Knockdown of TRIM66 Suppresses the Proliferation, Migration, Invasion and Glycolysis in Cholangiocarcinoma Cells

Background: The tripartite motif (TRIM) family proteins feature highly conserved order of domains in the RBCC motif and most of them play an essential role in various cellular processes. Recently, increasing evidence has shown association of TRIM proteins with cancer development. In this study, we examined the expression pattern and biological functions of TRIM66 in cholangiocarcinoma (CCA).Methods: Western blot was performed for the protein levels of TRIM66, E-cadherin, α-catenin, N-cadherin, vimentin, p-PI3K, PI3K, p-Akt and Akt. MTT assay, wound healing assay and transwell assay were conducted for cell proliferation, migration and invasion, respectively. Glucose uptake and lactate production were determined using specific kits.Results: TRIM66 was overexpressed in CCA tissues and cell lines. In addition, knockdown of TRIM66 significantly inhibited proliferation, migration, invasion and glycolysis of CCA cells. Moreover, TRIM66 silencing obviously decreased levels of phosphorylated PI3K and Akt in CCA cells.Conclusion: Our study provided a novel insight into the roles of TRIM66 in CCA and suggested TRIM66 as a promising therapeutic target for CCA treatment.

Mechanism of Action of the Sesquiterpene Compound Helenalin in Rhabdomyosarcoma Cells

Rhabdomyosarcoma (RMS) is the most frequent soft tissue sarcoma in paediatric patients. Relapsed or refractory RMS shows very low 5-year survival rates, which urgently necessitates new chemotherapy agents. Herein, the sesquiterpene lactone, helenalin, was investigated as a new potential therapeutic agent against the embryonal RMS (eRMS) and alveolar RMS (aRMS) cells. We have evaluated in vitro antiproliferative efficacy of helenalin on RMS cells by the MTT and wound healing assay, and estimated several cell death pathways by flow cytometry, confocal microscopy and immunoblotting. It was shown that helenalin was able to increase reactive oxygen species levels, decrease mitochondrial membrane potential, trigger endoplasmic reticulum stress and deactivate the NF-κB pathway. Confirmation was obtained through the use of antagonistic compounds which alleviated the effects of helenalin in the corresponding pathways. Our findings demonstrate that oxidative stress is the pivotal mechanism of action of helenalin in promoting RMS cell death in vitro.

Aberrant MEK5 Signalling Promotes Renal Cancer Development via mTOR Activation

Purpose: Renal cell carcinoma is one of the most incident malignancies globally. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma. However, comprehensive clinical treatment has some limitations. Therefore, exploring the pathogenesis and identifying novel therapeutic targets are required urgently. MEK5 has been reported to play an essential role in the development of various cancers. However, no study has evaluated its role and specific mechanisms in ccRCC. Methods: Using the ONCOMINE database, MEK5 expression in ccRCC and normal tissues was compared. ccRCC and adjacent normal tissues were collected from fourteen ccRCC patients, and ccRCC expression was assessed by qPCR and immunohistochemistry. MEK5 overexpression and knockdown plasmids were constructed and transfected into ccRCC cells. CCK8, wound-healing assay, and clone formation assay were performed to examine the cell proliferation, migration, and clone formation ability of ccRCC cells. Furthermore, a western blot was performed to verify the regulation and influence of MEK5 on the mTOR signaling pathway. The MEK5 small molecule inhibitor BIX was used to treat cells, followed by CCK8, wound-healing assay, clone formation, and flow cytometry assay to examine the cell proliferation, migration, clone formation ability, apoptosis, and cell cycle. Finally, a murine subcutaneous tumor model was constructed, and the effect and safety of BIX were evaluated in-vivo.Results: The ONCOMINE database indicated that the MEK5 expression in ccRCC was significantly higher than the normal tissues, further confirmed in clinical specimens. The knockdown of MEK5 markedly inhibited the ability of ccRCC cell proliferation, colony formation, and migration. In contrast, MEK5 overexpression promoted cell proliferation, colony formation, and migration. Western blotting showed that overexpression of MEK5 can further activate the mTOR signaling pathway. The MEK5 inhibitor, BIX treatment of ccRCC, significantly inhibited cell proliferation, arrested the cell cycle in the G0/G1 phase, induced apoptosis, and effectively inhibited cell migration and clone formation. BIX also showed an excellent anti-tumor effect and favorable safety profile in murine models.Conclusions: MEK5 regulated the mTOR signal pathway and regulated the cell proliferation, cycle, migration, clone formation of ccRCC. Targeted inhibition of MEK5 had a good anti-tumor effect and favorable safety profile, providing new directions for ccRCC therapy.

MiR-513a-5p Aggravates Atherosclerosis Phenotype by Downregulating TFPI2 in Vitro

Background: Atherosclerosis (AS) induced cardiology disease is largely associated with morbidity and mortality. The dysfunction of vascular smooth muscle cells (VSMCs) is considered to contribute to the etiology of AS. However, the mechanism underlying VSMCs dysfunction remains largely unclear. Our study aimed to explore novel molecules mediating VSMCs function. Methods: Bioinformatical analysis was applied to identify the key miRNAs that was aberrantly expressed in AS mouse and potentially targeted TFPI2. The AS-like cell model was generated by treating VSMCs with ox-LDL. The expression level of miR-513a-5p and TFPI2 in VSMCs and the serum of AS patients was evaluated by RT-qPCR, and the expression level of TFPI2 and PCNA was measured by western blot. The cell viability and migration capacity of VSMCs were determined by CCK-8 and wound healing assay, respectively. The target relationship between miR-513a-5p and TFPI2 was validated by dual-luciferase assay. Results: MiR-513a-5p was highly expressed while TFPI2 presented a low expression in AS patient serum. Treatment with 100 μg/mL ox-LDL overtly facilitated the cell viability and migration of VSMCs, also promoted miR-513a-5p expression while limit the expression of TFPI2. Moreover, silencing miR-513a-5p inhibited the cell viability, migration and the expression of proliferative marker in ox-LDL treated VSMCs, while inhibition of TFPI2 enhanced that. It was further found that miR-513a-5p could target TFPI2 and silencing miR-513a-5p compromised the aggresive effect of TFPI2 inhibition on the viability and migration ox-LDL treated VSMCs. Conclusion: miR-513a-5p could contribute to the dysfunction of VSMCs in AS through targeting and inhibiting TFPI2.

DeepSpot: a deep neural network for RNA spot enhancement in smFISH microscopy images

Detection of RNA spots in single molecule FISH microscopy images remains a difficult task especially when applied to large volumes of data. The small size of RNA spots combined with high noise level of images often requires a manual adaptation of the spot detection thresholds for each image. In this work we introduce DeepSpot, a Deep Learning based tool specifically designed to enhance RNA spots which enables spot detection without need to resort to image per image parameter tuning. We show how our method can enable the downstream accurate detection of spots. The architecture of DeepSpot is inspired by small object detection approaches. It incorporates dilated convolutions into a module specifically designed for the Context Aggregation for Small Object (CASO) and uses Residual Convolutions to propagate this information along the network. This enables DeepSpot to enhance all RNA spots to the same intensity and thus circumvents the need for parameter tuning. We evaluated how easily spots can be detected in images enhanced by our method, by training DeepSpot on 20 simulated and 1 experimental datasets, and have shown that more than 97% accuracy is achieved. Moreover, comparison with alternative deep learning approaches for mRNA spot detection (deepBlink) indicated that DeepSpot allows more precise mRNA detection. In addition, we generated smFISH images from mouse fibroblasts in a wound healing assay to evaluate whether DeepSpot enhancement can enable seamless mRNA spot detection and thus streamline studies of localized mRNA expression in cells.

miR-93-5p Suppresses Ovarian Cancer Malignancy and Negatively Regulate CCND2 By Binding To Its 3’UTR Region

Ovarian cancer is the most lethal gynecological cancer worldwide, but the underlying mechanism of ovarian cancer malignancy acquirement is largely unknown. miRNA is ubiquitously implicated in disease especially in cancer initiation and progression. In current study, we firstly detected the expression level of miR-93-5p in ovarian cancer patient samples and conducted a survival analysis. Our data revealed miR-93-5p is a favorable prognostic factor but is downregulated in ovarian cancer patients. Secondly, CCK8 assay wound healing assay and flow cytometry-based cell cycle analysis and apoptotic cell analysis were performed respectively to study the function of miR-93-5p. Functional analysis show miR-93-5p promotes ovarian cancer malignancy in term of cell proliferation, migration but reduce cell death. Bioinformatic analysis showed Cyclin-D2(CCND2) is a candidate gene of miR-93-5p with the binding site in its 3’UTR region. Furthermore, quantitive-PCR and western blot were utilized to measure miR-93-5p, CCND2 levels in tissues samples and cell lines. Our data suggested miR-93-5p is negatively correlated to the level of CCND2 mRNA and protein. Finally, Luciferase report assay was conducted, and we demonstrated miR-93-5p reduces CCND2 expression by binding to the 3’UTR region. Our study revealed the function of miR-93-5p in ovarian cancer malignancy and declaimed CCND2 as a target of miR-93-5p.

HPV16 E7 Nucleotide Variants Found in Cancer-Free Subjects Affect E7 Protein Expression and Transformation

The human papillomavirus (HPV) type 16 E7 oncogene is critical to carcinogenesis and highly conserved. Previous studies identified a preponderance of non-synonymous E7 variants amongst HPV16-positive cancer-free controls compared to those with cervical cancer. To investigate the function of E7 variants, we constructed full-length HPV16 E7 genes and tested variants at positions H9R, D21N, N29S, E33K, T56I, D62N, S63F, S63P, T64M, E80K, D81N, P92L, and P92S (found only in controls); D14E, N29H (CIN2), and P6L, H51N, R77S (CIN3). We determined the steady-state level of cytoplasmic and nuclear HPV16 E7 protein. All variants from the controls showed a reduced level of steady-state E7 protein, with 7/13 variants having deficient protein levels. In contrast, 2/3 variants from the CIN3 precancer group had near-normal E7 levels. We assayed the activity of representative variants in stably transfected NIH3T3 cells. The H9R, E33K, P92L, and P92S variants found in control subjects had lower transforming activity than D14E and N29H variants (CIN2); and the R77S (CIN3) had activity only slightly reduced from wildtype E7. In addition, R77S and WT E7 caused increased migration of NIH3T3 cells in a wound-healing assay as compared with H9R, E33K, P92L, and P92S (controls) and D14E (CIN2). These data provide evidence that the E7 variants found in HPV16-positive cancer-free women are partially defective for transformation and cell migration further demonstrating the importance of fully active E7 in clinical cancer development.

SPTBN2, a New Biomarker of Lung Adenocarcinoma

ObjectivesThe roles played by β-III-spectrin, also known as spectrin beta, non-erythrocytic 2 (SPTBN2), in the occurrence and development of lung adenocarcinoma (LUAD) have not been previously examined. Our study aimed to reveal the relationship between the SPTBN2 expression and LUAD.Materials and MethodsTwenty pairs of LUAD tissues and adjacent tissues were collected from patients diagnosed and treated at the Thoracic Surgery Department of The First Affiliated Hospital of Zhengzhou University from July 2019 to September 2020. RNA sequencing (RNA-seq) analysis determined that the expression of SPTBN2 was higher in LUAD samples than in adjacent normal tissues. The expression levels of SPTBN2 were examined in various databases, including the Cancer Cell Line Encyclopedia (CCLE), Gene Expression Omnibus (GEO), and Human Protein Atlas (HPA). The Search Tool for the Retrieval of Interacting Genes (STRING) online website was used to examine protein–protein interactions involving SPTBN2, and the results were visualized by Cytoscape software. The Molecular Complex Detection (MCODE) plug-in for Cytoscape software was used to identify functional modules of the obtained protein–protein interaction (PPI) network. Gene enrichment analysis was performed, and survival analysis was conducted using the Kaplan–Meier plotter. The online prediction website TargetScan was used to predict SPTBN2-targeted miRNA sequences by searching for SPTBN2 sequences. Finally, we verified the expression of SPTBN2 in the obtained tissue samples using real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The human lung cancer cell lines A549 and H1299 were selected for the transfection of small interfering RNA (siRNA) targeting SPTBN2 (si-SPTBN2), and the knockdown efficiency was evaluated by RT-qPCR. The cellular proliferation, migration, and invasion capacities of A549 and H1299 cells were determined using the cell counting kit-8 (CCK-8) proliferation assay; the wound-healing assay and the Transwell migration assay; and the Matrigel invasion assay, respectively.ResultsThe expression of SPTBN2 in non–small cell lung cancer (NSCLC) ranked 13th among cancer cell lines based on the CCLE database. At the mRNA and protein levels, the expression levels of SPTBN2 were higher in LUAD tissues than in normal lung tissues. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that proteins related to SPTBN2 were enriched in apoptotic and phagosomal pathways. Kaplan–Meier survival analysis revealed that SPTBN2 expression was significantly related to the prognosis of patients with LUAD. The TargetScan database verified that miR-16 was a negative regulator of SPTBN2 mRNA expression. The results of the CCK-8 cell proliferation assay revealed that SPTBN2 knockdown significantly inhibited the cell proliferation abilities of A549 and H1299 cells. The wound-healing assay indicated that SPTBN2 knockdown resulted in reduced migration after 48 h compared with the control group. The Transwell migration and invasion test revealed that the migration and invasion abilities were greatly decreased by SPTBN2 knockdown compared with control conditions.ConclusionWe uncovered a novel gene, SPTBN2, that was significantly upregulated in LUAD tissues relative to normal tissue expression. SPTBN2 is highly expressed in LUAD, positively correlated with poor prognosis, and can promote the proliferation, migration, and invasion of LUAD cells.

MicroRNA-200c-3p Functions as a Tumor Suppressor in Kidney Renal Clear Cell Carcinoma by Targeting Vascular Endothelial Growth Factor A

Background: MicroRNA(miR)-200c-3p is a tumor suppressor that helps inhibit the progression of various types of cancer. However, its role in kidney renal clear cell carcinoma (KRIC) is unknown. The purpose of this study was to explore the biological function and regulatory mechanism of miR-200c-3p in the development of KRIC.Methods: The relative levels of miR-200c-3p and vascular endothelial growth factor A (VEGFA) in KIRC tissues and cells were determined using the qRT-PCR technique. Transwell and wound healing assay methods were used to understand the effect of miR-200c-3p on the migration and invasion of 786-O and Caki-1 cells. The ability of miR-200c-3p to target VEGFA was determined using the Dual-Luciferase reporter assay system Results: MiR-200c-3p was downregulated in KIRC tissues and cell lines. The overexpression of miR-200c-3p attenuated the migratory capacities of 786-O and Caki-1 cells. The migratory ability increased under conditions of miR-200c-3p knockdown. Analysis of the results obtained using the wound healing assay revealed that overexpression of miR-200c-3p reduced the migratory abilities of Caki-1 and 786-O cells, and inhibition of miR-200c-3p accelerated the process of wound closure. Analysis of the results obtained from screening tests and by conducting the Dual-Luciferase reporter assay revealed that VEGFA was the direct target of miR-200c-3p. The VEGFA mRNA level was low in the 786-O and Caki-1 cells under conditions of overexpressed miR-200c-3p. High levels of VEGFA mRNA were observed in the cells post miR-200c-3p knockdon.Conclusions: The tumor suppression ability of miR-200c-3p in KIRC can be achieved by regulating the expression of VEGFA.

Zyxin Mediates Vascular Repair via Endothelial Migration Promoted by Forskolin in Mice

Background and Purpose: Endothelial repair upon vascular injury is critical for the protection of vessel integrity and prevention of the development of vascular disorders, but the underlying mechanisms remain poorly understood. In this study, we investigated the role of zyxin and its associated cyclic adenosine monophosphate (cAMP) signaling in the regulation of re-endothelialization after vascular injury.Experimental Approach: In zyxin-/- and wild-type mice, wire injury of the carotid artery was carried out, followed by Evans blue staining, to evaluate the re-endothelialization. Mice with endothelium-specific zyxin knockout were used to further determine its role. An in vitro wound-healing assay was performed in primary human endothelial cells (ECs) expressing zyxin-specific short-hairpin RNAs (shRNAs) or scrambled controls by measuring cell migration and proliferation. The effects of the cAMP signaling agonist forskolin were assessed.Key Results: The re-endothelialization of the injured carotid artery was impaired in zyxin-deficient mice, whereas the rate of cell proliferation was comparable with that in wild-type controls. Furthermore, endothelium-specific deletion of zyxin led to similar phenotypes. Knockdown of zyxin by shRNAs in primary human ECs significantly reduced cell migration in the wound-healing assay. Notably, forskolin enhanced endothelial migration in a dose-dependent manner, and this was dependent on zyxin through its interaction with vasodilator-stimulated phosphoprotein. In addition, forskolin promoted the re-endothelialization of the injured carotid artery, and this was compromised by zyxin deficiency.Conclusion and Implications: This study reveals zyxin as a new player in endothelial repair, which is promoted by forskolin, after vascular injury. Thus, zyxin-mediated signaling might be a potential treatment target for diseases involving vascular injury.