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2021 ◽  
Author(s):  
Jialei Yang ◽  
Xiaoxiao Song ◽  
Yibing Yang ◽  
Yan Yan ◽  
Baoyun Liang ◽  
...  

Abstract Previous studies reported that the SRC protein was involved in a variety of pathological mechanisms related to ischemic stroke (IS). In this study, we conducted a genetic association study between rs6017916 within the 5’UTR region of SRC gene and IS susceptibility. A total of 533 IS patients and 531 healthy controls were recruited to participate in the current study. The sequenom MassARRAY technology platform was used for genotyping. The quantitative polymerase chain reaction (qPCR) was conducted to detect SRC mRNA expression. The dual luciferase reporter system was used to verify the regulation of rs6017916 on SRC mRNA expression. Results showed that SRC mRNA expression was significantly increased in IS patients than that in controls (P<0.001). Receiver operating characteristic curve (ROC) analysis demonstrated that the signature of SRC mRNA expression differentiated between controls and IS patients with an area under the curve (AUC) of 0.935 corresponding to a specificity of 0.820 and sensitivity of 0.920. Genetic association analysis showed that rs6017916 was significantly associated with IS susceptibility under multiple genetic models, including additive [OR (95% CI)=0.76 (0.60,0.96), Padj=0.019] and dominant [OR (95% CI)=0.75(0.58,0.98), Padj=0.031]. In addition, the dual luciferase reporter system showed that the minor allele C of rs6017916 inhibited luciferase activity compared with the major allele A. In summary, we report that SRC mRNA expression was significantly increased in IS patients and was a potential diagnostic biomarker. Moreover, the 5’UTR variant rs6017916 of SRC was significantly associated with IS susceptibility. And rs6017916 might affect the pathological process of IS by regulating SRC expression.


2021 ◽  
Author(s):  
Marina Barba-Aliaga ◽  
Adriana Mena ◽  
Vanessa Espinoza ◽  
Nadezda Apostolova ◽  
Mercedes Costell ◽  
...  

Translation of mRNAs that encode peptide sequences with consecutive prolines (polyproline) requires the conserved and essential elongation factor eIF5A to facilitate the formation of peptide bonds. It has been shown that upon eIF5A depletion, yeast ribosomes stall in polyproline motifs, but also in tripeptide sequences that combine proline with glycine and charged amino acids. Mammalian collagens are enriched in putative eIF5A-dependent Pro-Gly-containing tripeptides. Here we show that depletion of active eIF5A in mouse fibroblasts reduced collagen 1 (Col1a1) content, which concentrated around the nuclei. Moreover, it provoked the up-regulation of endoplasmic reticulum (ER)-stress markers suggesting retention of partially synthesized Col1 in the ER. We confirmed that eIF5A is needed for heterologous collagen synthesis in yeast, and using a double luciferase reporter system we showed that eIF5A depletion interrupts translation at Pro-Gly-collagenic motifs. A dramatically lower level of Col1α1 protein was also observed in functional eIF5A-depleted human hepatic stellate cells treated with the profibrotic cytokine TGF-β1. In sum, our results show that collagen expression requires eIF5A and imply its potential as a target for regulating collagen production in fibrotic diseases.


2021 ◽  
Author(s):  
Zhongyuan Deng ◽  
Yuting Zhang ◽  
Leyao Li ◽  
Xingcheng Xie ◽  
Jinyong Huang ◽  
...  

2021 ◽  
Vol 11 ◽  
Author(s):  
Ji Li ◽  
Changjiang Lei ◽  
Bineng Chen ◽  
Qingfang Zhu

BackgroundLncRNA-FGD5-AS1, as an oncogene, participates in the development and progress of various cancers. However, the exact role and the molecular mechanisms by which FGD5-AS1 regulates radiosensitivity in breast cancer (BC) remains largely unknown.MethodsWe used X-Ray weekly-dose-increase method to establish radiation-resistance cell lines. Bioinformatics tools analyze the expression of FGD5-AS1 in breast cancer tissue and evaluated the relationship between FGD5-AS1 and clinic-pathological features. CCK-8 and colony formation were used to analyze cell proliferation. Western blotting and qPCR were applied to detect protein and gene expression, respectively. RNA interference was used to knock down the endogenous gene expression. Luciferase reporter system and immunoprecipitates were applied to verify the target of FGD5-AS1.ResultFGD5-AS1 was overexpressed in BC tissues and radiation-resistance cell lines. Higher levels of FGD5-AS1 predicted poorer clinical characteristics and prognosis. Loss-of-function FGD5-AS1 sensitized BC cells to X-ray, meanwhile, the cell gained radiation-resistance when exogenous FGD5-AS1 was expressed. FGD5-AS1 depletion arrested cells at G0/G1 and triggers cell apoptosis. The starBase database (ENCORI), predicted binding site of miR-497-5p in FGD5-AS1 sequence, and luciferase reporter system and immunoprecipitates verified miR-497-5p was the target of FGD5-AS1. Furthermore, MACC1 was predicted and verified as the target of miR-497-5p. Loss-of-function FGD5-AS1 sensitized ionizing radiation was rescued by the up-regulation of MACC1 and the inhibition of miR-497.ConclusionFGD5-AS1 displays an oncogene profile in CRC; patients with high expression of FGD5-AS1 should benefit less from radiotherapy and need a more frequent follow-up. Besides, FGD5-AS1 may be a potential therapeutic target for CRC.


2021 ◽  
Vol 12 ◽  
Author(s):  
Ruowu Liu ◽  
Jintao Du ◽  
Jiao Zhou ◽  
Bing Zhong ◽  
Luo Ba ◽  
...  

BackgroundCRSwNP is an inflammatory disease but the mechanism is not yet fully understood. MiR-21, a member of miRNAs, has been reported to play roles in mediating inflammation. However, the expression of miR-21 and its role in patients with CRSwNP remain elusive.MethodsTurbinates from control subjects, uncinate processes from CRSsNP, polyp tissues from CRSwNP, and nasal epithelial cells brushed from nasal mucosa were collected. The expression of miR-21 and cytokines in nasal tissues and epithelial cells were detected by qPCR. The localization of miR-21 was detected by ISH, and its target was identified by bioinformation analysis, qPCR, IHC, WB, and luciferase reporter system. The protein and mRNA of PDCD4 and NF-κB P65 were determined by WB and qPCR after miR-21 transfection in HNEpC. The role of miR-21 on cytokines was analyzed in HNEpC and nasal polyp explants.ResultsMiR-21 was upregulated in CRSwNP relative to control subjects by qPCR, which was determined mainly in nasal epithelial cells of CRSwNP by ISH. Both pro-inflammation cytokines (IL-1β, IL-6, IL-8, IL-25, and TSLP) and a suppressive cytokine (IL-10) were overexpressed in the epithelial cells of CRSwNP. The expression of miR-21 was positively correlated with IL-10 and negatively correlated with IL-6, IL-8, IL-33, and TSLP in the epithelial cells of CRSwNP. As a potential target of miR-21, the expression of PDCD4 was negatively correlated with miR-21 in CRSwNP. In HNEpC, miR-21 could reduce the expression of PDCD4 at both mRNA and protein levels, and bioinformation analysis and luciferase reporter system confirmed PDCD4 as one target of miR-21. Furthermore, miR-21 could decrease the activation of NF-κB and increase IL-10 mRNA. Both SEB and LPS could elevate miR-21, with IL-25, IL-33, TSLP induced by SEB and IL-1β, IL-6, IL-8 induced by LPS, while the miR-21 could regulate the expression of IL-33, TSLP, IL-1β, IL- 6 and IL-8 in vitro and ex vivo. Clinically, miR-21 expression was inversely correlated with the Lund-Mackay CT scores and the Lund-Kennedy scores in CRSwNP.ConclusionMiR-21 could be a prominent negative feedback factor in the inflammation process to attenuate the expression of pro-inflammatory cytokines, thereby playing an anti-inflammation role in CRSwNP.


2021 ◽  
Author(s):  
Marina Barba-Aliaga ◽  
Adriana Mena ◽  
Vanessa Espinoza ◽  
Nadezda Apostolova ◽  
Mercedes Costell ◽  
...  

AbstractThe evolutionary conserved elongation factor eIF5A is required for the translation of mRNAs that encode protein sequences with consecutive prolines or combined with glycine and charged amino acids. Mammalian collagens are enriched in putative eIF5A-dependent Pro-Gly-containing tripeptides. Here, we show that eIF5A is needed for heterologous expression of collagen in yeast, and using a dual luciferase reporter system we confirmed that eIF5A depletion interrupts translation at Pro-Gly-collagenic motifs. Using mouse fibroblasts, we showed that depletion of active eIF5A reduced collagen 1α (Col1a1) content, which became concentrated around the nuclei, in contrast to a stronger and all over the cell collagen signal in untreated cells. Active eIF5A-depleted mouse fibroblast showed upregulation of endoplasmic reticulum (ER) stress markers, suggesting retention of partially synthesized Col1a1 in the ER. A dramatically lower level of Col1α1 protein was also observed in functional eIF5A-depleted human hepatic stellate cells treated with the profibrotic cytokine TGF-β1. Our results show that collagen expression requires eIF5A and imply its potential as a target for regulating collagen production in fibrotic diseases.


2021 ◽  
Author(s):  
Jun Zhang ◽  
Xian Zhang ◽  
Shasha Yang ◽  
Yanqiu Bao ◽  
Dongyuan Xu ◽  
...  

Abstract Background: Forkhead box protein H1 (FOXH1) is upregulated in a variety of cancer types but its expression patterns and specific functions in lung cancer are unclear at present. The main objective of the present study was to establish whether FOXH1 plays a role in regulation of lung cancer progression. Methods: The TCGA and Kaplan-Meier plotter dataset was analyzed for possible association between FOXH1 expressions in lung cancer tissues and patient prognosis. A549 and PC9 cells were transfected with shRNA targeting FOXH1 mRNA. The Cell Counting Kit-8 (CCK8), plate clone formation, soft agar, wound healing, transwell invasion and flow cytometry assay were performed to detect the cell proliferation, migration and invasion in lung cancer cells. Tumorigenicity was examined in mouse model system. Western blot analysis was used to detect FOXH1, Matrix metalloproteinase 2 (MMP2), Vimentin, N-cadherin, E-cadherin, Snail, Slug, p-GSK-3β, β-catenin and Cyclin D1. The β-catenin activity was measured by luciferase reporter system assay. Results: FOXH1 expression in the lung cancer was determined using the TCGA and Kaplan-Meier plotter databases. Higher expression of FOXH1 was observed in tumor tissue relative to normal tissue, which was associated with reduced overall survival. FOXH1 knockdown resulted in significant inhibition of the proliferation, the cell cycle, the migration as well as the invasion of lung cancer cells. Then, we confirmed that FOXH1 knockdown can significantly slow down tumor growth and tumor cell proliferation in vivo using the mouse model. It also significantly reduced the migration and invasion capabilities of lung cancer cells. Using Western blot analyses, we found that FOXH1 depletion inhibited the epithelial-mesenchymal transition of lung cancer cells through downregulation of the mesenchymal cell markers Snail, Slug, MMP2, N-cadherin, and Vimentin, and upregulation of the epithelial marker E-cadherin. Moreover, FOXH1 knockdown significantly downregulated β-catenin and its downstream targets, such as p-GSK-3β and cyclin D1, and also led to direct suppression of β-catenin activity, as determined by the luciferase reporter system assay. Conclusion: In conclusion, FOXH1 promotes proliferation, migration, and invasion of lung cancer cells via regulation of Wnt/β-catenin signaling. FOXH1 is a prognostic marker and a potential therapeutic target for lung cancer treatment.


2021 ◽  
Vol 16 (1) ◽  
pp. 266-276
Author(s):  
Zhenfen Wang ◽  
Qing Liu ◽  
Ping Huang ◽  
Guohao Cai

Abstract Gastric cancer (GC) is ranked the fourth leading cause of cancer-related death, with an over 75% mortality rate worldwide. In recent years, miR-299-3p has been identified as a biomarker in multiple cancers, such as acute promyelocytic leukemia, thyroid cancer, and lung cancer. However, the regulatory mechanism of miR-299-3p in GC cell progression is still largely unclear. Cell viability and apoptosis tests were performed by CCK8 and flow cytometry assay, respectively. Transwell assay was recruited to examine cell invasion ability. The interaction between miR-299-3p and PAX3 was determined by the luciferase reporter system. PAX3 protein level was evaluated by western blot assay. The expression of miR-299-3p was downregulated in GC tissues and cell lines (MKN-45, AGS, and MGC-803) compared with the normal tissues and cells. Besides, overexpression of miR-299-3p significantly suppressed proliferation and invasion and promoted apoptosis in GC. Next, we clarified that PAX3 expression was regulated by miR-299-3p using a luciferase reporter system, qRT-PCR, and western blot assay. Additionally, downregulation of PAX3 repressed GC cell progression. The rescue experiments indicated that restoration of PAX3 inversed miR-299-3p-mediated inhibition on cell proliferation and invasion. miR-299-3p suppresses cell proliferation and invasion as well as induces apoptosis by regulating PAX3 expression in GC, representing desirable biomarkers for GC diagnosis and therapy.


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