MFN2-dependent mitochondrial dysfunction contributes to Relm-ß-induced pulmonary arterial hypertension via USP18/Twist1/miR-214 pathway.

Induction of resistin-like molecule ß (Relm-ß) and mitofusin 2 (MFN2) mediated aberrant mitochondrial fission have been found to be involved in the pathogenesis of pulmonary arterial hypertension (PAH). However, the molecular mechanisms underlying Relm-ß regulation of MFN2 therefore mitochondrial fission remain unclear. This study aims to address these issues. Primary cultured PASMCs and monocrotaline (MCT)-induced PAH rats were applied in this study. The results showed that Relm-ß promoted cells proliferation in PASMCs, this was accompanied with the upregulation of USP18, Twist1 and miR-214, and downregulation of MFN2. We found that Relm-ß increased USP18 expression which in turn raised Twist1 by suppressing its proteasome degradation. Elevation of Twist1 increased miR-214 expression and then reduced MFN2 expression and mitochondrial fragmentation leading to PASMCs proliferation. In vivo study, we confirmed that Relm-ß was elevated in MCT-induced PAH rat model, and USP18/Twist1/miR-214/MFN2 axis was altered similar as in vitro. Targeting this cascade by Relm-ß receptor inhibitor Calhex231, proteasome inhibitor MG-132, Twist1 inhibitor Harmine or miR-214 antagomiR prevented the development of pulmonary vascular remodeling and therefore PAH in MCT-treated rats. In conclusion, we demonstrate that Relm-ß promotes PASMCs proliferation and vascular remodeling by activating USP18/Twist1/miR-214 dependent MFN2 reduction and mitochondrial fission, suggesting that this signaling pathway might be a promising target for management of PAH.

NSC-38270 Exhibits Anti-invasive and Pro-apoptotic Effects on Hepatocellular Carcinoma Huh7 Cells.

BACKGROUND/AIM: Hepatocellular carcinoma (HCC) is a main type of liver cancer with high metastatic potential, and its incidence is steadily increasing worldwide. However, the development of new drugs for the treatment of HCC is still insufficient. This study aimed to determine the anticancer effect of NSC-38270, a natural product, on HCC. MATERIALS AND METHODS: After treating HCC Huh7 cells with NSC-38270, cell growth, wound healing, migration, and invasion assays were conducted. We investigated the effects of NSC-38270 on Twist1, a crucial epithelial-mesenchymal transition (EMT)-related transcription factor. In addition, apoptosis, histone H2A.X activation, and cell morphology assays were performed in Huh7 and immortalized normal liver cells following treatment with NSC-38270. RESULTS: NSC-38270 reduced the migration and invasion ability of Huh7 cells, accompanied by a decrease in Twist1. Furthermore, NSC-38270 induced apoptosis in Huh7 cells, whereas apoptosis was not observed in immortalized normal liver cells (THLE-2 cells and Chang liver cells). CONCLUSION: NSC-38270 exhibited significant inhibitory effects on the migration and invasion of Huh7 cells by repressing Twist1. Importantly, it induced cancer cell-specific apoptotic effects. These findings suggest that NSC-38270 holds promising potential as a therapeutic candidate for cancer treatment.

Huaier inhibits cholangiocarcinoma cells through the twist1/FBP1/Wnt/ß-catenin axis.

BACKGROUND: Although Huaier granules can be used as prospective anti-cholangiocarcinoma drugs, the mechanism of action of Huaier granules in cholangiocarcinoma is not clear. The anti-cholangiocarcinoma effect of Huaier granules was validated in cell line research. In vitro experiments were conducted to investigate the signalling pathways affected by Huaier in CCA cells. METHODS AND RESULTS: Real-time quantitative PCR (RT‒qPCR) and Western blot analysis were performed to analyse gene expression in CCA cells. MTT assays, scratch tests, and Transwell assays were used to explore the effects on the proliferation and metastasis of CCA cells. Chromatin immunoprecipitation assays were performed to reveal the potential underlying mechanisms involved. Twist1 was upregulated in human CCA tissues. In addition, its expression levels were negatively related to FBP1 expression levels. Mechanistically, Twist1 can bind to the region of the FBP1 promoter to reduce its expression. Huaier plays an indispensable role in suppressing Twist1 expression to inhibit the Twist1/FBP1/Wnt/ß-catenin axis. Then, we verified the effect of Huaier in vitro. CONCLUSIONS: These findings suggested that Huaier granules were capable of inhibiting CCA development through regulating the Twist1/FBP1/Wnt/ß-catenin signalling axis and provided a novel orientation for the development of novel anti-CCA drugs.

Intercellular interaction between FAP+ fibroblasts and CD150+ inflammatory monocytes mediates fibrostenosis in Crohn's disease.

Crohn's disease (CD) is marked by recurring intestinal inflammation and tissue injury, often resulting in fibrostenosis and bowel obstruction, necessitating surgical intervention with high recurrence rates. To elucidate the mechanisms underlying fibrostenosis in CD, we analyzed the transcriptome of cells isolated from the transmural ileum of patients with CD, including a trio of lesions from each patient: non-affected, inflamed, and stenotic ileum samples, and compared them with samples from patients without CD. Our computational analysis revealed that profibrotic signals from a subset of monocyte-derived cells expressing CD150 induced a disease-specific fibroblast population, resulting in chronic inflammation and tissue fibrosis. The transcription factor TWIST1 was identified as a key modulator of fibroblast activation and extracellular matrix (ECM) deposition. Genetic and pharmacological inhibition of TWIST1 prevents fibroblast activation, reducing ECM production and collagen deposition. Our findings suggest that the myeloid-stromal axis may offer a promising therapeutic target to prevent fibrostenosis in CD.

Craniofacial studies in chicken embryos confirm the pathogenicity of human FZD2 variants associated with Robinow syndrome.

Robinow syndrome is a rare disease caused by variants of seven WNT pathway genes. Craniofacial features include widening of the nasal bridge and jaw hypoplasia. We used the chicken embryo to test whether two missense human FZD2 variants (1301G>T, p.Gly434Val; 425C>T, p.Pro142Lys) were sufficient to change frontonasal mass development. In vivo, the overexpression of retroviruses with wild-type or variant human FZD2 inhibited upper beak ossification. In primary cultures, wild-type and variant human FZD2 significantly inhibited chondrogenesis, with the 425C>T variant significantly decreasing activity of a SOX9 luciferase reporter compared to that for the wild type or 1301G>T. Both variants also increased nuclear shuttling of ß-catenin (CTNNB1) and increased the expression of TWIST1, which are inhibitory to chondrogenesis. In canonical WNT luciferase assays using frontonasal mass cells, the variants had dominant-negative effects on wild-type FZD2. In non-canonical assays, the 425C>T variant failed to activate the reporter above control levels and was unresponsive to exogenous WNT5A. This is the first single amino acid change to selectively alter ligand binding in a FZD receptor. Therefore, FZD2 missense variants are pathogenic and could lead to the altered craniofacial morphogenesis seen in Robinow syndrome.

Inhibition of FBP1 expression by KMT5A through TWIST1 methylation is one of the mechanisms leading to chemoresistance in breast cancer.

Lysine methyltransferase 5A (KMT5A) is the sole mammalian enzyme known to catalyse the mono­methylation of histone H4 lysine 20 and non­histone proteins such as p53, which are involved in the occurrence and progression of numerous cancers. The present study aimed to determine the function of KMT5A in inducing docetaxel (DTX) resistance in patients with breast carcinoma by evaluating glucose metabolism and the underlying mechanism involved. The upregulation or downregulation of KMT5A­related proteins was examined after KMT5A knockdown in breast cancer (BRCA) cells by Tandem Mass Tag proteomics. Through differential protein expression and pathway enrichment analysis, the upregulated key gluconeogenic enzyme fructose­1,6­bisphosphatase 1 (FBP1) was discovered. Loss of FBP1 expression is closely related to the development and prognosis of cancers. A dual­luciferase reporter gene assay confirmed that KMT5A inhibited the expression of FBP1 and that overexpression of FBP1 could enhance the chemotherapeutic sensitivity to DTX through the suppression of KMT5A expression. The KMT5A inhibitor UNC0379 was used to verify that DTX resistance induced by KMT5A through the inhibition of FBP1 depended on the methylase activity of KMT5A. According to previous literature and interaction network structure, it was revealed that KMT5A acts on the transcription factor twist family BHLH transcription factor 1 (TWIST1). Then, it was verified that TWSIT1 promoted the expression of FBP1 by using a dual­luciferase reporter gene experiment. KMT5A induces chemotherapy resistance in BRCA cells by promoting cell proliferation and glycolysis. After the knockdown of the KMT5A gene, the FBP1 related to glucose metabolism in BRCA was upregulated. KMT5A knockdown expression and FBP1 overexpression synergistically inhibit cell proliferation and block cells in the G2/M phase. KMT5A inhibits the expression of FBP1 by methylating TWIST1 and weakening its promotion of FBP1 transcription. In conclusion, KMT5A was shown to affect chemotherapy resistance by regulating the cell cycle and positively regulate glycolysis­mediated chemotherapy resistance by inhibiting the transcription of FBP1 in collaboration with TWIST1. KMT5A may be a potential therapeutic target for chemotherapy resistance in BRCA.

Adipose-derived stem cells modified by TWIST1 silencing accelerates rat sciatic nerve repair and functional recovery.

The regeneration of peripheral nerves after injury is often slow and impaired, which may be associated with weakened and denervated muscles subsequently leading to atrophy. Adipose-derived stem cells (ADSCs) are often regarded as cell-based therapeutic candidate due to their regenerative potential. The study aims to assess the therapeutic efficacy of gene-modified ADSCs on sciatic nerve injury. We lentivirally transduced ADSCs with shRNA-TWIST1 and transplanted modified cells to rats undergoing sciatic nerve transection and repair. Results showed that TWIST1 knockdown accelerated functional recovery of rats with sciatic nerve injury as faster nerve conduction velocity and higher wire hang scores obtained by rats transplanted with TWIST1-silenced ADSCs than scramble ADSCs. Although the rats experienced degenerated axons and decreased myelin sheath thickness after sciatic nerve injury 8 weeks after operation, those transplanted with TWIST1-silenced ADSCs exhibited more signs of regenerated nerve fibers surrounded by newly formed myelin sheaths than those with scramble ADSCs. The rats transplanted with TWIST1-silenced ADSCs presented increased expressions of neurotrophic factors including neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and glial cell line-derived neurotrophic factor (GDNF) in the sciatic nerves than those with scramble ADSCs. These results suggest that genetically modifying TWIST1 in ADSCs could facilitate peripheral nerve repair after injury in a more efficient way than that with ADSCs alone.

Is epithelial-mesenchymal transition related to the biological behavior of salivary gland neoplasms?

OBJECTIVE: To evaluate and compare the expression of E-cadherin, Snail1 and Twist1 in pleomorphic adenomas (PAs), adenoid cystic carcinomas (AdCCa) and carcinoma ex-pleomorphic adenomas (CaexPA) of salivary glands, as well as investigate possible associations with clinicopathological parameters. STUDY DESIGN: E-cadherin, Snail1 and Twist1 antibody immunostaining were analyzed semiquantitatively in 20 PAs, 20 AdCCas and 10 CaexPAs. Cases were classified as low and high expression for analysis of the association with clinicopathological parameters. RESULTS: Compared to PAs, AdCCas and CaexPAs exhibited higher nuclear expression of Snail1 (p = 0.021 and p = 0.028, respectively) and Twist1 (p = 0.009 and p = 0.001). Membranous and cytoplasmic expression of E-cadherin were positively correlated in PAs, AdCCas and CaexPAs (r = 0.645, p = 0.002; r = 0.824, p < 0.001; r = 0.677, p = 0.031). In PAs, positive correlation was found between nuclear expression of Snail1 and membrane expression of E-cadherin (r = 0.634; p = 0.003), as well as between nuclear expression of Snail1 and Twist1 (r = 0.580; p = 0.007). Negative correlations were detected between membrane expression of E-cadherin and cytoplasmic expression of Snail1 in AdCCas (r = - 0.489; p = 0.029). CONCLUSIONS: E-cadherin, Twist1, and Snail1 may participate in modulating events related to cell differentiation and adhesion in PAs and to biological behavior in AdCCas and CaexPAs, which indicates the involvement of EMT in these processes. Furthermore, the expression of these proteins in these carcinomas may reflect the plasticity feature of EMT.

Targeting TGFß receptor-mediated snail and twist: WSG, a polysaccharide from Ganoderma lucidum, and it-based dissolvable microneedle patch suppress melanoma cells.

Cutaneous melanoma is a lethal skin cancer variant with pronounced aggressiveness and metastatic potential. However, few targeted medications inhibit the progression of melanoma. Ganoderma lucidum, which is a type of mushroom, is widely used as a non-toxic alternative adjunct therapy for cancer patients. This study determines the effect of WSG, which is a water-soluble glucan that is derived from G. lucidum, on melanoma cells. The results show that WSG inhibits cell viability and the mobility of melanoma cells. WSG induces changes in the expression of epithelial-to-mesenchymal transition (EMT)-related markers. WSG also downregulates EMT-related transcription factors, Snail and Twist. Signal transduction assays show that WSG reduces the protein levels in transforming growth factor ß receptors (TGFßRs) and consequently inhibits the phosphorylation of intracellular signaling molecules, such as FAK, ERK1/2 and Smad2. An In vivo study shows that WSG suppresses melanoma growth in B16F10-bearing mice. To enhance transdermal drug delivery and prevent oxidation, two highly biocompatible compounds, polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP), are used to synthesize a dissolvable microneedle patch that is loaded with WSG (MN-WSG). A functional assay shows that MN-WSG has an effect that is comparable to that of WSG alone. These results show that WSG has significant potential as a therapeutic agent for melanoma treatment. MN-WSG may allow groundbreaking therapeutic approaches and offers a novel method for delivering this potent compound effectively.

Epithelial-mesenchymal transition associated markers in sarcomatoid transformation of clear cell renal cell carcinoma.

Epithelial-mesenchymal transition (EMT) plays a pivotal role in the development and progression of many cancers. Partial EMT (pEMT) could represent a critical step in tumor migration and dissemination. Sarcomatoid renal cell carcinoma (sRCC) is an aggressive form of renal cell carcinoma (RCC) composed of a carcinomatous (sRCC-Ca) and sarcomatous (sRCC-Sa) component. The role of (p)EMT in the progression of RCC to sRCC remains unclear. The aim of this study was to investigate the involvement of (p)EMT in RCC and sRCC. Tissue samples from 10 patients with clear cell RCC (ccRCC) and 10 patients with sRCC were selected. The expression of main EMT markers (miR-200 family, miR-205, SNAI1/2, TWIST1/2, ZEB1/2, CDH1/2, VIM) was analyzed by qPCR in ccRCC, sRCC-Ca, and sRCC-Sa and compared to non-neoplastic tissue and between both groups. Expression of E-cadherin, N-cadherin, vimentin and ZEB2 was analyzed using immunohistochemistry. miR-200c was downregulated in sRCC-Ca compared to ccRCC, while miR-200a was downregulated in sRCC-Sa compared to ccRCC. CDH1 was downregulated in sRCC-Sa when compared to any other group. ZEB2 was downregulated in ccRCC and sRCC compared to corresponding non-neoplastic kidney. A positive correlation was observed between CDH1 expression and miR-200a/b/c. Our results suggest that full EMT is not present in sRCC. Instead, discreet molecular differences exist between ccRCC, sRCC-Ca, and sRCC-Sa, possibly representing distinct intermediary states undergoing pEMT.

Mechanical shear flow regulates the malignancy of colorectal cancer cells.

Colorectal cancer (CRC) is notable for its high mortality and high metastatic characteristics. The shear force generated by bloodstream provides mechanical signals regulating multiple responses of cells, including metastatic cancer cells, dispersing in blood vessels. We, therefore, studied the effect of shear flow on circulating CRC cells in the present study. The CRC cell line SW620 was subjected to shear flow of 12.5 dynes/cm2 for 1 and 2 h separately. Resulting elevated caspase-9 and -3 indicated that shear flow initiated the apoptosis of SW620. Enlarged cell size associated with a higher level of cyclin D1 was coincident with the flow cytometric results indicating that the cell cycle was arrested at the G1 phase. An elevated phosphor-eNOSS1177 increased the production of nitric oxide and led to reactive oxygen species-mediated oxidative stress. Shear flow also regulated epithelial-mesenchymal transition (EMT) by increasing E-cadherin and ZO-1 while decreasing Snail and Twist1. The migration and invasion of sheared SW620 were also substantially decreased. Further investigations showed that mitochondrial membrane potential was significantly decreased, whereas mitochondrial mass and ATP production were not changed. In addition to the shear flow of 12.5 dynes/cm2, the expressions of EMT were compared at lower (6.25 dynes/cm2) and at higher (25 dynes/cm2) shear flow. The results showed that lower shear flow increased mesenchymal characteristics and higher shear flow increased epithelial characteristics. Shear flow reduces the malignancy of CRC in their metastatic dispersal that opens up new ways to improve cancer therapies by applying a mechanical shear flow device.

Twist2 contributes to skin regeneration and hair follicle formation in mouse fetuses.

Unlike adult mammalian wounds, early embryonic mouse skin wounds completely regenerate and heal without scars. Analysis of the underlying molecular mechanism will provide insights into scarless wound healing. Twist2 is an important regulator of hair follicle formation and biological patterning; however, it is unclear whether it plays a role in skin or skin appendage regeneration. Here, we aimed to elucidate Twist2 expression and its role in fetal wound healing. ICR mouse fetuses were surgically wounded on embryonic day 13 (E13), E15, and E17, and Twist2 expression in tissue samples from these fetuses was evaluated via in situ hybridization, immunohistochemistry, and reverse transcription-quantitative polymerase chain reaction. Twist2 expression was upregulated in the dermis of E13 wound margins but downregulated in E15 and E17 wounds. Twist2 knockdown on E13 left visible marks at the wound site, inhibited regeneration, and resulted in defective follicle formation. Twist2-knockdown dermal fibroblasts lacked the ability to undifferentiate. Furthermore, Twist2 hetero knockout mice (Twist + /-) formed visible scars, even on E13, when all skin structures should regenerate. Thus, Twist2 expression correlated with skin texture formation and hair follicle defects in late mouse embryos. These findings may help develop a therapeutic strategy to reduce scarring and promote hair follicle regeneration.

DNA-PKcs/AKT1 inhibits epithelial-mesenchymal transition during radiation-induced pulmonary fibrosis by inducing ubiquitination and degradation of Twist1.

INTRODUCTION: Radiation-induced pulmonary fibrosis (RIPF) is a chronic, progressive, irreversible lung interstitial disease that develops after radiotherapy. Although several previous studies have focused on the mechanism of epithelial-mesenchymal transition (EMT) in lung epithelial cells, the essential factors involved in this process remain poorly understood. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) exhibits strong repair capacity when cells undergo radiation-induced damage; whether DNA-PKcs regulates EMT during RIPF remains unclear. OBJECTIVES: To investigate the role and molecular mechanism of DNA-PKcs in RIPF and provide an important theoretical basis for utilising DNA-PKcs-targeted drugs for preventing RIPF. METHODS: DNA-PKcs knockout (DPK-/-) mice were generated via the Cas9/sgRNA technique and subjected to whole chest ionizing radiation (IR) at a 20 Gy dose. Before whole chest IR, the mice were intragastrically administered the DNA-PKcs-targeted drug VND3207. Lung tissues were collected at 1 and 5 months after IR. RESULTS: The expression of DNA-PKcs is low in pulmonary fibrosis (PF) patients. DNA-PKcs deficiency significantly exacerbated RIPF by promoting EMT in lung epithelial cells. Mechanistically, DNA-PKcs deletion by shRNA or inhibitor NU7441 maintained the protein stability of Twist1. Furthermore, AKT1 mediated the interaction between DNA-PKcs and Twist1. High Twist1 expression and EMT-associated changes caused by DNA-PKcs deletion were blocked by insulin-like growth factor-1 (IGF-1), an AKT1 agonist. The radioprotective drug VND3207 prevented IR-induced EMT and alleviated RIPF in mice by stimulating the kinase activity of DNA-PKcs. CONCLUSION: Our study clarified the critical role and mechanism of DNA-PKcs in RIPF and showed that it could be a potential target for preventing RIPF.

Regulation of cellular and molecular markers of epithelial-mesenchymal transition by Brazilin in breast cancer cells.

Breast cancer is the most common invasive neoplasm and the leading cause of cancer death in women worldwide. The main cause of mortality in cancer patients is invasion and metastasis, where the epithelial-mesenchymal transition (EMT) is a crucial player in these processes. Pharmacological therapy has plants as its primary source, including isoflavonoids. Brazilin is an isoflavonoid isolated from Haematoxilum brasiletto that has shown antiproliferative activity in several cancer cell lines. In this study, we evaluated the effect of Brazilin on canonical markers of EMT such as E-cadherin, vimentin, Twist, and matrix metalloproteases (MMPs). By Western blot, we evaluated E-cadherin, vimentin, and Twist expression and the subcellular localization by immunofluorescence. Using gelatin zymography, we determined the levels of secretion of MMPs. We used Transwell chambers coated with matrigel to determine the in vitro invasion of breast cancer cells treated with Brazilin. Interestingly, our results show that Brazilin increases 50% in E-cadherin expression and decreases 50% in vimentin and Twist expression, MMPs, and cell invasion in triple-negative breast cancer (TNBC) MDA-MB-231 and to a lesser extend in MCF7 ER+ breast cancer cells. Together, these findings position Brazilin as a new molecule with great potential for use as complementary or alternative treatment in breast cancer therapy in the future.

Exosomal miRNA-92a derived from cancer-associated fibroblasts promote invasion and metastasis in breast cancer by regulating G3BP2.

Cancer-associated Fibroblasts (CAFs) exert a tumor-promoting effect in various cancers, including breast cancer. CAFs secrete exosomes containing miRNA and proteins, influencing the tumor microenvironment. In this study, we identified CAF-derived exosomes that transport functional miR-92a from CAFs to tumor cells, thereby intensifying the aggressiveness of breast cancer. CAFs downregulate the expression of G3BP2 in breast cancer cells, and a significant elevation in miR-92a levels in CAF-derived exosomes was observed. Both in vitro and in vivo experiments demonstrate that miR-92a enhances breast cancer cell migration and invasion by directly targeting G3BP2, functioning as a tumor-promoting miRNA. We validated that the RNA-binding proteins SNRPA facilitate the transfer of CAF-derived exosomal miR-92a to breast cancer cells. The reduction of G3BP2 protein by CAF-derived exosomes releases TWIST1 into the nucleus, promoting epithelial-mesenchymal transition (EMT) and further exacerbating breast cancer progression. Moreover, CAF-derived exosomal miR-92a induces tumor invasion and metastasis in mice. Overall, our study reveals that CAF-derived exosomal miR-92a serves as a promoter in the migration and invasion of breast cancer cells by reducing G3BP2 and may represent a potential novel tumor marker for breast cancer.

CD34+ synovial fibroblasts exhibit high osteogenic potential in synovial chondromatosis.

Synovial chondromatosis (SC) is a disorder of the synovium characterized by the formation of osteochondral nodules within the synovium. This study aimed to identify the abnormally differentiated progenitor cells and possible pathogenic signaling pathways. Loose bodies and synovium were obtained from patients with SC during knee arthroplasty. Single-cell RNA sequencing was used to identify cell subsets and their gene signatures in SC synovium. Cells derived from osteoarthritis (OA) synovium were used as controls. Multi-differentiation and colony-forming assays were used to identify progenitor cells. The roles of transcription factors and signaling pathways were investigated through computational analysis and experimental verification. We identified an increased proportion of CD34+ sublining fibroblasts in SC synovium. CD34+CD31- cells and CD34-CD31- cells were sorted from SC synovium. Compared with CD34- cells, CD34+ cells had larger alkaline phosphatase (ALP)-stained area and calcified area after osteogenic induction. In addition, CD34+ cells exhibited a stronger tube formation ability than CD34- cells. Our bioinformatic analysis suggested the expression of TWIST1, a negative regulator of osteogenesis, in CD34- sublining fibroblasts and was regulated by the TGF-ß signaling pathway. The experiment showed that CD34+ cells acquired the TWIST1 expression during culture and the combination of TGF-ß1 and harmine, an inhibitor of Twist1, could further stimulate the osteogenesis of CD34+ cells. Overall, CD34+ synovial fibroblasts in SC synovium have multiple differentiation potentials, especially osteogenic differentiation potential, and might be responsible for the pathogenesis of SC.

Clinical performance and utility of a noninvasive urine-based methylation biomarker: TWIST1/Vimentin to detect urothelial carcinoma of the bladder.

Traditional clinical modalities for diagnosing bladder urothelial carcinoma (BUC) remain limited due to their invasive nature, significant costs, discomfort associated with cystoscopy, and low sensitivity to urine cytology. Therefore, there is an urgent need to identify highly sensitive, specific, and noninvasive biomarkers for the early detection of this neoplasm. Hypermethylated TWIST1/Vimentin promoter may be a noninvasive biomarker using urine sample. We assessed the TWIST1/Vimentin promoter methylation status in urine samples using the Methylated Human TWIST1 and Vimentin Gene Detection Kit (Jiangsu MicroDiag Biomedicine Co., Ltd., China). The samples were collected from five groups: group 1 consisted of patients with BUC, group 2 contained other patients with urologic tumors, group 3 consisted of patients with benign diseases (e.g., urinary tract infections, lithiasis, and benign prostatic hyperplasia), Group 4 included UTUC (upper tract urothelial carcinoma) patients and group5 comprised healthy individuals. The study encompassed 77 BUC patients, and we evaluated the degree of methylation of the TWIST1/Vimentin gene in their urine samples. Notably, TWIST1/Vimentin positivity was significantly elevated in comparison to groups 2, 3 and 5 (all p < 0.001) at a rate of 77.9%, but no significant difference was observed when compared to group 4. In the relationship between TWIST1/Vimentin methylation and clinicopathological features of BC patients from our center, we found there was no significant association between TWIST1/Vimentin status and proteinuria and/or hematuria, and hypermethylation of TWIST1 / VIM genes was found in both high and low tumor grade and in both non-muscle invasive bladder cancer (stages Tis, Ta, or T1) and muscle-invasive bladder cancer (stage T2 or above). In the multivariable analysis for cancer detection, a positive TWIST1/Vimentin methylation were significantly linked to a heightened risk of BC. Moreover, TWIST1/Vimentin promoter methylation demonstrated an ability to detect BUC in urine samples with a sensitivity of 78% and a specificity of 83%. Our findings reveal that hypermethylation of the TWIST1/Vimentin promoter occurs in bladder urothelial carcinoma, and its high sensitivity and specificity suggest its potential as a screening and therapeutic biomarker for urothelial carcinoma of the bladder.

The possible correlation between miR-762, Hippo signaling pathway, TWIST1, and SMAD3 in lung cancer and chronic inflammatory diseases.

MicroRNAs are small RNA molecules that have a significant role in translational repression and gene silencing through binding to downstream target mRNAs. MiR-762 can stimulate the proliferation and metastasis of various types of cancer. Hippo pathway is one of the pathways that regulate tissue development and carcinogenesis. Dysregulation of this pathway plays a vital role in the progression of cancer. This study aimed to evaluate the possible correlation between miR-762, the Hippo signaling pathway, TWIST1, and SMAD3 in patients with lung cancer, as well as patients with chronic inflammatory diseases. The relative expression of miR-762, MST1, LATS2, YAP, TWIST1, and SMAD3 was determined in 50 lung cancer patients, 30 patients with chronic inflammatory diseases, and 20 healthy volunteers by real-time PCR. The levels of YAP protein and neuron-specific enolase were estimated by ELISA and electrochemiluminescence immunoassay, respectively. Compared to the control group, miR-762, YAP, TWIST1, and SMAD3 expression were significantly upregulated in lung cancer patients and chronic inflammatory patients, except SMAD3 was significantly downregulated in chronic inflammatory patients. MST1, LATS2, and YAP protein were significantly downregulated in all patients. MiR-762 has a significant negative correlation with MST1, LATS2, and YAP protein in lung cancer patients and with MST1 and LATS2 in chronic inflammatory patients. MiR-762 may be involved in the induction of malignant behaviors in lung cancer through suppression of the Hippo pathway. MiR-762, MST1, LATS2, YAP mRNA and protein, TWIST1, and SMAD3 may be effective diagnostic biomarkers in both lung cancer patients and chronic inflammatory patients. High YAP, TWIST1, SMA3 expression, and NSE level are associated with a favorable prognosis for lung cancer.

Luteolin Inhibits Lung Cancer Cell Migration by Negatively Regulating TWIST1 and MMP2 Through Upregulation of miR-106a-5p.

BACKGROUND: Luteolin, a common dietary flavonoid found in plants, has been shown to have anti-cancer properties. However, its exact mechanisms of action in non-small cell lung cancer (NSCLC) are still not fully understood, particularly its role in regulating broader genomic networks and specific gene targets. In this study, we aimed to elucidate the role of microRNAs (miRNAs) in NSCLC treated with luteolin, using A549 cells as a model system. MATERIALS AND METHODS: miRNA profiling was conducted on luteolin-treated A549 cells using Exiqon microarrays, with validation of selected miRNAs by qRT-PCR. Bioinformatic analysis identified the regulatory roles of miRNAs in biological processes and pathways following luteolin treatment. Computational algorithms were employed to identify potential target genes. A549 cells were transfected with miR-106a-5p mimic and inhibitor or their corresponding controls. The expression levels of 2 genes, twist basic helix-loop-helix transcription factor 1 (TWIST1) and matrix metallopeptidase 2 (MMP2), and cell migration were assessed. RESULTS: miRNA profiling identified 341 miRNAs, with 18 exhibiting significantly altered expression (P < 0.05). Subsequent qRT-PCR analysis confirmed altered expression of 6 selected miRNAs. KEGG and GO analyses revealed significant alterations in pathways and biological processes crucial for tumor biology. TWIST1 and MMP2, which both contain conserved miR-106a-5p binding sites, exhibited an inverse correlation with the expression levels of miR-106a-5p. Dual-luciferase reporter assays confirmed TWIST1 and MMP2 as direct targets of miR-106a-5p. Luteolin treatment led to a reduction in A549 cell migration, and this reduction was further amplified by the overexpression of miR-106a-5p. CONCLUSION: Luteolin inhibits A549 cell migration by modulating the miRNA landscape, shedding light on its mechanisms and laying the foundation for miRNA-based therapeutic approaches for NSCLC.

Combined DNA Methylation and Gastric Microbiome Marker Predicts Helicobacter pylori-Negative Gastric Cancer.

Background/Aims: While DNA methylation and gastric microbiome are each associated with gastric cancer (GC), their combined role in predicting GC remains unclear. This study investigated the potential of a combined DNA methylation and gastric microbiome signature to predict Helicobacter pylori-negative GC. Methods: In this case-control study, we conducted quantitative methylation-specific polymerase chain reaction to measure the methylation levels of DKK3, SFRP1, EMX1, NKX6-1, MIR124-3, and TWIST1 in the gastric mucosa from 75 H. pylori-negative patients, including chronic gastritis (CG), intestinal metaplasia (IM), and GC. A combined analysis of DNA methylation and gastric microbiome, using 16S rRNA gene sequencing, was performed in 30 of 75 patients. Results: The methylation levels of DKK3, SFRP1, EMX1, MIR124-3, and TWIST1 were significantly higher in patients with GC than in controls (all q<0.05). MIR124-3 and TWIST1 methylation levels were higher in patients with IM than those with CG and also in those with GC than in those with IM (all q<0.05). A higher methylation level of TWIST1 was an independent predictor for H. pylori-negative GC after adjusting for age, sex, and atrophy (odds ratio [OR], 15.15; 95% confidence interval [CI], 1.58 to 145.46; p=0.018). The combination of TWIST1 methylation and GC microbiome index (a microbiome marker) was significantly associated with H. pylori-negative GC after adjusting for age, sex, and atrophy (OR, 50.00; 95% CI, 1.69 to 1,476; p=0.024). Conclusions: The combination of TWIST1 methylation and GC microbiome index may offer potential as a biomarker for predicting H. pylori-negative GC.