Target-triggered enzyme-free amplification for highly efficient AND-gated bioimaging in living cells.

Rapid, simultaneous, and sensitive detection of biomolecules has important application prospects in disease diagnosis and biomedical research. However, because the content of intracellular endogenous target biomolecules is usually very low, traditional detection methods can't be used for effective detection and imaging, and to enhance the detection sensitivity, signal amplification strategies are frequently required. The hybridization chain reaction (HCR) has been used to detect many disease biomarkers because of its simple operation, good reproducibility, and no enzyme involvement. Although HCR signal amplification methods have been employed to detect and image intracellular biomolecules, there are still false positive signals. Therefore, a target-triggered enzyme-free amplification system (GHCR system) was developed, as a fluorescent AND-gated sensing platform for intracellular target probing. The false positive signals can be well avoided and the accuracy of detection and imaging can be improved by using the design of the AND gate. Two cancer markers, GSH and miR-1246, were used as two orthogonal inputs for the AND gated probe. The AND-gated probe only works when GSH and miR-1246 are the inputs at the same time, and FRET signals can be the output. In addition to the use of AND-gated imaging, FRET-based high-precision ratiometric fluorescence imaging was employed. FRET-based ratiometric fluorescent probes have a higher ability to resist interference from the intracellular environment, they can avoid false positive signals well, and they are expected to have good specificity. Due to the advantages of HCR, AND-gated, and FRET fluorescent probes, the GHCR system exhibited highly efficient AND-gated FRET bioimaging for intracellular endogenous miRNAs with a lower detection limit of 18 pM, which benefits the applications of ratiometric intracellular biosensing and bioimaging and offers a novel concept for advancing the diagnosis and therapeutic strategies in the field of cancer.

TRIM58 suppresses the tumor growth in gastric cancer by inactivation of ß-catenin signaling via ubiquitination.

Objective: To investigate and define the underlying molecular mechanism of tripartite motif-containing 58 (TRIM58) in regulating the tumor growth of gastric cancer (GC).Methods: TRIM58 expression in GC tissues and cells was detected by real-time PCR and Western blot, followed by lentiviral-induced overexpression or knockdown of TRIM58. Subsequently, CCK8, BrdU-ELISA, flow cytometry, immunoprecipitation, in vitro animal experiments and immunochemistry were performed to explore the function of TRIM58. Western blotting was used to detect ß-catenin, C-myc, Cyclin D1, and survivin expression.Results: TRIM58 expression was significantly reduced in tumor tissues of GC patients and GC cell lines, whereas ß-catenin, C-myc, Cyclin D1, and survivin were highly expressed. Overexpression of TRIM58 in GC cells resulted in decreases in ß-catenin, C-myc, Cyclin D1, and survivin protein expression and significantly suppressed proliferation by preventing cell-cycle progression and promoting cell apoptosis. Conversely, TRIM58 knockdown resulted in the opposite effects. Furthermore, the effect of TRIM58 knockdown on GC cells was potently reversed by a ß-catenin inhibitor, XAV939. Immunoprecipitations showed the interaction between TRIM58 and ß-catenin, and TRIM58 overexpression significantly enhanced ß-catenin degradation. In addition, we found a significant decrease in the growth and weight of tumors and an increase in tumor cell apoptosis in TRIM58-overexpression nude mice, which were also accompanied by reduced ß-catenin expression.Conclusions: These data suggest that TRIM58 may function as a tumor suppressor in GC and potentially suppress the tumor growth of gastric cancer by inactivation of ß-catenin signaling via ubiquitination.

Homeobox B7 accelerates the cancer progression of gastric carcinoma cells by promoting epithelial-mesenchymal transition (EMT) and activating Src-FAK pathway.

Aim: To study the carcinogenetic mechanism of HOXB7 in gastric cancer (GC) remains. Methods: Two human GC cell lines - SGC7901 and SNU1 - were used for this study. SGC7901 cells were transfected with siRNA-HOXB7 (siHOXB7) to knock down HOXB7 expression, whereas, SNU1 cells were transduced with pCDNA3.1-HOXB7 to overexpress HOXB7. After transfection, cancer progression was assessed by determining cell proliferation, wound-healing process, cell cycle, apoptosis, invasion, and migration. The effect of HOXB7 on epithelial-mesenchymal transition (EMT) was measured by observing changes in F-actin cytoskeleton and evaluating the expression of EMT markers. p-Scr and p-FAK were evaluated to assess the mechanism. Results: Knockdown of HOXB7 suppressed cell proliferation, alleviated the wound-healing process, inhibited cell migration and invasion, and arrested the cell cycle while promoting cell apoptosis, suggesting the tumor-suppressive effect of siHOXB7 in human GC cells. On the contrary, HOXB7 overexpression showed a tumor-promoting effect on human GC cells. Moreover, we confirmed an inhibitory effect of siHOXB7 on the EMT process by preventing epithelial cells from acquiring a mesenchymal phenotype and downregulating mesenchymal markers (vimentin, ß-catenin, N-cadherin, Twist) while upregulating epithelial markers (E-cadherin). Our data revealed that HOXB7 was associated with Src/FAK and favored the activation of the Src-FAK pathway in human GC cells. Conclusion: HOXB7 accelerated the malignancy of GC, by facilitating EMT and regulating the Scr-FAK pathway.

Effect of SOCS1 silencing on proliferation and apoptosis of melanoma cells: An in vivo and in vitro study.

This study aimed to investigate the effect of SOCS1 silencing on the proliferation and apoptosis of melanoma cells by in vivo and in vitro studies. Immunohistochemical staining was used to detect SOCS1 expression in melanoma tissues and pigmented nevi. Quantitative real-time polymerase chain reaction and western blotting were applied to detect the messenger RNA and protein expressions of SOCS1 in primary human melanocytes and malignant melanoma cell lines (A375, SK-MEL-5, M14, and MV3). Melanoma cells were assigned into mock, negative small interfering RNA, and SOCS1-small interfering RNA groups. The proliferation, cell cycle and apoptosis, and messenger RNA expression of SOCS1 in MV3 and A375 cells were detected using MTT assay, flow cytometry, and quantitative real-time polymerase chain reaction, respectively. The expressions of SOCS1 protein, extracellular signal-regulated kinase, and janus kinase signal transduction and activators of transcription signaling pathways-related proteins were detected using western blotting. After the establishment of subcutaneous xenograft tumor models in nude mice, the latent period, size, volume and growth speed of xenograft tumors in the mock, negative small interfering RNA, and SOCS1-small interfering RNA groups were examined and compared. The results indicated that positive expression rate of SOCS1 was higher in malignant melanoma tissues than in pigmented nevi. MV3 cells had the highest messenger RNA and protein expressions of SOCS1, followed by A357 cells. Compared with the mock and negative small interfering RNA groups, SOCS1-small interfering RNA group showed lower cell viability, elevated cell apoptosis, more cells in G0/G1 phase and less cells in S and G2/M phases, and decreased messenger RNA and protein expressions of SOCS1, p-ERK1/2, p-JAK2, p-STAT1, and p-STAT3. Compared with the mock and negative small interfering RNA groups, the SOCS1-small interfering RNA group showed longer latent period of tumor, smaller tumor size and volume, and smoother tumor growth curve. To conclude, SOCS1 silencing can inhibit proliferation and induce apoptosis of MV3 and A357 melanoma cells in vivo and in vitro by inhibiting extracellular signal-regulated kinase and janus kinase signal transduction and activators of transcription signaling pathways.

CtBP2 overexpression promotes tumor cell proliferation and invasion in gastric cancer and is associated with poor prognosis.

C-terminal binding protein-2 (CtBP2), a transcriptional corepressor, has been reported to correlate with tumorigenesis and progression and predict a poor prognosis in several human cancers. However, few studies on CtBP2 in gastric cancer (GC) have been performed. In this research, we evaluated the correlations between CtBP2 expression and the clinicopathological characteristics, as well as prognosis of GC patients. The effects of silencing CtBP2 expression on GC cells biology activity were also assessed. The results showed that CtBP2 was overexpressed in GC tissues and closely correlated with poor differentiation, advanced tumor stage and poor prognosis in GC patients. CtBP2 induced epithelial-to-mesenchymal transition (EMT) and repressed PTEN to increase proliferation rate, migration, and invasion in GC cells. Silencing CtBP2 inhibited GC growth in nude mice model. In conclusion, CtBP2 is overexpressed in GC and may accelerate GC tumorigenesis and metastasis, which could represent an independent prognostic marker and promising therapeutic target for GC.

High expression of WISP1 in colon cancer is associated with apoptosis, invasion and poor prognosis.

Colon cancer (CC) likes many epithelial-derived cancers, resulting from a complex tumorigenic process. However, the exactly mechanisms of development and progression of CC are still unknown. In this study, integrated analysis in the GSE33113 and Fudan University Shanghai Cancer Center Hospital datasets revealed that WISP1 expression was significantly increased in CC cases, positivity correlated with the advanced pathologic stage and a poor prognosis was more likely in CC patients with higher levels of WISP1. Downregulation of WISP1 inhibited cell proliferation and invasion through increasing apoptosis and blocking cell cycle at G1 phase in CC LOVO and RKO cells. Besides, Gene set enrichment analysis (GSEA) revealed that relative genes involved in the Cell adhesion molecules and Cytokine-cytokine receptor interaction pathways were enriched in WISP1-higher expression patients. Western blot analysis showed that Cell adhesion molecules pathway associated genes (ICAM- 1, VCAM-1, SDC2 and CDH2) and Cytokine-cytokine receptor interaction pathway associated genes (VEGFC, CCL18, CXCR4 and TGFBR1) were also modulated by WISP1 downregulation. Then, we found that the protein ß-catenin was identified as a binding partner of WISP1 and mediated the functions of WISP1 through promoting cell proliferation and invasion in LOVO and RKO cells. Further in vivo tumor formation study in nude mice indicated that inhibition of WISP1 delayed the progress of tumor formation and inhibited PCNA expression. These results indicate that WISP1 could act as an oncogene and may serve as a promising therapeutic strategy for colon cancer.