Potassium dehydroandrographolide succinate regulates the MyD88/CDH13 signaling pathway to enhance vascular injury-induced pathological vascular remodeling / 中国天然药物

Pathological vascular remodeling is a hallmark of various vascular diseases. Previous research has established the significance of andrographolide in maintaining gastric vascular homeostasis and its pivotal role in modulating endothelial barrier dysfunction, which leads to pathological vascular remodeling. Potassium dehydroandrographolide succinate (PDA), a derivative of andrographolide, has been clinically utilized in the treatment of inflammatory diseases precipitated by viral infections. This study investigates the potential of PDA in regulating pathological vascular remodeling. The effect of PDA on vascular remodeling was assessed through the complete ligation of the carotid artery in C57BL/6 mice. Experimental approaches, including rat aortic primary smooth muscle cell culture, flow cytometry, bromodeoxyuridine (BrdU) incorporation assay, Boyden chamber cell migration assay, spheroid sprouting assay, and Matrigel-based tube formation assay, were employed to evaluate the influence of PDA on the proliferation and motility of smooth muscle cells (SMCs). Molecular docking simulations and co-immunoprecipitation assays were conducted to examine protein interactions. The results revealed that PDA exacerbates vascular injury-induced pathological remodeling, as evidenced by enhanced neointima formation. PDA treatment significantly increased the proliferation and migration of SMCs. Further mechanistic studies disclosed that PDA upregulated myeloid differentiation factor 88 (MyD88) expression in SMCs and interacted with T-cadherin (CDH13). This interaction augmented proliferation, migration, and extracellular matrix deposition, culminating in pathological vascular remodeling. Our findings underscore the critical role of PDA in the regulation of pathological vascular remodeling, mediated through the MyD88/CDH13 signaling pathway.

Promotion effect of FOXCUT as a microRNA sponge for miR-24-3p on progression in triple-negative breast cancer through the p38 MAPK signaling pathway / 中华医学杂志(英文版)

BACKGROUND@#Triple-negative breast cancer (TNBC) is a type of highly invasive breast cancer with a poor prognosis. According to new research, long noncoding RNAs (lncRNAs) play a significant role in the progression of cancer. Although the role of lncRNAs in breast cancer has been well reported, few studies have focused on TNBC. This study aimed to explore the biological function and clinical significance of forkhead box C1 promoter upstream transcript (FOXCUT) in triple-negative breast cancer.@*METHODS@#Based on a bioinformatic analysis of the cancer genome atlas (TCGA) database, we detected that the lncRNA FOXCUT was overexpressed in TNBC tissues, which was further validated in an external cohort of tissues from the General Surgery Department of the First Affiliated Hospital of Nanjing Medical University. The functions of FOXCUT in proliferation, migration, and invasion were detected in vitro or in vivo. Luciferase assays and RNA immunoprecipitation (RIP) were performed to reveal that FOXCUT acted as a competitive endogenous RNA (ceRNA) for the microRNA miR-24-3p and consequently inhibited the degradation of p38.@*RESULTS@#lncRNA FOXCUT was markedly highly expressed in breast cancer, which was associated with poor prognosis in some cases. Knockdown of FOXCUT significantly inhibited cancer growth and metastasis in vitro or in vivo. Mechanistically, FOXCUT competitively bounded to miR-24-3p to prevent the degradation of p38, which might act as an oncogene in breast cancer.@*CONCLUSION@#Collectively, this research revealed a novel FOXCUT/miR-24-3p/p38 axis that affected breast cancer progression and suggested that the lncRNA FOXCUT could be a diagnostic marker and therapeutic target for breast cancer.

Hydroxysafflor Yellow A Promotes HaCaT Cell Proliferation and Migration by Regulating HBEGF/EGFR and PI3K/AKT Pathways and Circ_0084443 / 中国结合医学杂志

OBJECTIVE@#To investigate the effect and possible mechanism of hydroxysafflor yellow A (HSYA) on human immortalized keratinocyte cell proliferation and migration.@*METHODS@#HaCaT cells were treated with HSYA. Cell proliferation was detected by the cell counting kit-8 assay, and cell migration was measured using wound healing assay and Transwell migration assay. The mRNA and protein expression levels of heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF), EGF receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor-1α (HIF-1α) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. Circ_0084443-overexpressing HaCaT cells and empty plasmid HaCaT cells were constructed using the lentiviral stable transfection and treated with HSYA. The expression of circ_0084443 was detected by qRT-PCR.@*RESULTS@#HSYA (800 µmol/L) significantly promoted HaCaT cell proliferation and migration (P<0.05 or P<0.01). It also increased the mRNA and protein expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and increased the phosphorylation levels of PI3K and AKT (P<0.05 or P<0.01). Furthermore, HSYA promoted HaCaT cell proliferation and migration via the HBEGF/EGFR and PI3K/AKT/mTOR signaling pathways (P<0.01). Circ_0084443 attenuated the mRNA expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α (P<0.05). HSYA inhibited the circ_0084443 expression, further antagonized the inhibition of circ_0084443 on HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and promoted the proliferation of circ_0084443-overexpressing HaCaT cells (P<0.05 or P<0.01). However, HSYA could not influence the inhibitory effect of circ_0084443 on HaCaT cell migration (P>0.05).@*CONCLUSION@#HSYA played an accelerative role in HaCaT cell proliferation and migration, which may be attributable to activating HBEGF/EGFR and PI3K/AKT signaling pathways, and had a particular inhibitory effect on the keratinocyte negative regulator circ_0084443.

Inhibition of Proliferation, Migration, and Invasion of Keloid Fibroblasts By miR-183-5p Through Downregulating EGR1 / Inhibición de la Proliferación, Migración e Invasión de Fibroblastos Queloides por miR-183-5p Mediante la Regulación Negativa de EGR1

SUMMARY: Keloid scar is a unique benign fibroproliferative tumor of the human skin. Previously, it was reported that early growth response 1 (EGR1), a transcription factor, promotes keloid fibrosis; however, the mechanism by which EGR1 modulates keloid formation was not elaborated. In this research, the specific function and the microRNA (miRNA) regulatory network of EGR1 in keloids was examined. Keloid fibroblasts (KFs) were transfected with EGR1-small interfering RNA (siEGR1), EGR1-overexpression plasmid (pcDNA3.1-EGR1), and microRNA (miR-183-5p)-mimics to regulate the expression of EGR1 and miR-183-5p. The study employed dual-luciferase reporter assays to explore the targeting regulation of miR-183-5p on EGR1. Additionally, Western blotting, flow cytometry, qRT-PCR, cell count kit-8 (CCK-8), transwell, and wound healing assays, and RNA sequencing were conducted. EGR1 was upregulated in KFs, and EGR1 silencing diminished proliferation, fibrosis, migration, invasion, and apoptosis of cells. In KFs, the expression of miR- 183-5p was reduced, leading to the inhibition of cell proliferation, migration, and invasion. Conversely, it enhanced apoptosis. By targeting EGR1, miR-183-5p partially counteracted the impact of EGR1 on migration, invasion, and fibrosis in KFs. The findings imply that miR-183-5p suppresses keloid formation by targeting EGR1. As a result, EGR1 holds promise as a potential therapeutic target for preventing and treating keloids.

La cicatriz queloide es un tumor fibroproliferativo benigno único de la piel humana. Anteriormente, se informó que la respuesta de crecimiento temprano 1 (EGR1), un factor de transcripción, promueve la fibrosis queloide; sin embargo, no se explicó el mecanismo por el cual EGR1 modula la formación de queloides. En esta investigación, se examinó la función específica y la red reguladora de microARN (miARN) de EGR1 en queloides. Se transfectaron fibroblastos queloides (KF) con ARN de interferencia pequeño de EGR1 (siEGR1), plásmido de sobreexpresión de EGR1 (pcDNA3.1-EGR1) y miméticos de microARN (miR-183-5p) para regular la expresión de EGR1 y miR-183. -5p. El estudio empleó ensayos de indicador de luciferasa dual para explorar la regulación dirigida de miR-183-5p en EGR1. Además, se realizaron pruebas de transferencia Western, citometría de flujo, qRT-PCR, kit de recuento celular-8 (CCK-8), transwell y curación de heridas, y secuenciación de ARN. EGR1 estaba regulado positivamente en KF, y el silenciamiento de EGR1 disminuyó la proliferación, fibrosis, migración, invasión y apoptosis de las células. En KF, la expresión de miR- 183-5p se redujo, lo que llevó a la inhibición de la proliferación, migración e invasión celular. Por el contrario, mejoró la apoptosis. Al apuntar a EGR1, miR-183-5p contrarrestó parcialmente el impacto de EGR1 en la migración, invasión y fibrosis en KF. Los hallazgos implican que miR-183-5p suprime la formación de queloides al apuntar a EGR1. Como resultado, EGR1 es prometedor como objetivo terapéutico potencial para prevenir y tratar los queloides.

MiR-15b-5p Promotes Growth and Metastasis of Renal Clear Cell Carcinoma / MiR-15b-5p Promueve el Crecimiento y la Metástasis del Carcinoma Renal de Células Claras

SUMMARY: We investigated the expression and clinical significance of miR-15b-5p in clear cell renal cell carcinoma (RCC) through bioinformatics analysis and experimental verification. The differentially expressed miRNAs were screened in the GEO database. Venn diagram showed that there were 5 up-regulated miRNAs (has-miR-210, has-miR-142-3p, has-miR-142-5p, has-miR-15b-5p, and has-miR-193a-3p) and only 1 down-regulated miRNA (has-miR-532-3p) that were commonly expressed between GSE189331 and GSE16441 datasets. This was further confirmed in TCGA. Further analysis showed that the has-miR-193a-3p, has-miR-142-3p, has- miR-142-5p, and has-miR-15b-5p were closely related to tumor invasion, distant metastasis and survival probability. The expression of miR-15b-5p in ccRCC tissues was significantly higher than that in adjacent normal kidney tissues (P0.05). Following inhibition of miR-15b-5p expression, RCC cells had attenuated proliferation, increased apoptosis, and attenuated migration and invasion. has-miR-15b-5p-WEE1, has-miR-15b-5p-EIF4E, has-miR-15b-5p-PPP2R1B may be three potential regulatory pathways in ccRCC. miR-15b-5p is highly expressed in cancer tissues of ccRCC patients. It may promote proliferation, inhibit apoptosis and enhance cell migration and invasion of RCC cells. The has-miR-15b-5p-WEE1, has-miR-15b-5p-EIF4E, and has-miR-15b-5p-PPP2R1B may be three potential regulatory pathways in ccRCC.

Investigamos la expresión y la importancia clínica de miR-15b-5p en el carcinoma de células renales (CCR) de células claras mediante análisis bioinformático y verificación experimental. Los miARN expresados diferencialmente se examinaron en la base de datos GEO. El diagrama de Venn mostró que había 5 miARN regulados positivamente (has-miR-210, has-miR-142-3p, has-miR-142-5p, has-miR-15b-5p y has-miR-193a-3p). ) y solo 1 miARN regulado negativamente (has-miR-532-3p) que se expresaron comúnmente entre los conjuntos de datos GSE189331 y GSE16441. Esto fue confirmado aún más en TCGA. Un análisis más detallado mostró que has-miR-193a-3p, has-miR-142-3p, has- miR-142-5p y has-miR-15b-5p estaban estrechamente relacionados con la invasión tumoral, la metástasis a distancia y la probabilidad de supervivencia. La expresión de miR-15b-5p en tejidos ccRCC fue significativamente mayor que la de los tejidos renales normales adyacentes (P 0,05). Tras la inhibición de la expresión de miR-15b-5p, las células RCC tuvieron una proliferación atenuada, un aumento de la apoptosis y una migración e invasión atenuadas. has-miR-15b-5p-WEE1, has- miR-15b-5p-EIF4E, has-miR-15b-5p-PPP2R1B pueden ser tres posibles vías reguladoras en ccRCC. miR-15b-5p se expresa altamente en tejidos cancerosos de pacientes con ccRCC. Puede promover la proliferación, inhibir la apoptosis y mejorar la migración celular y la invasión de células RCC. has-miR-15b-5p-WEE1, has- miR-15b-5p-EIF4E y has-miR-15b-5p-PPP2R1B pueden ser tres posibles vías reguladoras en ccRCC.

YULINK regulates vascular formation in zebrafish and HUVECs

BACKGROUND: The distinct arterial and venous cell fates are dictated by a combination of various genetic factors which form diverse types of blood vessels such as arteries, veins, and capillaries. We report here that YULINK protein is involved in vasculogenesis, especially venous formation. METHODS: In this manuscript, we employed gene knockdown, yeast two-hybrid, FLIM-FRET, immunoprecipitation, and various imaging technologies to investigate the role of YULINK gene in zebrafish and human umbilical vein endothelial cells (HUVECs). RESULTS: Knockdown of YULINK during the arterial-venous developmental stage of zebrafish embryos led to the defective venous formation and abnormal vascular plexus formation. Knockdown of YULINK in HUVECs impaired their ability to undergo cell migration and differentiation into a capillary-like tube formation. In addition, the phosphorylated EPHB4 was decreased in YULINK knockdown HUVECs. Yeast two-hybrid, FLIM-FRET, immunoprecipitation, as well as imaging technologies showed that YULINK colocalized with endosome related proteins (EPS15, RAB33B or TICAM2) and markers (Clathrin and RHOB). VEGF-induced VEGFR2 internalization was also compromised in YULINK knockdown HUVECs, demonstrating to the involvement of YULINK. CONCLUSION: This study suggests that YULINK regulates vasculogenesis, possibly through endocytosis in zebrafish and HUVECs. Key points Knockdown of YULINK with morpholino in embryos of double transgenic zebrafish exhibited abnormal venous formation. Tube formation and phosphorylated EPHB4 were decreased in YULINK knockdown HUVECs. FLIM-FRET, immunoprecipitation, as well as other imaging technologies showed that YULINK colocalized with endosome related proteins (EPS15, RAB33B and TICAM2) and endosome markers (Clathrin and RHOB). Knockdown of YULINK decreased the internalization of VEGF and VEGFR2 in HUVECs.

Enrichment of miR-17-5p enhances the protective effects of EPC-EXs on vascular and skeletal muscle injury in a diabetic hind limb ischemia model

BACKGROUND/AIMS: Diabetes mellitus (DM) is highly susceptible to diabetic hind limb ischemia (DHI). MicroRNA (MiR)-17-5p is downregulated in DM and plays a key role in vascular protection. Endothelial progenitor cell (EPC)-released exosomes (EPC-EXs) contribute to vascular protection and ischemic tissue repair by transferring their contained miRs to target cells. Here, we investigated whether miR-17-5p-enriched EPC-EXs (EPC-EXsmiR-17-5p) had conspicuous effects on protecting vascular and skeletal muscle in DHI in vitro and in vivo. METHODS: EPCs transfected with scrambled control or miR-17-5p mimics were used to generate EPC-EXs and EPC-EXsmiR-17-5p. Db/db mice were subjected to hind limb ischemia. After the surgery, EPC-EXs and EPC-EXsmiR-17-5p were injected into the gastrocnemius muscle of the hind limb once every 7 days for 3 weeks. Blood flow, microvessel density, capillary angiogenesis, gastrocnemius muscle weight, structure integrity, and apoptosis in the hind limb were assessed. Vascular endothelial cells (ECs) and myoblast cells (C2C12 cells) were subjected to hypoxia plus high glucose (HG) and cocultured with EPC-EXs and EPC-EXsmiR-17-5p. A bioinformatics assay was used to analyze the potential target gene of miR-17-5p, the levels of SPRED1, PI3K, phosphorylated Akt, cleaved caspase-9 and cleaved caspase-3 were measured, and a PI3K inhibitor (LY294002) was used for pathway analysis. RESULTS: In the DHI mouse model, miR-17-5p was markedly decreased in hind limb vessels and muscle tissues, and infusion of EPC-EXsmiR-17-5p was more effective than EPC-EXs in increasing miR-17-5p levels, blood flow, microvessel density, and capillary angiogenesis, as well as in promoting muscle weight, force production and structural integrity while reducing apoptosis in gastrocnemius muscle. In Hypoxia plus HG-injured ECs and C2C12 cells, we found that EPC-EXsmiR-17-5p could deliver their carried miR-17-5p into target ECs and C2C12 cells and subsequently downregulate the target protein SPRED1 while increasing the levels of PI3K and phosphorylated Akt. EPC-EXsmiR-17-5p were more effective than EPC-EXs in decreasing apoptosis and necrosis while increasing viability, migration, and tube formation in Hypoxia plus HG-injured ECs and in decreasing apoptosis while increasing viability and myotube formation in C2C12 cells. These effects of EPC-EXsmiR-17-5p could be abolished by a PI3K inhibitor (LY294002). CONCLUSION: Our results suggest that miR-17-5p promotes the beneficial effects of EPC-EXs on DHI by protecting vascular ECs and muscle cell functions.

Synergistic effects of dendrosomal nanocurcumin and oxaliplatin on oncogenic properties of ovarian cancer cell lines by down-expression of MMPs

BACKGROUND: Contrary to the advantageous anticancer activities of curcumin (Cur), limited bioavailability and solubility hindered its efficacy. Here, nontoxic dendrosomal nano carrier with Cur was used to overcome these problems. Despite considerable antitumor properties of Oxaliplatin (Oxa), the limiting factors are drug resistance and adverse side-effects. The hypothesis of this study was to evaluate the possible synergism between dendrosomal nanocurcumin (DNC) and Oxa and these agents showed growth regulatory effects on SKOV3 and OVCAR3 cells. METHODS: and materials In the present study, colony formation, wound healing motility, cell adhesion, transwell invasion and migration assay and cell cycle arrest with or without DNC, Oxa and Combination were defined. In addition to, real time PCR and Western blot were used to analyze AKT, PI3K, PKC, JNK, P38 and MMPs mRNAs and proteins expressions. Docking of MMP-2-Cur, MMP-2-DNC and MMP-2-Oxa was performed and the results of all three complexes were simulated by molecular dynamics. RESULTS: Our findings illustrated that DNC had the greatest effect on cell death as compared to the Cur alone. Moreover, the growth inhibitory effects (such as cell death correlated to apoptosis) were more intense if Oxa was added followed by DNC at 4 h interval. However, insignificant effects were observed upon simultaneous addition of these two agents in both cell lines. Besides, a combination of agents synergistically alters the relative expression of MMP-9. CONCLUSIONS: The docking results showed that His70 and Asp100 may play a key role at the MMP-2 binding site. The matrigel invasion as well as cell viability of ovarian cancer cell lines SKOV3 and OVCAR3 by DNC alone or in combination with Oxa was inhibited significantly. The inhibitory effects of these agents were due to the differential expression levels of MMP 2 and MMP 9 regulated by multiple downstream signaling cascades. From the molecular dynamic simulation studies, it was confirmed that DNC established a strong interaction with MMP-2.

MiR-30e-5p overexpression promotes proliferation and migration of colorectal cancer cells by activating the CXCL12 axis via downregulating PTEN / 南方医科大学学报

OBJECTIVE@#To investigate the regulatory effects of miR-30e-5p on biological behaviors of colorectal cancer cells and the role of PTEN/CXCL12 axis in mediating these effects.@*METHODS@#Bioinformatic analysis was performed to explore the differential expression of miR-30e-5p between colorectal cancer tissues and normal tissues. RT-qPCR was used to detect the differential expression of miR-30e-5p in intestinal epithelial cells and colorectal cancer cells. Bioinformatics and dual luciferase assay were used to predict and validate the targeting relationship between miR-30e-5p and PTEN. Human and murine colorectal cancer cell lines were transfected with miR-30e-5p mimics, miR-30e-5p inhibitor, miR-30e-5p mimics+LV-PTEN, or miR-30e-5p inhibitor + si-PTEN. The changes in biological behaviors of the cells were detected using plate clone formation assay, CCK-8 assay, flow cytometry, scratch healing and Transwell assays. PTEN and CXCL12 expressions in the cancer cells were detected by Western blotting. The effects of miR-30e-5p inhibitor on colorectal carcinogenesis and development were observed in nude mice.@*RESULTS@#Bioinformatic analysis showed that miR-30e-5p expression was significantly elevated in colorectal cancer tissues compared with the adjacent tissue (P < 0.01). Higher miR-30e-5p expression was detected in colorectal cancer cell lines than in intestinal epithelial cells (P < 0.01). Dual luciferase assay confirmed the targeting relationship between miR-30e-5p and PTEN (P < 0.05). Transfection with miR-30e-5p mimics significantly enhanced proliferation and metastasis and inhibited apoptosis of the colorectal cancer cells (P < 0.05), and co-transfection with LV-PTEN obviously reversed these changes (P < 0.05). MiR-30e-5p mimics significantly inhibited PTEN expression and enhanced CXCL12 expression in the cancer cells (P < 0.01), and miR-30e-5p inhibitor produced the opposite effect. Transfection with miR-30e-5p inhibitor caused cell cycle arrest in the cancer cells, which was reversed by co-transfection with si-PTEN (P < 0.05). In the in vivo experiments, the colorectal cancer cells transfected with miR-30e-5p inhibitor showed significantly lowered tumorigenesis.@*CONCLUSION@#Overexpression of miR-30e-5p promotes the malignant behaviors of colorectal cancer cells by downregulating PTEN to activate the CXCL12 axis.

Colorectal cancer cells induce the formation of cancer-associated fibroblasts by activating the ERK signaling pathway in fibroblasts / 南方医科大学学报

OBJECTIVE@#To investigate the mechanism by which conditioned medium of colorectal cancer cells promotes the formation of cancer-associated fibroblasts (CAFs).@*METHODS@#Normal human colorectal fibroblasts (CCD-18Co cells) in logarithmic growth phase were treated with the conditioned media of colorectal cancer HCT116 cells (HCT116-CM) or Caco-2 cells (Caco-2-CM) alone or in combination with 300 nmol/L ERK inhibitor SCH772984. The expression levels of CAFs-related molecular markers were detected in the treated cells with real-time quantitative PCR (RT- qPCR) and immunofluorescence assay, and the changes in cell proliferation, colony formation and migration were assessed with RTCA, colony formation and wound healing assays; Western blotting was performed to detect the activated signaling pathways in the fibroblasts and the changes in CAFs formation after blocking of the signaling pathway.@*RESULTS@#HCT116-CM and Caco-2-CM significantly upregulated mRNA expression levels of CAFs markers (including α-SMA, FAP, FN and TGF-β) in CCD-18Co cells, and strongly promoted fibroblast transformation into CAFs (P < 0.05). The two conditioned media also promoted the proliferation, colony formation and migration of CCD-18Co cells (P < 0.05) and significantly increased the levels of α-SMA protein and ERK phosphorylation in the cells (P < 0.05). The ERK inhibitor SCH772984 obviously inhibited the expression of α-SMA and the transformation of CCD-18Co cells into CAFs induced by the conditioned medium of colorectal cancer cells (P < 0.05).@*CONCLUSION@#Colorectal cancer cells may induce the formation of colorectal CAFs by activating the ERK pathway in the fibroblasts.

Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/β-catenin pathway under hypoxia / 中西医结合学报

OBJECTIVE@#Jiedu Recipe (JR), a Chinese herbal remedy, has been shown to prolong overall survival time and decrease recurrence and metastasis rates in patients with hepatocellular carcinoma (HCC). This work investigated the mechanism of JR in HCC treatment.@*METHODS@#The chemical constituents of JR were detected using liquid chromatography-mass spectrometry. The potential anti-HCC mechanism of JR was screened using network pharmacology and messenger ribonucleic acid (mRNA) microarray chip assay, followed by experimental validation in human HCC cells (SMMC-7721 and Huh7) in vitro and a nude mouse subcutaneous transplantation model of HCC in vivo. HCC cell characteristics of proliferation, migration and invasion under hypoxic setting were investigated using thiazolyl blue tetrazolium bromide, wound healing and Transwell assays, respectively. Image-iT™ Hypoxia Reagent was added to reveal hypoxic conditions. Stem cell sphere formation assay was used to detect the stemness. Epithelial-mesenchymal transition (EMT) markers like E-cadherin, vimentin and α-smooth muscle actin, and pluripotent transcription factors including nanog homeobox, octamer-binding transcription factor 4, and sex-determining region Y box protein 2 were analyzed using Western blotting and real-time polymerase chain reaction. Western blot was performed to ascertain the anti-HCC effect of JR under hypoxia involving the Wnt/β-catenin pathway.@*RESULTS@#According to network pharmacology and mRNA microarray chip analysis, JR may potentially act on hypoxia and inhibit the Wnt/β-catenin pathway. In vitro and in vivo experiments showed that JR significantly decreased hypoxia, and suppressed HCC cell features of proliferation, migration and invasion; furthermore, the hypoxia-induced increases in EMT and stemness marker expression in HCC cells were inhibited by JR. Results based on the co-administration of JR and an agonist (LiCl) or inhibitor (IWR-1-endo) verified that JR suppressed HCC cancer stem-like properties under hypoxia by blocking the Wnt/β-catenin pathway.@*CONCLUSION@#JR exerts potent anti-HCC effects by inhibiting cancer stemness via abating the Wnt/β-catenin pathway under hypoxic conditions. Please cite this article as: Guo BJ, Ruan Y, Wang YJ, Xiao CL, Zhong ZP, Cheng BB, Du J, Li B, Gu W, Yin ZF. Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/β-catenin pathway under hypoxia. J Integr Med. 2023; 21(5): 474-486.

Long noncoding RNA LOC646029 functions as a ceRNA to suppress ovarian cancer progression through the miR-627-3p/SPRED1 axis / 医学前沿

Long noncoding RNAs (lncRNAs) play a crucial regulatory role in the development and progression of multiple cancers. However, the potential mechanism by which lncRNAs affect the recurrence and metastasis of ovarian cancer remains unclear. In the current study, the lncRNA LOC646029 was markedly downregulated in metastatic ovarian tumors compared with primary tumors. Gain- and loss-of-function assays demonstrated that LOC646029 inhibits the proliferation, invasiveness, and metastasis of ovarian cancer cells in vivo and in vitro. Moreover, the downregulation of LOC646029 in metastatic ovarian tumors was strongly correlated with poor prognosis. Mechanistically, LOC646029 served as a miR-627-3p sponge to promote the expression of Sprouty-related EVH1 domain-containing protein 1, which is necessary for suppressing tumor metastasis and inhibiting KRAS signaling. Collectively, our results demonstrated that LOC646029 is involved in the progression and metastasis of ovarian cancer, which may be a potential prognostic biomarker.

miRNA-128-3p inhibits malignant behavior of glioma cells by downregulating KLHDC8A expression / 南方医科大学学报

OBJECTIVE@#To determine whether miRNA-128-3p regulates malignant biological behavior of glioma cells by targeting KLHDC8A.@*METHODS@#Dual-luciferase reporter assays, qRT-PCR and Western blotting were used to verify the targeting of miRNA-128-3p to KLHDC8A. Edu assay, flow cytometry, Transwell assay and would healing assay were used to determine the effects of changes in miRNA-128-3p and KLHDC8A expression levels on malignant behavior of glioma cells. Rescue experiment was carried out to verify that miRNA-128-3p regulated glioma cell proliferation, apoptosis, invasion and migration by targeting KLHDC8A.@*RESULTS@#The expression level of KLHDC8A was significantly increased in high-grade glioma tissue and was closely related to a poor survival outcome of the patients. Overexpression of KLHDC8A promoted glioma cell proliferation, migration and invasion, and miRNA-128-3p overexpression inhibited proliferative and metastatic capacities of glioma cells. Mechanistically, KLHDC8A expression was directly modulated by miRNA-128-3p, which, by targeting KLHDC8A, inhibited malignant behavior of glioma cells.@*CONCLUSION@#Upregulation of miRNA-128-3p inhibits uncontrolled growth of glioma cells by negatively regulating KLHDC8A expression and its downstream effectors, suggesting that the miRNA-128-3p-KLHDC8A axis may serve as a potential prognostic indicator and a therapeutic target for developing new strategies for glioma treatment.

The effect of lncRNA ADPGK-AS1 on the proliferation and apoptosis of retinoblastoma cells by targeting miR-200b-5p / 中华肿瘤杂志

Objective: To explore the effect of lncRNA ADPGK-AS1 on the proliferation and apoptosis of retinoblastoma cells and its possible mechanism. Methods: The tumor tissues of 31 patients with retinoblastoma admitted to Henan Provincial Eye Hospital from February to June 2020 and their corresponding normal tissues adjacent to the cancer were collected. The expression levels of lncRNA ADPGK-AS1 and miR-200b-5p in retinoblastoma tissues and normal adjacent tissues were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Human retinal epithelial cell ARPE-19, human retinoblastoma cell Y-79 and WERI-Rb-1 were cultured in vitro. The expression levels of lncRNA ADPGK-AS1 and miR-200b-5p were detected by qRT-PCR. Y-79 cells were randomly divided into si-con group, si-lncRNA ADPGK-AS1 group, miR con group, miR-200b-5p group, si-lncRNA ADPGK-AS1+ anti-miR con group, and si-lncRNA ADPGK-AS1+ anti-miR-200b-5p group. The proliferation, cloning and apoptosis of cells in each group were detected by tetramethylazol blue method, plate cloning test and flow cytometry, respectively. The targeting relationship between lncRNA ADPGK-AS1 and miR-200b-5p was detected by double luciferase report test, and the expression level of cleaved-caspase-3 protein was detected by western blot. Results: Compared with the adjacent tissues, the expression of lncRNA ADPGK-AS1 in retinoblastoma tissues was increased (P<0.05), while the expression of miR-200b-5p was decreased (P<0.05). Compared with ARPE-19 cells, the expression of lncRNA ADPGK-AS1 in Y-79 and WERI-Rb-1 cells was increased (P<0.05), while the expression of miR-200b-5p was decreased (P<0.05). Compared with the si-con group, the cell viability of the si-lncRNA ADPGK-AS1 group was reduced (1.06±0.09 vs 0.53±0.05, P<0.05), the number of cell clone formation was reduced (114.00±8.03 vs 57.00±4.13, P<0.05), while the apoptosis rate [(7.93±0.68)% vs (25.43±1.94)%] and the protein level of cleaved-caspase-3 were increased (P<0.05). Compared with the miR-con group, the cell viability of the miR-200b-5p group was decreased (1.05±0.08 vs 0.57±0.05, P<0.05), the number of cell clone formation was decreased (118.00±10.02 vs 64.00±5.13, P<0.05), while the apoptosis rate [(7.89±0.71)% vs (23.15±1.62)%] and the protein level of cleaved-caspase-3 were increased (P<0.05). lncRNA ADPGK-AS1 could target the expression of miR-200b-5p. Compared with the si-lncRNA ADPGK-AS1+ anti-miR-con group, cell viability of the si-lncRNA ADPGK-AS1+ anti-miR-200b-5p group was increased (0.53±0.04 vs 1.25±0.10, P<0.05), and the number of cell clones was increased (54.00±4.39 vs 125.00±10.03, P<0.05), while the rate of apoptosis [(25.38±1.53)% vs (9.76±0.71)%] and the protein level of cleaved-caspase-3 were decreased (P<0.05). Conclusion: Interfering with the expression of lncRNA ADPGK-AS1 could inhibit the proliferation and clone formation and induce apoptosis of retinoblastoma cells by targeting the expression of miR-200b-5p.

Construction of TRAF6 ubiquitin site 331 mutant colorectal cancer cell stable line and its effect on biological behavior of colorectal cancer cells / 中华肿瘤杂志

Objective: To investigate the effect of ubiquitin mutation at position 331 of tumor necrosis factor receptor related factor 6 (TRAF6) on the biological characteristics of colorectal cancer cells and its mechanism. Methods: lentivirus wild type (pCDH-3×FLAG-TRAF6) and mutation (pCDH-3×FLAG-TRAF6-331mut) of TRAF6 gene expression plasmid with green fluorescent protein tag were used to infect colorectal cancer cells SW480 and HCT116, respectively. The infection was observed by fluorescence microscope, and the expressions of TRAF6 and TRAF6-331mut in cells was detected by western blot. Cell counting kit-8 (CCK-8) and plate cloning test were used to detect the proliferation ability of colorectal cancer cells in TRAF6 group and TRAF6-331mut group, cell scratch test to detect cell migration, Transwell chamber test to detect cell migration and invasion, immunoprecipitation to detect the ubiquitination of TRAF6 and TRAF6-331mut with ubiquitinof lysine binding sites K48 and K63. Western blot was used to detect the effects of TRAF6 and TRAF6-331mut over expression on the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinase mitogen-activated protein kinase (MAPK)/activating protein-1(AP-1) signal pathway. Results: The successful infection of colorectal cancer cells was observed under fluorescence microscope. Western blot detection showed that TRAF6 and TRAF6-331mut were successfully expressed in colorectal cancer cells. The results of CCK-8 assay showed that on the fourth day, the absorbance values of HCT116 and SW480 cells in TRAF6-331mut group were 1.89±0.39 and 1.88±0.24 respectively, which were lower than those in TRAF6 group (2.09±0.12 and 2.17±0.45, P=0.036 and P=0.011, respectively). The results of plate colony formation assay showed that the number of clones of HCT116 and SW480 cells in TRAF6-331mut group was 120±14 and 85±14 respectively, which was lower than those in TRAF6 group (190±21 and 125±13, P=0.001 and P=0.002, respectively). The results of cell scratch test showed that after 48 hours, the percentage of wound healing distance of HCT116 and SW480 cells in TRAF6-331mut group was (31±12)% and (33±14)%, respectively, which was lower than those in TRAF6 group [(43±13)% and (43±7)%, P=0.005 and 0.009, respectively]. The results of Transwell migration assay showed that the migration numbers of HCT116 and SW480 cells in TRAF6-331mut group were significantly lower than those in TRAF6 group (P<0.001 and P<0.002, respectively). The results of Transwell invasion assay showed that the number of membrane penetration of HCT116 and SW480 cells in TRAF6-331mut group was significantly lower than those in TRAF6 group (P=0.008 and P=0.009, respectively). The results of immunoprecipitation detection showed that the ubiquitin protein of K48 chain pulled by TRAF6-331mut was lower than that of wild type TRAF6 in 293T cells co-transfected with K48 (0.57±0.19), and the ubiquitin protein of K63 chain pulled down by TRAF6-331mut in 293T cells co-transfected with K63 was lower than that of wild type TRAF6 (0.89±0.08, P<0.001). Western blot assay showed that the protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-HCT116 cells were 0.63±0.08, 0.42±0.08 and 0.60±0.07 respectively, which were lower than those in TRAF6-HCT116 cells (P=0.002, P<0.001 and P<0.001, respectively). The expression level of AP-1 protein in TRAF6-HCT116 cells was 0.89±0.06, compared with that in TRAF6-HCT116 cells. The difference was not statistically significant (P>0.05). The protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-SW480 cells were 0.50±0.06, 0.51±0.04, 0.48±0.02, respectively, which were lower than those in TRAF6-SW480 cells (all P<0.001). There was no significant difference in AP-1 protein expression between TRAF6-331mut-SW480 cells and TRAF6-SW480 cells. Conclusion: The ubiquitin site mutation of TRAF6 gene at 331 may prevent the binding of TRAF6 and ubiquitin lysine sites K48 and K63, and then affect the expressions of proteins related to downstream NF-κB and MAPK/AP-1 signal pathways, and inhibit the proliferation, migration and invasion of colorectal cancer cells.

Effects of lncRNA-UCA1 targeting miR-204-5p on the proliferation, migration, apoptosis and immune escape of endometrial carcinoma cells / 中华肿瘤杂志

Objective: To investigate the effect of long non-coding RNA urothelial carcinoma-associated 1 (UCA1) gene on the proliferation, migration, apoptosis and immune escape of endometrial cancer cells and its molecular mechanism. Methods: Endometrial cancer tissues and adjacent normal tissues of patients with endometrioid adenocarcinoma who underwent total or partial hysterectomy in Henan Provincial People's Hospital from 2017 to 2019 were collected. The expressions of UCA1 and miR-204-5p were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), and the cell proliferation, migration and apoptosis were detected by cell counting kit 8 (CCK8) method, Transwell method, flow cytometry, and dual-luciferase reporter assay was used to explore the target relationship between UCA1 and miR-204-5p. HEC-1A-sh-NC or HEC-1A-sh-UCA1 cells were co-cultured with peripheral blood mononuclear cells (PBMC) or cytokine-induced killer cells in vitro to explore the role of UCA1 in immune escape. Results: The expression level of UCA1 in endometrial cancer tissue (17.08±0.84) was higher than that in adjacent normal endometrial tissue (3.00±0.37), and the expression level of miR-204-5p (0.98±0.16) was lower than that in adjacent normal endometrial tissue (2.00±0.20, P<0.05). Pearson correlation analysis showed that the expression of miR-204-5p was negatively correlated with the expression of UCA1 (r=-0.330, P=0.030). The expressions of UCA1 and miR-204-5p were associated with the International Federation of Gynecology and Obstetrics stage of endometrial cancer, lymph node metastasis and vascular invasion (P<0.05). The relative ratio of absorbance (0.58±0.11) and the number of cell migration [(199.68±18.44)] in the sh-UCA1 group were lower than those in the sh-NC group (1.24±0.17 and 374.76±24.83), respectively. The apoptosis rate of sh-UCA1 group [(28.64±7.80)%] was higher than that of sh-NC group [(14.27±4.38)%, P<0.05]. After different ratios of effector cells and target cells were cultured, the cell survival rate of HEC-1A-sh-UCA1 group was lower than that of HEC-1A-sh-NC group, and the difference was statistically significant (P<0.05). UCA1 had a binding site for miR-204-5p. The relative ratio of absorbance (1.74±0.08) and the number of cell migration (426.00±18.00) cells in the UCA1+ anti-miR-204-5p group were higher than those in the control group [1.00±0.03 and (284.00±8.00) cells, respectively]. The apoptosis rate of UCA1+ anti-miR-204-5p group [(5.42±0.93)%] was lower than that of control group [(14.82±1.48)%, P<0.05]. HEC-1A-sh-UCA1 cells could induce higher interferon gamma (IFN-γ) expression when co-cultured with PBMC, and the levels of IFN-γ expression in PHA group and PHA+ pre-miR-204-5p group cells were 2.42±0.49 and 1.88±0.26, which were higher than that in the PHA+ pre-NC group (0.85±0.10, P<0.05). When co-cultured with cytokine-induced killer cells (different ratios) in vitro, the HEC-1A-sh-UCA1 group and the HEC-1A-pre-miR-204-5p group had lower survival rates than that in the HEC-1A-pre-miR-204-5p group. In the HEC-1A-pre-NC group, the differences were statistically significant (P<0.05). Conclusion: UCA1/miR-204-5p may play an important role in human endometrial cancer.

Inhibition of CD36 and Nogo-B expression inhibited the proliferation and migration of triple negative breast cancer cells / 生物工程学报

Cluster of differentiation 36 (CD36) is a membrane glycoprotein receptor capable of binding and transporting fatty acid. Nogo-B regulates the metabolism of fatty acids in the liver and affects the development of liver cancer. To date, it remains unclear whether the interaction between CD36 and Nogo-B affects the proliferation and migration of breast cancer cells. In the current study, we aimed to determine whether the interference of CD36 and Nogo-B affects the proliferation and migration of triple-negative breast cancer (TNBC) cells. The results showed that inhibition of CD36 or Nogo-B alone can inhibit the proliferation and migration of TNBC cells, and the inhibitory effect was more pronounced when CD36 and Nogo-B were inhibited simultaneously. Meanwhile, it was found that inhibition of CD36 and Nogo-B expression can inhibit the expression of Vimentin, B-cell lympoma-2 (BCL2) and proliferating cell nuclear antigen (PCNA). In vivo, knockdown of CD36 or Nogo-B in E0771 cells reduced its tumorigenic ability, which was further enhanced by knockdown of CD36 and Nogo-B simultaneously. Mechanistically, inhibition of CD36 and Nogo-B expression can decrease fatty acid binding protein 4 (FABP4) and fatty acid transport protein 4 (FATP4) expression. Moreover, overexpression of CD36 and Nogo-B-induced cell proliferation was attenuated by FABP4 siRNA, indicating that inhibition of CD36 and Nogo-B expression could inhibit the absorption and transport of fatty acids, thereby inhibiting the proliferation and migration of TNBC. Furthermore, inhibition of CD36 and Nogo-B expression activated the P53-P21-Rb signaling pathway which contributed to the CD36 and Nogo-B-inhibited proliferation and migration of TNBC. Taken together, the results suggest that inhibition of CD36 and Nogo-B can reduce the proliferation and migration of TNBC, which provides new targets for the development of drugs against TNBC.

MiR-3653 blocks autophagy to inhibit epithelial-mesenchymal transition in breast cancer cells by targeting the autophagy-regulatory genes ATG12 and AMBRA1 / 中华医学杂志(英文版)

BACKGROUND@#Metastasis is the main cause of tumor-associated death and mainly responsible for treatment failure of breast cancer. Autophagy accelerates tumor metastasis. In our work, we aimed to investigate the possibility of microRNAs (miRNAs) which participate in the regulation of autophagy to inhibit tumor metastasis.@*METHODS@#MiRNA array and comprehensive analysis were performed to identify miRNAs which participated in the regulation of autophagy to inhibit tumor metastasis. The expression levels of miR-3653 in breast cancer tissues and cells were detected by quantitative real-time polymerase chain reaction. In vivo and in vitro assays were conducted to determine the function of miR-3653. The target genes of miR-3653 were detected by a dual luciferase reporter activity assay and Western blot. The relationship between miR-3653 and epithelial-mesenchymal transition (EMT) was assessed by Western blot. Student's t -test was used to analyze the difference between any two groups, and the difference among multiple groups was analyzed with one-way analysis of variance and a Bonferroni post hoc test.@*RESULTS@#miR-3653 was downregulated in breast cancer cells with high metastatic ability, and high expression of miR-3653 blocked autophagic flux in breast cancer cells. Clinically, low expression of miR-3653 in breast cancer tissues (0.054 ± 0.013 vs . 0.131 ± 0.028, t  = 2.475, P  = 0.014) was positively correlated with lymph node metastasis (0.015 ± 0.004 vs . 0.078 ± 0.020, t  = 2.319, P  = 0.023) and poor prognosis ( P  < 0.001). miR-3653 ameliorated the malignant phenotypes of breast cancer cells, including proliferation, migration (MDA-MB-231: 0.353 ± 0.013 vs . 1.000 ± 0.038, t  = 16.290, P  < 0.001; MDA-MB-468: 0.200 ± 0.014 vs . 1.000 ± 0.043, t  = 17.530, P  < 0.001), invasion (MDA-MB-231: 0.723 ± 0.056 vs . 1.000 ± 0.035, t  = 4.223, P  = 0.013; MDA-MB-468: 0.222 ± 0.016 vs . 1.000 ± 0.019, t  = 31.050, P  < 0.001), and colony formation (MDA-MB-231: 0.472 ± 0.022 vs . 1.000 ± 0.022, t  = 16.620, P  < 0.001; MDA-MB-468: 0.650 ± 0.040 vs . 1.000 ± 0.098, t  = 3.297, P  = 0.030). The autophagy-associated genes autophagy-related gene 12 ( ATG12 ) and activating molecule in beclin 1-regulated autophagy protein 1 ( AMBRA1 ) are target genes of miR-3653. Further studies showed that miR-3653 inhibited EMT by targeting ATG12 and AMBRA1 .@*CONCLUSIONS@#Our findings suggested that miR-3653 inhibits the autophagy process by targeting ATG12 and AMBRA1 , thereby inhibiting EMT, and provided a new idea and target for the metastasis of breast cancer.

USP25 promotes hepatocellular carcinoma progression by interacting with TRIM21 via the Wnt/β-catenin signaling pathway / 中华医学杂志(英文版)

BACKGROUND@#Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The ubiquitin-specific peptidase 25 (USP25) protein has been reported to participate in the development of several cancers. However, few studies have reported its association with HCC. In this study, we aimed to investigate the function and mechanism of USP25 in the progression of HCC.@*METHODS@#We analyzed USP25 protein expression in HCC based on The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) database cohorts. Then, we constructed USP25-overexpressing and USP25-knockdown HepG2, MHCC97H, and L-O2 cells. We detected the biological function of USP25 by performing a series of assays, such as Cell Counting Kit-8 (CCK-8), colony formation, transwell, and wound healing assays. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) analyses were performed to detect the interaction between USP25 and the Wnt/β-catenin signaling pathway. The relationship between USP25 and tripartite motif-containing 21 (TRIM21) was assessed through mass spectrometry and co-immunoprecipitation (Co-IP) analysis. Finally, we constructed a mouse liver cancer model with the USP25 gene deletion to verify in vivo role of USP25.@*RESULTS@#USP25 was highly expressed in HCC tissue and HCC cell lines. Importantly, high expression of USP25 in tissues was closely related to a poor prognosis. USP25 knockdown markedly reduced the proliferation, migration, and invasion of HepG2 and MHCC97H cells, whereas USP25 overexpression led to the opposite effects. In addition, we demonstrated that USP25 interacts with TRIM21 to regulate the expression of proteins related to epithelial-mesenchymal transition (EMT; E-cadherin, N-cadherin, and Snail) and the Wnt/β-catenin pathway (β-catenin, Adenomatous polyposis coli, Axin2 and Glycogen synthase kinase 3 beta) and those of their downstream proteins (C-myc and Cyclin D1). Finally, we verified that knocking out USP25 inhibited tumor growth and distant metastasis in vivo .@*CONCLUSIONS@#In summary, our data showed that USP25 was overexpressed in HCC. USP25 promoted the proliferation, migration, invasion, and EMT of HCC cells by interacting with TRIM21 to activate the β-catenin signaling pathway.

Long non-coding RNA colon cancer-associated transcript 1-Vimentin axis promoting the migration and invasion of HeLa cells / 中华医学杂志(英文版)

BACKGROUND@#Long non-coding RNA colon cancer-associated transcript 1 (CCAT1) is involved in transforming multiple cancers into malignant cancer types. Previous studies underlining the mechanisms of the functions of CCAT1 primarily focused on its decoy for miRNAs (micro RNAs). However, the regulatory mechanism of CCAT1-protein interaction associated with tumor metastasis is still largely unknown. The present study aimed to identify proteome-wide CCAT1 partners and explored the CCAT1-protein interaction mediated tumor metastasis.@*METHODS@#CCAT1-proteins complexes were purified and identified using RNA antisense purification coupled with the mass spectrometry (RAP-MS) method. The database for annotation, visualization, and integrated discovery and database for eukaryotic RNA binding proteins (EuRBPDB) websites were used to bioinformatic analyzing CCAT1 binding proteins. RNA pull-down and RNA immunoprecipitation were used to validate CCAT1-Vimentin interaction. Transwell assay was used to evaluate the migration and invasion abilities of HeLa cells.@*RESULTS@#RAP-MS method worked well by culturing cells with nucleoside analog 4-thiouridine, and cross-linking was performed using 365 nm wavelength ultraviolet. There were 631 proteins identified, out of which about 60% were RNA binding proteins recorded by the EuRBPDB database. Vimentin was one of the CCAT1 binding proteins and participated in the tumor metastasis pathway. Knocked down vimetin ( VIM ) and rescued the downregulation by overexpressing CCAT1 demonstrated that CCAT1 could enhance tumor migration and invasion abilities by stabilizing Vimentin protein.@*CONCLUSION@#CCAT1 may bind with and stabilize Vimentin protein, thus enhancing cancer cell migration and invasion abilities.