gluteal pain in lumbar disc hernias have rarely been reported. METHODS: All patients with lumbar disc herniation (LDH) in the Kanghua hospital from 2010 to 2014 were recruited. All patients underwent a lumbar spine MRI to clarify their LDH diagnosis, and patients were allocated to a GP group and a non-GP group. To determine the cause and effect relationship between LDH and GP, all of the patients were subjected to percutaneous endoscopic lumbar discectomy (PELD). RESULTS: A total of 286 cases were included according to the inclusive criteria, with 168 cases in the GP group and 118 cases in the non-GP group. Of these, in the GP group, 159 cases involved the L4/5 level and 9 cases involved the L5/S1 level, while in the non-GP group, 43 cases involved the L4/5 level and 48 cases involved the L5/S1 level. PELD was performed in both groups. Gluteal pain gradually disappeared after surgery in all of the patients. Gluteal pain recrudesced in a patient with recurrent disc herniation (L4/5). CONCLUSIONS: As a clinical finding, gluteal pain is related to low lumbar disc hernia. The L4/5 level is the main level responsible for gluteal pain in lumbar disc hernia. No patients with gluteal pain exhibited involvement at the L3/4 level.

ß3-adrenoceptor mediates metabolic protein remodeling in a rabbit model of tachypacing-induced atrial fibrillation.

BACKGROUND: The beta 3-adrenoceptor (ß3-AR) is closely associated with energy metabolism. This study aimed to explore the role of ß3-AR in energy remodeling in a rabbit model of pacing-induced atrial fibrillation (AF). METHODS: Rabbits with a sham-operation or pacing-induced AF were used for this study, and the latter group was further divided into three subgroups: 1) the pacing group, 2) the ß3-AR agonist (BRL37344)-treated group, and 3) the ß3-AR antagonist (SR59230A)-treated group. Atrial electrogram morphology and surface ECG were used to monitor the induction of AF and atrial effective refractory period (AERP). RT-PCR and western blot (WB) were used to show alterations in ß3-AR and metabolic-related protein. RESULTS: RT-PCR and WB results showed that ß3-AR was significantly upregulated in the pacing group, and that it corresponded with high AF inducibility and significantly decreased AERP200 and ATP production in this group. Inhibition of ß3-AR decreased the AF induction rate, reversed AERP200 reduction, and restored ATP levels in the AF rabbits. Further activation of ß3-AR using agonist BRL37344 exacerbated AF-induced metabolic disruption. Periodic acid Schiff (PAS) and Oil Red O staining showed ß3-AR-dependent glycogen and lipid droplet accumulation in cardiac myocytes with AF. Glucose transporter-4 (GLUT-4) and CD36, key transporters of glucose and fatty acids, were downregulated in the pacing group. Expression of carnitine-palmitoyltransferase I (CPT-1), a key regulator in fatty acid metabolism, was also significantly downregulated in the pacing group. Reduced glucose transportation and fatty acid oxidation could be restored by inhibition of ß3-AR. Furthermore, key regulators of metabolism, peroxisome proliferator-activated receptor-α (PPARα) and PPAR co-activator (PGC-1α) can be regulated by pharmacological intervention of the ß3-AR. CONCLUSIONS: ß3-AR is involved in metabolic protein remodeling in AF. PPARα/PGC-1α signaling pathway might be the relevant down-stream molecular machinery in response to AF-induced activation of ß3-AR. ß3-AR might be a novel target in AF treatment.

MiR-22-3p and miR-29a-3p synergistically inhibit hepatic stellate cell activation by targeting AKT3.

Hepatic fibrosis (HF) is a worldwide health problem for which there is no medically effective drug treatment at present, and which is characterized by activation of hepatic stellate cells (HSCs) and excessive extracellular matrix (ECM) deposition. The HF model in cholestatic rats by ligating the common bile duct was induced and the differentially expressed miRNAs in the liver tissues were analyzed by microarray, which showed that miR-22-3p and miR-29a-3p were significantly downregulated in bile-duct ligation (BDL) rat liver compared with the sham control. The synergistic anti-HF activity and molecular mechanism of miR-22-3p and miR-29a-3p by targeting AKT serine/threonine kinase 3 (AKT3) in HSCs were explored. The expression levels of miR-22-3p and miR-29a-3p were downregulated in activated LX-2 and human primary normal hepatic fibroblasts (NFs), whereas AKT3 was found to be upregulated in BDL rat liver and activated LX-2 cells. The proliferation, colony-forming, and migration ability of LX-2 were inhibited synergistically by miR-22-3p and miR-29a-3p. In addition, cellular senescence was induced and the expressions of the LX-2 fibrosis markers COL1A1 and α-SMA were inhibited by miR-22-3p and miR-29a-3p synergistically. Subsequently, these two miRNAs binding to the 3'UTR of AKT3 mRNA was predicted and evidenced by the luciferase reporter assay. Furthermore, the proliferation, migration, colony-forming ability, and the expression levels of COL1A1 and α-SMA were promoted and cellular senescence was inhibited by AKT3 in LX-2 cells. Thus, miR-22-3p/miR-29a-3p/AKT3 regulates the activation of HSCs, providing a new avenue in the study and treatment of HF.

Acupuncture Improves the Facial Muscular Function in a Case of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic muscle disorder in which muscles of the face, shoulder blades, and upper arms develop gradual and progressive weakness. There is no effective pharmacological treatment currently available for this disorder so far. We had an opportunity to treat a patient with FSHD using acupuncture. The patient was a 62-year-old female, who presented to us with symptoms such as weakness in her eyes, mouth, shoulder, and upper and lower limbs. Muscle atrophy could be found in multiple areas in her body including her face, shoulder, arm, chest, and lower limbs. Her diagnosis of FSHD muscular dystrophy was established a few years ago and was later genetically confirmed. After a long treatment course of about 10 months with acupuncture, this patient showed a significant restoration of her facial muscle function. However, acupuncture did not improve the function of other muscle groups. The potential mechanism that acupuncture improved the facial function but not the other muscles needs to be further investigated.

CFTR protects against vascular inflammation and atherogenesis in apolipoprotein E-deficient mice.

Atherosclerosis is a chronic inflammatory disease of the vascular wall. Dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to result in inflammatory responses in cystic fibrosis (CF) patients. However, little is known about the role of CFTR in vascular inflammation and atherogenesis. Our results showed that CFTR was dominantly expressed in macrophages of atherosclerotic plaque and reduced in aorta and aortic sinus from atherosclerotic apolipoprotein E-deficient (apoE-/-) mice. In vivo administration of adenovirus encoding CFTR (Ad-CFTR) with apoE-/- mice fed on high-fat diet (HFD) improved plaque stability by decreasing lipid accumulation and necrotic area and increasing smooth muscle cell content and collagen. The Ad-CFTR-treated mice also displayed reduced proinflammatory cytokines levels in aorta and peritoneal macrophages, whereas the anti-inflammatory M2 macrophage markers were increased. Confocal microscopy revealed that the infiltration of T lymphocytes, neutrophils, and macrophages in aortic sinus was markedly attenuated in Ad-CFTR-treated apoE-/- mice. Moreover, in vitro experiments showed that overexpression of CFTR inhibited ox-LDL-induced the migration of peritoneal macrophages. Finally, it was observed that CFTR up-regulation suppressed NFκB and MAPKs activity induced by ox-LDL. Inhibition of JNK or ERK abrogated CFTR down-regulation induced NFκB activation, whereas NFκB inhibitor had no effect on JNK or ERK activation. Taken together, these results demonstrate that CFTR prevents inflammation and atherogenesis via inhibition of NFκB and MAPKs activation. Our data suggest that CFTR may present a potential therapeutic target for the treatment of vascular inflammation and development of atherosclerotic disease.

[Inhibitory and apoptosis regulating effects of adriamycin on different human breast cancer cell lines].

OBJECTIVE: To investigate the inhibitory and apoptosis regulating effects of adriamycin (ADM) on different human breast cancer cell lines and to evaluate the value of apoptosis in breast cancer treatment. METHODS: Human breast cancer cells of the lines Bcap37 and MDA-MB-231 were cultured. ADM of different concentrations was added into the culture fluid. MTT method was used to detect the inhibition rate. Flow cytometry was used to detect the proliferation and apoptosis of the cells. To examine the expression of apoptosis-related molecules: Fas, mutant p53, and Bcl-2 proteins. RESULTS: ADM inhibited the proliferation of Bcap37 cells and MDA-MB-231 cells dose and time-dependently, however, the inhibitory effect of ADM was stronger on the Bcap37 cells than on the MDA-MB-231 cells. After being treated by ADM the expression of Fas was increased and the expression of Bcl-2 was decreased in the Bcap37 cells. However, after being treated by ADM the expressions of Fas, Bcl-2, and mutant p53 in the MDA-MB-231 cells remained almost unchanged. Treated by ADM for 24 hours the apoptotic rate of the Bcap37 cells was increased from 0% to 5.8% (P < 0.05), however, no apoptosis was detected in the MDA-MB-231 cells after treatment of ADM at any time pint. CONCLUSION: With different molecular and biological characteristics, different breast cancer lines are different in chemosensitivity and drug-resistance, which are related to their apoptotic abilities induced by chemotherapeutic drugs.