MiR-423-5p activated by E2F1 promotes neovascularization in diabetic retinopathy by targeting HIPK2

Abstract Background Diabetic retinopathy (DR) is a diabetic complication and the primary cause of blindness in the world. However, the treatments of DR are challenging given its complicated pathogenesis. Here, we investigated the molecular mechanisms of DR by focusing on the function of E2F1/miR-423-5p/HIPK2/HIF1α/VEGF axis. Methods Cultured retinal endothelial cells (hRMECs, hRECs) were treated with 25 mM glucose to mimic the high glucose-induced DR in vitro. Streptozotocin (STZ) was injected into mice to induce DR in mice. qRT-PCR, western blotting, immunohistochemistry, and ELISA were employed to measure levels of E2F1, miR-423-5p, HIPK2, HIF1α, and VEGF. H&E staining was utilized to examine retinal neovascularization. CCK-8 assay, transwell assay, and vascular tube formation assay were used to assess the cell viability, migration, and angiogenesis. Dual luciferase assay was performed to validate interactions between E2F1 and miR-423-5p, miR-423-5p and HIPK2. Results HG treatment increased the cell viability, migration, and angiogenesis accompanied by upregulation of E2F1, miR-423-5p, HIF1α, and VEGF levels, but reduction in HIPK2 expression. Knockdown of E2F1 or miR-423-5p suppressed the HG-induced increases in cell viability, migration, and angiogenesis. E2F1 transcriptionally activated miR-423-5p expression and miR-423-5p mimics blocked the effects of E2F1 knockdown on angiogenesis. Moreover, miR-423-5p directly targeted HIPK2 to disinhibit HIF1α/VEGF signaling. Knockdown of HIPK2 reversed the effects of miR-423-5p inhibitor on cell viability, migration, and angiogenesis. Knockdown of E2F1 suppressed neovascularization during DR in vivo. Conclusions E2F1 activates miR-423-5p transcription during DR to promote angiogenesis via suppressing HIPK2 expression to disinhibit HIF1α/VEGF signaling. Strategies targeting E2F1/miR-423-5p/HIPK2 axis could be potentially used for DR treatment.

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MiR-144-3p Aggravated Cartilage Injury in Rheumatoid Arthritis by Regulating BMP2/PI3K/Akt Axis

Modern Rheumatology ◽

10.1093/mr/roab105 ◽

2021 ◽

Author(s):

Mei-Li Mo ◽

Jin-Mei Jiang ◽

Xiao-Ping Long ◽

Li-Hu Xie

Keyword(s):

Extracellular Matrix ◽

Cell Viability ◽

Cell Injury ◽

Tissue Injury ◽

Cartilage Injury ◽

Luciferase Assay ◽

Tunel Staining ◽

Western Blots

Abstract Objectives Present study aimed to illustrate the role of miR-144-3p in RA. Methods N1511 chondrocytes were stimulated by IL-1β to mimic RA injury model in vitro. Rats were subjected to injection of type II collagen to establish an in vivo RA model and the arthritis index score was calculated. Cell viability was determined by CCK-8. The expression of cartilage extracellular matrix proteins (Collagen II and Aggrecan) and matrix metalloproteinases protein (MMP-13) were determined by qRT-PCR and western blots. Cell apoptosis was measured by Flow cytometry. ELISA was applied to test the secretion of pro-inflammatory cytokines (IL-1β and TNF-α). Tissue injury and apoptosis were detected by HE staining and TUNEL staining. Interaction of miR-144-3p and BMP2 was verified by dual luciferase assay. Results MiR-144-3p was dramatically increased in IL-1β induced N1511 cells. MiR-144-3p depletion elevated cell viability, suppressed apoptosis, pro-inflammatory cytokine releasing, and extracellular matrix loss in IL-1β induced N1511 cells. Moreover, miR-144-3p targeted BMP2 to modulate its expression negatively. Activation of PI3K/Akt signaling compromised inhibition of BMP2 induced aggravated N1511 cell injury with IL-1β stimulation. Inhibition of miR-144-3p alleviated cartilage injury and inflammatory in RA rats. Conclusion Collectively, miR-144-3p could aggravate chondrocytes injury inflammatory response in RA via BMP2/PI3K/Akt axis.

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DEPTOR Deficiency-Mediated mTORc1 Hyperactivation in Vascular Endothelial Cells Promotes Angiogenesis

Cellular Physiology and Biochemistry ◽

10.1159/000488619 ◽

2018 ◽

Vol 46 (2) ◽

pp. 520-531 ◽

Cited By ~ 7

Author(s):

Yan Ding ◽

Lanlan Shan ◽

Wenqing Nai ◽

Xiaojun Lin ◽

Ling Zhou ◽

...

Keyword(s):

Endothelial Cells ◽

Signaling Pathway ◽

Molecular Mechanisms ◽

Gene Knockout ◽

Umbilical Vein ◽

Vascular Endothelial Cell ◽

Tube Formation ◽

Vascular Endothelial

Background/Aims: The mechanistic target of rapamycin (mTOR) signaling pathway is essential for angiogenesis and embryonic development. DEP domain-containing mTOR-interacting protein (DEPTOR) is an mTOR binding protein that functions to inhibit the mTOR pathway In vitro experiments suggest that DEPTOR is crucial for vascular endothelial cell (EC) activation and angiogenic responses. However, knowledge of the effects of DEPTOR on angiogenesis in vivo is limited. This study aimed to determine the role of DEPTOR in tissue angiogenesis and to elucidate the molecular mechanisms. Methods: Cre/loxP conditional gene knockout strategy was used to delete the Deptor gene in mouse vascular ECs. The expression or distribution of cluster of differentiation 31 (CD31), vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1α) were detected by immunohistochemical staining or western blot. Tube formation assay was used to measure angiogenesis in vitro. Results: Deptor knockdown led to increased expression of CD31, VEGF and HIF-1α in heart, liver, kidney and aorta. After treatment with rapamycin, their expression was significantly down regulated. In vitro, human umbilical vein endothelial cells (HUVECs) were transfected with DEPTOR-specific small interfering RNA (siRNA), which resulted in a significant increase in endothelial tube formation and migration rates. In contrast, DEPTOR overexpression markedly reduced the expression of CD31, VEGF and HIF-1α. Conclusions: Our findings demonstrated that deletion of the Deptor gene in vascular ECs resulted in upregulated expression of CD31 and HIF-1α, and further stimulated the expression of VEGF which promoted angiogenesis, indicating that disruption of normal angiogenic pathways may occur through hyperactivation of the mTORC1/HIF-1α/VEGF signaling pathway.

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Nutraceuticals for the Treatment of Diabetic Retinopathy

Nutrients ◽

10.3390/nu11040771 ◽

2019 ◽

Vol 11 (4) ◽

pp. 771 ◽

Cited By ~ 20

Author(s):

Maria Grazia Rossino ◽

Giovanni Casini

Keyword(s):

Diabetic Retinopathy ◽

Molecular Mechanisms ◽

Treatment Options ◽

Vitreoretinal Surgery ◽

In Vivo Studies ◽

Early Diabetes ◽

Advanced Stages ◽

Endothelial Growth Factor

Diabetic retinopathy (DR) is one of the most common complications of diabetes mellitus and is characterized by degeneration of retinal neurons and neoangiogenesis, causing a severe threat to vision. Nowadays, the principal treatment options for DR are laser photocoagulation, vitreoretinal surgery, or intravitreal injection of drugs targeting vascular endothelial growth factor. However, these treatments only act at advanced stages of DR, have short term efficacy, and cause side effects. Treatment with nutraceuticals (foods providing medical or health benefits) at early stages of DR may represent a reasonable alternative to act upstream of the disease, preventing its progression. In particular, in vitro and in vivo studies have revealed that a variety of nutraceuticals have significant antioxidant and anti-inflammatory properties that may inhibit the early diabetes-driven molecular mechanisms that induce DR, reducing both the neural and vascular damage typical of DR. Although most studies are limited to animal models and there is the problem of low bioavailability for many nutraceuticals, the use of these compounds may represent a natural alternative method to standard DR treatments.

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Downregulation of Exosomal miR-26a-5p Promotes Lymphangiogenesis and Lymphatic Metastasis in Endometrial Cancer

10.21203/rs.3.rs-898526/v1 ◽

2021 ◽

Author(s):

Jing Wang ◽

Xiaodi Gong ◽

Linlin Yang ◽

Lijuan Li ◽

Xiaoyan Gao ◽

...

Keyword(s):

Endometrial Cancer ◽

Molecular Mechanisms ◽

Critical Role ◽

Cell Communication ◽

Lymphatic Endothelial Cell ◽

Tube Formation ◽

Downstream Target ◽

Ec Cells

Abstract Background: Endometrial cancer (EC) patients with lymph node (LN) metastasis have poor prognosis. However, the potential biomarkers that predict LN metastasis and the molecular mechanism of tumor-induced peritumoral lymphangiogenesis have not been well explored. Cancer-secreted exosomal miRNAs are emerging mediators of cell-cell communication in the tumor environment.Methods: Exosomes were isolated with a differential centrifugation method and confirmed by Transmission electron microscopy, NanoSight analysis, and Western blot. MicroRNA (miRNA) sequencing of exosomes derived from EC patients and healthy donors were performed. FISH and qRT-PCR were used to detect the indicated miRNA expression. Exosomal miRNA transferred to cells were confirmed by immunofluorescence and confocal microscope. siRNA and plasmid transfections as well as viral infection were performed to manipulate gene expression. A series of in vitro and in vivo phenotype experiments (tube formation, migration, and popliteal LN metastasis model) were performed to investigate the role of indicated miRNA in EC. RNA sequencing was used to select the underlying transcription factor, and luciferase activity assay and chromatin immunoprecipitation were performed to elucidate the molecular mechanisms. Results: Our data showed that serum exosomal miR-26a-5p was significantly reduced in EC patients, and its level was positively associated with LN metastasis. Loss of miR-26a-5p promoted the migratory and invasive abilities of EC cells, and miR-26a-5p could be transferred from EC cells-secreted exosomes into human lymphatic endothelial cell (HLEC). Mechanistically, miR-26a-5p could regulate LEF1/c-myc/VEGFA axis via binding to its direct downstream target lymphoid enhancer binding factor 1 (LEF1), consequently promoting HLEC tube formation and migration in vitro, facilitating lymphangiogenesis and LN metastasis in vivo. Re-expression and knockdown of LEF1 could respectively promote and rescue the effects induced by exosomal miR-26a-5p. Moreover, we demonstrated that transcriptional factor EB (TFEB) directly induced miR-26a-5p expression.Conclusions: Our results show that exosomal miR-26a-5p/LEF1/c-myc/VEGFA axis is dysregulated and plays a critical role in LN metastasis and exosomal miR-26a-5p may be used as a blood-based biomarker for EC patients with LN metastasis.

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CircRNA_0000392 Promotes Colorectal Cancer Progression by Sponging miR-193a-5p

10.21203/rs.3.rs-59369/v1 ◽

2020 ◽

Author(s):

Hanchen Xu ◽

Yujing Liu ◽

Peiqiu Cheng ◽

Chunyan Wang ◽

Yang Liu ◽

...

Keyword(s):

Colorectal Cancer ◽

Expression Profile ◽

High Throughput ◽

Molecular Mechanisms ◽

High Throughput Sequencing ◽

Malignant Progression ◽

Luciferase Assay ◽

Circular Rnas

Abstract Background: Circular RNAs (circRNAs), an important member of the non-coding RNA family, have been revealed the role in the pathogenic progression of diseases in recent years, particularly in the malignant progression of cancer. With the application of high-throughput sequencing technology, a large number of circRNAs have been found in tumor tissues, and some circRNAs have demonstrated the role as oncogenic genes. In this study, we analyzed the circRNA expression profile in colorectal cancer (CRC) tissues and normal adjacent tissues by high-throughput sequencing, focusing on the circRNA_0000392, a circRNA with significantly increased expression in colorectal cancer tissues, and further investigating its function in the progression of colorectal cancer.Methods: The expression profile of circRNAs in 6 pairs of CRC tissues and normal adjacent tissues was analyzed by RNA-sequencing. We verified the differential circRNAs with expanded samples by qRT-PCR, focused on circRNA_0000392, and evaluated its associations with clinicopathological features. Then we knocked down circRNA_0000392 in CRC cells and evaluated the effect in vitro and in vivo by functional experiments. The dual luciferase assay and RNA pull-down were performed to further explore the downstream potential molecular mechanisms.Results: CircRNA_0000392 was significantly up-regulated in CRC compared with normal adjacent tissues and cell line. The expression level of circRNA_0000392 was positively correlated with the malignant progression of CRC. Functional studies revealed that reducing the expression of circRNA_0000392 could inhibit the proliferation and invasion of CRC both in vitro and in vivo. Mechanistically, circRNA_0000392 could act as a sponge of miR-193a-5p and regulate the expression of PIK3R3, then affect the activation of the AKT-mTOR pathway in CRC cells.Conclusions: The circRNA_0000392 has the function as an oncogene through miR-193a-5p/PIK3R3-Akt axis in CRC cells, implying that circRNA_0000392 is a potential therapeutic target for the treatment of colorectal cancer and a predictive marker for CRC patients.

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LncRNA ST7-AS1, by regulating miR-181b-5p/KPNA4 axis, promotes the malignancy of lung adenocarcinoma

Cancer Cell International ◽

10.1186/s12935-020-01652-7 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Rong-Hang Hu ◽

Zi-Teng Zhang ◽

Hai-Xiang Wei ◽

Lu Ning ◽

Jiang-Shan Ai ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Noncoding Rna ◽

Molecular Mechanisms ◽

Epithelial Mesenchymal Transition ◽

Luciferase Assay ◽

Migration And Invasion ◽

Loss Of Function ◽

Mesenchymal Transition

Abstract Background Growing evidence suggests that suppressor of tumorigenicity 7 antisense RNA 1 (ST7-AS1) is an oncogenic long noncoding RNA (lncRNA). However, little is known on its clinical significance, biological functions, or molecular mechanisms in lung adenocarcinoma (LUAD). Methods The expression of ST7-AS1 and miR-181b-5p were examined by qRT-PCR. The correlations between ST7-AS1 level and different clinicopathological features were analysed. In vitro, LUAD cells were examined for cell viability, migration and invasion by MTT, wound healing and Transwell assay, respectively. Epithelial-mesenchymal transition (EMT) biomarkers were detected by Western blot. The regulations between ST7-AS1, miR-181b-5p, and KPNA4 were examined by luciferase assay, RNA immunoprecipitation, RNA pulldown. Both gain- and loss-of-function strategies were used to assess the importance of different signalling molecules in malignant phenotypes of LUAD cells. The in vivo effect was analysed using the xenograft and the experimental metastasis mouse models. Results ST7-AS1 was upregulated in LUAD tissues or cell lines, correlated with tumours of positive lymph node metastasis or higher TNM stages, and associated with shorter overall survival of LUAD patients. ST7-AS1 essentially maintained the viability, migration, invasion, and EMT of LUAD cells. The oncogenic activities of ST7-AS1 were accomplished by sponging miR-181b-5p and releasing the suppression of the latter on KPNA4. In LUAD tissues, ST7-AS1 level positively correlated with that of KPNA4 and negatively with miR-181b-5p level. In vivo, targeting ST7-AS1 significantly inhibited xenograft growth and metastasis. Conclusions ST7-AS1, by regulating miR-181b-5p/KPNA4 axis, promotes the malignancy of LUAD cells. Targeting ST7-AS1 and KPNA4 or up-regulating miR-181b-5p, therefore, may benefit the treatment of LUAD.

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Oxidatively Modified LDL Suppresses Lymphangiogenesis via CD36 Signaling

Antioxidants ◽

10.3390/antiox10020331 ◽

2021 ◽

Vol 10 (2) ◽

pp. 331

Author(s):

Bhupesh Singla ◽

Hui-Ping Lin ◽

WonMo Ahn ◽

Joseph White ◽

Gábor Csányi

Keyword(s):

Cell Cycle ◽

Molecular Mechanisms ◽

Oxidized Ldl ◽

Atherosclerotic Lesion ◽

Tube Formation ◽

Adventitial Layer ◽

Oxidatively Modified ◽

And Function

Arterial accumulation of plasma-derived LDL and its subsequent oxidation contributes to atherosclerosis. Lymphatic vessel (LV)-mediated removal of arterial cholesterol has been shown to reduce atherosclerotic lesion formation. However, the precise mechanisms that regulate LV density and function in atherosclerotic vessels remain to be identified. The aim of this study was to investigate the role of native LDL (nLDL) and oxidized LDL (oxLDL) in modulating lymphangiogenesis and underlying molecular mechanisms. Western blotting and immunostaining experiments demonstrated increased oxLDL expression in human atherosclerotic arteries. Furthermore, elevated oxLDL levels were detected in the adventitial layer, where LV are primarily present. Treatment of human lymphatic endothelial cells (LEC) with oxLDL inhibited in vitro tube formation, while nLDL stimulated it. Similar results were observed with Matrigel plug assay in vivo. CD36 deletion in mice and its siRNA-mediated knockdown in LEC prevented oxLDL-induced inhibition of lymphangiogenesis. In addition, oxLDL via CD36 receptor suppressed cell cycle, downregulated AKT and eNOS expression, and increased levels of p27 in LEC. Collectively, these results indicate that oxLDL inhibits lymphangiogenesis via CD36-mediated regulation of AKT/eNOS pathway and cell cycle. These findings suggest that therapeutic blockade of LEC CD36 may promote arterial lymphangiogenesis, leading to increased cholesterol removal from the arterial wall and reduced atherosclerosis.

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miR-142-5p regulates the progression of diabetic retinopathy by targeting IGF1

International Journal of Immunopathology and Pharmacology ◽

10.1177/2058738420909041 ◽

2020 ◽

Vol 34 ◽

pp. 205873842090904 ◽

Cited By ~ 2

Author(s):

Xiuming Liu ◽

Jianchang Li ◽

Xiaofeng Li

Keyword(s):

Cell Proliferation ◽

Diabetic Retinopathy ◽

Growth Factor ◽

Signaling Pathway ◽

Molecular Mechanisms ◽

Cell Counting ◽

Luciferase Reporter ◽

Igf1r Signaling

As one of leading causes of blindness, diabetic retinopathy (DR) is a progressive microvascular complication of diabetes mellitus (DM). Despite significant efforts have been devoted to investigate DR over the years, the molecular mechanisms still remained unclear. Emerging evidences demonstrated that microRNAs (miRNAs) were tightly associated with pathophysiological development of DR. Hence, this study was aimed to illustrate the role and molecular mechanisms of miR-412-5p in progression of DR. Streptozotocin (STZ) treatment in rats and human retinal endothelial cell (HREC) models were used to simulate DR conditions in vivo and in vitro. Hematoxylin-eosin (HE) staining was used to demonstrate the morphology of retinal tissues of rats. Qualitative real-time polymerase chain reaction (qRT-PCR) detected miR-142-5p and vascular endothelial growth factor (VEGF) expression levels. Cell counting kit-8 (CCK8) assay and immunofluorescence (IF) measured the cell proliferation rates. Western blot tested the expression status of IGF1/IGF1R-mediated signaling pathway. Dual-luciferase reporter assays demonstrated the molecular mechanism of miR-142-5p. miR-142-5p level was down-regulated in retinal tissues of DR rats and high glucose (HG)-treated HRECs. Insulin-like growth factor 1 (IGF1) was identified as a direct target of miR-142-5p. The reduced miR-142-5p level enhanced HRECs proliferation via activating IGF/IGF1R-mediated signaling pathway including p-PI3K, p-ERK, p-AKT, and VEGF activation, ultimately giving rise to cell proliferation. Either miR-142-5p overexpression or IGF1 knockdown alleviated the pathological effects on retinal tissues in DR rats. Collectively, miR-142-5p participated in DR development by targeting IGF1/p-IGF1R signaling pathway and VEGF generation. This miR-142-5p/IGF1/VEGF axis provided a novel therapeutic target for DR clinical treatment.

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Anti-angiogenesis and anti-metastasis effects of Polyphyllin VII on Hepatocellular carcinoma cells in vitro and in vivo

Chinese Medicine ◽

10.1186/s13020-021-00447-w ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Chao Zhang ◽

Qingrui Li ◽

Guozheng Qin ◽

Yi Zhang ◽

Chaoying Li ◽

...

Keyword(s):

Molecular Mechanisms ◽

Umbilical Vein ◽

Tube Formation ◽

Tumor Xenograft ◽

Migration And Invasion ◽

Transgenic Zebrafish ◽

Steroidal Saponin ◽

Potential Candidate

Abstract Background Polyphyllin VII (PP7), a steroidal saponin from P. polyphylla has been found to exert strong anticancer activity. Little is known about the anti-angiogenesis and anti-metastasis properties of PP7. In this study, the anti-angiogenic and anti-metastatic effects of PP7 on HCC and the molecular mechanisms were evaluated. Methods Effect of PP7 on angiogenesis was assessed by tube formation assay and applied a transgenic Tg(fli1:EGFP) zebrafish model. Effects of PP7 on tumor metastasis and invasion were examined in cell migration and invasion assay, zebrafish tumor xenograft models and lung metastasis mouse models. The protein levels were examined by Western blotting. Results PP7 significantly decreased the tube formation of human umbilical vein endothelial cells, the number and length of ISVs and SIVs of transgenic zebrafish, and the metastasis and invasion of cancer cells in vitro and in vivo. The anti-angiogenic and anti-metastatic effects of PP7 in HepG2 cells were attributable, at least partially, to downregulated NF-κB/MMP-9/VEGF signaling pathway. Conclusion This study demonstrates that PP7 possesses strong anti-angiogenesis and anti-metastasis activities, suggesting that PP7 could be a potential candidate agent for HCC treatment.

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N6-Methyladenosine Methyltransferase METTL3 Promotes Angiogenesis and Atherosclerosis by Upregulating the JAK2/STAT3 Pathway via m6A Reader IGF2BP1

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.731810 ◽

2021 ◽

Vol 9 ◽

Author(s):

Guo Dong ◽

Jiangbo Yu ◽

Gaojun Shan ◽

Lide Su ◽

Nannan Yu ◽

...

Keyword(s):

Molecular Mechanisms ◽

Chorioallantoic Membrane ◽

Tube Formation ◽

Luciferase Reporter ◽

Dependent Manner ◽

Stat3 Pathway ◽

Protein Levels ◽

Chick Chorioallantoic Membrane

Atherosclerosis (AS) is a life-threatening vascular disease. RNA N6-methyladenosine (m6A) modification level is dysregulated in multiple pathophysiologic processes including AS. In this text, the roles and molecular mechanisms of m6A writer METTL3 in AS progression were explored in vitro and in vivo. In the present study, cell proliferative, migratory, and tube formation capacities were assessed through CCK-8, Transwell migration, and tube formation assays, respectively. RNA m6A level was examined through a commercial kit. RNA and protein levels of genes were measured through RT-qPCR and western blot assays, respectively. VEGF secretion level was tested through ELISA assay. JAK2 mRNA stability was detected through actinomycin D assay. The relationship of METTL3, IGF2BP1, and JAK2 was investigated through bioinformatics analysis, MeRIP, RIP, RNA pull-down, and luciferase reporter assays. An AS mouse model was established to examine the effect of METTL3 knockdown on AS development in vivo. The angiogenetic activity was examined through chick chorioallantoic membrane assay in vivo. The results showed that METTL3 was highly expressed in ox-LDL-induced dysregulated HUVECs. METTL3 knockdown inhibited cell proliferation, migration, tube formation, and VEGF expression/secretion in ox-LDL-treated HUVECs, hampered AS process in vivo, and prevented in vivo angiogenesis of developing embryos. METTL3 positively regulated JAK2 expression and JAK2/STAT3 pathway in an m6A dependent manner in HUVECs. IGF2BP1 positively regulated JAK2 expression through directly binding to an m6A site within JAK2 mRNA in HUVECs. METTL3 knockdown weakened the interaction of JAK2 and IGF2BP1. METTL3 exerted its functions through JAK2/STAT3 pathway. In conclusion, METTL3 knockdown prevented AS progression by inhibiting JAK2/STAT3 pathway via IGF2BP1.

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