HOXA11-AS regulates diabetic arteriosclerosis-related inflammation via PI3K/AKT pathway.

OBJECTIVE: This study aims to explore whether homeobox A11 antisense RNA (HOXA11-AS) could regulate inflammation induced by diabetic arteriosclerosis (DAA) via PI3K/AKT pathway. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to detect expressions of HOXA11-AS and proinflammatory genes in carotid endarterectomy samples of symptomatic and asymptomatic atherosclerosis (AS) patients, diabetes mellitus (DM), and non-DM patients. The above-mentioned genes in DM animal model and non-DM animal model were also detected. We detected the expression of HOXA11-AS in vascular smooth muscle cells (VSMCs) treated with platelet-derived growth factor (PDGF) or PDGF inhibitor imatinib, respectively. Subsequently, we applied cell transfection technology to interfere with the expression of HOXA11-AS in VSMCs. In vascular endothelial cells (VECs) and VSMCs, we detected the effect of HOXA11-AS on the expressions of genes related to the proliferation, migration, and cell cycle. Then, VSMCs were treated with tumor necrosis factor-α (TNF-α), and the expression of HOXA11-AS was examined in VSMCs. The effect of HOXA11-AS on TNF-α-induced inflammation in VSMCs was detected as well. Finally, we analyzed the effect of HOXA11-AS on PDGF-induced activation of PI3K/AKT pathway in VSMCs and VECs. RESULTS: HOXA11-AS expression was markedly increased in carotid endarterectomy specimens of symptomatic AS patients compared to that of asymptomatic AS patients. Expression levels of HOXA11-AS and pro-inflammatory genes were significantly elevated in carotid endarterectomy specimens of DM patients. Similarly, HOXA11-AS expression was also significantly increased in carotid arteries of DM mice compared with that of non-DM mice. PDGF could upregulate HOXA11-AS expression in VSMCs, which was reversed by PDGF inhibitor imatinib. HOXA11-AS knockdown could reduce the expressions of the proliferation-associated gene (PCNA) and the cycle-related genes (p21, p53), and also inhibited the proliferation and migration of VSMCs induced by PDGF. HOXA11-AS was upregulated by TNF-α. HOXA11-AS knockdown remarkably downregulated expressions of inflammation-related genes in VSMCs induced by TNF-α. In VECs, low expression of HOXA11-AS can inhibit the expression of TNF-α-induced pro-inflammatory genes and PDGF-induced vascular inflammation-related genes. Low expression of HOXA11-AS inhibited PDGF-induced activation of PI3K/AKT pathway in VSMCs and VECs. CONCLUSIONS: HOXA11-AS may participate in DAA by activating the PI3K/AKT pathway to regulate inflammation in VSMCs and VECs.

Research on rat models of hypobaric hypoxia-induced pulmonary hypertension.

OBJECTIVE: Rat models of hypobaric hypoxia-induced pulmonary hypertension are commonly used in studies of chronic mountain sickness, while there are few researches specially focusing on these rats model. This study aims to exploring possible pathogenesis of hypobaric hypoxia-induced pulmonary hypertension by experimenting on hypobaric hypoxia-induced PH rat models at different simulate- altitudes. MATERIALS AND METHODS: 32 healthy male SD rats were randomly divided into six groups of different degree and time period of hypobaric hypoxia. The mean pulmonary arterial pressure (m PAP), right ventricular pressure (RVSP), the right ventricle (RV), left ventricular (LV), ventricular septal (S), the right ventricular hypertrophy index (RVHI) [calculated under the formula of RV / (LV + S)], hematoxylin-eosin staining, elastic fibers staining, the ratio of the thickness of vascular wall to its outer diameter (MT%), the ratio of the cross-sectional area of the middle vascular wall to the total vascular cross-sectional area (MA%); the α-SMA, and the Ki6 expressions were detected to evaluated the pulmonary hypertension. RESULTS: There were significant differences of the mPAP, RVSP and RVHI value between the hypobaric hypoxia groups and the control group (p < 0.05). The mPAP, RVSP, RVHI, MT%, MA%, α-SMA, and Ki6 of rats in model groups at an altitude of 3KM were higher than those of the control group, which raised gradually with the number of weeks increasing. The mPAP, RVSP, RV / (LV + S) value, MT%, MA%, α-SMA, and Ki67 of the 5KM-4W group were significantly higher than those of the control group (p < 0.05). CONCLUSIONS: Rat models with pulmonary hypertension at different altitudes have been successfully established by automatic adjusting hypobaric hypoxia chamber. Exposure to a low oxygen environment at a simulate-altitude of 3 km for 8 weeks have caused the pathological remodeling of pulmonary vascular walls and pulmonary hypertension, and further led to a series of pathological changes, including right ventricular hypertrophy. This model is easy to be replicated with good reproducibility and provides evidence for clinical trial of drugs.

Expression of RUNX3 gene in pancreatic adenocarcinoma and its clinical significance.

We investigated the clinical significance of RUNX3 gene expression in human pancreatic carcinoma. Five samples of pancreatic tissues and 30 samples of pancreatic cancer tissues and paracancerous tissues were collected. RUNX3 expression was detected by real-time PCR and immunohistochemistry. The relationships between clinicopathological findings and the expression of RUNX3 were analyzed. The relative quantification level of RUNX3 mRNA expression in human pancreatic carcinoma tissues and paracancerous tissues was 2.60 (0.42-12.82) and 1.02 (0.19-3.58), respectively (P < 0.05). The percentage of positive cells expressing RUNX3 protein in human pancreatic tissues and paracancerous tissues was 45.5 ± 26.2 and 6.9 ± 6.0%, respectively (P < 0.01). The high RUNX3 group (N = 9) with 45.5% or more of the cancer cells staining for RUNX3 and the low RUNX3 group (N = 21) with less than 45.5% cancer cells staining for RUNX3. Low expression of RUNX3 correlated significantly with an advanced TNM stage (χ(2) = 6.897, P = 0.045), lymph node metastasis (χ(2) = 4.739, P = 0.029) and neural invasion (χ(2) = 5.44, P = 0.020). On the other hand, no association could be found between RUNX3 expression and clinicopathological variables including age, gender, tumor location, tumor size, tumor differentiation or the serum concentration of CEA and CA199. The expression of RUNX3 in pancreatic cancer tissues was obviously higher than that in the paracancerous tissues. Low expression of RUNX3 may have an important role in aggressiveness, lymph node metastasis and neural invasion in pancreatic cancer. In pancreatic carcinoma tissues, low expression of RUNX3 may indicate a poor prognosis.

Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells.

Achieving transgene integration into preselected genomic sites is currently one of the central tasks in stem cell gene therapy. A strategy to mediate such targeted integration involves site-specific endonucleases. Two genomic sites within the MBS85 and chemokine (C-C motif) receptor 5 (CCR5) genes (AAVS1 and CCR5 zinc-finger nuclease (CCR5-ZFN) sites, respectively) have recently been suggested as potential target regions for integration as their disruption has no functional consequence. We hypothesized that efficient transgene integration maybe affected by DNA accessibility of endonucleases and therefore studied the transcriptional and chromatin status of the AAVS1 and CCR5 sites in eight human induced pluripotent stem (iPS) cell lines and pooled CD34+ hematopoietic stem cells (HSCs). Matrix chromatin immunoprecipitation (ChIP) assays demonstrated that the CCR5 site and surrounding regions possessed a predominantly closed chromatin configuration consistent with its transcriptional inactivity in these cell types. In contrast, the AAVS1 site was located within a transcriptionally active region and exhibited an open chromatin configuration in both iPS cells and HSCs. To show that the AAVS1 site is readily amendable to genome modification, we expressed Rep78, an AAV2-derived protein with AAVS1-specific endonuclease activity, in iPS cells after adenoviral gene transfer. We showed that Rep78 efficiently associated with the AAVS1 site and triggered genome modifications within this site. On the other hand, binding to and modification of the CCR5-ZFN site by a ZFN was relatively inefficient. Our data suggest a critical influence of chromatin structure on efficacy of site-specific endonucleases used for genome editing.

High efficiency reconstitution of a human-mouse chimeric Fab of CAb-1 antibody specific to human colon cancer.

Monoclonal antibody (mAb) has been widely applied in the treatment of human diseases, especially in malignant tumours. However, most antibodies produced in mouse by hybridoma technology might induce severe human anti-mouse reactions. We had reported a murine mAb CAb-1 of therapeutic interest for its specifically binding to a cell surface glycoprotein of human colon cancer. Here, we attempted to generate a reconstituted human-mouse chimeric Fab (cFab) of CAb-1 in vitro to reduce its antigenicity and increase its capacity of penetration. First, the genes of heavy and light chain variable region (VH, VL) of CAb-1 were cloned. Then, the chimeric light chain (cL) and Fd (cFd) were constructed and expressed in Escherichia coli. Finally, the reconstituted cFab was obtained by gradient dialysis of the mixture of cFd and cL. SDS-PAGE and western blot analysis showed the reconstituted cFab with a recovery rate of 70.2% when the initial total concentration of cL and cFd proteins to be 100 microg/ml. The reconstituted cFab maintained the affinity and specificity to colon cancer cells compared with its parental antibody as determined by immunostaining analysis, FACS and ELISA. Our results established a foundation for further application of the cFab in diagnosis and treatment of colon cancer.

Epitope mapping of series of monoclonal antibodies against the hepatocellular carcinoma-associated antigen HAb18G/CD147.

The hepatocellular carcinoma-associated antigen HAb18G/CD147, a member of CD147 family, could promote tumour invasion and metastasis via inducing the secretion of matrix metalloproteinases (MMP). Anti-CD147 monoclonal antibodies (MoAb) have exhibited obvious inhibitory effect on MMP induction. However, none of the epitopes of these MoAb has been reported. We previously prepared five MoAb against HAb18G/CD147, named HAb18, 3B3, 1B3, 5A5 and 4D2. To map the epitopes of these MoAb, a series of truncated fragments of extracellular region of HAb18G/CD147 was expressed in Escherichia coli and the MoAb-binding affinity to these fragments was examined with an enzyme-linked immunosorbent assay and Western blot. The residues (39)LTCSLNDSATEV(50), (36)KILLTCS(42) and (22)AAGTVFTTVEDL(33) were determined to be the epitopes of HAb18, 3B3 and 1B3, respectively, which were further proved by a dot-blot analysis with synthesized peptides and bioinformatics epitope prediction. The binding regions of MoAb 5A5 and 4D2 were located at residues E(120)-R(203). Then we constructed and expressed full-length HAb18G/CD147 and truncated HAb18G/CD147 without residues A(22)-V(50) in COS-7 cells. Gelatin zymography and Boyden chamber assay showed that the COS-7 cells expressing truncated HAb18G/CD147 failed to induce MMP production and enhance the cells' invasive potential, compared with the cells expressing full-length HAb18G/CD147. Taken together with the obviously inhibitory effects of HAb18 on the function of full-length HAb18G/CD147, these findings suggest that residues (22)AAGTVFTTVEDLGSKILLTCSLNDSATEV(50) may play a critical role in the functions of HAb18G/CD147 on MMP secretion and tumour invasion. These key residues can be used as potential drug target in cancer therapy.