Potential roles of microRNAs and long noncoding RNAs as diagnostic, prognostic and therapeutic biomarkers in coronary artery disease.

Coronary artery disease (CAD), which is mainly caused by atherosclerotic processes in coronary arteries, became a significant health issue. MicroRNAs (miRNAs), and long noncoding RNAs (lncRNAs), have been shown to be stable in plasma and could thereby be adopted as biomarkers for CAD diagnosis and treatment. MiRNAs can regulate CAD development through different pathways and mechanisms, including modulation of vascular smooth muscle cell (VSMC) activity, inflammatory responses, myocardial injury, angiogenesis, and leukocyte adhesion. Similarly, previous studies have indicated that the causal effects of lncRNAs in CAD pathogenesis and their utility in CAD diagnosis and treatment, has been found to lead to cell cycle transition, proliferation dysregulation, and migration in favour of CAD development. Differential expression of miRNAs and lncRNAs in CAD patients has been identified and served as diagnostic, prognostic and therapeutic biomarkers for the assessment of CAD patients. Thus, in the current review, we summarize the functions of miRNAs and lncRNAs, which aimed to identify novel targets for the CAD diagnosis, prognosis, and treatment.

Severe bleeding following off-pump coronary artery bypass grafting: predictive factors and risk model.

BACKGROUND: Severe bleeding following cardiac surgery remains a troublesome complication, but to date, there is a lack of comprehensive predictive models for the risk of severe bleeding following off-pump coronary artery bypass grafting (OPCABG). This study aims to analyze relevant indicators of severe bleeding after isolated OPCABG and establish a corresponding risk assessment model. METHODS: The clinical data of 584 patients who underwent OPCABG from January 2018 to April 2020 were retrospectively analyzed. We gathered the preoperative baseline data and postoperative data immediately after intensive care unit admission and used multifactor logistic regression to screen the potential predictors of severe bleeding, upon which we established a predictive model. Using the consistency index and calibration curve, decision curve, and clinical impact curve analysis, we evaluated the performance of the model. RESULTS: This study is the first to establish a risk assessment and prediction model for severe bleeding following isolated OPCABG. Eight independent risk factors were identified: male sex, aspirin/clopidogrel withdrawal time, platelet count, fibrinogen level, C-reactive protein, serum creatinine, and total bilirubin. Among the 483 patients in the training group, 138 patients (28.6%) had severe bleeding; among the 101 patients in the verification group, 25 patients (24.8%) had severe bleeding. Receiver operating characteristic (ROC) curve analysis for the internal training group revealed a convincing performance with a concordance index (C-index) of 0.859, while the area under the ROC curve for the external validation data was 0.807. Decision curve analysis showed that the model was useful for both groups. CONCLUSIONS: Although there are some limitations, the model can effectively predict the probability of severe bleeding following isolated OPCABG and is therefore worthy of further exploration and verification.

"Felodipine-indomethacin" co-amorphous supersaturating drug delivery systems: "Spring-parachute" process, stability, in vivo bioavailability, and underlying molecular mechanisms.

Amorphous solid dispersions (ASD) are one of most commonly used supersaturating drug delivery systems (SDDS) to formulate insoluble active pharmaceutical ingredients. However, the development of polymer-guided stabilization of ASD systems faces many obstacles. To overcome these shortcomings, co-amorphous supersaturable formulations have emerged as an alternative formulation strategy for poorly soluble compounds. Noteworthily, current researches around co-amorphous system (CAS) are mostly focused on preparation and characterization of these systems, but more detailed investigations of their supersaturation ("spring-parachute" process), stability, in vivo bioavailability and molecular mechanisms are inadequate and need to be clarified. In present study, we chose pharmacological relevant BCS II drugs to fabricate and characterize "felodipine-indomethacin" CAS. To enrich the current inadequate but key knowledge on CAS studies, we carried out following highlighted investigations including dissolution/solubility, semi-continuous "spring-parachute" process, long-term stability profile of amorphous state, in vivo bioavailability and underlying molecular mechanisms (molecular interaction, molecular miscibility and crystallization inhibition). Generally, the research provides some key information in the field of current "drug-drug" CAS supersaturable formulations.

MEF2C/miR-133a-3p.1 circuit-stabilized AQP1 expression maintains endothelial water homeostasis.

Aquaporin 1 (AQP1) plays an important role in endothelial functions and is regulated by MEF2C. However, how AQP1 level is stabilized to maintain endothelial water homeostasis is still not clear. Here, we show that AQP1 expression is significantly upregulated by MEF2C transcriptionally and inhibited by miR-133a-3p.1 post-transcriptionally. Meanwhile, MEF2C activates the expression of miR-133a1. Simultaneous overexpression of MEF2C and miR-133a-3p.1 suppresses the aptitude of changes in AQP1 expression caused by either MEF2C or miR-133a-3p.1. Accordingly, the changes in migration and tube formation of human umbilical vein endothelial cells (HUVECs) caused by MEF2C or miR133a-3p.1 are blunted by coexpression of both of them. These data demonstrate that the homeostasis and physiological function of AQP1 in endothelial health are maintained by the MEF2C and miR-133a-3p.1 regulatory circuit.

Hot melt extrusion technology for improved dissolution, solubility and "spring-parachute" processes of amorphous self-micellizing solid dispersions containing BCS II drugs indomethacin and fenofibrate: Profiles and mechanisms.

Many strategies have been employed to improve oral drug delivery. One such approach involves the use of supersaturable delivery systems such as amorphous self-micellizing solid dispersions (SmSDs). SmSDs have attracted more attention recently, but little is known regarding the impact of production methods on profiles and internal mechanisms of final SmSDs in spite of its importance. In this study, amorphous SmSDs containing self-micellizing Soluplus® and BCS II drug (either indomethacin (IND) or fenofibrate (FEN)) were generated using various methods: solvent evaporation (SOL), freeze-drying (FD), microwave radiation-quench cooling (MQC), and hot melt extrusion (HME). Microscopic morphology, amorphous state, thermal behavior, dissolution/solubility, and "spring-parachute" data were used to assemble physicochemical profiles for SmSD systems prepared using each method. Analysis of intermolecular interactions, solubilization, and crystallization inhibition further uncovered internal mechanisms explaining observed physicochemical properties. Generally, SmSD/IND and SmSD/FEN systems generated using HME exhibited superior dissolution, solubility, and spring-parachute profiles. The superior advantages of HME-generated SmSD/IND systems were attributed to relatively stronger intermolecular interactions than observed in SmSD/IND systems fabricated using other methods. Moreover, self-micellizing Soluplus® carrier was able to solubilize IND or FEN and suppress drug crystallization from a supersaturated state, which seemed to be an important mechanism for the properties enhancement caused by SmSD/FENHME. This knowledge should be useful for guiding further development of self-micellizing solid dispersions and for gaining deeper understanding of how HME technology can improve supersaturable drug delivery based on SmSDs strategy.

Prognostic Value of Lymph Node Ratio in Locally Advanced Rectal Cancer Patients After Preoperative Chemoradiotherapy Followed by Total Mesorectal Excision.

Although the absolute number of positive lymph nodes (LNs) has been established as 1 of the most important prognostic factors in rectal cancers, many researchers have proposed that the lymph node ratio (LNR) may have better predicted outcomes. We conducted a retrospective study to compare the predictive ability of LNR and ypN category in rectal cancer. A total of 264 locally advanced rectal cancer (LARC) patients who underwent preoperative chemoradiotherapy (CRT) followed by total mesorectal excision (TME) between 2005 and 2012 were reviewed. All patients were categorized into 3 groups or patients with metastatic LNs were categorized into 2 groups according to the LNR. The prognostic effect on overall survival (OS) and disease-free survival (DFS) was evaluated. With a median follow-up of 45 months, the OS and DFS were 68.4% and 59.3% for the entire cohort, respectively. The respective 5-year OS and DFS rates for the 3 groups (LNR = 0, 0 < LNR ≤ 0.20, and 0.20 < LNR ≤ 1.0) were as follows: 83.2%, 72.6%, and 49.4% (P < 0.001) and 79.5%, 57.3%, and 33.5% (P < 0.001), respectively. Multivariate analysis revealed that LNR and differentiation, but not the number of positive LNs, had independent prognostic value for OS (hazard ratio [HR] = 2.328, 95% confidence interval [CI]: 1.850-4.526, P < 0.001) and DFS (HR = 3.004, 95% CI: 1.616-5.980, P < 0.001). As for patients with positive LNs, the respective 5-year OS and DFS rates for the 2 groups (0 < LNR ≤ 0.20, and 0.20 < LNR ≤ 1.0) were 72.6% and 49.4% (P < 0.001) and 57.3% and 33.5% (P < 0.001), respectively. Multivariate analysis revealed that only LNR was an independent factor for OS (HR = 3.214, 95% CI: 1.726-5.986, P < 0.001) and DFS (HR = 4.230, 95% CI: 1.825-6.458, P < 0.001). Subgroups analysis demonstrated that the ypN category had no impact on survival whereas increased LNR was a significantly prognostic indicator for worse survival in the LNs < 12 subgroup. LNR is an independent prognostic factor in LARC patients treated with preoperative CRT followed by TME. It may be a better independent staging method than the number of metastatic LNs when <12 LNs are harvested after preoperative CRT.

Dl-3-n-butylphthalide-induced upregulation of antioxidant defense is involved in the enhancement of cross talk between CREB and Nrf2 in an Alzheimer's disease mouse model.

Synapse impairment in the Alzheimer's disease (AD) brain is an early event leading to cognitive dysfunction. Most oxidative stress localizes to the synapse, and synapse loss is the basis of cognitive decline in AD. Dl-3-n-butylphthalide (Dl-NBP), a small molecule compound has been shown to ameliorate oxidative stress. We evaluated the effects of a 5-month oral delivery with Dl-NBP on oxidative stress and cognitive function in APP/PS1 transgenic mice. Dl-NBP treatment reduced oxidative stress in the APP/PS1 mouse brain and alleviated learning and memory deficits. Dl-NBP supplementation meliorated synaptic plasticity, diminished soluble amyloid beta and amyloid beta oligomer in the APP/PS1 mouse brain. Dl-NBP administration caused an increase of cyclic adenosine monophosphate-response element binding protein (CREB)-binding protein (CBP)-associated Ser133-phosphorylated CREB (p-CREB) protein. Chromatin immunoprecipitation analysis revealed that Dl-NBP increased the recruitment of CBP to the promoters of best-characterized genes downstream of nuclear factor erythroid 2-related factor 2, nicotinamide adenine dinucleotide phosphate (NADPH) quinone oxidoreductase 1, and γ-glutamylcysteine synthetase modifier subunit. We demonstrate that the Dl-NBP-triggered upregulation of antioxidant defenses is involved in the enhancement of cross talk between CREB and nuclear factor erythroid 2-related factor 2 via CBP. Our results suggest that Dl-NBP may be a useful agent for the treatment of AD.

Clinical investigation of TROP-2 as an independent biomarker and potential therapeutic target in colon cancer.

Colon cancer is associated with a severe demographic and economic burden worldwide. The pathogenesis of colon cancer is highly complex and involves sequential genetic and epigenetic mechanisms. Despite extensive investigation, the pathogenesis of colon cancer remains to be elucidated. As the third most common type of cancer worldwide, the treatment options for colon cancer are currently limited. Human trophoblast cell­surface marker (TROP­2), is a cell­surface transmembrane glycoprotein overexpressed by several types of epithelial carcinoma. In addition, TROP­2 has been demonstrated to be associated with tumorigenesis and invasiveness in solid types of tumor. The aim of the present study was to investigate the protein expression of TROP­2 in colon cancer tissues, and further explore the association between the expression of TROP­2 and clinicopathological features of patients with colon cancer. The expression and localization of the TROP­2 protein was examined using western blot analysis and immunofluorescence staining. Finally, the expression of TROP­2 expression was correlated to conventional clinicopathological features of colon cancer using a χ2 test. The results revealed that TROP­2 protein was expressed at high levels in the colon cancer tissues, which was associated with the development and pathological process of colon cancer. Therefore, TROP­2 may be used as a biomarker to determine the clinical prognosis, and as a potential therapeutic target in colon cancer.

Cardioprotective effect of valsartan in mice with short-term high-salt diet by regulating cardiac aquaporin 1 and angiogenic factor expression.

Hypertension is the most common risk factor for various cardiovascular and cerebrovascular diseases that affects approximately 61 million, or 25% of the population in United States. The dietary salt intake is one of the most important but modifiable factors for hypertension. In the current study, we aim to elucidate the role of aquaporin 1 in high-salt-induced hypertension and cardiac injuries and whether angiotensin II receptor blocker valsartan could ameliorate the effect of high salt on blood pressure. Mice were fed with normal diet, high-salt diet in the presence or absence of valsartan for 4 weeks. The body weight gain, feeding behavior, blood pressure, and cardiac pathology changes were monitored after 4 weeks. The expression of aquaporin 1, vascular endothelial growth factor, transforming growth factor ß1, and basic fibroblast growth factor were analyzed using quantitative real-time polymerase chain reaction, Western blot, and immunohistochemical staining. Valsartan partially reversed the effects of high-salt diet on hypertension, cardiac injuries such as fibrosis and inflammatory cell infiltration, and inhibition of aquaporin 1 and angiogenic factors; valsartan alone did not exert such effects. The current data demonstrated that the reduction of cardiac aquaporin 1 and angiogenic factor expression level might be associated with high-salt-induced hypertension and cardiac injuries in mice, which could be ameliorated by angiotensin II receptor blocker treatment.

Peripheral blood miRNAs as a biomarker for chronic cardiovascular diseases.

Vascular diseases are the most prevalent diseases worldwide. This study intended to analyze peripheral blood miRNA levels and their correlation with NT-pro-BNP and cTN-I in patients with atherosclerosis or pre-atherosclerotic conditions to build a dynamic correlation between vascular diseases and their biomarkers. Serum NT-pro-BNP and cTN-I levels were measured by their respective ELISA kits. The miRNA levels were assayed by quantitative PCR. Unique miRNA signatures were identified for both atherosclerosis and pre-atherosclerosis. The levels of miR-92a, 126, 130a, 222, and 370 levels were decreased in the peripheral blood of pre-atherosclerotic subjects. In atherosclerosis, miR-21, 122, 130a, and 211 were significantly increased whereas miR-92a, 126, and 222 were markedly decreased. Serum levels of NT-pro-BNP and cTN-I correlated with each other and increased with the progression of atherosclerosis. Moreover, the levels of cTN-I and NT-pro-BNP were positively correlated with miR-21 and negatively correlated with miR-126. Integrating specific pattern of miRNA levels with NT-pro-BNP and/or cardiac troponin may improve the diagnosis of cardiovascular diseases.

CD36 upregulation mediated by intranasal LV-NRF2 treatment mitigates hypoxia-induced progression of Alzheimer's-like pathogenesis.

AIMS: There is extensive evidence that oxidative stress induces cellular dysfunction in the brain and plays a critical role in Alzheimer's disease (AD) pathogenesis. Hypoxia increases factors involved in oxidative stress injury and contributes to the onset and progression of AD. Nuclear factor erythroid 2-related factor 2 (NRF2), a major component regulating antioxidant response, is attenuated in the AD brain. Importantly, NRF2 directly regulates the alternative first exons of CD36, an important participant in oxidative and inflammatory processes. To explore the effects of hypoxia-induced deterioration of AD-like pathogenesis and investigate the correlation between hypoxia-induced NRF2 signal alterations and CD36 expression, we examined the NRF2 signaling, CD36, and oxidative stress events in hypoxia-treated APPswe/PSEN1dE9 (APP/PS1) mice brain. RESULTS: We observed that hypoxia treatment increased oxidative stress, exacerbated inflammation, and aggravated learning defects in aged APP/PS1 mice. Microglia from hypoxia-treated mice brain exhibited marked reduction in CD36 expression and inhibition of ß-amyloid (Aß) degradation. Accordingly, hypoxia treatment caused a decrease in transactivation of NRF2 target genes in the aging mouse brain. Intranasal administration with a lentiviral vector encoding human NRF2 increased CD36 expression, ameliorated the weak antioxidant response triggered by hypoxia, diminished Aß deposition, and improved spatial memory defects. INNOVATION: In this study, we demonstrated for the first time that NRF2 intranasal treatment-induced increases of CD36 could enhance Aß clearance in AD transgenic mouse. CONCLUSION: These results suggest that targeting NRF2-mediated CD36 expression might provide a beneficial intervention for cognitive impairment and oxidative stress in AD progression.

Tracking in vivo migration and distribution of antigen-specific cytotoxic T lymphocytes by 5,6-carboxyfluorescein diacetate succinimidyl ester staining during cancer immunotherapy.

BACKGROUND: Killing of targeted tumors during adoptive cell transfer therapy is associated with cytotoxic T lymphocyte (CTL) numbers, immunophenotype, tumor-specificity, and in vivo residence time, migration, and distribution. Therefore, tracing in vivo persistence, migration, and distribution of CTLs is important for cancer immunotherapy. METHODS: Optimal staining concentration for CTL proliferation was determined by cell counting kit-8 (CCK-8) assay and killing efficiencies of CTLs or carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled melanoma antigen-specific cytotoxic T lymphocytes (CFSE-CTLs) for malignant melanoma cells in vitro were compared. Additionally, CFSE-CTLs were intravenously transfused to mice receiving B16 melanoma, and their residence time, migration, and distribution in vivo were observed by measuring fluorescence intensities of CFSE-CTLs per gram of tissue (%FI/g) in various tissues and analyzing tumor/non-tumor (T/NT) values. Anti-tumor effects of transferred CTLs and correlation between %FI/g and D-value of tumor size were analyzed. RESULTS: Five-micromolar CFSE was optimal for labeling CTLs with minimal cytotoxicity. No significant difference occurred between CTLs and CFSE-CTLs for tumor cell killing (P = 0.849) or interleukin-2 (P = 0.318) and interferon-γ (P = 0.201) levels. Distribution of CTLs in vivo varied with time. A negative correlation between %FI/g in tumors and D-value of tumor sizes by Spearman correlation analysis was observed. CTLs were recruited to and killed tumors from 6 hours to 3 days after cell infusion. CTLs were observed up to three weeks later in the tumor, liver, kidneys, and spleen; this was related to the abundant blood supply or the nature of immune organs. CONCLUSIONS: CCK-8 assay is a novel method to select optimal CFSE staining concentrations. Fluorescence intensity of transferred CTLs reflects their killing efficiency of tumors. CFSE fluorescent markers can trace in vivo CTL persistence, migration, and distribution because of its stability, long half-life, and low toxicity.

Hypoxia-triggered m-calpain activation evokes endoplasmic reticulum stress and neuropathogenesis in a transgenic mouse model of Alzheimer's disease.

BACKGROUND: Previous studies have demonstrated that endoplasmic reticulum (ER) stress is activated in Alzheimer's disease (AD) brains. ER stress-triggered unfolded protein response (UPR) leads to tau phosphorylation and neuronal death. AIMS: In this study, we tested the hypothesis that hypoxia-induced m-calpain activation is involved in ER stress-mediated AD pathogenesis. METHOD: We employed a hypoxic exposure in APP/PS1 transgenic mice and SH-SY5Y cells overexpressing human Swedish mutation APP (APPswe). RESULTS: We observed that hypoxia impaired spatial learning and memory in the APP/PS1 mouse. In the transgenic mouse brain, hypoxia increased the UPR, upregulated apoptotic signaling, enhanced the activation of calpain and glycogen synthase kinase-3ß (GSK3ß), and increased tau hyperphosphorylation and ß-amyloid deposition. In APPswe cells, m-calpain silencing reduced hypoxia-induced cellular dysfunction and resulted in suppression of GSK3ß activation, ER stress and tau hyperphosphorylation reduction as well as caspase pathway suppression. CONCLUSION: These findings demonstrate that hypoxia-induced abnormal calpain activation may increase ER stress-induced apoptosis in AD pathogenesis. In contrast, a reduction in the expression of the m-calpain isoform reduces ER stress-linked apoptosis that is triggered by hypoxia. These findings suggest that hypoxia-triggered m-calpain activation is involved in ER stress-mediated AD pathogenesis. m-calpain is a potential target for AD therapeutics.

MicroRNA-451 inhibits growth of human colorectal carcinoma cells via downregulation of Pi3k/Akt pathway.

MicroRNAs (MiRNAs) play important roles in coordinating a variety of cellular processes and abnormal expression has been linked to the occurrence of several cancers. The miRNA miR-451 is downregulated in colorectal carcinoma (CRC) cells, suggested by several research groups including our own. In this study, synthetic miR-451 mimics were transfected into the SW620 human CRC cell line using Lipofectamine 2000 and expression of miR-451 was analyzed by real time PCR, while expression of CAB39, LKB1, AMPK, AKT, PI3K and Bcl2 was analyzed by Western blot, and cell growth was detected by MTT assay. In comparison to the controls, a significant increase in the expression of miR-451 was associated with significantly decreased expression of CAB39, LKB1, AMPK, AKT, PI3K and Bcl2. The capacity of cell proliferation was significantly decreased by miR-451 expression, which also inhibited cell growth. Our study confirmed that miR-451 has a repressive role in CRC cells by inhibiting cell growth through down-regulating the P13K/AKT pathway.

Trientine reduces BACE1 activity and mitigates amyloidosis via the AGE/RAGE/NF-κB pathway in a transgenic mouse model of Alzheimer's disease.

AIMS: There is mounting evidence that the transition metal copper may play an important role in the pathophysiology of Alzheimer's disease (AD). Triethylene tetramine dihydrochloride (trientine), a CuII-selective chelator, is a commonly used treatment for Wilson's disease to decrease accumulated copper, and thereby decreases oxidative stress. In the present study, we evaluated the effects of a 3-month treatment course of trientine (Trien) on amyloidosis in 7-month-old ß-amyloid (Aß) precursor protein and presenilin-1 (APP/PS1) double transgenic (Tg) AD model mice. RESULTS: We observed that Trien reduced the level of advanced glycation end products (AGEs), and decreased Aß deposition and synapse loss in brain of APP/PS1 mice. Importantly, we found that Trien blocked the receptor for AGEs (RAGE), downregulated ß-site APP cleaving enzyme 1 (BACE1), inhibited amyloidogenic APP cleavage, and subsequently reduced Aß levels. In vitro, in SH-SY5Y cells overexpressing Swedish mutant APP, Trien-mediated downregulation of BACE1 occurred via inhibition of the NF-κB signaling pathway. INNOVATION: In this study, we demonstrated for the first time that Trien inhibited amyloidogenic pathway including targeting the downregulation of RAGE and NF-κB. CONCLUSION: Trien might mitigate amyloidosis in AD by inhibiting the RAGE/NF-κB/BACE1 pathway. Our study demonstrates that Trien may be a viable therapeutic strategy for the intervention and treatment of AD and other AD-like pathologies.

[A procedure to assist achievement of total mesorectal excision through the extending intersphincteric plane].

OBJECTIVE: To investigate the value of assisted achievement total mesorectal excision (TME) through the extending intersphincteric plane. METHODS: From February 2006 to April 2010, 65 patients with low rectal cancer underwent assisted implementing TME through the extending intersphincteric plane under direct vision and achieved sphincter preservation. The clinical data was summarized and analyzed retrospectively. Follow-up visits were conducted on complications and oncological outcomes. RESULTS: The mean operation time was (245 ± 42) minutes, and the mean intraoperative blood loss was (114 ± 76) ml. There was no postoperative mortality. Postoperative complications included 2 cases of anastomotic leak, 13 cases of anastomotic stenosis, 2 cases of early postoperative inflammatory ileus, 1 case of urinary tract infection, and 1 case of incision infection. Distal margins and circumferential resection margin of all specimens were negative. For pathological stage, there were 26 cases at stage pTNMI, 17 cases at stage pTNMII and 22 cases at stage pTNMIII. The mean follow-up time was (47.9 ± 18.9) months. 10 patients were lost to follow up, 15 cases had distant metastasis or local recurrence in, and 8 cases died of tumor metastasis at the latest follow up. Local recurrence occurred in 3 cases, including recurrence in presacral region, metastasis of lymph node at the left side in pelvis cavity, and metastasis at the sacrum at 35, 36, and 52 months postoperatively. There was no anastomotic recurrence. Log-rank survival analysis showed 5-year cumulative survival rate was 100%, 93.3%, and 63.1% in TNM stage I, II, and III, respectively. The cumulative disease-free survival rate was 96.2%, 83.3%, 44.8% in TNM stage I, II, and III, respectively. CONCLUSION: It has a good oncological effect and was an advantageous procedure to assist achievement total mesorectal excision (TME) through the extending intersphincteric plane as surgeons encountered with difficulties from transabdominal TME.

Methods of isolation, expansion, differentiating induction and preservation of human umbilical cord mesenchymal stem cells.

OBJECTIVE: This literature review aims to summarize the methods of isolation, expansion, differentiation and preservation of human umbilical cord mesenchymal stem cells (hUCMSCs), for comprehensive understanding and practical use in preclinical research and clinical trials. DATA SOURCES: All the literature reviewed was published over the last 10 years and is listed in PubMed and Chinese National Knowledge Infrastructure (CNKI). Studies were retrieved using the key word "human umbilical cord mesenchymal stem cells". RESULTS: Explants culture and enzymatic digestion are two methods to isolate hUCMSCs from WJ and there are modifications to improve these methods. Culture conditions may affect the expansion and differentiating orientations of hUCMSCs. In addition, hUCMSCs can maintain their multi-potential effects after being properly frozen and thawed. CONCLUSION: Considering their multi-potential, convenient and non-invasive accessibility, low immunogenicity and the reported therapeutic effects in several different preclinical animal models, hUCMSCs have immense scope in regeneration medicine as a substitute for MSCs derived from bone marrow or umbilical cord blood.

Effective adoptive transfer of haploidentical tumor-specific T cells in B16-melanoma bearing mice.

BACKGROUND: Adoptive transfer of allogeneic tumor-specific T cells often results in severe graft-versus-host disease (GVHD). Here, we sought to maximize graft-versus-tumor and minimize GVHD by using haploidentical T cells in pre-irradiated B16-melanoma bearing mice. METHODS: C57BL/6 mice bearing B16-melanoma tumors were irradiated with 0, 5, or 7 Gy total body irradiation (TBI), or 7 Gy TBI plus bone marrow transplantation. Tumor areas were measured every 3 days to assess the influence of irradiation treatment on tumor regression. B16-melanoma bearing mice were irradiated with 7 Gy TBI; sera and spleens were harvested at days 1, 3, 5, 7, 9, 11, and 13 after irradiation. White blood cell levels were measured and transforming growth factor ß1 (TGF-b1) and interleukin 10 (IL-10) levels in serum were detected using enzyme-linked immunosorbent assay (ELISA) kits. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry were performed to test TGF-b1, IL-10 and Foxp3 mRNA levels and the proportion of CD4+CD25+Foxp3+ T-regulatory cells (Tregs) in spleens. B16-melanoma bearing C57BL/6 mice were irradiated with 7 Gy TBI followed by syngeneic (Syn1/Syn2) or haploidentical (Hap1/Hap2), dendritic cell-induced cytotoxic T lymphocytes (DC-CTLs) treatment, tumor areas and system GVHD were observed every 3 days. Mice were killed 21 days after the DC-CTLs adoptive transfer; histologic analyses of eyes, skin, liver, lungs, and intestine were then performed. RESULTS: Irradiation with 7 Gy TBI on the B16-melanoma-bearing mice did not influence tumor regression compared to the control group; however, it down-regulated the proportion of Tregs in spleens and the TGF-b1 and IL-10 levels in sera and spleens, suggesting inhibition of autoimmunity and intervention of tumor microenvironment. Adoptive transfer of haploidentical DC-CTLs significantly inhibited B16-melanoma growth. GVHD assessment and histology analysis showed no significant difference among the groups. CONCLUSION: Adoptive transfer of haploidentical tumor-specific T cells in irradiation-pretreated B16-melanoma bearing mice preserved antitumor capacity without causing a GVHD response.

Therapeutic effects of signal transducer and activator of transcription 3 siRNA on human breast cancer in xenograft mice.

BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) is usually constitutively activated in a variety of malignancies. It directly contributes to tumorigenesis, invasion, and metastasis. The surgical treatment of breast cancer has made no breakthroughs in terms of treatment effect, in spite of its long history. Current biotherapies bring a note of optimism to breast cancer treatment. To explore the possibility of a siRNA targeted STAT3 blocking treatment for over-activated tumor cells, we evaluated the efficacy of a STAT3 siRNA on human breast cancer cells in vitro and in vivo. METHODS: Three MCF-7 human breast cancer cell lines were tested: control MCF-7 cells, non-specific siRNA transfected MCF-7 cells and STAT3 siRNA transfected MCF-7 cells. Expression of STAT3 in MCF-7 cells was inhibited by RNA interference (RNAi). The STAT3 mRNA and protein levels were detected by semi-quantity RT-PCR and Western blotting. Cell proliferation and apoptosis were determined by MTT method and flow cytometry. The three groups of MCF-7 cells mentioned above were transplanted subcutanuously into nude mice and their tumorgenic ability observed. The STAT3 mRNA and protein levels of the samples from tumors in different groups were determined by semi-quantity RT-PCR and Western blotting and compared. RESULTS: In STAT3 siRNA transfected MCF-7 cells, the expressions (STAT3/ß-actin) of STAT3 mRNA (0.327 ± 0.020) and protein (0.153 ± 0.006) were significantly lower than that in control MCF-7 cells (mRNA 1.093 ± 0.018, protein 1.374 ± 0.022) and non-specific siRNA transfected MCF-7 cells (mRNA 1.035 ± 0.050, protein 1.320 ± 0.033) (P < 0.05). MTT showed that cell proliferation was significantly reduced and the cell growth inhibition ratio in the STAT3-siRNA group was (44.00 ± 5.10)%, significantly higher than that in non-specific siRNA transfected MCF-7 cells ((16.10 ± 1.05)%, P < 0.05). Flow cytometry results showed that more apoptosis was observed in the STAT3-siRNA group. The rate of apoptosis was (14.79 ± 0.22)%, much higher than in control MCF-7 cells (7.06 ± 0.71) and non-specific siRNA transfected MCF-7 cells (8.45 ± 0.43) (P < 0.05). The tumor growth in the STAT3 siRNA transfected MCF-7 cells was significantly slower than in the two control groups. On the 22th day after transplantation the tumor weight ((21.40 ± 10.57) mg) and volume ((41.15 ± 12.17) mm(3)) in the STAT3 siRNA transfected group were significantly lower than in control group (weight (88.60 ± 12.16) mg, volume (118.45 ± 24.68) mm(3)) and non-specific siRNA transfected group (weight (57.20 ± 21.86) mg, volume (101.36 ± 21.90) mm(3)) (P < 0.05). Both the STAT3 mRNA and protein levels in the tumors from the STAT3 siRNA transfected group were significantly lower than in the tumors from the two control groups. CONCLUSIONS: STAT3 siRNA can effectively silence the STAT3 gene in vitro and in vivo, increase cell apoptosis rate and significantly decrease cell proliferation, which inhibits the growth of breast cancer cell in vitro. Tumor growth of xenograft mice is significantly inhibited. The results obtained in vivo are in consistency with those in vitro. STAT3 may be a novel therapeutic target for breast cancer and RNA interference has potential clinical application.