Fano resonance sensing based on coupling of a sub-wavelength grating and an all-dielectric multilayer film under angle modulation.

Based on the diffraction effect of the waveguide grating, a hybrid structure of an all-dielectric multilayer film and a sub-wavelength dielectric grating is proposed. The discrete state provided in the sub-wavelength waveguide grating couples with the continuous state provided in the Fabry-Perot cavity (F-P cavity) containing the photonic crystal to form a Fano resonance. The relationship between the structural parameter and the incident angle is analyzed by numerical simulation. The result shows that the figure of merit (FOM) value is 533.2RIU-1. This structure provides an effective theoretical basis for the realization of Fano resonance in the all-dielectric sensing structure.

γ-Synuclein is Closely Involved in Autophagy that Protects Colon Cancer Cell from Endoplasmic Reticulum Stress.

BACKGROUND: In previous studies, we provided evidence suggesting the involvement of γ-synuclein in growth, invasion, and metastasis of colon cancer cells in vitro and in vivo. Among γ-synuclein downstream genes, the microtubule-associated protein 1 Light Chain 3 (LC3), an autophagy gene, was screened by gene expression profile chip analysis. OBJECTIVE: We planned to investigate the functional effects of γ-synuclein on autophagy induced by ER stress in colon cancer cells. METHODS: We investigated the functional effects of γ-synuclein on autophagy and apoptosis induced by Thapsigargin (TG), ER stress-inducing agent, in colon cancer cell lines using immunofluorescence staining, RT-PCR, western blot, CCK8 test, flow cytometry analysis, and transmission electron microscopy. To further determine how γ-synuclein regulated autophagy and apoptosis, PD98059 (ERK inhibitor), SP600125 (ERK inhibitor), anisomycin (JNK activator), and c-Jun siRNA were used respectively in γ-synuclein siRNA transfected HCT116 cells. Then, autophagy proteins, apoptosis proteins, and pathway proteins were detected by western blot analysis. The expression of autophagy genes was assessed by RT-PCR. RESULTS: Our data showed that ER stress-induced colon cancer cells autophagy mainly in the early stage (0-24h) and apoptosis mainly in the late stage (24-48h). ER stress up-regulated γ-synuclein gene and protein expression in colon cancer cells, accompanied by autophagy. γ-synuclein protected HCT116 cells by enhancing autophagy in the early stage (0-24h) through activation of ERK and JNK pathway and inhibiting apoptosis in the late stage (24-48h) through inhibition of the JNK pathway. γ-synuclein could promote autophagy via the JNK pathway activation of ATG genes, LC3, Beclin 1, and ATG7. γ-synuclein may play a role in the transition between autophagy and apoptosis in our model. CONCLUSION: Overall, we provided the first experimental evidence to show that γ-synuclein may play an important role in autophagy that protects colon cancer cells from ER stress. Therefore, our data suggest a new molecular mechanism for γ-synuclein-mediated CRC progression.

CILP2 overexpression correlates with tumor progression and poor prognosis in patients with colorectal cancer in The Cancer Genome Atlas (TCGA) study.

BACKGROUND: Genetic alterations play an important role in the progression of colorectal cancer (CRC). Identifying new biomarkers to assess the prognosis of patients with CRC is critical. Cartilage intermediate layer protein 2 (CILP2) gene, screened from TCGA database by bioinformatics, may be closely related to the progression of CRC. CILP2 was barely reported with clinical features of tumors. MATERIALS AND METHODS: Clinical information and RNA-seq data were derived from TCGA colorectal carcinoma cohort. CILP2 expression at mRNA level was estimated by bioinformatical analysis of TCGA cases. Tissue microarray (TMA) was constructed containing paraffin-embedded 64 pairs of CRC and matched adjacent normal tissues. The expression at the protein level was detected in 64 pairs of CRC and matched adjacent normal tissues by immunohistochemical analysis. CILP2 expression level and its clinical value were estimated by bioinformatical analysis with linear and logistic regression. Survival analysis was performed between high and low groups of CILP2 expression by Cox regression analysis, and the P value was calculated by the log-rank test. The Kaplan-Meier curves were tested by the log-rank test. RESULTS: CILP2 was statistically significantly higher expressed in the CRC tissues when compared with paired adjacent normal tissues in TCGA cohort (P < 0.001) and in the TMA cohort (P = 0.001). Also, CILP2 high expression was strongly correlated with T3/4 stage (P = 0.001), N1/2/3 stage (P = 0.005), M1 stage (P = 0.048), and higher clinical stage (UICC 2010 stage) (P < 0.001) in TCGA cohort, and also positively associated with T3/4 stage (P = 0.022) and higher clinical stage (UICC 2010 stage) (P = 0.03) in TMA cohort. Furthermore, CILP2 overexpression predicted poor prognosis and could be an independent prognostic factor (P = 0.003). CONCLUSION: We revealed that CILP2 is associated with advanced stages and could play a role as an independent predictor of poor survival in CRC.

Effect of PD-1 knockout by CRISPR/Cas9 system on proliferation and IFN-γ secretion in human T lymphocytes / 中国肿瘤生物治疗杂志

@#Objective: : To explore the effect of PD-1 gene knockout by CRISPR/Cas9 system on the proliferation and IFN-γ secretion in human T cells. Methods: : The sequence of sgRNA targeting PD-1 was designed. The PD-1-sgRNA and Cas9 mRNA were synthesized by T7 RNApolymerase in vitro, and then the mixture of PD-1-sgRNAand Cas9 mRNAwas delivered into activated T cells by nucleofection. The efficiency of gene knockout was confirmed by sequencing. The phenotypes of T lymphocytes and the expression of PD-1 after gene knockout were analyzed by Flow cytometry. The proliferation of T lymphocytes was calculated by trypan blue counting. The level of IFN-γ secreted by T lymphocytes was detected by ELISA. Results： ：PD-1-sgRNA and Cas9 mRNA were successfully synthesized in vitro and delivered into T cells by nucleofection. Sequencing technology confirmed that the PD-1 gene sequence was edited and the editing efficiency was 58.3%. The expression of PD-1 on T lymphocyte surface was down-regulated successfully by CRISPR/Cas9 system [(9.6±1.85)% vs (16.2±2.05)%, P<0.05]. The knockout of PD-1 gene did not affect the proliferation and phenotype of T lymphocytes(P<0.05); However, compared with the control group, the level of IFN-γ secreted by T lymphocytes in the PD-1sgRNA group was significantly increased (P<0.01). Conclusion: : CRISPR/Cas9 system can successfully ablate PD-1 gene in human T lymphocytes, which could block the negative regulation of PD-1／PD-L1 and further promote the IFN-γ secretion in T cells.

Prognostic significance of infiltrating immune cell subtypes in invasive ductal carcinoma of the breast.

PURPOSE: To explore the correlation between tumor-infiltrating immune cell subsets and breast cancer prognosis. MATERIALS AND METHODS: Specimens of 102 patients with invasive ductal carcinoma of the breast were analyzed for immune-related markers (CD8, CD20, FOXP3 and CD68). The number of positive cells in the 3 most highly stained intratumoral stroma areas of the primary tumor was counted. The mean number was calculated and used to divide patients into 2 groups for each marker (CD8-high/CD8-low, CD20-high/CD20-low, FOXP3-high/FOXP3-low, and CD68-high/CD68-low). RESULTS: Kaplan-Meier survival analysis showed (a) for all patients that high tumor-infiltrating CD8+ and CD20+ B lymphocytes, low tumor-infiltrating FOXP3+ regulatory T cells (Tregs), and CD68+ macrophages all increased OS and DFS (p<0.05); (b) for both the 35 ER-negative and 45 lymph-node-negative patients, high CD8+ cytotoxic T lymphocytes (CTLs) increased OS and DFS (p<0.05). Multivariate analysis of OS and DFS showed that for all patients high CD8+ CTLs and low FOXP3+ Tregs were related to good OS and DFS (p<0.05). CONCLUSION: High numbers of tumor-infiltrating CD8+ and low numbers of FOXP3+ T lymphocytes both could function as potential independent prognostic markers for invasive ductal breast carcinoma.

The cardioprotection of dihydrotanshinone I against myocardial ischemia-reperfusion injury via inhibition of arachidonic acid ω-hydroxylase.

Arachidonic acid (AA) is a precursor that is metabolized by several enzymes to many biological eicosanoids. Accumulating data indicate that the ω-hydroxylation metabolite of AA, 20-hydroxyeicosatetraenoic acid (20-HETE), is considered to be involved in the myocardial ischemia-reperfusion injury (MIRI). The inhibitors of AA ω-hydroxylase, however, are demonstrated to exhibit protective effects on MIRI. Dihydrotanshinone I (DI), a bioactive constituent of danshen, is proven to be a potent inhibitor of AA ω-hydroxylase by our preliminary study in vitro. The purpose of the present study was to investigate the cardioprotection of DI against MIRI and its effects on the concentrations of 20-HETE in vivo. Rats subjected to 30 min of ischemia followed by 24 h of reperfusion were assigned to intravenously receive vehicle (sham and ischemia-reperfusion), low (1 mg/kg), middle (2 mg/kg), or high (4 mg/kg) doses of DI before reperfusion. The results demonstrated that DI treatment could improve cardiac function, reduce infarct size, ameliorate the variations in myocardial zymogram and histopathological disorders, decrease 20-HETE generation, and regulate apoptosis-related protein in myocardial ischemia-reperfusion rats. These findings suggested DI could exert considerable cardioprotective action on MIRI by the attenuation of 20-HETE generation, subsequent myocardial injury, and apoptosis through inhibition on AA ω-hydroxylase.

[Construction of lentiviral vector of siRNA specific for γ-synuclein and its inhibition effect on colorectal carcinoma cell line SW1116].

OBJECTIVE: To construct a lentiviral vector carrying the γ-synuclein(SNCG) gene and establish a human colorectal carcinoma cell line SW1116 stably expressing this gene, and then investigate the inhibition of the growth and invasion capacity of SW1116 cells. METHODS: RNA interference fragment was designed according to the SNCG sequence (GenBank: No.NM003087.2), and then SNCG RNAi effective target genes were screened. After the Oligo DNA of target sequences was synthesized, the lentiviral vectors carrying LV-SNCG-RNAi-EGFP (RNAi group) and LV-SNCG-NC-EGFP (NC group) were constructed and packaged to produce lentivirus venom. The supernatants of different virus-producing cells were used to transfect SW1116 cells respectively. Wild SW1116 cells were used as blank control (CON group) EGFP fluorescence was detected by fluorescent microscopy and the differential expression of SNCG mRNA and protein was detected by real-time PCR and Western blot. CCK-8, soft agar assay and Transwell chamber were employed to estimate the inhibiting effect on growth and invasion of SW1116 respectively. RESULTS: Recombinant lentiviral vectors respectively carrying the SNCG-RNAi-EGFP and SNCG-NC-EGFP were successfully constructed and the supernatants of lentivirus could effectively infect SW1116 cells. The titer of the virus carrying LV-SNCG-RNAi-EGFP or LV-SNCG-NC-EGFP was 8×10(8) TU/ml. Real-time PCR and Western blot confirmed that compared with the NC group, SNCG-RNAi group had lower SNCG expression (1.009±0.161 vs. 0.114±0.030, P=0.009), and showed tremendous silencing effect as 76.8%(P<0.05). SNCG protein expression was also significantly reduced (RNAi:12.001±2.884, NC:32.443±4.731, CON:34.308±6.920, P<0.05). After SNCG knockdown, the number of proliferation cells was obviously reduced at 48, 72, 96 and 120 hours respectively(P=0.036). In soft agar assay, clones in RNAi group were smaller[RNAi:(0.582±0.103) mm, NC:(1.863±0.316) mm, CON:(1.749±0.525) mm]. Colony formation rate of RNAi group was down to (17.1±3.5)%, which was significantly lower than (36.5±4.3)% in NC group and (33.8±3.9)% in CON group. In migration test, the number of invasion cell was 37.4±9.3 in RNAi group, which was significantly less than 112.3±8.6 in NC group and 100±0.0 in CON group. CONCLUSION: Expression of SNCG mRNA and protein plays an important role in the growth and the invasion capacity of SW1116 cells.

Protective Effect of Astragaloside IV Against Paraquat-Induced Lung Injury in Mice by Suppressing Rho Signaling.

The purpose of the present study was to evaluate the protective effects of astragaloside IV (AS IV) against paraquat (PQ)-induced pulmonary injury in vivo. Fifty BALB/C mice were randomized into five groups: (1) control, (2) PQ, (3) PQ + dexamethasone (Dex, 5 mg/kg), (4) PQ + AS IV (50 mg/kg), and (5) PQ + AS IV (100 mg/kg). A single dose of PQ (50 mg/kg, i.p.) was intraperitoneally given to induced acute lung injury. Then, mice were treated with AS IV (50 and 100 mg/kg/day, orally) for 5 days. At the end of the experiment, animals were euthanized; then, the bronchoalveolar lavage fluid (BALF) and lung tissues were collected for histological observation, biochemical assay, and Western blot analysis. Malondialdehyde (MDA), myeloperoxidase (MPO), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) in lung tissues, and interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α) levels in BALF were determined. Histological examination indicated that AS IV attenuated lung damage caused by PQ. Biochemical results showed that AS IV treatment significantly reduced the levels of MDA, MPO, and inflammatory cytokines while increased the levels of SOD, CAT, and GSH-Px compared with those in PQ group. Western blot results revealed that AS IV attenuated the Txnip/Trx expressions and inhibited Rho/ROCK/nuclear factor kappaB (NF-κB) signaling pathway in PQ-challenged mice. These findings suggested the protective effect of AS IV as a natural product on PQ-induced pulmonary injury.

miRNA-148b suppresses hepatic cancer stem cell by targeting neuropilin-1.

The existence of cancer stem cells (CSCs) is considered as a direct reason for the failure of clinic treatment in hepatocellular carcinoma (HCC). Growing evidences have demonstrated that miRNAs play an important role in regulation of stem cell proliferation, differentiation and self-renewal and their aberrances cause the formation of CSCs and eventually result in carcinogenesis. We recently identified miRNA-148b as one of the miRNAs specifically down-regulated in side population (SP) cells of PLC/PRF/5 cell line. However, it remains elusive how miRNA-148b regulates CSC properties in HCC. In the present study, we observed that overexpression or knockdown of miR-148b through lentiviral transfection could affect the proportion of SP cells as well as CSC-related gene expression in HCC cell lines. In addition, miR-148b blocking could stimulate cell proliferation, enhance chemosensitivity, as well as increase cell metastasis and angiogenesis in vitro. More importantly, miR-148b could significantly suppress tumorigenicity in vivo. Further studies revealed that Neuropilin-1 (NRP1), a transmembrane co-receptor involved in tumour initiation, metastasis and angiogenesis, might be the direct target of miRNA-148b. Taking together, our findings define that miR-148b might play a critical role in maintenance of SP cells with CSC properties by targeting NRP1 in HCC. It is the potential to develop a new strategy specifically targeting hepatic CSCs (HCSCs) through restoration of miR-148b expression in future therapy.

[Construction of γ-synuclein eukaryotic expression vector and its effect on invasion and metastasis of colon cancer cell line SW1116 in vitro].

OBJECTIVE: To construct γ-synuclein gene eukaryotic expression vector, and to study its effect on the invasion of colon cancer cell line SW1116 and the adhesion between SW1116 and human umbilical vein endothelial cells(HUVECs) in vitro. METHODS: Total RNA was extracted from colon cancer cell line HT29 and the cDNA of γ-synuclein was amplified using RT-PCR. The digested fragment of cDNA coding sequence was linked to the eukaryotic expression vector pEGFP-C1 containing the GFP gene. After identification by sequence analysis, the recombinant plasmid was transfected into colon cancer cell line SW1116 via lipofectamine. The stable cell line was selected with G-418. The invasion in vitro was tested by Transwell invasion chamber assay. HUVECs were previously seeded onto 96-well plates before SW1116 cells seeded, and fluorescence intensity of GFP was detected to represent the amount of adhesion cells by ELISA. RESULTS: Human γ-synuclein eukaryotic expression vector was successfully constructed, which was stably expressed in SW1116 cells and could translate the GFP-γ-synuclein protein in vitro. γ-synuclein facilitated SW1116 cell passing through matrigel and filter membrane(198.4±20.7 vs. 98.8±13.2, P<0.05) and elevated the adherence of SW1116 cells to HUVECs(3.08±0.36 vs. 1.22±0.21, P<0.05). CONCLUSION: Expression of γ-synuclein can strengthen colon cancer cell SW1116 potentiality of invasion and metastasis in vitro.

Identification of cancer stem cells from hepatocellular carcinoma cell lines and their related microRNAs.

The aim of this study was to identify cancer stem cells (CSC) from three hepatocellular carcinoma (HCC) cell lines and to screen for specific microRNAs (miRNAs) regulating CSCs. Side population (SP) phenotype analysis was used. Four factors in the staining process, the incubation time, shaking interval, culture time and Hoechst 33342 concentration were explored, respectively, to define the SP subtype. CSC characteristics of SP cells were verified by sphere-forming assay and tumorigenic ability in NOD/SCID mice. QPCR assay for 370 miRNAs was performed to identify the differential miRNA expression between SP and Non-SP (NSP) cells in the PLC/PRF/5 cell line. The selected miRNAs were tested again in SP and NSP cells from Huh-7 and Hep-3B cell lines by qPCR assay. All four factors influenced SP percentage, when the other three conditions were fixed, the optimal Hoechst 33342 concentrations determined were 11 µg/ml for PLC/PRF/5 cells, 4 µg/ml for Huh-7 and 5 µg/ml for Hep-3B cells. The resultant SP percentage was 0.73±0.12%, 0.49±0.04% and 0.63±0.08%, respectively. The purity of sorted SP cells was >85%. Floating spheres were formed by SP cells from all three cell lines, while NSP cells did not form a single floating sphere. Mice injected with SP cells on the right side formed more tumor masses compared to their counterpart NSP at the same injection dosage; qPCR profiling identified 27 differentially expressed miRNAs in PLC/PRF/5 cells. Subsequent qPCR assay showed that miR-9* and miR-194 were also downregulated in SP cells from Huh-7 and Hep-3B. The present study identified CSCs via SP and sphere-forming assay from three liver cancer cell lines. Altogether, 27 CSC-specific miRNAs were determined in PLC/PRF/5; miR-9* and miR-194 were identified as the common CSC-specific miRNAs across the three HCC cell lines.

Effects of γ-synuclein on the tumorigenicity and metastasis of colon cancer SW1116 cells in vitro and in vivo.

Recent evidence suggests the involvement of γ-synuclein in tumorigenesis and tumor progression. The present study was designed to further clarify the effects of γ-synuclein on the biological features of colon cancer cells in vitro and in vivo. We constructed the eukaryotic expression vector and siRNA vector and selected stable transfectants to respectively upregulate and downregulate γ-synuclein expression in SW1116 cells. we found that silencing of γ-synuclein significantly attenuated SW1116 cell growth and colony formation in vitro (P<0.05), and overexpression of γ-synuclein moderately enhanced cell growth and colony formation, but not significantly when compared with the parental SW1116 cells and empty vector-transfected cells (P>0.05). Meanwhile, overexpression of γ-synuclein significantly facilitated SW1116 cell migration, invasion and adhesion to human liver sinusoidal endothelial cells (HLSECs) in vitro (P<0.05), and the effects were less attenuated by γ-synuclein knockdown (P>0.05). Furthermore, γ-synuclein promoted these malignant phenotypes in a γ-synuclein expression quantity-dependent manner not only in vitro but also in the in vivo expression. stable cells were injected subcutaneously into the right flank, and injected intrasplenically in nude mice. γ-synuclein knockdown suppressed the tumorigenicity of SW1116 cells in mice, which presented significantly smaller tumor masses on day 6 over a 30-day period, compared with empty vector cells (P<0.05). Meanwhile, overexpression of γ-synuclein led to a profound augmentation of liver metastasis in nude mice, not only in macroscopic appearance but also in the size and weight of livers (P<0.05). These results provide strong evidence that suggests γ-synuclein plays a positive role in the progression of colorectal cancer.

Floating cells with stem cell properties in gastric cell line SGC-7901.

AIMS: To obtain floating spheres from the adherent gastric cancer cell line SGC-7901 and to analyze the properties of the spheres. METHODS: Serum-free medium was applied to cultured SGC-7901 cells. Limiting dilution assay, tumor formation assay, microarray analysis, and real-time fluorescent quantitative PCR were used to test the stem cell properties of the spheres. RESULTS: A subpopulation of SGC-7901 cells formed floating spheres in serum-free medium. These cells showed enhanced tumorigenic ability and also highly expressed certain stem-cell-associated proteins. CONCLUSIONS: We successfully propagated floating spheres with stem cell properties in the SGC-7901 gastric cell line.