ULK2 suppresses ovarian cancer cell migration and invasion by elevating IGFBP3.

Background: Ovarian cancer is an aggressive malignancy with high mortality known for its considerable metastatic potential. This study aimed to explore the expression and functional role of Unc-51 like autophagy activating kinase 2 (ULK2) in the progression of ovarian cancer. Methods: ULK2 expression patterns in ovarian cancer tissues as well as benign tumor control samples obtained from our institution were evaluated using immunohistochemistry. Cell counting kit 8 and Transwell assays were applied to assess the effects of ULK2 overexpression on cell proliferation, migration and invasion, respectively. RNA sequencing was performed to explore potential mechanisms of action of ULK2 beyond its classical autophagy modulation. Results: Our experiments showed significant downregulation of ULK2 in ovarian cancer tissues. Importantly, low expression of ULK2 was markedly correlated with decreased overall survival. In vitro functional studies further demonstrated that overexpression of ULK2 significantly suppressed tumor cell proliferation, migration, and invasion. RNA sequencing analysis revealed a potential regulatory role of ULK2 in the insulin signaling pathway through upregulation of insulin-like growth factor binding protein-3 (IGFBP3) in ovarian cancer cells. Conclusions: In summary, the collective data indicated that ULK2 acted as a tumor suppressor in ovarian cancer by upregulating the expression of IGFBP3. Our study underscores the potential utility of ULK2 as a valuable prognostic marker for ovarian cancer.

FUS-stabilized USP35 promotes growth, invasion and angiogenesis in NSCLC through deubiquitinating VEGFA.

Vascular endothelial growth factor A (VEGFA) is an important regulator for non-small cell lung cancer (NSCLC). Our study aimed to reveal its upstream pathway to provide new ideas for developing the therapeutic targets of NSCLC. The mRNA and protein levels of VEGFA, ubiquitin-specific peptidase 35 (USP35), and FUS were determined by quantitative real-time PCR and Western blot. Cell proliferation, apoptosis, invasion and angiogenesis were detected using CCK8 assay, EdU assay, flow cytometry, transwell assay and tube formation assay. The interaction between USP35 and VEGFA was assessed by Co-IP assay and ubiquitination assay. Animal experiments were performed to assess USP35 and VEGFA roles in vivo. VEGFA had elevated expression in NSCLC tissues and cells. Interferences of VEGFA inhibited NSCLC cell proliferation, invasion, angiogenesis, and increased apoptosis. USP35 could stabilize VEGFA protein level by deubiquitination, and USP35 knockdown suppressed NSCLC cell growth, invasion and angiogenesis via reducing VEGFA expression. FUS interacted with USP35 to promote its mRNA stability, thereby positively regulating VEGFA expression. Also, USP35 silencing could reduce NSCLC tumorigenesis by downregulating VEGFA. FUS-stabilized USP35 facilitated NSCLC cell growth, invasion and angiogenesis through deubiquitinating VEGFA, providing a novel idea for NSCLC treatment.

CRISPR/Cas9-mediated knock out of ITGB6 in human OSCC cells reduced migration and proliferation ability.

BACKGROUND: The treatment of oral squamous cell carcinoma (OSCC) remains challenging and survival rates have not been improved significantly over the past decades. Integrins have been recognized driving the cancer progression and high expression levels cause poor outcomes in patients afflicted with OSCC. Integrin αvß6 and its subunit integrin beta 6 (ITGB6) were discovered to enhance the invasiveness by providing beneficial effects on downstream pathways promoting the cancer progression. The objective of this study was to establish a CRISPR/Cas9-mediated knock out of ITGB6 in the human OSCC cell line HN and investigate the effects on the migration and proliferation ability. METHODS: ITGB6 knock out was performed using the CRISPR/Cas9-system, RNPs, and lipofection. Monoclonal cell clones were achieved by limiting dilution and knock out verification was carried out by sanger sequencing and FACS on protein level. The effects of the knock out on the proliferation and migration ability were evaluated by using MTT and scratch assays. In addition, in silico TCGA analysis was utilized regarding the effects of ITGB6 on overall survival and perineural invasion. RESULTS: In silico analysis revealed a significant impact of ITGB6 mRNA expression levels on the overall survival of patients afflicted with OSCC. Additionally, a significantly higher rate of perineural invasion was discovered. CRISPR/Cas9-mediated knock out of ITGB6 was performed in the OSCC cell line HN, resulting in the generation of a monoclonal knock out clone. The knock out clone exhibited a significantly reduced migration and proliferation ability when compared to the wildtype. CONCLUSIONS: ITGB6 is a relevant factor in the progression of OSCC and can be used for the development of novel treatment strategies. The present study is the first to establish a monoclonal CRISPR/Cas9-mediated ITGB6 knockout cell clone derived from an OSCC cell line. It suggests that ITGB6 has a significant impact on the proliferative and migratory capacity in vitro.

CRISPRi screen of long non-coding RNAs identifies LINC03045 regulating glioblastoma invasion.

INTRODUCTION: Glioblastoma (GBM) invasion studies have focused on coding genes, while few studies evaluate long non-coding RNAs (lncRNAs), transcripts without protein-coding potential, for role in GBM invasion. We leveraged CRISPR-interference (CRISPRi) to evaluate invasive function of GBM-associated lncRNAs in an unbiased functional screen, characterizing and exploring the mechanism of identified candidates. METHODS: We implemented a CRISPRi lncRNA loss-of-function screen evaluating association of lncRNA knockdown (KD) with invasion capacity in Matrigel. Top screen candidates were validated using CRISPRi and oligonucleotide(ASO)-mediated knockdown in three tumor lines. Clinical relevance of candidates was assessed via The Cancer Genome Atlas(TCGA) and Genotype-Tissue Expression(GTEx) survival analysis. Mediators of lncRNA effect were identified via differential expression analysis following lncRNA KD and assessed for tumor invasion using knockdown and rescue experiments. RESULTS: Forty-eight lncRNAs were significantly associated with 33-83% decrease in invasion (p<0.01) upon knockdown. The top candidate, LINC03045, identified from effect size and p-value, demonstrated 82.7% decrease in tumor cell invasion upon knockdown, while LINC03045 expression was significantly associated with patient survival and tumor grade(p<0.0001). RNAseq analysis of LINC03045 knockdown revealed that WASF3, previously implicated in tumor invasion studies, was highly correlated with lncRNA expression, while WASF3 KD was associated with significant decrease in invasion. Finally, WASF3 overexpression demonstrated rescue of invasive function lost with LINC03045 KD. CONCLUSION: CRISPRi screening identified LINC03045, a previously unannotated lncRNA, as critical to GBM invasion. Gene expression is significantly associated with tumor grade and survival. RNA-seq and mechanistic studies suggest that this novel lncRNA may regulate invasion via WASF3.

LINC00520 promotes colorectal cancer progression through miRNA-195-3p / NAT2 axis.

In this study, we investigated the role of LINC00520 in colorectal cancer (CRC) progression. We analyzed LINC00520 expression in 15 pairs of CRC tissues and adjacent tissues using qRT-PCR, revealing significantly elevated levels in CRC tissues and cell lines. Lentivirus-mediated up/down-regulation of LINC00520 in CRC cell lines demonstrated that increased LINC00520 expression enhanced cell invasiveness, as confirmed by transwell and wound healing assays. Bioinformatics analysis identified a regulatory axis involving LINC00520, microRNA-195-3p, and NAT2. Luciferase assays confirmed direct binding between LINC00520 and microRNA-195-3p, as well as microRNA-195-3p and NAT2. Overexpression of NAT2 reversed the inhibitory effects on invasion and migration induced by LINC00520 silencing. This suggests that LINC00520, highly expressed in CRC tissues, may modulate tumor biological functions through the microRNA-195-3p/NAT2 axis. Our findings provide insights into the mechanism underlying CRC progression, highlighting the potential of LINC00520 as a therapeutic target.

Transcriptome Analysis in Patients With Muscle-invasive Bladder Cancer.

BACKGROUND/AIM: Bladder cancer (BC) is the most prevalent malignant tumor in the urinary tract, classified mainly into muscle-invasive BC (MIBC) and non-MIBC (NMIBC). Recent studies highlight the important role of changes in transcriptome activity in carcinogenesis, aiding in the identification of additional differentially regulated candidate genes, improving our understanding of the molecular basis of gene regulation in BC. This study aimed to evaluate the transcriptome of MIBC patients compared with normal subjects. MATERIALS AND METHODS: mRNA sequencing was conducted using the Illumina NovaSeq 6000 Dx system in a case series comprising 11 subjects with MIBC and 19 healthy controls matched for age and sex. For functional analysis, the pathfindR package was utilized to comprehensively identify pathways enriched in omics data within active subnetworks. RESULTS: Our results demonstrated the presence of differentiated pathways, including spliceosome activity, oxidative phosphorylation, and chemical carcinogenesis due to reactive oxygen species, in MIBC patients compared with controls. CONCLUSION: The identification of novel molecular pathways in MIBC patients could prove useful in defining cancer predisposition factors and exploring potential therapeutic options.

ß-Sitosterol alleviates the malignant phenotype of hepatocellular carcinoma cells via inhibiting GSK3B expression.

To explore the effects of ß-Sitosterol upon hepatocellular carcinoma cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition (EMT), and to investigate the underlying mechanism using network pharmacology. Human hepatocellular carcinoma cell lines (Huh-7 and HCCLM3) were expose to gradient concentrations of ß-Sitosterol (5 µg/mL, 10 µg/mL, and 20 µg/mL). Cell viability and proliferation were assessed using MTT, CCK-8, colony formation, and EdU assays.Flow cytometry was employed to evaluate cell cycle and apoptosis. Scratch and Transwell assays were performed, respectively, to detect cell migration and invasion. The levels of apoptosis-associated proteins (BAX, BCL2, and cleaved caspase3) as well as EMT-associated proteins (E-cadherin, N-cadherin, Snail, and Vimentin) were detected in Huh-7 and HCCLM3 cell lines using Western blot analysis. The drug target gene for ß-Sitosterol was screened via PubChem and subsequently evaluated for expression in the GSE112790 dataset. In addition, the expression level of glycogen synthase kinase 3 beta (GSK3B) within the Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database was analyzed, along with its correlation to the survival outcomes of patients with hepatocellular carcinoma. The diagnostic efficiency of GSK3B was assessed by analyzing the ROC curve. Subsequently, Huh-7 and HCCLM3 cell lines were transfected with the overexpression vector of GSK3B and then treated with ß-Sitosterol to further validate the association between GSK3B and ß-Sitosterol. GSK3B demonstrated a significantly elevated expression in patients with hepatocellular carcinoma, which could predict hepatocellular carcinoma patients' impaired prognosis based on GEO dataset and TCGA database. GSK3B inhibitor (CHIR-98014) notably inhibited cell proliferation and invasion, promoted cell apoptosis and cell cycle arrest at G0/G1 phase in hepatocellular carcinoma cells. ß-Sitosterol treatment further promoted the efffects of GSK3B inhibitor on hepatocellular carcinoma cells. GSK3B overexpression has been found to enhance the proliferative and invasive capabilities of hepatocellular carcinoma cells. Furthermore it has been observed that GSK3B overexpression, it has been obsear can partially reverse the inhibitory effect of ß-Sitosterol upon hepatocellular. ß-Sitosterol suppressed hepatocellular carcinoma cell proliferation and invasion, and enhanced apoptosis via inhibiting GSK3B expression.

IL-6 regulates epithelial ovarian cancer EMT, invasion, and metastasis by modulating Let-7c and miR-200c through the STAT3/HIF-1α pathway.

Interleukin-6 (IL-6) and hypoxia-inducible factor-1α (HIF-1α) play important roles in epithelial-mesenchymal transformation (EMT) and tumor development. Previous studies have demonstrated that IL-6 promotes EMT, invasion, and metastasis in epithelial ovarian cancer (EOC) cells by activating the STAT3/HIF-1α pathway. MicroRNA (miRNA) is non-coding small RNAs that also play an important role in tumor development. Notably, Let-7 and miR-200 families are prominently altered in EOC. However, whether IL-6 regulates the expression of Let-7 and miR-200 families through the STAT3/HIF-1α signaling to induce EMT in EOC remains poorly understood. In this study, we conducted in vitro and in vivo investigations using two EOC cell lines, SKOV3, and OVCAR3 cells. Our findings demonstrate that IL-6 down-regulates the mRNA levels of Let-7c and miR-200c while up-regulating their target genes HMGA2 and ZEB1 through the STAT3/HIF-1α signaling in EOC cells and in vivo. Additionally, to explore the regulatory role of HIF-1α on miRNAs, both exogenous HIF blockers YC-1 and endogenous high expression or inhibition of HIF-1α can be utilized. Both approaches can confirm that the downstream molecule HIF-1α inhibits the expression and function of Let-7c and miR-200c. Further mechanistic research revealed that the overexpression of Let-7c or miR-200c can reverse the malignant evolution of EOC cells induced by IL-6, including EMT, invasion, and metastasis. Consequently, our results suggest that IL-6 regulates the expression of Let-7c and miR-200c through the STAT3/HIF-1α pathway, thereby promoting EMT, invasion, and metastasis in EOC cells.

Long Non-coding RNA PCED1B Antisense RNA 1 Promotes Cell Proliferation and Invasion in Hepatocellular Carcinoma by Regulating the MicroRNA-34a/CD44 Axis.

OBJECTIVE: This study aimed to examine the role of long non-coding RNA PCED1B antisense RNA 1 (PCED1B-AS1) in the development of hepatocellular carcinoma (HCC). METHODS: A total of 62 pairs of HCC tissues and adjacent non-tumor tissues were obtained from 62 HCC patients. The interactions of PCED1B-AS1 and microRNA-34a (miR-34a) were detected by dual luciferase activity assay and RNA pull-down assay. The RNA expression levels of PCED1B-AS1, miR-34a and CD44 were detected by RT-qPCR, and the protein expression level of CD44 was determined by Western blotting. The cell proliferation was detected by cell proliferation assay, and the cell invasion and migration by transwell invasion assay. The HCC tumor growth after PCED1B-AS1 was downregulated was determined by in vivo animal study. RESULTS: PCED1B-AS1 was highly expressed in HCC tissues, which was associated with poor survival of HCC patients. Furthermore, PCED1B-AS1 interacted with miR-34a in HCC cells, but they did not regulate the expression of each other. Additionally, PCED1B-AS1 increased the expression level of CD44, which was targeted by miR-34a. The cell proliferation and invasion assay revealed that miR-34a inhibited the proliferation and invasion of HCC in vitro, while CD44 exhibited the opposite effects. Furthermore, PCED1B-AS1 suppressed the role of miR-34a. Moreover, the knockdown of PCED1B-AS1 repressed the HCC tumor growth in nude mice in vivo. CONCLUSION: PCED1B-AS1 may play an oncogenic role by regulating the miR-34a/CD44 axis in HCC.

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

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

Pseudogene CSPG4P12 inhibits colorectal cancer progression by attenuating epithelial-mesenchymal transition.

Colorectal cancer is one of the most common malignant cancers. Pseudogenes have been identified as oncogenes or tumor suppressor genes in the development of various cancers. However, the function of pseudogene CSPG4P12 in colorectal cancer remains unclear. Therefore, the aim of this study was to investigate the potential role of CSPG4P12 in colorectal cancer and explore the possible underlying mechanism. The difference of CSPG4P12 expression between colorectal cancer tissues and adjacent normal tissues was analyzed using the online Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database. Cell viability and colony formation assays were conducted to evaluate cell viability. Transwell and wound healing assays were performed to assess cell migration and invasion capacities. Western blot was used to measure the expression levels of epithelial-mesenchymal transition-related proteins. Colorectal cancer tissues had lower CSPG4P12 expression than adjacent normal tissues. The overexpression of CSPG4P12 inhibited cell proliferation, invasion, and migration in colorectal cancer cells. Overexpressed CSPG4P12 promoted the expression of E-cadherin, whereas it inhibited the expression of vimentin, N-cadherin, and MMP9. These findings suggested that CSPG4P12 inhibits colorectal cancer development and may serve as a new potential target for colorectal cancer.

Radiation-induced upregulation of FGL1 promotes esophageal squamous cell carcinoma metastasis via IMPDH1.

BACKGROUND: While radiation therapy remains pivotal in esophageal squamous cell carcinoma (ESCC) treatment, the perplexing phenomenon of post-radiation metastasis presents a formidable clinical challenge. This study investigates the role of fibrinogen-like protein 1 (FGL1) in driving ESCC metastasis following radiation exposure. METHODS: FGL1 expression in post-radiation ESCC cells was meticulously examined using qRT-PCR, western blotting, and immunofluorescence. The impact of FGL1 on ESCC cell invasion and migration was assessed through Transwell and wound healing assays. In vivo, the metastatic potential of ESCC in response to FGL1 was scrutinized using nude mice models. Comprehensive RNA sequencing and functional experiments elucidated the intricate mechanism associated with FGL1. RESULTS: Radiation induced upregulation of FGL1 in ESCC cells through FOXO4, intensifying ESCC cell invasion and migration. Targeted knockdown of FGL1 effectively alleviated these characteristics both in vitro and in vivo. FGL1 depletion concurrently suppressed IMPDH1 expression. Rescue experiments underscored that IMPDH1 knockdown robustly reversed the pro-invasive effects induced by FGL1 in ESCC cells. ESCC tissues exhibited heightened IMPDH1 mRNA levels, demonstrating a correlation with patient survival. CONCLUSIONS: Radiation-induced upregulation of FGL1 propels ESCC metastasis through IMPDH1, proposing a potential therapeutic target to mitigate post-radiotherapy metastasis in ESCC patients.

Methylation-regulated tumor suppressor gene PDE7B promotes HCC invasion and metastasis through the PI3K/AKT signaling pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) has a high mortality rate, and the mechanisms underlying tumor development and progression remain unclear. However, inactivated tumor suppressor genes might play key roles. DNA methylation is a critical regulatory mechanism for inactivating tumor suppressor genes in HCC. Therefore, this study investigated methylation-related tumor suppressors in HCC to identify potential biomarkers and therapeutic targets. METHODS: We assessed genome-wide DNA methylation in HCC using whole genome bisulfite sequencing (WGBS) and RNA sequencing, respectively, and identified the differential expression of methylation-related genes, and finally screened phosphodiesterase 7B (PDE7B) for the study. The correlation between PDE7B expression and clinical features was then assessed. We then analyzed the changes of PDE7B expression in HCC cells before and after DNA methyltransferase inhibitor treatment by MassArray nucleic acid mass spectrometry. Furthermore, HCC cell lines overexpressing PDE7B were constructed to investigate its effect on HCC cell function. Finally, GO and KEGG were applied for the enrichment analysis of PDE7B-related pathways, and their effects on the expression of pathway proteins and EMT-related factors in HCC cells were preliminarily explored. RESULTS: HCC exhibited a genome-wide hypomethylation pattern. We screened 713 hypomethylated and 362 hypermethylated mCG regions in HCC and adjacent normal tissues. GO analysis showed that the main molecular functions of hypermethylation and hypomethylation were "DNA-binding transcriptional activator activity" and "structural component of ribosomes", respectively, whereas KEGG analysis showed that they were enriched in "bile secretion" and "Ras-associated protein-1 (Rap1) signaling pathway", respectively. PDE7B expression was significantly down-regulated in HCC tissues, and this low expression was negatively correlated with recurrence and prognosis of HCC. In addition, DNA methylation regulates PDE7B expression in HCC. On the contrary, overexpression of PDE7B inhibited tumor proliferation and metastasis in vitro. In addition, PDE7B-related genes were mainly enriched in the PI3K/ATK signaling pathway, and PDE7B overexpression inhibited the progression of PI3K/ATK signaling pathway-related proteins and EMT. CONCLUSION: PDE7B expression in HCC may be regulated by promoter methylation. PDE7B can regulate the EMT process in HCC cells through the PI3K/AKT pathway, which in turn affects HCC metastasis and invasion.

Genomic progression for local invasion of cutaneous squamous cell carcinoma from the superficial to the deep portion.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer. While many treatments exist, our understanding of its genomic progression, especially from the epidermis to the deep dermis, remains limited. This study aims to identify genetic mutations associated with the progression of cSCC into the deep dermis, providing insights into its aggressive behavior and high-risk features. We performed high-depth whole-exome sequencing on 12 cSCC tissues, along with paired normal tissues from six patients, using microdissection techniques. The mutational analysis focused on identifying alterations enriched during cSCC progression. Gene Ontology enrichment analysis, immunohistochemical assays, and external single-cell RNA data were utilized for validation. A total of 8863 non-synonymous somatic mutations were identified in 4092 genes across the superficial and deep portions of cSCCs. Analysis of deep portion mutations revealed a significant correlation with gene ontology biological processes, particularly cell junction organization, and cell-cell adhesion. Clonal mutations in these processes were more prevalent in the deep portions, indicating their impact on the cSCC mutation landscape. Genetic evolution analysis identified 29 causal genes associated with dermal invasion in cSCC. We highlight somatic mutations in cSCC, revealing heterogeneity between superficial and deep regions. Altered genes in cell junction organization and cell-cell adhesion emerged as pivotal in dermal invasion. We identified 29 causal genes primarily in deep tumor regions. Our findings emphasize analyzing multiple tumor regions to capture varied mutational landscapes. These insights advance our understanding of cSCC progression, emphasizing genetic and cellular changes during tumor evolution.

Evaluation of Pm2.5 Influence on Human Lung Cancer Cells Using a Microfluidic Platform.

In this study, we developed a microfluidic device that is able to monitor cell biology under continuous PM2.5 treatment. The effects of PM2.5 on human alveolar basal epithelial cells, A549 cells, and uncovered several significant findings were investigated. The results showed that PM2.5 exposure did not lead to a notable decrease in cell viability, indicating that PM2.5 did not cause cellular injury or death. However, the study found that PM2.5 exposure increased the invasion and migration abilities of A549 cells, suggesting that PM2.5 might promote cell invasiveness. Results of RNA sequencing revealed 423 genes that displayed significant differential expression in response to PM2.5 exposure, with a particular focus on pathways associated with the generation of reactive oxygen species (ROS) and mitochondrial dysfunction. Real-time detection demonstrated an increase in ROS production in A549 cells after exposure to PM2.5. JC1 assay, which indicated a loss of mitochondrial membrane potential (ΔΨm) in A549 cells exposed to PM2.5. The disruption of mitochondrial membrane potential further supports the detrimental effects of PM2.5 on A549 cells. These findings highlight several adverse effects of PM2.5 on A549 cells, including enhanced invasion and migration capabilities, altered gene expression related to ROS pathways, increased ROS production and disruption of mitochondrial membrane potential. These findings contribute to our understanding of the potential mechanisms through which PM2.5 can impact cellular function and health.

Ethanol responsive lnc171 promotes migration and invasion of HCC cells via mir-873-5p/ZEB1 axis.

BACKGROUNDS: Long nonconding RNAs (lncRNAs) have been found to be a vital regulatory factor in the development process of human cancer, and could regarded as diagnostic or prognostic biomarkers for human cancers. Here, we aim to confirm the expression and molecular mechanism of RP11-171K16.5 (lnc171) in hepatocellular carcinoma (HCC). METHODS: Screening of differentially expressed lncRNAs by RNA sequencing. Expression level of gene was studied by quantitative real-time PCR (qRT-PCR). The effects of lnc171, mir-873-5p, and ethanol on migration and invasion activity of cells were studied used transwell assay, and luciferase reporter assay was used to confirm the binding site. RESULTS: RNA sequencing showed that lnc171 was markedly up-regulated in HCC. siRNA-mediated knockdown of lnc171 repressed the migration and invasion ability of HCC cells. Bioinformatic analysis, dual luciferase reporter assay, and qRT-PCR indicated that lnc171 interacted with mir-873-5p in HCC cells, and Zin-finger E-box binding homeobox (ZEB1) was a downstream target gene of mir-873-5p. In addition, lnc171 could enhance migration and invasion ability of HCC cells by up-regulating ZEB1 via sponging mir-873-5p. More interestingly, ethanol stimulation could up-regulate the increase of lnc171, thereby regulating the expression of competing endogenous RNA (ceRNA) network factors which lnc171 participated in HCC cells. CONCLUSIONS: Our date demonstrates that lnc171 was a responsive factor of ethanol, and plays a vital role in development of HCC via binding of mir-873-5p.

Cancer-associated fibroblasts-derived exosomal METTL3 promotes the proliferation, invasion, stemness and glutaminolysis in non-small cell lung cancer cells by eliciting SLC7A5 m6A modification.

Cancer-associated fibroblasts (CAFs) can promote the crosstalk between cancer cells and tumor microenvironment by exosomes. METTL3-mediated N6-methyladenine (m6A) modification has been proved to promote the progression of non-small cell lung cancer (NSCLC). Here, we focused on the impacts of CAFs-derived exosomes and METTL3-mediated m6A modification on NSCLC progression. Functional analyses were conducted using Cell Counting Kit-8, EdU, colony formation, sphere formation and transwell assays, respectively. Glutamine metabolism was evaluated by detecting glutamate consumption, and the production of intercellular glutamate and α-ketoglutarate (α-KG). qRT-PCR and western blotting analyses were utilized to measure the levels of genes and proteins. Exosomes were isolated by kits. The methylated RNA immunoprecipitation assay detected the m6A modification profile of Amino acid transporter LAT1 (SLC7A5) mRNA. The NSCLC mouse model was established to conduct in vivo experiments. We found that CAFs promoted the proliferation, invasion, stemness and glutaminolysis in NSCLC cells. METTL3 was enriched in CAFs and was packaged into exosomes. After knockdown of METTL3 in CAF exosomes, it was found the oncogenic effects of CAFs on NSCLC cells were suppressed. CAFs elevated m6A levels in NSCLC cells. Mechanistically, exosomal METTL3-induced m6A modification in SLC7A5 mRNA and stabilized its expression in NSCLC cells. Moreover, SLC7A5 overexpression abolished the inhibitory effects of exosomal METTL3-decreased CAFs on NSCLC cells. In addition, METTL3 inhibition in CAF exosomes impeded NSCLC growth in vivo. In all, CAFs-derived exosomal METTL3 promoted the proliferation, invasion, stemness and glutaminolysis in NSCLC cells by inducing SLC7A5 m6A modification.

Hsa_circ_0079875 functions as a competitive endogenous RNA to promote hepatocellular carcinoma progression.

Circular RNA (circRNA) can influence the development of hepatocellular carcinoma (HCC) as a competitive endogenous RNA (ceRNA). However, there are still many circRNAs whose functions are unknown. Our research explores the role of a novel circRNA, hsa_circ_0079875, in HCC. The expression of hsa_circ_0079875 in HCC was verified by next-generation sequencing, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and fluorescence in situ hybridization (FISH). The distribution of hsa_circ_0079875 in HCC cells was investigated by RNA subcellular isolation and FISH assays. The functional effects on HCC proliferation, invasion, migration, cell cycle, and apoptosis were verified by overexpression and knockdown of hsa_circ_0079875. Moreover, xenograft mouse models and immunohistochemistry experiments were used to assess the function of hsa_circ_0079875 in vivo. Hsa_circ_0079875 was up-regulated in HCC tissues and mainly distributed in the cytoplasm. Higher hsa_circ_0079875 leads to larger tumor tissue, more microvascular invasion(MVI) and higher AFP levels, which in turn leads to a poor prognosis. Overexpression of hsa_circ_0079875 can promote the proliferation, migration, and invasion of HCC cells and inhibit apoptosis in vitro and in vivo. Knocking down hsa_circ_0079875 has the opposite effect. Sequencing and biological information predicted the target miRNA and mRNA of hsa_circ_0079875. Further bioinformatics and clinical correlation analysis revealed that hsa_circ_0079875 promote the malignant biological behaviors of HCC through hsa_circ_0079875/miR-519d-59/NRAS ceRNA net. Therefore, hsa_circ_0079875 can be a potential prognostic marker and therapeutic target for HCC.

Keratin 6A (KRT6A) promotes radioresistance, invasion, and metastasis in lung cancer via p53 signaling pathway.

BACKGROUND: It is reported that the incidence rate and mortality of lung cancer are very high. Therefore, early diagnosis and identification of specific biomarkers are crucial for the clinical treatment of lung cancer. This study aims to comprehensively investigate the prognostic significance of KRT6A in human lung cancer. METHODS: The GEO2R online tool was utilized to analyze the differential expression of mRNA between lung carcinoma tissues and radioresistant tissues in the GSE73095 and GSE197236 datasets. DAVID database was used to perform GO and KEGG enrichment analyses on target genes. The Kaplan-Meier plotter tool was used to analyze the impact of key messenger ribonucleic acid on the survival status of lung cancer. In addition, quantitative real-time polymerase chain reaction (qPCR) was used to investigate the impact of key genes on the phenotype of lung cancer cells. After the knockout, we conducted cell migration and CCK-8 experiments to detect their effects on cell proliferation and invasion. RESULTS: 40 differentially expressed genes (DEGs) were chosen from GSE73095 and 118 DEGs were chosen from GSE197236. Kaplan-Meier map analysis showed that the overall cancer survival rate of the high-expression KRT6A group was higher than that of the low-expression group (P < 0.05). Besides, cell experiments have shown that when the KRT6A gene is downregulated, the proliferation and invasion ability of lung cancer cells is weakened. CONCLUSIONS: Our research concluded that KRT6A may take part in the radioresistance and progression of lung cancer and can be a potential biomarker for lung cancer patients.

Biological functions of Circular RNA_LARP4/ Upstream frameshift 1 in development of gastric cancer.

Recently, the progression of gastric cancer (GC), as one of the most ordinary malignant tumors, has been reported to be associated with circular RNAs. This study aimed to identify the role of circular RNA_LARP4 in GC. We performed real-time quantitative polymerase chain reaction (RT-qPCR) in 46 paired GC patients and GC cell lines to detect the expression of circular RNA_LARP4. Moreover, the role of circular RNA_LARP4 in GC proliferation was identified through proliferation assay and colony formation assay, while the role of circular RNA_LARP4 in GC metastasis was measured through scratch wound assay and transwell assay. Furthermore, the potential targets of circular RNA_LARP4 were predicted through bioinformatics methods and further identified by western blot assay and RT-qPCR. Circular RNA_LARP4 expression was remarkably lower in GC tissues compared with that in adjacent samples. Besides, cell proliferation of GC was inhibited after overexpression of circular RNA_LARP4, while cell migration and invasion of GC was inhibited after overexpression of circular RNA_LARP4. Furthermore, Upstream frameshift 1 (UPF1) was predicted as the potential target of circular RNA_LARP4 and was upregulated via overexpression of circular RNA_LARP4 in GC. Circular RNA_LARP4 inhibits GC cell proliferation and metastasis via targeting UPF1 in vitro, which might provide a new tumor suppressor in GC development.