Gene Expression In Cell Lines Research Articles

Study on the effect and mechanisms of SIGLEC-15 on laryngeal squamous cell carcinoma

Objective: To investigate the effect of sialic acid combined with immunoglobulin-like lectin 15 (SIGLEC-15) on laryngeal squamous cell carcinoma (LSCC) and underlying mechanisms. Methods: The Cancer Genome Atlas (TCGA), Cancer Cell Line Encyclopedia (CCLE) and Gene Expression Profiling Interactive Analysis (GEPIA2) databases were used for bioinformatics analysis. Cell Counting Kit-8 (CCK8), flow cytometry, and Transwell method were used respectively to detect proliferation, apoptosis, cell cycle, metastasis and invasion behaviors of the cells. Gene chip method was used for detecting up-regulated and down-regulated genes and performing enrichment analysis of differential genes. Western Blotting (WB) and Quantitative Real-time PCR (qPCR) were used to detect the expressions of proteins. Tumor formation experiments in nude mice were used to detect the effect of SIGLEC-15 on the growth of transplanted tumors. Wilcoxon rank sum test, t-test and Log-rank test were used for statistical analysis. Results: SIGLEC-15 was highly expressed in laryngeal squamous cell carcinoma and closely related to life in being. TU686SIGLEC-15-with low expression of SIGLEC-15 was constructed. Compared to TU686SIGLEC-15+, TU686SIGLEC-15-showed significantly reduced activities of proliferation (48 h: 1.32±0.23 vs. 2.56±0.37, t=6.59, P<0.05), migration (1 036.52±51.22 vs. 1 819.62±180.24, t=7.22, P<0.05) and invasion (469.21±112.25 vs. 961.45±102.03, t=7.85, P<0.05); early increased apoptosis ((23.27±1.12)% vs. (5.64±1.61)%, t=11.32, P<0.05); blocked cell cycle at G0/G1 ((59.32±3.65)% vs. (35.46±3.57)%, t=9.85, P<0.05); the knockdown of SIGLEC-15 resulted in up-regulation of 864 genes, down-regulation of 357 genes, with significant changes in molecules of cell cycle, apoptosis and JAK/STAT signal pathways, and the expressions of p-JAK2, p-STAT3, Caspase-3, Bad, Bcl-2, and Cyclin d1 proteins. Tumor formation experiments in nude mice showed that at 8 weeks after the tumors were implanted, the growth transplanted tumors of TU686 SIGLEC-15-cell group was slower than that of TU686 SIGLEC-15+cell group, with significant difference in the mean tumor weights between two groups ((0.382±0.054) g vs. (1.277±0.126) g, t=8.44, P<0.05), while the expression of SIGLEC-15 was lower in the transplanted tumors of SIGLEC-15 knockdown group compared to control group, with significant difference(11.29±2.17 vs. 36.25±7.56, t=9.28, P<0.05). Conclusion: SIGLEC-15 is highly expressed in LSCC and can promote the progression of LSCC through the JAK2-STAT3 pathway.

EMT-Related Gene Signature: A New Method for Personalized Risk Assessment in Patients with Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death worldwide, accounting for more than 700,000 deaths annually. Epithelial-mesenchymal transition (EMT) is posited to contribute to HCC progression. We, therefore, aimed to elucidate the relationship between EMT-related gene (ERG) expression and prognosis in patients with HCC. Methods: Univariate and multivariate Cox regression analyses were performed to identify prognostic-related differentially expressed EMT-related genes (DE-ERGs). Time-dependent receiver operating characteristic and Kaplan-Meier curves were used to assess the prognostic performance of the EMT-related signature. pRRophetic R package was used to evaluate sorafenib sensitivity in the GSE14520 cohort. Gene expression in Huh7 and L02 cell lines was detected by real-time PCR and Western blotting. Differential expression of the three genes between HCC tissues and normal tissues was validated using immunohistochemical analyses. Results: Of the 76 identified DE-ERGs, 29 were associated with overall survival. Three prognosis-related ERGs (GOLM1, SOX4, and CD14) were screened out by multivariate Cox regression. A gene signature was identified based on the three prognostic-related ERGs. HCC patients with a low-risk score had a better prognosis and were more sensitive to sorafenib compared to those with a high-risk score. Moreover, we further confirmed increased expression of GOLM1 and SOX4, and decreased expression of CD14, in liver cancer cell line and HCC tissue. Conclusions: The results of the present study demonstrate the utility of an ERG signature as a potential biomarker informing prognosis in patients with HCC, which may contribute to the implementation of personalized therapies.

Newly identified breast luminal progenitor and gestational stem cell populations likely give rise to HER2-overexpressing and basal-like breast cancers

Breast Cancer (BrC) is a common malignancy with genetically diverse subtypes. There is evidence that specific BrC subtypes originate from particular normal mammary cell populations. However, the cell populations that give rise to most BrC subtypes are unidentified. Several human breast scRNAseq datasets are available. In this research, we utilized a robust human scRNAseq dataset to identify population-specific marker genes and then identified the expression of these marker genes in specific BrC subtypes. In humans, several BrC subtypes, HER2-enriched, basal-like, and triple-negative (TN), are more common in women who have had children. This observation suggests that cell populations that originate during pregnancy give rise to these BrCs. The current human datasets have few normal parous samples, so we supplemented this research with mouse datasets, which contain mammary cells from various developmental stages. This research identified two novel normal breast cell populations that may be the origin of the basal-like and HER2-overexpressing subtypes, respectively. A stem cell-like population, SC, that expresses gestation-specific genes has similar gene expression patterns to basal-like BrCs. A novel luminal progenitor cell population and HER2-overexpressing BrCs are marked by S100A7, S100A8, and S100A9 expression. We bolstered our findings by examining SC gene expression in TN BrC scRNAseq datasets and S100A7-A9 gene expression in BrC cell lines. We discovered that several potential cancer stem cell populations highly express most of the SC genes in TN BrCs and confirmed S100A8 and A9 overexpression in a HER2-overexpressing BrC cell line. In summary, normal SC and the novel luminal progenitor cell population likely give rise to basal-like and HER2-overexpressing BrCs, respectively. Characterizing these normal cell populations may facilitate a better understanding of specific BrCs subtypes.

Expression of the hedgehog signalling pathway and the effect of inhibition at the level of smoothened in canine osteosarcoma cell lines.

Osteosarcoma (OSA) is the most common malignant bone cancer in dogs. Canine and human OSA share several features, including tumour environments, response to traditional treatment, and several molecular pathways. Hedgehog (Hh) signalling is known to contribute to tumorigenesis and progression of various cancers, including human OSA. This study aimed to identify the role of the Hh signalling pathway in canine OSA cell lines, including Abrams, D17, and Moresco, focusing on the signal transducer Smoothened (SMO). mRNA and protein levels of Hh pathway components, including SHH, IHH, SMO, and PTCH1, were aberrant in all examined OSA cell lines compared with canine osteoblast cells. The SMO inhibitor cyclopamine significantly decreased cell viability and colony-forming ability in the canine OSA cell lines in a dose-dependent manner. Moresco cells, which expressed the highest level of SMO protein, were the most sensitive to the anticancer effect of cyclopamine among the three canine OSA cell lines tested. Hh downstream target gene and protein expression in canine OSA cell lines were downregulated after cyclopamine treatment. In addition, cyclopamine significantly increased apoptotic cell death in Abrams and Moresco cells. The findings that Hh/SMO is activated in canine OSA cell lines and cyclopamine suppresses OSA cell survival via inhibition of SMO suggest that the Hh/SMO signalling pathway might be a novel therapeutic target for canine OSA.

Response of Osteosarcoma Cell Lines to a Novel CHD1L Inhibitor

Osteosarcoma (OSA) is a primary tumor of bone that occurs in both humans (hOSA) and dogs (cOSA) and has been recognized as being very similar across these species. In fact, OSA is probably the best example where surgical, radiological and drug trials have been translated from testing in pet dogs with OSA to trials in human patients. In both humans and dogs treated for OSA, the primary treatment modalities are surgery and cytotoxic chemotherapy with doxorubicin and platinum‐based drugs standard for both and methotrexate also commonly added in humans. The treatment of OSA in both dogs and humans has been virtually unchanged in the last 40 years leading to stagnation in outcome improvement with lung metastasis and drug resistance being the primary mechanism of disease progression and patient morbidity and mortality. Thus, drug treatments that can effectively treat OSA and inhibit disease progression at metastatic sites are badly needed. Chromodomain Helicase DNA‐binding protein 1‐Like (CHD1L), a.k.a. Amplified in Liver Cancer (ALC1), is an oncogene that was originally identified in hepatocellular carcinoma. It has recently gained more attention with regards to resistance to PARP inhibitors in BRCA‐mutant cancers as well as a potential target for anticancer drug therapy. Studies have also suggested that CHD1L plays a role in resistance to platinum‐based chemotherapy agents through the modulation of DNA repair processes. This has led to a great deal of interest into drugs that can inhibit CHD1L. The first and only known small molecule CHD1L inhibitors (CHD1Li) have recently been reported by Dr. LaBarbera’s lab in the CU School of Pharmacy (Mol Cancer Ther 19:1598, 2020) who has an active medicinal chemistry program developing these molecules. Preliminary analysis of CHD1L gene expression in cOSA cell lines and tumors shows that expression levels are significantly higher in cOSA cell lines as opposed to other canine cell lines and that CHD1L expression is higher in cOSA tumors as opposed to normal bone. Treatment of cOSA and hOSA cell lines with a CHD1Li show a steep dose‐response of inhibition of cell proliferation and induction of cell death with IC50 values ranging between 2 and 3 μM and LD50values between 3 and 4 μM. These μM drug concentrations are safely achievable in mice dosed daily with this drug based on in vivo studies. The results suggest that CHD1L inhibition should be further pursued for therapeutic development in OSA and that cOSA and hOSA cell lines have a similar response to these agents.

Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells.

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.

Gene expression analysis of Aerva javanica and Parkinsonia aculeata synthesized gold nanoparticles in Cervical and Liver cancer cell lines

Chemotherapy is effective in many cancer treatments compare to surgery and radiation. Due to its side effects and multidrug resistance, Nanoparticles synthesized from some of the medicinal plants are one of the prominent alternatives for the treatment of various diseases. In our present study, Synthesized the AuNPs from Aerva javanica and Parkinsonia aculeata extract and studied the p53, caspase-3, Caspase-9 and NF-kB gene expression in HeLa and HepG2 cell lines by Real-time PCR. p53 gene down regulation was observed in Aerva javanica AuNPs treated HeLa cells compared to HepG2 cells and caspase-3, Caspase-9 and NF-kB gene expression levels were not changed in both cell lines. Parkinsonia aculeata AuNPs treated HeLa and HepG2 cells not showed any changes in gene expression levels of studied genes. Based on the present results concluding the Aerva javanica AuNPs are effective anti-cancer activity on cervical cancer cells.

Influence of Periodontal Ligament Stem Cell-Derived Conditioned Medium on Osteoblasts.

Mesenchymal stem cells (MSC) are involved in the regeneration of various missing or compromised periodontal tissues, including bone. MSC-derived conditioned medium (CM) has recently been explored as a favorable surrogate for stem cell therapy, as it is capable of producing comparable therapeutic effects. This study aimed to evaluate the influence of periodontal ligament stem cells (PDLSC)-CM on osteoblasts (OB) and its potential as a therapeutic tool for periodontal regeneration. Human PDLSC were isolated and characterized, and CM from these cells was collected. The presence of exosomes in the culture supernatant was observed by immunofluorescence and by transmission electron microscopy. CM was added to a cultured osteoblastic cell line (Saos-2 cells) and viability (MTT assay) and gene expression analysis (real-time PCR) were examined. A cell line derived from the periodontal ligament and showing all the characteristics of MSC was successfully isolated and characterized. The addition of PDLSC-CM to Saos-2 cells led to an enhancement of their proliferation and an increased expression of some osteoblastic differentiation markers, but this differentiation was not complete. Saos-2 cells were involved in the initial inflammation process by releasing IL-6 and activating COX2. The effects of PDLSC-CM on Saos-2 appear to arise from a cumulative effect of different effective components rather than a few factors present at high levels.

CRLF2 overexpression defines an immature-like subgroup which is rescued through restoration of the PRC2 function in T-cell precursor acute lymphoblastic leukemia.

CRLF2 overexpression has been described as a biomarker of poor prognosis in T-cell acute lymphoblastic leukemia (T-ALL). In the present study, we aimed to unravel the genomic profile underlying CRLF2 overexpression (CRLF2-high) by analyzing RNA-seq, WES, and SNP-array data from 264 T-ALL patients and five cell lines deposited on the TARGET initiative, Cancer Cell Line Encyclopedia and Gene Expression Omnibus. These data allowed us to delineate the genomic landscape of CRLF2-high in T-ALL, which was associated with PTEN, JAK3, PHF6, EZH2, and RUNX1 mutations. We also observed an enrichment of CRLF2-high in early T-precursor (ETP)-ALL (23.08% vs. 4.02%, P=7.579e-06 ) and a very similar gene upregulation profile between these two entities. The inhibition of BET (iBET) proteins is a strategy previously demonstrated to reverse the gene upregulation pattern of ETP cells through restoration of polycomb repressive complex 2 (PRC2) activity. While CRLF2 expression was rescued by using this strategy in LOUCY (untreated vs. iBET P =0.0095, DMSO vs. iBET P=0.0286), a classical ETP-ALL cell line, PRC2 loss was not sufficient to promote CRLF2 upregulation in JURKAT, a more mature T-ALL cell line. Considering the role of IKZF1 in CRLF2 regulation and in recruitment of PCR2, we evaluated IKZF1 status according to CRLF2-expression subgroups. We identified that IKZF1 transcripts with intron retention were upregulated in the CRLF2-high subgroup. Here, we delineated the gene expression profile of CRLF2-high T-ALL samples and unraveled the crucial role of PRC2 in CRLF2 regulation in ETP-ALL.

Nanosized paclitaxel-loaded niosomes: formulation, in vitro cytotoxicity, and apoptosis gene expression in breast cancer cell lines.

In this study, the optimized niosomal formulation containing paclitaxel using non-ionic surfactants and cholesterol was designed and its cytotoxic effects against different breast cancer cell lines and apoptosis gene expression analysis were also investigated. Due to enhancing equation variables, the Box-Behnken method has been applied. Lipid/drug molar ratio, the amounts of Span 60, and cholesterol were selected as the target for optimization. The particle size of niosome loaded paclitaxel and entrapment efficiency proportion have been considered in the role of dependent variables. Then the cytotoxic activity of the optimized formulation was evaluated using an MTT assay against different breast cancer cell lines including MCF-7, T-47D, SkBr3, and MDA-MB-231. The expression level of Bax and Bcl-2 apoptosis genes was determined by Real-Time PCR. In this study, the optimized niosomal formulation revealed that the synthesized niosomes had a spherical appearance and had an average size of 192.73 ± 5.50nm so that the percentage of drug loading was 94.71 ± 1.56%. Moreover, this formulation showed a controlled and slowed release of paclitaxel at different pH (7.4, 6.5, and 5.4). The cytotoxicity results demonstrated that cell viability in all concentrations of niosome loaded paclitaxel had profound cytotoxic effects on all studied breast cancer cell lines compared to the free paclitaxel (p < 0.05). In addition, the expression of apoptosis genes was much higher than that of free paclitaxel indicating the susceptibility of cells to apoptosis. As a result, niosomal formulations containing paclitaxel can be used as a new drug delivery system to increase cytotoxicity and treatment of breast cancer in the upcoming future.

PEG-4000 formed polymeric nanoparticles loaded with cetuximab downregulate p21 &stathmin-1 gene expression in cancer cell lines

Cetuximab (CTX) is known to have cytotoxic effects on several human cancer cells in vitro; however, as CTX is poorly water soluble, there is a need for improved formulations can reach cancer cells at high concentrations with low side effects. We developed (PEG-4000) polymeric nanoparticles (PEGNPs) loaded with CTX and evaluated their in vitro cytotoxicity and anticancer properties against human lung (A549) and breast (MCF-7) cancer cells. CTX-PEGNPs were formulated using the solvent evaporation technique, and their morphological properties were evaluated. Further, the effects of CTX-PEGNPs on cell viability using the MTT assay and perform gene expression analysis, DNA fragmentation measurements, and the comet assay. CTX-PEGNP showed uniformly dispersed NPs of nano-size range (253.7 ± 0.3 nm), and low polydispersity index (0.16) indicating the stability and uniformity of NPs. Further, the zeta potential of the preparations was −17.0 ± 1.8 mv. DSC and FTIR confirmed the entrapping of CTX in NPs. The results showed IC50 values of 2.26 μg/mL and 1.83 μg/mL for free CTX and CTX-PEGNPs on the A549 cancer cell line, respectively. Moreover, CTX-PEGNPs had a lower IC50 of 1.12 μg/mL in MCF-7 cells than that of free CTX (2.28 μg/mL). The expression levels of p21 and stathmin-1 were significantly decreased in both cell lines treated with CTX-PEGNPs compared to CTX alone. The CTX-PEGNP-treated cells also showed increased DNA fragmentation rates in both cancer cell lines compared with CTX alone. The results indicated that CTX-PEGNP was an improved formulation than CTX alone to induce apoptosis and DNA damage and inhibit cell proliferation through the downregulation of P21 and stathmin-1 expression.

DNA Methylation Mediates EMT Gene Expression in Human Pancreatic Ductal Adenocarcinoma Cell Lines.

Due to abundant stroma and extracellular matrix, accompanied by lack of vascularization, pancreatic ductal adenocarcinoma (PDAC) is characterized by severe hypoxia. Epigenetic regulation is likely one of the mechanisms driving hypoxia-induced epithelial-to-mesenchymal transition (EMT), responsible for PDAC aggressiveness and dismal prognosis. To verify the role of DNA methylation in this process, we assessed gene expression and DNA methylation changes in four PDAC cell lines. BxPC-3, MIA PaCa-2, PANC-1, and SU.86.86 cells were exposed to conditioned media containing cytokines and inflammatory molecules in normoxic and hypoxic (1% O2) conditions for 2 and 6 days. Cancer Inflammation and Immunity Crosstalk and Human Epithelial to Mesenchymal Transition RT² Profiler PCR Arrays were used to identify top deregulated inflammatory and EMT-related genes. Their mRNA expression and DNA methylation were quantified by qRT-PCR and pyrosequencing. BxPC-3 and SU.86.86 cell lines were the most sensitive to hypoxia and inflammation. Although the methylation of gene promoters correlated with gene expression negatively, it was not significantly influenced by experimental conditions. However, DNA methyltransferase inhibitor decitabine efficiently decreased DNA methylation up to 53% and reactivated all silenced genes. These results confirm the role of DNA methylation in EMT-related gene regulation and uncover possible new targets involved in PDAC progression.

MiR-612, miR-637, and miR-874 can Regulate VEGFA Expression in Hepatocellular Carcinoma Cell Lines.

MicroRNAs (miRNAs) are short non-coding RNA molecules acting as important posttranscriptional gene and protein expression regulators in cancer. The study goal was to examine VEGFA (vascular endothelial growth factor A) expression in hepatocellular carcinoma (HCC) cell lines upon transfection miR-612, miR-637, or miR-874. Methods: MiR-612 mimics, miR-637 mimics, or miR-874 inhibitors were transfected using Lipofectamine RNAiMax in both HCC cell lines, HepG2 and HuH-7. Real-time PCR, Western blotting, and ELISA methods were used to evaluate VEGFA regulation by the miRNAs. Results: Gene and protein expression levels of VEGFA were down-expressed in both cell lines, HepG2 and HuH-7, transfected with miR-612 or miR-637. Transfection with miR-874 inhibitor showed an increase in VEGFA gene expression in HepG2 and HuH-7 cell lines; however, no regulation was observed on VEGFA protein expression by miR-874 inhibition. Correlation analysis between miRNAs and VEGFA protein expression showed that miR-637 and miR-874 expression present inversely correlated to VEGFA protein expression. Conclusions: VEGFA was down-regulated in response to hsa-miR-612 or hsa-miR-637 overexpression; however, the modulation of VEGFA by miR-874 was observed only at the gene expression and thus, needs further investigation.

Ferroptosis Markers Predict the Survival, Immune Infiltration, and Ibrutinib Resistance of Diffuse Large B cell Lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common hematological malignancy in adults. Ferroptosis is an iron-dependent programmed cell death caused by lipid peroxidation. However, the potential functions of ferroptosis in the DLBCL prognosis, immune infiltration, and drug resistance remain unknown. Data of DLBCL patients were downloaded from public GEO databases and TCGA cohort. R software was used for analysis. Ferroptosis-related risk score model was constructed using LASSO Cox regression analysis. The prognosis of the model and its association with immune cells infiltration and ibrutinib-resistance were studied by single-sample gene set enrichment analysis (ssGSEA) and correlation analysis. Ferroptosis-related risk score model was constructed with 11 ferroptosis-related genes. DLBCL patients can be divided into high- or low-risk groups with this model. High-risk patients had significant shorter survival (p < 0.001). The area under curve at 3-year was 0.779. Functional enrichment analysis was mainly associated with the immune response. High score patients were positively correlated with immunosuppressive cell infiltration, including macrophages and regulatory T cells, and immunoevasion checkpoints, such as CTLA4, PD-L1, LAG-3, and TIM-3. We also found that tumors with high risk would resist to ibrutinib treatment and uncovered that acetaminophen, as a ferroptosis inducer, inhibited the defined high-risk gene expression in the ibrutinib-resistant DLBCL cell lines. Ferroptosis-related risk score model can predict the overall survival (OS) of DLBCL patients and ibrutinib resistance of ABC-DLBCL cells, which was associated with immunosuppression status within the tumor microenvironment.

Profiling the regulatory interplay of BET bromodomains and Sirtuins in cancer cell lines

Alterations in epigenetic marking, due to changes in expression or activity of epigenetic regulators, may affect cancer development and progression and thus, targeting epigenetic regulators provides potential avenues for cancer treatment. Bromodomain and extra terminal domain (BET) proteins, epigenetic readers recognizing histone acetylation, and Sirtuins (SIRT1–7), histone deacetylases or erasers, affect the chromatin acetylation status, and thus have a vital role in transcriptional regulation of a variety of cancer-related genes. Here, the effects of three BET inhibitors on SIRT expression were screened in a broad set of cancer cell lines to study the potential interplay of these distinct epigenetic factors in gene regulation. We show that BET inhibitors have distinct effects on SIRTs and their target gene expression in cancer cell lines derived from several solid tumour cancers. This functional link may open further avenues for epigenetic combination therapies for different cancers.

Differential Gene Expression and Pathway Analysis in Melanoma Cell Lines Following MET and PD-L1 Downregulation

Differential Gene Expression and Pathway Analysis in Melanoma Cell Lines Following MET and PD-L1 Downregulation

LncRNA DSCAM-AS1 Negatively Interacts with miR-124 to Promote Hepatocellular Carcinoma Proliferation.

It is known that the development of resistance of breast cancer cells to chemotherapy is at least partially mediated by the upregulation of long non-coding RNA DSCAM antisense RNA 1 (DSCAM-AS1). Therefore, DSCAM-AS1 may play a role in cancer biology. The differential expression of DSCAM-AS1 and miR-124 in hepatocellular carcinoma (HCC) was analyzed by quantitative polymerase chain reaction. One-way analysis of variance (ANOVA) Tukey test was used to analyze the differences in gene expression and cell proliferation in HCC cell lines. Expression levels of DSCAM-AS1 and miR-124 were compared between HCC and paired non-tumor tissues by paired t-test. The effects of transfections on SNU-449 and SNU-398 cell proliferation were analyzed by CCK-8 assay. DSCAM-AS1 was upregulated, while miR-124 was downregulated in HCC tissues. DSCAM-AS1 and miR-124 were inversely correlated across HCC tissues but not normal tissues. DSCAM-AS1 level increased, while miR-124 level decreased along with the clinical stage increasing. miR-124 overexpression increased DSCAM-AS1 level while DSCAM-AS1 overexpression decreased miR-124 level. In addition, miR-124 overexpression decreased HCC cell proliferation, while DSCAM-AS1 increased HCC cell proliferation. Moreover, DSCAM-AS1 overexpression reduced the effects of miR-124 overexpression. DSCAM-AS1 interacts with miR-124 to promote HCC proliferation.

The CBS-H2S axis promotes liver metastasis of colon cancer by upregulating VEGF through AP-1 activation.

The main therapy for colon cancer with liver metastasis is chemotherapy based on 5-fluorouracil combined with targeted drugs. However, acquired drug resistance and severe adverse reactions limit patients' benefit from standard chemotherapy. Here, we investigate the involvement of endogenous hydrogen sulfide (H2S) in liver metastasis of colon cancer and its potential value as a novel therapeutic target. We used the CRISPR/Cas9 system to knockdown CBS gene expression in colon cancer cell lines. PCR arrays and proteome arrays were applied to detect the transcription and protein expression levels, respectively, of angiogenesis-related genes after knockdown. The molecular mechanism was investigated by western blot analysis, RT-qPCR, immunofluorescence staining, ChIP assays and dual-luciferase reporter assays. A liver metastasis mouse model was adopted to investigate the effect of targeting CBS on tumour metastasis in vivo. Knockdown of CBS decreased the metastasis and invasion of colon cancer cells and inhibited angiogenesis both in vivo and in vitro. Tissue microarray analysis showed a positive correlation between CBS and VEGF expression in colon cancer tissues. Further analysis at the molecular level validated a positive feedback loop between the CBS-H2S axis and VEGF. Endogenous H2S promotes angiogenesis and metastasis in colon cancer, and targeting the positive feedback loop between the CBS-H2S axis and VEGF can effectively intervene in liver metastasis of colon cancer.

Specificity protein 1/microRNA-92b forms a feedback loop promoting the migration and invasion of head and neck squamous cell carcinoma

ABSTRACT In this study we report a novel specificity protein 1 (SP1)/microRNA-92b (miR-92b) feedback loop regulating the migration and invasion of head and neck squamous cell carcinoma (HNSCC). Microarray and real-time Polymerase Chain Reaction (PCR) were used to detect gene expression in HNSCC tissues and cell lines. Transwell migration, invasion, wound healing and cell counting kit – 8 (CCK-8) cell assays were used to compare cell migration, invasion and proliferation abilities. Chromatin Immunoprecipitation (ChIP) assays were used to detect SP1 binding to the miR-92b promoter. Western blot was used to detect protein levels. An in vivo tumorigenesis experiment was used to evaluate the effect of SP1 knockdown on tumor growth and protein levels were evaluated by immunohistochemistry. We found that the miR-92b expression level was elevated in HNSCC primary focus tissue compared with adjacent normal tissue, and a higher level of miR-92b was related to a higher clinical stage and worse prognosis of HNSCC patients. MiR-92b and SP1 mutually promoted each expression and cooperatively facilitated the migration, invasion and proliferation of HNSCC cells. A decreased level of SP1/miR-92b resulted in a restraint of in vivo tumor growth. In conclusion, our results suggest that the SP1/miR-92b feedback loop generally promotes HNSCC invasion and metastasis, thus presenting a possible therapeutic target in the treatment of HNSCC patients.

Effects of galectin-1 inhibitor OTX008 on oral squamous cell carcinoma cells in vitro and the role of AP-1 and the MAPK/ERK pathway

ObjectiveTo investigate the in vitro effects of inhibiting galectin-1 using the small-molecule inhibitor OTX008 on oral squamous cell carcinoma (OSCC) cell lines and the role of the MAPK pathway. MethodsOne normal oral keratinocyte (NOK) and three OSCC cell lines were cultured in vitro and the expression of galectin-1 protein by each quantified using ELISA. Cell lines were treated with galectin-1 (50, 100 and 150 ng/mL) or OTX008 (12.5, 25, 50 and 100 μg/mL) and cell viability assayed (n = 3). OSCC cell lines with and without 25 μg/mL OTX008 (n = 3) treatment for 48 h, were analysed using qRT2-PCR with a custom array, to assess relative gene expression. ResultsAll cell lines were found to express galectin-1 protein. Exogenous galectin-1 significantly reduced cell viability in one OSCC cell line over time while the others were only minimally affected. OTX008 treatment reduced cell viability in a dose and time-dependent manner in all cell lines and this was associated with significant regulation of FOS gene expression in the OSCC cell lines. ConclusionOTX008 decreased the viability of OSCC and NOK cells in a dose-dependent manner. The significant regulation of FOS suggests OTX008 causes early induction of the MAPK pathway via the immediate response gene FOS as a subunit of the AP-1 complex.