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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 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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@#[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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@#To investigate the apoptotic effect of flavonoid compound GL-V9 on human gastric cancer cells and its potential mechanism, MGC-803 and BGC-823 cells were treated with GL-V9. MTT assay was performed to assess the growth inhibition effects on MGC-803 and BGC-823 cells under different concentrations of GL-V9. Annexin V-FITC/PI staining assay was employed to observe the apoptotic rate of GL-V9 cells with the treatment of GL-V9. DAPI staining was performed to observe the nuclear morphological changes using fluorescence microscopy. Activation of caspase-9 and caspase-3 was analyzed by Western blotting. Ca2+ concentration in gastric cancer cells was detected by Fluo-3 AM staining assay. Results showed that GL-V9 could inhibitcell viability, change the nuclear morphologyl, activate caspase-9 and caspase-3 and induce the apoptosis in gastric cancer cells. The mechanism of the induction of apoptosis in MGC-803 cells under the treatment of GL-V9 may aetivate the Ca2+ associated mitochondrial apoptosis pathway.
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Objective To study the effects of MACC1 down-regulation on the growth of gastric adenocarcinoma cells. Methods siRNA (MACC1-siRNA1 and MACC1-siRNA2) that can transiently silenced MACC1 was designed, syn?thesized and transfected into MGC-803 cells by lipofectamine 2000. Non-specific siRNA was transfected to be used as nega?tive control. The efficiency of MACC1 depletion was determined by Real-time quantitative PCR. MTT, colony formation and flow cytometry assay were performed to examine cell proliferation. The expressions of MACC1, P21,CDK4, CCND1 and c-myc were determined by Western blot. Results Compared with cells in negative control group, transiently silencing MACC1 decreased the expression of MACC1 in MGC-803 cells shown by Real-time PCR. MACC1 downregulation drastical?ly changed the proliferation, colony formation and cell cycle of gastric adenocarcinoma cells in vitro ( P<0.05). The expres?sions of MACC1 , CDK4, CCND1 and c-myc proteins in cells of MACC1 silence group were much lower while P21 expres?sion level was much higher than those in negative control. Conclusion Down-regulation of MACC1 result in blocking cell cycle, inhibiting proliferation of MGC-803 cells. So it may serve as a promising target in the treatment of gastric cancer.
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This study was aimed to provide experimental evidence for clinical application of curcumin to strengthen the effect of 5-Fluorouracil (5-FU) in gastric cancer treatment, so as to reduce the dose of 5-FU and its side-effect in the future treatment. MTT assay was used to detect medication effect on MGC-803 cell growth inhibition; AO/EB double staining fluorescence microscopic observation and FITC/PI double staining flow cytometry were used to detect medication effect on cell apoptosis. Flow cytometry was based on PI staining used to detect medication effect on MGC-803 cell cycle. The results showed that curcumin (25μmol·L-1) with low-dose (2.4μmol·L-1) and middle-dose (4.8μmol·L-1) 5-FU can effectively inhibit MGC-803 cell growth, induce cell apoptosis and block cell cycle in S phase. Which showed dose-time dependent manner. The combined use of curcumin with low-dose 5-FU was more effective than the middle-dose (4.8μmol·L-1) 5-FU alone (P < 0.01); similarly, the combined use of curcumin with middle-dose 5-FU was more effective than the use of high-dose (9.6μmol·L-1) 5-FU alone (P < 0.01). It indicated that curcumin can enhance the antitumor effect of 5-FU against MGC-803 cells in a dose-time dependent manner. It was concluded that the study provided a preliminary experimental basis for using curcumin as an adjuvant to 5-FU with the benefit of reducing its dose and toxicity in the clinical treatment of gastric cancer.
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Objective: To investigate the effect of total glycosides from Ranunculus japonicus (TGRJ) on vasculogenic mimicry of human gastric cancer MGC803 cell line in vitro and its mechanisms. Methods: The effect of TGRJ on the cell adhesion of MGC803 was observed by cell attachment assay. The effect of TGRJ on the cell migration of MGC803 was studied by cell wound healing assay. The function of TGRJ on the tube-like structures formation of MGC803 in vitro was verified by tube formation assay. The effect of TGRJ on the expression of vasculogenic mimicry related genes was assessed by semi-quantitative RT-PCR assay. Results: TGRJ effectively inhibited the cell adhesion, cell migration of MGC803, and the MGC803-mediated vasculogenic mimicry in vitro in a dosage-dependent manner. Furthermore, the semi-quantitative RT-PCR showed that TGRJ markedly inhibited the expression of MGC803 vasculogenic mimicry-related genes VE-cadherin, sema 4D, and integrin β5. Conclusion: TGRJ could effectively inhibit the cell attachment and cell migration of MGC803, and the MGC803-mediated vasculogenic mimicry by down regulating vasculogenic mimicry-related genes VE-cadherin, sema 4D, and integrin β5.
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Objective To investigate the proliferation inhibition and apoptosis induction effect of thalidomide(Thal) and its joint effect with 5-Fu on MGC-803 cell line. Methods The morphological changes of MGC-803 cells with AO/EB stain were observed under fluorescence microscope. The proliferation inhibition effeet was evaluated with MTT. The apoptosis induction effect was determined by FAM. Results The results of MTr array were as below: the difference was significant between Thai groups of 25 mg/L or above concentration and the control group (P＜0.05), 5-Fu of all testing concentrations showed significant difference compared with the control gToup(P＜0.01). For combined groups with the same concentration of 25 mg/L 5-Fu,combined groups showed distinct difference from the corresponding 5-Fu group (P＜0.01), but two combined groups showed no distinct difference with each other (P＞0.05). For combined groups with the same concentration of 5-Fu 12.5 mg/L, 5-Fu plus 50 mg/L or 100 mg/L Thai showed no distinct difference from 5-Fu group of 25 mg/L(P＞0.05). With FAM study, all test groups showed significant difference compared with the control group (P＜0.01). There was also significant difference between two test groups arbitrarily (P＜0.01).Conclusion Both Thai and 5-Fu could inhibit the proliferation and induce the apoptosis of MGC-803 cells.The effect is reinforced with the combination significantly. Combination with Thai could decrease the concentration of 5-Fu but have the similar effect.
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Objective To study the effects of ganoderma applanatum polysaccharides(GAPS) on cell morphology, proliferation and PDGFR -?expression in cell lines MGC - 803 , and to explore its potential mechanisms of anti - tumor of GAPS. Methods Cell morphology was observed by inverting microscope. MTT assay was used to investigate the inhibitory effect of GAPS on MGC -803.Expressions of PDGFR -?was analyzed by flow cytometry (FCW). The fluorescence intensity of expressions of PDGFR -?was observed by fluorescence microscope. Results Cells of GAPS group were irregular shaped and grew poorly. GAPS inhibited the proliferation of MGC -803 cells in dose - dependent and time - dependent manners . After MGC - 803 cells were treated with GAPS for 72h, GAPS could down - regulate expression of PDGFR -?observed by fluorescence microscope which was consistented with the results of FCW with statistic significance difference as compared with control group (P
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Aim To investigate whether DATS induce MGC803 cell apoptosis and the relationship betweenapoptosis and Ca~(2+) disruption. Methods MGC-803 cell growth inhibition was measured by MTT assay. Tunnel and flow cytometry methods were used to determine the induction of apoptosis and Ca2+ homeostasis disruption. Result MTT assay showed that the inhibitory rates on MGC-803 cell growth of different concentrations of DATS 4,8,12,16 and 24 mg?L-1 were 0.231?0.037,0.305?0.036,0.455?0.029,0.607?0.058,0.751?0.019 respectively. Flow cytometry analysis showed that treating MGC803 cell with DATS significantly increased the percentage of apoptosis cells and intracellular Ca2+. Treatment of cells with 1,2-bis(2-aminophenoxye-thane)-N,N,N-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), cellular Ca2+ chelator, resulted in abolishment of the elevation of intracellular Ca~(2+) and blockage of DATS induced apoptotic of MGC-803. Conclusoin DATS could induce apoptosis of MGC-803 cells through the mechanism of Ca~(2+) homeostasis disruption.
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Aim To study the effect of As_2O_3(arsenic trioxide) on the expressions of telomeric repeat binding factor1,2 and telomeric stability of human gastric cancer MGC803 cells, and explore the mechanism of cell apoptosis. Method MGC803 cell growth inhibition was measured with MTT assay. Apoptosis was analyzed with flow cytometry. Influence on chromosome distal end was analyzed with chromosome end-end fusion analysis. Expressions of TRF1 and TRF2 were determined with Western blot analysis. Results MTT assay showed that As_2O_3 clearly inhibited the growth of MGC803 cells, depending on time and dosage. The apoptosis rates were significantly higher than those of the control group in a concentration and time-dependent manner. These changes were not found in the control group. After disposal with 5 ?mol?L -1 As_2O_3, chromosome fusion rate was obviously increased in 48 h. After 48 h of disposal with 5 ?mol?L -1 As_2O_3, the TRF1 of MGC803 cells was up-regulated while TRF2 was down-regulated. Conclusion As_2O_3 induced chromosome fusion and MGC803 cells apoptosis through up-regulating expressin of TRF1 and down-regulating expressin of TRF2.
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AIM: To study the effects of growth inhibition of different concentrations of diallyl trisulfide(DATS) on gastric cancer MGC-803 cell line in vitro. METHODS: The influence of different concentrations of DATS were examined by MTT assay, clonal formation rates and cell growth curve. RESULTS: Suppression and decrease of MGC-803 cell proliferation was found after treatment by DATS in vitro. The inhibitory rates on MGC-803 cell growth of different concentration of DATS, 4, 8, 12, 16 and 24 mg·L-1, were 26%, 46%, 65%, 76% and 89% respectively, and its half inhibitory concentration (IC50) was 8.2 mg·L-1. The clonal formation rates and clonal formation relative counts of 8, 12, 16 and 24 mg·L-1 were 32.4% and 58.7%, 24.8% and 42.5%, 19.0% and 33.5%, 8.8% and 15.1% respectively. There was significant correlation between dose and effect in all, and the cell growth culve became lower and flatter when concentration of DATS increase gradually. CONCLUSION: The effect of growth inhibition of DATS for gastric cancer MGC-803 cell in vitro is remarkable.
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AIM To study the effects of growth inhibition of different concentrations of diallyl trisulfide(DATS) on gastric cancer MGC 803 cell line in vitro. METHODS The influence of different concentrations of DATS were examined by MTT assay, clonal formation rates and cell growth curve. RESULTS Suppression and decrease of MGC 803 cell proliferation was found after treatment by DATS in vitro. The inhibitory rates on MGC 803 cell growth of different concentration of DATS,4, 8, 12, 16 and 24 mg?L -1 , were 26%,46%,65%,76% and 89% respectively, and its half inhibitory concentration (IC 50 ) was 8 2 mg?L -1 . The clonal formation rates and clonal formation relative counts of 8, 12, 16 and 24 mg?L -1 were 32 4% and 58 7%,24 8% and 42 5%,19 0% and 33 5%?8 8% and 15 1% respectively.There was significant correlation between dose and effect in all, and the cell growth culve became lower and flatter when concentration of DATS increase gradually. CONCLUSION The effect of growth inhibition of DATS for gastric cancer MGC 803 cell in vitro is remarkable.