Small extracellular vesicles derived from cerebral endothelial cells with elevated microRNA 27a promote ischemic stroke recovery

Axonal remodeling is a critical aspect of ischemic brain repair processes and contributes to spontaneous functional recovery.Our previous in vitro study demonstrated that exosomes/small extracellular vesicles(sEVs)isolated from cerebral endothelial cells(CEC-sEVs)of ischemic brain promote axonal growth of embryonic cortical neurons and that microRNA 27a(miR-27a)is an elevated miRNA in ischemic CEC-sEVs.In the present study,we investigated whether normal CEC-sEVs engineered to enrich their levels of miR-27a(27a-sEVs)further enhance axonal growth and improve neurological outcomes after ischemic stroke when compared with treatment with non-engineered CEC-sEVs.27a-sEVs were isolated from the conditioned medium of healthy mouse CECs transfected with a lentiviral miR-27a expression vector.Small EVs isolated from CECs transfected with a scramble vector(Scra-sEVs)were used as a control.Adult male mice were subjected to permanent middle cerebral artery occlusion and then were randomly treated with 27a-sEVs or Scra-sEVs.An array of behavior assays was used to measure neurological function.Compared with treatment of ischemic stroke with Scra-sEVs,treatment with 27a-sEVs significantly augmented axons and spines in the peri-infarct zone and in the corticospinal tract of the spinal grey matter of the denervated side,and significantly improved neurological outcomes.In vitro studies demonstrated that CEC-sEVs carrying reduced miR-27a abolished 27a-sEV-augmented axonal growth.Ultrastructural analysis revealed that 27a-sEVs systemically administered preferentially localized to the pre-synaptic active zone,while quantitative reverse transcription-polymerase chain reaction and Western Blot analysis showed elevated miR-27a,and reduced axonal inhibitory proteins Semaphorin 6A and Ras Homolog Family Member A in the peri-infarct zone.Blockage of the Clathrin-dependent endocytosis pathway substantially reduced neuronal internalization of 27a-sEVs.Our data provide evidence that 27a-sEVs have a therapeutic effect on stroke recovery by promoting axonal remodeling and improving neurological outcomes.Our findings also suggest that suppression of axonal inhibitory proteins such as Semaphorin 6A may contribute to the beneficial effect of 27a-sEVs on axonal remodeling.

Akt1/p27^(kip1) Pathway Mediates Inhibition of LXA_4 on TNF-α-induced Proliferation of Rat Mesangial Cells

Objective:To examine whether lipoxin A 4(LXA 4) has an inhibitory effect on tumor necrosis factor-α(TNF-α)-induced proliferation of glomerular mesangial cells of rat, and explore the molecular mechanisms of signal pathway in LXA 4 actions. Methods: Glomerular mesangial cells of rat were cultured and treated with TNF-α(10 ng/ml), with or without preincubation with LXA 4 at different concentrations. Cell proliferation was evaluated by monotetrazolium (MTT) colorimetric assay. The expression of cyclin E mRNA was measured by RT-PCR. Phosphorylated Akt1(Thr308) and p27 kip1 were analyzed by Western blotting. Results: TNF-α-stimulated proliferation of mesangial cells was inhibited by LXA 4 in a dose-dependent manner. The marked increments in cyclin E mRNA expression induced by TNF-α during proliferation of mesangial cells were down-regulated by LXA 4. Threonine phosphorylated Akt1 proteins at 308 site stimulated by TNF-α was reduced by LXA 4. TNF-α-induced decrements in expression of p27 kip1 proteins was ameliorated by LXA 4 in a dose-dependent manner. Conclusion: TNF-α-induced proliferation of rat mesangial cells can be inhibited by TXA 4 through the mechanism of Akt 1/p27 kip1 pathway-dependent signal transduction.

Influence of paeonol on expression of COX-2 and p27 in HT-29 cells

AIM： To investigate the effect of paeonol on controlling the proliferation of colorectal cancer cell line HT-29 and to discuss its possible mechanism. METHODS： The inhibitory effect of paeonol on proliferation of HT-29 cells was detected by M-I-I- assay. The results of apoptosis were measured by flow cytometry. Immunocytochemical staining was performed to detect the expression of cyclooxygenase-2 （COX-2） and protein p27 in HT-29 cells treated with paeonol at different concentrations. Reverse transcription-polymerase chain reaction （RT- PCR） was used for mRNA analysis. RESULTS： From the data of both MTT and flow cytometry, we observed that cell proliferation was inhibited by different concentrations of paeonol. By immunocytochemical staining, we found that HT-29 cells treated with paeonol （0.024-1.504 mmol/L） reflected reduced expression of COX-2 and increased expression of p27 in a dose-dependent manner. RT-PCR showed that paeonol down-regulated COX-2 and up-regulated p27 in a dose- and time-dependent manner in HT-29 cells. CONCLUSION： One of the apoptotic mechanisms of paeonol is down-regulation of COX-2. p27 is upregulated simultaneously and plays an important part in controlling cell proliferation and is a crucial factor in the Fas/FasL apoptosis pathway.

Effects of Sodium Salicylate on the Expression of HSP27 Protein during Oxidative Stress in Tissue-cultured Human Lens Epithelial Cells

The effects of sodium salicylate on the expression of heat shock protein 27 （HSP27） during oxidative stress in tissue-cultured human lens epithelial cells were investigated. Cultured human lens epithelial cells （HLB-3） were divided into 3 groups： control group （group A）, oxidation injury group （group B） and sodium salicylate group （group C）. Apoptosis of human lens epithelial cells cultured in vitro was induced in the presence of 150 μmol/L H2O2. Cells viability and the expression of HSP27 were analyzed. Viability of the cells was measured by methyl thiazole tetrazolium （MTT） chromatometry. The expression of HSP27 in HLB-3 cells was detected by using immunohistochemistry and image analysis system, Sodium salicylate could induce the expression of HSP27, and the cells viability in group C was significantly higher than in group B （0.2667±0.01414 vs 0.2150±0.01080, P=0.012〈0.05）. The average gray value of HSP27 in group B was less than that in group C （P=0.000〈0.05）. The increased expression of HSP27 by sodium salicylate might play an important role in the protection of hydrogen peroxide-induced injury of human lens epithelial cells, suggesting that sodium salicylate could suppress, at least in part, the apoptosis of human lens epithelial cells.

Effect of p27^(KIP1) on cell cycle and apoptosis in gastric cancer cells

AIM： To elucidate the effect of p27^KIP1 on cell cycle and apoptosis regulation in gastric carcinoma cells. METHODS： The whole length of p27^KIP1 cDNA was transfected into human gastric cancer cell line SCG7901 by lipofectamine. Expression of p27^KIP1 protein or mRNA was analyzed by Western blot and RNA dot blotting, respectively. Effect of p27^KIP1 on cell growth was observed by MTT assay and anchorage-independent growth in soft agar. Tumorigenicity in nude mice was used to assess the in vivo biological effect of p27^KIP1. Flow cytometry, TUNEL, and electron microscopy were used to assess the effect of p27^KIP1 on cell cycle and apoptosis. RESULTS： Expression of p27^KIP1 protein or mRNA increased evidently in SCG7901 cells transfected with p27^KIP1. The cell growth was reduced by 31% at 48 h after induction with zinc determined by cell viability assay. The alteration of cell malignant phenotype was evidently indicated by the loss of anchorage-independent growth ability in soft agar. The tumorigenicity in nude mice was reduced evidently （0.55±0.14 cm vs 1.36±0.13cm, P〈0.01）. p27^KIP1 overexpression caused cell arrest with 36% increase （from 33.7% to 69.3%, P〈0.01） in G1 population. Prolonged p27^KIP1 expression induced apoptotic cell death reflected by pre-G1 peak in the histogram of FACS, which was also confirmed by TUNEL assay and electron microscopy. CONCLUSION： p27^KIP1 can prolong cell cycle in G1 phase and lead to apoptosis, p27^KIP1 may be a good candidate for cancer gene therapy.

Exogenous p27KIP1 expression induces anti-tumour effects and inhibits the EGFR/PI3K/Akt signalling pathway in PC3 cells

p27 is a cyclin-dependent kinase inhibitor that regulates the progression of cells from G1 to S phase of the cell cycle. Loss of p27 has been associated with disease progression and with an unfavourable outcome in prostate cancer. In this study, we investigated whether exogenous p27 expression in the human androgen-independent prostate cancer PC3 cell line had any effect on cell growth, and we studied the molecular mechanisms involved. p27 expression was restored in PC3 cells by plasmid delivery. Cell proliferation and apoptosis were assessed in PC3 cells transfected with p27. We also investigated the effects of p27 on the epidermal growth factor receptor （EGFR）/ phosphatidylinositol 3-kinase （PI3K）/Akt signalling pathway in PC3 cells. By restoring p27 expression in PC3 cells, we observed that p27 reduced proliferation and induced arrest in G0/G1 phase. Moreover, p27-transfected PC3 cells underwent apoptosis, as shown by flow cytometric analysis and western blotting analysis of Bcl-2, Bax, Bad, caspase-3 and poly（ADP-ribose）polymerase expression. Furthermore, the p27-induced anti-tumour action corre- lated with inhibition of the EGFR/PI3K/Akt signalling pathway, as confirmed by western blotting analysis and densitometry of EGFR, PI3K （p85）, Akt and p-Akts473 expression. Our results suggest that exogenous expression of p27 inhibits the proliferation of PC3 cells through induction of G1 arrest and apoptosis, and this process correlates with inhibition of the EGFR/PI3K/Akt signalling pathway.

Skp2-RNAi suppresses proliferation and migration of gallbladder carcinoma cells by enhancing p27 expression

AIM:To explore the role of S-phase kinase-associated protein-2(Skp2)in gallbladder carcinoma and to identify whether depletion of Skp2 by Skp2-RNAi could attenuate proliferation and migration of gallbladder carcinoma.METHODS:Skp2-RNAi was transduced into cells of the gallbladder carcinoma cell line GBC-SD,using a lentiviral vector.The effect of Skp2-RNAi on the proliferation,migration,invasion and cell cycle of GBC-SD cells was studied using in vitro assays for cell proliferation,colony formation,wound healing and cell cycle.The expression of Skp2 and p27 was detected by real-time polymerase chain reaction and Western immunoblotting.The effect of Skp2-RNAi on the proliferation of GBC-SD cells in vivo was investigated by tumorigenicity experiments in nude mice.RESULTS:Lentivirus-mediated RNAi reduced the expression of Skp2 in cultured cells.The expression of the p27 protein increased along with the down-regulation of Skp2,although no significant difference was found in p27 mRNA expression.Flow cytometry revealed that Skp2-RNAi transfection significantly increased the proportion of cells in the S phase and significantly decreased the proportion of cells in the G 2 /M phase.No significant difference in the frequency of cells in the G0/G1 phase was observed.The results from the cell proliferation,colony formation and wound healing assays revealed that Skp2-RNAi transfection markedly inhibited the proliferation and migration of GBC-SD cells in vitro.Additionally,tumorigenicity experiments showed that suppression of Skp2 significantly decreased the weights of the tumors(0.56 ± 0.11 and 0.55 ± 0.07 g in the control and Scr-RNAi groups vs 0.37 ± 0.09 and 0.35 ± 0.08 g in the Skp2-RNAi-L and Skp2-RNAi-H groups).CONCLUSION:The expression of Skp2 in GBC-SD cells was inhibited following Skp2-RNAi transfection.Silencing of the Skp2 gene inhibited proliferation,migration and invasiveness of GBC-SD cells by mechanisms dependent on enhanced expression of the p27 protein.

Malarial pigment enhances heat shock protein-27 in THP-1 cells:new perspectives for in vitro studies on monocyte apoptosis prevention

Objective:To investigate the effect of malarial pigment(hemozoin,HZ) on expression of heat shock proteins(HSPs) and cell viability in human monocytes by using a stable cell line(THP-1 cells).Methods:THP-1 cells were fed with native HZ or treated with pro-apoptotic molecule gliotoxin for 9 h.Thereafter,the protein expression of HSP-27 and HSP-70 was evaluated by western blotting.Alternatively,HZ-fed cells were cultured up to 72 h and cell viability parameters(survival,apoptosis and necrosis rates) were measured by flow cytometric analysis. Results:HZ increased basal protein levels of HSP-27 without altering those of HSP-70 in THP-1 cells,and promoted long-term cell survival without inducing apoptosis.As expected,gliotoxin inhibited HSP-27 protein expression and promoted long-term cell apoptosis.Conclusions: Present data show that HZ prevents cell apoptosis and enhances the expression of anli-apoptotic HSP-27 in THP-1 cells,confirming the previous evidences obtained from HZ-fed immunopurified monocytes.Since the use of a stable cell line is pivotal to perform HSP-27 silencing experiments, monocytic THP-1 cells could be a good candidate line for such an approach,which is heavily required to clarify the role of HSP-27 in survival of impaired HZ-fed monocytes during falciparum malaria.

Sensitized heat shock protein 27 induces retinal ganglion cells apoptosis in rat glaucoma model

AIM: To investigate the relationships between the changes of heat shock protein 27 antibody(anti-HSP27) in serum/cerebrospinal fluid(CSF), intraocular pressure(IOP), retinal ganglion cell(RGC) apoptosis in a rat glaucoma model and disclose the underlying pathogenesis of glaucoma.METHODS: A total of 115 Wistar rats were randomly divided into 4 groups. Group 1 was the ocular hypertension group by condensing 3 episcleral & limbal veins or episcleral area of right eye(HP group, n=25) and sham operation group with conjunctiva incision without coagulation(n=25). Group 2: HSP27 or dose-matched PBS was injected into the vitreous(V-HSP27 group, n=15;V-PBS group, n=15). Group 3: HSP27 and complete Freund's adjuvant or dosematched PBS was injected subcutaneously into the hind limb accompanied intraperitoneal injection of pertussis toxin [sensitized group(I-HSP27 group), n=15;I-PBS group, n=15)]. Group 4 was normal group without any treatment(n=5). IOPs of the rats were measured before, day 3, weeks 1, 2, 4, 6, and 8 after treatment. Paraffin-embedded sections were prepared for HE staining and RGCs apoptosis were detected by TUNEL. Anti-HSP27 level in serum and CSF were examined by ELISA. RESULTS: IOPs were elevated significantly in HP and V-HSP27, V-PBS groups(P<0.01) and positively related to anti-HSP27 levels in serum and CSFs. Anti-HSP27 levels in serum and CSF were elevated significantly in I-HSP27group compared to other groups(P<0.05). However, the IOPs did not show any relationship with the high-level antiHSP27 in serum and CSFs. RGC apoptosis were all elevated significantly in the HP, V-HSP27, V-PBS and I-HSP27 groups and also positively relative with anti-HSP27 level in serum and CSFs except that high-level of anti-HSP27 in the serum of I-HSP group.CONCLUSION: The increases of anti-HSP27 levels in serum and CSFs both promote IOP escalation and the increase of RGC apoptosis in retina when anti-HSP27 is at low level. The case of high-level anti-HSP27 is opposite and shows protective function in preventing IOP increase and RGC apoptosis.

Influence of human mutant p27 gene on the biological behaviors of colon cancer cells

Objective： To observe the influence of human mutant p27 gene （p27mt） on the growth and apoptosis of colon cancer cells so as to investigate the function mechanism of p27mt in gene therapy for colon cancer. Methods： Colon cancer cell line SW480 was infected with recombinant replication defective adenovirus Ad-p27mt, and expression of p27mt protein was detected by Western blot; the inhibition effect of p27mt on SW480 cells was detected with cytometry. Cell cycle was decided with flow cytometer, and DNA fragment analytic process identified the occurrence of apoptosis. Results： After transfected SW480 cells with Ad-p27mt, high expression of p27 protein was identified with immunoblotting assay. PI staining and flow cytometer assay showed 77.96% colon cancer cells was blocked in phase G0/G1, while in Ad-LacZ group and blank control group, 27.57% and 25.29% cells were blocked in the same phase, respectively. Growth curve showed Ad-p27mt has an obvious inhibition effect on the growth of SW480 cells, DNA fragment assay demonstrated that p27mt was able to induce the apoptosis of colon cancer cells. Conclusion： p27mt has an obvious blocking effect on colon cancer cell cycle, and most cells were blocked in phase G0/G1. This blockage is related with the growth inhibition and apoptosis induction effect of p27mt.

The Expression of IL-27, Th17 Cells and Treg Cells in Peripheral Blood of Patients with Allergic Rhinitis

Objective: To explore the expression of IL-27, Th17 cells and CD4+CD25+ regulatory T cells (Treg) as well as its associated cytokines in peripheral blood of patients with allergic rhinitis (AR). Method: From March 2012 to May, the peripheral blood of 24 cases of AR patients (AR group) and 16 cases of healthy volunteers (control group) was collected, and the percentage of Th17 cells and Treg cells in the peripheral blood was detected by flow cytometry (FCM);the levels of IL-27, IL-17 and IL-10 in serum was detected by ELISA. Result: The percentage of Th17 cells in AR group and the control group was 1.76% ± 0.60% and 0.59% ± 0.17%, respectively. It was higher in AR group than in control group, and the difference between two groups was statistically significant (P 0.05). Conclusion: In the peripheral blood of AR patients there was a reduction of IL-27 level and imbalance of Th17/Treg cell function. IL-27 on Th17/Treg cells adjustment may play an important role on the pathogenesis of the AR.

Effect of p27Kip1 Inhibition on Proliferation of Bovine Corneal Endothelial Cells by RNA Interference

Three plasmids （pGenesil-P1, pGenesil-P2, pGenesil-P3） with different p27Kip1-shRNA sequences were designed and synthesized. Their effects on the proliferation of bovine corneal endothelial cells （bCEC） were investigated. Plasmid expressing irrelevant shRNA with a random combination was used as negative control （pGenesil-HK）. The recombination of four plamids was confirmed by restrictive enzyme digestion and sequence analysis. The expression of mRNA and protein of p27Kip1 was detected by RT-PCR and Western blotting after stable transfection. The expressions of p27Kip1 mRNA and p27Kip1 protein of pGenesil-P1 group, pGenesil-P2 group and pGenesil-P3 group were all lower than those in the pGenesil-HK group and the blank group （non-transfected group）, pGenesil-P3 had the strongest inhibitory effect and was selected for the next steps. The proliferation rates of the pGenesil-P3 group, the pGenesil-HK group and the blank group were assessed by MTT. The influence of shRNA-p27Kip1 on bCEC cell cycle was detected by flow cytometry （FCM）. Compared with the control groups, the proliferation rate of the pGenesil-P3 group was increased significantly, and the ratio of S-phase also increased. It is concluded that shRNA-p27Kip1 could down-regulate the expression of p27Kip1 effectively and increase the proliferation of bCEC. RNA interference （RNAi） may be an effective means to promote the proliferation of CEC.

Effect and mechanism of high glucose on apoptosis and cell cycle arrest of islet β cells via p27 pathway

Objective:To study the effect of mechanism of high glucose on apoptosis and cell cycle arrest of isletβcells via p27 pathway.Methods:Islet INS-1 cells were cultured and divided into groups.The control group was treated with ordinary medium,the high glucose group was treated with high glucose medium containing 25mmol/L glucose,the high glucose+si-NC group was treated with high glucose medium and transfected with NC siRNA,and the high glucose+si-P27 group was treated with high glucose medium and transfected with p27 siRNA.After 24 hours treatment,MTS assay was used to detect the cell viability A490,TUNEL assay was used to detect apoptosis rate,flow cytometry was used to detect the cell cycle distribution and western blot was used to detect the expression levels of P27,caspase-8 and cyclinD1.Results:Compared with those in the control group,the A490,the ratio of S phase and G2/M phase as well as the expression level of CyclinD1 decreased,while the apoptosis rate,the ratio of G0/G1 phase as well as the expression levels of P27 and caspase-8 increased in the high glucose group(P<0.05);compared with those in the high glucose group,the A490,cell cycle as well as the expression levels of P27,caspase-8 and cyclinD1 were not different from those in the high glucose+si-NC group(P>0.05);compared with those in the high glucose group and high glucose+si-NC group,the A490,the ratio of S phase and G2/M phase as well as the expression levels of cyclinD1 increased,while the apoptosis rate,the ratio of G0/G1 phase as well as the expression levels of p27 and caspase-8 decreased in the high glucose+si-P27 group(P<0.05).Conclusion:The apoptosis and cell cycle arrest induced by high glucose are related to P27 pathway activation.

CacyBP/SIP nuclear translocation regulates p27Kip1 stability in gastric cancer cells

AIM: To investigate the mechanism of calcyclin binding protein/Siah-1 interacting protein(Cacy BP/SIP) nuclear translocation in promoting the proliferation of gastric cancer(GC) cells. METHODS: The effect of Cacy BP/SIP nuclear translocation on cell cycle was investigated by cell cycle analysis. Western blot analysis was used to assess the change in expression of cell cycle regulatory proteins and proteasome-mediated degradation of p27Kip1. Coimmunoprecipitation(co-IP) analysis was performed to examine the binding of Cacy BP/SIP with Skp1. A Cacy BP/SIP truncation mutant which lacked the Skp1 binding site was constructed and fused to a fluorescent protein. Subsequently, the effect on Skp1 binding with the fusion protein was examined by co-IP, while localization of fluorescent fusion protein observed by confocal laser microscopy, and change in p27Kip1protein expression assessed by Western blot analysis.RESULTS: Cacy BP/SIP nuclear translocation induced by gastrin promoted progression of GC cells from G1 phase. However, while Cacy BP/SIP nuclear translocation was inhibited using si RNA to suppress Cacy BP/SIP expression, cell cycle was clearly inhibited. Cacy BP/SIP nuclear translocation significantly decreased the level of cell cycle inhibitor p27Kip1, increased Cyclin E protein expression whereas the levels of Skp1, Skp2, and CDK2 were not affected. Upon inhibition of Cacy BP/SIP nuclear translocation, there were no changes in protein levels of p27Kip1 and Cyclin E, while p27Kip1 decrease could be prevented by the proteasome inhibitor MG132. Moreover, Cacy BP/SIP was found to bind to Skp1 by immunoprecipitation, an event that was abolished by mutant Cacy BP/SIP, which also failed to stimulate p27Kip1 degradation, even though the mutant could still translocate into the nucleus.CONCLUSION: Cacy BP/SIP nuclear translocation contributes to the proliferation of GC cells, and Cacy BP/SIP exerts this effect, at least in part, by stimulating ubiquitin-mediated degradation of p27Kip1.

红景天苷对人舌鳞状细胞癌CAL-27细胞增殖、凋亡、周期及迁移的影响

目的研究红景天苷对人舌鳞状细胞癌CAL-27细胞体外增殖及迁移的影响。方法用四甲基偶氮唑盐(MTT)法、流式细胞检测术、Western?blot、Transwell小室实验等探究红景天苷对人舌鳞状细胞癌CAL-27细胞增殖、凋亡、周期及迁移的影响。结果红景天苷对CAL-27细胞的增殖抑制作用与浓度-时间呈正相关(P<0.05);不同浓度红景天苷处理组凋亡发生率的差异均有意义(P<0.05),且与浓度呈正相关;红景天苷对CAL-27细胞周期产生显著影响,诱导其发生G_0/G_1期阻滞;随着红景天苷浓度的增加,CAL-27细胞迁移能力显著降低(P<0.05)。结论红景天苷对人舌鳞状细胞癌CAL-27细胞的增殖、凋亡、周期及迁移产生影响,具有显著的体外抑制作用。

FAK基因沉默对人舌鳞癌细胞CAL-27增殖与迁移能力的影响

目的探讨运用RNA干扰技术沉默FAK基因表达对人舌鳞癌细胞株CAL-27增殖与迁移能力的影响。方法使用si RNA干扰技术瞬时转染构建FAK si RNA。将实验细胞分为空白对照组、阴性对照组和实验组。运用q PCR和Western blotting法检测FAK m RNA和蛋白的表达情况,MTT法检测细胞的增殖情况,Transwell小室法研究细胞的迁移能力。结果 q PCR和Western blotting实验结果显示,实验组细胞FAK m RNA和蛋白表达量明显低于空白对照组和阴性对照组(Pm RNA<0.01,P蛋白<0.05);MTT实验结果显示在48和72 h时,空白对照组和阴性对照组的吸光度值均明显高于实验组(P<0.01);Transwell实验结果显示实验组穿膜迁移细胞数明显低于空白对照组和阴性对照组(P<0.05)。结论沉默FAK基因表达可有效抑制人舌鳞癌CAL-27细胞的增殖和迁移能力。

人脐带间充质干细胞对舌鳞状细胞癌Cal-27细胞侵袭和迁移能力的影响

目的:探讨人脐带间充质干细胞(human umbilical cord blood mesenchymal stem cells,HUC-MSCs)对舌鳞状细胞癌Cal-27细胞侵袭和迁移能力的影响。方法:细胞划痕实验检测HUC-MSCs分泌的细胞因子对Cal-27细胞迁移能力的影响;Transwell小室比较共培养前后Cal-27细胞的侵袭和迁移能力;逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测共培养前后Cal-27细胞的侵袭、迁移相关基因的表达水平;Western blot技术检测肿瘤侵袭转移相关蛋白分子基质金属蛋白酶(matrix metalloproteinase,MMP)-2和MMP-9的蛋白表达的情况。结果:条件培养基培养的Cal-27细胞的迁移距离短于对照组;共培养后Cal-27细胞的侵袭和迁移细胞数目减少;侵袭迁移相关基因、蛋白的表达水平下调。结论:HUC-MSCs与Cal-27细胞共培养可以抑制Cal-27细胞的侵袭和迁移。

三氧化二砷对人舌鳞癌细胞CAL-27侵袭能力的影响

目的检测三氧化二砷对人舌鳞癌细胞CAL-27体内、外侵袭能力的影响并探讨其相关作用机制。方法体外采用黏附实验、划痕实验、Transwell侵袭实验、免疫荧光染色和蛋白质印迹法检测三氧化二砷对CAL-27细胞黏附、迁移及侵袭能力的影响;体内采用免疫组织化学和蛋白质印迹法检测三氧化二砷对裸鼠移植瘤中CD44和基质金属蛋白酶(MMP)-2和MMP-9表达的影响。结果三氧化二砷作用后,实验组CAL-27细胞的黏附、迁移及侵袭能力明显低于对照组(P<0.05),细胞骨架微丝解聚,微管结构模糊、紊乱,MMP-2和MMP-9的表达下降;三氧化二砷降低了裸鼠移植瘤中CD44、MMP-2和MMP-9的表达。结论低浓度的三氧化二砷可降低人舌鳞癌细胞CAL-27细胞黏附能力,改变细胞骨架排列,下调CD44、MMP-2和MMP-9的表达,进而抑制癌细胞的侵袭能力。

miR-30a-5p对口腔鳞状细胞癌CAL-27细胞的影响及分子机制

目的:探讨miR-30a-5p对人口腔鳞状细胞癌(OSCC) CAL-27细胞的增殖、迁移、侵袭及上皮间质转化(EMT)的影响,并探讨其分子机制。方法:人OSCC CAL-27细胞转染后,设置为miR-30a-5p mimic组(miR-30a-5p的模拟物)、NC mimic组(阴性对照模拟物)、NC inhibitor组(阴性对照抑制剂)及miR-30a-5p inhibitor组(miR-30a-5p的抑制剂),同时设未转染CAL-27细胞为Control组;采用实时荧光定量PCR检测各组细胞miR-30a-5p基因表达,采用Western blot实验检测各组细胞EMT上皮标志物E-cadherin和间质标记物N-cadherin的蛋白表达,采用3-(4,5二甲基噻唑-2)-2,5二苯基四氮唑溴盐(MTT)法、划痕实验及Transwell实验分别检测各组细胞的细胞活力、迁移能力和侵袭能力。结果:miR-30a-5p mimic组CAL-27细胞miR-30a-5p表达较NC mimic组明显上调,miR-30a-5p inhibitor组较NC inhibitor组表达明显下降(P <0. 01);miR-30a-5p mimic组CAL-27细胞OD值较NC mimic组下降,miR-30a-5p inhibitor组较NC inhibitor组上调(P <0. 05);miR-30a-5p mimic组CAL-27细胞的迁移率较NC mimic组明显下降,miR-30a-5p inhibitor组较NC inhibitor组明显上调(P <0. 01);miR-30a-5p mimic组CAL-27细胞侵袭数较NC mimic组明显下降(P <0. 01),miR-30a-5p inhibitor组较NC inhibitor组上调(P <0. 05);miR-30a-5p mimic组CAL-27细胞EMT上皮标志物E-cadherin表达较NC mimic组明显上调、间质标志物N-cadherin的表达较NC mimic组明显下调(P <0. 01),miR-30a-5p inhibitor组E-cadherin表达较NC inhibitor组明显下调、N-cadherin表达较NC inhibitor组明显上调(P <0. 01)。结论:MiR-30a-5p可抑制OSCC CAL-27细胞增殖、迁移及侵袭能力,其机制可能与miR-30a-5p抑制CAL-27细胞EMT转化有关。

沉默Icmt基因对舌鳞癌CAL-27和SCC-4细胞增殖、凋亡及细胞周期的影响

目的 :探讨异戊二烯基半胱氨酸羧基甲基转移酶(isoprenylcysteine carboxyl methyltransferase,Icmt)对舌鳞癌CAL-27和SCC-4细胞增殖、凋亡和细胞周期的影响及其相关机制。方法:针对人Icmt基因序列设计并构建3条小干扰RNA(small interfering RNA,siRNA),采用脂质体载体瞬时转染Icmt-siRNA抑制舌鳞癌CAL-27和SCC-4细胞Icmt表达,将实验组分为Icmt-siRNA-1组、Icmt-siRNA-2组、Icmt-siRNA-3组;同时将脂质体转染NC-siRNA作为阴性对照组,只加转染试剂作为空白对照组。采用实时荧光定量PCR(qRT-PCR)和蛋白质免疫印记法(Western blot)检测转染后各组细胞Icmt、K-Ras的mRNA和蛋白表达及K-Ras膜蛋白的表达;Western免疫印迹检测Cyclin D1、p21、Akt、p-Akt蛋白表达;细胞增殖活性检测试剂盒和流式细胞术检测细胞的增殖活性、周期变化和凋亡能力。应用GraphPad Prism 8.2.1软件对实验数据进行统计学分析。结果:qRT-PCR和Western免疫印迹检测结果显示,与对照组相比,实验组Icmt mRNA和蛋白表达显著下降(P<0.05),K-Ras mRNA和蛋白表达无显著差异(P>0.05),K-Ras膜蛋白表达显著下降(P<0.05);周期相关蛋白Cyclin D1表达显著下调,p21表达显著上调(P<0.05);Ras/PI3K/Akt/mTOR信号通路相关蛋白Akt表达无统计学差异(P>0.05),但p-Akt表达显著下降(P<0.05)。细胞增殖活性检测结果表明,与对照组相比,实验组细胞增殖能力显著下降(P<0.05);流式细胞术检测结果表明,实验组细胞凋亡水平较对照组显著增加,细胞周期被阻滞在G1/S期(P<0.05)。结论:体外沉默Icmt基因可有效抑制CAL-27和SCC-4细胞增殖且诱导凋亡,其作用可能是通过影响K-Ras膜蛋白靶向膜定位,负性调控细胞周期和下调Ras/PI3K/Akt/mTOR信号通路而实现。