Overexpression SPRY2 Suppresses Esophageal Squamous Cell Carcinoma Progression Via Inactivation of ERK/AKT Signaling / La Sobreexpresión SPRY2 Suprime la Evolución del Carcinoma de Células Escamosas de Esófago Mediante la Inactivación de la Señalización ERK/AKT

SUMMARY: Esophageal cancer is one of the most aggressive gastrointestinal cancers. Invasion and metastasis are the main causes of poor prognosis of esophageal cancer. SPRY2 has been reported to exert promoting effects in human cancers, which controls signal pathways including PI3K/AKT and MAPKs. However, the expression of SPRY2 in esophageal squamous cell carcinoma (ESCC) and its underlying mechanism remain unclear. In the present study, we aimed to investigate the detailed role of SPRY2 in the regulation of cell proliferation, invasion and ERK/AKT signaling pathway in ESCC. It was identified that the expression level of SPRY2 in ESCC was remarkably decreased compared with normal tissues, and it was related to clinicopathologic features and prognosis ESCC patients. The upregulation of SPRY2 expression notably inhibited the proliferation, migration and invasion of Eca-109 cells. In addition, the activity of ERK /AKT signaling was also suppressed by the SPRY2 upregulation in Eca-109 cells. Our study suggests that overexpression of SPRY2 suppress cancer cell proliferation and invasion of by through suppression of the ERK/AKT signaling pathways in ESCC. Therefore, SPRY2 may be a promising prognostic marker and therapeutic target for ESCC.

El cáncer de esófago es uno de los cánceres gastrointestinales más agresivos. La invasión y la metástasis son las principales causas de mal pronóstico del cáncer de esófago. Se ha informado que SPRY2 ejerce efectos promotores en los cánceres humanos, que controla las vías de señales, incluidas PI3K/AKT y MAPK. Sin embargo, la expresión de SPRY2 en el carcinoma de células escamosas de esófago (ESCC) y su mecanismo subyacente aún no están claros. En el presente estudio, nuestro objetivo fue investigar el papel detallado de SPRY2 en la regulación de la proliferación celular, la invasión y la vía de señalización ERK/AKT en ESCC. Se identificó que el nivel de expresión de SPRY2 en ESCC estaba notablemente disminuido en comparación con los tejidos normales, y estaba relacionado con las características clínico-patológicas y el pronóstico de los pacientes con ESCC. La regulación positiva de la expresión de SPRY2 inhibió notablemente la proliferación, migración e invasión de células Eca-109. Además, la actividad de la señalización de ERK/AKT también fue suprimida por la regulación positiva de SPRY2 en las células Eca-109. Nuestro estudio sugiere que la sobreexpresión de SPRY2 suprime la proliferación y la invasión de células cancerosas mediante la supresión de las vías de señalización ERK/AKT en ESCC. Por lo tanto, SPRY2 puede ser un marcador de pronóstico prometedor y un objetivo terapéutico para la ESCC.

SPRY4-IT1 and breast cancer / 国际肿瘤学杂志

Long non-coding RNA (lncRNA) is an RNA molecule that does not code to express proteins, and plays an important role in the occurrence and development of a variety of tumors. As an lncRNA, SPRY4-IT1 is highly expressed in breast cancer tissues, and can be used as an upstream and downstream regulator of breast cancer, promoting the progression of breast cancer, and is closely related to breast cancer stage and prognosis. In-depth study of the molecular mechanism associated with SPRY4-IT1 and breast cancer can provide new ideas for discovering biomarkers for early diagnosis of breast cancer, assessing disease prognosis and finding targeting sites.

Can SpRY recognize any PAM in human cells? / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)‍-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.

Role of MicroRNA-21 in Proliferation and Migration of Pancreatic Cancer Cells / 胃肠病学

Background: Studies have shown that microRNAs are closely related to the occurrence and development of tumors. As one of the most common carcinogenic microRNAs, the specific role and mechanism of miR-21 in the proliferation and migration of pancreatic cancer cells have not been fully elucidated. Aims: To investigate the effect and specific molecular mechanism of miR-21 on proliferation and migration of pancreatic cancer cells. Methods: PANC-1 and MIA PaCa-2 cells were transfected with lentiviral vectors to construct stable cell lines with overexpression and knockdown of miR-21, respectively. The transfection efficiency was verified by qRT-PCR. CCK-8 assay was used to detect cell proliferation, scratch test was used to detect cell migration ability. The expressions of Spry2 mRNA and protein were determined by qRT-PCR and Western blotting, respectively, and luciferase report assay was used to detect the regulatory effect of miR-21 on Spry2. Results: Overexpression of miR-21 significantly promoted PANC-1 cells proliferation and migration (P<0.05). Knockdown of miR-21 significantly inhibited MIA PaCa-2 cells proliferation and migration (P<0.05). Compared with controls, expressions of Spry2 mRNA and protein were significantly decreased after up-regulation of miR-21 (P<0.05), however, expressions of Spry2 mRNA and protein were significantly increased after down-regulation of miR-21 (P<0.05). Overexpression of miR-21 could inhibit luciferase activity of plasmids containing wild-type Spry2 3'-UTR sequence (P<0.05), and knockdown of miR-21 could enhance luciferase activity (P<0.05). Spry2 could reverse miR-21-mediated cell proliferation and migration (P<0.05). Conclusions: MiR-21 can affect biological function of pancreatic cancer cells by targeted regulating the expression of Spry2.

The effect of FSD1 protein on the invasion of glioma stem cells / 国际药学研究杂志

Objective To investigate the effect of fibronectin type Ⅲ and SPRY domain containing 1 (FSD1) protein on the invasion of glioma stem cells (GSCs), so as to probe into the new biomarkers or potential therapeutic targets for gliomas. Methods The Cancer Genome Altas (TCGA) database data were used to analyze and compare the FSD1 gene expression (the FSD1 mRNA level) in the glioblatoma (also known as glioblastoma multiforme, GBM) and normal brain tissues as well as in the different grade glioma tissues, and the correlation of the FDS1 gene expression (mRNA level) with the survival prognosis of patients was also analyzed using the TCGA database data. The lentivirus was used to overexpress the FSD1 protein in the GSCs, T4121 and D456. The effect of the overexpressed FSD1 protein on the invasive ability of the GSCs, T4121 and D456 was evaluated by Transwell invasion assay. Results The FSD1 gene expression (mRNA level) was significantly lower in GBM than in normal brain (P＜0.01). The FSD1 gene expression (the mRNA level) in gliomas significantly decreased with the increase of the gliomas grade (gradeⅡvs Ⅲ, P＜0.05;gradeⅢvs Ⅳ, P＜0.01). The survival prognosis of patients with gilomas was well associated with the level of FSD1 gene expression (the FSD1 mRNA level), as indicated by the overall survival rate of the patients, which was significantly lower in the patients with the low FSD1 mRNA level than in the patients with the high FSD1 mRNA level (P＜0.01). In the Transwell invasion assay, the count of the invasive cell numbers significantly decreased in the FSD1 protein-overexpressed T421 and D456 groups than in the corresponding control group (P＜0.01 in both T4121 and D456 cell lines). Conclusion There is a clinical relevance of the FSD1 expression for the malignant progression of gliomas (the grade of gliomas). The low level FSD1 is favorable for keeping the invasive ability in GSCs.

The Bidirectional Effects of Arsenic on miRNA-21: A Systematic Review and Meta-analysis / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Arsenic is a metalloid environmental carcinogen involved in the occurrence and development of many cancers. miRNA-21 plays a crucial role in arsenic-induced carcinogenesis. We aimed to elucidate the mechanism by which miRNA-21 influences arsenic-induced cancer.</p><p><b>METHODS</b>We used meta-analysis of published studies to determine how arsenic induces cancerous cells through miRNA-21.</p><p><b>RESULTS</b>Low-dose arsenic exposure (⪕ 5 μmol/L) can increase miRNA-21 and phosphorylated signal transducter and activator of transcription 3 (pSTAT3) expression, and decrease programmed cell death protein 4 (PDCD4) and protein sprouty homolog 1 (Spry1) expression. High-dose arsenic exposure (> 5 μmol/L), can increase miRNA-21 expression, and decrease Spry1 and E-cadherin expression. Short-term arsenic exposure (⪕ 24 h) can increase miRNA-21 and pSTAT3 expression, and decrease PDCD4 expression. Moreover, long-term arsenic exposure (> 24 h) can increase the miRNA-21, STAT3, and pSTAT3 expression, and decrease PDCD4 expression. We found that activation of miRNA-21 and pSTAT3 were most pronounced following long-term arsenic exposure at low doses, and the effects on PDCD4 expression were most pronounced following short-term arsenic exposure at low doses. miRNA-21 inhibitors increased the expression of tumor suppressor genes PDCD4, PTEN, and Spry1 and miRNA-21-mimics suppressed the expression of these tumor suppressor genes.</p><p><b>CONCLUSION</b>Arsenic can cause cancer by activating miRNA-21 and inhibiting the expression of PDCD4, PTEN, and Spry1.</p>

The impact of Spry1 on adipocyte differentiation / 天津医药

Objective To investigate the effect of Spry1 on adipocyte differentiation from ST2 cells by using siRNA. Methods Synthesized siRNA targeting Spry1 was used as experimental group, and control siRNA was used as control group. Spry1 siRNA and control siRNA were transfected into ST2 cells, then treating with adipogenic medium to induce adipocyte differentiation. The mRNA expression levels of Spry1 and adipocyte differentiation-specific genes PPARγ(peroxisome proliferator-activated receptor gamma), C/EBP α (CCAAT enhancer binding protein α), FABP4 (adipocyte marker gene fatty acid binding protein 4) and adipsin were examined by quantitative real-time PCR. The mature adipocytes were stained with oil red O, the staining adipocytes were observed by microscope, then understanding the effect of Spry 1 siRNA on adipocyte differentiation. In addition, oil red O of the staining adipocytes was extracted with isopropanol, optical density (OD) values of oil red O were measured at a wavelength of 520 nm. Results Spry1 siRNA was transfected into ST2 cells. Compared with control group, the mRNA expression level of Spry1 was significantly reduced. The number of differentiated adipocytes from ST2 cells was decreased after staining with oil red O. And the OD value was lower than that of control group. The mRNA expression levels of adipocyte differentiation-specific genes PPARγ, C/EBP α, FABP4 and adipsin were significantly reduced compared with those of control group (P<0.05). Conclusion Spry1 siRNA can effectively suppresse adipogenic differentiation from progenitor cells.

Upregulated long noncoding RNA SPRY4‑IT1 contributes to increased cell viability by activating zinc finger 703 expression in esophageal squamous cell carcinoma.

OBJECTIVES: The function of long noncoding RNA SPRY4‑IT1 in human esophageal squamous cell carcinoma (ESCC) has been showed in the former studies. The purpose of this study was to further analyze the underlined mechanisms responsible for its role in ESCC cells. MATERIALS AND METHODS: Quantitative reverse transcriptase polymerase chain reaction was firstly used to measure the expression of SPRY4‑IT1 in 50 ESCC patients of different clinical stages. Loss of function approach was then applied to confirm the biological function, especially cell viabilities in cultured ESCC cells, by cell counting kit‑8 and clonogenic assay. We further used western blot to reveal the activation of zinc finger 703 (ZNF703) by SPRY4‑IT1. RESULTS: We validated that SPRY4‑IT1 was upregulated in ESCC tissues of advanced clinical stages. In vitro function assays demonstrated that SPRY4‑IT1 cause promotion of cell viability in ESCC cells. We further verified that SPRY4‑IT1 could also activate the expression of ZNF703 in ESCC cells, which might contribute to the role of SPRY4‑IT1 in ESCC cells. CONCLUSION: SPRY4‑IT1 is a vital regulator in ESCC progression, and the SPRY4‑IT1/ZNF703 axis might provide novel clues for future ESCC therapy.

Induction of Resistance to BRAF Inhibitor Is Associated with the Inability of Spry2 to Inhibit BRAF-V600E Activity in BRAF Mutant Cells

The clinical benefits of oncogenic BRAF inhibitor therapies are limited by the emergence of drug resistance. In this study, we investigated the role of a negative regulator of the MAPK pathway, Spry2, in acquired resistance using BRAF inhibitor-resistant derivatives of the BRAF-V600E melanoma (A375P/Mdr). Real-time RT-PCR analysis indicated that the expression of Spry2 was higher in A375P cells harboring the BRAF V600E mutation compared with wild-type BRAF-bearing cells (SK-MEL-2) that are resistant to BRAF inhibitors. This result suggests the ability of BRAF V600E to evade feedback suppression in cell lines with BRAF V600E mutations despite high Spry2 expression. Most interestingly, Spry2 exhibited strongly reduced expression in A375P/Mdr cells with acquired resistance to BRAF inhibitors. Furthermore, the overexpression of Spry2 partially restored sensitivity to the BRAF inhibitor PLX4720 in two BRAF inhibitor-resistant cells, indicating a positive role for Spry2 in the growth inhibition induced by BRAF inhibitors. On the other hand, long-term treatment with PLX4720 induced pERK reactivation following BRAF inhibition in A375P cells, indicating that negative feedback including Spry2 may be bypassed in BRAF mutant melanoma cells. In addition, the siRNA-mediated knockdown of Raf-1 attenuated the rebound activation of ERK stimulated by PLX4720 in A375P cells, strongly suggesting the positive role of Raf-1 kinase in ERK activation in response to BRAF inhibition. Taken together, these data suggest that RAF signaling may be released from negative feedback inhibition through interacting with Spry2, leading to ERK rebound and, consequently, the induction of acquired resistance to BRAF inhibitors.

Long non-coding RNA SPRY4-IT1 expression in esophageal squamous cell carcinoma and its effects on cell growth / 中国肿瘤临床

Objective:This study aimed to clarify the correlation of SPRY4-IT1 expression with the clinicopathological character-istics and prognosis of patients with esophageal squamous cell carcinoma (ESCC), as well as the role of SPRY4-IT1 in promoting ES-CC cell growth. Methods:Quantitative real-time polymerase chain reaction for SPRY4-IT1 expression was performed on 50 paired can-cerous and adjacent non-cancerous esophageal specimens. Small interfering RNA was used to suppress SPRY4-IT1 expression to fur-ther explore its role in tumor progression. Cell viability was tested in vitro by MTT assay (OD=490 nm), and cell apoptosis and cell cy-cle were investigated by flow cytometry. Results:We found markedly elevated SPRY4-IT1 expression in cancerous tissues compared with adjacent non-cancerous tissues (90%, P0.05). Further experiments showed that SPRY4-IT1 expression levels were significantly higher in three ESCC cell lines than in the normal human esophageal epithelial cell line Het-1A. In vitro assays of the ESCC cell line KYSE30 demonstrated that knockdown of SPRY4-IT1 expression by small interfering RNA reduced cell growth, mediated cell cycle arrest at the G0-G1 phase, and promoted cell apoptosis (all P<0.01). Conclusion:SPRY4-IT1 was overexpressed in ESCC tissues and ESCC cell lines and promoted the growth of ESCC cells. The dysregulated expression of long non-coding RNA SPRY4-IT1 may play an important role in the process of ESCC development and may be developed as a useful biomarker for the diagnosis and prognosis of ESCC.