NPFs-mediated actin cytoskeleton: a new viewpoint on autophagy regulation.

Macroautophagy/autophagy is a lysosome-dependent catabolic process induced by various cellular stress conditions, maintaining the homeostasis of cells, tissues and organs. Autophagy is a series of membrane-related events involving multiple autophagy-related (ATG) proteins. Most studies to date have focused on various signaling pathways affecting ATG proteins to control autophagy. However, mounting evidence reveals that the actin cytoskeleton acts on autophagy-associated membranes to regulate different events of autophagy. The actin cytoskeleton assists in vesicle formation and provides the mechanical forces for cellular activities that involve membrane deformation. Although the interaction between the actin cytoskeleton and membrane makes the role of actin in autophagy recognized, how the actin cytoskeleton is recruited and assembles on membranes during autophagy needs to be detailed. Nucleation-promoting factors (NPFs) activate the Arp2/3 complex to produce actin cytoskeleton. In this review, we summarize the important roles of the actin cytoskeleton in autophagy regulation and focus on the effect of NPFs on actin cytoskeleton assembly during autophagy, providing new insights into the occurrence and regulatory mechanisms of autophagy. Video Abstract.

Independent prognostic value of CLDN6 in bladder cancer based on M2 macrophages related signature.

M2 macrophages are associated with the prognosis of bladder cancer. CLDN6 has been linked to immune infiltration and is crucial for predicting the prognosis in multi-tumor. The effect of CLDN6 on M2 macrophages in bladder cancer remains elusive. Here, we compared a total of 40 machine learning algorithms, then selected optimal algorithm to develop M2 macrophages-related signature (MMRS) based on the identified M2 macrophages related module. MMRS predicted the prognosis better than other models and associated to immunotherapy response. CLDN6, as an important variable in MMRS, was an independent factor for poor prognosis. We found that CLDN6 was highly expressed and affected immune infiltration, immunotherapy response, and M2 macrophages polarization. Meanwhile, CLDN6 promoted the growth of bladder cancer and enhanced the carcinogenic effect by inducing polarization of M2 macrophages. In total, CLDN6 is an independent risk factor in MMRS to predict the prognosis of bladder cancer.

Claudins and hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a high incidence and dismal prognosis, making it a significant global health burden. To change this, the development of new therapeutic strategies is imminent. The claudin (CLDN) family, as key components of tight junctions (TJs), plays an important role in the initiation and development of cancer. Dysregulated expression of CLDNs leads to loss of intercellular adhesion and aberrant cell signaling, which are closely related to cancer cell invasion, migration, and epithelial-mesenchymal transition (EMT). CLDN1, CLDN3, CLDN4, CLDN5, CLDN6, CLDN7, CLDN9, CLDN10, CLDN11, CLDN14, and CLDN17 are aberrantly expressed in HCC, which drives the progression of the disease. Consequently, they have tremendous potential as prognostic indicators and therapeutic targets. This article summarizes the aberrant expression, molecular mechanisms, and clinical application studies of different subtypes of CLDNs in HCC, with a particular emphasis on CLDN1.

New insights into the regulation of METTL3 and its role in tumors.

As one of the most abundant epigenetic modifications in RNA, N6-methyladenosine (m6A) affects RNA transcription, splicing, stability, and posttranscriptional translation. Methyltransferase-like 3 (METTL3), a key component of the m6A methyltransferase complex, dynamically regulates target genes expression through m6A modification. METTL3 has been found to play a critical role in tumorigenesis, tumor growth, metastasis, metabolic reprogramming, immune cell infiltration, and tumor drug resistance. As a result, the development of targeted drugs against METTL3 is becoming increasingly popular. This review systematically summarizes the factors that regulate METTL3 expression and explores the specific mechanisms by which METTL3 affects multiple tumor biological behaviors. We aim to provide fundamental support for tumor diagnosis and treatment, at the same time, to offer new ideas for the development of tumor-targeting drugs.

CLDN6 inhibits colorectal cancer proliferation dependent on restraining p53 ubiquitination via ZO-1/PTEN axis.

Colorectal cancer (CRC) is one of the most common cancers in the world. Abnormal proliferation is a chief characteristic of cancer and is the initiation of CRC progression. As an important component of tight junctions, CLDN6 regulates the proliferation of multiple tumors. Our previous study showed that CLDN6 was low expressed in CRC, and CLDN6 overexpression inhibited CRC proliferation. However, the specific mechanism of how CLDN6 works remains unclear. This research aimed to reveal the relationship between CLDN6 and clinical features, as well as the molecular mechanism by which CLDN6 inhibited CRC proliferation. We found that low expression of CLDN6 was associated with pathological grade and prognosis of CRC patients, and confirmed that CLDN6 inhibited CRC proliferation dependent on p53. Mechanically, we elucidated that CLDN6 regulated ubiquitination to enhance p53 stability and nuclear import by PTEN/AKT/MDM2 pathway. Through the PDZ-binding motif (PBM), CLDN6 bound to ZO-1 to interact with PTEN, and regulate AKT/MDM2 pathway. Collectively, our data enriched the theoretical basis for CLDN6 as a potential biomarker for diagnosis, therapy and prognosis of CRC.

CLDN6 inhibits breast cancer metastasis through WIP-dependent actin cytoskeleton-mediated autophagy.

BACKGROUND: As a breast cancer suppressor gene, CLDN6 overexpression was found to inhibit breast cancer metastasis in our previous studies, but the specific mechanism remains unclear. This study aimed to clarify the role and mechanism of CLDN6 in inhibiting breast cancer metastasis. METHODS: Western blot, immunofluorescence and transmission electron microscopy were performed to detect autophagy. Wound healing, transwell assays and lung metastasis mouse models were used to examine breast cancer metastasis. Phalloidin staining and immunofluorescent staining were used to observe actin cytoskeleton. mRNA seq, RT-PCR, western blot, chromatin immunoprecipitation, dual luciferase reporter assay, co-immunoprecipitation and immunofluorescence were performed to define the molecular mechanism. The expression levels and clinical implication of CLDN6, WIP and LC3 in breast cancer tissues were evaluated using immunohistochemistry. RESULTS: We demonstrated that CLDN6 inhibited breast cancer metastasis through autophagy in vitro and vivo. We unraveled a novel mechanism that CLDN6 regulated autophagy via WIP-dependent actin cytoskeleton assembly. Through its PDZ-binding motif, overexpressed CLDN6 interacted with JNK and upregulated JNK/c-Jun pathway. C-Jun promoted WIP expression at the transcriptional level. Notably, we observed c-Jun transcriptionally upregulated CLDN6 expression, and there was a positive feedback loop between CLDN6 and JNK/c-Jun. Finally, we found that CLDN6, WIP and LC3 expression correlated with each other, and WIP expression was significantly associated with lymph node metastasis of breast cancer patients. CONCLUSIONS: The data provide a new insight into the inhibitory effects of CLDN6-mediated autophagy on breast cancer metastasis, and revealed the new mechanism of CLDN6 regulating autophagy through WIP-dependent actin cytoskeleton. Our findings enrich the theoretical basis for CLDN6 as a potential biomarker for breast cancer diagnosis and therapy.

The expression and the tumor suppressor role of CLDN6 in colon cancer.

As a member of the tight junction family, CLDN6 is a tumor suppressor in breast cancer, but its role in colon cancer is unknown. In this research, we aimed at revealing the function of CLDN6 in colon cancer. We found that colon cancer tissues lowly expressed CLDN6, and the expression of CLDN6 was negatively correlated with lymph node metastasis. Similarly, CLDN6 was lowly expressed in the colon cancer cell line SW1116, and overexpression of CLDN6 inhibited cell proliferation in vitro and in vivo. Consistently, the migration and invasion abilities of cells were significantly inhibited after CLDN6 overexpression. In addition, we demonstrated that CLDN6 may inhibit the migration and invasion abilities by activating the TYK2/STAT3 pathway. Therefore, our data indicated that CLDN6 acted as a tumor suppressor and had the potential to be regarded as a biomarker for the progression of colon cancer.

CLDN6 inhibits breast cancer cell malignant behavior by suppressing ERK signaling.

Claudin 6 (CLDN6) is an important component of tight junctions. Through the PDZ binding motif, CLDN6 binds to a variety of signaling proteins that contain the PDZ domain to regulate different signaling pathways, and plays an important role in the occurrence and development of tumors. Our previous work showed that CLDN6 was expressed at low levels in breast cancer cells, and overexpression of CLDN6 inhibited breast cancer cell proliferation, migration and invasion. However, the mechanism of how CLDN6 works remains unclear. In this study, we aimed to explore the mechanism by which CLDN6 inhibits breast cancer cell malignant behavior. As a result, overexpression of CLDN6 inhibited the proliferation of breast cancer cells along with the downregulation of cyclin D1, which plays an important role in regulating cell proliferation. After overexpression of Sp1 in CLDN6-overexpressing cells, the expression of cyclin D1 was upregulated. On the other hand, CLDN6 inhibited breast cancer cell migration and invasion along with the downregulation of IL-8, CXCR2 and FAK. When treated with IL-8, the migration and invasion ability were promoted along with the upregulation of CXCR2 and p-FAK, and the cytoskeleton was rearranged in CLDN6-overexpressing cells. Furthermore, when treated with the ERK signaling activator PMA, the proliferation, migration and invasion abilities were promoted along with the upregulation of Sp1, cyclin D1 and IL-8 in CLDN6-overexpressin cells. In conclusion, CLDN6 suppressed ERK/Sp1/cyclin D1 and ERK/IL-8 signaling to inhibit proliferation, migration and invasion in breast cancer cells. The mechanism may provide experimental evidence for the treatment of breast cancer targeting CLDN6.

CLDN6: From Traditional Barrier Function to Emerging Roles in Cancers.

Claudins (CLDNs) are the most important tight junction proteins, which are mainly expressed in endothelial cells or epithelial cells in a tissue-specific manner. As a member of the CLDNs family, CLDN6 is highly expressed in fetal tissues such as the stomach, pancreas, lung, and kidney, but is not expressed in corresponding adult tissues. The expression of CLDN6 is regulated by a variety of factors, including but not limited to stimuli and transcription factors, DNA methylation, and post-translational modifications. CLDN6 has been found to have a key role in the formation of barriers, especially the lung epithelial barrier and the epidermal permeability barrier (EPB). Importantly, the roles of CLDN6 in cancers have gained focus and are being investigated in recent years. Strong evidence indicates that the altered expression of CLDN6 is linked to the development of various cancers. Malignant phenotypes of tumors affected by CLDN6 include proliferation and apoptosis, migration and invasion, and drug resistance, which are regulated by CLDN6-mediated key signaling pathways. Given the important role in tumors and its low or no expression in normal tissues, CLDN6 is an ideal target for tumor therapy. This review aims to provide an overview of the structure and regulation of CLDN6, and its traditional barrier function, with a special emphasis on its emerging roles in cancers, including its impact on the malignant phenotypes, signal-modulating effects, the prognosis of tumor patients, and clinical applications in cancers.

Splicing factor Srsf5 deletion disrupts alternative splicing and causes noncompaction of ventricular myocardium.

The serine/arginine-rich (SR) family of splicing factors plays important roles in mRNA splicing activation, repression, export, stabilization, and translation. SR-splicing factor 5 (SRSF5) is a glucose-inducible protein that promotes tumor cell growth. However, the functional role of SRSF5 in tissue development and disease remains unknown. Here, Srsf5 knockout (Srsf5 -/- ) mice were generated using CRISPR-Cas9. Mutant mice were perinatally lethal and exhibited cardiac dysfunction with noncompaction of the ventricular myocardium. The left ventricular internal diameter and volume were increased in Srsf5 -/- mice during systole. Null mice had abnormal electrocardiogram patterns, indicative of a light atrioventricular block. Mechanistically, Srsf5 promoted the alternative splicing of Myom1 (myomesin-1), a protein that crosslinks myosin filaments to the sarcomeric M-line. The switch between embryonic and adult isoforms of Myom1 could not be completed in Srsf5-deficient heart. These findings indicate that Srsf5-regulated alternative splicing plays a critical role during heart development.

The Expression of CLDN6 in Hepatocellular Carcinoma Tissue and the Effects of CLDN6 on Biological Phenotypes of Hepatocellular Carcinoma Cells.

CLDN6, a member of claudin (CLDN) family, was found to be a breast cancer suppressor gene in our early experiments. However, CLDN6 was highly expressed in human hepatocellular carcinoma (hHCC) (TCGA database), and the role of CLDN6 in hHCC is still unclear. To investigate the expression of CLDN6, immunohistochemical staining was performed in hHCC tissues. As a result, hHCC tissues highly expressed CLDN6, and the expression was related to the degree of tumor's differentiation. To research the role of CLDN6 in hHCC cells, CLDN6 was silenced in HepG2 and Hep3B cells which highly expressed CLDN6 through liposome transfection. Results showed that after silencing of CLDN6, the proliferation, migration and invasion abilities of hHCC cells were inhibited. Meanwhile, the expression of E-cadherin was upregulated, and the expression of N-cadherin and Vimentin was downregulated. All the results above indicated that CLDN6 promoted the development of hHCC, and could be a potential target for the treatment of it.

MiR-124 and Small Molecules Synergistically Regulate the Generation of Neuronal Cells from Rat Cortical Reactive Astrocytes.

Irreversible neuron loss caused by central nervous system injuries usually leads to persistent neurological dysfunction. Reactive astrocytes, because of their high proliferative capacity, proximity to neuronal lineage, and significant involvement in glial scarring, are ideal starting cells for neuronal regeneration. Having previously identified several small molecules as important regulators of astrocyte-to-neuron reprogramming, we established herein that miR-124, ruxolitinib, SB203580, and forskolin could co-regulate rat cortical reactive astrocyte-to-neuron conversion. The induced cells had reduced astroglial properties, displayed typical neuronal morphologies, and expressed neuronal markers, reflecting 25.9% of cholinergic neurons and 22.3% of glutamatergic neurons. Gene analysis revealed that induced neuron gene expression patterns were more similar to that of primary neurons than of initial reactive astrocytes. On the molecular level, miR-124-driven neuronal differentiation of reactive astrocytes was via targeting of the SOX9-NFIA-HES1 axis to inhibit HES1 expression. In conclusion, we present a novel approach to inducing endogenous rat cortical reactive astrocytes into neurons through co-regulation involving miR-124 and three small molecules. Thus, our research has potential implications for inhibiting glial scar formation and promoting neuronal regeneration after central nervous system injury or disease.

CLDN6 Suppresses c-MYC-Mediated Aerobic Glycolysis to Inhibit Proliferation by TAZ in Breast Cancer.

Claudin 6 (CLDN6) was found to be a breast cancer suppressor gene, which is lowly expressed in breast cancer and inhibits breast cancer cell proliferation upon overexpression. However, the mechanism by which CLDN6 inhibits breast cancer proliferation is unclear. Here, we investigated this issue and elucidated the molecular mechanisms by which CLDN6 inhibits breast cancer proliferation. First, we verified that CLDN6 was lowly expressed in breast cancer tissues and that patients with lower CLDN6 expression had a worse prognosis. Next, we confirmed that CLDN6 inhibited breast cancer proliferation through in vitro and in vivo experiments. As for the mechanism, we found that CLDN6 inhibited c-MYC-mediated aerobic glycolysis based on a metabolomic analysis of CLDN6 affecting cellular lactate levels. CLDN6 interacted with a transcriptional co-activator with PDZ-binding motif (TAZ) and reduced the level of TAZ, thereby suppressing c-MYC transcription, which led to a reduction in glucose uptake and lactate production. Considered together, our results suggested that CLDN6 suppressed c-MYC-mediated aerobic glycolysis to inhibit the proliferation of breast cancer by TAZ, which indicated that CLDN6 acted as a novel regulator of aerobic glycolysis and provided a theoretical basis for CLDN6 as a biomarker of progression in breast cancer.

Enhanced O-linked Glcnacylation in Crohn's disease promotes intestinal inflammation.

BACKGROUND: Treatment of Crohn's disease (CD) remains to be a challenge due to limited insights for its pathogenesis. We aimed to determine the role of O-Linked ß-N-acetylglucosamine (O-GlcNAc) in the development of CD and evaluate therapeutic effects of O-GlcNAc inhibitors on CD. METHODS: O-GlcNAc in intestinal epithelial tissues of CD, adherent-invasive Escherichia coli (AIEC) LF82-infected cells and mice was determined by immunoblot and immunohistochemistry. AIEC LF82 and dextran sulfate sodium were administrated into C57BL/6 mice for estabolishing inflammatory bowel disease model and for therapeutic study. FINDINGS: O-GlcNAc was increased in intestinal epithelial tissues of CD patients and AIEC LF82-infected mice. Infection of AIEC LF82 up-regulated the level of UDP-GlcNAc and increased O-GlcNAc in human colon epithelial HCT116 and HT-29 cells. We identified that IKKß and NF-κB were O-Glycosylated in AIEC LF82-treated cells. Mutations of IKKß (S733A) and p65 (T352A) abrogated the O-GlcNAc in IKKß and NF-κB and inhibited AIEC LF82-induced activation of NF-κB. Application of 6-diazO-5-oxO-L-norleucine, an agent that blocks the production of UDP-GlcNAc and inhibits O-GlcNAc, inactivated NF-κB in AIEC LF82-infected cells, enhanced the formation of autophagy, promoted the removal of cell-associated AIEC LF82, alleviated intestinal epithelial inflammation, and improved the survival of the colitis mice. INTERPRETATION: Intestinal inflammation in CD is associated with increased O-GlcNAc modification, which is required for NF-κB activation and suppression of autophagy. Targeting O-GlcNAc could be an effective therapy for inflammatory bowel disease. FUNDING: National Natural Science Foundation of China (Nos. 81573087 and 81772924) and International Cooperation Foundation of Jilin Province (20190701006GH).

A SUMOylation-dependent HIF-1α/CLDN6 negative feedback mitigates hypoxia-induced breast cancer metastasis.

BACKGROUND: We have previously described CLDN6 as a tumor suppressor gene in breast cancer. Here, a new finding is that CLDN6 was upregulated under hypoxia, a commonly recognized factor that promotes tumor metastasis. In this study, we aim to explain this confusing finding and delineate the role of CLDN6 in the breast cancer metastasis induced by hypoxia. METHODS: RNAi and ChIP assays were used to confirm that CLDN6 is transcriptional regulated by HIF-1α. mRNA seq and KEGG analysis were performed to define the downstream pathways of CLDN6. The roles of the CLDN6/SENP1/HIF-1α signaling on tumor metastasis were evaluated by function experiments and clinical samples. Finally, the possible transcription factor of SENP1 was suspected and then validated by ChIP assay. RESULTS: We demonstrated a previously unrecognized negative feedback loop exists between CLDN6 and HIF-1α. CLDN6 was transcriptionally up-regulated by HIF-1α under hypoxia. On the other hand, in cytoplasm CLDN6 combines and retains ß-catenin, a transcription factor of SENP1, causing ß-catenin degradation and preventing its nuclear translocation. This process reduced SENP1 expression and prevented the deSUMOylation of HIF-1α, ultimately leading to HIF-1α degradation and breast cancer metastasis suppression. CONCLUSIONS: Our data provide a molecular mechanistic insight indicating that CLDN6 loss may lead to elevated HIF-1α-driven breast cancer metastasis in a SUMOylation-dependent manner.

Predictive Value of Pin1 in Cervical Low-Grade Squamous Intraepithelial Lesions and Inhibition of Pin1 Exerts Potent Anticancer Activity against Human Cervical Cancer.

Many oncogenes are involved in the progression from low-grade squamous intraepithelial lesions (LSILs) to high-grade squamous intraepithelial lesions (HSILs); which greatly increases the risk of cervical cancer (CC). Thus, a reliable biomarker for risk classification of LSILs is urgently needed. The prolyl isomerase Pin1 is overexpressed in many cancers and contributes significantly to tumour initiation and progression. Therefore, it is important to assess the effects of cancer therapies that target Pin1. In our study, we demonstrated that Pin1 may serve as a biomarker for LSIL disease progression and may constitute a novel therapeutic target for CC. We used a the novel Pin1 inhibitor KPT-6566, which is able to covalently bind to Pin1 and selectively target it for degradation. The results of our investigation revealed that the downregulation of Pin1 by shRNA or KPT-6566 inhibited the growth of human cervical cancer cells (CCCs). We also discovered that the use of KPT-6566 is a novel approach to enhance the therapeutic efficacy of cisplatin (DDP) against CCCs in vitro and in vivo. We showed that KPT-6566-mediated inhibition of Pin1 blocked multiple cancer-driving pathways simultaneously in CCCs. Furthermore, targeted Pin1 treatment suppressed the metastasis and invasion of human CCCs, and downregulation of Pin1 reversed the epithelial-mesenchymal transition (EMT) of CCCs via the c-Jun/slug pathway. Collectively, we showed that Pin1 may be a marker for the risk of progression to HSIL and that inhibition of Pin1 has anticancer effects against CC.

Estrogen receptor ß inhibits breast cancer cells migration and invasion through CLDN6-mediated autophagy.

BACKGROUND: Estrogen receptor ß (ERß) has been reported to play an anti-cancer role in breast cancer, but the regulatory mechanism by which ERß exerts this effect is not clear. Claudin-6 (CLDN6), a tight junction protein, acts as a tumor suppressor gene in breast cancer. Our previous studies have found that 17ß-estradiol (E2) induces CLDN6 expression and inhibits MCF-7 cell migration and invasion, but the underlying molecular mechanisms are still unclear. In this study, we aimed to investigate the role of ERß in this process and the regulatory mechanisms involved. METHODS: Polymerase chain reaction (PCR) and western blot were used to characterize the effect of E2 on the expression of CLDN6 in breast cancer cells. Chromatin immunoprecipitation (ChIP) assays were carried out to confirm the interaction between ERß and CLDN6. Dual luciferase reporter assays were used to detect the regulatory role of ERß on the promoter activity of CLDN6. Wound healing and Transwell assays were used to examine the migration and invasion of breast cancer cells. Western blot, immunofluorescence and transmission electron microscopy (TEM) were performed to detect autophagy. Xenograft mouse models were used to explore the regulatory effect of the CLDN6-beclin1 axis on breast cancer metastasis. Immunohistochemistry (IHC) was used to detect ERß/CLDN6/beclin1 expression in breast cancer patient samples. RESULTS: Here, E2 upregulated the expression of CLDN6, which was mediated by ERß. ERß regulated CLDN6 expression at the transcriptional level. ERß inhibited the migration and invasion of breast cancer cells through CLDN6. Interestingly, this effect was associated with CLDN6-induced autophagy. CLDN6 positively regulated the expression of beclin1, which is a key regulator of autophagy. Beclin1 knockdown reversed CLDN6-induced autophagy and the inhibitory effect of CLDN6 on breast cancer metastasis. Moreover, ERß and CLDN6 were positively correlated, and the expression of CLDN6 was positively correlated with beclin1 in breast cancer tissues. CONCLUSION: Overall, this is the first study to demonstrate that the inhibitory effect of ERß on the migration and invasion of breast cancer cells was mediated by CLDN6, which induced the beclin1-dependent autophagic cascade.

ERα is required for suppressing OCT4-induced proliferation of breast cancer cells via DNMT1/ISL1/ERK axis.

OBJECTIVE: POU5F1 (OCT4) is implicated in cancer stem cell self-renewal. Currently, some studies have shown that OCT4 has a dual function in suppressing or promoting cancer progression. However, the precise molecular mechanism of OCT4 in breast cancer progression remains unclear. MATERIALS AND METHODS: RT-PCR and Western blot were utilized to investigate OCT4 expression in breast cancer tissues and cells. Cell proliferation assays and mouse models were applied to determine the effects of OCT4 on breast cancer cell proliferation. DNMT1 inhibitors, ChIP, CoIP, IHC and ERα inhibitors were used to explore the molecular mechanism of OCT4 in breast cancer. RESULTS: OCT4 was down-regulated in breast cancer tissues, and the overexpression of OCT4 promoted MDA-MB-231 cell proliferation and inhibited the proliferation of MCF-7 cells in vitro and in vivo, respectively. Two DNMT1 inhibitors (5-aza-dC and zebularine) suppressed OCT4-induced MDA-MB-231 cell proliferation through Ras/Raf1/ERK inactivation by targeting ISL1, which is the downstream of DNMT1. In contrast, OCT4 interacted with ERα, decreased DNMT1 expression and inactivated the Ras/Raf1/ERK signalling pathway in MCF-7 cells. Moreover, ERα inhibitor (AZD9496) reversed the suppression of OCT4-induced proliferation in MCF-7 cells via the activation of ERK signalling pathway. CONCLUSIONS: OCT4 is dependent on ERα to suppress the proliferation of breast cancer cells through DNMT1/ISL1/ERK axis.

OCT4 maintains self-renewal and reverses senescence in human hair follicle mesenchymal stem cells through the downregulation of p21 by DNA methyltransferases.

BACKGROUND: Self-renewal is dependent on an intrinsic gene regulatory network centered on OCT4 and on an atypical cell cycle G1/S transition, which is also regulated by OCT4. p21, a gene negatively associated with self-renewal and a senescence marker, is a member of the universal cyclin-dependent kinase inhibitors (CDKIs) and plays critical roles in the regulation of the G1/S transition. The expression of p21 can be regulated by OCT4-targeted DNA methyltransferases (DNMTs), which play distinct roles in gene regulation and maintaining pluripotency properties. The aim of this study was to determine the role of OCT4 in the regulation of self-renewal and senescence in human hair follicle mesenchymal stem cells (hHFMSCs) and to characterize the molecular mechanisms involved. METHODS: A lentiviral vector was used to ectopically express OCT4. The influences of OCT4 on the self-renewal and senescence of hHFMSCs were investigated. Next-generation sequencing (NGS) was performed to identify the downstream genes of OCT4 in this process. Methylation-specific PCR (MSP) analysis was performed to measure the methylation level of the p21 promoter region. p21 was overexpressed in hHFMSCsOCT4 to test its downstream effect on OCT4. The regulatory effect of OCT4 on DNMTs was examined by ChIP assay. 5-aza-dC/zebularine was used to inhibit the expression of DNMTs, and then self-renewal properties and senescence in hHFMSCs were detected. RESULTS: The overexpression of OCT4 promoted proliferation, cell cycle progression, and osteogenic differentiation capacity of hHFMSCs. The cell senescence of hHFMSCs was markedly suppressed due to the ectopic expression of OCT4. Through NGS, we identified 2466 differentially expressed genes (DEGs) between hHFMSCsOCT4 and hHFMSCsEGFP, including p21, which was downregulated. The overexpression of p21 abrogated the proliferation and osteogenic differentiation capacity of hHFMSCsOCT4 and promoted cell senescence. OCT4 enhanced the transcription of DNMT genes, leading to an elevation in the methylation of the p21 promoter. The inhibition of DNMTs reversed the OCT4-induced p21 reduction, depleted the self-renewal of hHFMSCsOCT4, and triggered cell senescence. CONCLUSIONS: OCT4 maintains the self-renewal ability of hHFMSCs and reverses senescence by suppressing the expression of p21 through the upregulation of DNMTs.

Identification of genes and pathways associated with MDR in MCF-7/MDR breast cancer cells by RNA-seq analysis.

Multidrug resistance (MDR) is a major problem in the treatment of breast cancer. In the present study, next-generation sequencing technology was employed to identify differentially expressed genes in MCF­7/MDR cells and MCF­7 cells, and aimed to investigate the underlying molecular mechanisms of MDR in breast cancer. Differentially expressed genes between MCF­7/MDR and MCF­7 cells were selected using software; a total of 2085 genes were screened as differentially expressed in MCF­7/MDR cells. Furthermore, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the DAVID database. Finally, a protein­protein interaction network was constructed and the hub genes in the network were analyzed using the STRING database. GO annotation demonstrated that the differentially expressed genes were enriched in various biological processes, including 'regulation of cell differentiation', 'cell development', 'neuron development', 'movement of cell or subcellular component' and 'cell morphogenesis involved in neuron differentiation'. Cellular component analysis by GO revealed that differentially expressed genes were enriched in 'plasma membrane region' and 'extracellular matrix' terms. Furthermore, KEGG analysis demonstrated that the target genes were enriched in various pathways, including 'cell adhesion molecules (CAMs)', 'calcium signaling pathway', 'tight junction', 'Wnt signaling pathway' and 'pathways in cancer' terms. A protein­protein interaction network demonstrated that certain hub genes, including cyclin D1, nitric oxide synthase 3 (NOS3), NOTCH3, brain­derived neurotrophic factor (BDNF), paired box 6, neuropeptide Y, phospholipase C ß (PLCB) 4, PLCB2 and actin α cardiac muscle 1, may be associated with MDR in breast cancer. Subsequently, RT­qPCR confirmed that the expression of these 9 hub genes was higher in MCF­7/MDR cells compared with MCF­7 cells, consistent with the RNA­sequencing analysis. Additionally, a Cell Counting Kit­8 assay demonstrated that specific inhibitors of NOS3 and BDNF/neurotrophic receptor tyrosine kinase, type 2 signaling reduced the IC50 of MCF­7/MDR cells in response to various anticancer drugs, including adriamycin, cisplatin and 5­fluorouracil. The results of the present study provide novel insights into the mechanism underlying MDR in MCF­7 cells and may identify novel targets for the treatment of breast cancer.