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Objective To select bronchial epithelial BEAS-2B cell line as control, cellular uptake and localization of Hematoporphyrin derivatives (HPD) were compared between normal bronchial epithelial cells (BEAS-2B) and different lung cancer cell lines (lung adenocarcinoma A549, lung squamous carcinoma H520 and lung small cell carcinoma H446), as well as among lung cancer cell lines. Methods The standard curve of HPD was drawn based on the fluorescence intensity of different concentrations of HPD measured by a multifunctional microplate reader. Cellular uptake of HPD was quantified by fluorescence intensity with a multifunctional microplate reader and flow cytometry. Cellular distribution of HPD was observed and compared by laser scanning confocal microscopy. Results In a certain concentration range, HPD concentration was positively correlated with fluorescence intensity. Cellular uptake of HPD increased with time. HPD was rapidly accumulated in BEAS-2B, A549 and H446 cells after incubating with HPD for 12h, while the uptake rate of HPD was slow in H520 cells and accelerated after 24h. In the 24h and 48h experimental groups, the average fluorescence intensity in the four cells was significantly different (P P = 0.0766 > 0.05), and both were higher than those of H520 cells (P

Background: Choroidal melanoma (CM) accounts for 70% of uveal melanomas and is prone to metastasize and invade. Previous studies have reported that forkhead box protein 3 (FOXP3) is associated with carcinogenesis, however, the effect of FOXP3 on CM remains unclear. The purpose of the study is to explore the role of FOXP3 in the progression of CM and to elucidate its related mechanisms. Methods: FOXP3 protein expression was detected in CM clinical specimens and CM cells. We then established a cell line with stable FOXP3 knockout as well as a cell line that transiently overexpressed FOXP3, and their transfection efficiencies were detected by Western blotting (WB). The effects of FOXP3 on cell biological functions and epithelial-mesenchymal transition (EMT) in CM were verified via the CCK-8 assay, monoclonal formation assay, migration and invasion assays, WB and tumorigenesis assay in nude mice in vivo. We also demontrated that FOXP3 promoted CM development through the Wnt5a/CaMKII signaling pathway. Results: The level of FOXP3 was found to be upregulated in CM clinical specimens and CM cells. The overexpression of FOXP3 promoted the proliferation, migration, invasion, and EMT process of CM cells in vitro, while the knockdown of FOXP3 inhibited these cell functions in vitro and tumor growth in vivo. In addition, FOXP3 was found promoting the progression of CM, including EMT, through the Wnt5a/CaMKII signaling pathway. Conclusion: This study demonstrated that FOXP3 promoted the development of choroidal melanoma through the Wnt5a/CaMKII signaling pathway as an oncogenic factor of CM, and thereby provides a novel potential target for the pathogenesis of CM.

We reported a delayed diagnosised Chinese JATD case with mild skeletal phenotype, and presented with renal insufficiency as the initial symptom of disease onset. Novel bilateral c.2789C>T (p.S930L) mutations in WDR60 gene were identified. Our report will help to improve our awareness and diagnosibility for this disease in China.

All mutations in exon 9 of the Cullin3 gene associated with pseudohypoaldosteronism type II (PHA II) contribute to exon skipping to different degrees, but the specific molecular mechanism of this aberrant splicing is still unclear. The aims of this study were to investigate the regulatory mechanism underlying two synonymous splicing events, c.1221A > G (p. Glu407Glu) and c.1236G > A (p. Leu412Leu), and to discover a therapeutic strategy for correcting this aberrant splicing by targeting potential regulatory sites. Through a series of RNA pulldown, silver staining, western blotting, small interfering RNA knockdown, in vitro overexpression and single or double site-directed mutagenesis experiments, we first explored the pathogenesis of exon 9 skipping caused by mutations in the CUL3 gene and verified that the main splicing regulators associated with the synonymous c.1221A > G and c.1236G > A mutations were heterogeneous nuclear ribonucleoproteins. In addition, we verified that introducing another synonymous mutation, c.1224A > G (A18G), significantly rescued the abnormal splicing caused by c.1221A > G and c.1236G > A, highlighting the therapeutic potential for the treatment of PHA II.

The molecular mechanism underlying hyperuricemia-induced lipid metabolism disorders is not clear. The purpose of the current study was to investigate the mechanism of lipid disturbances in a hyperuricemia mice model. RNA-Seq showed that differentially expressed genes (DEGs) in the fatty acid synthesis signaling pathway were mainly enriched and CXCL-13 was significantly enriched in protein-protein interaction networks. Western blotting, Q-PCR, and immunofluorescence results further showed that hyperuricemia upregulated CXCL-13 and disturbed lipid metabolism in vivo and in vitro. Furthermore, CXCL-13 alone also promoted the accumulation of lipid droplets and upregulated the expression of FAS and SREBP1, blocking AMPK signaling and activating the PKC and P38 signaling pathways. Silencing CXCL-13 reversed uric-acid-induced lipid droplet accumulation, which further downregulated FAS and SREBP1 expression, inhibited the p38 and PKC signaling, and activated AMPK signaling. In conclusion, hyperuricemia induces lipid metabolism disorders via the CXCL-13 pathway, making CXCL-13 a key regulatory factor linking hyperuricemia and lipid metabolism disorders. These results may provide novel insights for the treatment of hyperuricemia.

Angiogenesis and vasculogenic mimicry (VM) are considered to be the main processes to ensure tumor blood supply during the proliferation and metastasis of choroidal melanoma (CM). The traditional antimalarial drug artesunate (ART) has some potential anti-CM effects; however, the underlying mechanisms remain unclarified. Recent studies have shown that the Wnt5a/calmodulin-dependent kinase II (CaMKII) signaling pathway has a close correlation with angiogenesis and VM formation. This study demonstrated that ART eliminated VM formation by inhibiting the aforementioned signaling pathway in CM cells. The microvessel sprouting of the mouse aortic rings and the microvessel density of chicken chorioallantoic membrane (CAM) decreased significantly after ART treatment. VM formation assay and periodic acid schiff (PAS) staining revealed that ART inhibited VM formation in CM. Moreover, ART downregulated the expression levels of the angiogenesis-related proteins vascular endothelial growth factor receptor (VEGFR) 2, platelet-derived growth factor receptor (PDGFR) and vascular endothelial growth factor (VEGF) A, and VM-related proteins ephrin type-A receptor (EphA) 2 and vascular endothelial (VE)-cadherin. The expression of hypoxia-inducible factor (HIF)-1α, Wnt5a, and phosphorylated CaMKII was also downregulated after ART treatment. In addition, we further demonstrated that ART inhibited the proliferation, migration, and invasion of OCM-1 and C918 cells. Collectively, our results suggested that ART inhibited angiogenesis and VM formation of choroidal melanoma likely by regulating the Wnt5a/CaMKII signaling pathway. These findings further supported the feasibility of ART for cancer therapy.

To investigate the effect and mechanism of 630-nm laser on human lung adenocarcinoma cell xenograft model in nude mice mediated by hematoporphyrin derivatives (HPD) and provide theoretical basis for clinical photodynamic therapy (PDT). Human lung adenocarcinoma cell xenograft model in nude mice was established and randomly divided into four groups: control group, pure photosensitizer group, pure irradiation group, and photodynamic treatment group. The tumor volume growth was compared, and the tumor growth inhibition rate was calculated. HE staining was used for routine pathological observation of tumor sections, and gross conditions of cells, interstitium, and blood vessels in several groups of tumor tissues were observed. TUNEL staining was used to observe and compare the apoptosis induced by photodynamic therapy. Real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression level of angiogenesis-related factors VEGF, HIF-1α and apoptosis-related factors Bax and Bcl-2 mRNA in the transplanted tumor tissues. Western blot was employed to detect the expression of angiogenesis-related proteins VEGF, HIF-1α and apoptosis-related proteins Bax, Caspase-3, and Bcl-2. Compared with the other three groups, the tumor growth inhibition rate of the photodynamic treatment group was significantly increased and the difference was statistically significant (P < 0.05). HE staining showed that the animal model of lung adenocarcinoma A549 was successfully established. TUNEL staining revealed that more apoptotic cells were found in the photodynamic treatment group, and the apoptosis index was calculated. Compared with the other three groups, the difference was statistically significant (P < 0.05). RT-PCR results showed that compared with the other three groups, the mRNA expressions of VEGF, HIF-1α, and Bcl-2 in the photodynamic treatment group decreased, while the expression of Bax mRNA increased(P < 0.05), and the differences were statistically significant. Western blot results showed that protein expressions of VEGF, HIF-1α, and Bcl-2 decreased in the photodynamic treatment group, while protein expression level of Bax and Caspase-3 increased (P < 0.05), indicating statistically significant differences. The 630-nm laser mediated by hematoporphyrin derivatives can significantly inhibit the growth of human lung adenocarcinoma xenograft tumor in nude mice, the mechanism of which is related to the inhibition of tumor angiogenesis by down-regulating VEGF and HIF-1α gene expression, and the promotion of tumor apoptosis by up-regulating Bax, Caspase-3, and down-regulating Bcl-2 gene expression.

Objective To examine the effects of gingival mesenchymal stem cells (GMSCs) on inflammatory macrophages upon oxidized low-density lipoprotein (ox-LDL) stimulation and evaluate therapeutic potential of GMSCs on mouse model of periodontitis associated with hyperlipidemia. Methods in vitro, GMSCs were co-cultured with macrophages for 48 h in the absence or presence of M1 polarizing conditions and oxidized low-density lipoprotein in the transwell system. The supernatants were collected for ELISA. M1 and M2 markers of macrophages were analyzed by flow cytometry and PCR, and lipid accumulation was assessed by oil red O staining. in vivo, eighteen mice were divided into three groups (n = 6): Group A (periodontally healthy mice as control), Group B (periodontitis mice with hyperlipidemia), Group C (periodontitis mice with hyperlipidemia with the transplantation of GMSCs). The serum levels of cholesterol and inflammatory factors were measured by automatic analyzer. Bone regeneration was evaluated by Masson staining. Results When co-cultured with GMSCs, the M1 markers of Tumor Necrosis Factor (TNF) -α, Interleukin (IL) -6, Interleukin (IL) -1β, CD86, and Human Leukocyte Antigen (HLA) -DR were significantly reduced. In contrast, M2 markers such as Interleukin(IL) -10 and CD206 were moderately increased. Similar results were obtained in the cell culture supernatants. In animal experiment, GMSCs suppressed the expression of sterol regulatory element binding transcription factor 1c (SREBP-1c) and elevated the levels of peroxisome proliferator-activated receptor alpha (PPARα) and peroxisome proliferator activator receptor- coactivator 1(PGC-1α) in the liver, attenuated cholesterol dysfunction via the downregulation of low-density lipoprotein (LDL) and total cholesterol (TC), and the upregulation of high-density lipoprotein (HDL), and decreased the levels of TNF-α and IL-6. Moreover, GMSC treatment improved bone regeneration. Conclusion GMSCs inhibit the activation of M1 macrophages, regulate lipid metabolism and reduce inflammatory response, and promote bone regeneration in mouse model of periodontitis associated with hyperlipidemia.

Background Gut microbiota has been proposed to be related to the pathogenesis of pulmonary diseases such as asthma and lung cancer, according to the gut-lung axis. However, little is known about the roles of gut microbiota in the pathogenesis of bronchopulmonary dysplasia (BPD). This study was designed to investigate the changes of gut microbiota in neonatal mice with BPD. Methods BPD model was induced through exposure to high concentration of oxygen. Hematoxylin and eosin (H&E) staining was utilized to determine the modeling efficiency. Stool samples were collected from the distal colon for the sequencing of V3–V4 regions of 16S rRNA, in order to analyze the gut microbiota diversity. Results Alpha diversity indicated that there were no statistical differences in the richness of gut microbiota between BPD model group and control group on day 7, 14 and 21. Beta diversity analysis showed that there were statistical differences in the gut microbiota on day 14 (R = 0.368, p = 0.021). Linear discriminant analysis effect size (LEfSe) showed that there were 22 markers with statistical differences on day 14 (p < 0.05), while those on day 7 and 21 were 3 and 4, respectively. Functional prediction analysis showed that the top three metabolic pathways were signal transduction (PFDR = 0.037), glycan biosynthesis and metabolism (PFDR = 0.032), and metabolism of terpenoids and polyketides (PFDR = 0.049). Conclusions BPD mice showed disorder of gut microbiota, which may involve specific metabolic pathways in the early stage. With the progression of neonatal maturity, the differences of the gut microbiota between the two groups would gradually disappear.

Xiao Zhang,1,&ast; Juntao Wang,1,&ast; Aihua Sui,2 Nannan Zhang,1 Qiulan Lv,2 Zhenfang Liu3 1Department of Anesthesiology, The Affiliated Hospital of Qingdao University, Qingdao, 266000, People’s Republic of China; 2Medical Research Center, The Affiliated Hospital of Qingdao University, Qingdao, 266000, People’s Republic of China; 3Department of Emergency, The Affiliated Hospital of Qingdao University, Qingdao, 266000, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Zhenfang Liu Email liuzhenfangzf@outlook.comBackground and Objective: Neuropathic pain remains a clinical challenge with limited effective treatments. Previous studies have found that magnolol (Mag), an ingredient existing in some herbs, showed neuroprotective effect. However, it remains unclear whether Mag can alleviate neuropathic pain.Methods: Chronic constriction injury (CCI) is used as the neuropathic pain model. Mice were randomly divided into 5 groups: Sham, CCI, CCI + 5, 10, 30 mg/kg Mag groups. Thermal and mechanical paw withdrawal threshold were performed at baseline and on the 3rd, 5th, 7th, 14th days post-surgery. Lumbar spinal cord and blood samples were collected on the 14th day. Blood lipid profile, kidney and liver functions, as well as the activation of microglia were evaluated, along with the related signal pathway examined using multiple methods including immunohistochemistry, RT-PCR and Western blot.Results: Mag alleviated thermal and mechanical hypersensitivity in CCI mice. CCI activated microglia and upregulated the expression of P2Y12, while Mag inhibited microglial activation, and downregulated the expression of P2Y12. Mag also blocked the activation of p38 mitogen-activated protein kinase (MAPK) and other pain-related cytokines such as IL-6, TNF-α and IL-1β.Conclusion: The findings indicate that Mag has antinociceptive effect on neuropathic pain, probably mediated through P2Y12 receptors and p38 MAPK mediated pathways. With its relatively safe profile, Mag may be a potential therapeutic agent for neuropathic pain.Keywords: magnolol, neuropathic pain, P2Y12, CCI, MAPK, cytokines

Sea cucumber is a kind of food. Holothurian glycosaminoglycan (hGAG) is extracted from the body wall of the sea cucumber. Administration of hGAG and cisplatin (DDP) together to treat lung cancer was investigated. Lung adenocarcinoma A549 cells were cultured and divided into 4 groups: control group, hGAG 100 µg/mL group, DDP 3 µg/mL group, and hGAG 100 µg/mL + DDP 3 µg/mL group. Cell inhibition and apoptosis was evaluated by CCK8 and Hoechst33258 staining. Cell cycle was tested by Annexin V-FITC/PI (propidium iodide) double-staining and flow cytometry. The expression of mRNA and protein of Bcl-2, Bax, caspase-3, and survivin were detected by reverse transcriptase-polymerase chain reaction and Western blot, respectively. The results showed that hGAG combined with DDP enhanced the inhibitory effect of DDP on A549 lung cells through apoptosis pathway. The mechanism of apoptosis may be related to the reduction of Bcl-2 and survivin, as well as the ascension of Bax and caspase-3. hGAG could promote A549 cell cycle arrest in G1 and G2 phase and improve the DDP chemotherapy effects on A549 cells.

Increasing evidence indicates that miR-429 is involved in tumor suppression in various human cancers. however, its role in hepatocellular carcinoma (HCC) remains unclear. In the present study, we found that miR-429 was significantly downregulated in HCC tissue samples and cell lines. Upregulation of miR-429 markedly suppressed proliferation and migration of HCC cells. Moreover, we identified TRAF6 as a direct target of miR-429. Downregulation of TRAF6 partially attenuated the oncogenic effect of anti‑miR-429 on HCC cells. Ectopic expression of miR-429 in HCC cells inhibited TCF-4 activity as well as nuclear accumulation of P65 and expression of the NF-κB targets c-Myc and phosphorylation of TAK1. In a nude xenograft model, miR-429 upregulation significantly decreased HCC growth. In conclusion, by targeting TRAF6, miR-429 is downregulated in HCC and inhibits HCC cell proliferation and motility. Our data suggest that miR-429 may serve as a potential anticancer target for the treatment of HCC.

Three dimensional (3D) culture has gradually become a research hotspot in the field of drug screening, stem cell research, and tissue engineering due to its more physiological-like morphology and function. In this study, we compared the differences of cell proliferation, population, protein expression and chemoresistance profiles between two dimensional (2D) and 3D culture of acute lymphoblastic leukemia (ALL) Jurkat cell line. Polycaprolactone (PCL) is used for 3D culture owing to its biochemical properties and compatibility. Culturing of ALL Jurkat cell line in collagen type I coated polycaprolactone scaffold for 168 h increased cell proliferation, attachment, as well as the drug resistance to cytarabine (Ara-C) and daunorubicin (DNR) without changing the original CD2+CD3+CD4+dimCD8−CD34−CD45+dim phenotype, compared to uncoated PCL scaffold and tissue culture plate systems. Molecularly, increased chemoresistance is associated with the upregulation of discoidin domain receptor 1 (DDR1) and transcription factor STAT3. Inhibition of DDR1 activity by DDR1-specific inhibitor DDR-IN-1 accelerated cell death in the presence of Ara-C, DNR or their combination. These results demonstrated that 3D culture enhances chemoresistance of ALL Jurkat cell line by increasing DDR1 expression. Importantly, the cell adhesion-mediated drug resistance induced by DDR1 in the scaffold was similar to the clinical situation, indicating the 3D culture of cancer cells recapitulate the in vivo tumor environment and this platform can be used as a promising pre-clinic drug-screen system.

In this study, we investigated the association between genetic polymorphisms of XRCC1 Arg399Gln (G→A) and response to oxaliplatin-based chemotherapy in advanced colorectal cancer. XRCC1 genotypes of totally 99 patients (37 stage III and 62 stage IV) with advanced colorectal cancer treated with oxaliplatin-based chemotherapy were detected by the TaqMan-MGB probe allelic discrimination method, and clinical response of 62 patients in stage IV after 2 to 3 cycles of chemotherapy were evaluated. Also, time to progression (TTP) of all patients was evaluated. The results showed that of the genotype frequencies in all patients, up to 52.53% were G/G genotype, 9.09% were A/A genotype, and 38.38% were G/A genotype. The response rate (CR + PR) of 62 patients in stage IV was 61.29% (19/31). Patients with G/G genotype showed enhanced response to chemotherapy compared with those with G/A + A/A (x(2) = 5.6, p = .029; Odds Ratio (OR) = 3.845, 95% Confidence Interval (CI) = 1.231 ∼ 12.01, p = .018). Individuals with the G/G genotype had a TTP of 10.0 (8.88-11.12) months, and those with the G/A + A/A genotype had a TTP of 5.0 (4.26-5.74) months. The Log-Rank test was marginally significant (x(2) = 29.20, p < .01). The Cox proportional hazards model, adjusted for stage, performance status, and chemotherapy regimen showed that only XRCC1 G/G genotype increases the OR significantly (OR = 3.555; 95% CI, 2.119 ∼ 5.963; p < .01). These results indicate that XRCC1 Arg399Gln polymorphism is associated with response to oxaliplantin-based chemotherapy and TTP in advanced colorectal cancer in Chinese population. It is proposed that the XRCC1 Arg399Gln polymorphism should be routinely detected to screen patients who are more likely to benefit from oxaliplantin-based treatment.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer therapeutics that exhibits the ability to preferentially induce apoptosis in malignant cells. RGD peptides bind to the integrins, ανβ3 and ανβ5, which are selectively expressed in tumor neovasculature and at the surface of certain tumor cells. To enhance the antitumor effect, an RGD-TRAIL protein, in which TRAIL was fused with the RGD motif-containing cell adhesive sequence, GRGDNP (gly-arg-gly-asp-asn-pro), was constructed and evaluated for bioactivity. The soluble TRAIL and RGD-TRAIL proteins were expressed in Escherichia coli BL21 (DE3) and purified using a non-denaturing method. The antitumor effect of the purified RGD-TRAIL on cell proliferation was evaluated in vitro using MTT and wound healing assays and cell apoptosis was assessed by Hoechst 33342 staining and PARP expression analysis. The results revealed that RGD-TRAIL inhibited the proliferation of multiple tumor cell lines (A549, NCI-H1299 and HCC827). Western blot analysis demonstrated that the treatment of tumor cells with RGD-TRAIL activated the apoptotic pathway by the cleavage of PARP, in the same way as wild-type TRAIL (wtTRAIL). These results demonstrate that RGD-TRAIL possesses more potent antitumor effects than wtTRAIL and, therefore, merits further investigation in preclinical and clinical studies.

In this work, the full-length open reading frame of the human interleukin-10 (hIL-10) gene was amplified through reverse transcription–polymerase chain reaction (RT-PCR), and then PCR products were inserted into pcDNA4/HisMax to construct an eukaryotic expression vector. After optimization by green fluorescent protein (GFP), recombinant hIL-10 genes were transfected and expressed in rabbit synovial cells compounded with liposome in vitro. In cell culture supernatant, rhIL-10 was detected using enzyme-linked immunosorbent assay (ELISA) at time intervals of 12 hours, 24 hours, 48 hours, 72 hours, 7 days, and 14 days. After 12 hours of transfection, ELISA showed that transgene expression of hIL-10 in rabbit synovial cells was elevated; at 72 hours, hIL-10 expression reached its peak value; and then it declined gradually until 7 days, compared with the control. After 14 days, transgene expression ceased. Gene cloning of hIL-10 and its transgene expression in synovial cells therefore gives a basis for the gene therapy of rheumatoid arthritis.

Nucleophosmin (NPM) and human epidermal growth factor receptor-2 (Her-2) are abnormally expressed in various types of human malignant tumors, including gastric cancer, and have been closely associated with cancer chemoresistance. However, their interaction and roles in oxaliplatin resistance are not fully understood. Therefore, the present study aimed to elucidate the relationship between NPM and Her-2 in gastric cancer cell lines and clinical samples, and further investigated their role in the resistance of gastric cancer to oxaliplatin. Western blotting and reverse transcription-quantitative polymerase chain reaction confirmed that NPM and Her-2 expression were significantly upregulated in gastric cancer cells and clinical samples, and that their expression levels were strongly correlated. However, Her-2 expression was not affected by upregulation or downregulation of NPM expression in gastric cancer cells. Cell counting kit-8 assays demonstrated that the cell sensitivity to oxaliplatin decreased simultaneously with an increase in NPM expression. Furthermore, inhibition of Her-2 expression using trastuzumab significantly increased the sensitivity of the cells to oxaliplatin, which occurred simultaneously with the downregulation of NPM. These results indicated that inhibition of NPM, as a Her-2 downstream signal, may be a novel strategy to overcome oxaliplatin-resistant gastric cancer, and that trastuzumab and oxaliplatin may exhibit a synergistic antitumor effect in Her-2-positive gastric cancer cells.

Triple-negative breast cancer (TNBC), characterized by distinct biological and clinicopathological features, has a poor prognosis due to lack of effective therapeutic targets. Our previous data revealed that high levels of USP39 were selectively present in TNBC samples compared with their normal breast tissue samples and USP39 was also expressed at different levels in cultured TNBC cells and normal breast cells. Yet, the underlying cellular and molecular mechanisms of USP39 remain unclear. In the present study, we describe a doxycycline (DOX)-regulated lentiviral vector system expressing shRNA or cDNA of the USP39 gene in the TNBC cell line MDA-MB-231. USP39 expression was knocked down by the miR-30-based inducible lentiviral short hairpin RNA (shRNA) delivery system or overexpressed by the inducible cDNA system. The inducible shRNA-mediated downregulation of USP39 expression markedly reduced the proliferation and colony-forming ability of MDA-MB-231 cells, while overexpression of USP39 by the inducible system did not promote cancer cell proliferation. The lentiviral vector-mediated Tet-on system demonstrated efficient and inducible knockdown of USP39 or overexpression of USP39 in TNBC cells, facilitating a wide variety of applications for gene knockdown and overexpression experiments in gene functional studies in vitro and in vivo.

The aim of this study was to construct RNA interference (RNAi) lentiviral vector particles targeting the mouse tumor necrosis factor-α (TNF-α) gene. Three types of small interfering RNA (siRNA) targeting the mouse TNF-α gene were designed, synthesized and transfected into RAW264.7 cells. Screening was performed to identify the siRNA sequence exhibiting the highest inhibition efficiency; based on this, recombinant lentiviral plasmids were constructed and co-transfected into 293T cells with packaging plasmids for the production of lentiviral particles. The screening results showed that the TNF-α mRNA expression levels of the three siRNA groups were significantly lower than those of the negative control group, with the highest inhibition rate in the siRNA2 group (83.09%). Similarly, the expression levels of TNF-α protein in the three siRNA groups were significantly lower than those of the negative control group, and the highest inhibition rate was found in the siRNA2 group (51.16%). The mRNA expression of interleukin (IL)-1β and IL-6 showed no significant difference among the siRNA groups and the negative control. The recombinant lentiviral shuttle plasmid was constructed, and electrophoresis revealed the polymerase chain reaction product to be 343 bp, while that of the empty vector was 306 bp; DNA sequencing showed partial insertion. The virus titer was calculated to be 2×106 TU/µl. In conclusion, RNAi lentiviral vector particles targeting the mouse TNF-α gene were successfully obtained in the present study. This method may be used to produce lentiviral vector for the in vivo study of RNAi gene therapy targeting TNF-α.

Although the survival of lung cancer patients has improved significantly due to the development of early detective tools, lung cancer remains a leading cause of cancer-related death after curative surgery. So it is extremely important for cancer patients to predict early metastasis, especially pleural dissemination, the most frequent type of recurrence in patients after surgery. Based on a retrospective study of 86 curatively resected lung cancer patients (training set), we determined a cutoff value of NSE mRNA using receiver-operating characteristic curve. Then, we prospectively used this cutoff value to validate the risk of pleural recurrence in a new cohort of 81 lung cancer patients (validation set) between April 2009 and June 2010 by real-time reverse transcriptase-polymerase chain reaction. During the median 27 months of postoperative surveillance, 16 of the 81 patients died, and 9 of the 16 developed pleural metastases. Multivariate analysis with the Cox proportional hazards model showed that positive NSE mRNA was a significant independent risk factor with both overall survival and pleural recurrence-free survival (both P < 0.0001) as end points which were significantly worse in patients with positive NSE mRNA (P < 0.0001). These results indicate that quantitative NSE mRNA in pleural fluid is a reliable prognostic indicator of pleural recurrence in the clinical setting.

Recent diagnostic procedure advances have considerably improved early lung cancer detection. However, the invasive, unpleasant, and inconvenient nature of current diagnostic procedures limits their application. There is a great need for novel noninvasive biomarkers for early lung cancer diagnosis. In the present study, we aimed to determine whether microRNA (miRNA) blood signatures are suitable for early detection of lung cancer. Using quantitative reverse transcriptase PCR analysis, we first selected and identified three aberrant plasma expression miRNAs (miR-21, miR-145, and miR-155) in a training set of 62 patients and 60 healthy smokers to define a panel that had high diagnostic efficiency for lung cancer. Then, we validated the detective ability of this miRNA panel in a testing set of 34 malignant tumor patients, 30 patients with benign pulmonary nodules and 32 healthy smokers. In the training set, miR-21 and miR-155 showed higher plasma expression levels, whereas miR-145 showed a lower expression level in patients with malignant cancer, compared with healthy controls (P ≤ 0.001). The three miRNAs used in combination produced the area under receiver operating characteristic curve at 0.847, which helped distinguish lung cancer from healthy smokers with 69.4% sensitivity and 78.3% specificity. A logistic regression model with the best prediction was constructed on the basis of miR-21, miR-145, and miR-155. Validation of the miRNA panel in the testing set confirmed their diagnostic value, which yields a significant improvement over any single one. Plasma miR-21, miR-145, and miR-155 have strong potential as novel noninvasive biomarkers for early detection of lung cancer.

Increased knowledge of molecular alterations involved in gastric carcinogenesis has provided useful information for diagnosis and prediction. In this study, we investigated the clinicopathological significance of TFF3 expression and Her-2/neu status in gastric adenocarcinoma and explored the correlation between TFF3 expression and Her-2/neu status. A hundred and twenty-six (126) patients having undergone curative gastrectomy were enrolled. Immunohistochemistry for TFF3 was performed on tumor tissues and non-neoplastic resection margins. Her-2/neu status was assessed by immunohistochemical staining and fluorescence in situ hybridization (FISH) on tumor tissues. As a result, TFF3 expression and Her-2/neu positivity were detected in 46.8% and 11.9% of the cases, respectively. Patients with negative TFF3 exhibited longer survival than the positive group (P=0.0142), while patients with positive Her-2/neu only showed a tendency toward longer overall survival (P>0.05). However, Her-2/neu was significantly associated with improved disease-free survival (P=0.0234). Multivariate analysis demonstrated Her-2/neu and TFF3 to be independent prognostic indicators of recurrence, and a significantly poor prognosis for expression of TFF3 in patients with Her-2/neu negative tumors. TFF3 is an independent indicator for overall survival in gastric cancer, while Her-2/neu, as a marker of long time survival prediction, seems to be limited.

The aim of the study was to investigate the expression levels of ‘NPM’/nucleophosmin/B23 and human epidermal growth factor receptor 2 (Her-2)/neu in gastric cancer (GC) and corresponding non-malignant tissues, correlation with their clinicopathological parameters and the relationship of nucleophosmin/B23 and Her-2/neu in the occurrence and development of GC. A total of 131 postoperative patients were examined for nucleophosmin/B23 expression by immuno-histochemistry and for Her-2/neu expression by fluorescence in situ hybridization with the median follow-up period of 38 months. The positive expression rates of nucleophosmin (NPM) in neoplastic tissues and adjacent gastric mucosa were 65.6% and 52.7%, respectively. Nucleophosmin/B23 levels were linked to more advanced tumor stages, poor prognosis, and likelihood of recurrence (p

Hepatocellular carcinoma (HCC) overexpresses insulin-like growth factor-I receptor (IGF-IR), as compared with normal hepatocytes. Since IGF-1R-mediated signaling promotes survival, oncogenic transformation and tumor growth and spread, it represents a potential target for treating HCC. Here, we have generated a murine anti-IGF-1R antibody, 4F2, that recognizes the IGF-IRα subunit and blocks in vitro IGF-I and IGF-II-induced cell proliferation of SMMC-7721 and Bel-7402 HCC cell lines. 4F2 can inhibit IGF-IR autophosphorylation, IRS-1 phosphorylation and the activation of the major downstream signaling molecules AKT and mitogen-activated protein kinase. Additionally, we observed a moderate increase in apoptosis as demonstrated by detection of changes in the expression of the pro-apoptotic and anti-apoptotic proteins Bax and Bcl-2 after 4F2 treatment. Combined treatment with 4F2 and doxorubicin was more effective in reducing cell proliferation and promoting apoptosis than either agent alone. These data support that therapeutic anti-IGF-IR antibodies are potential new agents for treating HCC.