Knockdown of calponin 2 suppressed cell growth in gastric cancer cells.

Calponin family members are actin filament-associated regulatory proteins with distinct expression patterns. Previous studies on CNN2 (calponin 2) have demonstrated that CNN2 is expressed in a broad range of tissues and cell types, exhibiting potential regulatory roles in a number of cellular activities, including cell proliferation, cell migration, and platelet adhesion. In this work, we found that both messenger RNA and protein expression levels of CNN2 were remarkably upregulated in 60%-70% of gastric cancer tissues by comparison with those of neighboring non-tumorous mucosa. By utilizing specific shCNN2 (small hairpin RNA targeting CNN2), the potential role of CNN2 in regulating AGS gastric cancer cell growth was then further investigated. AGS cells infected with shCNN2 exhibited significantly decreased cell growth ability by comparison with control cells in vitro. Moreover, while there was no obvious difference in cell cycle distribution between two groups, enhanced cell apoptosis was detected in cells with reduced CNN2 expression. Consistently, caspase 3/7 activity was also remarkably activated upon shCNN2 lentivirus infection. Taken together, our results demonstrated that knockdown of endogenous CNN2 in AGS cells could significantly activate cell apoptosis pathway and therefore suppress cell growth in vitro. The deletion of CNN2 might be a potential therapeutic approach to inhibit aggressive growth of gastric cancer.

[The effect of inhaled glucocorticoid therapy on serum proteomics of asthmatic patients].

OBJECTIVE: To screen the biomarkers which may play important roles in the pathogenesis and therapy of asthma by using serum comparative proteomics. METHODS: From June 2011 to September 2012, 30 chronic persistent asthmatic patients (asthma group) and 30 healthy controls (control group) were selected for study in our hospital. All the asthmatic patients were given 8 week-treatment with inhaled glucocorticoids (ICS). Then comparative proteomics were employed to identify differential proteins in serum samples from the control group, the asthma pre-treatment group and the asthma post-treatment group. The differential proteins which had significant differences before and after ICS therapy were selected for Western blot analysis and ELISA detection. Besides, the correlation between the differential proteins and IgE, eosinophils (EOS), neutrophil percentage(NEUT%), and FEV1% were analyzed. RESULTS: Eleven differential proteins were identified. Among them, heat shock protein 70 (HSP70), eosinophil chemotactic protein (Eotaxin) and vitamin D binding protein (VDBP) had significant differences in protein abundance before and after treatment. The differential expression of the 3 proteins in each group was confirmed by Western blot. ELISA data showed that the serum levels of HSP70 and Eotaxin were significantly higher in the asthma pre-treatment group [(439 ± 103)ng/L, (183 ± 79)ng/L] than those in the control group [(209 ± 58)ng/L, (91 ± 46)ng/L] (t = 5.281, 4.972, all P < 0.01), but significantly lower in the asthma post-treatment group[(247 ± 96) ng/L, (105 ± 58)ng/L] than those in the pre-treatment group (t = 4.157, 3.892, all P < 0.01). However, the serum level of VDBP was significantly lower in the asthmatics pre-treatment group [(318 ± 115)mg/L] than that in the control group [(541 ± 98)mg/L] (t = 3.878, P < 0.01), but significantly higher in the asthma post-treatment group[(479 ± 132)mg/L] than that in the pre-treatment group(t = 3.572, P < 0.01). The correlation analysis showed that HSP70 was correlated positively with IgE and NEUT%, but negatively with FEV1% (r = 0.568, 0.613, -0.516, all P < 0.01). Eotaxin was correlated positively with IgE and EOS, but negatively with FEV1% (r = 0.752, 0.826, -0.618, all P < 0.01). VDBP was correlated negatively with NEUT%, but positively with FEV1% (r = -0.537, 0.426, all P < 0.05). CONCLUSIONS: HSP70, Eotaxin, and VDBP may participate in the pathogenesis of asthma, and may become the potential targets for ICS therapy.

Diagnostic accuracy of risk assessment and fecal immunochemical test in colorectal cancer screening: Results from a population-based program and meta-analysis.

BACKGROUND: Fecal immunochemical test (FIT) is a commonly used initial test for colorectal cancer (CRC) screening. Parallel use of FIT with risk assessment (RA) could improve the detection of non-bleeding lesions, but at the expense of compromising sensitivity. In this study, we evaluated the accuracy of FIT and/or RA in the Shanghai CRC screening program, and systematically reviewed the relevant evaluations worldwide. METHODS: RA and 2-specimen FIT were used in parallel in the Shanghai screening program, followed by a colonoscopy among those with positive results. Sensitivity, specificity, detection rate of CRC, positive predictive value (PPV), and other measures with their 95% confident intervals were calculated for each type of tests and several assumed combined tests. We further searched PubMed, Embase, Web of Science, and Cochrane Library for relevant studies published in English up to January 5, 2022. RESULTS: By the end of 2019, a total of 1,901,360 participants of the screening program completed 3,045,108 tests, with 1,901,360 first-time tests and 1,143,748 subsequent tests. Parallel use of RA and 2-specimen FIT achieved a sensitivity of 0.78 (0.77-0.80), a specificity of 0.78 (0.78-0.78), PPV of 0.89% (0.86-0.92), and a detection rate of 1.99 (1.93-2.05) for CRC per 1000 among participants enrolled in the first screening round, and performed similarly among those who participated for several times. A meta-analysis of 103 published observational studies demonstrated a higher sensitivity [0.76 (0.36, 0.94)] but a much lower specificity [0.59 (0.28, 0.85)] of parallel use of RA and FIT for detecting CRC in average-risk populations than in our subjects. One-specimen FIT, the most commonly used initial test, had a pooled specificity comparable to the Shanghai screening program (0.92 vs. 0.91), but a much higher pooled sensitivity (0.76 vs. 0.57). CONCLUSION: Our results indicate the limitation of FIT only as an initial screening test for CRC in Chinese populations, and highlight the higher sensitivity of parallel use of RA and FIT. Attempts should be made to optimize RA to improve effectiveness of screening in the populations.

PACAP38 improves airway epithelial barrier destruction induced by house dust mites allergen.

PURPOSE: This study aimed to investigate the mechanism of PACAP38 on house dust mite (HDM)-induced asthmatic airway epithelial barrier destruction. METHODS: The HDM-induced asthma mice model and 16HBE cell model was established respectively. The enzyme linked immunosorbent assay (ELSIA), cell count and immunohistochemical assay were performed on mice in control group, HDM group and PACAP38 + HDM group.The cAMP/PKA activity, p-CREB and total CREB expression, TEER and the FITC-DX were investigated on cells in control-16HBE group, HDM-16HBE group and PACAP38 + HDM-16HBE group. RESULTS: The levels of IL-4 and IL-5 in the HDM group were significantly higher than those in the control group (P < 0.05), while the above indexes in the PACAP38 + HDM group were lower than those in the HDM group (P < 0.05). E-cadherin, ß-catenin, ZO-1 and occludin in the control group were highly immunoreactive in airway epithelial cells, whereas connexin staining was attenuated after HDM induction. The TEER level, cAMP levels and PKA activity were decreased, while FITC-DX transmittance was increased in HDM-16HBE group (P < 0.05) compared with the control-16HBE group. CONCLUSION: PACAP38 could reduce the airway inflammation, weaken the AJC protein heterotopia and activate cAMP/PKA signaling pathway in HDM-induced asthma, which indicate that PACAP38 may be an important contributor in HDM-induced asthma.

BRMS1 participates in regulating cell sensitivity to DNA interstrand crosslink damage by interacting with FANCI.

Breast cancer metastasis suppressor 1 (BRMS1) is a tumor metastasis suppressor implicated in multiple steps during the metastatic cascade. Many proteins interacting with BRMS1 have been identified to unravel the intracellular signaling mechanisms. In the present study, we report that FANCI is a novel interacting protein of BRMS1 as determined by co­immunoprecipitation assay. The linker region between two coiled­coil motifs of BRMS1 is required for BRMS1­FANCI interaction. FANCI is an essential protein in the Fanconi anemia (FA) pathway responsible for the repair of DNA interstrand crosslinks (ICLs). We demonstrated that knockdown or knockout of BRMS1 significantly diminished the monoubiquitination of FANCI and FANCD2 in response to DNA ICL damage. BRMS1­deficient cells exhibited suppressed FANCD2 foci formation and hypersensitivity to ICLs. Moreover, rescue assays by utilizing different BRMS1 constructs suggested that BRMS1­FANCI interaction is necessary for the regulatory role of BRMS1 in the FA pathway. Overall, our findings characterize BRMS1 as a novel regulatory protein functioning in the DNA repair pathway via protein interaction.

Knockdown of ZNF233 suppresses hepatocellular carcinoma cell proliferation and tumorigenesis.

Zinc finger proteins (ZNFs) are one of the most abundant proteins in eukaryotic genomes with extraordinarily diverse functions. ZNF233 is located on 19q13.31 and encodes a 670-amino acid protein belonging to the Krüppel C2H2-type ZNF family. However, little is known about the role of ZNF233 in cancer progression. In this study, we reported for the first time that ZNF233 mRNA was remarkably up-regulated in hepatocellular carcinoma (HCC) tissues in comparison with corresponding non-tumorous normal liver tissues. ZNF233 expression level was correlated with tumor grade, tumor stage and prognosis of HCC patients. We further investigated the effect of ZNF233 on HCC cell growth. It is found that overexpression of ZNF233 in SMMC-7721 could promote G1/S transition and thus accelerate cell growth ratio. Consistently, knockdown of ZNF233 in QGY-7701 cells successfully suppressed cell proliferation in vitro and in vivo. Further immunohistochemical staining revealed a reduced Ki-67-positive cell percentage in xenografted tumor derived from ZNF233-knocking down cells. Taken together, these results demonstrate a positive role of ZNF233 in regulating HCC cell growth. ZNF233 might be developed as a novel biomarker and a potential therapeutic target for HCC.

Scinderin is a novel transcriptional target of BRMS1 involved in regulation of hepatocellular carcinoma cell apoptosis.

Tumor metastasis suppressor factor BRMS1 can regulate the metastasis of breast cancer and other tumors. Here we report scinderin (SCIN) as a novel transcriptional target of BRMS1. SCIN protein belongs to the cytoskeletal gelsolin protein superfamily and its involvement in tumorigenesis remains largely illusive. An inverse correlation between the expression levels of BRMS1 and SCIN was observed in hepatocellular carcinoma (HCC) cells and tissues. On the molecular level, BRMS1 binds to SCIN promoter and exerts a suppressive role in regulating SCIN transcription. FACS analysis and caspase 9 immunoblot reveal that knockdown of SCIN expression can sensitize HCC cells to chemotherapeutic drugs, leading to suppression of tumor growth in vivo. Consistently, overexpression of SCIN protects cells from apoptotic death, contributing to increased xenografted HCC cell growth. In summary, our study reveals SCIN as a functional apoptosis regulator as well as a novel target of BRMS1 during HCC tumorigenesis. Inhibition of SCIN might bring a potential cancer therapy approach.

Characterization of DAPK1 as a novel transcriptional target of BRMS1.

Breast cancer metastasis suppressor 1 (BRMS1) can specifically regulate tumor metastasis in many cancers. Our previous studies have demonstrated that BRMS1 can promote cell apoptosis through regulating osteopontin (OPN) expression in hepatocellular carcinoma (HCC) cells. However, the transcriptional targets of BRMS1 have not been thoroughly studied. In this study, death-associated protein kinase 1 (DAPK1), a tumor suppressor gene with multiple roles in regulating cell death, was identified as a potential transcriptional target of BRMS1 in the whole genome expression microarray. Quantitative real-time PCR and western blot analysis of HCC cells overexpressing BRMS1 further confirmed the transcriptional regulation relationship between BRMS1 and DAPK1. Moreover, DAPK1 expression was frequently decreased or even lost in HCC tissue samples by comparison with neighboring pathologically normal liver tissue, which was consistent with the decreased BRMS1 expression pattern. To unravel the molecular mechanism of BRMS1 in regulating DAPK1, a series of deletion mutants of DAPK1 promoter was subjected to luciferase assay. The luciferase units of -200 to -80 bp region, with two tandem putative NF-κB binding sites, were specifically enhanced by BRMS1 expression. Site-directed mutants of NF-κB binding sites blocked the transcriptional activation effect. In addition, the binding capability of BRMS1 and the putative NF-κB binding sites were demonstrated in the chromatin immunoprecipitation (ChIP) assay. In conclusion, our study characterized DAPK1 as a novel transcriptional target of BRMS1. Transcriptional activation of DAPK1 might be another important mechanism accounting for the metastasis suppressive activity of BRMS1.

Breast cancer metastasis suppressor 1 modulates SIRT1-dependent p53 deacetylation through interacting with DBC1.

Breast cancer metastasis suppressor 1 (BRMS1) is a specific tumor metastasis suppressor implicated in the regulation of chromatin modification and gene transcription. However, the molecular mechanism of BRMS1 remains to be elucidated. Here, we report that DBC1 (deleted in breast cancer 1), is a novel interacting protein of BRMS1. The imperfect leucine zipper motifs of BRMS1 and the N-terminal domain of DBC1 are required for the interaction. DBC1 is identified as an important negative regulator of SIRT1's activity and genotoxic stress response. We demonstrated that BRMS1 is able to interrupt endogenous DBC1-SIRT1 association. Consistently, SIRT1-dependent p53 acetylation under genotoxic stress is also affected by BRMS1. Overall, our results identify BRMS1 as a novel regulator of DBC1-SIRT1 complex and SIRT1-dependent p53 deacetylation.