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@#[摘 要] 目的：探究桃叶珊瑚苷（AU）调控RhoA/ROCK信号通路对胃癌MGC803细胞上皮间质转化（EMT）进程和血管生成拟态（VM）形成的影响。方法：常规培养人胃癌MGC803细胞，将其随机分为对照组、AU-L组（20 μmol/L AU）、AU-M组（40 μmol/L AU）、AU-H组（80 μmol/L AU）、AU-H+RhoA激活剂水仙环素（Nar）组（AU-H+Nar组，80 μmol/L AU+30 μmol/L Nar）。采用CCK-8法、Transwell实验、细胞划痕实验分别检测不同浓度AU对细胞增殖、迁移和侵袭的影响，三维细胞培养法观察不同浓度AU对细胞体外VM管腔结构形成的影响，WB法检测AU对各组细胞RhoA、ROCK、VM与EMT相关蛋白表达的影响。结果：与对照组相比，AU-M组、AU-H组MGC803细胞增殖率（48、72 h时）、细胞迁移率、细胞侵袭数目、VM管腔结构数，以及RhoA、ROCK1、N-cadherin、vimentin、VE-cadherin的蛋白表达均显著降低（均P<0.05），E-cadherin表达显著升高（P<0.05）；同时，使用Nar处理显著减弱了AU对MGC803细胞EMT和VM形成的抑制作用（均P<0.05）。结论：AU通过下调RhoA/ROCK信号通路抑制胃癌MGC803细胞的增殖、迁移、侵袭、EMT和VM形成过程。
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Aim To investigate the effect of RORα antagonist T0901317 promoting EMT (epithelial-mesenchymal transition) of human gastric cancer MGC803 cells by RORα/β-catenin signal. Methods Cell proliferation was detected by MTT. Cell migration and invasion were detected by cell scratch and Transwell assay respectively. RORα/β-catenin signaling molecules were detected by Western blot and immunofluorescence. RORα binding to β-catenin protein was detected by immunoprecipitation. Results MTT assay showed that the proliferation ability of T0901317 cells increased in a time-dependent manner compared with MGC803 cells (P < 0. 05). Cell scratches and Transwell experiments showed that the migration and invasion ability of T0901317 cells were significantly enhanced compared with MGC803 cells (P < 0. 05). Western blot analysis showed that RORα protein was significantly down-regulated after T0901317 compared with untreated group (P < 0. 05), and total β-catenin protein and nuclear β-catenin in MGC803 cells were up-regulated after T0901317 (P < 0. 05). Compared with the control group, RORα protein binding to β-catenin protein significantly decreased after T0901317 treatment (P < 0. 05). Compared with MGC803 cells treated with T0901317, the long spindle cells increased and the heteromorphism was more obvious. T0901317 significantly up-regulated the expression of Rac1, TGFβ1 and Vimentin in MGC803 cells (P < 0. 05), and inhibited the expression of E-cadherin (P < 0. 05). Conclusion T0901317 can promote the proliferation, migration, invasion and EMT in human gastric cancer MGC803 cells by RORα/β-catenin signal.
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@#[摘 要] 目的：探讨DJ-1基因过表达对人胃癌MGC803细胞增殖、迁移、侵袭与上皮间质转化（EMT）的影响及其机制。方法：利用基因转染技术构建DJ-1基因过表达MGC803细胞，实验分为MGC803、空载体和DJ-1过表达组。采用MTT、平板克隆形成、细胞划痕和Transwell实验分别检测DJ-1过表达对MGC803细胞增殖、克隆形成、迁移与侵袭的影响；qPCR和WB法检测DJ-1过表达对各组细胞DJ-1、PTEN、Akt、p-Akt、Snail、vimentin、E-cadherin、MMP-9与TIMP-3表达的影响，相差显微镜下观察MGC803细胞形态学的变化。裸鼠荷瘤实验检测DJ-1过表达对MGC803细胞移植瘤体内生长的影响。结果：成功构建DJ-1基因稳定过表达的MGC803细胞。与MGC803组和空载体组比较，DJ-1过表达组细胞的增殖能力与克隆形成数均显著增加（均P<0.05），细胞迁移距离明显增加、划痕距离明显缩短（均P<0.05），迁移与侵袭细胞数显著增多（均P<0.05），DJ-1 mRNA与蛋白表达明显上调、PTEN mRNA与蛋白表达下调（均P<0.05），Akt总蛋白各组比较无明显差异（均P>0.05），p-Akt蛋白表达明显上调（P<0.05），Snail、vimentin与MMP-9表达上调、E-cadherin与TIMP-3表达下调（均P<0.05）。相差显微镜下见长梭形细胞数目增多，圆形与椭圆形细胞减少，异型性更为明显。荷瘤裸鼠体内实验结果表明，与MGC803组相比较，DJ-1过表达组MGC803细胞移植瘤生长速度明显加快、移植瘤质量显著增加（均P<0.05）。结论：DJ-1过表达可通过PTEN/Akt通路在体内外抑制MGC803细胞的增殖、迁移、侵袭与EMT。
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Objective:This studu aims to investigate the effect of aqueous extract of modified Xiao Xianxiongtang on the epithelial mesenchymal transition（EMT） and the change of its invasion and migration ability of human gastric cancer MGC-803 cells mediated by transforming growth factor-<italic>β</italic><sub>1</sub>（TGF-<italic>β</italic><sub>1</sub>），and to explore the mechanism of regulating Wnt5a/Ca<sup>2+</sup>/ activated T-cell nuclear factor（NFAT） signaling pathway to inhibit EMT and invasion and metastasis of MGC-803 cells. Method:TGF-<italic>β</italic><sub>1</sub>（10 μg·L<sup>-1</sup>）was used to induce EMT and the invasion and metastasis model of human gastric cancer MGC-803 cells. Transwell chamber experiment， scratchhealing experiment， Western blot and immunofluorescence assay were used to detect cell invasion and migration ability， expression of EMT marker protein and key protein expression of Wnt5a/Ca<sup>2+</sup>/NFAT signaling pathway， and intracellular Ca<sup>2+</sup> concentration. Result:Compared with the blank group， TGF-<italic>β</italic><sub>1</sub> could significantly enhance the invasion and migration ability of MGC-803 cells（<italic>P</italic><0.01）， down-regulate the level of E-cadherin（<italic>P</italic><0.01）， up-regulate protein expressions of N-cadherin， Snail and Vimentin（<italic>P</italic><0.01）， and induce cell Wnt5a， calcineurin （CaN）， total protein of activated T-cell nuclear factor 1（NFAT1）， up-regulation of phosphorylated proteins p-NFAT1 and NFAT1 nucleoprotein and intracellular accumulation of Ca<sup>2+</sup>（<italic>P</italic><0.01）. Compared with the TGF-<italic>β</italic><sub>1</sub> group， modified Xiao Xianxiongtang （10， 20， 40 mg·L<sup>-1</sup>） could significantly inhibit this phenomenon，and 40 mg·L<sup>-1</sup> had the best effect（<italic>P</italic><0.05，<italic>P</italic><0.01）.The specific inhibitors of Wnt5a/Ca<sup>2+</sup>/NFAT signaling pathway （<italic>R</italic>）-（+）-Bay-K-8644 and modified Xiao Xianxiongtang （40 mg·L<sup>-1</sup>） could significantly inhibit theinvasion and migration of MGC-803 cells mediated by TGF-<italic>β</italic><sub>1</sub>， up-regulate the level of E-cadherin， and down-regulate expressions of N-cadherin， Snail， Vimentin， Wnt5a， CaN and NFAT1 proteins and reduce the intracellular accumulation of Ca<sup>2+</sup>（<italic>P</italic><0.05，<italic>P</italic><0.01）.Moreover， （<italic>R</italic>）-（+）-Bay-K-8644 combined with modified Xiao Xianxiongtang （40 mg·L<sup>-1</sup>） had stronger inhibitory effect（<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:These results suggest that modified Xiao Xianxiongtang can inhibit the EMT mediated by TGF-<italic>β</italic><sub>1</sub> via Wnt5a/Ca<sup>2+</sup>/NFAT signaling pathway，thereby reducing the invasion and migration ability of MGC-803 cells.
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@#[Abstract] Objective: To investigate the effect of apatinib (APA) combined with cisplatin (DDP) on the proliferation, invasion and migration capacity of gastric carcinoma (GC) cells and its molecular mechanism. Methods: Cancer and para-cancerous tissue samples resected from 50 GC patients, who were surgically treated in Wuwei People's Hospital from January 2016 to June 2019, were collected for this study; in addition, GC cell lines MGC803 and SGC7901 were also collected. qPCR was used to detect the HMGA2 expression in tissues and mRNA expressions of molecules related to cell proliferation, migration and invasion in GC cell lines. MGC803 and SGC7901 cells were transfected with pcHMGA2 by liposome transfection technology. After treatment with DDP and APA at different concentrations, the cells were divided into NC, pcHMGA2, pcHMGA2+DDP and pcHMGA2+DDP+APA groups. Protein expression of HMGA2 in GC cells was detected by Western blotting, and proliferation, migration and invasion of the cells were detected by MTT and Transwell assay, respectively. Results: The mRNA expression of HMGA2 in GC tissues was higher than that in para-cancerous tissues (P<0.05), and the survival rate of GC patients in the high expression group was significantly reduced (P<0.01). DDP significantly inhibited the proliferation, invasion and migration of MGC803 and SGC7901 cells (all P<0.01); the proliferation, invasion and migration of MGC803 and SGC7901 cells in DDP+APA group significantly decreased (all P<0.01) as compared with DDP group; APA significantly enhanced the inhibitory effect of DDP on HMGA2 expression in GC cells (P<0.01); APA enhanced the anticancer activity of DDP against GC by down-regulating HMGA2 expression. Conclusion: APA promotes the anticancer activity of DDP against GC, and its molecular mechanism is the promotion of the inhibitory effect of DDP on HMGA2 expression.
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@# Objective: To observe the effect of ursolic acid (UA) on autophagy and apoptosis of gastric cancer cell line MGC-803, and to explore the mechanism of UA-induced autophagy of MGC-803 cells based on PI3K/AKT/mTOR signaling pathway. Methods: Human gastric cancer cell line MGC-803 was cultured in vitro and divided into blank control group, UAintervention group and UA+3-MA group. The cell apoptosis in each group was detected by flow cytometry. Cell autophagy was detected by double fluorescence mRFPeGFP-LC3 plasmid transfection method. The mRNA expression levels of LC3B, BAX and Bcl-2 were detected by qPCR. The protein expression levels of PI3K type I, p-AKT, p-mTOR, ULK1, LC3B, BAX and Bcl-2 were detected by WB. Results: Flow cytometry showed that the cell apoptotic rate of UA intervention group was significantly higher than that of blank control group (P<0.05). Compared with UAintervention group, the apoptotic rate in UA+ 3-MAgroup was significantly reduced (P<0.05). The double fluorescence mRFP-eGFP-LC3 plasmid transfection method showed that the green and red fluorescent bright spots in UA intervention group increased significantly compared with the blank control group (P<0.05), and the green and red fluorescent bright spots in UA+3-MA group were significantly reduced compared with UA intervention group (P<0.05). Real-time quantitative PCR and WB method showed that compared with the blank control group, the mRNAand protein expressions of BAX and LC3B, and ULK1 protein were significantly increased in UA intervention group, while the mRNA and protein expressions of Bcl-2, and the protein expressions of PI3K, p-AKT and p-mTOR were significantly decreased in UA intervention group (all P<0.05); Compared with UA intervention group, mRNA and protein expressions of BAX and LC3B were significantly down-regulated and the mRNAand protein expressions of Bcl-2 were significantly up-regulated in UA+3-MA group (all P<0.05), while protein levels of PI3K, p-AKT, p-mTOR and ULK1 were not significantly changed in UA+3-MA group (P>0.05). Conclusion: UA can promote apoptosis of MGC-803 cells via inducing autophagy, which may be related to UA's involvement in regulating the expressions of PI3K/AKT/mTOR signaling pathway-related proteins.
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@# Objective: To investigate the effect of ELMOD2 over-expression on the malignant biological behaviors of gastric cancer MGC803 cells, and to study its related molecular mechanism. Methods: GV141-ELMOD2 expression vector was transfected into human gastric cancer MGC803 cells. The mRNA and protein expressions of ELMOD2 were detected by Real-time fluorescent quantitative PCR and WB, respectively. The cell proliferation ability was detected by CCK-8 method. Apoptosis rate was detected by flow cytometry. The cell migration ability was detected by Transwell method. The protein expressions of PCNA, BAX and Bcl-2 and Vimentin were detected by WB. Results:After transfection of ELMOD2 expression vector, the mRNAand protein expressions of ELMOD2 were significantly increased in MGC803 cells (P<0.05). Further studies showed that over-expression of ELMOD2 increased the proliferation and migration ability but reduced the apoptosis rate of MGC803 cells significantly (P<0.05 or P<0.01). The protein levels of PCNA, Vimentin and Bcl-2 in MGC803 cells increased, while the protein level of BAX decreased (P<0.05 or P<0.01). Conclusion: Over-expression of ELMOD2 can promote the proliferation and migration of MGC803 cells and inhibit cell apoptosis. These effects may be achieved by increasing the protein level of PCNA, Vimentin and Bcl-2, and reducing the protein level of BAX.
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@#Objective: To explore the influence of circ_0023642 on the proliferation and metastasis of gastric cancer GMC-803 cells by modulating miR-508-3p/ERBB4 axis. Methods: Cancer tissues and corresponding para-cancer normal tissues from 31 gastric cancer patients, who underwent surgical resection at the Second People's Hospital of Nantong City from May 2015 to March 2018, were collected for this study; meanwhile, gastric cancer cell lines (MGC-803, MKN-45 and MKN-28) were also collected. qPCR was performed to determine the expression levels of circ_0023642 and miR-508-3p in above mentioned tissues and cell lines. WB was applied to measure the expressions of ERBB4, E-cadherin, N-cadherin and Vimentin in MGC-803 cells. CCK-8 assay and Transwell assay were used to evaluate the effects of circ_0023462 and miR-508-3p expression on proliferation, migration and invasion of MGC-803 cells. Dual-luciferase reporter gene was carried out to validate whether miR-508-3p could bind to the 3' UTR of circ_0023642 and ERBB4. Results: Compared with para-cancer tissues or normal gastric mucosal cells, the expression of circ_0023642 was significantly up-regulated in gastric cancer tissues and cells lines, and the expression was highest in MGC-803 cells (P<0.05 or P<0.01). Silencing circ_0023642 dramatically decreased the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of MGC-803 cells (P<0.05 or P<0.01). Both circ_0023642 and ERBB4 could target the binding sites of miR-508-3p. Further experiments confirmed that circ_ 0023642 promoted the proliferation, migration, invasion and EMT of MGC-803 cells by sponging miR-508-3p (P<0.05 or P<0.01). Conclusion: circ_0023642, by competing ERBB4 to bind with miR-508-3p, promotes the proliferation and metastasis of gastric cancer MGC-803 cells, thus could be used as a marker for the clinical diagnosis of gastric cancer.
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Aim: To explore the apoptosis and mechanisms of human gastric cancer cells MGC-803 induced by RAA-11. Methods: MTT and colony assays were used to detect the survival of MGC-803 cells treated with RAA-11. The effect of RAA-11 on the apoptotic morphology of MGC-803 cells was observed by Hoechst 33258 staining. The effect of RAA-11 on the apoptosis rate of MGC-803 cells was detected with flow cytometry. The effects of RAA-11 on apoptosis related protein expression of caspase-3, Bcl-2 and Bax as well as pathway proteins ERK and p-ERK in MGC-803 cells were observed by Western blot. Results: MTT assay showed that the proliferation of MGC-803 cells was effectively inhibited in a time- and concentration-dependent manner (P <0. 01). The result of colony suggested that RAA-11 could inhibit the proliferation of MGC-803 cells. Hoechst 33258 staining indicated that the nucleus of MGC-803 cells had a typical apoptotic morphological change after intervention with RAA-11. The result of flow cytometry suggested that RAA-11 had a significant apoptotic effect on MGC-803 cells. The expressions of Bax and caspase-3 were significantly up-regulated (P <0. 01), and the expressions of Bcl-2, ERK and p-ERK were down-regulated (P <0. 01) in MGC- 803 cells treated with RAA-11. Conclusions: RAA-11 inhibits the proliferation and induces apoptosis in MGC-803 cells, which may be related to the inhibition of ERK/MAPK pathway.
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Aim: To investigate the expression and i-dentification of differential miRNAs in human gastric cancer MGC803 cells induced by diallyl disulfide (DADS). Methods: Differential miRNAs expression in human gastric cancer MGC803 cells induced by DADS was detected and identified by miRNA chip and qPCR. Results: MiRNAs chip detection showed upregulation of miR-200b, miR-22, miR-7, miR-143, miR-138, miR-34a and miR-150, and down-regulation of miR-222, miR-21, miR-15b, miR-182 and miR-18a in differential miRNAs of MGC803 cells treated with 3 0 mg · IT-1 DADS at 24 h(P<0.05). And qPCR demonstrated that the expressions of miR-200b, miR-22, miR-7, miR-143, miR-138, miR-34a and miR-150 was up-regulated in MGC803 cells treated with 30 mg · L-1 DADS(P <0. 05). Moreover, qPCR showed that the expressions of miR-200b and miR-22 in various human gastric cancer cells including MGC803, BGC823, MKN28, SGC7901 and HGC27 cells were lower than normal human gastric cancer GES-1 cells (P <0. 05). The expression of miR-200b and miR-22 in gastric cancer tissues was significantly lower than that in normal gastric tissues (P < 0. 05). Conclusions: The expression of down-regulation of 7 miRNA and up-regulation of 5 miRNA in differential miRNAs in MGC803 cells induced by DADS. The expression of down-regulation of miR-200b and miR-22 in gastric cancer tissues and cells. DADS could up-regulate the expression of miR-200b and miR-22 in gastric cancer cells.
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OBJECTIVE： To study the effects of ivermectin on the migration and invasion of human gastric cancer cell lines BGC-823 and MGC-803 and its mechanism. METHODS： After treated with 0， 2.5， 5， 10， 20， 40 μmol/L ivermectin for 24 h， inhibitory rate of human gastric cancer cell lines BGC-823 and MGC-803 were detected by MTT assay. Effects of 5 μmol/L ivermectin and phosphate buffercontaining 0.67‰ dimethyl sulfoxide （control group） for 24 h on the migration and invasion of` gastric cancer cells BGC-823 and MGC-803 were observed by Transwell chamber invasion assay.Western blot assay was used to detect the protein expression of TGF-β1， TGF-βR， Smad2 and Smad3 in epithelial-mesenchymal transition （EMT） markers E-cadherin， N-cadherin， Vimentin， Snail and EMT transduction pathway TGF-β/smad of BGC-823 and MGC-803 cells after treated with 5， 10 μmol/L ivermectin and phosphate buffercontaining 0.67‰ dimethyl sulfoxide （control group） for 24 h. RESULTS： Ivermectin could inhibit the growth of BGC-823 and MGC-803， inhibitory rate of it was positively correlated with its concentration. Compared with control group， the number of migration and invasion BGC-823 and MGC-803 cells were decreased significantly after treated with 5 μmol/L ivermectin （P＜0.01 or P＜0.001）； the expression of E-cadherin protein was enhanced significantly in BGC-823 and MGC-803 cells after treated with 5 and 10 μmol/L ivermectin （P＜0.05 or P＜0.01 or P＜0.001）； the protein expression of N-cadherin， Vimentin， Snail， TGF-βR， Smad2 and Smad3 were decreased significantly （P＜0.05， P＜0.01 or P＜0.001）； protein expression of TGF-β1 was decreased significantly after treated with 10 μmol/L ivermectin （P＜0.05）. CONCLUSIONS： Ivermectin can significantly inhibit the migration and invasion of gastric cancer cells BGC-823 and MGC-803， and inhibiting the biological activity of EMT by reducing the expression of TGF-β/smad pathway is one of the mechanisms that inhibit the migration and invasion of gastric cancer cells.
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@#[Abstract] Objective: To study the effects of twisted gastrulation protein homolog 1 (TWSG1) gene on proliferation and apoptosis of gastric cancer MGC-803 cells. Methods: Three siRNAs for TWSG1 gene were designed.The MGC-803 cells at logarithmic phase were divided into blank control group, negative control group (siRNA-NC), siRNA1 interference group, siRNA2 interference group and siRNA3 interference group by transfecting with relevant vectors. The mRNAand protein expressions of TWSG1 in each group were identified by qPCR and Western blotting, respectively; and the stable cell line with highest interference efficiency was screened.The proliferation of cells in each group was detected by CCK-8 assay, and the apoptosis of three groups was detected by flow cytometry. Results: The results of qPCR and Western blotting showed siRNA1 exhibited highest interference efficiency. Compared with the blank control group and the negative control group, the expression of TWSG1 in siRNAinterference cell group was lower (P<0.05), the cell proliferation significantly increased (P<0.05), and apoptosis significantly reduced (P<0.05). Conclusion: siRNA interfering TWSG1 expression in MGC-803 cells can promote cell proliferation, inhibit cell apoptosis.
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@# Objective: To investigate the mechanism of miR-29c modulating apatinib resistance of gastric cancer tissues and cells MGC-803 via regulating TNRC18. Methods: A total of 39 gastric cancer patients with complete clinical data, who were treated in the Central Hospital of Wuhan from Feb. 2015 to Oct. 2017, were collected for this study. The expression of miR-29c was detected by qRTPCR in gastric cancer tissues and cell lines. The effect of miR-29c over-expression/knockdown on the proliferation, invasion and apoptosis of MGC-803/AP cells in vitro was measured by CCK-8 assay, Transwell and Annexin V-FITC/PI double staining flow cytometry assay. Western blotting was used to detect the regulation of miR-29c on TNRC18. Moreover, the relationship between miR-29c and TNRC18 was examined by dual luciferase reporter gene assay. Results: qRT-PCR revealed that miR-29c was low expressed in gastric cancer cell lines and gastric cancer tissues from patients resistant to apatinib. Moreover, dual luciferase reporter gene assay confirmed that miR-29c directly binds to the 3′UTR of TNRC18 mRNAto suppress its expression in MGC-803/AP cells. Furthermore, miR-29c inhibited the apatinib resistance in gastric cancer MGC-803/AP cells via inhibiting cell proliferation, invasion and promoting cell apoptosis by targeted down-regulating TNRC18. Additionally, in vivo experiment also confirmed that miR-29c modulated apatinib-resistance in gastric cancer cells by targeted inhibiting TNRC18. Conclusion: miR-29c/TNRC18 axis plays a certain role in the resistance of gastric cancer tissues and MGC-803/AP cells to apatinib, and over-expression of miR-29c may reverse the resistance of MGC-803/AP cells to apatinib.
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@#Objective: To investigate the effects of nuclear factor 5 of activated T cells (NFAT5) on proliferation and apoptosis of human gastric cancer MGC803 cells and to explore the possible mechanisms. Methods: Three siRNAs targeting NFAT5 gene (siRNA2567, siRNA2714 and siRNA4562) and one negative control siRNA were designed and chemically synthesized before transfected into human gastric cancer cell line MGC803 by liposome. Real-time PCR was used to detect the changes of N F AT 5 mRNA expression in MGC803 cells to further pick out the siRNA that most effectively inhibit the expression of N F AT 5 . Further, Real-time PCR and Western blotting assay were carried out to test mRNAand protein levels of NFAT5 and S100A4 in cells 48 h after N F AT 5 -siRNAtransfection. Then, CCK-8 assay and FCM assay were used to detect the influence of silencing N F AT 5 on cell proliferation and apoptosis, respectively. Results: siRNA2567 was the most effective siRNA that significantly inhibited the expression of N F AT 5 mRNA ( P <0.01), and thus was validated as NFAT5-siRNA. Real-time PCR and Western blotting assay confirmed that both mRNA and protein levels of NFAT5 and S100A4 were down-regulated in cells 48 h after N F AT 5 -siRNAtransfection. Compared with NC-siRNAgroup, the proliferation ability of MGC803 cells in the N F AT 5 siRNAgroup was significantly down-regulated at 72 h and 96 h ( P <0.01).And FCM assay showed that compared with NC-siRNA group, cell apoptosis rate of N F AT 5 -siRNA group was significantly increased from (2.7±0.2)% to (7.9±0.2)%, ( P <0.01) 48 h after N F AT 5 -siRNA transfection. Conclusion: N F AT 5 -siRNA transfection can silence N F AT 5 gene expression in gastric cancer MGC803 cells effectively. N F AT 5 may inhibit proliferation and promote cell apoptosis of gastric cancer cells possibly through regulating S100A4 expression.
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@# Objective: To investigate the molecular mechanism of lncRNA FOXD2-AS1 participating in apatinib resistance in gastric cancer cells by regulating miR-185-5p/CCND2 axis. Methods: The gastri cancer tissues and corresponding paracancerous tissues of 25 patients with gastric cancer were collected from April 2016 to December 2017 in the Fifth People’s Hospital of Wuxi City. The expressions of FOXD2-AS1, miR-185-5p, and cyclin D2 (CCND2) in gastric cancer tissues or cell lines were examined by quantitative realtime polymerase chain reaction (qRT-PCR). CCK-8 assay, Transwell assay and Annexin V-FITC/PI double staining flow cytometry assay were applied to assess the sensitivity of gastric cancer cells to apatinib. The interaction between FOXD2-AS1, miR-185-5p and CCND2 was explored by dual luciferase reporter gene assay, which was then confirmed by qRT-PCR, and Western blotting. Results: FOXD2-AS1 was highly expressed in gastric cancer tissues and apatinib-resistant gastric cancer cells. Over-expression of FOXD2-AS1 promoted apatinib-resistance of MGC-803/AP cells. Dual luciferase reporter gene assay confirmed that FOXD2-AS1 directly interacted with miR-185-5p and suppressed its expression. miR-185-5p significantly abolished the promotion effect of FOXD2-AS1 on apatinibresistance via inhibiting cell proliferation, invasion and promoting apoptosis of gastric MGC-803/AP cells. miR-185-5p could negatively regulate CCND2 expression; and FOXD2-AS1 promoted the cell proliferation, invasion and inhibited apoptosis of MGC-803/AP cells via down-regulating the inhibition effect of miR-185-5p on CCND2, thus further enhanced the apatinib-resistance of gastric cancer cells. Conclusion: FOXD2-AS1 induced apatinib-resistance of gastric cancer cells by regulating miR-185-5p/CCND2 axis.
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Aim To study the apoptosis-inducing effect of rosmarinic acid derivative RAD-9 on gastric cancer MGC-803 cells and the underlying mechanisms.Methods MTT assay was taken to detect the survival of gastric cancer MGC-803 cells effected by RAD-9.Cell apoptosis was detected by flow cytometry.The apoptotic morphology of MGC-803 cells was observed by Hoechst 33258 staining.The protein expression levels of Bcl-2,Bax,caspase-3,Akt,p-Akt,p38 MAPK and p-p38 MAPK were measured by Western blot.Results The results of MTT assay showed that RAD-9 inhibited the viability of gastric cancer MGC-803 cells in a time and concentration-dependent manner.Flow cytometry showed that RAD-9 significantly promoted apoptotic cell percentage in gastric cancer MGC-803 cells (P ＜ 0.01).Hoechst 33258 staining showed that the nucleus of MGC-803 cells could be observed with typical apoptotic morphological changes after RAD-9 administration.Compared with the control group,the protein expression levels of Bcl-2,Akt,p-Akt were significantly down-regulated (P ＜ 0.01),while those of Bax,caspase-3,p38 MAPK,p-p38 MAPK were significantly up-regulated (P ＜ 0.01).Conclusion RAD-9 can inhibit the growth and further induce apoptosis in gastric cancer MGC-803 cells,which may involve inhibiting PI3K/Akt and activating p38 MAPK signaling pathway.
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Objective To investigate the expression of FTO in gastric cancer tissues and the functional significance of FTO in MGC-803 cell line.Methods The FTO mRNA was detected by RT-qPCR in 54 cases of gastric cancer samples and their paired adjacent normal control tissues.The effect of FTO overexpression in MGC-803 on cell proliferation,cell migration and invasion were detected by CCK-8,wound heal and ranswell assays,respectively.Results The expression of FTO mRNA was significantly lower than that of adjacent tissues(P＜O.05).Furthermore,overexpression of FTO in MGC-803 cells inhibited cell proliferation,cell migration and invasion.Conclusions FTO is low expressed in gastric cancer tissues and inhibits gastric cancer cell line MGC-803 proliferation,migration and invasion.FTO is closely associated with the development of gastric cancer.
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OBJECTIVE To investigate the effect of furanodiene(FDE),a diterpene derived from the medicinal plant Zedoary,on apoptosis of human gastric cancer MGC-803 cells induced in vitro. METHODS MGC-803 cells were treated with FDE 46.29~740.74μmol·L-1 for 24,48 and 72 h,and the cell viability was detected with MTT assay. Cell morphology was observed by light microscopy and Hoechst33342 staining. Flow cytometry was used to detect cell apoptotic rate and cell cycle. Rh123 staining and fluorescence probe DCFH-DA were employed to detect the changes in mitochondrial membrane potential (MMP) and reactive oxygen species(ROS). RESULTS MTT Results showed that FDE 46.29-740.74μmol · L-1 exhibited significantly higher cytotoxicity to gastric cancer MGC-803 cells. IC50 for MGC-803 of 24,48 and 72 h treatment was 347.91,257.41 and 101.01μmol·L-1,respectively. Treatment with FDE 92.58-370.32μmol·L-1 for 24 h also caused significant morphological changes in MGC-803 cells. AnnexinⅤ-FITC/PI double staining showed that the apoptotic rate increased after FDE 92.58-370.32μmol·L-1 treatment for 24 h(P<0.05). FDE enabled MGC-803 cell cycle arrest in S phase. DCFH-DA staining showed that FDE resulted in an increase in intracellular ROS levels(P<0.05) when PDE concentration was 370.37μmol·L-1(P<0.05). MMP decreased after FDE treatment when PDE concen?tration was 370.37μmol·L-1(P<0.05). CONCLUSION FDE Possesses potent tumor selected toxicity and can induce apoptosis of MGC-803 cells through cell cycle arresting,which is related to inhibition of DNA biosynthesis.
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OBJECTIVE: To investigate the effects of combination treatment with L-carnitine and 5-fluorouracil on the proliferation and cell apoptosis of gastric cancer MGC803 cells. METHODS: MGC803 cells were divided into control group, 5-fluorouracil group and the combination of L-carnitine and 5-fluorouracil group (L-carnitine +/→5-fluorouracil group) in vitro. The inhibitory rate of cells was measured by MTT assay. The apoptosis rate and cell cycle of cells were detected by FLOW. Western blot was used to analyzed the expression of Bcl-2, Bax, adenine nucleotide translocator1 (ANT1) and cleaved-PARP. RESULTS: Compared with 5-fluorouracil group, the inhibition rate of MGC803 cells was increased when cells were treated with the combination of L-carnitine and 5-fluorouracil. The apoptosis rate of cells was raised and the cells were blocked at S phase. In addition, the combination group can decrease the expression of Bcl-2 and increase the expression of Bax, ANT1 and cleaved-PARP. At the same time, the apoptosis rate of cells and the cell cycle were different with the different dosage regimen when treated with the combination. Compared with the L-carnitine + 5-fluorouracil group, the apoptosis rate of cells was increased to (24.17 ± 3.12)% from (19.60 ± 1.06)% (P < 0.05). The G0/G1 phase proportion of cells was decreased to (62.62 ± 1.04)% from (72.95 ± 0.91)%, and the S phase proportion of cells was increased to (37.35 ± 1.03)% from (27.05 ± 0.91)% (P < 0.001). CONCLUSION: Treatment with L-carnitine and 5-fluorouracil could enhance the inhibitory effect of 5-fluorouracil on MGC803 cells. The possible mechanism of action is achieved by regulating the expression of Bcl-2 protein family and influencing the cell cycle.
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OBJECTIVE: To screen the best combination of extractum of Robinia-living trametes and chemotherapy and investigate the action mechanism of Robinia-living trametes against the apoptosis of human gastric cancer cell MGC803. METHODS: MGC803 Cells were treated with different concentrations of Robinia-living trametes and chemotherapy drugs (5-Fu and paclitaxel) in vitro. The inhibitory rate of cells was measured by MTT assay. Morphological changes were observed with inverted microscope. The apoptosis rate of MGC803 cells which were treated with combination of Robinia-living trametes (0.2 mg·mL-1) and 5-Fu (2.5 μg·mL-1) was detected by FCM. The protein expression of P53 and p-Akt in MGC803 cells which were treated with combination of Robinia-living trametes (0.2 mg·mL-1) and 5-Fu (2.5 μg·mL-1) was detected by Western blot. RESULTS: The viability of MGC803 cells was reduced by Robinia-living trametes and chemotherapy drugs (5-Fu and paclitaxel) in a concentration- and time-dependent manner (P<0.01). Under reverse microscopy, cell body shrinking, nuclear pyrosis, and nuclear fragmentation were observed. The higher concentration, the longer treatment time, the more cells died. Compared with monotherapy, the combination of Robinia-living trametes and chemotherapy could reduce the survival rate of MGC803 cells. The protein expressions of P53 in MGC803 cells treated with combination of drugs was up-regulated, while that of P-Akt was down-regulated. CONCLUSION: The apoptosis of MGC803 cells in vitro may be induced by the inhibitory effect of the combination of Robinia-living trametes and 5-Fu on PI3K/Akt signaling pathway. Combination therapy of Robinia-living trametes and 5-Fu is potentially more effective in inhibition of tumor cells than monotherapy of Robinia-living trametes.