Screening DNA aptamers that control the DNA cleavage, homology-directed repair, and transcriptional regulation of the CRISPR-(d)Cas9 system.

Accurate genome editing based on various molecular tools has always been the focus of gene-editing research and the primary goal for therapeutic application. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is a well-established gene-editing method that is preferred due to its simplicity and high efficiency. In this study, a group of single-stranded DNA aptamers with high affinity and high specificity for the Cas9 protein were obtained by the systematic evolution of ligands through the exponential enrichment method. Their binding affinity and possible binding domains to the Cas9 protein were analyzed. In addition, we demonstrated the effectiveness of aptamers in regulating dCas9-modulated gene transcription, in terms of both transcriptional activation and repression. Additionally, the aptamers successfully reduced the off-target effect and improved the efficiency of gene homologous recombination repair mediated by CRISPR-Cas9. The findings suggest a potential method to better control precise gene editing and enrich the diversity of modulating tools for the CRISPR-Cas9 system.

Long noncoding RNA ALOX12-AS1 inhibits cervical cancer cells proliferation via targeting miR-3171.

Cervical cancer is a common female malignancy worldwide, and the molecular mechanism of cervical tumorigenesis remains poorly understood. A large piece of evidence have demonstrated the important roles of long noncoding RNAs (lncRNAs) in tumorigenesis, cancer progression and drug resistance. In this study, we comprehensively analyzed the lncRNAs expression pattern in cervical cancer using RNA sequencing and microarray data from the cancer genome atlas, gene expression omnibus and Genotype Tissue Expression. Moreover, we assessed the correlation between lncRNA expression levels and cervical cancer patient's survival. We uncovered hundreds of lncRNAs that are upregulated or downregulated in cervical cancer tissues. Among these aberrantly lncRNAs, some are significantly associated with cervical patients' poorer prognosis, such as ALOX12-AS1 and LINC00173. ALOX12-AS1 expression is downregulated in cervical cancer, and over-expression of ALOX12-AS1 could inhibit cervical cancer cells proliferation in vitro. Further, mechanistically investigation revealed that ALOX12-AS1 could interact with AGO2 and sponge miR-3171, thereby antagonizing its' repression of tumor suppressor phosphatase and tensin homolog expression in cervical cancer cell. Taken together, this study provides lncRNA candidates in cervical cancer and highlights the critical role of ALOX12-AS1 in cervical cancer.

Silencing of AFAP1-AS1 lncRNA impairs cell proliferation and migration by epigenetically promoting DUSP5 expression in pre-eclampsia.

As a unique and common obstetric complication of pregnant women, pre-eclampsia (PE) has been the first leading cause of maternal and perinatal morbidity and mortality in the world. Mounting studies have demonstrated that an abnormality of long noncoding RNA (lncRNA) expression was related to the pathological process of PE. Here, we showed that lncRNA AFAP1-AS1 was markedly downregulated in pre-eclamptic placentas. We further investigated the mechanism underlying the regulatory role of AFAP1-AS1 in PE using human trophoblast cells. In vitro functional assays revealed that AFAP1-AS1 knockdown inhibited trophoblast proliferation, migration, and invasion. Moreover, AFAP1-AS1 interacts with EZH2 and inhibits DUSP5 expression through modulating H3K27m3 in the DUSP5 promoter of trophoblast cells, thus being involved in PE pathogenesis. Overall, these findings suggest that AFAP1-AS1 could potentially become a prognostic biomarker as well as a new therapeutic target for PE.

PUM1 modulates trophoblast cell proliferation and migration through LRP6.

Preeclampsia is a severe pregnancy complication characterized by hypertension and may cause maternal morbidity and mortality. A better understanding of the essential genes involved in preeclampsia pathophysiology is urgently needed. This study investigated the function and molecular mechanisms of pumilio RNA binding family member 1 (PUM1) in extravillous trophoblast cells (EVTs). The interaction between protein and mRNA was verified by RNA pull-down assays, RNA immunoprecipitation assays, and luciferase reporter assays. The mRNA and protein levels of the genes involved were determined by RT-qPCR and western blot assays, respectively. Our results demonstrated that PUM1 could bind to the 3'-untranslated region of low-density lipoprotein receptor-related protein 6 (LRP6) mRNA, resulting in reduced expression of LRP6 mRNA and protein. Repression of PUM1 resulted in enhanced colony formation, cell proliferation, migration, and invasion of EVTs. The PUM1-depletion-mediated promotion effects on EVTs could be abrogated by LRP6 knockdown. PUM1 regulates the growth and mobility of EVTs by modulating LRP6 expression. Developing strategies to balance PUM1 and LRP6 levels may be beneficial for the management of preeclampsia patients.

Upregulated long noncoding RNAs LINC02163 and FEZF1-AS1 exert oncogenic roles in colorectal cancer.

A growing number of evidence has revealed that aberrantly expressed long noncoding RNAs (lncRNAs) are involved in the development of a variety of malignancies, including colorectal cancer (CRC). However, the clinical relevance of most lncRNAs and their potential biological functions in CRC remains poorly understood. The aim of this study was to identify the key lncRNAs related to patient prognosis as well as their biological function and underlying mechanism in CRC. Therefore, five independent datasets containing CRC and normal tissue RNA sequencing, microarray data and the corresponding clinical data from The Cancer Genome Atlas and Gene Expression Omnibus were screened. Hundreds of significantly differentially expressed lncRNAs in CRC were determined, and Kaplan-Meier analyses revealed that some of these lncRNAs were related to the overall survival and progression-free survival of patients with CRC, such as RP11-108K3.2, FOXD3-AS1, H19 and AP001469.9. Among these dysregulated lncRNAs, LINC02163 and FEZF1-AS1 were significantly upregulated in CRC tissues, suggesting that they may have oncogenic roles in CRC. Furthermore, loss of function assays revealed that downregulation of LINC02163 and FEZF1-AS1 impaired CRC cell proliferation. In addition, RNA Immunoprecipitation and Chromatin Immunoprecipitation assays determined that FEZF1-AS1 regulates CRC cell growth via interacting with LSD1 and repressing KLF2 expression. Collectively, hundreds of dysregulated lncRNAs and their associated biological roles identified in this study may provide potentially useful biomarkers and therapeutic targets for CRC.

Resveratrol promotes trophoblast invasion in pre-eclampsia by inducing epithelial-mesenchymal transition.

Impairment spiral arteries remodelling was considered to be the underlying cause of pathogenesis of pre-eclampsia (PE). Resveratrol (RE) was reported that it could modulate cellar phenotype to ameliorate diverse human diseases. However, the biological function of RE in PE remains poorly understood. In this report, we investigated the effect of RE on trophoblast phenotype both in vivo and in vitro. We conducted MTT and transwell assays to explore cell proliferation and invasion events in HTR-8/SVneo. In mice model, the clinical characteristics of PE were established through the injection of NG-nitro-l-arginine methyl ester (L-NAME). Furthermore, related experiments were performed to detect cellar phenotype-associated signalling pathway, including epithelial-mesenchymal transition (EMT) and Wnt/ß-catenin. Cell assays indicated that RE could increase trophoblasts migration and invasion. In addition, hypertension and proteinuria were markedly ameliorated by RE compared with the controls in PE mice model. Moreover, treatment by RE in trophoblasts or in PE model, we found that RE activated EMT progress through the regulation of E-cadherin, ß-catenin, N-cadherin, vimentin expression, and further altered the WNT-related gene expression, including WNT1, WNT3 and WNT5B. Our findings demonstrated that RE might stimulate the invasive capability of human trophoblasts by promoting EMT and mediating the Wnt/ß-catenin pathway in PE.

lncRNA GHET1 has effects in development of pre-eclampsia.

To explain long noncoding RNA (lncRNA) gastric carcinoma high expressed transcript 1 (GHET1) affects the mechanism in development of pre-eclampsia. The pathological changes of normal, mild, and severe pre-eclampsia were evaluated by hematoxylin and eosin staining and measured the lncRNA GHET1 expression in different tissues by reverse-transcription polymerase chain reaction. In the cell experiment, the BeWo cells were randomly divided into three groups: normal control (NC) group, model group, and lncRNA group. The JEG3 cells of the model and lncRNA groups were cultured in the hypoxia condition. The JEG3 cells invasion and migration abilities were measured by Tanswell and wound-healing assays. The relative protein expressions of different groups were evaluated by Western blot (WB) assay. Compared with normal puerperal, the lncRNA GHET1 gene expression of pre-eclampsia was significantly downregulated (P < 0.05, respectively). In the cell experiment, the invasion cell number and wound-healing rate of the model group were significantly suppressed compared with the NC group (P < 0.05, respectively). However, the invasion cell number and wound-healing rate of lncRNA group were enhanced by lncRNA GHET1 overexpression (P < 0.05, respectively). In WB assay, the E-cadherin, fibronectin, and vimentin proteins expression showed significant differences between the model and lncRNA groups (P < 0.05, respectively). lncRNA GHET1 overexpression had restored cell invasion and migration abilities reduced by hypoxia in pre-eclampsia.

Promotion of trophoblast invasion by lncRNA MVIH through inducing Jun-B.

Preeclampsia (PE), a pregnancy-specific disorder, is associated with impaired uterine spiral artery remodelling, which is related to the dysfunction of trophoblast cells. Lately, mounting evidence has indicated that aberrant expression of long non-coding RNAs (lncRNAs) is associated with various human diseases. The lncRNA MVIH transcript has been shown to decrease the severity of several diseases. However, the biological function of MVIH, which is down-regulated in placental tissues in PE, has not yet been clarified. Here, we report that MVIH may act as a vital factor in the pathogenesis of PE. In this study, functional analysis revealed that the silencing of MVIH expression via transfection with small interfering RNA (siRNAs) inhibited cell growth, migration, invasion, and angiogenesis in various trophoblast cell lines, and stimulation with MVIH could promote these functions. Mass spectrometry analysis revealed that MVIH could modulate Jun-B protein expression, which has been reported to potentially regulate cell growth and angiogenesis. Further cotransfection assays were performed, revealing that MVIH and Jun-B have a synergistic effect on the regulation of angiogenesis and cell proliferation. Taking these findings together, MVIH could be associated with PE and may be a candidate biomarker for its diagnosis and treatment.

MicroRNA-223 is involved in the pathogenesis of atopic dermatitis by affecting histamine-N-methyltransferase.

Atopic dermatitis (AD) is one of the most prevalent skin diseases around the world. Excessive histamine plays a critical role as an inflammatory factor in the pathogenesis of AD. Deregulated microRNAs (miRNAs) were involved in atopic dermatitis by targeting various genes. MiR-223 had been reported to play a vital role in hematopoiesis. In this study, we identified upregulated miR-223 in the whole blood cells of a large group of AD patients. What's more, we found for the first time that one of the major histamine degradation enzymes, histamine-N-methyltransferase (HNMT), was increased in AD patients and AD model mice. Although there was one miR-223 binding site in the 3'- untranslated region of the HNMT gene, HNMT were not inhibited by miR-223. Taken together, it suggested that miR-223 participates in AD through upregulating HNMT indirectly to degrade the excessive histamine.

Down-regulated long non-coding RNA MEG3 and its effect on promoting apoptosis and suppressing migration of trophoblast cells.

Preeclampsia is characterized by hypertension and proteinuria twenty weeks into pregnancy. Failure of uterine spiral artery remodeling contributes to preeclampsia's development. The development might be associated with trophoblast cells functioning abnormally. Long non-coding RNAs (lncRNAs) are aberrantly expressed in many diseases. Maternally expressed gene 3 (MEG3), one of these lncRNAs, might function as a tumor suppressor. Aberrant expression of MEG3 induces prenatal death, and little is known of MEG3's role in preeclampsia. This study aims to identify the role of lncRNA MEG3 on apoptosis and the migration of human trophoblast cells, and to investigate the involvement of lncRNA MEG3 in pathogenic mechanisms underlying preeclampsia. In this study, we found MEG3 levels were down-regulated by approximately 80% in placental samples collected from preeclamptic patients (n = 30) compared to samples collected from normotensive patients (n = 30) by qRT-PCR analysis. By designing RNA interference species to suppress MEG3 and specific plasmids designed to over-express MEG3, we explored the role of MEG3 on the functions of two trophoblast cell-lines, HTR-8/SVneo and JEG3 cells. Over-expression of MEG3 reduced apoptosis and promoted migration of HTR-8/SVneo and JEG3 cells. Furthermore, inhibition of endogenous MEG3 increased apoptosis and decreased migration of HTR-8/SVneo and JEG3 cells. Additionally, lncRNA MEG3 influenced expression of NF-κB, Caspase-3, and Bax protein expressions in trophoblast cells. Our findings highlight that abnormal levels of lncRNA MEG3 might lead to aberrant conditions in HTR-8/SVneo and JEG3 trophoblast cells, which might be associated with uterine spiral artery remodeling failure and its contribution to preeclampsia.

A Role of sFlt-1 in Oxidative Stress and Apoptosis in Human and Mouse Pre-Eclamptic Trophoblasts.

Pre-eclampsia (PE) is a hypertensive disorder that occurs during pregnancy, and is a multifactorial disease. The antiangiogenic factor, soluble fms-like tyrosine kinase 1 (sFlt-1), has been reported to be important in the pathogenesis of PE, but the mechanism of its involvement remains unknown. To test the effects of sFlt-1 on pregnancy, we injected pregnant mice with exogenous mouse sFlt-1. After 18 days of gestation, higher blood pressure, proteinuria, and histological differences were observed compared with controls. Mitochondrial swelling inside the trophoblast cells in the placenta of sFlt-1-treated pregnant mice was observed by electron microscopy, which suggested a role of sFlt-1 in oxidative stress in trophoblasts in PE. Furthermore, apoptosis markers were upregulated in sFlt-1-treated mice. In conclusion, sFlt-1 appears to play a role in oxidative stress, which promotes apoptosis of trophoblasts. This may be an important mechanism in the development of PE.

Role of SOCS3 in the Jak/stat3 pathway in the human placenta: different mechanisms for preterm and term labor.

INTRODUCTION: To identify changes in interleukin (IL)-6 levels and its pathway (Jak/stat3) in the human placenta during preterm and term labor, placental tissues were collected from primiparous women who underwent vaginal deliveries or cesarean sections in our hospital. The women were divided into three groups: preterm labor (n = 15), term labor (n = 15), and term not in labor (n = 15). MATERIAL AND METHODS: IL-6 levels were detected by ELISA in placental supernatant, and p-STAT3 and SOCS3 protein was detected by Western blot. TUNEL was used to detect apoptosis in trophoblasts. HTR-8/SVneo cells were cultured after stimulation with IL-6, and we measured p-STAT3, SOCS3, and the rate of apoptosis. RESULTS: Expression of p-STAT3 and SOCS3 in the placenta and trophoblast cells showed that IL-6 levels were highest in the preterm labor group and lowest in the term not in labor group. The highest expression of placental SOCS3 protein was observed in the preterm labor group. More apoptotic cells were found in the preterm labor group than in the other two groups by TUNEL. SOCS3 and p-STAT3 expression was significantly upregulated after stimulation by IL-6 in trophoblast cells in a dose-dependent manner. However, p-STAT3 was significantly decreased after 50 ng/mL and 100 ng/mL IL-6 for 72 h. A significant increase of apoptosis was observed with treatment of 50 ng/mL IL-6 in HTR-8/SVneo cells. CONCLUSIONS: The role of the SOCS3 protein in the Jak/stat3 pathway is to mediate different mechanisms for preterm and term labor processes in the placenta.

[Long noncoding RNA HOTAIR modulates the function of trophoblast cells in pre-eclampsia].

OBJECTIVE: To investigate the expression of long noncoding RNA (lncRNA) HOTAIR in pre-eclampsia (PE) placentas and its effect on trophoblast cells proliferation, invasion, and apoptosis. METHODS: The expression of HOTAIR was determined by qPCR for 23 severe PE and 23 normal pregnant women. qRT-PCR was used to detect the efficiency of overexpression and knock-down after the HTR-8/SVneo cells were transfected with HOTAIR overexpression and siRNAs targeting HOTAIR for 24-48 h, respectively. MTT and colony formation assays were used to test the proliferation of HTR-8/SVneo cells transfected. Transwell assay was used to show the migration and invasion ability of HTR-8/SVneo cells transfected. Flow cytometry assay was used to detect the cell apoptosis rate after treatment. Western blot assay was applied to detect the expression level of apoptotic proteins Caspase-3 and BCL-2. RESULTS: The level of HOTAIR in severe pre-eclampsia groups was significantly increased compared to normal pregnant placentas (P<0.05). The expression of HOTAIR in HTR-8/SVneo cells after transfected with pcDNA-HOTAIR was 51. 27-fold than that of control; while inhibition of HOTAIR was more than 95% in si-HOTAIR than that of control. According to the MTT and colony formation assays, we found that cells proliferation rate of cells were significantly decreased in overexpression HOTAIR group while increased in si- HOTARI group when compared with control, respectively. The transwell assay showed that the invasive capacity of HTR-8/SVneo cells in cells transfected with pcDNA-HOTAIR decreased, while increased in si-HOTAIR transfected cells when compared with that of control. The apoptosis rate in cells transfected with HOTAIR overexpression was apparently more than that of control, while less in cells treated with si-HOTAIR. Western blot assay showed that the Caspase-3 showed an obvious increase in HOTAIR overexpression group while decreased in si-HOTAIR group. And BCL-2 presented an opposite trend. CONCLUSION: HOTAIR is probably involved in the onset of preeclampsia by regulating proliferation, invasion and apoptosis of trophoblast cells.

Decorin-mediated inhibition of cholangiocarcinoma cell growth and migration and promotion of apoptosis are associated with E-cadherin in vitro.

Emerging evidences have shown that decorin expression is significantly reduced in many cancer tissues and cancer cells. However, its biological role and clinical significance in cholangiocarcinoma development and progression are unknown. In this study, immunohistochemistry was conducted to investigate the expression of decorin in cholangiocarcinomas. The results showed that decorin levels markedly decreased in 44 cholangiocarcinoma tissues compared to 40 adjacent normal tissues. The analysis between decorin expression and clinicopathological characteristics in cholangiocarcinoma patients showed that patients with low levels of decorin expression had a relatively poor prognosis. Moreover, recombinant human decorin treatment and overexpression of decorin in cholangiocarcinoma cells could inhibit cell proliferation, migration, and invasion and promote apoptosis. Furthermore, the E-cadherin expression significantly increased after decorin overexpression or use of recombinant human decorin in cholangiocarcinoma cells. Our findings indicated that downregulation of decorin may be identified as a poor prognostic biomarker in cholangiocarcinomas. Also, decorin-mediated inhibition of cholangiocarcinoma cell growth, migration, and invasion and promotion of cell apoptosis might be through regulation of the expression of E-cadherin in vitro.

Long non-coding RNA MVIH indicates a poor prognosis for non-small cell lung cancer and promotes cell proliferation and invasion.

Long non-coding RNAs (lncRNAs) have emerged as major players in governing fundamental biological processes, and many of which are misregulated in multiple cancers and likely to play a functional role in tumorigenesis. Therefore, identification of cancer-associated lncRNAs and investigation of their biological functions and molecular mechanisms are important for understanding the development and progression of cancer. lncRNA associated with microvascular invasion in HCC (lncRNA MVIH) was found to be generally upregulated in HCC. Moreover, MVIH overexpression could serve as an independent risk factor to predict poor RFS and promote tumor growth and metastasis via activating angiogenesis. However, its biological role and clinical significance in non-small cell lung cancer (NSCLC) development and progression is unknown. In this study, we found that lncRNA MVIH levels were increased in NSCLC tissues compared with adjacent normal tissues. Its expression level was significantly correlated with TNM stages, tumor size, and lymph node metastasis. Moreover, patients with high levels of MVIH expression had a relatively poor prognosis. Furthermore, knockdown of MVIH expression by siRNA could inhibit cell proliferation and invasion, while ectopic expression of MVIH promoted cell proliferation and invasion in NSCLC cells partly via regulating MMP2 and MMP9 protein expression. Our findings present that increased lncRNA MVIH could be identified as a poor prognostic biomarker in NSCLC and regulate cell proliferation and invasion.

Resveratrol inhibits trophoblast apoptosis through oxidative stress in preeclampsia-model rats.

Resveratrol has been shown to be a therapeutic agent for cardiovascular disorders by maintaining a lower redox level in vivo through its anti-oxidant properties. Resveratrol can prevent cells from p53- and reactive oxygen species-dependent apoptosis induced by interleukin-1b. We identified an inhibitory effect of resveratrol against oxidative stress and apoptosis using the TUNEL assay in NG-Nitro-l-arginine methyl ester-induced preeclampsia in rats. To investigate a possible association between resveratrol and the apoptosis caused by oxidative stress in vitro, assays for superoxide dismutase and malondialdehyde as well as flow cytometric analyses were conducted in HTR-8/SVneo cells after hypoxic treatment with or without resveratrol for 24 h. These data suggest that resveratrol significantly opposes the effects of oxidative stress in vivo and in vitro.

Recombinant hirudin attenuates pulmonary hypertension and thrombosis in acute pulmonary embolism rat model.

Background: Acute pulmonary embolism (APE) is classified as a subset of diseases that are characterized by lung obstruction due to various types of emboli. Current clinical APE treatment using anticoagulants is frequently accompanied by high risk of bleeding complications. Recombinant hirudin (R-hirudin) has been found to have antithrombotic properties. However, the specific impact of R-hirudin on APE remains unknown. Methods: Sprague-Dawley (SD) rats were randomly assigned to five groups, with thrombi injections to establish APE models. Control and APE group rats were subcutaneously injected with equal amounts of dimethyl sulfoxide (DMSO). The APE+R-hirudin low-dose, middle-dose, and high-dose groups received subcutaneous injections of hirudin at doses of 0.25 mg/kg, 0.5 mg/kg, and 1.0 mg/kg, respectively. Each group was subdivided into time points of 2 h, 6 h, 1 d, and 4 d, with five animals per point. Subsequently, all rats were euthanized, and serum and lung tissues were collected. Following the assessment of right ventricular pressure (RVP) and mean pulmonary artery pressure (mPAP), blood gas analysis, enzyme-linked immunosorbnent assay (ELISA), pulmonary artery vascular testing, hematoxylin-eosin (HE) staining, Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, immunohistochemistry, and Western blot experiments were conducted. Results: R-hirudin treatment caused a significant reduction of mPAP, RVP, and Malondialdehyde (MDA) content, as well as H2O2 and myeloperoxidase (MPO) activity, while increasing pressure of oxygen (PaO2) and Superoxide Dismutase (SOD) activity. R-hirudin also decreased wall area ratio and wall thickness to diameter ratio in APE rat pulmonary arteries. Serum levels of endothelin-1 (ET-1) and thromboxaneB2 (TXB2) decreased, while prostaglandin (6-K-PGF1α) and NO levels increased. Moreover, R-hirudin ameliorated histopathological injuries and reduced apoptotic cells and Matrix metalloproteinase-9 (MMP9), vascular cell adhesion molecule-1 (VCAM-1), p-Extracellular signal-regulated kinase (ERK)1/2/ERK1/2, and p-P65/P65 expression in lung tissues. Conclusion: R-hirudin attenuated pulmonary hypertension and thrombosis in APE rats, suggesting its potential as a novel treatment strategy for APE.

Integrative analysis of bulk and single-cell RNA-seq reveals the molecular characterization of the immune microenvironment and oxidative stress signature in melanoma.

Background: The immune microenvironment and oxidative stress of melanoma show significant heterogeneity, which affects tumor growth, invasion and treatment response. Single-cell and bulk RNA-seq data were used to explore the heterogeneity of the immune microenvironment and oxidative stress of melanoma. Methods: The R package Seurat facilitated the analysis of the single-cell dataset, while Harmony, another R package, was employed for batch effect correction. Cell types were classified using Uniform Manifold Approximation and Projection (UMAP). The Secreted Signaling algorithm from CellChatDB.human was applied to elucidate cell-to-cell communication patterns within the single-cell data. Consensus clustering analysis for the skin cutaneous melanoma (SKCM) samples was executed with the R package ConsensusClusterPlus. To quantify immune infiltrating cells, we utilized CIBERSORT, ESTIMATE, and TIMERxCell algorithms provided by the R package Immuno-Oncology Biological Research (IOBR). Single nucleotide variant (SNV) analysis was conducted using Maftools, an R package specifically designed for this purpose. Subsequently, the expression levels of PXDN and PAPSS2 genes were assessed in melanoma tissues compared to adjacent normal tissues. Furthermore, in vitro experiments were conducted to evaluate the proliferation and reactive oxygen species expression in melanoma cells following transfection with siRNA targeting PXDN and PAPSS2. Results: Malignant tumor cell populations were reclassified based on a comprehensive single-cell dataset analysis, which yielded six distinct tumor subsets. The specific marker genes identified for these subgroups were then used to interrogate the Cancer Genome Atlas Skin Cutaneous Melanoma (TCGA-SKCM) cohort, derived from bulk RNA sequencing data, resulting in the delineation of two immune molecular subtypes. Notably, patients within the cluster2 (C2) subtype exhibited a significantly more favorable prognosis compared to those in the cluster1 (C1) subtype. An alignment of immune characteristics was observed between the C2 subtype and unique immune functional tumor cell subsets. Genes differentially expressed across these subtypes were subsequently leveraged to construct a predictive risk model. In vitro investigations further revealed elevated expression levels of PXDN and PAPSS2 in melanoma tissue samples. Functional assays indicated that modulation of PXDN and PAPSS2 expression could influence the production of reactive oxygen species (ROS) and the proliferative capacity of melanoma cells. Conclusion: The constructed six-gene signature can be used as an immune response and an oxidative stress marker to guide the clinical diagnosis and treatment of melanoma.

miR-206 is down-regulated in breast cancer and inhibits cell proliferation through the up-regulation of cyclinD2.

MicroRNAs act as important gene regulators in human genomes, and their aberrant expression is linked to many malignancies. Aberrant expression of miR-206 has been frequently reported in cancer studies; however, the role and mechanism of its function in breast cancer remains unclear. Quantitative real-time PCR was performed to detect the relative expression levels of miR-206 in breast cancer and normal breast tissues. Lower expression of miR-206 in breast cancer tissues was associated with larger tumour size and a more advanced clinical stage. Further in vitro observations showed that the enforced expression of miR-206 in MCF-7 breast cancer cells inhibited cell growth by blocking the G1/S transition and suppressed cell proliferation and colony formation, implying that miR-206 functions as a tumour suppressor in the progression of breast cancer. Interestingly, Luciferase assays first revealed that miR-206 inhibited cyclinD2 expression by targeting two binding sites in the 3'-untranslated region of cyclinD2 mRNA. qRT-PCR and Western blot assays verified that miR-206 reduced cyclinD2 expression at both the mRNA and protein levels. A reverse correlation between miR-206 and cyclinD2 expression was noted in breast cancer tissues. Altogether, our results identify a crucial tumour suppressive role of miR-206 in the progression of breast cancer, at least partly via up-regulation of the expression of cyclinD2, and suggest that miR-206 might be a candidate prognostic predictor or an anticancer therapeutic target for breast cancer patients.

[Effect of transforming growth factor ß1 on the expression of matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 1 and nuclear factor kappa B signalling pathway in the human amniotic cells WISH].

OBJECTIVE: To investigate the effect of transforming growth factor ß1 (TGF-ß1) on the expression of matrix metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), nuclear factor kappa B (NF-κB) and the possible signalling pathways in human amniotic cells WISH. METHODS: The WISH cell line was cultured. WISH cells were added with TGF-ß1 of different concentrations (0, 2, 10 and 20 ng/ml, respectively) for 24 hours. Then, reverse transcription (RT) PCR and western blotting were used to analyze the protein and mRNA expression of TIMP-1 and MMP-9; and the expression of NF-κB was analyzed by western blot. RESULTS: (1) The profile of TIMP-1 mRNA (0.413 ± 0.036, 0.623 ± 0.058, 1.392 ± 0.124, 1.387 ± 0.102) in WISH cells elevated when the concentration of TGF-ß1 increased (0, 2, 10, 20 ng/ml). In accordance with TIMP-1 mRNA, the expression of TIMP-1 also elevated with the increase of TGF-ß1 (0.357 ± 0.031, 0.596 ± 0.048, 1.243 ± 0.097 and 1.359 ± 0.121, respectively). And when 2, 10 or 20 ng/ml of TGF-ß1 was added, the TIMP-1 mRNA and protein were significantly higher than the TIMP-1 mRNA and protein when no TGF-ß1 was added (P < 0.05). (2) In contrast with TIMP-1, MMP-9 mRNA (1.325 ± 0.056, 0.987 ± 0.081, 0.610 ± 0.034, 0.347 ± 0.023) in WISH cells decreased when the concentration of TGF-ß1 increased (0, 2, 10, 20 ng/ml). The MMP-9 protein (1.119 ± 0.064, 1.008 ± 0.052, 0.578 ± 0.041, 0.401 ± 0.015) also decreased with the increase of TGF-ß1. And when 2, 10 or 20 ng/ml of TGF-ß1 was added, the MMP-9 mRNA and protein were significantly lower than the MMP-9 mRNA and protein when no TGF-ß1 was added (P < 0.05). (3) The NF-κB protein (1.423 ± 0.065, 1.116 ± 0.045, 0.796 ± 0.041, 0.359 ± 0.021) was significantly reduced with the increase of TGF-ß1 (0, 2, 10, 20 ng/ml;P < 0.05). CONCLUSIONS: The mRNA and protein expression of TIMP-1 decreased when TGF-ß1 was low in WISH cells, whereas those of MMP-9 elevated when TGF-ß1 was low. The unbalance of TIMP-1 and MMP-9 was related to the pathology of the premature rupture of membrane. And the NF-κB singalling pathway might be an important mechanism in the regulation of TIMP-1 and MMP-9 system.