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【Objective】 To investigate the expression profile of circRNA in nuclear receptor NURR1 overexpressed prostate cancer (PCa) cells, so as to provide reference for revealing the mechanism of PCa progression. 【Methods】 The expression of NURR1 in PCa was analyzed with UALCAN and TNMplot. The distinct circRNAs in NURR1 overexpressed PCa cells were screened with RNA-sequencing. The functions and signal pathways of differentially expressed circRNA molecules were analyzed with GO and KEGG. 【Results】 The circ_0000915 was significantly downregulated in DU145, LNCaP and PC3 cells. In NURR1 overexpressed DU145 cells, circ_0005991 was up-regulated, while circ_0001460 and circ_0001315 were down-regulated. In NURR1 overexpressed LNCaP cells, circ_0040729 and circ_0000722 were significantly up-regulated. In NURR1 overexpressed PC3 cells, circ_0001577, circ_0000854 and circ_0018168 were up-regulated, while circ_013035, circ_0003028, circ_0082096 and circ_0005320 were down-regulated. KEGG analysis revealed that the differentially expressed circRNAs were significantly associated with dorsal/ventral neural tube patterns, protein folding chaperones, disordered domain specific binding, positive regulation of BMP signaling pathways, and neural tube patterning functions. 【Conclusion】 CircRNAs play an important role in NURR1 mediated PCa progression, but there are certain differences among different prostate cancer cell types. The regulatory mechanism between NURR1 and circ_0000915 in the progression of PCa needs further investigation.
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Aim To prepare prostate cancer exosomes containing melittin and observe their uptake by prostate cancer cells. Methods Cells treated with starvation for different time were screened for exosome extraction. Exosomes from PC-3 cells were extracted by ultracentrifugation, and the extracted particles were examined by transmission electron microscopy, nanoparticle tracking analyzer(NTA), and Western blot. Melittin exosome system was prepared by repeated freeze-thaw method, incubation at room temperature as well as electroporation, and the size of encapsulation efficiency was measured by centrifugation. A high-performance liquid chromatography(HPLC)method was applied to assay the content of melittin exosomes(exo-mel). Fluorescence inverted microscopy was employed to evaluate the uptake of melittin exosomes by PC-3 cells, DU145 cells as well as LNCaP cells. Results The results of starvation treatment showed that 24 h starvation treatment was the optimal time point. TEM results showed that the exosomes were round or oval in shape with a distinct membranous structure, and the diameter was around 100 nm. The reagent protein concentration for NTA analysis of exosomes was 0.222 g·L-1. The results of Western blot for the marker proteins of exosomes showed that Alix and CD63 were positively expressed, which indicated that the exosomes could be obtained by starvation culture of PC-3 cells and ultracentrifugation. The results of entrapment efficiency showed that the entrapment efficiency of electroporation method was 17.51% ± 2.39%, that of repeated freeze-thaw method was 11.46% ± 1.02%, and that of room temperature incubation method was 3.93% ± 2.44%. The encapsulation efficiency of electroporation was the highest with significant difference(P<0.05). The uptake assay showed that PC-3 cells could efficiently take up exo-mel in a time-dependent manner, and DU145 cells and LNCaP cells also could take up exo-mel over time. Conclusions Exosomes can be accessed by starvation treatment and high-speed centrifugation, and the prostate cancer melittin exosome system prepared by electroporation method could be taken up by prostate cancer cells.
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Objective: To determine whether the adenine base editor (ABE7.10) can be used to fix harmful mutations in the human G6PC3 gene. Methods: To investigate the safety of base-edited embryos, off-target analysis by deep sequencing was used to examine the feasibility and editing efficiency of various sgRNA expression vectors. The human HEK293T mutation models and human embryos were also used to test the feasibility and editing efficiency of correction. Results: ①The G6PC3(C295T) mutant cell model was successfully created. ②In the G6PC3(C295T) mutant cell model, three distinct Re-sgRNAs were created and corrected, with base correction efficiency ranging from 8.79% to 19.56% . ③ ABE7.10 could successfully fix mutant bases in the human pathogenic embryo test; however, base editing events had also happened in other locations. ④ With the exception of one noncoding site, which had a high safety rate, deep sequencing analysis revealed that the detection of 32 probable off-target sites was <0.5% . Conclusion: This study proposes a new base correction strategy based on human pathogenic embryos; however, it also produces a certain nontarget site editing, which needs to be further analyzed on the PAM site or editor window.
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Humans , Gene Editing , CRISPR-Cas Systems , Adenine , HEK293 Cells , Mutation , Glucose-6-Phosphatase/metabolismABSTRACT
ABSTRACT Background: Severe congenital neutropenia type 4 (SCN4) is a rare autosomal recessive granulopoiesis disorder caused by G6PC3 gene pathogenic variants. The estimated prevalence is 1/10,000,000 people. Over 90% of patients present a syndromic form with variable multisystemic involvement, including congenital heart defects, increased visibility of superficial veins (IVSV), inflammatory bowel disease, and congenital urogenital defects as prominent symptoms. Objectives: The objective of the study was to study non-hematological phenotypic findings that suggest a clinical diagnosis of SCN4. Methods: We examined medical records of patients diagnosed with neutropenia from January 2000 to December 2020, selecting cases with non-hematologic manifestations for phenotypic description and G6PC3 gene sequencing. Results: We found 11 cases with non-hematologic features: congenital heart defects in 8, IVSV in 6, inflammatory bowel disease in 4, urogenital defects in 4, and similar facial appearance. In addition, Sanger sequencing confirmed 3 homozygous cases for the c.210delC variant, a compound heterozygous harboring this variant, and a c.199_218+1 deletion. Conclusions: Our findings of the c.210delC variant in very close geographical settings, to date, have only been reported among Mexicans, and a mutual uncommon surname in two families strongly supports a founder effect for the variant in the studied population. Furthermore, the described non-hematologic symptoms in patients with severe primary neutropenia should be explored, confirming SCN4 by investigating G6PC3 gene mutations.
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@#目的:通过 CRISPR/Cas9 技术构建前列腺癌 PC3 细胞 TFDP3 基因敲除的稳转株,探讨抑制 TFDP3 表达对 PC3 细 胞周期、凋亡、迁移和侵袭能力的影响。方法:通过生物信息学筛选 sgRNA,通过 CRISPR/Cas9 技术、构建抑制 TFDP3 基因表达 的 sgRNA-Cas9 共转染慢病毒,感染 PC3 细胞后筛选获取稳转细胞株。通过流式细胞术对 TFDP3 基因敲除的实验组与空白对照 组进行细胞周期和凋亡检测,并进一步通过划痕实验和 Transwell 实验进行细胞迁移和侵袭能力检测。结果:通过生物信息学 筛选获得 3 条 sgRNA,其中 sgRNA2 有明显的抑制前列腺癌细胞基因表达的功能;通过 CRISPR/Cas9 技术成功构建了基于 CRISPR/Cas9 介导的 TFDP3 低表达的 PC3 细胞稳转株。抑制 TFDP3 基因表达后,相比于对照组,KO 组中 G0/G1 期细胞 百分比增加、G2/M 期细胞百分比下降(P<0.05 或 P<0.01),细胞凋亡率显著升高(P<0.05),细胞迁移率明显下降 [24 h 迁移率: (44.00±1.60)% vs (65.00±4.40)%,P<0.01],穿过聚碳酸酯膜的侵袭细胞数明显下降 [(185.89±11.71)vs (248.33±11.95)个, P<0.01]。结论:通过 CRISPR/Cas9 技术抑制 TFDP3 基因表达后,PC3 细胞发生周期阻滞、凋亡率也有所增加、迁移和侵袭能力 显著减弱,提示 TFDP3 是一个前列腺癌促癌基因。
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OBJECTIVE:To study the effe cts of the water extract from Carpesium cernuum (AECC)on the proliferation , metastasis and invasion of prostate cancer PC 3 cells. METHODS :Cells were divided into control group and different concentration groups of AECC (5,10,20,40,80 μg/L),and then treated with relevant medicine or medium for different time (24,48,72 h). The survival rates of cells were detected. Cells were divided into control group ,and AECC low ,medium and high concentration groups(20,40,80 μg/L). After cultured for 24 h,Hoechst 33258 staining and flow cytometry were used to detect the apoptosis of cells. The number of cell metastasis and invasion were detected by Transwell assay. RT-qPCR and Western blot assay were applied to detect the mRNA and protein expression of β-catenin signaling pathway related migration and apoptosis proteins (β-catenin, MMP-7,c-Myc,caspase-3,Bcl-2 and Bax )in AECC low and medium concentration groups. RESULTS :With the increase of the concentration and culture time ,the survival rates of cells in AECC different concentration groups were significantly lower than control group (P<0.05 or P<0.01),and showed a decreasing trend. Compared with control group ,the early apoptosis rate (except the medium concentration group )and the number of cell metastasis and invasion in AECC groups ,the mRNA and protein expression of MMP- 7,c-Myc(except for the low concentration group )and Bcl- 2(except for mRNA of the low concentration group)in AECC low and medium concentration groups were decreased significantly (P<0.05 or P<0.01). Late apoptosis rate of AECC groups ,the mRNA and protein expression of β-catenin,caspases-3(except for the low concentration group ),Bax(except for mRNA of the low concentration group )in AECC low and medium concentration groups were increased significantly (P<0.05 or P<0.01). CONCLUSIONS :AECC could inhibit the proliferation ,metastasis and invasion of PC 3 cells;the mechanism of which may be associated with regulating the expression of β-catenin signaling pathway related migration and apoptotic factors.
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@#[摘 要] 目的:探讨lncRNA FLJ26245在前列腺癌组织和细胞中的表达及其对PC-3细胞增殖与迁移能力的影响及其分子机制。方法:选用2017年3月至2019年5月在洛阳中心医院手术切除的52例前列腺癌及相应的癌旁组织标本,以及前列腺细胞系LNCaP、C4-2B、PC-3、DU-145和正常前列腺上皮细胞RWPE-1,用qPCR法检测前列腺癌组织和细胞中FLJ26245的表达水平。分别将FLJ26245 mimic和阴性对照质粒(lncR-NC)转染到PC-3细胞中,用MTT法、细胞划痕愈合实验分别检测细胞的增殖和迁移能力。生物信息学技术预测和双荧光素酶基因报告实验验证FLJ26245与miR-200a-3p、酪氨酸磷酸酶受体G(PTPRG)三者之间的靶向关系。用qPCR和WB法分别检测FLJ26245下游基因及增殖与迁移相关蛋白的表达。结果:FLJ26245在前列腺癌组织和细胞中的表达水平分别显著低于癌旁组织(P<0.01)和RWPE-1细胞(P<0.01或P<0.05),以PC-3细胞中的表达水平为最低(P<0.01)。FLJ26245过表达可明显抑制PC-3细胞的增殖和迁移能力(P<0.05或P<0.01)。FLJ26245可与miR-200a-3p靶向结合,miR-200a-3p可与PTPRG靶向结合(均P<0.01)。FLJ26245过表达的PC-3细胞中miR-200a-3p表达水平显著降低(P<0.01)、PTPRG mRNA和蛋白表达水平均明显升高(均P<0.01),细胞中增殖和迁移相关蛋白cyclin A、CDK2和Twist、N-cadherin均显著降低(均P<0.01)。结论:FLJ26245在前列腺癌组织及细胞中均低表达,其可能通过miR-200a-3p/PTPRG轴调控前列腺癌PC-3细胞的增殖与迁移能力。
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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 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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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 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 investigate the inhibitory effect of Coicis Semen oil on human prostate cancer PC-3 cell. Methods A Nude mice xenograft model was established with human prostate cancer PC-3 cells. Meanwhile, intragastric administration with Coicis Semen oil 2 mL/kg and 6 mL/kg was run for once daily and growth-curve was drawn through measuring the tumor volume until 26 d. At the end of the experiment, tumor weights were measured and the tumor inhibition rate of each group was calculated. The activity of fatty acid synthase (FAS) was determined through UV-VIS spectrophotometry. PC-3 cells were cultured in vitro. SYBR Green I real-time quantitative RT-PCR was used to determine the relative expression of FAS mRNA. Results The tumor inhibition rate of Coicis Semen oil 6 mL/kg group was 43.9%. Compared with model group, FAS activity in this tumor tissue decreased by 44.7% (P < 0.05). The expression of FAS mRNA was significantly decreased at 20 μL/mL of Coicis Semen oil group (P < 0.05). Conclusion Coicis Semen oil on human prostate cancer PC-3 cells has a significant inhibitory effect. FAS activity and mRNA expression decline may be related to its main anti-cancer mechanism.
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Objective To study the effects of S-adenosyl-homocysteine hydrolase inhibitor DZNep (3-Dea zaneplanocin A) on human prostatic cancer PC3 cells growth and further explore its potential value in anti-human prostatic cancer treatment.Methods MTT method was used to analyze the effects of EZH2 gene silencing,EZH2 over-expression and DZNep treatment on cell proliferation.Gene expression of EZH2,Bax and Bcl-2 in PC3 cells was detected with western blot.Results DZNep could significantly inhibit PC3 cell growth and induce apoptosis which was identified with Bax expression up-regulation and Bcl-2 expression down-regulation.EZH2 knock-down inhibited PC3 cells growth,and over-expression of EZH2 partially counteract the inhibitory effects of DZNep on PC3 cells growth.Conclusions DZNep can inhibit PC3 cells growth by targeting EZH2 inhibition which leads to endogenous apoptosis.DZNep has the potential value in the treatment of human prostatic cancer.
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Objective To investigate the effect of lentivirus carrying shRNA-VDR vector on GLi1 in pros-tate cancer PC-3 cells. Methods The cells were cultured according to the culture conditions of PC-3 cells. Expression of VDR and GLi1 in PC-3 cells was detected by fluorescence quantitative PCR and immunocytochemistry SP method.The efficiency of PC-3 cell virus infection was evaluated.The effect of VDR gene interference and GLi1 transcription level on PC-3 cells was detected by RT-PCR.Results Cell culture,cell status was recorded and PC-3 cells were in good condition and the passages was 4 days. Fluorescence quantitative and immunocytochemi-cal SP showed that VDR and GLi1 were expressed in PC-3 cells.The virus infection efficiency showed that the in-fection efficiency was about 95% when adding LV3-NC lentivirus to PC-3 cells at 1:10 ratio. RT-PCR showed that VDR-shRNA lentivirus successfully disturbed VDR expression and decreased by 85%(P < 0.05)compared with the control group after 72 days of VDR-shRNA lentivirus transfection. Transcription level of GLi1 gene in the experimental group increased by 9% compared with the control group(P < 0.05). The transcription level of GLi1 gene in the experimental group increased by 248% compared with the control group(P < 0.05). Conclusion The cultured PC-3 cells were in good condition. VDR and GLi1 genes were expressed in PC-3 cells. Lentivirus showed highest efficiency in infecting PC-3 at 1:10 ratio. When VDR was disturbed,the expression of GLi1 in-creased.In prostate cancer cells,vitamin D can inhibit the Hh signaling pathway and cause GLi1 expression down expression.
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Objective@#To study the expression of long noncoding RNA (lncRNA) H19 in human prostate cancer tissue and its effect on the glycometabolism and growth of human prostate cancer cells.@*METHODS@#Realtime quantitative RTPCR (qRTPCR) was employed to detect the expression of lncRNA H19 in human prostate tissues from 20 patients with prostate cancer (10 cases of highGleason score prostate cancer [HGPC] and 10 cases of lowGleason score prostate cancer [LGPC]) and another 5 with benign prostatic hyperplasia (BPH). After transfection of H19 siRNA into the DU145 and PC3 prostate cancer cells, the growth of the cells and the H19 expression in the cells were determined by MTT and qRTPCR respectively, and the changes in the glycometabolism of the prostate cancer cells were analyzed by measuring the contents of glucose and lactate in the culture medium. Nontransfected and transfected negative vectors were used as blank and negative controls respectively.@*RESULTS@#The relative expression of H19 was significantly increased in both the HGPC and LGPC tissues (0.725±0.385 and 2.086±0.542) as compared with that in the BPH tissue (0.210±0.068) (P< 0.01), even higher in the HGPC than in the LGPC tissue (P< 0.01). After transfection of H19 siRNA, the expressions of H19 were remarkably decreased in the DU145 and PC3 prostate cancer cells in comparison with those in the blank control and negative control groups (P< 0.01), and so were the proliferation of and the glucose and lactate levels in the DU145 and PC3 cells (P< 0.01).
Subject(s)
Humans , Male , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Glucose , Metabolism , Lactic Acid , Metabolism , Prostate , Metabolism , Prostatic Hyperplasia , Metabolism , Prostatic Neoplasms , Metabolism , Pathology , RNA, Long Noncoding , Genetics , Metabolism , RNA, Small Interfering , TransfectionABSTRACT
Objective@#To investigate the anti-prostate cancer (PCa) effect of roemerine in vitro and in vivo in the mouse model of PCa.@*METHODS@#We detected the effects of roemerine on the proliferation, apoptosis and migration of PCa cells DU145, LNCaP, PC-3 and 22RV1, screened out the sensitive cell line and constructed a tumor-bearing model in mice for verification of the antitumor efficacy of roemerine in vivo.@*RESULTS@#Roemerine inhibited the proliferation and migration of the DU145, LNCaP, PC-3 and 22RV1 cells and induced their apoptosis in different degrees, particularly those of the LNCaP cells. The average tumor weight was less in the roemerine intervention group ([1.99±0.95] g) than in the control ([2.95±1.04] g), the least in the high-dose roemerine (30 mg/kg) plus paclitaxel intervention group ([0.90±0.16] g). The mean heart, liver, and kidney indexes were markedly lower in the roemerine (0.58±0.06, 6.20±0.42 and 1.49±0.33) than in the paclitaxel group (0.66±0.04, 6.99±0.72 and 1.95±0.34), while the mean spleen and thymus indexes were remarkably higher in the former (0.54±0.11 and 0.06±0.01) than in the latter (0.41±0.09 and 0.05±0.01). Pathological staining showed a lower degree of malignancy and metastasis in both the roemerine and the roemerine + paclitaxel intervention group than in the control, as well as a lower degree of visceral injury in the roemerine and roemerine + paclitaxel groups than in the paclitaxel group.@*CONCLUSIONS@#Roemerine has some anti-PCa effect and alleviates adverse reactions in paclitaxel combination administration.
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Animals , Male , Mice , Alkaloids , Therapeutic Uses , Antineoplastic Agents, Phytogenic , Therapeutic Uses , Apoptosis , Cell Line, Tumor , Cell Movement , Cell Proliferation , Disease Models, Animal , Drug Therapy, Combination , Methods , Drugs, Chinese Herbal , Therapeutic Uses , Mice, Nude , Paclitaxel , Therapeutic Uses , Prostatic Neoplasms , Drug TherapyABSTRACT
Objective@#To investigate the effect of small interfering RNA silencing the vitamin D receptor (VDR) on the biological behavior of prostate cancer PC-3 cells.@*METHODS@#We constructed the VDR-shRNA lentiviral vector and determined the mRNA and protein expressions of VDR by RT-PCR and Western blot. Using scratch wound healing and Transwell chamber assays, we detected the changes in the migration and invasiveness of the PC-3 cells after silencing VDR.@*RESULTS@#The VDR-shRNA plasmid significantly interfered the VDR expression and successfully screened the cell lines with stable VDR-shRNA interference. The rate of scratch wound healing was markedly lower in the VDR interference group than in the blank control and LV3 negative control groups (59% vs 73.6% and 77.8%, P 0.05), and so was the count of permeable cells (P 0.05). The migration ability and invasiveness of the VDR-treated cells were remarkably decreased as compared with those of the control cells.@*CONCLUSIONS@#Down-regulated expression of the VDR gene may reduce the migration and invasiveness of prostate cancer cells.
Subject(s)
Humans , Male , Cell Line, Tumor , Cell Movement , Genetics , Cell Proliferation , Down-Regulation , Gene Silencing , Lentivirus , Neoplasm Invasiveness , Genetics , Plasmids , Prostatic Neoplasms , Genetics , Pathology , RNA, Messenger , Metabolism , RNA, Small Interfering , Receptors, Calcitriol , Genetics , Metabolism , Transfection , Wound Healing , GeneticsABSTRACT
AIM:To explore the mitochondrial pathway in the apoptosis of PC3 cells induced by PXD101 (also named as belinostat).METHODS:PC3 cells were treated with PXD101 at different doses or stimulated with PXD101 for different time.The effect of PXD101 on the viability of PC3 cells was measured by CCK-8 assay.The apoptotic rates and the mitochondrial membrane potential (MMP) were analyzed by flow cytometry.The protein levels of Bcl-2, Bax and cytochrome C (Cyt C) were determined by Western blot.The caspase-3 activity were tested by caspase-3 activity assay kit.RESULTS:The viability of the PC3 cells was inhibited by PXD101 in a dose-and time-dependent manner.Flow cytometric analysis showed that the apoptotic rates were increased in the cells treated with PXD101 (P<0.01).At the same time, PXD101 induced the decreases in MMP and Bcl-2, the release of Cyt C, and the increase in caspase-3 activity.CONCLUSION:PXD101 induces the apoptosis of human prostate cancer cell line PC3 by mitochondrial signal pathway.
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Objective To observe the effect of β-asarone on the proliferation, cycle, apoptosis and migration of tumor cells A549, PC3, and PC9-R, thus to provide experimental basis for the application of β-asarone to the treatment of cancer. Methods After A549, PC3, and PC9-R were cultured with different concentrations ofβ-asarone for 24 h, 48 h, and 72 h respectively, CCK-8 was used to detect the optical density (D) of cell proliferation, and then the cell proliferation rate was calculated. The flow cytometry was used to measure the cell DNA cycle and cell apoptosis, and Transwell Chambers were used to detect the cell migration. Results After treatment with different concentrations of β-asarone for 24 h, 48 h, and 72 h respectively, the growth of A549, PC3, and PC9-R showed declining trend in concentration- and time-dependent manner. The proportion of A549, PC3, and PC9-R at G0/G1 phase was increased, the proportion of the three kinds of cells at S phase and the proliferation indexes were declined, the apoptotic rate of A549, PC3, and PC9-R was increased, and the migration of A549, PC3, and PC9-R was reduced (P<0.05 or P<0.01 compared with those of the normal control group). Conclusion β-asarone has certain effects on suppressing proliferation, blocking G0/G1 phase developing into S phrase, inhibiting DNA synthesis, promoting the apoptosis, and inhibiting the migration of A549, PC3 and PC9-R.
ABSTRACT
<p><b>Objective</b>To investigate the potential role of the RhoA/Rock signaling pathway in the formation of prostate cancer and the effects of the Rock inhibitor fasudil on the invasion, migration and apoptosis of human prostate cancer cells.</p><p><b>METHODS</b>Human prostate cancer cell lines PC3 and DU145 were treated with fasudil at the concentrations of 5, 10, 20, 40, 80, and 160 μmol/L, respectively, and those as negative controls cultured in the Ham's-F12 medium, all for 24 hours. Then, MTT assay was used to measure the cell inhibition rate and half maximal inhibitory concentration (IC50) value of fasudil, with 1/4 of IC50 as the medication dose for further investigation. The expressions of RhoA, RockⅠ, and RockⅡ proteins in the PC3 and DU145 cells were detected by Western blot and immunohistochemistry, and the invasion, migration and apoptosis of the cells were determined using the Transwell chamber, scratch wound healing assay and flow cytometry.</p><p><b>RESULTS</b>Fasudil inhibited the proliferation of the PC3 cells from (9.29±1.23)% at 5 μmol/L to (81.37±3.97)% at 160 μmol/L and that of DU145 from (7.59±1.54)% to (76.53±2.67)%, both in a dose-dependent manner (P<0.05 ). Significantly fewer PC3 and DU145 cells migrated into the lower compartment in the experimental group (39.2±8.4 and 34.2±6.7) than in the negative control (116.8±9.3 and 112.5±10.8) (P<0.05 ). The wound healing rates of the PC3 and DU145 cells were remarkably lower in the former ([37.26±1.17]% and [32.38±2.73]%) than in the latter ([78.12±4.16]% and [69.47±6.71]%) (P<0.05 ). Annexin V-FITC/PI double staining showed markedly increased apoptosis rates of PC3 and DU145 cells treated with fasudil ([31.88±2.49]% and [28.65±2.99]%) as compared with the negative controls ([7.51±2.28]% and [7.13±1.61]%) (P<0.05 ). The expressions of RockⅠ and RockⅡ were significantly reduced in the fasudil-treated cells in comparison with those of the control group (P<0.05 ) while that of RhoA showed no significant difference between the two groups (P>0.05 ).</p><p><b>CONCLUSIONS</b>The RhoA/Rock signaling pathway may play an important role in the formation of prostate cancer. Fasudil can significantly inhibit the proliferation, migration, and invasion and promote the apoptosis of human prostate cancer PC3 and DU145 cells by reducing RhoA/Rho kinase activity.</p>