Expression Levels Of CD44 Research Articles

Contribution of Interstitial Cells of Cajal to Gastrointestinal Stromal Tumor Risk.

BackgroundGastrointestinal stromal tumors (GISTs), which originate from interstitial cells of Cajal (ICCs), are one of most common mesenchymal tumors of the gastrointestinal tract. This study explored the impact of ICCs and immunological markers on GIST risk.Material/MethodsA total of 122 patients diagnosed with GISTs who underwent surgery were recruited for the study. Demographic and clinical information, including modified NIH criteria, sex, age, tumor site, and tumor size, of all patients were collected. GIST risk was assessed using the modified NIH risk classification for primary GISTs. Paraffin-embedded GIST specimens were evaluated by hematoxylin-eosin staining and ICCs immunohistochemistry.ResultsAccording to the modified NIH criteria, most GIST cases (44 cases, 36.07%) were at very low risk. Females had greater incidence of high-risk GISTs (P<0.05). The mean age at GIST diagnosis was 58.69±9.90 years and had no impact on GIST risk (P>0.05). Most GISTs were located in the stomach (87 cases, 71.73%), and the size of the tumors varied (0.5–20 cm). CD117/c-kit and CD34 were specific immuno-markers for ICCs and GIST. Most patients with GIST were CD117-positive (115 cases, 94.26%), 111 cases (90.98%) were CD34-positive, and 109 cases (89.34%) were positive for both CD117/c-kit and CD34. With increasing GIST risk, CD117 (also named c-k0it) and CD34 expression levels increased, as well as the number of ICCs (all P<0.05).ConclusionsICCs have a great impact on GISTs incidence. CD117/c-kit and CD34 expression, as well ICCs levels, appear to affect GIST risk.

CD44 enhances adriamycin resistance in chronic myelogenous leukaemia cells K562.

To investigate CD44 effects on the adriamycin-resistant in chronic myelogenous leukaemia cells K562, we explored the role of CD44 in the K562 cells migration and apoptosis. GeneChip® screening is used for elucidating various chemoresistance-related gene expression in the adriamycin-resistant leukaemia cells K562/ADR. We constructed K562/CD44 cells by transfection of an EGFP-SV40-CD44 plasmid, and adriamycin-resistant ability was confirmed by detecting migration and apoptosis-related proteins and mRNA expression using Western blotting and Real-time PCR respectively. K562/CD44 cells were generated by the transfection of an EGFP-SV40-CD44 plasmid with high CD44 expression. mRNA expression levels of CD44 and P-glycoprotein (P-gp), along with the proliferation rate, were increased, while the apoptosis rate of K562/CD44 cells was decreased. Migration-associated proteins such as MMP-2 and MMP-9 were upregulated, whereas apoptosis-related protein Bax was downregulated and Bcl-2 protein was not significantly altered in the K562/CD44 cells. CD44 might be involved in adriamycin resistance via regulation of P-gp, MMP-2, MMP-9, and Bcl-2/Bax.

B7-H4 induces epithelial–mesenchymal transition and promotes colorectal cancer stemness

B7-H4 is a unique negative regulator of T cells that is typically significantly overexpressed in various carcinomas and is associated with poor prognosis. However, the effects of B7-H4 expression on epithelial–mesenchymal transition (EMT) and cancer stemness of colorectal cancer (CRC) are not entirely clear. In the present study, we used tissue samples from 98 patients with CRC and CRC cell lines to determine the clinicopathological significance of B7-H4 in CRC and its effects on CRC stemness. We performed immunohistochemical staining; immunofluorescence imaging; western blotting; and tumor sphere formation, wound healing, transwell migration, and in vivo tumorigenesis assays. B7-H4 expression was upregulated in CRC tissues and was associated with lymph node metastasis, distant metastasis, clinical stage, a shorter overall survival rate, and disease-free survival rate. Cox regression analyses indicated that B7-H4 is an independent poor prognostic factor for CRC. In addition, B7-H4 expression was correlated with the expression of EMT-related proteins and cancer stemness-related proteins. Moreover, immunohistochemical and immunofluorescence analyses revealed that B7-H4 was correlated with CD133 and CD44 expression levels in both CRC tissues and HT29 and HCT116 cell lines. Conversely, B7-H4 knockdown downregulated the expression of EMT- and cancer stemness-related proteins, while inhibiting tumor spheroid formation, cell migration, and invasion of CRC cell lines. These results indicate that B7-H4 can promote EMT and may be a novel stem cell marker, suggesting its potential as a prognostic biomarker for CRC.

Co-expression of CD24 and Hsp70 as a prognostic biomarker for lung cancer

Lung cancer is one of the most common malignant neoplasms worldwide. CD24 is a marker of tumor stem cells that plays an important role in tumorigenesis. Hsp70 is an important molecular chaperone. However, the co-expression and interaction of CD24 and Hsp70, as well as the significance for the prognosis of lung cancer are still unclear. The expression levels of CD24 and Hsp70 were detected by immunohistochemistry and their correlation was analyzed. The expression levels of CD24 mRNA and protein were examined using qRT-PCR and western blotting in SPCA1, A549, H1975, and H1650 cell lines. A CD24-overexpressing cell model was established. The interaction between CD24 and Hsp70 was verified by co-immunoprecipitation and western blotting. CD24 and Hsp70 expression were significantly higher in lung cancer tissues than in adjacent tissues (CD24: p=0.008; Hsp70: p<0.001). CD24 protein expression showed a positive correlation with lymph node metastasis, TNM stage, and vascular cancer thrombus. Hsp70 protein expression showed a positive correlation with differentiation, lymph node metastasis, and TNM stage. CD24 and Hsp70 high expression were also correlated with poor survival. The positive co-expression rate of CD24 and Hsp70 in lung cancer tissues was 52.7% (49/93). CD24 and Hsp70 expression in lung cancer were positively correlated (r=0.368, p<0.001), and co-immunoprecipitation was verified that both endogenous and exogenous CD24 co-precipitated with Hsp70 directly or indirectly. When Hsp70 inhibitor VER15508 was added to A549 cells, Hsp70 and CD24 protein expression were significantly decreased. The present study demonstrated that CD24 and Hsp70 were highly expressed in lung cancer tissues, and associated with invasion, metastasis, and poor survival. Hsp70 may regulate CD24 expression. Co-expression of CD24 and Hsp70 may be a prognostic biomarker for lung cancer.

Expression of Biomarkers (CD44, COX-2, TWIST, VEGF-C and LOX) in Esophageal Cancer: Implication for Prophylactic Radiation Field

<i>Background and objective</i>: If it becomes possible to reduce the prophylactic radiation field by using a personalized radiation field, radiation therapy of esophageal cancer may become safer. In this study, we examined the relationship between expression levels of biomarkers (CD44, COX-2, TWIST, VEGF-C and LOX) and lymph node metastasis and distant metastasis. <i>Materials and methods</i>: We performed retrospective analysis of 14 patients who underwent radical surgery for esophageal cancer between 2009 and 2011 in Yamagata University Hospital. Stain index was used for assessment of biomarker expression. The relationships of stain index with regional recurrence and distant metastasis were evaluated. <i>Results</i>: All patients had squamous cell carcinoma. The median follow-up duration was 38 months. Distant failure was significantly related to low COX-2 expression in the deep part of the tumor (stain index <2.8; p=0.0479). Pathological node-positive or regional recurrence was significantly related to low VEGF-C expression (stain index <0.3; p=0.0418). Patients with low CD44 expression in the surface of the tumor tended to have better reginal control (stain index <0.3; p=0.0902). <i>Conclusions</i>: COX-2, VEGF-C, and CD44 expression levels might have potential to enable omission of the prophylactic radiation field. Further analysis using biopsy samples is required for application to radical radiotherapy.

Effect of short-term in vitro antibiotic administration into the human umbilical cord tissue on cfu count and mesenchymal stem cell immunophenotype

Mesenchymal stem cells play important roles in regenerative and reparative medicine applications. Their differentiation and proliferation abilities both in the human body and in the laboratory, environment have been revealed through many clinical and pre-clinical trials. Umbilical cord tissue is an important source of mesenchymal stem cells and has become a popular option in cord blood banking, especially in the last decade. As done for the cord blood, the steps of obtaining, transferring, processing, and cryopreserving umbilical cord tissue are defined by standard procedures. However, there is limited information about the microbiological contamination rate of tissues and their decontamination before the procedure. This study aimed to assess the effect of in vitro short-term antibiotic treatment applied to the umbilical cord tissue obtained via superficial ethanol disinfection immediately after birth on the formation of colony-forming units (CFUs) and immunophenotype of the mesenchymal stem cells. For this purpose, tissues obtained from 10 individuals were evaluated in three different groups that included the control group and groups of tissues with 5- and 10-min antibiotic treatment. There was no significant difference among the groups in the expression levels of CD45, CD34, CD73, CD105, and CD44, which are the markers used in immunophenotyping. Similarly, no difference was found in CFU formation either. The present study has revealed for the first time that an antibiotic preparation used in in vitro cell culture for cord tissue banking can be used safely in tissue preparation and banking stages. Our results show that short-term in vitro antibiotic administration can contribute to the standard formation during decontamination/disinfection stages in cord tissue banking.

Correlation between inflammatory cytokines and liver cancer stem cell markers in DEN-induced liver cancer rats.

To study the association of inflammatory factors and hepatocarcinoma stem cells of induced liver cancer rats. 30 SD male healthy rats were selected. 10 rats were given water as normal control group. 10 rats only were implemented laparotomy as sham operation group. The remaining 10 rats were the liver cancer model group and treated with diethylnitrosamine (DEN) to induce liver cancer. Real-time quantitative PCR was used to detect the related inflammatory factors in HCC tissues, including interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-β1 (TGF-β), human interleukin-1α (IL-1α), human interleukin 1β (IL-1β) and levels of hepatocarcinoma stem cells indicators CD90, CD133, Alpha-fetoprotein (AFP). Correlation analysis was used to analyze the correlation between inflammatory factors and hepatocarcinoma stem cells markers CD90 and CD133. The expression levels of IL-6, MCP-1 and TGF-β of HCC tissues in liver cancer model group were significantly higher than those in the control group and the sham operation group. The expression levels of CD90 and CD133 of tissues in the liver cancer model group were significantly higher than those in the control group and the sham operation group. The differences were statistically significant (p<0.001). By inhibiting related inflammatory factors, the growth, migration and invasion of liver cancer cells were significantly inhibited, and apoptosis was promoted. Correlation analysis results showed that the expression changes of IL-6, MCP-1 and TGF-β were significantly positively correlated with CD90 up-regulation (p<0.05), while the expression changes of IL-6, MCP-1 and TGF-β were significantly positively correlated with CD133 up-regulation (p<0.05). The inflammatory factors IL-6, MCP-1 and TGF-β are closely related to hepatocarcinoma stem cells, which play an important role in promoting the occurrence and deterioration of liver cancer.

Targeting Breast Cancer Using Hyaluronic Acid-Conjugated Liposomes Triggered with Ultrasound.

The successful targeting of tumors can be achieved by conjugating targeting moieties to nanoparticles. These modifications allow nannocarriers to achieve greater targeting specificity through binding to specific receptors overexpressed on the surface of the tumor cells. In this study, pegylated liposomes encapsulating the model drug/dye calcein and conjugated to hyaluronic acid (HA) molecules were successfully synthesized, and their ability to target HA receptors overexpressed on a breast cancer cell line was investigated in vitro. Low-frequency ultrasound (LFUS), applied at three different power densities (6.2, 9, and 10 mW/cm²) were used to trigger the release of the entrapped calcein. Both the control and HAconjugated liposomes showed similar release profiles. HA conjugation to the liposomes resulted in a significant increase in calcein uptake by the breast cancer cell line MDA-MB-231 known for its CD44 (HA receptor) overexpression, while such an effect was not recorded with NIH-3T3, an embryonic mouse fibroblast, with low levels of CD44 expression. The application of low LFUS showed a significant enhancement of calcein uptake by MDA-MB-231 cells from our liposome compared to calcein uptake without cell exposure to ultrasound. These findings suggest that combining HA-conjugated liposomes with ultrasound is a promising drug delivery platform in breast cancer treatment.

Confirmation of differentiation clusters’ and endoglin markers preset in porcine buccal mucosa cells

Abstract Several genes, namely CD44, CD90, CD105 and PCNA may be important in differentiation of porcine mucosa cell cultures. These genes are, inter alia, responsible for cell adhesion to extracellular matrix and its constituent secretion, cytoskeleton organization, epithelial to mesenchymal transition or proper course of DNA replication. A total of 20 pubertal crossbred Landrace gilts bred on commercial farms were used to produce buccal mucosa cultures, which were harvested on the 7th, 15th and 30th day after initiation of the culture. Expression levels of CD44, CD90, CD105 and PCNA were evaluated employing Real-Time Quantitative Polymerase Chain Reaction. CD44, CD90 and PCNA showed an unchanged expression pattern. Expression of CD44 on day 7 was the highest of all factors measured. The greatest difference between the measurement on 7th and 30th day was found in the PCNA gene. These results broaden the understanding of the transcriptome changes in porcine buccal mucosa cells for the duration of in vitro cultivation. Nevertheless, it is very important to consider that the in vitro conditions do not fully reflect the changes taking place in the living organism. It appears that tissues of the oral cavity possess high regenerative potential, and constitute suitable model for wound healing investigation. Running title: Confirmation of differentiation clusters’ and endoglin markers preset in porcine buccal mucosa cells

ESM1/HIF‑1α pathway modulates chronic intermittent hypoxia‑induced non‑small‑cell lung cancer proliferation, stemness and epithelial‑mesenchymal transition.

Obstructive sleep apnea (OSA) is a sleep‑related disorder characterized by chronic intermittent hypoxia (CIH). Previous studies have found that intermittent hypoxia promotes drug resistance, cell proliferation, migration and invasion in non‑small cell lung cancer (NSCLC). Endothelial cell‑specific molecule‑1 (ESM1) is a molecule shown to be overexpressed in several types of tumors. The purpose of this study was to investigate the correlation between CIH and ESM1 and their potential roles in the progression of NSCLC. Tumorspheres, cell viability and colony formation assays were used to evaluate cell proliferation. The expression levels of cancer stem cell (CSC) markers CD44, CD133, OCT4 and SOX2 were measured with western blotting and/or RT‑qPCR. Transwell assays were applied to assess cell migration and invasion. Changes in the expression levels of epithelial‑mesenchymal transition (EMT)‑associated proteins were also detected by western blotting. The results indicated that CIH enhanced lung cancer stem cell (LCSC) NSCLC progression by promoting stemness, drug resistance, cell proliferation, migration and invasion via the ESM1/HIF‑1α pathway. Unexpectedly, inhibition of ESM1 reversed the CIH‑involved negative effects on LCSCs and in a mouse model. ESM1 therefore appears to be crucial mediator of CIH‑mediated lung cancer progression.

Site-Directed Mutations of Calcium-Binding Sites Contribute to Reducing the Immunoreactivity of the EF-Hand Sarcoplasmic Calcium-Binding Protein in Scylla paramamosain.

In order to reduce the immunoreactivity of sarcoplasmic calcium-binding protein (SCP), site-directed mutations were used to replace key amino acids in the conformational epitopes and calcium-binding sites. The mutant SCPs (mSCPs) were expressed in Escherichia coli, and their immunoreactivities were analyzed using iELISA and basophil activation assays. Furthermore, the structural changes of mSCPs were determined from the circular dichroism spectra. The iELISA results showed that mSCPs could effectively inhibit the binding of wild-type SCP (wtSCP) to sensitive serum, with inhibition rates that reached 90%. Moreover, mSCPs could downregulate the expression levels of CD63 and CD203c on the basophil surface. Compared with wtSCP, the peak values were significantly changed, and the calcium binding ability was impaired, which explained the decline in immunoreactivities of the mSCPs. All of the data confirmed that this approach was effective in reducing the immunoreactivity of SCP and could be applied to other shellfish allergens.

Mir-193a-5p Replacement Can Alter Metastasis Gene Expression in Breast Adenocarcinoma Cells In Vitro

Background: Recent evidence presented the significant role of the microRNA-193 (miR-193) family in biological processes by the contribution of specific targeting, which mainly display as a tumor suppressor in various cancers. In the present study, we evaluated the effect of miR-193a-5p replacement on some metastasis gene expression in metastatic breast cancer (BC) cells. Methods: For this purpose, firstly, the quantitative real-time polymerase chain reaction (qRTPCR) was used to detect the miR-193a-5p expression in the MDA-MB-231 BC cell line. Subsequently, miR-193a-5p was transfected into the cells, and the expression levels of ROCK1 (Rho‑associated, coiled‑coil containing protein kinase 1), CXCR4 (Chemokine Receptor-4), CD44, and vimentin genes were evaluated by qRT-PCR. Results: The expression level of miR-193a-5p strongly reduced in MDA-MB-231 cells. Interestingly, the ROCK1 (P &lt; 0. 001), CD44 (P &lt; 0.0001), CXCR4 (P &lt; 0. 001) and vimentin (P &lt; 0. 001) expression levels significantly decreased following miR-193a-5p transfection in MDA-MB-231 BC cells. Conclusion: To conclude, it seems that miR-193a-5p restoration can attenuate the metastatic behavior of BC cells in vitro through decreased expression level of metastasis-related genes and may constitute an effective novel therapeutic strategy in miRNA-replacement therapy and treatment of metastatic breast adenocarcinoma in the future.

Changes in Human Fat Injected Alongside Hyaluronic Acid in the Backs of Nude Mice.

Cross-linked hyaluronic acid (HA) is an active anti-aging cosmetic filler. The combination of cross-linked HA and preadipocytes or adipose-derived stem cells has been previously investigated, but the effects of agglomerated cross-linked HA injection on the vascularization of fat grafts remain unclear. The aim of this study was to explore the effects of agglomerated cross-linked HA injection on the vascularization of fat grafts. The backs of nude mice were divided into 4 regions that received different treatments: nothing (control group), agglomerated Biohyalux (HA group), agglomerated fat (FAT group), and lumps formed by the sequential injection of Biohyalux and fat (HA/FAT group). Samples were collected after 1 month for weighing and hematoxylin and eosin staining, immunohistochemistry, image analysis, and Western blotting. The weight of fat and the mean number of adipocytes in the HA/FAT group did not significantly differ from those in the FAT group. No living tissue was found in agglomerated HA. Some tiny HA particles were surrounded by tissue rich in blood vessels. The expression levels of CD31 and vascular endothelial growth factor (VEGF) in the HA/FAT group were higher than those in the FAT group, but the difference was only significant for VEGF expression. Cross-linked HA had minimal effect on the early retention rate of surrounding fat grafts, but enhanced their vascularization. Fat grafts should be not injected into lumps of cross-linked HA. Therefore, agglomerated cross-linked HA should be dissolved before fat transplantation.

Limited Mitochondrial Activity Coupled With Strong Expression of CD34, CD90 and EPCR Determines the Functional Fitness of ex vivo Expanded Human Hematopoietic Stem Cells

Ex vivo expansion strategies of human hematopoietic stem cell (HSC) grafts with suboptimal stem cell dose have emerged as promising strategies for improving outcomes of HSC transplantation in patients with hematological malignancies. While exposure of HSCs to ex vivo cultures expands the number of phenotypically identifiable HSCs, it frequently alters the transcriptomic and metabolic profiles, therefore, compromising their long-term (LT) hematopoietic reconstitution capacity. Within the heterogeneous pool of expanded HSCs, the precise phenotypic, transcriptomic and metabolic profile and thus, the identity of HSCs that confer LT repopulation potential remains poorly described. Utilizing valproic acid (VPA) in ex vivo cultures of umbilical cord blood (UCB)-CD34+ cells, we demonstrate that expanded HSCs phenotypically marked by expression of the stem cell markers CD34, CD90 and EPCR (CD201) are highly enriched for LT-HSCs. Furthermore, we report that low mitochondrial membrane potential, and, hence, mitochondrial activity distinguishes LT-HSCs within the expanded pool of phenotypically defined HSCs. Remarkably, such reduced mitochondrial activity is restricted to cells with the highest expression levels of CD34, CD90 and EPCR phenotypic markers. Together, our findings reveal that high expression of CD34, CD90 and EPCR in conjunction with low mitochondrial activity is critical for identification of functional LT-HSCs generated within ex vivo expansion cultures.

Identification of regulatory T cell molecules associated with severity of multiple sclerosis

Background: Regulatory CD4+ T cells (Tregs) exhibit functional alterations in patients with multiple sclerosis (MS). Transforming growth factor (TGF)-β is a key regulator of Treg development and function. Objective: The objective of this study is to determine whether the expression of functionally relevant TGF-β-regulated molecules is altered in Tregs from patients with MS. Methods: Expression of nine Treg markers was analyzed by multi-color flow cytometry in CD4+ T cells and Treg subpopulations of 31 untreated MS patients and age- and sex-matched healthy donors (HDs). Correlations between Treg marker expression and clinical variables were sought. Results: Expression of the transcription factor Helios, which defines thymic-derived Tregs, was decreased in this Treg subpopulation. The frequency of peripherally generated Tregs was increased in patients with MS, particularly in patients with progressive MS. Low frequencies of thymic-derived Tregs were associated with magnetic resonance imaging (MRI) lesion-burden and a high relapse rate. Four surface markers associated with TGF-β signaling (ABCA1, BTLA, DNAM-1, and GARP) were differentially expressed on Tregs from patients with MS and HDs. Expression levels of CD73, CD103, ABCA1, and PAR2 showed strong correlations with disease severity. Conclusion: We have identified novel markers abnormally expressed on Tregs from patients with MS that could detect patients with severe disease.

HER2 overexpression triggers the IL-8 to promote arsenic-induced EMT and stem cell-like phenotypes in human bladder epithelial cells

Arsenic is a natural chemical element that is strongly associated with bladder cancer. Understanding the underlying mechanisms behind the association between arsenic and bladder cancer as well as identifying effective preventive interventions will help reduce the incidence and mortality of this disease. The epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties play key roles in cancer development and progression. Here, we reported that chronic exposure to arsenic resulted in EMT and increased levels of the CSC marker CD44 in human uroepithelial cells. Furthermore, IL-8 promoted a mesenchymal phenotype and upregulated CD44 by activating the ERK, AKT and STAT3 signaling. Phosphorylation of the human epidermal growth factor receptor 2 (HER2) was key for arsenic-induced IL-8 overexpression and depended on the simultaneous activation of the MAPK, JNK, PI3K/AKT and GSK3β signaling pathways. We also found that genistein inhibited arsenic-induced HER2 phosphorylation and downregulated its downstream signaling pathways, thereby inhibiting progression of EMT, and reducing CD44 expression levels. These results demonstrate that the HER2/IL-8 axis is related to the acquisition of an EMT phenotype and CSCs in arsenic-treated cells. The inhibitory effects of genistein on EMT and CSCs provide a new perspective for the intervention and potential chemotherapy against arsenic-induced bladder cancer.

Simvastatin accelerates the healing process of burn wound in Wistar rats through Akt/mTOR signaling pathway

Statins, apart from cholesterol-lowering properties, have wound healing effects. Hereby, we aimed to assess the impact of Simvastatin (SMV), one of the most commonly used statins, on Akt/mTOR signaling pathway during burn wound healing process. After creating a second-degree burn on the dorsal area of adult male Wistar rats (n = 60), they were randomly divided into the control, SMV, vehicle of Simvastatin (SMV-Veh), Rapamycin (RM), vehicle of Rapamycin (RM-Veh), and combined SMV and RM (SMV + RM) groups. The animals were sacrificed on the 7th and 14th post-burn days and wound tissue samples were collected for histologic, immunohistochemical, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot investigations. Rapamycin (RM) was also used to treat animals as an mTOR inhibitor. Topical administration of SMV resulted in a faster healing rate, elevated collagen deposition, and increased myofibroblast population compared to other experimental groups. Moreover, qRT-PCR findings showed that the wounds treated with SMV alone had the highest expression levels of CD31, VEGF, Akt, mTOR, and p70S6K after 7 and 14 days of burn model (p < 0.001). According to western blot findings, daily topical treatment with SMV further increased protein levels of P-AktThr308, P-mTORSer2448, and P-p70S6 KThr389 compared with other treatments, at both follow-up time points (p < 0.001). In contrast, inhibition of Akt/mTOR signaling pathway by RM reduced SMV-induced wound healing process. Seemingly, SMV promotes burn wound healing, at least in part, through activating Akt/mTOR signaling pathway, suggesting topically applied SMV as an alternative therapeutic approach for managing burn wound healing.

MiR-326 inhibits the cell proliferation and cancer stem cell-like property of cervical cancer in vitro and oncogenesis in vivo via targeting TCF4.

BackgroundCervical cancer ranks as one of the most prevalent female malignancies globally, and its treatment with new targets has been the focus of current research. The present study set out to investigate the function of microRNA-326 (miR-326) in vitro and in vivo and to verify the direct targeting of transcription factor 4 (TCF4) by miR-326.MethodsThe detection of messenger RNA (mRNA) expressing miR-326 and TCF4 in cervical cancer cell lines and tumor samples was conducted using quantitative real-time polymerase chain (qRT-PCR). A dual-luciferase reporter assay was carried out to detect the target relationship of miR-326 with TCF4. A Cell Counting Kit-8 (CCK-8) assay was employed to detect the effect of miR-326 on CasKi cell viability. Flow cytometry and western blotting were employed to examine the effects of miR-326 on cancer stem cell (CSC)-like property. Tumor weight was measured in orthotopic xenograft mouse models. Immunohistochemistry was employed to analyze the protein expression levels of Ki-67, proliferating cell nuclear antigen (PCNA), CD44, and SRY-box 4 (SOX4).ResultDownregulation of the mRNA expression levels of miR-326 was observed in cervical cancer cell lines and tumor tissue, while the levels of TCF4 were upregulated. The dual-luciferase reporter assay revealed binding of miR-326 to the three prime untranslated region (3'-UTR) of TCF4. In vitro assays demonstrated that miR-326 inhibited CasKi cell proliferation through regulating TCF4. miR-326 also suppressed the CSC-like property of CasKi cells by targeting TCF4. Furthermore, the protein expression levels of cyclin D1, β-catenin, and c-Myc were decreased when miR-326 was added to TCF4-transfected cells. In vivo assays demonstrated that miR-326 inhibited tumor weight, growth, and the protein expression levels of Ki-67, PCNA, CD44, SOX4, and β-catenin.ConclusionsmiR-326 acted in a tumor-suppressive manner through its regulation of TCF4, and has potential as a biomarker or therapeutic target for cervical cancer.

Extracellular vesicles-encapsulated microRNA-10a-5p shed from cancer-associated fibroblast facilitates cervical squamous cell carcinoma cell angiogenesis and tumorigenicity via Hedgehog signaling pathway.

Cancer-associated fibroblast (CAF) secretes extracellular vesicle (EV)-encapsulated microRNAs (miRNAs) which have been underlined great promise for therapeutic target for diseases and cancers. Our study aimed to explore the role of EV-encapsulated miR-10a-5p from CAFs in angiogenesis in cervical cancer. Expression of miR-10a-5p in clinical samples of cervical cancer and cancer cells was detected by in situ hybridization and RT-qPCR. Results demonstrated that miR-10a-5p expression was upregulated in both cancer tissues and cells. CAFs and normal fibroblasts (NFs) from cervical cancer patient tissues were characterized under transmission electron microscopy, followed by EV isolation from CAFs. The EVs labeled with PKH67 were cultured with cervical squamous cell carcinoma (CSCC) cell line (SiHa) and HUVECs. Data indicated that CAF-EVs were internalized by cancer cells and promoted cell proliferation and tube formation. CAF-EVs then were transfected with miR-10a-5p inhibitor and then injected into nude mice. While injection of CAF-EVs promoted tumor growth and increased VEGFR and CD31 expression level, miR-10a-5p inhibitor-treated CAF-EVs resulted in decreased tumor volume and amount of vessel around tumor. Of note, dual-luciferase reporter gene assay and bioinformatic analysis indicated TBX5 as a target gene of miR-10a-5p. Moreover, EV-derived miR-10a-5p promoted angiogenesis in vivo and in vitro through activation of Hedgehog signaling via downregulation of TBX5. Taken altogether, miR-10a-5p in CAF-EVs promoted CSCC cell angiogenesis and tumorigenicity via activation of Hh signaling by inhibition of TBX5, providing insight into novel treatment based on miR-10a-5p against CSCC.

SiRNA-mediated silencing of CD44 delivered by Jet Pei enhanced Doxorubicin chemo sensitivity and altered miRNA expression in human breast cancer cell line (MDA-MB468).

CD44, as a superficial cellular glycoprotein, is an essential factor in cell-cell and cell-matrix interaction. The CD44 expression level has been substantially up-regulated in breast cancer, and this upregulation facilitates tumor proliferation and angiogenesis. This study aims to evaluate the combination therapy of Jet Pei/CD44-specific-siRNA/doxorubicin in breast cancer MDA-MB468 cell line. The MTT assay, wound healing test, colony formation assay, DAPI staining, and flow cytometry were performed to investigate the tumoral cell viability, migration, clonogenesis, and apoptosis progression. The quantitative real-time PCR (qRT-PCR) was performed to demonstrate the CD44 expression level. Finally, the effect of CD44 silencing on the expression of VEGF, CXCR4, MMP9, and MiR-142-3p was measured. The combination of CD44-specific-siRNA with doxorubicin decreased tumoral metastasis, proliferation, invasion, and migration, and increased apoptosis in MDA-MB468 cells. In conclusions,CD44 can serve as a therapeutic target in breast cancer. Moreover, the combination therapy of CD44-specific-siRNA with doxorubicin can be a promising treatment for patients with breast cancer.