scholarly journals NF-kB-dependent activation of STAT3 by H. pylori is suppressed by TFF1

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Mohammed Soutto ◽  
Nadeem Bhat ◽  
Shayan Khalafi ◽  
Shoumin Zhu ◽  
Julio Poveda ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Nuclear Localization ◽  
Luciferase Reporter ◽  
Western Blots ◽  
In Vivo Models ◽  
Reporter Assays ◽  
H Pylori ◽  
Trefoil Factor 1

Abstract Background H. pylori infection is the main risk factor for gastric cancer. In this study, we investigated H. pylori-mediated activation of STAT3 and NF-κB in gastric cancer, using in vitro and in vivo models. Methods To investigate the activation of NF-κB and STAT3 by H. pylori strains we used in vitro and in vivo mouse models, western blots, immunofluorescence, ChIP Assay, luciferase and quantitative real-time PCR assays. Results Following infection with H. pylori in vitro, we found an earlier phosphorylation of NF-kB-p65 (S536), followed by STAT3 (Y705). Immunofluorescence, using in vitro and in vivo models, demonstrated nuclear localization of NF-kB and STAT3, following H. pylori infection. NF-kB and STAT3 luciferase reporter assays confirmed earlier activation of NF-kB followed by STAT3. In vitro and in vivo models demonstrated induction of mRNA expression of IL-6 (p < 0.001), VEGF-α (p < 0.05), IL-17 (p < 0.001), and IL-23 (p < 0.001). Using ChIP, we confirmed co-binding of both NF-kB-p65 and STAT3 on the IL6 promoter. The reconstitution of Trefoil Factor 1 (TFF1) suppressed activation of NF-kB with reduction in IL6 levels and STAT3 activity, in response to H. pylori infection. Using pharmacologic (BAY11-7082) and genetic (IκB super repressor (IκBSR)) inhibitors of NF-kB-p65, we confirmed the requirement of NF-kB-p65 for activation of STAT3, as measured by phosphorylation, transcription activity, and nuclear localization of STAT3 in in vitro and in vivo models. Conclusion Our findings suggest the presence of an early autocrine NF-kB-dependent activation of STAT3 in response to H. pylori infection. TFF1 acts as an anti-inflammatory guard against H. pylori-mediated activation of pro-inflammatory networks.

scholarly journals Circular RNA Circ-0002570 Accelerates Cancer Progression by Regulating VCAN via MiR-587 in Gastric Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Lei Yang ◽  
Yong-ning Zhou ◽  
Miao-miao Zeng ◽  
Nan Zhou ◽  
Bin-sheng Wang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Progression ◽  
Circular Rna ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Circular Rnas ◽  
Functional Roles ◽  
Reporter Assays

BackgroundCircular RNAs (circRNAs) are closely associated with the occurrences and progress of gastric cancer (GC). We aimed to delve into the function and pathological mechanism of Circular RNA-0002570 (circ-0002570) in GC progression.MethodsCircRNAs differentially expressed in GC were screened using bioinformatics technology. The expression of circ-0002570 was detected in GC specimens and cells via qRT-PCR, and the prognostic values of circ-0002570 were determined. The functional roles of circ-0002570 on proliferation, migration, and invasion in GC cells were explored in vitro and in vivo. Interaction of circ-0002570, miR-587, and VCAN was confirmed by dual-luciferase reporter assays, Western blotting, and rescue experiments.ResultsCirc-0002570 expression was distinctly increased in GC tissues compared to adjacent normal specimens, and GC patients with higher circ-0002570 expressions displayed a short survival. Functionally, knockdown of circ-0002570 resulted in the inhibition of cell proliferation, migration, and invasion, and suppressed tumor growth in vivo. Mechanistically, miR-587 was sponged by circ-0002570. VCAN expression in NSCLC was directly inhibited by miR-587. Overexpression of circ-0002570 prevented VCAN from miR-587-mediated degradation and thus facilitated GC progression.ConclusionThe circ-0002570-miR-587-VCAN regulatory pathway promoted the progression of GC. Our findings provided potential new targets for the diagnosis and therapy of GC.

scholarly journals Circular RNA MCTP2 Inhibits Cisplatin Resistance in Gastric Cancer by miR-99a-5p-Mediated Induction of MTMR3 Expression

2020 ◽  
Author(s):  
Guangli Sun ◽  
Zheng Li ◽  
Zhongyuan He ◽  
Weizhi Wang ◽  
Sen Wang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Circular Rna ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Transmission Electron ◽  
Reporter Assays ◽  
Mirna Sponges ◽  
High Level

Abstract Background: Cisplatin (CDDP) is the first-line chemotherapy for gastric cancer (GC). Poor prognosis of GC patients is partially due to development of CDDP resistance. Circular RNAs (circRNAs) are a subclass of non-coding RNAs that function as microRNA (miRNA) sponges. The role of circRNAs in CDDP resistance in GC has not been evaluated. Methods: RNA-sequencing was used to identify the differentially expressed circRNAs between the CDDP-resistant and CDDP-sensitive GC cells. qRT-PCR was used to detect the expression of circMCTP2 in GC tissues. The effects of circMCTP2 on CDDP resistance were investigated in vitro and in vivo. Pull-down assays and Luciferase reporter assays were performed to confirm the interaction among circMCTP2, miR-99a-5p, and myotubularin related protein 3 (MTMR3). The protein expression levels of MTMR3 were detected by western blotting. Autophagy was evaluated by confocal microscopy and transmission electron microscopy (TEM).Results: CircMCTP2 was found to be downregulated in the CDDP-resistant GC cells and tissues as compared to that of the CDDP-sensitive ones. A high level of circMCTP2 was found to be a favorable factor for the prognosis of patients with GC. CircMCTP2 inhibited cell proliferation and autophagy while promoting apoptosis of CDDP-resistant GC cells in response to CDDP treatment. CircMCTP2 upregulated MTMR3 by sponging miR-99a-5p, and knockdown of MTMR3 could reverse the effects of circMCTP2 on CDDP resistance and autophagy of GC cells. Conclusions: CircMCTP2 sensitizes GC to CDDP through the upregulation of MTMR3 by sponging miR-99a-5p. Overexpression of CircMCTP2 could be a new therapeutic strategy for counteracting CDDP resistance in GC.

scholarly journals HOXC-AS1-MYC regulatory loop contributes to the growth and metastasis in gastric cancer

2019 ◽  
Vol 38 (1) ◽  
Author(s):  
Yangyang Dong ◽  
Xinyu Li ◽  
Zhibin Lin ◽  
Wenbing Zou ◽  
Yan Liu ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Growth ◽  
Luciferase Reporter ◽  
Gene Expressions ◽  
In Vivo Experiments ◽  
Promising Target ◽  
Reporter Assays ◽  
Regulatory Loop

Abstract Background Gastric cancer (GC) is one of the most prevalent and deadly malignancies worldwide. Accumulating reports have indicated the participation of long non-coding RNAs (lncRNAs) in the onset and progression of GC. Methods GSE109476 data was utilized to screen out lncRNAs dysregulated in GC. Gene expressions were determined by qRT-PCR and western blot. Both in vitro and in vivo experiments were carried out to assess the function of HOXC-AS1 in GC. The association between genes was verified via RIP, ChIP, CoIP, RNA pull down and luciferase reporter assays, as appropriate. Results HOXC-AS1 was discovered to be upregulated in GC and located both in cytoplasm and in nucleus in GC cells. Functionally, inhibition of HOXC-AS1 restrained GC cell growth and metastasis both in vitro and in vivo. Moreover, HOXC-AS1 was proved to be trans-activated by c-MYC in GC. In return, HOXC-AS1 positively regulated MYC expression in GC through targeting miR-590-3p/MYC axis in cytoplasm and modulating BRG1/β-catenin complex-activated MYC transcription in nucleus. Furthermore, the rescue assays verified that MYC mediated HOXC-AS1-affected GC progression. Conclusion Our research illustrated a feedback loop of HOXC-AS1-MYC in aggravating GC cell growth and metastasis, highlighting HOXC-AS1 as a promising target for GC diagnosis and treatment.

scholarly journals TEAD4 modulated LncRNA MNX1-AS1 contributes to gastric cancer progression partly through suppressing BTG2 and activating BCL2

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
You Shuai ◽  
Zhonghua Ma ◽  
Weitao Liu ◽  
Tao Yu ◽  
Changsheng Yan ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Mechanistic Model ◽  
Ectopic Expression ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Tissue Samples ◽  
Reporter Assays

Abstract Background Gastric cancer (GC) is the third leading cause of cancer-related mortality globally. Long noncoding RNAs (lncRNAs) are dysregulated in obvious malignancies including GC and exploring the regulatory mechanisms underlying their expression is an attractive research area. However, these molecular mechanisms require further clarification, especially upstream mechanisms. Methods LncRNA MNX1-AS1 expression in GC tissue samples was investigated via microarray analysis and further determined in a cohort of GC tissues via quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays. Cell proliferation and flow cytometry assays were performed to confirm the roles of MNX1-AS1 in GC proliferation, cell cycle regulation, and apoptosis. The influence of MNX1-AS1 on GC cell migration and invasion was explored with Transwell assays. A xenograft tumour model was established to verify the effects of MNX1-AS1 on in vivo tumourigenesis. The TEAD4-involved upstream regulatory mechanism of MNX1-AS1 was explored through ChIP and luciferase reporter assays. The mechanistic model of MNX1-AS1 in regulating gene expression was further detected by subcellular fractionation, FISH, RIP, ChIP and luciferase reporter assays. Results It was found that MNX1-AS1 displayed obvious upregulation in GC tissue samples and cell lines, and ectopic expression of MNX1-AS1 predicted poor clinical outcomes for patients with GC. Overexpressed MNX1-AS1 expression promoted proliferation, migration and invasion of GC cells markedly, whereas decreased MNX1-AS1 expression elicited the opposite effects. Consistent with the in vitro results, MNX1-AS1 depletion effectively inhibited the growth of xenograft tumour in vivo. Mechanistically, TEAD4 directly bound the promoter region of MNX1-AS1 and stimulated the transcription of MNX1-AS1. Furthermore, MNX1-AS1 can sponge miR-6785-5p to upregulate the expression of BCL2 in GC cells. Meanwhile, MNX1-AS1 suppressed the transcription of BTG2 by recruiting polycomb repressive complex 2 to BTG2 promoter regions. Conclusions Our findings demonstrate that MNX1-AS1 may be able to serve as a prognostic indicator in GC patients and that TEAD4-activatd MNX1-AS1 can promote GC progression through EZH2/BTG2 and miR-6785-5p/BCL2 axes, implicating it as a novel and potent target for the treatment of GC.

scholarly journals Tumor Suppressive Role of miR-342-5p in Human Chondrosarcoma Cells and 3D Organoids

2021 ◽  
Vol 22 (11) ◽  
pp. 5590
Author(s):  
Clément Veys ◽  
Abderrahim Benmoussa ◽  
Romain Contentin ◽  
Amandine Duchemin ◽  
Emilie Brotin ◽  
...  
Keyword(s):  
Luciferase Reporter ◽  
Functional Screening ◽  
Western Blots ◽  
Egfr Expression ◽  
Reporter Assays ◽  
Direct Target ◽  
Induced Apoptosis ◽  
First Time

Chondrosarcomas are malignant bone tumors. Their abundant cartilage-like extracellular matrix and their hypoxic microenvironment contribute to their resistance to chemotherapy and radiotherapy, and no effective therapy is currently available. MicroRNAs (miRNAs) may be an interesting alternative in the development of therapeutic options. Here, for the first time in chondrosarcoma cells, we carried out high-throughput functional screening using impedancemetry, and identified five miRNAs with potential antiproliferative or chemosensitive effects on SW1353 chondrosarcoma cells. The cytotoxic effects of miR-342-5p and miR-491-5p were confirmed on three chondrosarcoma cell lines, using functional validation under normoxia and hypoxia. Both miRNAs induced apoptosis and miR-342-5p also induced autophagy. Western blots and luciferase reporter assays identified for the first time Bcl-2 as a direct target of miR-342-5p, and also Bcl-xL as a direct target of both miR-342-5p and miR-491-5p in chondrosarcoma cells. MiR-491-5p also inhibited EGFR expression. Finally, only miR-342-5p induced cell death on a relevant 3D chondrosarcoma organoid model under hypoxia that mimics the in vivo microenvironment. Altogether, our results revealed the tumor suppressive activity of miR-342-5p, and to a lesser extent of miR-491-5p, on chondrosarcoma lines. Through this study, we also confirmed the potential of Bcl-2 family members as therapeutic targets in chondrosarcomas.

scholarly journals Let-7a regulates EV secretion and mitochondrial oxidative phosphorylation by targeting SNAP23 in colorectal cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
You Dong Liu ◽  
Xiao Peng Zhuang ◽  
Dong Lan Cai ◽  
Can Cao ◽  
Qi Sheng Gu ◽  
...  
Keyword(s):  
Oxidative Phosphorylation ◽  
Metabolic Reprogramming ◽  
Luciferase Reporter ◽  
Mitochondrial Oxidative Phosphorylation ◽  
In Vivo Assays ◽  
Reporter Assays ◽  
Extracellular Mirna

Abstract Background MicroRNAs (miRNAs) are abundant in tumor-derived extracellular vesicles (EVs) and the functions of extracellular miRNA to recipient cells have been extensively studied with tumorigenesis. However, the role of miRNA in EV secretion from cancer cells remains unknown. Methods qPCR and bioinformatics analysis were applied for determining extracellular let-7a expression from CRC patient serum and cells. Nanosight particle tracking analysis was performed for investigating the effect of let-7a on EV secretion. Luciferase reporter assays was used for identifying targeted genes synaptosome-associated protein 23 (SNAP23). In vitro and in vivo assays were used for exploring the function of let-7a/SNAP23 axis in CRC progression. Bioenergetic assays were performed for investigating the role of let-7a/SNAP23 in cellular metabolic reprogramming. Results let-7a miRNA was elevated in serum EVs from CRC patients and was enriched in CRC cell-derived EVs. We determined that let-7a could suppress EV secretion directly targeting SNAP23. In turn, SNAP23 promotes EV secretion of let-7a to downregulate the intracellular let-7a expression. In addition, we found a novel mechanism of let-7a/SNAP23 axis by regulating mitochondrial oxidative phosphorylation (OXPHOS) through Lin28a/SDHA signaling pathway. Conclusions Let-7a plays an essential role in not only inhibiting EV secretion, but also suppressing OXPHOS through SNAP23, resulting in the suppression of CRC progression, suggesting that let-7a/SNAP23 axis could provide not only effective tumor biomarkers but also novel targets for tumor therapeutic strategies.

Radiation activates the NORAD expression to promote ESCC radioimmunotherapy resistance via EEPD1/ATR/Chk1 signaling by inhibiting the pri-miR-199 processing and exosomal transfer of miR-199a-5p

2021 ◽  
Author(s):  
Yuchen Sun ◽  
Jizhao Wang ◽  
Xuanzi Sun ◽  
Jing Li ◽  
Xu Zhao ◽  
...  
Keyword(s):  
Its Region ◽  
Luciferase Reporter ◽  
In Vivo Experiments ◽  
Cycle Arrest ◽  
Non Coding Rna ◽  
Irradiation Treatment ◽  
Recombination Repair ◽  
Reporter Assays

Abstract Background Radioresistance, a poorly understood phenomenon, results in the failure of radiotherapy and consequent local recurrence, threatening a large proportion of ESCC patients. To date, lncRNAs have been found to be involved in diverse biological processes, including radioresistance.Methods ELISA was used to evaluated the H3 modifications in radio-resistant ESCC cells. FISH and qRT-PCR were adopted to examine the expression and localization of lncRNA-NORAD, pri-miR-199a and miR-199a. Electron microscopy and Nanoparticle tracking analysis (NTA) was conducted to observe and identify exosomes. High-throughput RNA sequencing and TMT mass spectrometry were performed to identify the functional lncRNAs and proteins involved in ESCC radioresistance. A series of in vitro and in vivo experiments were performed to investigate the biological effect of NORAD. CHIP, qPCR-RIP, co-IP and dual-luciferase reporter assays were used to explore the interaction of related RNAs and proteins. Results We show here that a DNA damage activated non-coding RNA-NORAD, which is critical for ESCC radio-resistance. NORAD was highly expressed in radio-resistant ESCC cells and tissues. Irradiation treatment promotes NORAD expression via enhancing H3K4me2 enrichment on its region. NORAD knockdown cells exhibit significantly hypersensitivity to irradiation in vivo and in vitro. NORAD is required for initiating repair and restart of stalled forks, G2 cycle arrest and homologous recombination repair upon irradiation treatment. Mechanistically, NORAD inhibits miR-199a expression by competitively binding PUM1 from pri-miR-199a, inhibiting the process of pri-miR-199a. Mature miR-199a in NORAD-knockdown cells can be packaged into exosomes; miR-199a restores the radiosensitivity of radioresistant cells by targeting EEPD1, then inhibiting ATR/Chk1 signaling pathway. Simultaneously, NORAD knockdown blocks the ubiquitination of PD-L1, leads to the better response for radiation and anti-PD-1 treatment in mouse model.Conclusion This study raises the possibility that LncRNA-NORAD could be a potential treatment target for improving the efficiency of immunotherapy in combination with radiation in ESCC.

scholarly journals Has-miR-875-5p Inhibits Tumorigenesis by Targeting USF2 via TGF-β Signaling Pathway in Gastric Cancer

2021 ◽  
Author(s):  
Shenshuo Gao ◽  
Zhikai Zhang ◽  
Xubin Wang ◽  
Yan Ma ◽  
Chensheng Li ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Signaling Pathway ◽  
Research Direction ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Gastric Cancer Cells ◽  
New Research

Abstract Background: Gastric cancer (GC) is one of the most common malignancies, and more and more evdiences show that the pathogenesis is regulated by various miRNAs.In this study, we investigated the role of miR-875 in GC. Methods:The expression of miR-875-5p was detected in human GC specimens and cell lines by miRNA RT-PCR. The effect of miR-875-5p on GC proliferation was determined by CCK-8 proliferation assay and EDU assay. Migration and invasion were examined by transwell migration and invasion assay and wound healing assay. The interaction between miR-875-5p and its target gene USF2 was verified by a dual luciferase reporter assay. The effects of miR-875-5p in vivo were studied in xenograft nude mice models.Related proteins were detected by Western blot.Results:The results showed that miR-875-5p inhibited the proliferation, migration and invasion of gastric cancer cells in vitro, and inhibited tumorigenesis in vivo. USF2 proved to be a direct target of miR-875-5p. Knockdown of USF2 partially counteracts the effects of miR-875-5p inhibitors.Overexpression of miR-875-5p can inhibit proliferation, migration, and invasion through the TGF-β signaling pathway by down-regulation of USF2 in GC, providing a new research direction for the diagnosis and targeted therapy of GC.Conclusions: MiR-875-5pcan inhibited the progression of GC by directly targeting USF2 and negatively regulating TGF-β signaling pathway.In the future, miR-875-5p is expected to be used as a potential therapeutic target for GC therapy.

scholarly journals YY1-modulated Long Non-coding RNA SNHG12 Predicts and Promotes Gastric Cancer Metastasis via Activating miR-218-5p/YWHAZ-dependent β-catenin: A Bed to Bench Approach

2020 ◽  
Author(s):  
Tian Qi Zhang ◽  
Qingqiang Dai ◽  
Maneesh Kumarsing Beeharry ◽  
Zhenqiang Wang ◽  
Liping Su ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Transcription Factor ◽  
Peritoneal Carcinomatosis ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Reverse Transcription ◽  
Reverse Transcription Pcr ◽  
Reporter Assays

Abstract Background: Gastric Cancer (GC) is one of the leading causes of cancer-related deaths and mortality. Long non-coding RNAs (lncRNAs) such as SNHG12 play important roles in the pathogenesis and progression of cancers. However, the role and significanve of SNHG12 in the metastasis of GC has not yet been thoroughly investigated.Methods: The SNHG12 expression pattern was detected in GC tissue samples from our faculty and cell lines using quantitative reverse transcription PCR. In vivo and in vitro gain and loss assays were conducted to observe the effects of SNHG12 regulation on GC cell metastasis potential. The underlying mechanisms of SNHG12 regulation on EMT and metastatic potential of GC cells were further determined by quantitative reverse transcription PCR, western blotting, dual luciferase reporter assays, co-immunoprecipitation, immunoprecipitation, RIP assays, TOPFlash/FOPFlash reporter assays and Ch-IP assays.Results: SNHG12 was upregulated in GC tissues and cell lines. The expression levels of SNHG12 in GC samples was significantly related to tumor invasion depth, TNM staging and lymph node metastasis, and was associated with poorer DFS and OS in the GC patients. SNHG12 was significantly highly expressed in peritoneal metastatic tissues from GC patients and mice subjects, suggesting a possible role of SNHG12 in peritoneal carcinomatosis from GC. Further in vivo and in vitro gain and loss assays indicated that SNHG12 promoted GC metastasis and EMT. Based on hypothetical bioinformatic analysis findings, our mechanistic analyses revealed that miR-218-5p was a direct target of SNHG12 and suggested that both SNHG12 and miR-218-5p could collectively regulate YWHAZ, forming the SNHG12/ miR-218-5p/YWHAZ axis, hereby decreasing the ubiquitination of β-catenin, thus activating the β-catenin signaling pathway and facilitating metastasis and EMT. Further analysis also revealed that the transcription factor YY1 could negatively modulate SNHG12 transcription.Conclusions: Our findings demonstrate that SNHG12 is be a potential prognostic marker and therapeutic target for GC. Negatively modulated by transcription factor YYI, SNHG12 promotes GC metastasis and EMT by regulating the miR-218-5p/YWHAZ axis and hence activating the β-catenin signaling pathway. Furthermore, we discovered high SNHG12 expression could be related to peritoneal carcinomatosis from GC but this requires further validation.

scholarly journals Circular RNA CDR1as Inhibits the Metastasis of Gastric Cancer through Targeting miR-876-5p/GNG7 Axis

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Jiajia Jiang ◽  
Rong Li ◽  
Junyi Wang ◽  
Jie Hou ◽  
Hui Qian ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Epithelial Mesenchymal Transition ◽  
Circular Rna ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Mesenchymal Transition ◽  
Underlying Mechanisms

Circular RNA CDR1as has been demonstrated to participate in various cancer progressions as miRNA sponges. The exact underlying mechanisms of CDR1as on gastric cancer (GC) metastasis remain unknown. Here, we found that CDR1as knockdown facilitated GC cell migration and invasion while its overexpression inhibited the migration and invasion abilities of GC cells in vitro and in vivo. Moreover, epithelial-mesenchymal transition- (EMT-) associated proteins and MMP2 and MMP9 were downregulated by CDR1as. Bioinformatics analysis combined with dual-luciferase reporter gene assays, western blot, RT-qPCR analysis, and functional rescue experiments demonstrated that CDR1as served as a miR-876-5p sponge and upregulated the target gene GNG7 expression to suppress GC metastasis. In summary, our findings indicate that CDR1as suppresses GC metastasis through the CDR1as/miR-876-5p/GNG7 axis.

Sign in / Sign up

Export Citation Format

Share Document