Dysfunction of proteasome inhibits the proliferation of hBMSCs via activation of P53

AIM: To investigate the role of P53 in decreased cell proliferation and proteasomal activity during culture expansion of human bone marrow mesenchymal stem cells (hBMSCs). METHODS: The proteasomal activity and expression level of P53 in early-passage (passage 3~4) and late-passage (passage≥14) hBMSCs were observed. Early-passage hBMSCs were divided into DMSO control group, MG132 group (treated with 10 μmol/L MG132 for 4 h) and pifithrin-α(PET-α) + MG132 group (pretreatment with 20 μmol/L PFT-α for 1 h then exposure to MG132 for 4 h). The proliferation and cell cycle distribution of the cells were measured by BrdU incorporation assay and PI staining. To further confirm the effect of P53 inhibitor on late-passage hBMSCs, the cells were incubated with 20 μmol/L PFT-α and the BrdU-positive cells were counted. RESULTS: Accompanied by reduced proteasomal activity, the expression level of P53 in late-passage hBMSCs was up-regulated by 16.89%±4.44% compared with early-passage cells. Application of the proteasome inhibitor MG132 reduced the proliferation of early-passage hBMSCs, and the percentage of BrdU-positive cells dropped to 33.36%±2.24%. However, MG132-induced decrease in cell proliferation was partially reversed by pretreatment with PFT-α. BrdU-positive cells in PFT-α + MG132 group were increased to 49.23%±2.67%. The cell cycle distribution had no significant difference between PFT-α + MG132 group and DMSO group, and the higher proliferation index was found in PFT-α + MG132 group but not in MG132 group. Inhibition of P53 activity in late-passage hBMSCs by PFT-α promoted the cell proliferation, and the percentage of BrdU-positive cells was higher than that in control group. CONCLUSION: Long-term expansion of hBMSCs in vitro reduces proteasomal activity, which in turn activates P53 to suppress cell proliferation through blocking cell cycle progression.     

Relationship between apoptosis and change of P53 expression in mouse renal tissues after acute kidney injury

AIM: To investigate the relationship between renal cell apoptosis induced by ischemia/reperfusion injury and the activation of P53. METHODS: Eighteen mice were randomly divided into 3 groups: sham operation group, acute kidney injury (AKI) group and pifithrin-alpha(PFT-α) treatment group. The AKI model was established by clamping bilateral renal arteries for 45 min and then performing reperfusion. The mice in PFT-α group were intraperitoneally injected with PFT-α at dose of 2.2 mg/kg 5 min before AKI model was established. The changes of serum creatinine and urea nitrogen were determined and renal pathological changes were observed 48 h after AKI. The P53 expression in the kidney was evaluated by Western blotting and immunofluorescence methods. Apoptosis of the renal cells was observed by TUNEL assay. The protein expression of tumor necrosis factor receptor (TNFR), caspase-3 and Bcl-2 was detected by immunohistochemical method. RESULTS: The levels of serum creatinine and urea nitrogen in AKI group and PFT-α group were higher than those in sham operation group. Compared with AKI group, the levels of serum creatinine and urea nitrogen were significantlydecreased in PFT-α group. No pathological change of the kidney was observed in sham operation group. In AKI group, the pathological changes such as shedding of brush border, vacuolus and dropwise degeneration in the renal tissues were observed. These pathological changes were attenuated in PFT-α group as compared with AKI group. The protein was expression level of P53 and the apoptotic cells were much higher in AKI group than those in sham operation group, and P53 protein was mainly expressed in the renal cortex, while those were significantly decreased in PFT-α group as compared with AKI group. Compared with sham operation group, the expression levels of TNFR and caspase-3 were increased and the Bcl-2 levels was decreased. Compared with AKI group, the expression level of TNFR and caspase-3 decreased and Bcl-2 expression was increased. CONCLUSION: P53 protein is mainly expressed in the renal cortex and induces apoptosis by increasing the expression of caspase-3 and regulating the expression of TNFR and Bcl-2 in the kidney following ischemia/reperfusion injury.     

Caspase-8 small hairpin RNA attenuates apoptosis of human bone marrow mesenchymal stem cells under conditions of serum deprivation and hypoxia

AIM：To investigate the effect of caspase-8 small hairpin RNA (shRNA) on attenuating apoptosis of human mesenchymal stem cells (hMSCs). METHODS：Two recombinant plasmids for over-expression of caspase-8 shRNA, pAd-Cap8 shRNA1 and pAd-Cap8 shRNA2, were constructed. Caspase-8 mRNA was determined in pAd-Cap8 shRNA-transfected human HEK293 cells by Q-PCR. The screened pAd-Cap8 shRNA was used to construct the recombinant adenovirus plasmid, which was linearized and transfected into HEK293 cells for packaging and amplification of the recombinant adenovirus rAd-Cap8 shRNA. The expression of caspase-8 at mRNA and protein levels was determined by Q-PCR and Western blotting. Annexin V/PI staining and determination of caspase-8 activity were performed to assess apoptosis of hMSCs under the conditions of serum deprivation and hypoxia. The mRNA expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), Bcl-2 and Bcl-xL was analyzed by Q-PCR. RESULTS：The pAd-Cap8 shRNA, which efficiently inhibited caspase-8 expression, was screened by Q-PCR. The recombinant adenovirus plasmid for caspase-8 shRNA was constructed and used to package and amplify the recombinant adenovirus (rAd)-Cap8 shRNA successfully. rAd-Cap8 shRNA-mediated caspase-8 shRNA markedly inhibited caspase-8 expression in hMSCs. Over-expression of caspase-8 shRNA by infection of rAd-Cap8 shRNA also efficiently decreased the apoptotic rate and caspase-8 activity in hMSCs under the conditions of serum deprivation and hypoxia, with up-regulation of the mRNA expression of HGF, IGF-1 and Bcl-2. CONCLUSION：Caspase-8 shRNA attenuates hMSC apoptosis under the conditions of serum deprivation and hypoxia.     

Effects of rs35100176 polymorphism in IPO8 promoter on gene expression

AIM: To investigate the effect of rs35100176 CCT insertion/deletion polymorphism in the promoter region of importin 8 (IPO8) gene on its mRNA expression. METHODS: A 342-bp fragment of IPO8 gene promoter containing the rs35100176 polymorphism was amplified from 49 DNA samples and sequenced. The IPO8 promoter fragments containing CCT 3-nucleotide insertion or deletion were amplified using the corresponding homozygote DNA samples. The PCR products were sequenced and inserted into the luciferase reporter vector pGL3-Basic. Recombinant vectors were transfected into the cells by Fugene 6.0 and the expression of the reporter gene was detected by a dual-luciferase reporter assay system. The mRNA expression level of IPO8 was detected by real-time PCR in 3-nucleotide insertion or deletion homozygote cells. RESULTS：The sequencing results showed that there were 3 kinds of genotypes in the rs35100176 polymorphism, CCT/CCT,CCT/- and -/-, and the gene frequencies were 1837%, 5510% and 2653%, respectively. The recombinant expression vectors pGL3-3N Insertion and pGL3-3N Deletion were successfully constructed. The luciferase assay showed that pGL3-3N Insertion produced significantly lower luciferase activity than that by pGL3-3N Deletion. Real-time PCR showed that HEK293 cells with 3-nucleotide insertion homozygote expressed relative lower IPO8 mRNA than Saos-2 cells with 3-nucleotide deletion homozygote. CONCLUSION：The CCT 3-nucleotide insertion variant decreases the promoter activity of IPO8, thus affecting the gene expression.     

Roles of microRNA-29a in expression of Bcl-2 and Mcl-1 in rat cardiomyocytes

AIM: To investigate the regulatory mechanisms of microRNA-29a (miR-29a) on the expression of Bcl-2 and Mcl-1 in rat cardiomyocytes (CM cells). METHODS: The CM cells were isolated from the hearts of newborn rats and transfected with miR-29a mimic (100 nmol/L) by Lipofectamine RNAiMAX. The expression of Bcl-2 and Mcl-1 at mRNA and protein levels was detected by real-time fluorescence quantitative PCR and Western blotting. The luciferase assay was performed in HEK293T cells and CM cells, which were co-transfected with plasmid DNA and miRNA using Lipofectamine 2000. RESULTS: Transfection of miR-29a mimics significantly reduced the expression levels of Bcl-2 and Mcl-1 in CM cells as compared with the control cells (P<0.05). In addition, HEK293T cells co-transfected with miR-29a mimic and Bcl-2-3’UTR-WT or Mcl-1-3’UTR-WT plasmid significantly reduced the luciferase activity as compared with control group (P<0.05). While CM cells transfected with miR-29a inhibitor and Bcl-2-3’UTR-WT or Mcl-1-3’UTR-WT plasmid in succession, the luciferase activity was increased inversely (P<0.05). CONCLUSION: miR-29a may regulate apoptosis by targeting the bcl-2 and mcl-1 genes.     

Overexpressed human FAT10 protein induces the apoptosis in the starved HEK293 cells

AIM: To study the effect of human FAT10 on the apopotosis of HEK293 cells using flag-tagged human FAT10 protein.METHODS: The fragment of FAT10 gene was cloned into the pcDNA3-flag vector,which was identified by PCR,enzyme digestion and sequencing.The reconstructed plasmids were transfected into HEK293 cells.The expression of introduced FAT10 in the normal cultured and starved cells was detected respectively by Western blotting.XTT assay and DNA ladder method were used to analyze the effect of FAT10 on the apoptosis of starved HEK293 cells.RESULTS: The reconstructed plasmids were highly expressed in HEK293 cells with different expression mode at the mormal cultured and starved state.The livability of starved FAT10 overexpressed HEK293 cells was significantly lower than that of normal cultured cells.DNA ladder was observed in the starved FAT10 overexpressed cells,but not in the normal cultured cells.CONCLUSION: The eukaryotic expressed plasmids of flag-tagged FAT10 were constructed successfully,and highly expressed in HEK293.Overexpressed FAT10 enhances the apoptosis in the starved HEK293 cells.     

Effect of NOD8 on production of nitric oxide,IL-1β and TNF-α in LPS-treated macrophages

AIM: To investigate the effect of NOD8 on lipopolysaccharide (LPS)-induced releases of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 cells. METHODS: The plasmids of pEGFP-C2 and pEGFP-NOD8 were transfected into RAW264.7 cells respectively. The transfected and non-transfected cells were stimulated by LPS for 0, 6, 12 and 24 h. NO production was evaluated by Griess reagent assay, and the levels of IL-1β and TNF-α were measured by ELISA. The protein expression of NOD8 and the nuclear translocation of nuclear factor κB (NF-κB) p65 subunit were detected by Western blotting. The level of activated caspase-1 was determined by fluorimetric method. RESULTS: Compared with pEGFP-C2 group, the protein expression of NOD8 was significantly elevated in pEGFP-NOD8+LPS group. The releases of NO, IL-1β and TNF-α were obviously increased after RAW264.7 cells were treated with LPS for 6 h, 12 h and 24 h, and while the secretion of NO was significantly reduced in the cells transfected with pEGFP-NOD8 and induced by LPS for 12 h and 24 h, and the release of IL-1β was also significantly reduced at 6 h, 12 h and 24 h. However, no significant difference of TNF-α release was observed between pEGFP-C2+LPS group and pEGFP-NOD8+LPS group. The activation of caspase-1 in RAW264.7 cells stimulated with LPS for 6 h, 12 h and 24 h was markedly increased, and the expression of NF-κB p65 subunit in the cytoplasm was significantly decreased, indicating that p65 nuclear translocation was increased. In addition, the activation of caspase-1 and the nuclear translocation of p65 were significantly inhibited in pEGFP-NOD8+LPS group. CONCLUSION: NOD8 suppresses the releases of LPS-induced NO and IL-1β in RAW264.7 cells by inhibiting the activation of caspase-1 and NF-κB.     

NOD8 inhibits autophagy in human pancreatic cancer cells and effect of apoptosis on autophagy regulatd by NOD8

AIM: To explore whether NOD8 inhibits autophagy in human pancreatic cancer cells and its underlying mechanisms, and to investigate the effect of apoptosis on the autophagy regulated by NOD8. METHODS: The empty plasmid pEGFP-C2 and recombinant plasmid pEGFP-NOD8 were transfected into the Panc-1 cells using JetPRIME reagent.The untransfected cells served as control group. The protein levels of NOD8, autophagy-related proteins beclin-1 and LC3-II, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway-related proteins Akt, p-Akt, mTOR and p-mTOR were determined by Western blot 48 h after transfection. Meanwhile, the number of LC3 spots was quantified by immunofluorescence staining. Furthermore, after a broad caspase inhibitor Z-VAD-FMK was applied to NOD8-over-expressing cells, the protein expression levels of beclin-1 and LC3-II were detected by Western blot and the number of LC3 spots was observed by immunofluorescence staining. RESULTS: The protein level of NOD8 in pEGFP-NOD8 group was significantly higher than that in control group and pEGFP-C2 group (P<0.01). The protein expression of beclin-1 and LC3-II, and the number of LC3 spots in pEGFP-NOD8 group were significantly decreased as compared with control group and pEGFP-C2 group. Moreover, the protein levels of p-AKT and p-mTOR in pEGFP-NOD8 group were higher than those in control group and pEGFP-C2 group, while no significant difference of mTOR and AKT protein expression was found among these 3 groups. Furthermore, the protein levels of beclin-1 and LC3-II, and the number of LC3 spots in pEGFP-NOD8+Z-VAD-FMK group were significantly increased compared with pEGFP-NOD8 group. CONCLUSION: NOD8 inhibits autophagy in the Panc-1 cells and its mechanism may be related to the activation of PI3K/Akt/mTOR pathways. Apoptosis enhances the inhibitory effect of NOD8 on autophagy.     

PI3K/Akt regulates endoplasmic reticulum stress-mediated GRP78 induction

AIM: To investigate the effect of PI3K/Akt pathway on endoplasmic reticulum (ER) stress-mediated glucose-regulated protein 78 (GRP78) induction in human embryonic kidney 293 cells (HEK293) cells.METHODS: PI3K inhibitor LY294002, dominant negative kinase-dead mutant vector for HA-Akt (K179M) and Akt1 siRNAs were used to block the PI3K/Akt pathway under ER stress. Constitutively active expression vectors for Akt (myr-HA-Akt) were used to up-regulate Akt activity under ER stress. The effects of PI3K/Akt on ER stress-mediated GRP78 induction in HEK293 cells were determined by Western blotting and RT-PCR. RESULTS: GRP78 induction was inhibited by LY294002, Akt1 (K179M) and Akt1 siRNA, but was increased by myr-Akt1 in dithiothreitol-and thapsigargin-treated HEK293 cells. However, both myr-Akt2/3 and Akt2/3 siRNA had no effect on GRP78 induction in HEK293 cells under ER stress. Furthermore, the PI3K/Akt pathway didnt regulated GRP78mRNA induction but increased GRP78 protein stability.CONCLUSION: PI3K/Akt promotes GRP78 accumulation through increasing the stability of GRP78 protein in HEK293 cells under ER stress.     

miR-204 inhibits proliferation of Hodgkin lymphoma cells by targeting Sirt1/p53 signaling pathway

AIM: To investigate the effect of microRNA-204 (miR-204) on the proliferation of Hodgkin lymphoma cells and the underlying mechanism. METHODS: The expression of miR-204 and Sirt1 mRNA in Hodgkin lymphoma tissues was detected by RT-qPCR. After transfection with miR-204 mimic, Sirt1 siRNA and miR-204 mimic+pcDNA3.1-Sirt1 into the L428 cells, the cell viability and BrdU incorporation were measured by CCK-8 assay and BrdU assay, respectively. The protein levels of Sirt1 and acetylated p53 (ac-p53) were determined by Western blot.The targeting relationship between miR-204 and Sirt1 was verified by double luciferase reporter assay. RESULTS: The low expression of miR-204 and the high mRNA expression of Sirt1 were found in the Hodgkin lymphoma tissues. Compared with control group, the cell viability, BrdU incorporation and the protein levels of Sirt1 and ac-p53 were significantly decreased after L428 cells were transfected with miR-204 mimic or Sirt1 siRNA (P<0.05). Compared with miR-204 mimic alone group, the cell viability, BrdU incorporation and the protein levels of Sirt1 and ac-p53 were increased after L428 cells were co-transfected with miR-204 mimic and pcDNA3.1-Sirt1 (P<0.05). The results of double luciferase reporter assay confiermed that Sirt1 was the target gene of miR-204. CONCLUSION: The inhibitory effect of miR-204 on the proliferation of L428 cells may be achieved by inhibiting the expression of Sirt1 and promoting the up-regulation of ac-p53.     

Construction of pNTAP-PRAK eukaryotic expression plasmid and its expression in HEK293 cells

AIM: To construct pNTAP-PRAK eukaryotic expression plasmid and to establish a stable HEK293 cell line expressing tandam affinity purification (TAP)-tagged PRAK. METHODS: Human PRAK coding region was subcloned into pNTAP vector to construct a recombinant plasmid called pNTAP-PRAK, then DH5α E.coli was transformed with the recombinant plasmid. After identified by PCR, digestion with restriction endonuclease and sequencing, the correct recombinant expression plasmid was transfected with PolyFect liposome transfection reagent to HEK293 cells. The cell line with stable expression of exogenous TAP tagged-PRAK gene was established by screening of antibiotic G418. The expression and localization of the fusion protein TAP tagged-PRAK were detected by Western blotting and immunofluorescence assay. RESULTS: All the results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the recombinant eukaryotic expression plasmid pNTAP-PRAK was constructed correctly. The result of Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection followed by G418 screening. The result of immunofluorescence assay showed that the expression product TAP tagged-PRAK distributed mainly in the nucleus. CONCLUSION: The eukaryotic expression vector pNTAP-PRAK was successfully constructed and the cell line stably expressing TAP tagged-PRAK was established. TAP tag didnt influence the localization of exogenous PRAK.     

Eukaryotic expression of human Arresten gene and its effect on the proliferation and migration of vascular smooth muscle cells

AIM: To express human Arresten gene in eukaryotic cell,and to investigate its effect on the proliferation and migration in vitro of rat primary cultured thoracic aortic vascular smooth cells (VSMCs).METHODS: COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome.48 hours after transfection,polymerase chain reaction (RT-PCR) was used to detect the expression of Arresten mRNA in the cells,while Western blotting assay was applied to detect expressed Arresten protein in concentrated supernatants.VSMCs were then co-cultured with the concentrated supernatants;and its proliferation was detected using cell counting kit-8 (CCK-8) in vitro.Migration of VSMCs was assayed by a microchemotaxis chamber and a polycarbonate filter (Transwell's chamber) with pores of 8 μm in diameter.RESULTS: RT-PCR revealed that the genome of Arresten-transferred cells contained a 449bp specific fragment of Arresten gene.Successful protein expression in supernatants was confirmed by Western blotting.CCK-8 assay showed that the proliferation of VSMCs was inhibited significantly by Arresten protein as compared with control group (P<0.01).Transwell's chamber showed that the number of control group,pSecTag2 transfected group and pSecTag2-AT transfected group were 28.70±3.97,26.10±4.53 and 14.00±3.33 (P<0.01).CONCLUSION: Arresten protein expressed in eukaryotic cells inhibits the proliferation and migration of VSMCs effectively in vitro.     

Effects of xeroderma pigmentosum group D and p53 on replication of HBV

AIM:To investigate the effects of xeroderma pigmentosum D (XPD) and p53 on the replication of hepatitis B virus (HBV). METHODS:Recombinant plasmid pEGFP-N2/XPD and vacant vector plasmid pEGFP-N2 were transfected into HepG2.2.15 cells by liposome. On the next day, these cells were incubated with pifithrin-α, a p53 inhibitor, at a concentration of 20 μmol/L for 24 h. The cells were divided into 5 groups: blank control group, pEGFP-N2 group, pEGFP-N2/XPD group, pEGFP-N2/XPD+pifithrin-α group and pifithrin-α group. The mRNA expression of XPD, hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and hepatitis B virus X protein (HBx) was detected by RT-PCR. The content of HBsAg and HBeAg in the supernatants of culture medium was measured by ELISA. The content of HBV-DNA in the supernatants of culture medium was examined by fluorescence quantitative PCR. Using the method of bDNA, the content of HBV-DNA in the core particles was assessed. RESULTS:The expression of XPD mRNA was elevated by the transfection of recombinant plasmid pEGFP-N2/XPD. The increase in XPD expression significantly down-regulated the mRNA expression of HBsAg, HBeAg and HBx. The content of HBsAg and HBeAg in the supernatants of culture medium was significantly decreased by the increase in XPD expression. The results of fluorescence quantitative PCR showed that the content of HBV-DNA in the supernatants of culture medium was significantly down-regulated by the increase in XPD expression. bDNA results showed that the content of HBV-DNA in the core particles was significantly decreased by the increase in XPD expression.Pifithrin-α abolished the above-mentioned effects of XPD (all P<0.01). CONCLUSION: XPD inhibits the replication of HBV through p53 pathway. Therefore, XPD and p53 may be the targets for antiviral therapy of hepatitis B.     

Effects of phosphorylation-defective retinoic acid receptor α1 on proli-feration of human multiple myeloma cells in vitro

AIM: To investigate the effect of phosphorylation-defective retinoic acid receptor α1 (RARα1) on the proliferation of human multiple myeloma cells. METHODS: The mRNA expression of RARα subtypes in U266 cells was detected by RT-PCR. Lentiviral plasmid construction, viral production, titer determination and cell transfection were carried out by the general methods of molecular biology. Proliferation analysis was performed with CCK-8 assay. The U266 cells were treated with all-trans retinoic acid (ATRA,0~100 μmol/L) or transfected with lentivirus RARαS77A. The expression levels of proliferation-related proteins, P53 and Rb, in U266 cells treated with ATRA or transfected with lentivirus RARαS77A were detected by Western blotting. RESULTS: RARα1 was positively expressed in U266 cells and RARα2 expression was negative. ATRA significantly inhibited the proliferation of U266 cells in a dose- and time-dependent manner. Proliferation of U266 cells was significantly inhibited 48 h after transfection with lentivirus RARαS77A, and the inhibitory rate was 15.16%±3.84%. The up-regulated expression of Rb and down-regulated expression of P53 were detected in U266 cells not only in the cells treated with ATRA, but also in the cells transfected with lentivirus RARαS77A. CONCLUSION: Phosphorylation-defective RARα1 (RARαS77A) mimics the growth inhibitory effect of ATRA on U266 cells that express RARα1 (+) and RARα2 (-) via down-regulating the expression of P53 and up-regulating the expression of Rb, suggesting that the antiproliferative effect of ATRA is mainly mediated by decreasing the phosphorylation of RARα1.     

The expression of 53BP1 and 53BP2 gene in nasopharyngeal carcinoma

AIM:To investigate the expression map of two p53 binding proteins 53BP1 and 53BP2 in nasopharyngeal carcinoma (NPC) tissue. METHODS:The expression of 53BP1 and 53BP2 mRNA in NPC biopsy and control group are tested by RT-PCR. The expression of two mRNA in NPC paraffin section are examined by in situ hybridization. RESULTS:No expression of 53BP1 mRNA was found in NPC tissue and control group. However, expression of 53BP2 was detected in NPC biopsy and control group by RT-PCR, specific expressoin found cancerous nest in NPC paraffin section by in situ hybridization. CONCLUSION:The high expression of 53BP2 may be related to the development of NPC.