ABSTRACT
This study aimed to examine the effect of long non-coding RNA (LncRNA) MEG3 on the biological behaviors of renal cell carcinoma (RCC) cells 786-0 and the possible mechanism. MEG3 expression levels were detected by RT-qPCR in tumor tissues and adjacent non-tumor tissues from 29 RCC patients and in RCC lines 786-0 and SN12 and human embryonic kidney cell line 293T. Plasmids GV144-MEG3 (MEG3 overexpression plasmid) and GV144 (control plasmid) were stably transfected into 786-0 cells by using lipofectamine 2000. Cell viabilities were determined by MTT, cell apoptosis rates by flow cytometry following PE Annexin V and 7AAD staining, apoptosis-related protein expressions by Western blotting, and Bcl-2 mRNA by RT-qPCR in the transfected cells. The results showed that MEG3 was evidently downregulated in RCC tissues (P<0.05) and RCC cell lines (P<0.05). The viabilities of 786-0 cells were decreased significantly after transfection with GV144-MEG3 for over 24 h (P<0.05). Consistently, the apoptosis rate was significantly increased in 786-0 cells transfected with GV144-MEG3 for 48 h (P<0.05). Furthermore, overexpression of MEG3 could reduce the expression of Bcl-2 and procaspase-9 proteins, enhance the expression of cleaved caspase-9 protein, and promote the release of cytochrome c protein to cytoplasm (P<0.05). Additionally, Bcl-2 mRNA level was declined by MEG3 overexpression (P<0.05). It was concluded that MEG3 induces the apoptosis of RCC cells possibly by activating the mitochondrial pathway.
Subject(s)
Humans , Apoptosis , Carcinoma, Renal Cell , Genetics , Metabolism , Pathology , Cell Line, Tumor , Cell Survival , Gene Expression Regulation, Neoplastic , HEK293 Cells , Kidney Neoplasms , Genetics , Metabolism , Pathology , Mitochondria , Genetics , RNA, Long Noncoding , Genetics , Metabolism , Signal TransductionABSTRACT
<p><b>OBJECTIVE</b>To study the effect of silencing pyruvate kinase M2 (PKM2) on gambogic acid (GA)-induced apoptosis of human prostate cancer PC3 cells.</p><p><b>METHODS</b>Three specific PKM2 siRNAs and one negative control siRNA (si-NC) were transfected into PC3 cells. The silencing effect of PKM2 siRNAs was determined by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, and the effects of PKM2 siRNA on the vitality and apoptosis of GA-stimulated PC3 cells detected by MTT and AO/EB double staining, respectively. The mRNA and protein levels of c-myc and cyclin D1 were analyzed by qRT-PCR and Western blot, respectively.</p><p><b>RESULTS</b>All the 3 PKM2 siRNAs effectively reduced the mRNA and protein expressions of PKM2, and PKM2 siRNA-1 exhibited the strongest silencing effect. At 24 h after transfection, the expression levels of PKM2 mRNA and protein were reduced by 70% and 85%, respectively (P < 0.05). Twenty-four hours of treatment with GA (0.5 micromol/L) following transfection with PKM2 siRNA-1 inhibited the vitality of the PC3 cells by 68%, increased their apoptosis, and significantly down-regulated the mRNA and protein levels of c-myc (50% and 35%) and cyclin D1 (60% and 20%) (P < 0.05).</p><p><b>CONCLUSION</b>Inhibition of PKM2 sensitized PC3 cells to GA-induced apoptosis, suggesting that PKM2 may be a potential therapeutic target for sensitizing human prostate cancer to GA.</p>
Subject(s)
Humans , Male , Apoptosis , Carrier Proteins , Genetics , Metabolism , Cell Line, Tumor , Membrane Proteins , Genetics , Metabolism , Prostatic Neoplasms , Genetics , Metabolism , Pathology , RNA Interference , RNA, Small Interfering , Thyroid Hormones , Genetics , Metabolism , Xanthones , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To clone the mouse testis specific gene TSEG-2 via a bioinformatic approach.</p><p><b>METHODS</b>The expressed sequence tags (EST) in the normal mouse testis were obtained from the online EST database ZooDDD. Their highly homologous EST sequences were retrieved through the dbEST database to construct contigs and spliced with the biomedical software Biolign. The corresponding exons and introns within the genome sequences were predicted with the software GeneScan. Primers were designed according to the open reading frame. RT-PCR was applied in cloning the cDNA of the novel gene from the mouse testis tissue and analyzing its expression patterns in the undescended testis and various organ tissues as well as in different developmental stages of the mouse testis. The sequencing results of TSEG-2 underwent bioinformatic analyses.</p><p><b>RESULTS</b>The novel mouse testis gene TSEG-2 was successfully cloned, with full-length sequence of 451 bp. The open reading frame was 267 bp, coding a protein of 88 amino acid residues, and demonstrated to be correct by RT-PCR. The expression of TSEG-2 was high in the mouse testis, regular in the testis cDNA samples of different postnatal days, and down-regulated in the cryptorchidism model. No obvious homology with other mouse cDNA was found for TSEG-2. The GenBank accession number EU079025 was achieved. Function prediction showed that mouse TSEG-2 was probably a soluble non-secretary protein located at chromosome 15qE3, or a nucleoprotein with 2 phosphorylation sites of protein kinase C (PKC) and 1 of casein kinase II (CK2).</p><p><b>CONCLUSION</b>A novel mouse testis specific gene TSEG-2 was successfully cloned, which could be down-regulated by cryptorchidism-inducible 17-beta estradiol. This has prepared the ground for further researches on the biological function and expression regulation of TSEG-2.</p>
Subject(s)
Animals , Female , Male , Mice , Pregnancy , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Expressed Sequence Tags , Gene Expression , Mice, Inbred Strains , Molecular Sequence Data , Open Reading Frames , Proteins , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Testis , MetabolismABSTRACT
This study is to explore the inhibitory effect of methyl jasmonate on cell proliferation and expression of XIAP and survivin of human neuroblastoma cell line BE(2)-C. After cultivation of 1 - 2 mmol x L(-1) jasmonates with BE (2) -C cells for 6 - 24 h, the growth inhibiting rates of BE (2) -C cells were studied by MTT colorimetry. Cell proliferation was detected by colony formation assay. Cell cycle phases were assayed by propidium iodide staining flow cytometery. Cell apoptosis was inspected by acridine orange-ethidium bromide fluorescent staining, Hoechst 33258 fluorescent staining, and Annexin V-FITC and propidium iodide staining flow cytometry. Expressions of cyclin D1, XIAP and survivin were determined by RT-PCR and real-time RT-PCR. Methyl jasmonate inhibited the growth of BE(2)-C cells in a dose- and time-dependent manner. After addition of 1, 1.5 and 2 mmol x L(-1) of methyl jasmonate for 24 h, the inhibiting rates of cell growth reached 20.6% - 85.5% (P < 0.01), and the IC50 was 1.35 mmol x L(-1). The cell cycles were arrested at S phase. A part of cells presented the characteristic morphological changes of apoptosis. The early apoptotic rates were 13.51%, 17.32%, 24.59% (P < 0.01) and the cell death rates were 29.36% , 54.73% , 75.52% (P < 0.01), respectively. The expression of XIAP and survivin mRNA were downregulated by 18.5% - 68.9% , 22.4% - 48.7% (P < 0.05), respectively, without change in that of cyclin D1. The results indicated that methyl jasmonate could significantly inhibit the growth of BE(2) -C cells through inducing cell cycle arrest and apoptosis, downregulating the expression of XIAP and survivin might be one of its molecular mechanisms of action.
Subject(s)
Humans , Acetates , Pharmacology , Antineoplastic Agents, Phytogenic , Pharmacology , Apoptosis , Cell Line, Tumor , Cell Proliferation , Cyclin D1 , Genetics , Cyclopentanes , Pharmacology , Dose-Response Relationship, Drug , Down-Regulation , Inhibitor of Apoptosis Proteins , Microtubule-Associated Proteins , Genetics , Neuroblastoma , Metabolism , Pathology , Oxylipins , Pharmacology , RNA, Messenger , Metabolism , S Phase , X-Linked Inhibitor of Apoptosis Protein , GeneticsABSTRACT
Background and purpose:Smac/DIABLO was the only apoptosis-related protein that could inhibit IAPs directly and simultaneously.The four amino-residual AVPI(Ala-Val-Pro-lie)in its N-terminal was the very important domain that could stimulate apoptosis.This study investigated the effect of synthetic Smac peptide (SmacN7) on chemotherapy sensitivity of bladder cancer cells.Methods:SmacN7 penetratin peptide was synthesized and delivered into T24 cells.MTT assay was adopted to evaluate the viability of T24 cells induced by low-dosage of MMC. Flow cytometry was applied to analyze the proportion of apoptosis and Western blot was used to detect the expression of XIAP and caspase-3;The activity of caspase-3 was measured and the effect of SmacN7 combined with MMC on T24 cell lines was also determined.Results:SmacN7 penetratin peptide could successfully interact with endogenous XIAP and increase the proportions of apoptosis of T24 cell lines induced by low-dosage of MMC in a dose-and time- dependent manner.An obvious down-regulation of XIAP expression and up-regulation of caspase-3 was identified by Western blot.The activity of caspase-3 in experimental group was significantly increased as compared with that in the control group;Combining the treatment with SmacN7 penetratin peptide,the viability of T24 cells decreased to 55% and 72.7% in 24 hrs and 48 hrs respectively.Conclusion:SmacN7 penetratin peptide could act as a cell-permeable IAP inhibitor,inhibit the proliferation,induce apoptosis and enhance the chemo-sensitivity of bladder cancer cells to MMC. When combined with chemotherapy,it may be a very promising strategy for bladder cancer therapy.