Cell Invasion Capacity Research Articles

ABCF1/CXCL12/CXCR4 Enhances Glioblastoma Cell Proliferation, Migration, and Invasion by Activating the PI3K/AKT Signal Pathway

Glioblastoma (GBM) is the most prevalent and fatal form of brain tumor, which is associated with a poor prognosis. ATP-binding cassette subfamily F member 1 (ABCF1) is an E2 ubiquitin-conjugating enzyme, which is implicated in regulating immune responses and tumorigenesis. Aberrant E3 ubiquitylation has been evidenced in GBM. However, the role of ABCF1 in GBM needs to be further explored. The expression of ABCF1, CXC chemokine ligand 12 (CXCL12), and CXC chemokine receptor 4 (CXCR4) in GBM tissues was examined by the GEPIA tool, real-time PCR and Western blotting. HMC3, U251MG, and LN-229 cells were cultured and transfected with shRNA targeting ABCF1 and ABCF1 plasmids. The proliferative, migrative, and invasive ability of cells was detected. Western blotting was used to detect the levels of phosphorylated phosphatidylinositol 3-kinase (PI3K) and phosphorylated protein kinase B (AKT). We observed that GBM tissues had higher ABCF1, CXCL12, and CXCR4 expression levels. The expression levels of CXCL12 and CXCR4 were enhanced by ABCF1 overexpression, which were significantly reversed by silence of ABCF1 in GBM cells. Silencing ABCF1 or CXCR4 inhibition weakened the capacity of GBM cell growth, migration, and invasion, while ectopic ABCF1 expression or CXCL12 treatment enhanced the cellular function of GBM cells. Furthermore, p-PI3K and p-AKT protein levels were downregulated by ABCF1 knockdown or CXCR4 blockade, which were prompted by ABCF1 overexpression or CXCL12 supplement. The ABCF1-CXCL12-CXCR4 axis was identified as a key player in GBM cell survival and metastasis by activating the PI3K/AKT signaling pathway in GBM cells.

Protective effects of a new generation of probiotic Bacteroides fragilis against colitis in vivo and in vitro

Bacteroides fragilis, one of the potential next-generation probiotics, but its protective mechanism is not yet known. We aimed to characterize the anti-inflammatory effect of B. fragilisATCC25285 and to elucidate its mechanism through in vivo and in vitro experiments. An in vitro model of inflammation by induction of colonic cells with TNF-a, and co-cultured with B. fragilis to detect cell viability, apoptosis and invasive capacity. Furthermore, critical proteins of the TLR/NF-κB pathway and the inflammatory cytokines were measured. For animal trials, C57BL/6 J male mice were orally administered B. fragilis or PBS once daily for 21 days. Colitis was induced by drinking 2.5% DSS from days 0 to 7. The mice were weighed daily and rectal bleeding, stool condition and blood in the stool were recorded. We found that B. fragilis treatment alone was harmless and had no effect on cell viability or apoptosis. While predictably TNF-α decreased cell viability and increased apoptosis, B. fragilis attenuated this deterioration. The NF-κB pathway and inflammatory cytokines IL-6 and IL-1β activated by TNF-α were also blocked by B. fragilis. Notably, the metabolic supernatant of B. fragilis also has an anti-inflammatory effect. Animal studies showed that live B. fragilis rather than dead strain ameliorated DSS-induced colitis, as evidenced by weight loss, shortened colon length and enhanced barrier function. The colonic tissue levels of inflammatory cytokines (TNF-α, IL-1β, IL-6) were decreased and IL-10 was increased as a result of B. fragilis administration. In conclusion, B. fragilis ATCC25285 exhibited anti-inflammatory effects whether in vivo or in vitro, and it may be a potential probiotic agent for improving colitis.

ZNF746 promotes M2 macrophage polarisation and favours tumour progression in breast cancer via the Jagged1/Notch pathway

Breast cancer (BC) is a major threat to women's health. BC is a heterogeneous disease and treatment strategies and outcomes differ between subtypes. Investigating the molecular mechanisms of BC will help to identify potential therapeutic targets and develop new therapies. Here we report that zinc finger protein 746 (ZNF746), a Krüppel-associated box and zinc finger protein, exhibits tumour-promoting properties in BC. Functional experiments (cell growth, colony formation, cell cycle analysis, and transwell analysis) were used to evaluate the proliferation, migration, and invasion capacity of BC cells. Immunohistochemistry was performed to detect the expression of ZNF746, CD163 (M2 macrophage marker), and HES1 (Notch target) in BC tissues. ZNF746 was highly expressed in BC tissues compared to adjacent paired non-tumour tissues. Patients with M1 BC had higher expression of ZNF746 compared to patients with non-metastatic (M0) BC, and higher expression of ZNF746 was associated with poorer overall survival. The immunohistochemical results showed a positive correlation between the expression of ZNF746 and the expression of CD163 or HES1. ZNF746 promoted BC cell proliferation, migration, and invasion and increased the expression of molecules essential for monocyte recruitment and differentiation (CCL2 and CSF1). Furthermore, THP-1 monocytes cultured in the conditioned medium derived from BC cells overexpressing ZNF746 exhibited enhanced M2 polarisation. In contrast, ZNF746 knockdown reduced BC cell proliferation, migration, and invasion and suppressed M2 polarisation. Mechanistically, ZNF746 promoted the activation of the Jagged1/Notch pathway, and the Jagged1 siRNA-mediated blockade of this pathway prevented the tumour-promoting functions of ZNF746. In conclusion, this study uncovers the role of ZNF746 in promoting M2 macrophage polarisation and suggests that ZNF746 may be a promising therapeutic target for limiting BC progression.

Upregulation of ubiquitin carboxy‑terminal hydrolase 47 (USP47) in papillary thyroid carcinoma exvivo and reduction of tumor cell malignant behaviors after USP47 knockdown by stabilizing SATB1 expression invitro.

Aberrant ubiquitination contributes to cancer development, including thyroid carcinoma. The present study assessed the expression of ubiquitin carboxy-terminal hydrolase 47 (USP47) and underlying molecular events in the development of papillary thyroid carcinoma (PTC). The effects of USP47 on PTC cell invasion and migration were analyzed by Transwell assays, while. the effects of USP47 and SATB1on PTC cell gene expression and changes in tumor cell metabolism were assayed by reverse transcription-quantitative PCR, western bolt, or ELISA, respectively. The expression of USP47 mRNA and protein was upregulated in PTC tissue and associated with the PTC tumor size. Knockdown of USP47 expression in PTC cell lines (TPC-1 and K1), decreased the cell proliferation mobility and invasion capacities, whereas USP47 overexpression in these cell lines showed an inverse effect and promoted cell glycolysis and glutamine metabolism. Moreover, expression of special AT-rich sequence-binding protein-1 (SATB1) was high in PTC tissue and was associated with USP47 expression. SATB1 expression promoted tumor cell glycolysis and glutamine metabolism, while USP47 protein bound to and deubiquitinated SATB1 to increase its intracellular levels, thus promoting glycolysis and glutamine metabolism. USP47 promotion of PTC development may be due to its stabilization of SATB1 protein, suggesting that targeting the USP47/SATB1 signaling axis may serve as a therapeutic intervention for PTC.

P300/SP1 complex mediating elevated METTL1 regulates CDK14 mRNA stability via internal m7G modification in CRPC

BackgroundN7-methylguanosine (m7G) modification is, a more common epigenetic modification in addition to m6A modification, mainly found in mRNA capsids, mRNA interiors, transfer RNA (tRNA), pri-miRNA, and ribosomal RNA (rRNA). It has been found that m7G modifications play an important role in mRNA transcription, tRNA stability, rRNA processing maturation, and miRNA biosynthesis. However, the role of m7G modifications within mRNA and its “writer” methyltransferase 1(METTL1) in tumors, particularly prostate cancer (PCa), has not been revealed.MethodsThe differential expression level of METTL1 between hormone-sensitive prostate cancer (HSPC) and castrate-resistant prostate cancer (CRPC) was evaluated via RNA-seq and in vitro experiments. The effects of METTL1 on CRPC progression were investigated through in vitro and in vivo assays. The upstream molecular mechanism of METTL1 expression upregulation and the downstream mechanism of its action were explored via Chromatin Immunoprecipitation quantitative reverse transcription polymerase chain reaction (CHIP-qPCR), Co-immunoprecipitation (Co-IP), luciferase reporter assay, transcriptome-sequencing, m7G AlkAniline-Seq, and mRNA degradation experiments, etc.Results and conclusionHere, we found that METTL1 was elevated in CRPC and that patients with METTL1 elevation tended to have a poor prognosis. Functionally, the knockdown of METTL1 in CRPC cells significantly limited cell proliferation and invasive capacity. Mechanistically, we unveiled that P300 can form a complex with SP1 and bind to the promoter region of the METTL1 gene via SP1, thereby mediating METTL1 transcriptional upregulation in CRPC. Subsequently, our findings indicated that METTL1 leads to enhanced mRNA stability of CDK14 by adding m7G modifications inside its mRNA, ultimately promoting CRPC progression.

CXCL12/CXCR4/CXCR7 axis in placenta tissues of patients with placenta previa.

CXCR4 and CXCR7 have been revealed to be receptors of CXCL12. This research was designed to probe the expression of chemokine CXCL12 and its receptors CXCR4 and CXCR7 in placental tissues of patients with placenta previa and the effect of CXCL12/CXCR4/CXCR7 axis on the biological functions of human trophoblast cells. CXCL12, CXCR4, and CXCR7 expression in placental tissue from patients with placenta previa and healthy puerperae was detected. CXCL12, CXCR4, and CXCR7 expression in human trophoblast cell lines (HTR8/SVneo cells) was assessed after suppression or overexpression of CXCL12, CXCR4, and CXCR7. The cell proliferative, invasive, and migratory capacities were also evaluated in HTR8/SVneo cells after suppression or overexpression of CXCL12, CXCR4, and CXCR7. CXCL12, CXCR4, and CXCR7 expression was elevated in placental tissues from patients with placenta previa. Downregulation of CXCL12, CXCR4, and CXCR7 could lead to decreased mRNA levels of CXCL12, CXCR4, and CXCR7 in HTR-8/SVneo cells, which was accompanied by diminished cell proliferative, migratory, and invasive capabilities. Overexpression of CXCL12, CXCR4, and CXCR7 genes presented an opposite tendency. CXCL12, CXCR4, and CXCR7 are highly expressed in placental tissues of patients with placenta previa and induce the biological activities of HTR8/SVneo cells.

Human neural stem cells repress glioma cell progression in a paracrine manner by downregulating the Wnt/β-catenin signalling pathway.

Neural stem cells (NSCs) play crucial roles in neurological disorders and tissue injury repair through exerting paracrine effects. However, the effects of NSC-derived factors on glioma progression remain unclear. This study aimed to evaluate the effects of human NSC-conditioned medium (NSC-CM) on the behaviour of glioma cells using an in vitro co-culture system. Cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays revealed that NSC-CM inhibited glioma cell proliferation and growth in a foetal bovine serum (FBS)-independent manner. In addition, our wound healing assay demonstrated that NSC-CM repressed glioma cell migration, while results from transwell and 3D spheroid invasion assays indicated that NSC-CM also reduced the invasion capacity of glioma cells. Flow cytometry showed that NSC-CM prevented cell cycle progression from the G1 to S phase and promoted apoptosis. Western blotting was used to show that the expression of Wnt/β-catenin pathway-related proteins, including β-catenin, c-Myc, cyclin D1, CD44, and Met, was remarkably decreased in NSC-CM-treated glioma cells. Furthermore, the addition of a Wnt/β-catenin pathway activator, CHIR99021, significantly induced the expression of β-catenin and Met and increased the proliferative and invasive capabilities of control medium-treated glioma cells but not those of NSC-CM-treated glioma cells. The use of enzyme-linked immunosorbent assays (ELISA) revealed the secretion of some anti-tumour factors in human and rat NSCs, including interferon-α and dickkopf-1. Our data suggest that NSC-CM partially inhibits glioma cell progression by downregulating Wnt/β-catenin signalling. This study may serve as a basis for developing future anti-glioma therapies based on NSC derivatives.

UCA1 executes an oncogenic role in pancreatic cancer by regulating miR-582-5p/BRCC3.

As a fatal disease, the mechanism of pancreatic cancer is unclear. Urothelial carcinoma antigen 1(UCA1), a long noncoding RNA (lncRNA) that was first reported in bladder cancer, acts as an oncogene. However, the regulatory role and mechanism of UCA1 in pancreatic cancer remain unknown. This study aims to investigate the expression level and prognostic value of UCA1 in pancreatic cancer tissues, the effects and mechanism of UCA1 in regulating cell proliferation, apoptosis and metastasis. UCA1 expression levels in tissues were detected by in situ hybridization (ISH) and the prognostic value was evaluated by univariate and multivariate survival analysis. For in vitro experiments, proliferation was evaluated by a cell count kit assay, Edu experiments, and a clone formation assay. Apoptosis was evaluated by fluorescence-activated cell sorting flow-cytometry. Cell migration and invasion capacities were detected by wound healing and transwell assays. Western blots were performed to detect apoptotic associated molecules and epithelial-mesenchymal transition (EMT) markers. For the in vivo experiment, subcutaneous transplantation models of pancreatic cancer in nude mice were established to observe the tumor growth. The regulatory mechanism of UCA1 was explored by proteomics, bioinformatic analysis, luciferase reporter assays, and rescue experiments. ISH staining revealed that UCA1 levels between cancer tissues (n=94) and tumor-adjacent tissues (n=73) did not show significant differences. Survival analysis indicated that high expression of UCA1 was an unfavorable prognosis factor for pancreatic cancer. Downregulation of UCA1 by siRNA significantly inhibited cell proliferation, decreased the capacities of cell migration and invasion, induced cell apoptosis, and inhibited EMT. Furthermore, we demonstrated that UCA1 positively regulated the expression of BRCC3 by inhibiting miR-582-5p. Rescue experiments indicated that either inhibiting the expression of miR-582-5p or enhancing expression of BRCC3 could partly attenuate the antitumor effects of downregulation of UCA1. UCA1 acted as an oncogene in pancreatic cancer by partly regulating miR-582-5p/BRCC3, which could be a new therapeutic target for pancreatic cancer.

Sauchinone inhibits the proliferation and immune invasion capacity of colorectal cancer cells through the suppression of PD-L1 and MMP2/MM9.

Colorectal cancer (CRC) is one of the most common tumors globally and a leading cause of cancer-related death. In China, CRC is the third most common cancer type. Sauchinone is known to exhibit anti-tumor and anti-inflammatory activity, but its effects on CRC have not been investigated to-date Objective: To investigate the effects of Sauchinone on CRC development and metastasis and its underlying mechanism(s) of action. SW480 and HCT116 cells were treated with a range of concentrations of Sauchinone. Cell proliferation was measured using EDU assays and flow cytometry. Treatment with 50 µM Sauchinone decreased the expression of MMP2 and MMP9 and downregulated PD-L1 expression (PD-1/PD-L1) leading to checkpoint inhibition. Sauchinone treatment also enhanced the cytotoxicity of SW840 and HCT116 cells co-cultured with CD8+ T cells. The overexpression of PD-L1 rescued the anti-proliferative and cytotoxic effects of Sauchinone in both types. We show that Sauchinone suppresses CRC cell growth through the downregulation of MMP2 and MM9 expression and PD-1/PD-L1 mediated checkpoint inhibition. Collectively, these data highlight the promise of Sauchinone as a future anti-CRC therapeutic.

Mechanistic insights into genomic structure and functions of a novel oncogene YEATS4.

As a novel oncogene, the role of YEATS domain-containing protein 4 (YEATS4) in the occurrence, development, and treatment of tumors is now beginning to be appreciated. YEATS4 plays an important role in regulating DNA repair during replication. The upregulation of YEAST4 promotes DNA damage repair and prevents cell death, whereas its downregulation inhibits DNA replication and induces apoptosis. Additionally, accumulating evidence indicates that the aberrant activation of YEATS4 leads to changes in drug resistance, epithelial-mesenchymal transition and also in the migration and invasion capacity of tumor cells. Therefore, specific inhibition of the expression or activity of YEATS4 protein may be an effective strategy for inhibiting the proliferation, motility, differentiation, and/or survival of tumor cells. Taken together, YEATS4 has emerged as a potential target for multiple cancers and is an attractive protein for the development of small-molecule inhibitors. However, research on YEAST4 in tumor-related fields is limited and its biological functions, metabolism, and the regulatory mechanism of YEATS4 in numerous cancers remain undetermined. This review comprehensively and extensively summarizes the functions, structure and oncogenic roles of YEATS4 in cancer progression and aims to further contribute to the study of its underlying molecular mechanism and targeted drugs.

MicroRNA-378a-3p plays a regulatory role in trophoblast cell function in preeclampsia by targeting CMTM3

BackgroundPreeclampsia (PE) is a potential multisystemic disease in the middle and late pregnancy. Although its precise etiology and pathogenesis remain unknown, it is a significant cause of morbidity and mortality in both pregnant women and newborns. This study explored the effects of the miR-378a-3p/CKLF-like MARVEL transmembrane domain containing 3 (CMTM3) upon the trophoblast biological functions in PE. MethodsThe placental pathology of PE were identified by hematoxylin-eosin (HE) staining, and miR-378a-3p expression in placental tissues of PE was verified by RT-qPCR. Trophoblast cells (HTR-8/SVneo and JEG-3) were treated with lipopolysaccharide (LPS), and cell counting kit-8 (CCK-8) assay, flow cytometry, scratch assay, and Transwell assay were carried out to measure cell viability, apoptosis, migratory and invasive capacities, respectively. Western blot was performed to determine the expression levels of the cell migration-related proteins. The binding of miR-378a-3p to CMTM3 was verified through a dual-luciferase reporter gene assay. ResultsmiR-378a-3p expression levels were down-regulated in placental tissues and primary trophoblast cells from women with PE compared to the control group. The overexpression of miR-378a-3p promoted the capabilities of LPS-treated trophoblast cells to proliferate, migrate and invade. In contrast, it impeded cell apoptosis, promoted matrix metallopeptidase (MMP)-2 and MMP-9 expression and inhibiting TIMP metallopeptidase inhibitor (TIMP)-1 and TIMP-2 expression. Regarding the molecular mechanism, miR-378a-3p was chosen as the target to modulate the expression level of CMTM3. CMTM3 expression was increased in placental tissues and primary trophoblast cells from women with PE compared to the control group. CMTM3 overexpression could partially neutralize the effects of the overexpressed miR-378a-3p on trophoblast cell function and the expression levels of migration-associated proteins. ConclusionOur study provides a foundation for miRNA-targeted therapy for preeclampsia by establishing for the first time a potential role for the miR-378a-3p/CMTM3 axis in regulating trophoblast cell activities by altering the expression of migration-related proteins.

P-316 Involvement of collective cell migration in endometrial invasiveness in uterine adenomyosis and the role of M2 macrophages

Abstract Study question How do endometrial cells manage to invade surrounding myometrium to establish adenomyotic lesions and what is the role of macrophages in this process? Summary answer M2 macrophage-mediated invasiveness and resistance of endometrial cells to physiological cell death appear to contribute to the pathogenesis of adenomyosis. What is known already Despite the high prevalence and debilitating symptoms of uterine adenomyosis, its pathogenesis has not yet been elucidated. To date, the most acceptable theory to explain disease development suggests invasion of the myometrium by eutopic endometrial cells. Recent data suggest aberrant infiltration of immune cells but not platelets in adenomyosis, eventually leading to enhanced cell motility via collective cell migration (CCM) and resistance to physiological cell death during disease pathogenesis. Study design, size, duration A retrospective immunohistochemistry-based study was conducted on formalin-fixed paraffin-embedded uterine tissue from 17 women (8 adenomyosis patients and 9 healthy subjects). Fresh endometrial biopsies were retrieved from 16 women (6 adenomyosis patients and 10 healthy subjects) for in vitro culture of epithelial and stromal cells, and co-culture with THP-1 monocyte-derived M2 macrophages. Participants/materials, setting, methods Double immunofluorescence was performed to detect macrophages of M1 and M2 phenotypes. Invasiveness of endometrial cells upon interaction with macrophages was assessed by in vitro invasion assays and quantitative PCR for genes involved in cell motility and epithelial-mesenchymal transition (EMT). Mechanisms of disease progression were investigated by immunohistochemistry against E-cadherin, N-cadherin and matrix metalloproteinase 9 (MMP9). Caspase 3 and microtubule-associated protein light chain 3 beta (LC3B) were studied as markers of apoptosis and autophagy respectively. Main results and the role of chance Only M2 macrophages were found to accumulate in adenomyosis, in higher numbers in both endometrium and ectopic lesions from adenomyosis patients than healthy controls. Co-culture with M2 macrophages significantly increased invasion capacity in endometrial epithelial and stromal cells from both adenomyosis patients and healthy controls. Epithelial cells from adenomyosis patients were nonetheless more invasive than their healthy counterparts in the presence of M2 macrophages. No gene expression differences pointing to EMT were noted, either between co-cultured and control cells, or between adenomyotic and healthy cells, refuting the implication of this mechanism in adenomyosis invasiveness. E- and N-cadherin protein expression did not differ significantly between eutopic endometrium from adenomyosis subjects and healthy tissue, but N-cadherin expression was stronger in ectopic lesions. In adenomyosis, both E- and N-cadherin were more extensively expressed in basal (also known as invasive) glands than functional glands, suggesting maintenance of intercellular junctions and subsequent CCM. Extracellular matrix-degrading enzyme MMP9 was more abundantly expressed in eutopic stroma from adenomyosis patients compared to healthy tissue. Lower levels of caspase 3 were detected in both endometrium and lesions from adenomyosis patients, while LC3B was found significantly decreased in adenomyotic stroma compared to that of healthy subjects. Limitations, reasons for caution A limitation of the study is the 2D nature of in vitro cultures, which cannot fully reconstitute in vivo cell arrangement and cell-cell interactions, nor allow to observe CCM in real time. Wider implications of the findings M2 macrophages accumulating in endometrium and ectopic lesions from adenomyosis patients boost invasion capacity of endometrial epithelial and stromal cells, as shown by in vitro co-culture experiments. These findings indicate that M2 macrophages play a key role in disease pathogenesis and could be targeted to develop novel therapeutic options. Trial registration number not applicable

PITX2 in pancreatic stellate cells promotes EMT in pancreatic cancer cells via the Wnt/β-catenin pathway.

Since the prognosis of patients with pancreatic cancer is very poor and there is a lack of treatment methods, this study is performed to investigate the function of PITX2 in pancreatic stellate cells (PSCs) in the progression of pancreatic cancer. Scientific hypotheses are proposed according to bioinformatics analysis and tissue microarray analysis. Stable knockdown of PITX2 in PSCs is achieved through lentiviral infection. The relative expressions of PITX2, α-SMA, vimentin, CTNNB1, AXIN1 and LEF1 are measured in wild-type PSCs and PITX2-knockdown PSCs. Proliferative capacity is measured by EdU assay. After coculture with PSCs, the proliferation, invasion and migration capacity of pancreatic cancer cells are tested. EMT and Wnt/β-catenin downstream genes of pancreatic cancer cells are investigated to reveal the potential mechanism. Bioinformatics analysis reveals that the PITX2 gene is highly expressed in stromal cells in pancreatic cancer and is correlated with squamous-type PDAC. Analysis of PDAC tissue microarray further demonstrates that high PITX2 level in stromal cells is correlated with poor prognosis in PDAC. After stable knockdown of PITX2 in PSCs, the relative protein levels of α-SMA, vimentin, CTNNB1, AXIN1 and LEF1 are decreased, and the proliferative capacity of PSCs is also decreased. After coculture with PSCs, in which PITX2 expression is downregulated, the proliferation, invasion and migration capacities of pancreatic cancer cells are inhibited. Thus, our results show that PITX2-silenced PSCs inhibit the growth, migration and invasion of pancreatic cancer cells via reduced EMT and Wnt/β-catenin signaling.

Oridonin promotes endoplasmic reticulum stress via TP53-repressed TCF4 transactivation in colorectal cancer

BackgroundThe incidence of colorectal cancer and cancer death rate are increasing every year, and the affected population is becoming younger. Traditional Chinese medicine therapy has a unique effect in prolonging survival time and improving the prognosis of patients with colorectal cancer. Oridonin has been reported to have anti-cancer effects in a variety of tumors, but the exact mechanism remains to be investigated.MethodsCell Counting Kit-8 assay (CCK8) and 5-Ethynyl-2'-deoxyuridine (EdU) staining assay, Tranwell, and Wound healing assays were performed to measure cell proliferation, invasion, and migration capacities, respectively. The protein and mRNA expression levels of various molecules were reflected by Western blot and Reverse Transcription quantitative Polymerase Chain Reaction (qRT-PCR). Transcription Factor 4 (TCF4) and its target genes were analyzed by Position Weight Matrices (PWMs) software and the Gene Expression Omnibus (GEO) database. Immunofluorescence (IF) was performed to visualize the expression and position of Endoplasmic Reticulum (ER) stress biomarkers. The morphology of the ER was demonstrated by the ER tracker-red. Reactive Oxygen Species (ROS) levels were measured using a flow cytometer (FCM) or fluorescent staining. Calcium ion (Ca2+) concentration was quantified by Fluo-3 AM staining. Athymic nude mice were modeled with subcutaneous xenografts.ResultsOridonin inhibited the proliferation, invasion, and migration of colorectal cancer, and this effect was weakened in a concentration-dependent manner by ER stress inhibitors. In addition, oridonin-induced colorectal tumor cells showed increased expression of ER stress biomarkers, loose morphology of ER, increased vesicles, and irregular shape. TCF4 was identified as a regulator of ER stress by PWMs software and GEO survival analysis. In vitro and in vivo experiments confirmed that TCF4 inhibited ER stress, reduced ROS production, and maintained Ca2+ homeostasis. In addition, oridonin also activated TP53 and inhibited TCF4 transactivation, further exacerbating the elevated ROS levels and calcium ion release in tumor cells and inhibiting tumorigenesis in colorectal cancer cells in vivo.ConclusionsOridonin upregulated TP53, inhibited TCF4 transactivation, and induced ER stress dysregulation in tumor cells, promoting colorectal cancer cell death. Therefore, TCF4 may be one of the important nodes for tumor cells to regulate ER stress and maintain protein synthesis homeostasis. And the inhibition of the TP53/TCF4 axis plays a key role in the anti-cancer effects of oridonin.

Delta/Notch-like Epidermal Growth Factor-Related Receptor (DNER), a Potential Prognostic Marker of Gastric Cancer Regulates Cell Survival and Cell Cycle Progression.

Upregulation of the expression of Delta/notch-like epidermal growth factor-related receptor (DNER) and its oncogenic role have been reported in several cancers, including gastric, breast, and prostate cancers. This study aimed to investigate the oncogenic role of DNER and the mechanisms behind its oncogenic role in gastric cancer. Analysis of the RNASeq data of gastric cancer tissues obtained from the TCGA database revealed that the expression of DNER was associated with the pathology of advanced gastric cancer and the prognosis of patients. DNER expression was increased upon stem cell-enriching cancer spheroid culture. Knockdown of DNER expression inhibited cell proliferation and invasion, induced apoptosis, enhanced chemosensitivity, and decreased spheroid formation of SNU-638 gastric cancer cells. DNER silencing elevated the expression of p53, p21cip/waf, and p27, and increased G1 phase cells at the expense of S phase cells. Knockdown of p21cip/waf expression in the DNER-silenced cells partially restored cell viability and S phase progression. DNER silencing also induced the apoptosis of SNU-638 cells. While both cleaved caspases-8 and 9 were detected in adherent cells, only cleaved caspase-8 was found to have increased in spheroid-cultured cells, suggesting a distinct activation pattern of caspase activation depending on the growth condition. Knockdown of p53 expression rescued the DNER-silenced cells from apoptosis and partially restored cell viability. In contrast, overexpression of the Notch intracellular domain (NICD) decreased the expression of p53, p21cip/waf, and cleaved caspase-3 in DNER-silenced cells. Moreover, NICD expression fully reverted the cell viability reduction, arrest in the G1 phase, and elevated apoptosis caused by DNER silencing, thereby suggesting activation of Notch signaling by DNER. Expression of a membrane-unbound mutant of mDNER also decreased cell viability and induced apoptosis. On the other hand, TGF-β signals were found to be involved in DNER expression in both adherent and spheroid-cultured cells. DNER could therefore be a link connecting TGF-β signaling to Notch signaling. Taken together, DNER regulates cell proliferation, survival, and invasive capacity of the gastric cancer cells through the activation of Notch signaling, which may facilitate tumor progression into an advanced stage. This study provides evidences suggesting that DNER could be a potential prognostic marker, a therapeutic target, and a drug candidate in the form of a cell-free mutant.

Esomeprazole Alleviates Cisplatin Resistance by Inhibiting the AKT/mTOR Pathway in Ovarian Cancer Cells.

Ovarian cancer is the most lethal malignancy in gynecology. Due to limited treatment strategies and platinum resistance, newer drugs and therapeutic options are needed. Esomeprazole (ESO) has been reported to have multiple anticancer activities in preclinical and clinical research. Therefore, this study aimed to explore the anticancer effects of esomeprazole on ovarian cancer and its underlying molecular mechanisms. CCK-8 and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to detect cell viability and proliferation. The Transwell assay was used to evaluate cell migration and invasion capacity. Flow cytometry was used to detect cell apoptosis. Western blotting and immunofluorescence were used to detect protein expression. ESO effectively inhibited the cell viability, proliferation, invasion, migration, and induced apoptosis of ovarian cancer cells in a concentration-dependent manner. Treatment with ESO decreased the expression of c-MYC, SKP2, E2F1, N-cadherin, vimentin, and matrix metalloproteinase 2 (MMP2), while it increased E-cadherin, caspase3, p53, BAX, and cleaved poly (ADP-ribose) polymerase (PARP) expression, and downregulated the PI3K/AKT/mTOR signaling pathway. Furthermore, ESO combined with cisplatin showed synergistic effects in inhibiting proliferation, invasion, and migration of cisplatin-resistant ovarian cancer cells. The mechanism may be related to the increased inhibition of c-MYC, epithelial-mesenchymal transition (EMT), and the AKT/mTOR signaling pathway and enhanced the upregulation of the pro-apoptotic protein BAX and cleaved PARP levels. Moreover, ESO combined with cisplatin synergistically upregulated the expression of the DNA damage marker γH2A.X. ESO exerts multiple anticancer activities and has a synergistic effect in combination with cisplatin on cisplatin-resistant ovarian cancer cells. This study provides a promising strategy to improve chemosensitivity and overcome resistance to cisplatin in ovarian cancer.

Streptococcus agalactiae virulence factors isolated from bovine mastitis and antibiotic treatment response.

This study aims to characterize the virulence factors of Streptococcus agalactiae strains isolated from bovine mastitis and their potential association with the antibiotic treatment response. Four different profiles were identified among the 181 S. agalactiae strains using polymerase chain reaction, of which hyl B and cylE were found in 100% of the isolated strains, followed by cfb (99.5%), scpB (40.3%), and lmb (15.5%). The cell invasion assay showed that lmb-positive strains exhibited higher invasion ability than lmb-negative. There was a significant association (P=0.018) between lmb gene and cows that required more than one treatment cycle. These results showed that intramammary infections caused by lmb-carrier S. agalactiae strains did not adequately respond to the antibiotics administered, likely because of the presence of this virulence factor, which potentially favours the cell invasion capacity of these strains.

LINC01614 Promotes Colorectal Cancer Cell Growth and Migration by Regulating miR-217-5p/HMGA1 Axis.

Colorectal cancer (CRC) substantially contributes to cancer-related deaths worldwide. Recently, a long non-coding RNA (lncRNA), LINC01614, has emerged as a vital gene regulator in cancer progression. Yet, how LINC01614 affects CRC progression remains enigmatic. Here, we defined LINC01614 expression in CRC, investigated the performance of CRC cells lacking LINC01614, and elucidated the underpinning mechanism. We observed that LINC01614 was upregulated in both CRC tissues and cell lines. LINC01614 knockdown repressed the proliferation and metastasis capacity of CRC cell lines. Consistently, an in vivo LINC01614 deficiency model exhibited slow tumor growth rate. Moreover, luciferase reporter assay, RNA pull-down, and immunoprecipitation confirmed that LINC01614 targeted miR-217-5p. LINC01614 knockdown reduced the expression of HMGA1 and N-cadherin, while increasing that of E-cadherin, resulting in decreased viability, proliferation, migration, and invasion capacity of CRC cells. Our results demonstrate that LINC01614 regulates CRC progression by modulating the miR-217-5p/HMGA1 axis, thus holding great potential as a prognostic biomarker for CRC diagnosis and treatment.

RPA3 promotes the proliferation, migration, and invasion of gliomas by activating the PI3K-AKT-mTOR pathway.

Gliomas are the most common primary malignant brain tumors, with a poor prognosis and high mortality, and there is no effective treatment regimen. A number of studies have shown that replication protein A3 (RPA3) can regulate DNA replication and that the abnormal expression of RPA3 can lead to genomic instability and induce the development of a variety of tumors. However, the relationship between RPA3 and gliomas and the mechanism of action remains unclear. In this study, we investigated the role of RPA3 in the development of gliomas and the possible mechanism. The Chinese Glioma Genome Atlas (CGGA), The Cancer Genome Atlas (TCGA), and the Gene Expression Omnibus (GEO) databases were used to analyze the expression level of RPA3 and its correlation with clinical prognosis. A univariate Cox regression model was established to predict the prognosis of glioma patients and analyze the correlation between RPA3 and immune cell infiltration and activation. Immunohistochemistry, RT-PCR, and Western blot (WB) were used to detect the expression of RPA3 in glioma specimens. After knocking down and overexpressing RPA3 with plasmids, effects on glioma cell proliferation, migration and invasive capacity were investigated in vitro. The possible molecular mechanisms were analyzed using WB. Results showed that the expression of RPA3 in glioma tissue and cells was significantly higher than that in normal glial cells and was positively correlated with the poor prognosis of patients with gliomas. The overexpression of RPA3 expression activated the phosphatidylinositol 3-kinase (PI3K)-AKT-mammalian target of the rapamycin (mTOR) pathway by promoting the phosphorylation of PI3K, AKT, and mTOR, thereby promoting the proliferation, migration and invasion of glioma cells. In conclusion, RPA3 is highly expressed in gliomas and promotes the proliferation, migration and invasion of gliomas by activating the PI3K-AKT-mTOR pathway. Therefore, RPA3 may be a prognostic biomarker and therapeutic target for gliomas.

Loss of Caveolin-1 Expression in Tumor Cells is Associated with Increased Aggressiveness and Cell Invasion in Oral Squamous Cell Carcinoma.

Changes in Caveolin-1 (CAV-1) expression are related to tumorigenesis. The aim of this study was to evaluate the role of CAV-1 in tumor progression in oral squamous cell carcinoma (SCC) tissue samples and the effect of CAV-1 silencing on two oral tongue SCC (OTSCC) cell lines (SCC-25, from a primary tumor, and HSC-3 from lymph node metastases). Mycroarray hybridization, mRNA expression, and immunohistochemistry were performed on OSCC tissue samples and corresponding non-tumoral margin tissues. The effects of CAV-1 silencing (siCAV-1) on cell viability, membrane fluidity, on the expression of epithelial to mesenchymal transition (EMT) markers and on cell migration and invasion capacity of OTSCC cell lines were evaluated. Microarray showed a greater CAV-1 expression (1.77-fold) in OSCC tumors than in non-tumoral tissues and 2.0-fold more in less aggressive OSCCs. However, significant differences in CAV-1 gene expression were not seen between tumors and non-tumoral margins nor CAV-1 with any clinicopathological parameters. CAV-1 protein was localized both in carcinoma and in spindle cells of the tumor microenvironment (TME), and CAV-1 positive TME cells were associated with smaller/more aggressive tumors, independent of the carcinoma cells' expression. Silencing of CAV-1 increased cell viability only in SCC-25 cells. It also stimulated the invasion of HSC-3 cells and increased ECAD and BCAT mRNA in these cells; however, the protein levels of the EMT markers were not affected. Decreased expression of CAV-1 by tumor cells in OSCC and an increase in the TME were associated with increased cell invasiveness and tumor aggressiveness.