Functions Of Gastric Cancer Cells Research Articles

Long non-coding RNA HCG18 promotes gastric cancer progression by regulating miRNA-146a-5p/tumor necrosis factor receptor-associated factor 6 axis

ABSTRACT Although long non-coding RNAs (lncRNAs) have been demonstrated to be dysregulated in gastric cancer (GC), the function of lncRNA HCG18 (HCG18) in GC is elusive. Therefore, the study was designed to evaluate the underlying mechanism of HCG18 in GC. HCG18 and microRNA 146a-5p (miR-146a-5p) levels in GC were evaluated by RT-qPCR. The effects of miR-146a-5p and HCG18 on GC cell function were examined using Transwell assay, colony formation, and CCK-8 assays. Tumor necrosis factor receptor-associated factor 6 (TRAF6) and p65 expression levels were detected by Western blot. HCG18 and miR-146a-5p target genes were identified using luciferase reporter and bioinformatics assays. HCG18 expression was increased in GC. HCG18 overexpression significantly increased GC cell proliferation, invasion, and migration. Furthermore, HCG18 overexpression inhibited miR-146a-5p and upregulated TRAF6 and p65 expression. Finally, miR-146a-5p/TRAF6 was found to be involved in the role of HCG18 in GC progression in vivo. Altogether, HCG18 promotes GC progression via the miR-146a-5p/TRAF6 axis and could be a GC treatment target.

Long noncoding RNA NR2F1-AS1 plays a carcinogenic role in gastric cancer by recruiting transcriptional factor SPI1 to upregulate ST8SIA1 expression

ABSTRACT Gastric cancer (GC) is a highly malignant solid tumor of the digestive tract, which is associated with a high mortality rate. Long non-coding RNA (lncRNA) nuclear receptor subfamily 2 group F member 1 antisense RNA 1 (NR2F1-AS1) has been reported to exert a tumor-promoting effect in some types of cancer. The present study aimed to investigate the role of NR2F1-AS1 in GC. The expression levels of NR2F1-AS1 and its potential target gene were measured in GC cell lines. Bioinformatics analysis, an RNA immunoprecipitation assay and a chromatin immunoprecipitation assay were used to determine the binding relationship between NR2F1-AS1 and downstream genes. The effect of NR2F1-AS1 regulatory axis on AGC cell viability, proliferation, migration, invasion and epithelial-mesenchymal transition was evaluated. The results of the present study revealed that the knockdown of NR2F1-AS1 inhibited the proliferation, invasion and migration of GC cells. NR2F1-AS1 also upregulated the expression levels of ST8SIA1 by recruiting transcriptional factor SPI1. Thus, the effects of the knockdown of NR2F1-AS1 on GC cell functions were suggested to occur via regulation of ST8SIA1. In conclusion, the findings of the current study indicated that NR2F1-AS1 may promote the proliferation, invasion and migration of GC cells by recruiting SPI1, to upregulate ST8SIA1 expression. Thus, the regulation of their expression levels may provide a novel direction for the treatment of GC.

LncRNA SLCO4A1-AS1 Accelerates Growth and Metastasis of Gastric Cancer via Regulation of the miR-149/XIAP Axis.

ObjectiveRecently, long noncoding RNA SLCO4A1 antisense RNA 1 (SLCO4A1-AS1) has been shown to act as an oncogene in several cancer types; however, its role in gastric cancer (GC) and its underlying molecular mechanisms are yet to be elucidated.MethodsUsing the ENCORI database, we identified SLCO4A1-AS1, miR-149-5p (miR-149), and the X-linked inhibitor of apoptosis (XIAP) whose expressions were obviously changed in GC samples, and analyzed the correlation between their expressions in GC samples. Moreover, we explored the expression of SLCO4A1-AS1, miR-149, and XIAP in clinical samples and GC cell lines using RT-qPCR and western blotting assay; the correlation between them was analyzed using RNA immunoprecipitation and dual-luciferase reporter. CCK-8, colony formation, and Transwell assays were conducted to determine the effects of SLCO4A1-AS1, miR-149, and XIAP expression on cell proliferation, migration, and invasion, respectively. A nude mouse xenograft model was used to explore their function in xenograft growth.ResultsSLCO4A1-AS1 was significantly upregulated in the GC samples and cell lines, and a high level of SLCO4A1-AS1 was associated with an advanced tumor stage and shortened patient survival. Mechanistically, SLCO4A1-AS1 post-transcriptionally regulated XIAP by functioning as competing endogenous RNA in GC to sponge miR-149. Further functional assays revealed that the overexpression of miR-149 and knockdown of XIAP considerably inhibited GC cell viability and its migratory and invasive characteristics in vitro. SLCO4A1-AS1 knockdown also determined the function of GC cells but was diminished by the miR-149 inhibitor in vitro. Finally, we demonstrated that the deletion of SLCO4A1-AS1 suppressed tumor growth and metastasis in vivo.ConclusionsAltogether, these findings suggest that SLCO4A1-AS1 functions as a crucial oncogenic lncRNA in GC and it can facilitate GC tumor growth and metastasis by interacting with miR-149 and enhancing XIAP expression. Therefore, SLCO4A1-AS1 is a potential novel therapeutic target in GC treatment.

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.

HnRNPA2B1 regulates the alternative splicing of BIRC5 to promote gastric cancer progression

BackgroundSystematic profiling studies have implicated regulators of pre-mRNA splicing as important disease determinants in gastric cancer (GC), but the underlying mechanisms have remained elusive. Here we focused on hnRNPA2B1 splicing factor-dependent mechanisms governing GC development.MethodsThe expression of hnRNPA2B1 was analyzed among the Cancer Genome Atlas (TCGA) datasets of GC and validated at mRNA level. The function of hnRNPA2B1 in GC cells was analyzed and its downstream gene was identified using RNA immunoprecipitation. Further, effect of hnRNPA2B1 on BIRC5 alternative splicing was investigated.ResultsWe show that overexpression of hnRNPA2B1 in GC is correlated with poor survival, and hnRNPA2B1 is required for maintaining GC malignant phenotype by promoting cell proliferation, inhibiting cell apoptosis and increasing cell metastasis. Mechanistically, hnRNPA2B1 co-expressed with several core spliceosome components and controls alternative splicing of anti-apoptotic factor BIRC5. BIRC5 isoform 202 (BIRC5-202) played the oncogenic function in GC cells, and overexpression of the BIRC5-202 transcript partly rescued the decrease in cisplatin resistance induced by downregulation of hnRNPA2B1.ConclusionsWe demonstrate that hnRNPA2B1 regulates BIRC5 splicing and might act as a therapeutic target of chemo-resistant GC cells.

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.

Calycosin Induces Gastric Cancer Cell Apoptosis via the ROS-Mediated MAPK/STAT3/NF-κB Pathway.

BackgroundCalycosin, an active compound in plants, can promote the apoptosis of various cancer cells; however, the mechanism by which it regulates reactive oxygen species (ROS) in gastric cancer (GC) cells remains unclear.PurposeIn this study, we investigated the effects of calycosin on apoptosis, the cell cycle, and migration in GC cells under ROS regulation.ResultsThe results of the Cell Counting Kit-8 assay suggested that calycosin had significant cytotoxic effects on 12 gastric cancer cells, but no significant cytotoxic effects on normal cells. Hoechst 33342/propidium iodide (PI) double staining and flow cytometry showed that calycosin had clear pro-apoptotic effects on AGS cells. Western blotting revealed that the expression of cytochrome C and pro-apoptotic proteins B-cell lymphoma 2 (Bcl-2)-associated agonist of cell death (Bad), cleaved (cle)-caspase-3, and cle-poly (ADP-ribose) polymerase gradually increased, and the expression of anti-apoptotic protein Bcl-2 gradually decreased. Calycosin also decreased the expression of extracellular signal-regulated kinase, nuclear factor kappa B (NF-κB), and signal transducer and activator of transcription 3 (STAT3), and increased the phosphorylation levels of p38, c-Jun N-terminal kinase, and inhibitor of NF-κB. In addition, calycosin markedly increased ROS accumulation, and pretreatment with active oxygen scavenger n-acetyl-l-cysteine (NAC) clearly inhibited apoptosis. Calycosin downregulated the cell cycle proteins cyclin-dependent kinase 2 (CDK2), CDK4, CDK6, cyclin D1, and cyclin E; upregulated p21 and p27; and arrested cells in the G0/G1 phase. Similarly, calycosin also downregulated Snail family transcriptional repressor 1, E-cadherin, and β-catenin and inhibited cell migration. However, pretreatment with NAC inhibited the calycosin-induced effects of cycle arrest and migration.ConclusionIn summary, calycosin induces apoptosis via ROS-mediated MAPK/STAT3/NF-κB pathways, thereby exerting its anti-carcinogenic functions in GC cells.

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.

LncRNA SNHG4 promotes cell proliferation, migration, invasion and the epithelial-mesenchymal transition process via sponging miR-204-5p in gastric cancer.

Long non-coding (lnc)RNAs and microRNAs (miRNAs/miRs) have physiological and pathological functions in various diseases, including gastric cancer (GC). The current study explored the association between lncRNA small nucleolar RNA host gene 4 (SNHG4) and miR-148a-3p, and their functions in GC cells. SNHG4 expression and overall survival data were analyzed using bioinformatics, and the interaction of SNHG4 and miR-148a-3p was predicted using starBase and confirmed via a dual-luciferase reporter assay. Cell viability, colony formation ability and apoptosis rate were detected using Cell Counting Kit-8, colony formation and flow cytometry assays, respectively. Cell migration and invasion were determined via wound-healing and Transwell assays. mRNA and protein expression levels were determined via reverse transcription-quantitative PCR and western blotting. The results demonstrated that in GC tissues and cell lines, SNHG4 was highly expressed, while miR-204-5p expression was decreased, and that the expression levels of SNHG4 and miR-204-5p were negatively correlated. The downregulated expression of SNHG4 decreased the effects of miR-204-5p inhibitor on promoting cell proliferation, migration, invasion and epithelial-mesenchymal transition, but enhanced the inhibitory effect of miR-204-5p on GC cell apoptosis. The findings of the current study revealed the potential mechanism of the SNHG4-miR-204-5p pathway in GC, which may be conducive to the development of novel drugs against GC growth.

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.

LncRNA RPPH1 predicts poor prognosis and regulates cell proliferation and migration by repressing P21 expression in gastric cancer.

The purpose of this study was to explore the expression and biological functions of long non-coding ribonucleic acid (lncRNA) ribonuclease P RNA component H1 (RPPH1) in gastric cancer (GC), and to analyze the correlations of lncRNA expression with the clinical features and prognosis of GC patients. The relative expression of RPPH1 in tissue specimens from 60 GC patients was measured via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), and the correlations of RPPH1 expression with tumor-node-metastasis (TNM) stage, lymph node metastasis, etc. in GC patients were analyzed. Then, qRT-PCR was performed to detect the relative expression level of RPPH1 in GC cells. Moreover, colony formation assay, 5-Ethynyl-2'-deoxyuridine (EdU) staining, wound-healing assay, and transwell assay were employed to investigate the influence of RPPH1 on GC cell functions. After interfering in the expression of RPPH1, the changes in p21 (CDKN1A, cyclin dependent kinase inhibitor 1A) expression were determined through qRT-PCR and Western blotting. It was shown in qRT-PCR assay results that the expression of RPPH1 was upregulated in 60 cases of GC tissues. Statistical analysis revealed that RPPH1 expression was positively correlated with the TNM stage, lymph node metastasis, and infiltration depth in GC patients. Besides, highly expressed lncRNA RPPH1 suggested poor prognosis of GC patients. Based on the results of qRT-PCR assay, the expression of RPPH1 in GC cells was upregulated. After interfering in RPPH1 expression, both colony formation assay and EdU staining indicated that the proliferative capacity of GC cells was repressed. Furthermore, it was manifested in the results of wound-healing and transwell assays that the migratory and invasive abilities of GC cells were weakened. Finally, the qRT-PCR and Western blotting assay results demonstrated that p21 expression was upregulated after interfering in the expression of RPPH1 in GC cells. The expression of lncRNA RPPH1 is upregulated in GC, suggesting that the prognosis of the patients is poor. Highly expressed RPPH1 promotes the proliferation and metastasis of GC cells by regulating p21 expression.

GALNT10 promotes the proliferation and metastatic ability of gastric cancer and reduces 5-fluorouracil sensitivity by activating HOXD13.

To investigate the expression and potential mechanism of GALNT10 in gastric cancer (GC). A total of 60 cases of GC tissues, as well as normal tissues were collected. The total RNA of GC specimens and cells were extracted by TRIzol method and the level of GALNT10 was examined by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the relationship between GALNT10 and clinical parameters and prognosis of GC patients was analyzed. Subsequently, Lentivirus was used to construct GALNT10 knockdown GC cell lines, and cell counting kit-8 (CCK-8) and transwell assays were applied to analyze the influence of GALNT10 on GC cell function. Bioinformatics and Luciferase assay was used to evaluate the relationship between GALNT10 and HOXD13. Furthermore, 5-fluorouracil (5-Fu)-resistant cells were used to detect the relationship between GALNT10 and 5-Fu sensitivity of GC cells. qRT-PCR results revealed that GALNT10 level was markedly increased in tissues, as well as cell lines of GC. Statistical analysis suggested that GALNT10 expression was in close relation with the incidence of lymph node and distant metastasis along with poor prognosis in GC patients, but not with other indicators. CCK-8 and transwell migration experiment results indicated that GALNT10 silencing can inhibit the proliferative and migration ability of GC cells. Western blot results displayed that the HOXD13 level was remarkably decreased after GALNT10 knocking down. In addition, Luciferase gene assay indicated that GALNT10 could bind to HOXD13. Further rescue experiments showed that HOXD13overexpression can synergistically reverse the inhibitory effect of GALNT10 knockdown on GC cell proliferative and migration ability, which further demonstrated that GALNT10 could promote GC cell metastasis ability and reduce the sensitivity to 5-Fu by regulating HOXD13. GALNT10 could regulate the proliferative and migration ability of GC cells and reduce the sensitivity to 5-Fu by enhancing the expression of HOXD13. Therefore, GALNT10 was expected to be a new therapeutic target for diagnosis of 5-fluorouracil resistance in GC.

SNHG10 Promotes Cell Proliferation and Migration in Gastric Cancer by Targeting miR-495-3p/CTNNB1 Axis.

Long non-coding RNAs have been acknowledged as the crucial regulators in the progression of human cancers, including gastric cancer (GC). Small nucleolar RNA host gene 10 (SNHG10) has been identified as an oncogene in several cancer types. Nonetheless, it is unclear whether SNHG10 exerts functions in GC cells. The aims of the current study were to explore the function and underlying mechanism of SNHG10 in GC. The expression levels of SNHG10, miR-495-3p and catenin beta 1 (CTNNB1) were detected by RT-qPCR. Loss-of-function assays, including CCK-8, colony formation assay, flow cytometry analysis and transwell assays, were conducted to verify the effect of SHNG10 on the proliferation, apoptosis, migration and invasion of GC cells. Mechanism experiments were performed to identify the downstream molecular mechanism of SNHG10. SNHG10 was expressed at a high level in GC cells. Knockdown of SNHG10 inhibited the proliferation, migration and invasion of GC cells. Silencing of SNHG10 led to the downregulation of core factors of WNT signaling pathway. Knockdown of SNHG10 could decline the expression of CTNNB1 through sequestering miR-495-3p. SNHG10 promotes the procession of GC through targeting miR-495-3p/CTNNB1 and activating WNT signaling pathway.

HOTTIP Predicts Poor Survival in Gastric Cancer Patients and Contributes to Cisplatin Resistance by Sponging miR-216a-5p.

Gastric cancer (GC) is a significant public health burden worldwide, and cisplatin resistance is the leading cause for the failure of chemotherapy in this disease. Previous studies have revealed that HOXA transcript at the distal tip (HOTTIP) is involved in the pathology of GC and is associated with poor overall survival. However, the functional role of HOTTIP in GC chemoresistance remains unclear. In this study, quantitative real-time PCR was used to analyze HOTTIP expression in GC cell lines and in tissues of GC patients who received cisplatin-based chemotherapy. The mechanism of HOTTIP-mediated chemoresistance was assessed using cell viability, apoptosis, and autophagy assays. The relationships among HOTTIP, miR-216a-5p, and Bcl-2 were determined using luciferase reporter and western blot assays. HOTTIP was markedly upregulated in the tissues of GC patients who were treated with gastrectomy and cisplatin chemotherapy, especially in those with recurrent tumors. Further, HOTTIP was increased in the cisplatin-resistant cell line, SGC7901/DDP, compared to the parental cells, SGC7901. Functional assays demonstrated that HOTTIP expression promoted cisplatin resistance and inhibited apoptosis and autophagy in GC cells. Mechanistic investigations revealed that HOTTIP may regulate the functions of GC cells by sponging miR-216a-5p. MiR-216a-5p overexpression decreased Bcl-2 expression, enhanced Beclin1 expression, and active autophagy. Taken together, our study demonstrated that HOTTIP is closely associated with recurrence in GC patients. HOTTIP expression confers cisplatin resistance by regulating the miR-216a-5p/BCL-2/Beclin1/autophagy pathway, which provides a novel strategy to overcome resistance to chemotherapy in GC.

MicroRNA-216a-5p inhibits the development of gastric cancer through target combination with TCTN1.

We aimed at investigating microRNA-216a-5p expression in gastric cancer (GC) tissues and further exploring whether microRNA-216a-5p suppresses GC progression through interacting with TCTN1. microRNA-216a-5p expression in 60 pairs of GC tissues and adjacent ones was studied by quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and the relationship between microRNA-216a-5p and clinical indicators as wells as prognosis of GC patients was also analyzed. At the same time, qRT-PCR was conducted to further verify microRNA-216a-5p level in GC cells. The impacts of microRNA-216a-5p on GC cell functions were evaluated using cell counting kit-8, plate cloning and Transwell experiments. Meanwhile, we studied the specific regulatory relationship between microRNA-216a-5p and TCTN1 in depth. Our data showed that microRNA-216a-5p level in GC tumor specimens was remarkably lower than that in adjacent ones. In comparison to patients in group of high microRNA-216a-5p expression, patients in group of low expression showed an increased metastasis incidence and a lower survival rate. Cell functional experiments suggested that microRNA-216a-5p mimics markedly attenuated the proliferative and migratory capacities of GC cells. Bioinformatics analysis suggest that microRNA-216a-5p can bind to its target gene TCTN1, which was confirmed by luciferase assay. Further, qPCR results revealed a negative correlation between the expression of TCTN1 and microRNA-216a-5p in GC tumor tissues. Finally, in vitro cell experiments suggested that overexpression of TCTN1 could reverse the inhibitory impact of upregulation of microRNA-216a-5p on GC cell functions. microRNA-216a-5p, abnormally lowly expressed in GC tissues, is markedly relevant to the high metastasis incidence and the poor prognosis of GC patients; in addition, microRNA-216a-5p inhibited GC's migration and proliferation capabilities through regulating TCTN1.

CircRBM33 regulates IL-6 to promote gastric cancer progression through targeting miR-149

There is increasing evidence of the vital role played by circular RNAs (circRNAs) in the progression of gastric cancer (GC). A circRNA, hsa_circ_0001772, was generated from the RBM33 gene and named circRBM33. The aim of this study was to investigate the role of circRBM33 in GC. Quantitative real-time PCR (qRT-PCR) was used to quantify the expression of circRBM33 in 79 pairs of GC tissues and paracancerous tissues and 4 GC cell lines (MGC-803, BGC-823, SGC-7901, and AGS). Bioinformatics databases were used to predict downstream targets of circRNA and micro RNA (miRNA). Dual luciferase reporter assay was used to verify whether miR-149 was a direct binding target for circRBM33. Cell Counting Kit-8 (CCK-8) assay, 5-Ethynyl-2´-deoxyuridine (EDU) assay, transwell assay, and flow-cytometric analyses were performed to determine the role of circRBM33 in the biological functioning of GC cells. Western blot technique was used to quantify the levels of interleukin-6 (IL-6). CircRBM33 was distinctly upregulated in GC specimens and cell lines and a close correlation between circRBM33 expression and clinical characteristics of GC was observed. After silencing circRBM33, the apoptosis of GC cells increased, while proliferation, migration, and invasion decreased. Rescue experiments indicated that circRBM33 manipulates biological function in GC cells through the circRBM33/miR-149/IL-6 axis. CircRBM33 can be used as a tumor biomarker and a possible therapeutic target in the future.

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.

OGDH promotes the progression of gastric cancer by regulating mitochondrial bioenergetics and Wnt/β-catenin signal pathway.

Background/aims2-oxoglutarate dehydrogenase (OGDH) is the first rate-limiting E1 subunit of OGDH complex (OGDHC), which plays as a regulatory point in the cross-road of TCA cycle and glutamine metabolism. Until now, the role of OGDH in carcinogenesis has been unclear.MethodsIn the present study, we determined the expression of OGDH in human gastric cancer (GC) tissues and cell lines by RT-qPCR, Western blotting and immunohistochemical staining respectively. The biological impacts of OGDH on cell growth and migration were explored through modulation OGDH expression in GC cells. Furthermore, mitochondrial functions and Wnt/β-catenin signal were analyzed to elucidate the mechanism by which OGDH was involved in GC progression.ResultsThe results showed that the levels of OGDH mRNA and protein were significantly higher in GC tissues, which was positively correlated with clinicalpathological parameters of GC patients. OGDH inhibitor SP significantly suppressed GC cell viability. Modulation of OGDH had distinct effects on cell proliferation, cell cycle and cell migration in the GC cell lines AGS and BGC823. Overexpression of OGDH resulted in the downregulation of the EMT molecular markers E-cadherin and ZO-1, the upregulation of N-cadherin and claudin-1. OGDH deficiency had the opposite outcomes in GC cells. Meantime, OGDH knockdown cells showed decreased mitochondrial membrane potential, oxygen consumption rate, intracellular ATP product, and increased ROS level and NADP+/NADPH ratio. Consistently, overexpression of OGDH enhanced the mitochondrial function in GC cells. Furthermore, OGDH knockdown reduced the expressions of β-catenin, slug and TCF8/ZEB1, and the downstream targets cyclin D1 and MMP9 in GC cells. OGDH overexpression facilitated the activation of Wnt/β-catenin signal pathway. Additionally, overexpression of OGDH promoted tumorigenesis of GC cells in nude mice.ConclusionTaken together, these results indicate that OGDH serves as a positive regulator of GC progression through enhancement of mitochondrial function and activation of Wnt/β-catenin signaling.