SOX8 promotes cetuximab resistance via HGF/MET bypass pathway activation in colorectal cancer.

AIM: Cetuximab is an essential drug for the treatment of wild-type K-RAS colorectal cancer (CRC). It improves the overall survival of patients. However, acquired resistance prevents its clinical efficacy. Tumor heterogeneity may be a nonnegligible reason for cetuximab resistance. We attempted to explore the corresponding molecular mechanism. METHODS: Cetuximab-resistant CRC cell RKO and cetuximab-sensitive CRC cell Caco-2 were applied in this study. Cells were centrifuged to determine the concentration in the culture supernatant (CS). MTT, EdU, and colony formation assays were utilized to evaluate cell survival and proliferation. Chromatin immunoprecipitation (ChIP) and promoter-luciferase reporter assays were employed to confirm the direct binding of transcription factors. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) assays were used to detect the expression of molecular markers in the pathway. RESULTS: Hepatocyte growth factor (HGF) was up-regulated in RKO cell culture supernatant and induced cetuximab resistance in Caco-2 cells. SRY-Box Transcription Factor 8 (SOX8) bound to the promoter sequence of HGF. HGF activated the HGF/MET bypass pathway and induced cetuximab resistance in Caco-2 cells. CONCLUSION: The SOX8/HGF/MET axis played a crucial role in the communication between cetuximab-resistant cells and cetuximab-sensitive cells, inducing treatment resistance.

Hypoxia associated lncRNA HYPAL promotes proliferation of gastric cancer as ceRNA by sponging miR-431-5p to upregulate CDK14.

Gastric cancer (GC) is a common malignant solid tumor that is characterized by high hypoxia. The transcription of genes associated with hypoxia affects tumor occurrence and development. Long non-coding RNAs (lncRNAs) have been reported to play important roles in cancer development. In this study, we screened for differentially expressed ncRNAs (non-coding RNA) and mRNAs between hypoxia-inducible factor-1 (HIF-1α) knockdown GC cells and scrambled GC cells. Microarray data revealed that HIF-1α regulated the expression of LINC01355 (Hypoxia Yield Proliferation Associated LncRNA, HYPAL). HYPAL was found to be significantly upregulated in GC cells and tissues and was correlated with poor GC prognosis. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays revealed that HIF-1α promoted HYPAL expression by binding the promoter region. A regulatory network for the competing endogenous RNA (ceRNA) was constructed using bioinformatics tools. Mechanistic studies revealed that HYPAL acted as a ceRNA of miR-431-5p to regulate CDK14 expression. Carcinogenic effects of HYPAL were evaluated in vitro and in vivo. The HIF-1α/HYPAL/miR-431-5p/CDK14 (Cyclin-dependent kinase 14) axis activated the Wnt/ß-catenin signaling pathway and induced GC cell proliferation while inhibiting apoptosis. In conclusion, HYPAL is a potential molecular target for GC therapy.

LINC00184 involved in the regulatory network of ANGPT2 via ceRNA mediated miR-145 inhibition in gastric cancer.

Background: Disrupted gene levels are intimately correlated with the occurrence and prognosis of gastric cancer (GC). As genes do not function in isolation, we set out to investigate the possible relationship among mRNA and non-coding RNAs (ncRNAs). Materials and methods: RNA sequencing from 406 cases of GC was acquired through the TCGA database. R packages were utilized to assess differential RNA expression. The competing endogenous RNA (ceRNA) network was predicted using miRcode, miRDB, mirTarBase, Target Scan and constructed by Cytoscape 3.6.1. GO enrichment analysis, KEGG pathway analysis, GSEA, and WGCNA were applied for pathway analysis. The expression of select candidate molecules was confirmed using western blot and RT-PCR in GC cells and tissues. CCK-8, EdU staining, and Transwell assays were conducted to assess the influence of candidate molecules on proliferation and invasion. The gain and loss-of-function were achieved by co-culture with sh-lncRNA, mimics and sh-mRNA. Luciferase reporters were created using the psiCHECK2 vector, and the relative luciferase activity was calculated. Results: Using data from TCGA, we determined differentially expressed RNAs and created a ceRNA regulatory network. Interestingly, we identified a regulatory complex surrounding ANGPT2. We detected that ANGPT2 was highly expressed in GC, which correlated with a worse prognosis. Our findings indicated that ANGPT2 encourages growth, invasion, and epithelial-mesenchymal transition (EMT) in GC. Importantly, miR-145 inhibits ANGPT2 and abrogates its effects. Furthermore, LINC00184, a ceRNA, blocks miR-145, thereby improving ANGPT2-mediated carcinogenesis. Conclusions: Our findings indicate that the LINC00184/miR-145/ANGPT2 pathway has a crucial function in the development of GC and can act as a possible biomarker and targets for GC therapy.

Development and validation of a three-long noncoding RNA signature for predicting prognosis of patients with gastric cancer.

BACKGROUND: Gastric cancer (GC) is one of the most frequently diagnosed gastrointestinal cancers throughout the world. Novel prognostic biomarkers are required to predict the prognosis of GC. AIM: To identify a multi-long noncoding RNA (lncRNA) prognostic model for GC. METHODS: Transcriptome data and clinical data were downloaded from The Cancer Genome Atlas. COX and least absolute shrinkage and selection operator regression analyses were performed to screen for prognosis associated lncRNAs. Receiver operating characteristic curve and Kaplan-Meier survival analyses were applied to evaluate the effectiveness of the model. RESULTS: The prediction model was established based on the expression of AC007991.4, AC079385.3, and AL109615.2 Based on the model, GC patients were divided into "high risk" and "low risk" groups to compare the differences in survival. The model was re-evaluated with the clinical data of our center. CONCLUSION: The 3-lncRNA combination model is an independent prognostic factor for GC.

To Develop and Validate the Combination of RNA Methylation Regulators for the Prognosis of Patients with Gastric Cancer.

BACKGROUND: Gastric cancer (GC) accounts for high mortality. RNA methylation has recently gained interest as markers in specific tumors. This study aimed to uncover the function of the roles of 25 RNA methylation regulators in GC. METHODS: RNA sequence and clinical data were downloaded from The Cancer Genome Atlas (TCGA) database. "STRING" and R were performed to analyze the correlation among the methylase. COX and LASSO were performed to screen for prognostic associated RNA methylation regulators. A prognostic model was established based on the expression of methylase. RT-PCR and immunohistochemistry detected the expression of methylase in GC cells and tissue. Kaplan-Meier curve and Cox analysis were applied to evaluate the effectiveness of the model. RESULTS: The prediction model was established based on the expression of m6A RNA methylation regulators FTO (fat mass and obesity-associated) and RBM15 (RNA binding motif protein 15). Based on the model, GC patients were divided into "high risk" and "low risk" groups to compare the differences in survival. The model was re-evaluated with the clinical data of our center. CONCLUSION: The two-methylase combination model was an independent prognostic factor of GC.

LncRNA PCGEM1 enhances metastasis and gastric cancer invasion through targeting of miR-129-5p to regulate P4HA2 expression.

AIM: Aberrantly expressed long non-coding RNAs (lncRNAs) are critical instigators of gastric cancer (GC) progression and metastasis. The ceRNA (competing endogenous RNAs) network is well-known in modulating tumor pathological and physiological processes. This research aims to determine the more effective molecular mechanisms of lncRNA PCGEM1 (prostate cancer gene expression marker 1). METHODS: Bioinformatics database and Ago2-RIP were performed to predict and verify the potential targets of lncRNA PCGEM1. Both gain- and loss-of-function experiments were carried out to dissect the biological functions of RNAs. Fluorescence in situ hybridization, dual-luciferase reporter assays, western blot, and real-time PCR (RT-PCR) experiments were utilized to determine the pathophysiological pathways of competitive endogenous RNAs (ceRNAs). RESULTS: GC cells expressed high levels of cytoplasmic PCGEM1. Loss-of-function experiments displayed that the silencing of PCGEM1 suppressed metastatic and invasive cell qualities. PCGEM1 was also found to have associations with miR-129-5p. Subsequently, luciferase reporter and RIP experiments, together with RT-PCR, verified that PCGEM1 functioned as a ceRNA of P4HA2 (Prolyl 4-Hydroxylase Subunit Alpha 2) via sponging miR-129-5p to up-regulate P4HA2 expression. Finally, the rescue assays determined that P4HA2 overexpression rescued the inhibited cell invasion and metastasis caused by PCGEM1 down-regulation. CONCLUSION: These findings found that an over-expression of PCGEM1 in GC acts as a miR-129-5p sponge, leading to higher levels of P4HA2. The PCGEM1/miR-129-5p/P4HA2 axis was confirmed to possess a crucial role in GC metastasis and invasion, suggesting its utility as a potential diagnostic and therapeutic biomarker.

Exosomal Long Non-Coding RNA CEBPA-AS1 Inhibits Tumor Apoptosis and Functions as a Non-Invasive Biomarker for Diagnosis of Gastric Cancer.

AIM: Traditional non-invasive diagnostic markers for gastric cancer (GC) exhibit insufficient sensitivity and specificity. Circulating exosomes are clinically useful non-invasive biomarkers for tumor diagnosis. In addition to their potential role in cancer biology, circulating long non-coding RNAs (lncRNAs) are a new class of promising cancer biomarkers. In the present study, we aimed to identify lncRNAs in circulating exosomes with potential as biomarkers for GC detection. METHODS: We compared the expression of CEBPA-AS1 between GC cells and gastric epithelial cells. The biological function of exosomal CEBPA-AS1 was determined by cell phenotype experiments and rescue assays. We also compared the expression of CEBPA-AS1 in cancerous tissue from GC patients and corresponding adjacent normal tissues, as well as the expression of CEBPA-AS1 in plasma exosomes of GC patients and healthy controls. Diagnostic accuracy was assessed by the receiver operating characteristic (ROC) curve and area under the curve (AUC). RESULTS: CEBPA-AS1 was highly expressed in both GC cells and in exosomes secreted by GC cells. In addition, CEBPA-AS1-containing exosomes secreted by GC cells could promote cell proliferation and inhibit apoptosis, thereby inducing the malignant behavior of GC cells. The level of CEBPA-AS1 was also significantly increased in tissues and plasma exosomes of GC patients. Stability tests showed that most plasma CEBPA-AS1 was encased in exosomes, thus avoiding degradation by RNases. We evaluated the diagnostic accuracy of exosome-derived CEBPA-AS1. The AUC value of CEBPA-AS1 in discriminating GC patients from healthy controls was 0.824, which was higher than the diagnostic accuracy of other traditional tumor biomarkers. CONCLUSION: CEBPA-AS1-containing exosomes secreted from GC cells could promote cell proliferation, inhibit apoptosis, and induce GC progression, indicating that exosomal CEBPA-AS1 is involved in cell-to-cell communication in GC carcinogenesis. Exosomal CEBPA-AS1 is a promising new biomarker for clinical diagnosis of GC.

LINC00163 inhibits the invasion and metastasis of gastric cancer cells as a ceRNA by sponging miR-183 to regulate the expression of AKAP12.

BACKGROUND: Gastric cancer (GC) is the most common and aggressive cancer of the digestive system and poses a serious threat to human health. Since genes do not work alone, our aim was to elucidate the potential network of mRNAs and noncoding RNAs (ncRNAs) in this study. METHODS: Transcriptome data of GC were obtained from TCGA. R and Perl were used to obtain the differentially expressed RNAs and construct a competing endogenous RNA (ceRNA) regulatory network. To investigate the biological functions of differentially expressed RNAs, loss-of-function and gain-of-function experiments were performed. Real-time PCR (RT-qPCR), western blot analysis, dual-luciferase reporter assays and fluorescence in situ hybridization were conducted to explore the underlying mechanisms of competitive endogenous RNAs (ceRNAs). RESULTS: Based on TCGA data and bioinformatics analysis, we identified the LINC00163/miR-183/A-Kinase Anchoring Protein 12 (AKAP12) axis. We observed that AKAP12 was weakly expressed in GC and suppressed invasion and metastasis in GC cells, which could be abolished by miR-183. In addition, LINC00163 can be used as a ceRNA to inhibit the expression of miR-183, thus enhancing the anticancer effect of AKAP12. CONCLUSION: Our results demonstrated that weak LINC00163 expression in GC can sponge miR-183 to promote AKAP12. We established that the LINC00163/miR-183/AKAP12 axis plays an important role in GC invasion and metastasis and may be a potential biomarker and target for GC treatment.

HNRNPAB-regulated lncRNA-ELF209 inhibits the malignancy of hepatocellular carcinoma.

Our previous study demonstrated that heterogeneous nuclear ribonucleoprotein AB (HNRNPAB) is a key gene that facilitates metastasis of hepatocellular carcinoma (HCC). However, the molecular mechanisms behind this relationship are not fully understood. In our study, we utilized long-noncoding RNA (lncRNA) microarrays to identify a HNRNPAB-regulated lncRNA named lnc-ELF209. Our findings from chromatin immunoprecipitation assays indicate that HNRNPAB represses lnc-ELF209 transcription by directly binding to its promoter region. We also analyzed clinical samples from HCC patients and cell lines with quantitative real-time polymerase chain reactions, RNA in situ hybridization and immunohistochemistry, and found that there is a negative relationship between HNRNPAB and lnc-ELF209 expression. Up/downregulation assays and rescue assays indicate that lnc-ELF209 inhibits cell migration, invasion and epithelial-mesenchymal transition regulated by HNRNPAB. This suggests a new regulatory mechanism for HNRNPAB-promoted HCC progression. RNA pull-down and LC-MS/MS were used to determine triosephosphate isomerase, heat shock protein 90-beta and vimentin may be involved in the tumor-suppressed function of lnc-ELF209. Furthermore, we found lnc-ELF209 could stabilize TPI protein expression. We also found that lnc-ELF209 overexpression in HCCLM3 cell resulted in a lower rate of lung metastatic, which suggested a less aggressive HCC phenotype. Collectively, these findings offer new insights into the regulatory mechanisms that underlie HNRNPAB cancer-promoting activities and demonstrate that lnc-ELF209 is a HNRNPAB-regulated lncRNA that may play an important role in the inhibition of HCC progression.

Long noncoding RNA NALT1-induced gastric cancer invasion and metastasis via NOTCH signaling pathway.

BACKGROUND: Long noncoding RNAs (lncRNAs) are aberrant and play critical roles in gastric cancer (GC) progression and metastasis. Searching for coexpressed lncRNA clusters or representative biomarkers related to malignant phenotypes of GC may help to elucidate the mechanism of tumor development and predict the prognosis of GC. AIM: To investigate the prognostic value of NOTCH1 associated with lncRNA in T cell acute lymphoblastic leukemia 1 (NALT1) in GC and the mechanism of its involvement in GC invasion and metastasis. METHODS: RNA sequencing and corresponding clinical data were downloaded from The Cancer Genome Atlas database. The significance module was studied by weighted gene coexpression network analysis. A total of 336 clinical samples were included in the study. Gene silencing, reverse transcription polymerase chain reaction, western blotting, scrape motility assay, and Transwell migration assay were used to assess the function of hub-lncRNAs. RESULTS: At the transcriptome level, 3339 differentially expressed lncRNAs were obtained. weighted gene coexpression network analysis was used to obtain 15 lncRNA clusters and observe their coexpression. Pearson's correlation showed that blue module was correlated with tumor grade and survival. NALT1 was the hub-lncRNA of blue module and was an independent risk factor for GC prognosis. NALT1 was overexpressed in GC and its expression was closely related to invasion and metastasis. The mechanism may involve NALT1 regulation of NOTCH1, which is associated with lncRNA in T cell acute lymphoblastic leukemia, through cis regulation, thereby affecting the expression of the NOTCH signaling pathway. CONCLUSION: NALT1 is overexpressed and promotes invasion and metastasis of GC. The mechanism may be related to regulation of NOTCH1 by NALT1 and its effect on NOTCH signaling pathway expression.

Circulating long non-coding RNA PCGEM1 as a novel biomarker for gastric cancer diagnosis.

AIM: Previous studies have confirmed that overexpression of the long non-coding RNA prostate cancer gene expression marker 1 (PCGEM1) contributes to the invasion and metastasis of gastric cancer (GC) cells. However, the expression of circulating PCGEM1 in the plasma of GC patients and its clinical value remain unclear. METHODS: A total of 317 patients with GC and 100 healthy subjects were enrolled in this study. Circulating PCGEM1 was detected by reverse transcription-polymerase chain reaction. The diagnostic value of plasma PCGEM1 was evaluated by receiver operating characteristic curves and the area under the curve (AUC) value. RESULTS: The expression level of PCGEM1 in the GC group was significantly higher than that in the healthy control subjects. In addition, the PCGEM1 expression level was associated with tumor differentiation and TNM stage. The AUC value of PCGEM1 was higher than that of other conventional tumor markers (CEA, CA12-5, CA72-4, AFP, and CA19-9), although the combination of all markers showed the highest predictive value. CONCLUSION: Plasma PCGEM1 may be a potential novel circulating biomarker for GC diagnosis and prognosis.

ALKBH5 promotes invasion and metastasis of gastric cancer by decreasing methylation of the lncRNA NEAT1.

N6-Methyladenosine (m6A) is the most common posttranscriptional modification of RNA and plays critical roles in cancer pathogenesis. However, the biological function of long noncoding RNA (lncRNA) methylation remains unclear. As a demethylase, ALKBH5 (alkylation repair homolog protein 5) is involved in mediating methylation reversal. The purpose of this study was to investigate lncRNA m6A modification and its role in gastric cancer (GC). Bioinformatics predicted interactions of ALKBH5 with lncRNAs. Five methods were employed to assess the function of nuclear paraspeckle assembly transcript 1 (NEAT1), including gene silencing, RT-PCR, separation of nuclear and cytoplasmic fractions, scrape motility assays, and transwell migration assays. Then, m6A RNA immunoprecipitation and immunofluorescence were used to detect methylated NEAT1 in GC cells. Rescue assays were performed to define the relationship between NEAT1 and ALKBH5. NEAT1 is a potential binding lncRNA of ALKBH5. NEAT1 was overexpressed in GC cells and tissue. Additional experiments confirmed that knockdown of NEAT1 significantly repressed invasion and metastasis of GC cells. ALKBH5 affected the m6A level of NEAT1. The binding of ALKBH5 and NEAT1 influences the expression of EZH2 (a subunit of the polycomb repressive complex) and thus affects GC invasion and metastasis. Our findings indicate a novel mechanism by which ALKBH5 promotes GC invasion and metastasis by demethylating the lncRNA NEAT1. They may be potential therapeutic targets for GC.