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Aim To investigate the insulinotropic effect of telmisartan anrl the underlying electrophysiological mechanism.Methods Islets and cells were isolated from Wistar rats.Islets were incubated with drugs un¬der different conditions, then supernatant liquid was collected for insulin secretion.Intracellular Ca" + ( Ca'+ j) levels of (3-cells were measured by calcium imaging technology.Patch-clamp technology was ap¬plied to detect effects on voltage-gated potassium chan¬nel ( Kv ) , and voltage-gated calcium channel ( VGCC ).Results Not affecting insulin secretion un¬der low glucose condition, telmisartan dose-dependent- ly stimulated insulin secretion under high glucose con¬ dition, and stimulation was enhanced with increasing glucose concentration.Acute increases of Ca' + concentration were elicited by telmisartan under high glucose condition.Telmisartan decreased current den¬sity of Kv channel, and increased VGCC current densi¬ty.Conclusions Telmisartan enhanced Ca~+ ; lev¬els of p-cells through its action on Kv channel and VGCC, thereby amplifying glucose-stimulated insulin secretion.
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@#[Abstract] Objective: To explore the anti-tumor activity of MUC16-targeted chimeric antigen receptor modified NK-92 (CARNK-92) cells against ovarian cancer. Methods: The expression of MUC16 in surgically resected tumor tissues of 15 patients with ovarian cancer treated in the Department of Obstetrics and Gynecology of Qingyang Hospital of Traditional Chinese Medicine and 4 ovarian tumor cell lines was detected by Immunohistochemistry and Flow cytometry. MUC CAR sequence was synthesized by gene synthesis, and its lentivirus expression vector were constructed. CARNK-92 cells targeting MUC16 (MUC-BBz) were obtained by lentivirus infection. The expression of CD107a in MUC-BBz was detected by Flow cytometry. The activation of MUC-BBz cells and its cytotoxicity against SKOV3 target cells were characterized by the release of LDH assay. The xenograft nude mouse model of SKOV3 cells was established to verify the in vivo anti-tumor activity of MUC-BBz cells. Results: MUC16 was highly expressed in ovarian cancer tissues and human ovarian cancer cells. MUC-BBz was successfully constructed by infecting NK-92 cells with lentivirus, with a positive rate of (42.79±2.58)%. MUC-BBz could be specifically activated by MUC16 over-expressing tumor cells. After co-incubation of effector cells and target cells, the expression of CD107a on MUC-BBz was upregulated significantly (P<0.01), and the ability of MUC-BBz secreting cytokines IFN-γ and perforin also increased (all P<0.01). The LDH test indicated that with the increase of effector-target ratio, the cytotoxicity of MUC-BBz against 4 ovarian cancer cells (hey, COC1, SKOV3 and A2780) also significantly enhanced. The results of transplanted tumor model showed that transfusion of MUC-BBz could significantly inhibit the growth of SKOV3 xenograft in mice (P<0.01). Conclusion: The CARNK-92 cells can significantly inhibit the growth of ovarian cancer cells in vitro and in vivo, which provides an important basis for further evaluation of its clinical application.
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@#[Abstract] Objective: To investigate the effects of silencing monocarboxylate transporter 4 (MCT4) on the proliferation, migration and invasion of prostate cancer PC3 cells and its possible molecular mechanism. Methods: RNA interference technology was used to transfect siRNA-MCT4 (si-MCT4) and negative control plasmid (si-NC) into PC3 cells, respectively. The content of lactic acid in the cell culture medium of transfected PC3 cells was detected by lactic acid assay after culturing for 96 h. The proliferation, migration and invasion ability of PC3 cells were detected by CCK-8 and Transwell assay, respectively. Western blotting was used to detect the silencing effect and the expressions of integrin β 4-FAK-SRC-MEK-ERK signaling pathway associated proteins (integrin β4, p-FAK, p-SRC, p-ERK1/2, p-MEK1/2) and EMT associated proteins (E-cadherin and N-cadherin). Results: PC3 cell line with silenced MCT4 was successfully constructed. Compared with the control group, the content of extracellular lactic acid in the PC3 cell culture medium of the si-MCT4 group was significantly decreased (P<0.01), and the proliferation, migration and invasion of cells were significantly decreased (P<0.05 or P<0.01). Compared with the control group, the protein expressions of integrin β4, p-FAK, p-SRC, p-MEK1/2, p-ERK1/2 and N-cadherin were significantly decreased (all P<0.01), while the protein expression of E-cadherin was significantly increased (P<0.01). Conclusion: Silencing MCT4 can significantly inhibit the proliferation, migration and invasion of PC3 cells, the mechanism of which may be related to the inhibition of lactic acid level in cell culture medium and suppression of integrin β4-FAK-SRC-MEKERK signaling pathway associated proteins as well as EMT associated proteins.
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@#[Abstract] Objective: To investigate the effect of HMGB1 gene on the growth of human epithelial ovarian cancer xenografts in nude mice, and to lay a foundation for finding new targets for the treatment of ovarian cancer. Methods: Human epithelial ovarian cancer SKOV3 cells in logarithmic growth phase were selected to establish a human epithelial ovarian cancer xenograft model in nude mice. Nude mice with successful model establishment were randomly divided into control group and HMGB1-siRNA group. On the 7th, 9th, 11th, 14th, and 16th days after cell inoculation, the same amount of saline and HMGB1-siRNA were respectively injected into two groups of mice under the armpit.After 3 weeks, the nude mice were sacrificed by cervical dislocation, the tumor tissues were separated, and the volume of the tumor was measured. The apoptosis of transplanted tumor cells was detected by Tunnel staining. The expressions of HMGB1, STAT3 and p-STAT3 were detected by Western blotting. The expression of vascular endothelial growth factorA(VEGF-A) and microvascularization were detected by immunohistochemistry. Results: Compared with the control group, the growth of tumor volume slowed down in HMGB1 siRNA group, and on the 21st day, the tumor volume of HMGB1-siRNA group was significantly smaller than that of the control group (P<0.05). HMGB1-siRNA successfully knocked down the expression of HMGB1 mRNA in transplanted tumor tissue. The apoptosis rate of tissue cells in HMGB1-siRNA group was significantly increased ([34±8]% vs [6±2]%, P=0.04), and the expressions of HMGB1 and p-STAT3 were significantly reduced (P<0.05). The expression of VEGF-Aand the number of microvessels were significantly lower than those of the control group (both P<0.05). Conclusion: Knockdown of HMGB1 gene reduces the expression of VEGF-A and microvessel formation possibly by inhibiting the HMGB1/STAT3 signaling pathway, thereby promoting the apoptosis of tumor tissues and slowing the growth of xenografts.
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@#[Abstract] Objective: To explore the effect of lncRNA HOTAIR/miR-519d-3p/cyclin D1 (CCND1) axis on the proliferation and metastasis of breast cancer cells and its underlying mechanism. Methods: A total of 50 pairs of breast cancer tissues and corresponding para-cancer tissues resected from breast cancer patients in the Department of Breast Surgery, the Third Hospital of Nanchang from March 2017 to February 2019 were collected for this study. The expression level of HOTAIR in breast cancer tissues and paired paracancer tissues was detected by qPCR, in addition, the expressions of HOTAIR and miR-519d-3p in normal breast epithelial cells and breast cancer cell lines were also detected. Breast cancer SKBR3 cells were divided into NC group (without any treatment), si-HOTAIR group, mir-519d-3p mimics group, miR-519d-3p mimic+pcHOTAIR group, miR-519d-3p mimic+pcCCND1 group, and si-HOTAIR+ pcCCND1 group. The proliferation ability of SKBR3 cells was detected by CCK-8. Invasion and migration of SKBR3 cells were detected by Transwell. The expression levels of E-cadherin, N-cadherin, Vimentin and CCND1 in SKBR3 cells were detected by Western blotting. The targeting relationship between HOTAIR and miR-519d-3p, miR-519d-3p and CCND1 was detected by Dualluciferase reporter gene system. Results: HOTAIR was highly expressed in breast cancer tissues and cell lines, with the highest expression in SKBR3 cells. HOTAIR knockdown significantly inhibited the proliferation, invasion and migration of SKBR3 cells, as well as increased the expression level of E-cadherin and decreased the expression levels of N-cadherin and Vimentin. Dual-luciferase reporter gene assay showed that HOTAIR targetedly down-regulated the expression of miR-519d-3p, and miR-519d-3p targetedly downregulated the expression of CCND1. Further studies showed that knockout of HOTAIR inhibited the EMT, proliferation, invasion and migration of SKBR3 cells through enhancing the inhibitory effect of miR-519d-3p on CCND1 expression (all P<0.05). Conclusion: HOTAIR knockdown inhibits proliferation and metastasis of SKBR3 cells by regulating the axis of miR-519d-3p/CCND1.
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@#[Abstract] Objective: To investigate the effects of exosome originated from bone marrow mesenchymal stem cell (BMSCs) on proliferation, migration and invasion of prostate cancer PC-3 cell and its mechanism. Methods: qPCR was used to detect the expression level of miR-21-5p in prostate cancer cell lines. The morphology of exosomes isolated from BMSCs was observed with an electron microscope. Western blotting was used to detect the expressions of exosome surface markers and the epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, N-cadherin and Vimentin). Dual luciferase reporter gene experiment was used to detect the targeted regulation relationship between miR-21-5p and PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2). PC-3 cells were co-cultured with 10 μl BMSCs exosomes suspension (Exo group), transfected with sh-PHLPP2 or antagomiR, then CCK-8 and Transwell experiments were used to detect changesinproliferation,migrationandinvasionofPC-3cell.Results: miR-21-5p was highly expressed in prostate cancer PC-3 cell line. The exosomes in the supernatant of BMSCs culture fluid were successfully isolated, and the typical vesicle-like structures of exosomes were observed under transmission electron microscope. Exosomes expressed specific proteins such as CD9, CD63 and CD81. In the Exo group, the proliferation, invasion, migration, as well as the expressions of N-cadherin, Vimentin and miR-21-5p in PC-3 cells were significantly higher than those in the control group (all P<0.05). PHLPP2 is a target gene of miR-21-5p. Compared with the control group, the expression of PHLPP2 in PC-3 cells of Exo group and sh-PHLPP2 group was significantly reduced (0.66±0.09, 0.42±0.05 vs 1.09±0.08, all P<0.01); cell viability, invasion and migration were significantly improved (all P<0.01); and E-cadherin expression level was significantly reduced while N-cadherin and Vimentin expressions were significantly increased (both P<0.05). Conclusion: miR-21-5p is highly expressed in prostate cancer PC-3 cell line. BMSC exosome miR-21-5p can increase the proliferation, migration and invasion ability of PC-3 cells through targeted down-regulation of PHLPP2.
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@#[Abstract] Objective:To explore the targeting relationship between long-chain noncoding RNA HOXA-AS2 (lncRNA HOXA-AS2) and microRNA-520a-3p (miR-520a-3p) and their effects on the proliferation, migration and invasion of ovarian cancer SKOV3 cells. Methods： ：qPCR was used to detect the expression levels of lncRNA HOXA-AS2 and miR-520a-3p in various ovarian cancer cell lines (SKOV3, HO8910, OVCAR3 cells) and normal ovarian epithelial cell line HOSE. Bioinformatics methods were used to predict the targeting relationship between HOXA-AS2 and miR-520a-3p, which was then verified by Dual luciferase reporter gene assay. si-HOXA-AS2, miR-520a-3p mimic, anti-miR-520a-3p and corresponding control fragments were transfected into SKOV3 cells separately or in combination. MTT, Transwell and Western blotting were used to detect the proliferation, migration, invasion and expressions of related proteins (CyclinD1, p21, p27, MMP-2, MMP-9, MMP-14) of SKOV3 cells in each group. Results: Compared with HOSE cells, HOXA-AS2 was over-expressed while miR-520a-3p was under-expressed in ovarian cancer cell lines (all P<0.05). HOXA-AS2 could targetedly down-regulate the expression of miR-520a-3p. Compared with the NC group, the proliferation, migration and invasion of SKOV3 cells in the si-HOXA-AS2 and miR-520a-3p mimics groups were significantly reduced (all P<0.01), and the protein expressions of p21 and p27 were significantly increased, while protein expressions of CyclinD1, MMP-2, MMP-9, MMP-14 were significantly reduced (all P<0.01). The proliferation, migration and invasion of SKOV3 cells in the si-HOXA-AS2+antimiR-520a-3p group were significantly enhanced compared with those in si-HOXA-AS2 and si-HOXA-AS2+anti-miR-NC groups (all P<0.05). Conclusion: lncRNA HOXA-AS2 enhances the proliferation, migration and invasion of ovarian cancer SKOV3 cells by targetedly inhibiting the expression of miR-520a-3p.
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OBJECTIVE：To investigate the effects and m echanism of oleanolic acid on inhibiting the proliferation ，invasion and metastasis of human ovarian cancer SKOV 3 cells. METHODS ：CCK-8 assay was used to detect the effects of different concentrations of oleanolic acid （10，20，40，60，80，100 μmol/L）on the proliferation of ovarian cancer SKOV 3 cells at 12，24， 36 and 48 h. The effects of low-dose and high-dose of oleanolic acid （20，40 μmol/L）on the metastasis and invasion ability of SKOV3 cells for 24 h were observed in Transwell assay. Western blotting assay was used to detect the effects of low-dose and high-dose of oleanolic acid on the protein expression of NF-κB p65，PRL-3，TNF-α，IL-6 and E-cadherin in SKOV 3 cells. Through LPS induction and NF-κB p65 plasmid transfection ，Western blotting and RT-qPCR assay were used to investigate the effects of low-dose and high-dose oleanolic acid on the expression of NF-κB/PRL-3 pathway related proteins and their mRNA. RESULTS ： With the increase of the concentration and action time of oleanolic acid ，the proliferation capacity of ovarian cancer SKOV 3 cells was decreased ，the surval rates of administration groups were significantly lower than that of the control group （P＜0.05 or P＜ 0.01）. Low-dose and high-dose of oleanolic acid could significantly reduce the number of migrating and invading cells （P＜0.05 or P＜0.01）. The protein relative expression of NF-κB p65，PRL-3，TNF-α and IL-6 in SKOV 3 cells were significantly decreased ， while the protein relative expression of E-cadherin was significantly increased （P＜0.05 or P＜0.01）. After LPS induction ，protein and mRNA relative expression of NF-κB p65，PRL-3，TNF-α and IL-6 were increased significantly in LPS model group ，while protein and mRNA relative expression of E-cadherin were significantly decreased （P＜0.05 or P＜0.01）. The protein and mRNA relative expression of NF-κ B p65，PRL-3，TNF-α and IL-6 were significantly decreased ，and protein and mRNA relative expression of E-cadherin were significantly increased in low-dose and high-dose of oleanolic acid group （P＜0.05 or P＜0.01）. In SKOV3 cells with over-expressed NF-κB p65，low-dose and high-dose of oleanolic acid c ould significantly down-regulat the proteinexpression of NF-κ B p65，PRL-3，TNF-α and IL-6，while upregult the protein relative expression of E-cadherin （P＜0.05 E-mail：122821905@qq.com or P＜0.01）. CONCLUSIONS ： Oleanolic acid can inhibit SKOV3 cells proliferation，invasion and metastasis by regulating NF-κB/PRL-3 signaling pathway.
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@# Objective: To explore the action mechanism of miR-139-5p inhibiting proliferation and invasion of epithelial ovarian cancer (EOC) cells by targetedly regulatingNotch1.Methods: A total of 24 pairs of EOC tissues and its corresponding para-cancerous tissues from patients, who underwent surgical resection in the DepartmentofGynecology,Nanyang Central Hospital of Henan Province, were collected for this study; in addition, human ovarian cancer cell lines (SKOV3, ES2, HEY-T30) and human ovarian epithelial cell line IOSE80 were also collected. Real-time quantitative PCR (qPCR) was applied to detectmRNAexpressionofmiR-139-5pandNotch1 in EOC tissues and cell lines. The miR-139-5p over-expression vector and recombinant plasmid pLV-Notch1 were transfected into SKOV3 cells. Blank control group (Ctrl group) and negative control group (NC group) were set up. Dual luciferase reporter gene assay was applied to verify the targeting relationship between miR-139-5p and Notch1 3'-UTR. CCK-8, Transwell and Scratch healing experiments were applied to detect cell proliferationinvasionandmigration, respectively. Western blotting was applied to detect expressions of proliferation and migration related proteins in cells. Results: Compared with para-cancerous tissues and IOSE80 cells, the expression of miR-139-5p was significantly decreased in EOC tissues and cell lines, while the expression of Notch1 mRNA was significantly increased (all P<0.01). The results of Dual luciferase reporter showed that Notch1 was the downstream target gene of miR-139-5p. Compared with NC group, cell proliferation, invasion and migration ability, expression levels of Notch1, NICD, Cyclin D1, Cyclin A1, Snail1, β-catenin and N-cadherin were all significantly decreased on 3 d in miR-139-5p mimic group (all P<0.01), while expression of E-cadherin was significantly increased (P<0.01); meanwhile, over-expression of Notch1 could reverse the inhibitory effect of miR-1395p on proliferation, invasion and migration of SKOV3 cells. Conclusion: miR-139-5p can targetedly regulate Notch1 to inhibit proliferation, invasion and migration of EOC cells, which may be related to its down-regulation of NICD, Cyclin D1, Cyclin A1, Snail1, βcatenin and N-cadherin, and up-regulation of E-cadherin.
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Objective: To study the role of dopamine D2 receptor (D2R) on the regulation of prolactin (PRL) secretion by malt total alkaloids. Methods: MMQ and GH3 cells of pituitary adenoma were divided into control group, bromocriptine (5 μg/mL), malt total alkaloids (4.4, 8.8, 35.2, 70.4 μg/mL), haloperidol (10, 20, 40 μg/mL), and combined administration group of total malt alkaloids and haloperidol. Cell viability was detected by CCK-8; The expressions of PRL and D2R were detected by western blotting; The level of PRL was detected by ELISA; The level of PRL and D2R mRNA were detected by qRT-PCR. Results: Compared with control group, malt alkaloids (35.2, 70.4 μg/mL) significantly reduced the expression levels of PRL protein and mRNA, and the level of PRL in the supernatant of MMQ cells (P < 0.05). Malt alkaloids (35.2, 70.4 μg/mL) significantly increased the expression levels of D2R protein and mRNA in MMQ cells. Haloperidol significantly inhibited the downregulation of malt alkaloids on the expression levels of PRL protein and mRNA, and the expression level of PRL in supernatant of MMQ cells (P < 0.05). Haloperidol significantly inhibited the upregulation of malt alkaloids on the levels of D2R protein and mRNA (P < 0.05). The level of PRL in GH3 cells had no change by malt alkaloids. Conclusion: Malt alkaloids could inhibit the expression and secretion of PRL in MMQ cell by upregulating D2R.
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A UPCC-Q-TOF-MS method was established to analyze the components of polyoxyethylene 35 castor oil. The separation was performed at 50 ℃ on a Waters Acquity UPCC system by an Torus Diol column (3.0 mm × 100 mm, 1.7 μm) with gradient elution of CO2 and methanol - acetonitrile (50∶50); the flow rate was 1.0 mL·min-1, the back pressure was 2 000 psi, and methanol containing 2.5 mmol·L-1 ammonium formate was used as ionization reagent, whose flow rate was 0.2 mL·min-1. Positive ion electrospray ionization (ESI) mode and MSE technology were used. The qualitative analyses were carried out by using precise mass information of the parent and product ions and a PCA model was established by UPCC-Q-TOF-MS. L-02 cells and RBL-2H3 cells were used to study the cytotoxicity and histamine release of CrEL samples in vitro. A total of 13 kinds of CrEL components were obtained and their structures were identified by UPCC-Q-TOF-MS, with 255 compounds in total. The percentage content of 13 types of components was calculated by the normalization method. The content of polyoxyethylene glycerol tri-ricinoleate (PGTri-ricinoleate) in all samples was 0.36% - 2.80% and the main components were polyethylene glycol, polyethylene glycerol and polyoxyethylene glycerol mono-ricinoleate. All samples have different degrees of cytotoxicity and histamine release, which is negatively correlated with the content of PGTri-ricinoleate and positively correlated with the content of polyoxyethylene glycol fatty acid esters. The UPCC-Q-TOF-MS method is simple and rapid, has strong separation ability and high accuracy. It is suitable for the analysis of CrEL components. It is suggested that the fatty acid composition should be included in the monograph of CrEL for injection to increase the content of PGTri-ricinoleate and decrease the content of polyoxyethylene glycol fatty acid esters, so as to improve the product safety.
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@#[Abstract] Objective: To investigate the effect of long non coding RNA (lncRNA) LINC00308 on proliferation, invasion and migration of prostate cancer cells and its related mechanism. Methods: lncRNAs and mRNAs differentially expressed in prostate cancer tissues and adjacent control tissues were screened by gene chip, and LINC00308 and TRIP13 (thyroid hormone receptor interactor13) were identified as the research objects. The effects of LINC00308 on the proliferation, invasion and migration of prostate cancer cells were detected by MTT assay, plate cloning, Transwell and scratch test. The above effects were verified in nude mice xenografts. The effect of LINC00308 on expression of TRIP13 in tumor tissues and cancer cells was detected by Western blotting and immunohistochemistry. Bioinformatics analysis, RIP (RNA immunoprecipitation), qPCR and Double luciferase gene reporter experiments were used to predict and explore the interaction mechanism between miR-361-5p and LINC00308 as well as TRIP13, and plate cloning and Transwell invasion test were used to verify the biological behaviors of cancer cells. Results: Both the microarray results and qPCR confirmed that the expressions of LINC00308 (P<0.01) and TRIP13 (P<0.05) were abnormally high in prostate cancer tissues and four cell lines; cell function test results showed that overexpression of LINC00308 could promote the proliferation, invasion and migration of prostate cancer PC3 cells (all P<0.05), while down-regulation of LINC00308 in prostate cancer cells had the opposite effect. In nude mice. LINC00308 could promote the tumorigenesis of prostate cancer cells in vivo, and increase the expression of TRIP13 both in vivo and in vitro (P<0.05). Bioinformatics analysis, RIP, qPCR and Double luciferase gene reporter results confirmed that miR-361-5p could bind to 3'-UTR of LINC00308 and TRIP13 respectively, and LINC00308 could act as a competing endogenous RNA (ceRNA) by sponging miR-361-5p to regulate the expression of TRIP13. In addition, MTT, plate cloning and Transwell assay confirmed the regulatory interaction among LINC00308 miR-361-5p and TRIP13 from the levels of proliferation, colony formation and invasion in cancer cells. Conclusion: LINC00308, which is abnormally highly expressed in prostate cancer tissues and cells, can inhibit the expression of miR-361-5p and enhance the expression of TRIP13 by exerting its ceRNA function, thus promoting the proliferation, invasion and migration of prostate cancer.
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OBJECTIVE：To st udy the inhibitory mechanism of pomegranate peel polyphenols （PPP）on the proliferation of human prostate cancer PC 3 cells based on autophagy and apoptosis pathway. METHODS ：CCK-8 assay was used to investigate the effects of PPP with different concentrations （25-300 μg/mL）on PC 3 cell activity after culturing for 24，48，72 h，so as to screen the drug concentration and treatment time. PC 3 cells were divided into control group （complete culture medium ），PPP low- ，medium- and high-concentration groups. After treated for 48 h，flow cytometry and Annexin V-FITC/PI staining were used to detect cell cycle distribution and apoptosis of PC 3 cells. Western blotting assay was used to detect the expression of apoptosis-related protein as Bax ，Bcl-2，as well as the expression of autophagy-related proteins as LC 3，Beclin-1，p62，Atg12 and Atg 16. RESULTS ：The culturing time was chosen as 48 h. IC 50 of PPP was 110 μg/mL，and 50，100，200 μg/mL were chosen as low，medium，high concentrations of PPP. Compared with control group ，the percentage of PC 3 cells at phase G 0/G1 decreased significantly in PPP low- and medium-concentration groups while increased significantly at phase S ；that of PC 3 cells at phase G 0/G1 increased significantly in PPP high-concentration group ； while that of PC 3 cells at phase G 2/M decreased significantly in PPP medium- and high-concentration groups （P＜0.05 or P＜0.01）. The apoptosis rate of PC 3 cells was increased significantly in PPP groups （P＜ 0.05 or P＜0.01）. Compared with control group ，protein expression of anti-apoptosis protein Bcl- 2 and autophagy-related promoting protein p 62 were decreased in PPP groups to different extents ，while protein expression of promoting-apoptosis protein Bax as wells as autophagy-related protein LC 3-Ⅱ/LC3-Ⅰ ration and protein expression of Beclin- 1，Atg5，Atg12 and Atg 16 were increased to different extents ；there was statistical significance in above indexes in PPP high-concentration group and some of above indexes in PPP low- and medium-concentration groups （P＜0.05 or P＜0.01）. CONCLUSIONS ：PPP can inhibit the proliferation of human prostate cancer PC 3 cells，mechanism of which may be related to inducing autophagy and promoting apoptosis.
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@#[Abstract] Objective: To investigate the effects of forkhead box transcription factor (FOXK2) overexpression on the proliferation, migration, invasion and adhesion of human ovarian cancer SK-OV-3 cells and its related molecular mechanism. Methods: The open reading frame (ORF) of FOXK2 was cloned into lentivirus expression vector, which was then enveloped in HEK293T cells and transfected into human ovarian cancerSK-OV-3cells.TheoverexpressionefficiencywasdetectedbyqPCRandWesternblotting.Theproliferation, migration, invasion and adhesion of SK-OV-3 cells were detected by CCK-8, Scratch-healing, Transwell and Cell adhesion assays respectively, and the expressions of epithelial-mesenchymal transition (EMT) markers were detected by qPCR. Results: The FOXK2 overexpression vector was constructed successfully and packaged into lentivirus, which was then transfected into SK-OV-3 cells. After transfection, the expression of FOXK2 was significantly increased (P<0.01); the proliferation, migration and invasion of SK-OV-3 cells were significantly reduced while the adhesion ability was significantly increased (P<0.05 or P<0.01); and the expression levels of E-cadherin and β-catenin were significantly increased while that of vimentin and fibronection were significantly decreased (all P<0.01). Conclusion: Overexpression of FOXK2 in SK-OV-3 cells leads to a significant decrease in proliferation, migration and invasion but increase in adhesion. The molecular mechanism may be related to the reversion of the EMT process in tumor cells, suggesting that FOXK2 may be a potential target for the diagnosis and treatment of ovarian cancer.
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OBJECTIVE：To investigate the effects of metformin on malignant phenotype of pancreatic cancer BxPC- 3 cells. METHODS：Using human pancreatic cancer BxPC- 3 cells with natural deletion of Smad4 gene as reaserch objects ，CCK-8 assay and flow cytometry were used to detect the proliferation and apoptosis of BxPC- 3 cells after treated with different doses of metformin（5，10，20 mmol/L）for 24 h. The cell survival rate and apoptosis rate were calculated. Transwell assay was used to test the migration of cells after treated with different doses of metformin （10，20 mmol/L）for 24 h. The number of migrating cells was recorded. qRT-PCR and Western blotting assay were performed to determine mRNA and protein expression of E-cadherin ，Vimentin and RGC- 32 in cells. RESULTS ：Compared with control group and 5 mmol/L metformin group ，survival rate of cells were decreased significantly in 10，20 mmol/L metformin groups ，while apoptosis rate was increased significantly ；the apoptosis rate in 20 mmol/L metformin group was significantly higher than 10 mmol/L metformin group （P＜0.05）. Compared with control group ， the number of migrating cells was decreased significantly in 10，20 mmol/L metformin groups ，and the 20 mmol/L metformin group was significantly lower than 10 mmol/L metformin group （P＜0.05）. Relative mRNA and protein expression of E-cadherin were increased significantly in 10，20 mmol/L metformin groups ，and relative mRNA expression of E-cadherin in 20 mmol/L metformin group was significantly higher than 10 mmol/L metformin group. Relative mRNA expression of Vimentin in 10 mmol/L metformin group ，relative mRNA and protein expression of Vimentin in 20 mmol/L metformin group ，relative mRNA and protein expression of RGC- 32 in 10，20 mmol/L metformin groups were decreased significantly ；relative mRNA and protein expression of Vimentin as well as mRNA expression of RGC- 32 in 20 mmol/L metformin group were significantly lower than 10 mmol/L metformin group （P＜0.05 or P＜0.01）. CONCLUSIONS ：Metformin can inhibit the proliferation and migration of pancreatic cancer cells through smael-independent pathways in a dose- dependent manner ，and promote their apoptosis ，which is associated with the inhibition epithelial- mesenchymal transition and the expression of RGC- 32 of pancreatic cancer.
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Objective To investigate the permeability of brain microvascular endothelial cells under the condition of high glucose exposure. Methods The bEnd.3 cell line was chosen to detect the value of trans- endothelial electrical resistance (TEER), the activity of alkaline phosphatase (ALP) and γ-glutamyl transferase (γ-GT).Hence, the characteristics of blood-brain barrier in cell model were identified.The permeability of brain microvascular endothelial cells on high glucose exposure was evaluated by cell morphology, cell viability, intracellular lactate dehydrogenase activity and relative expression of ZO-1 and Occludin genes. Results The value of TEER, the activity of ALP and γ-GT increased gradually with increasing incubation time.The observation of cell morphology showed that the number of cells decreased significantly under high glucose exposure, and the adherence was unstable.Cell viability decreased with higher concentration of glucose or longer exposure time under high glucose exposure.The activity of lactate dehydrogenase was also decreased, and there were significant differences among the dose groups (P < 0.05).In addition, the expression levels of tight junction protein ZO-1 and Occludin were further detected.It was found that high glucose exposure inhibited the expression of ZO-1 and Occludin genes in a dose-dependent manner. Conclusion The bEnd.3 cell line has the characteristics of blood-brain barrier.High glucose exposure inhibited the expression of tight junction protein ZO-1 and Occludin. The results might be related to the change of the permeability in brain microvascular endothelial cells
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Objective To investigate the permeability of brain microvascular endothelial cells under the condition of high glucose exposure. Methods The bEnd.3 cell line was chosen to detect the value of trans- endothelial electrical resistance (TEER), the activity of alkaline phosphatase (ALP) and γ-glutamyl transferase (γ-GT).Hence, the characteristics of blood-brain barrier in cell model were identified.The permeability of brain microvascular endothelial cells on high glucose exposure was evaluated by cell morphology, cell viability, intracellular lactate dehydrogenase activity and relative expression of ZO-1 and Occludin genes. Results The value of TEER, the activity of ALP and γ-GT increased gradually with increasing incubation time.The observation of cell morphology showed that the number of cells decreased significantly under high glucose exposure, and the adherence was unstable.Cell viability decreased with higher concentration of glucose or longer exposure time under high glucose exposure.The activity of lactate dehydrogenase was also decreased, and there were significant differences among the dose groups (P < 0.05).In addition, the expression levels of tight junction protein ZO-1 and Occludin were further detected.It was found that high glucose exposure inhibited the expression of ZO-1 and Occludin genes in a dose-dependent manner. Conclusion The bEnd.3 cell line has the characteristics of blood-brain barrier.High glucose exposure inhibited the expression of tight junction protein ZO-1 and Occludin. The results might be related to the change of the permeability in brain microvascular endothelial cells
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Objective To investigate the effect of tamoxifen (TAM) on the invasiveness of breast cancer SK-BR-3 cells and its mechanism.Methods The SK-BR-3 breast cancer cells in logarithmic growth period were cultured for 24 h by theserum-free RPMI 1640 culture medium,and then were divided into control group (serumfree RPMI 1640 medium) and TAM group(serum-freeRPMI 16400.5,2.5,12.5 mol·L-1TAM).Western blot was used to detect matrix metalloproteinase-9 (MMP-9),matrix metalloproteinase-2 (MMP-2),and vascular endothelial growth factor (VEGF) in groups.Scratch and transwelhests were used to detect the invasiveness of SK-BR-3 cells.Results The results of scratch test and Transwell experiment showed that the relative invasive ability of SKM-BR-3 cells in TAM (0.5,2.5,12.5 mol/L) group was significantly higher than that in the control group,and the difference was statistically significant (P＜0.05).The invasive ability of SK-BR-3 cells increased with the increase of TAM con centration.The results of Western blot showed that the expression levels of VEGF,MMP-9 and MMP-2 in SK-BR-3 cells of TAM group were average.It was significantly higher than the control group,and the expression levels of VEGF,MMP-9 and MMP-2 in SK-BR-3 cells increased gradually with the increase of TAM concentration.Conclusion Tamoxifen may play the role of estrogens and promote the invasiveness of SK-BR-3 breast cancer cells,via increasing the expression levels of VEGF,MMP-9 and MMP-2.
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@#Objective: To investigate the effect of human epididymal protein 4 (HE4) and paired box gene 8 (PAX8) gene knockdown on proliferation, migration, invasion and apoptosis of human epithelial ovarian cancer OVCAR3 cells treated with TC regimen (paclitaxel+carboplatin). Methods: Sequences of single-target siRNA (HE4-siRNA or PAX8-siRNA) and double-target siRNA (HE4+PAX8siRNA) as well as negative siRNAwere respectively designed and synthesized, and then linked with plasmid vector pGCsi-H1 to obtain the recombinant plasmids. The obtained recombinant plasmids were then transfected into human epithelial ovarian cancer OVCAR3 cells, namely HE4-siRNA group, PAX8-siRNA group, HE4+PAX8-siRNA group and siRNA-NC group, respectively. The blank control group was also set up (without any treatment). The cells in above five groups were treated with TC regimen (paclitaxel 3.13 g/ml+carboplatin 2.82 µg/ml), and the changes in proliferation, migration, invasion and apoptosis of the cells were detected by MTT, wound-healing assay, Transwell chamber assay, and flow cytometry, respectively. Results: After knocking down the HE4 and PAX8 genes, compared with siRNA-NC group and blank control group, the proliferation, migration and invasion abilities of OVCAR3 cells in HE4-siRNA group, PAX8-siRNA group and HE4+PAX8-siRNA group significantly decreased (all P<0.01), and the apoptosis rate significantly increased (P<0.01), especially in HE4+PAX8-siRNA group. Conclusion: Knockout of either HE4 or PAX8 can enhance the effect of TC regimen on inhibiting proliferation, migration and invasion as well as promoting apoptosis of epithelial ovarian cancer cells, and the effect of simultaneous down-regulation of HE4 together with PAX8 is better.
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@#Objective: To investigate the potential effects of miR-455-3p on proliferation, invasion and epithelial-mesenchymal transition (EMT) process of ovarian cancer cells, and explore its molecular mechanism. Methods: The IOSE80, SKOV-3 and A2780 cells were transfected with miR-455-3p mimics and negative controls (NC) by using LipofectamineTM 2000. Quantitative polymerase chain reaction (qPCR) assay was performed to detect the mRNA expressions of miR-455-3p and fatty acid-binding protein 4 (FABP4) in IOSE80, SKOV-3 and A2780 cells. The expression levels of FABP4 and EMT-associated proteins were detected by Wb. CCK-8 assay was applied to measure cell proliferation. Cell migration was analyzed by using Transwell assay. Bioinformatics analysis was used to predict the potential target of miR-455-3p, and the targeting effect of miR-455-3p on FABP4 was verified by the dual-luciferase reporter assay system. Results: The expression of miR-455-3p was declined (all P<0.05), while the expression of FABP4 was elevated (all P< 0.05) in ovarian cancer cells (SKOV-3 and A2780) in comparison with normal ovarian IOSE80 cells. Additionally, over-expression of miR-455-3p obviously inhibited cell proliferation and migration capacity of SKOV-3 cells (all P<0.05). Furthermore, over-expression of miR-455-3p impeded EMT progress by up-regulating E-cadherin expression and down-regulating N-cadherin and vimentin expression (all P<0.05). Importantly, the dual-luciferase reporter system, qPCR and Wb validated that FABP4 was a specific target gene of miR-455-3p, and miR-455-3p showed specific binding with FABP4 3’-UTR and negatively regulated the expression of FABP4 at both mRNA and protein levels. Mechanistically, over-expression of FABP4 apparently reversed the inhibitory effects of miR-455-3p on cell proliferation and migration of SKOV-3 cells (all P<0.05). Conclusion: miR-455-3p, acting as a tumor suppressor protein, can inhibit ovarian cancer cell proliferation, migration and EMT process by targeting FABP4, suggesting that miR-455-3p may be a new potential therapeutic target for ovarian cancer treatment.