Biological Functions Of Gastric Cancer Cells Research Articles

RNF6 promotes gastric cancer progression by regulating CCNA1/CREBBP transcription

ABSTRACT Ring finger protein 6 (RNF6) is a member of the E3 ubiquitin ligase family. Previous studies have reported the involvement of RNF6 as a ubiquitin ligase in the progression of gastric cancer (GC). However, this study found that RNF6 has a clear localization in the nucleus of GC, indicating a role other than ubiquitin ligase. Further chromatin immunoprecipitation sequencing (ChIP-seq) analysis revealed that RNF6 has DNA binding and transcriptional regulatory effects and is involved in important pathways such as tumor cell cycle and apoptosis. Cyclin A1 (CCNA1) and CREB binding protein (CREBBP) are downstream targets for RNF6 transcription regulation in GC. RNF6 binds to the promoter region of CCNA1/CREBBP and is actively regulating their expression in GC cells. Silencing CCNA1/CREBBP partially reversed the promoting effect of RNF6 overexpression on the biological function of GC cells. Our study suggests that RNF6 promotes the progression of GC by regulating CCNA1/CREBBP transcription.

ZFP36L1 Promotes Gastric Cancer Progression via Regulating JNK and p38 MAPK Signaling Pathways.

The RNA-binding protein Zinc Finger Protein 36 like 1(ZFP36L1) plays an important role in regulating the AU-rich elements (AREs) in the 3' untranslated region (3' UTR) of mRNAs, indicating a potential link between its expression and cancers. However, the role and mechanism of ZFP36L1 in gastric cancer (GC) are unclear. This study aimed to explore the role and mechanism of ZFP36L1 in gastric cancer. GC tissue samples and matched normal gastric tissues were collected, and the ZFP36L1 expression in these samples was evaluated by immunohistochemistry analysis. GC cells with different differentiation were selected for in vitro experiments. The ZFP36L1 expression in GC cells was examined by quantitative real-time polymerase chain reaction (qRTPCR) and Western blot analysis. The viability and invasiveness of GC cells were assayed by 5- Ethynyl-2-deoxyuridine (EdU) and Transwell assays, respectively. Western blot assay was used to detect the expression of epithelial-to-mesenchymal transition (EMT) related proteins and proteins of the c-Jun N-terminal kinase (JNK) and p38 Mitogen-Activated Protein Kinase (MAPK) signaling pathways. ZFP36L1 is overexpressed in GC tissues. Patients with high ZFP36L1 expression have a poor prognosis. Moreover, ZFP36L1 is overexpressed in the cell lines with a high degree of malignancy. ZFP36L1 increases cell proliferation, invasion, and migration in vitro. Furthermore, ZFP36L1 induces EMT. The JNK inhibitor and p38 inhibitor alone or in combination affect the biological function of GC cells. Furthermore, ZFP36L1 promotes GC progression by inhibiting JNK and p38 MAPK signaling pathways. RNA-binding protein ZFP36L1 exerts a role in the occurrence of gastric cancer by the regulation of the JNK and p38 MAPK signaling pathways. The combination of inhibitors of the JNK and p38 MAPK signaling pathways could be a novel treatment strategy for gastric cancer.

Helicobacter pylori CagA Protein Regulating the Biological Characteristics of Gastric Cancer through the miR-155-5p/SMAD2/SP1 axis.

Helicobacter pylori (Hp) is a grade Ι carcinogen of gastric cancer (GC), and its high infection rate seriously affects human health. Cytotoxin-associated gene A (CagA) plays a key role in the carcinogenesis of Hp as one of its main virulence factors. miR-155-5p is abnormally expressed in patients with GC, associated with the occurrence and development of cancer. However, little is known about the association between CagA and miR-155-5p. (1) Background: This study explored the association and mechanism of CagA and miR-155-5p in GC. (2) Methods: The CagA sequence was obtained from the NCBI. After sequence optimization, it was connected to the pcDNA3.1 vector to construct a CagA eukaryotic expression plasmid (pcDNA-CagA). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate the expression of miR-155-5p and CagA in GC cells. The function of CagA on GC cells was detected by CCK8, wound healing, and Transwell assays. Similarly, the function of miR-155-5p was also studied through the above functional experiments after the miR-155-5p overexpression and knockdown models had successfully been constructed. The associations among CagA, miR-155-5p, and SMAD2/SP1 were evaluated using RNA immunoprecipitation (RIP) and rescue experiments. (3) Results: The expression of miR-155-5p was significantly reduced in GC cells, and the expression of miR-155-5p was further reduced after CagA induction. Both overexpressed CagA and knockdown miR-155-5p cell models enhanced malignant transformation, whereas overexpressed miR-155-5p inhibited malignant transformation in vitro. The function of miR-155-5p on GC cells could be influenced by CagA. We also found that the influence of miR-155-5p on SMAD2 and SP1 could be regulated by CagA. (4) Conclusions: CagA potentially regulates the biological function of GC cells through the miR-155-5p/SMAD2/SP1 axis. miR-155-5p could be a therapeutic target for GC related to CagA.

Enhancer of zeste homolog 2 exerts functions in gastric cancer development via modulating microRNA-222-3p methylation and WEE1 expression.

Enhancer of zeste homolog 2 (EZH2) has been studied in gastric cancer (GC), while the role of EZH2 in GC via binding to microRNA (miR)-222-3p remains obscure. This research aims to unravel the regulatory mechanism of EZH2 in GC progression via the modulation of miR-222-3p/WEE1 axis. Initially, EZH2, miR-222-3p, and WEE1 levels in GC cells and tissues were examined. Thereafter, constructs altering EZH2, miR-222-3p, or WEE1 expression were transfected into GC cells to determine the malignant behaviors involved in tumorigenesis of GC cells. Finally, the targeting relations among EZH2, miR-222-3p, and WEE1 were validated. EZH2 and WEE1 were upregulated while miR-222-3p was down-regulated in GC tissues and cells. The decreased EZH2, silenced WEE1, or restored miR-222-3p constrained the malignant behaviors involved in tumorigenesis of GC cells. Deletion of miR-222-3p could reverse the effect of silenced EZH2 on suppressing the biological functions of GC cells. EZH2 could bind to the promoter of miR-222-3p, and there was a targeting relationship between miR-222-3p and WEE1. Our study demonstrates that EZH2 promotes GC development via the modulation of miR-222-3p/WEE1 axis, thus providing promising therapeutic targets for GC therapy.

Hsa-circ_0000064 accelerates the malignant progression of gastric cancer via sponging microRNA-621.

Gastric cancer (GC) is one of the most common digestive system tumors in the world. Many circular RNAs (circRNAs) are involved in the progression of GC. The purpose of this study was to delve into the expression characteristics and biological functions of circ_0000064 in GC, and further study its mechanisms. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect circ_0000064 expression in 61 GC tissues and cell lines. Circ_0000064 knockdown was successfully modeled with siRNA. The effects of circ_0000064 on the biological functions of GC cells were analyzed by CCK-8, BrdU, and Transwell assays. Bioinformatics and dual-luciferase reporter gene assay were adopted to explore the relations between circ_0000064 and microRNA-621 (miR-621). Western blot was used to examine the regulatory function of circ_0000064 and miR-621 on SYF2 pre-mRNA splicing factor 2. Cric_0000064 expression was elevated in GC tissues and cell lines. Knocking down cric_0000064 could inhibit the viability, migration, and invasion of GC cells. Dual-luciferase reporter gene assay showed that miR-621 could bind circ_0000064 and SYF2 3'UTR; in addition, miR-621 overexpression or SYF2 knockdown could partially weaken the cancer-promoting effect of circ_0000064 on GC cells. Circ_0000064 expression was negatively correlated with miR-621 expression in GC tissues while positively with SYF2 expression. Circ_0000064 can participate in the GC progression via modulating miR-621/SYF2 axis. This implies that circ_0000064 may be a new diagnosed biomarker or a new therapeutic target of GC.

Upregulated hsa_circRNA_100269 inhibits the growth and metastasis of gastric cancer through inactivating PI3K/Akt axis.

The pathogenesis of GC involves the complex networking of multiple signaling pathways; however, the detailed mechanisms of tumorigenesis of GC remains largely unknown. Therefore, it is necessary to explore novel diagnostic/prognostic biomarkers for GC. In this study, the levels of hsa_circRNA_100269 in gastric cancer (GC) samples and cells were examined, and its effects on the biological functions of GC cells were elucidated. The levels of hsa_circRNA_100269 in specimens/cell lines were examined using RT-qPCR. Cell models with hsa_circRNA_100269 overexpression or knockdown were generated using lentiviral vectors. Cell viability was determined by MTT assay; cell migratory/invasive activity was evaluated using wound healing/Transwell assay. Cell cycle arrest and apoptosis were assessed by flow cytometry; expression of associated markers involved in cell apoptosis, EMT and the PI3K/Akt signaling were determined by RT-qPCR/immunoblotting. In vivo study was also performed using hsa_circRNA_100269 knockout mice. Our findings revealed downregulation of hsa_circRNA_100269 in GC tissues compared to non-cancerous control. Additionally, the levels of PI3K were remarkably elevated in GC tissues, where hsa_circRNA_100269 and PI3K was negatively correlated. Moreover, the expression of hsa_circRNA_100269 was associated with histology grade and occurrence of metastasis in GC patients. In addition, hsa_circRNA_100269 was downregulated in GC cells compared to normal gastric epithelial cells. Overexpressed hsa_circRNA_100269 notably inhibited the proliferation, migration, invasion and EMT of GC cells, whereas cell cycle arrest at G0/G1 phase was promoted and cell apoptosis was enhanced. Moreover, the PI3K/Akt signaling was involved in hsa_circRNA_100269-regulated GC cell proliferation, migration, invasion, EMT and apoptosis. Knockdown of hsa_circRNA_100269 also remarkably induced tumor growth in mouse model. In summary, our findings indicated that the levels of hsa_circRNA_100269 were reduced in GC. Furthermore, hsa_circRNA_100269 could suppress the development of GC by inactivating the PI3K/Akt pathway. More importantly, hsa_circRNA_100269/PI3K/Akt axis may be a novel therapeutic candidate for GC treatment.

IL‑33/ST2 promotes the malignant progression of gastric cancer via the MAPK pathway.

Gastric cancer (GC) remains one of the commonest malignant tumors and the second leading cause of cancer-related deaths worldwide. IL-33 is highly expressed in tumor tissues and serum of patients with GC. However, the function of the IL-33 and IL-33 receptor ST2 in the malignant progression of GC is yet to be elucidated. The present study aimed to explore the effect of the IL-33/ST2 axis on the biological functions of GC cells. The expression of ST2 in GC tissues was measured by immunohistochemistry. GC cell lines (AGS and MKN45) were treated with IL-33, and the expression of ST2 was downregulated by using specific siRNA. The effects of the IL-33/ST2 axis on cell proliferation, migration, invasion, cell cycle and apoptosis was detected by CCK8, Transwell, wound healing, flow cytometry and western blotting assays. The present study found that ST2 was highly expressed in GC tissues compared with normal tissues. IL-33 promoted the proliferation and cell cycle progression of GC cells, and upregulated the expression levels of CDK4, CDK6 and cyclin D1. Furthermore, IL-33 inhibited the apoptosis of GC cells and regulated the expression of apoptosis-associated proteins. In addition, IL-33 stimulated the invasion and migration of GC cells. However, the transfection of ST2 small interfering (si)RNA attenuated the effects of IL-33. Finally, IL-33 stimulation increased the phosphorylation levels of ERK1/2, JNK and p38. The transfection of ST2 siRNA could significantly inhibit the IL-33-induced ERK1/2, JNK and p38 activation. In conclusion, it was found that ST2 was highly expressed in GC tissues. IL-33/ST2 promoted the malignant progression of GC cells by inducing the activation of ERK1/2, JNK and p38.

N6-Methyladenosine Demethylase Fto Promotes Growth and Metastasis of Gastric Cancer <i>via</i> M <sup>6</sup>A Modification of Caveolin-1 and Metabolic Regulation of Mitochondrial Dynamics

Background: Gastric cancer (GC) is the fifth most common tumor and the third most deadly cancer worldwide. N6-methyladenosine (m6A) modification has been reported to play a regulatory role in human cancers. However, the exact role of m6A in GC remains largely unknown, and the dysregulation of m6A on mitochondrial metabolism has never been studied. Method: The expression level of FTO in the GC tissue was analyzed by real-time quantitative PCR, Western blotting, IF and IHC. Colorimetric m6A RNA methylation assay kit and Dot blotting were used to evaluate the level of m6A methylated RNA. In vitro assays, including cell viability, colony formation assay, wound healing assay and invasion assay were performed to detect the effects of FTO and caveolin-1 manipulation on the biological functions of GC cells. Xenografts models were constructed to evaluate the effects of FTO and caveolin-1 manipulation on the biological function of GC cell in vivo. HiSeq RNA-Seq was used to screen the potential targets of FTO. Real-time integrated cellular oxygen consumption rate (OCR) was measured using the Seahorse XF24 Extracellular Flux Analyzer. Finding: We demonstrated that FTO, a key demethylase for RNA m6A modification, was up-regulated in GC tissues, especially in tissues with liver metastasis. Functionally, FTO acted as a promoter for the proliferation and metastasis in GC. Moreover, FTO enhanced the degradation of caveolin-1 mRNA via its demethylation, which regulated the mitochondrial fission/fusion and metabolism. Interpretation: Our current findings provided some valuable insights into FTO-mediated m6A demethylation modification and could be used as a new strategy for more careful surveillance and aggressive therapeutic intervention. Funding: This project was supported by grants from the National Key R&D Plan (2018YFC1313400), National Natural Science Foundation of China (31701111), Young Talent Development Plan of Changzhou Health Commission (CZQM2020008, CZQM2020018) and Youth Talent Science and Technology Project of Changzhou Health Commission (QN202014). Declaration of Interest: None to declare. Ethical Approval: All animal experiments were approved by the Animal Care Committee of the Third Affiliated Hospital of Soochow University. All patients have provided written informed consent, and our study was preformed after formal approval by the Institutional Review Board of the Third Affiliated Hospital of Soochow University.

Apatinib regulates the growth of gastric cancer cells by modulating apoptosis and autophagy.

Apatinib is a novel, highly selective small-molecule inhibitor of the tyrosine kinase VEGFR-2. Although its safety and efficacy in the treatment of advanced gastric cancer (GC) and other solid tumors have been confirmed, the precise molecular mechanism underlying its efficacy remains unclear. The purpose of this study was to investigate the mechanism by which apatinib regulates the biological functions of GC cells in vitro. The CCK-8 assay was used to detect the inhibitory effect of apatinib at different concentrations on the proliferation of SGC7901 and MKN45 human GC cells. The effects of apatinib on apoptosis, autophagy, and cell cycle-related genes in SGC7901 and MKN45 cells were detected by Western blotting and real-time quantitative PCR (RT-qPCR). JC-1 staining, flow cytometry, Hoechst 33342 staining, dansylcadaverine (MDC) staining, and Transwell assays were used to detect the effects of apatinib on apoptosis, the cell cycle, autophagy, and invasion and migration capacities, respectively, in SGC7901 and MKN45 cells. The inhibitory effect of apatinib on the proliferation of GC cells was dependent on concentration. Apatinib significantly promoted apoptosis and autophagy. It also altered the cell cycle distribution and inhibited the invasion and migration of GC cells. In general, apatinib inhibited the proliferation of GC cells by promoting apoptosis and autophagy, regulating the cell cycle and inhibiting the invasion and migration capacities of GC cells.

LINC01857 promotes the development of gastric cancer by regulating microRNA-200b.

The aim of this study was to investigate the expression characteristics of LINC01857 in gastric cancer (GCa), and to further study whether it could promote GCa development by modulating microRNA-200b. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to examine LINC01857 expression in 60 pairs of GCa tissues and adjacent tissues. The interplay between LINC01857 level and clinical indexes and the prognosis of GCa patients was analyzed. Meanwhile, qRT-PCR was used to verify the expression level of LINC01857 in GCa cell lines. LINC01857 knockdown model was constructed by lentivirus transfection in GCa cell lines. Subsequently, the effect of LINC01857 on the biological function of GCa cells was analyzed by Cell Counting Kit 8 (CCK8), wound healing, and transwell assays. Furthermore, the in-depth relationship between LINC01857 and microRNA-200b was explored. QRT-PCR results showed that LINC01857 level in GCa tissues was remarkably higher than that of adjacent tissues, and the difference was statistically significant (p<0.05). Compared with patients with a low level of LINC01857, the rate of lymph node and distant metastasis in patients with a high level of LINC01857 was remarkably higher, while the overall survival rate was lower (p<0.05). In vitro experiments showed that LINC01857 knockdown remarkably decreased the invasion, migration, and crawling ability of GCa cells (p<0.05). Subsequent qRT-PCR results demonstrated that the level of microRNA-200b was remarkably upregulated after the silence of LINC01857. In addition, the silence of microRNA-200b could reverse the biological function of GCa cells induced by the knockout of LINC01857. LINC01857 was highly expressed in GCa, and was associated with lymph node metastasis, distant metastasis, and poor prognosis of patients with GCa. In addition, LINC01857 enhanced the metastatic ability of GCa cells by regulating microRNA-200b.

ETV4 promotes the progression of gastric cancer through regulating KDM5D.

This study was aimed to investigate the expression characteristics of ETS variant 4 (ETV4) in gastric cancer (GCa), and to further explore whether it promotes the development of GCa by regulating KDM5D. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was performed to examine the expression of ETV4 in 35 pairs of tumor tissue and paracancerous tissue specimens collected from GCa patients, and the interplay between ETV4 expression and clinical indexes, as well as the prognosis of GCa patients, were analyzed. Meanwhile, the expression of ETV4 in GCa cell lines was verified using qRT-PCR assay. Furthermore, ETV4 knockdown model was constructed using lentivirus in GCa cell lines including AGS and BGC-823, and then, the transwell invasion and cell wound healing assays were applied to analyze the effect of ETV4 on the biological function of GCa cells. In addition, an in-depth study of the relationship between ETV4 and KDM5D was conducted. The results of qRT-PCR showed that the expression level of ETV4 in GCa tissue samples was remarkably higher than that in adjacent tissues, and the difference was statistically significant. Compared with patients with low expression of ETV4, the patients with high ETV4 expression had a higher occurrence rate of lymph node or distant metastasis and a lower overall survival rate. Similarly, the metastasis ability of GCa cells in the ETV4 expression knockdown group (sh-ETV4) was remarkably decreased when compared with the sh-NC group. In addition, qRT-PCR results indicated that the protein expression of KDM5D was significantly increased after the knockdown of ETV4. Therefore, it was demonstrated that ETV4 might be able to regulate the malignant progression of GCa via modulating KDM5D expression. Finally, the results of the cell reverse experiment confirmed that the silence of ETV4 could reverse the malignant progression of GCa induced by the downregulation of KDM5D. ETV4 expression was found remarkably elevated in GCa tissues and was significantly associated with the occurrence of lymph node or distant metastasis and poor prognosis. In addition, ETV4 might promote GCa cell metastasis by modulating KDM5D.

LINC01116 promotes the proliferation and inhibits the apoptosis of gastric cancer cells.

To detect the relative expression of long intergenic non-protein coding ribonucleic acid (LINC) 01116 in gastric cancer (GC) tissues and cells and analyze the correlations of LINC01116 expression with the clinicopathologic characteristics of patients and investigate the biological functions of LINC01116 via in vitro experiments. The quantitative Real Time Fluorescence-Polymerase Chain Reaction (qRT-PCR) was applied to detect the relative expression level of LINC01116 in 73 cases of tissues and cells in GC patients. The patients were divided into LINC01116 high expression group and LINC01116 low expression group, and the correlations of LINC01116 with patient's pathological characteristics were statistically analyzed. In vitro experiments [cell counting kit-8 (CCK-8) assay, colony formation assay, and flow cytometry] were adopted to investigate the influences of LINC01116 on the biological functions of GC cells. According to the results of qRT-PCR, the expression of LINC01116 was upregulated in 54 out of 73 cases of tissues (fold change >1), and it was upregulated in GC cells compared with that in the normal gastric mucosal epithelial cells (GES-1). The statistical analysis manifested that the highly expressed LINC01116 was positively correlated with the tumor-node-metastasis (TNM) stage (p=0.008), lymph node metastasis (p=0.005), and depth of invasion (p=0.007) of the GC patients. The patients with high expression of LINC01116 in the GC tissues had a shorter survival time than those with low expression (p=0.017). After interference in the expression of LINC01116, it was shown in CCK-8 assay and colony formation assay that the proliferative capacity of the cells was decreased. The results of flow cytometry indicated that the cell cycle was arrested at the G1/G0 phase, and the apoptosis rate was increased. LINC01116 is highly expressed in GC tissues and cells, and highly expressed LINC01116 indicates poor prognosis of the patients, promotes the proliferation, and inhibits the apoptosis of GC cells.

CYP2E1 changes the biological function of gastric cancer cells via the PI3K/Akt/mTOR signaling pathway.

The present study investigated the role of cytochrome P450 family 2 subfamily E polypeptide 1 (CYP2E1) in the development and progression of gastric cancer (GC). The expression levels of CYP2E1 in MGC-803 GC cells and normal GES-1 cells were investigated via western blotting, and it was identified that the expression of CYP2E1 was different between GES-1 and MGC-803 cells. CYP2E1 was overexpressed in MGC-803 cells using a lentiviral vector GV358. Cell Counting Kit-8, flow cytometry, cell migration and Matrigel invasion assays suggested that overexpression of CYP2E1 promoted the proliferation and invasion, and inhibited the apoptosis of GC cells. The relationship between CYP2E1 expression and key signaling molecules in the PI3K/Akt/mTOR signaling pathway was assessed. Reverse transcription-quantitative PCR analysis showed that mTOR mRNA expression was significantly increased after overexpression of CYP2E1 (P<0.05). Western blotting results showed that overexpression of CYP2E1 upregulated the expression of phosphorylated (p)-Akt, p-mTOR and p-p70 ribosomal protein S6 kinase (P70S6K; Ser371) proteins (P<0.05). To further investigate the relationship between CYP2E1 and the PI3K/Akt/mTOR signaling pathway in GC cells, MGC-803 cells were treated with the PI3K inhibitor LY294002, and changes in the expression levels of PI3K, AKT, mTOR, P70S6K and CYP2E1 were observed. The present results showed that LY294002 downregulated the expression of PI3K, CYP2E1, AKT, mTOR and P70S6K (P<0.05). Therefore, changes in the biological function of GC cells induced by CYP2E1 overexpression may be via the PI3K/Akt/mTOR signaling pathway.

MicroRNA-4513 Promotes Gastric Cancer Cell Proliferation and Epithelial-Mesenchymal Transition Through Targeting KAT6B.

The purpose of this study was to investigate the expression level of microRNA-4513 (miR-4513) in gastric cancer (GC), and to elucidate the mechanisms underlying its regulation of GC progression. Quantitative real-time PCR (qRT-PCR) was performed to measure the expression level of miR-4513 in GC cells. Transfection efficacy of synthetic miRNAs was examined by qRT-PCR. After synthetic miRNA transfection, cell counting kit-8 assay and transwell invasion assay were conducted to measure biological changes in these groups. The key molecular expression level involved in epithelial-mesenchymal transition (EMT) was analyzed by Western blot. Bioinformatic analysis and Western blot were performed to investigate the connection between miR-4513 and lysine acetyltransferase 6B (KAT6B). qRT-PCR results showed that miR-4513 expression level was upregulated in GC cell lines. Downregulation of miR-4513 expression inhibited GC cell proliferation, invasion, and EMT. KAT6B was validated as a direct target of miR-4513. In addition, KAT6B expression level can be upregulated by miR-4513 inhibitor. Collectively, we showed that miR-4513 is involved in regulating the biological function of GC cells via KAT6B. In addition, miR-4513 may serve as a potential target for the molecular therapy of GC.