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Objective:To observe the effects of "pushing patella and extending knee" manipulation on the protein and mRNA expression levels of integrin β1 (ITGβ1) and phosphorylated adhesion plaque kinase (p-FAK) in rabbit knee osteoarthritis (KOA) model, and to investigate the mechanism of manipulations in the treatment of KOA.Methods:Twenty healthy 6-month-old New Zealand rabbits were divided into the normal group, the model group, the acupuncture group, and the manipulation group according to the random number table method. Among them, the model group, the acupuncture group, and the manipulation group were modeled using the modified Hulth method for KOA. After 7 d of successful modeling, the normal group and the model group did not receive any intervention, while the acupuncture group and the manipulation group received one acupuncture intervention and one "pushing patella and extending knee" manipulation intervention daily, respectively. After 2 weeks of treatment, the rabbit KOA model was executed by air embolization, and the protein and mRNA expression levels of ITGβ1 and p-FAK in knee cartilage were measured by Western blot and real-time polymerase chain reaction (PCR), respectively.Results:Compared with the normal group, the ITGβ1 protein expression level was decreased ( P<0.05) and p-FAK protein expression level was increased ( P<0.05), and the mRNA expression levels of ITGβ1 and p-FAK did not change significantly (all P>0.05) in the model group. Compared with the model group, the ITGβ1 protein expression level was increased ( P<0.05), the p-FAK protein expression level decreased ( P<0.05), and the mRNA expression levels of both ITGβ1 and p-FAK increased (all P<0.05) in the acupuncture group. Compared with the acupuncture group, ITGβ1 protein expression level increased ( P<0.05), p-FAK protein expression level decreased ( P<0.05), and mRNA expression levels of both ITGβ1 and p-FAK increased (all P<0.01) in the manipulation group. Conclusions:The "pushing patella and extending knee" manipulation can optimize the protein and mRNA expression levels of ITGβ1 and p-FAK in the articular cartilage of the rabbit KOA model.
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We investigated the inhibitory effect and mechanism of action of bruceantin (BCT) on the proliferation, invasion and migration of non-small cell lung cancer (NSCLC) cells. The cytotoxic activity of BCT was measured by MTT assay; a colony forming assay, wound healing assay, and a Transwell assay were used to investigate the anti-proliferative, anti-migration, and anti-invasion effects, respectively; immunoblotting and RT-qPCR were used to detect the expression of related proteins, miRNA, and mRNA, respectively, that were involved in cell proliferation, migration, and invasion. Two gene prediction websites were used to predict the downstream target gene of miRNA. Our results show that BCT has a potent cytotoxic effect on NSCLC cell lines, with a half maximal inhibitory concentration (IC50) of BCT against H1299, PC-9, and A549 of 0.12 ± 0.02, 0.31 ± 0.20, and 2.07 ± 0.70 μmol·L-1, respectively. When H1299 cells were treated with 0.03, 0.15, and 0.75 μmol·L-1 BCT for 24 h, the proliferation, migration, and invasive ability were inhibited in a concentration-dependent manner. It is worth noting that the expression level of miRNAs related to cell migration and invasion, such as miR-29a-3p, miR-21-3p, miR-183-5p, and miR-34b-5p increased with the concentration of BCT, especially for miR-29a-3p. Using the two gene prediction websites, we predict that integrin β1 (ITGB1) may be the target gene of miR-29a-3p; immunoblot results further show that a variety of proteins related to cell proliferation, migration, and invasion, such as various proteins of the integrin family, β-catenin, p-Src, and vascular endothelial growth factor, all decreased in a concentration-dependent manner, among which the reduction of ITGB1 protein was the most obvious. RT-qPCR results showed that there was no change in ITGB1 mRNA expression. We speculate that BCT might inhibit the expression of ITGB1 protein by up-regulating miR-29a-3p independent of its mRNA level. The in-depth mechanism needs to be further explored. This study suggests that BCT has the potential for further development in the treatment of NSCLC.
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Objective To investigate the effect of erythropoietin (EPO) on the expression of integrin-β1 in hippocampus of neonatal rats after hypoxic-ischemic brain damage (HIBD) and to discuss the neuroprotective mechanisms for EPO.Methods Neonatal Sprague-Dawley rats of 7 days old were randomly divided into 3 groups by random number table method (24 cases in each group):sham-operated group,HIBD group and EPO treated group(EPO group),then each group was further divided into 4 subgroups (n=6) based on different time points following the injection of 9 g/L or EPO (6 h,24 h,3 d,7 d).The expressions of integrin-β1 and integrin-β1 gene in hippocampus were determined by Western blot and real-time fluorescent quantitative PCR(RT-qPCR).Results By Western blot method:integrin-β1 had low expression in the hippocampus of the sham-operated group at each time point,which had no significant difference(0.47±0.22,0.45±0.18,0.54±0.24,0.53±0.22,F=.223,P=0.879).The expression of integrin-β1 in HIBD group was increased at first and then decreased,which had a significant difference(0.62±0.16,1.42±0.32,1.13±0.37,0.81±0.21,F=11.809,P=0.000).In the EPO group the expression of integrin-β1 was rising significantly at 24 h and 7 d and showed two peaks (0.64±0.21,1.10±0.37,0.96±0.30,1.19±0.31,F=3.741,P=0.028).Compared with the sham-operated group at corresponding time points,the integrin-β1 protein expression showed significant increase at 24 h and on 3 d in HIBD group and 24 h,3 d,and 7 d in EPO group (all P<0.05).By RT-qPCR:there was no significant difference in the gene expression of integrin-β1 in ippocampus of the sham-operated group at different time points (0.31±0.13,0.33±0.16,0.34±0.15,0.32±0.17,F=0.036,P=0.990).Gene expression of integrin-β1 in HIBD group was increased at first and then decreased and the summit was at 24 h point(0.72±0.35,1.39±0.33,0.95±0.24,0.61±0.25).The difference between each time point was statistically significant (F=8.022,P=0.001).Integrin-β1 gene expression of EPO group showed two peaks at 24 h and on 7 d(0.77±0.30,1.09±0.27,0.90±0.38,1.17±0.36),and there was a significant difference between 6 h group and 7 d group (P<0.05).Compared with the sham-operated group at corresponding time points,the expression of integrin-β1 gene in between HIBD group and EPO group was extremely higher(all P<0.05).Besides,the gene expression of integrin-β1 in EPO group was higher than that in the HIBD group on 7 d with statistically significant difference (P<0.05).Conclusion Erythropoietin can upregulate the expression of integrin-β1 in the recovery phase of HIBD,which may be one of the beneficial effects of EPO for HIBD.
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Objective To observe the effect of low intensity pulsed ultrasound (LIPUS) on chondrocytes co-cultured with infrapatellar fat pads.Methods Twenty-four infrapatellar fat pads and cartilages from female knee trauma patients aged between 25 and 35 were cut and stained using hematoxylin-eosin staining.Chondrocytes were isolated from part of the integrated surface of the cartilages to be cultured in vitro.They were then randomly divided into a normal chondrocyte group (the control group),a normal chondrocyte+FCM (fat conditioned medium) group (the model group),a normal chondrocyte+ FCM + LIP US group (the treatment group),and a normal chondrocyte+ FCM + LIPUS + GRGDSP (an integrin inhibitor) group (the inhibited group).The treatment group and inhibited group received LIPUS at 40 mW/cm2 for 20 min once a day,while the other groups received sham LIPUS treatment.Five days later,the cells were collected and western blotting was used to examine the expression of type Ⅱ collagen (COL2),aggrecan (Acan),matrixmetalloproteinase (MMP)-13,integrin β1,phosphorylation-focal adhesion kinase (p-FAK) and phosphorylation p38 (p-p38).Results Western blotting showed that compared with the control group,the expression of COL2 and Acan was significantly lower in the model group,but that of MMP-13,integrin β1,p-FAK and p-p38 was significantly higher.As compared with the model group,the expression of COL2,Acan,integrin β1 and p-FAK was significantly higher,and that of MMP-13 and p-p38 was significantly lower in the treatment group.The expression of COL2,Acan,MMP-13,integrin β1,p-FAK and p-p38 showed no significant difference between the inhibited and model groups,but that of COL2,Acan,MMP-13,integrin β1,p-FAK and p-p38 was significantly different between the control and treatment groups.Conclusions LIPUS provides a protective effect on chondrocytes through inhibiting the expression of MMP-13 induced by adipokines and the degradation of COL2 and Acan through activating the integrin-FAK-p38 MAPK pathway.
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Objective To investigate the association of lovastatin overcoming gefitinib resistance with the levels of integrin β1 and Survivin in human non-small cell lung cancer (NSCLC) cell line PC9 in vitro, and to explore the possible mechanism. Methods The NSCLC cell line PC9 with acq uired gefitini-resistance were divided into 4 groups; control group (RPM1 1640), gefitinib group (1 /μmol/L gefitinib), lovastatin group (5 μmol/L lovastatin), and gefitinib combined with lovastatin group (1 μmol/L gefitinib+5 /μmol L lovastatin). After treatment with different drugs in each group, the inhibition of cell proliferation was detected by cell counting kit-8 (CCK-8) test, the levels of integrin ft 1 and Survivin mRNA were detected by PCR, and the expressions of integrin (31 and Survivin protein were detected by Western blotting analysis. Results Compared with the control group, lovastatin group and gefitinib group, lovastatin combined with gefitinib treatment had significantly inhibited proliferation of PC9 cells with acquired gefitinib-resistance (P<0. 01), and the expressions of integrin)31. Survivin protein and mRNA in PC9 cells were significantly decreased in the three groups (P<0. 05, P<0. 01). Conclusion Mechanisms of lovastatin combined with gefitinib in overcoming gefitinib resistance may be through blocking integrin β1-p-Akt-Survivin signaling pathway, indicating that the combination treatment might be an effective strategy for gefitinib resistance.
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Steroid sulfatase (STS) is an enzyme responsible for the hydrolysis of aryl and alkyl sulfates. STS plays a pivotal role in the regulation of estrogens and androgens that promote the growth of hormone-dependent tumors, such as those of breast or prostate cancer. However, the molecular function of STS in tumor growth is still not clear. To elucidate the role of STS in cancer cell proliferation, we investigated whether STS is able to regulate the integrin signaling pathway. We found that overexpression of STS in HeLa cells increases the protein and mRNA levels of integrin β1 and fibronectin, a ligand of integrin α5β1. Dehydroepiandrosterone (DHEA), one of the main metabolites of STS, also increases mRNA and protein expression of integrin β1 and fibronectin. Further, STS expression and DHEA treatment enhanced phosphorylation of focal adhesion kinase (FAK) at the Tyr 925 residue. Moreover, increased phosphorylation of ERK at Thr 202 and Tyr 204 residues by STS indicates that STS activates the MAPK/ERK pathway. In conclusion, these results suggest that STS expression and DHEA treatment may enhance MAPK/ERK signaling through up-regulation of integrin β1 and activation of FAK.
Subject(s)
Humans , Androgens , Breast , Cell Proliferation , Dehydroepiandrosterone , Estrogens , Fibronectins , Focal Adhesion Protein-Tyrosine Kinases , HeLa Cells , Hydrolysis , Phosphorylation , Prostatic Neoplasms , RNA, Messenger , Steryl-Sulfatase , Sulfates , Up-Regulation , Uterine Cervical NeoplasmsABSTRACT
AIM:To study the effect of integrin β1 on multidrug resistance in gastric cancer and its possible mechanisms .METHODS:The expression of integrin β1 at mRNA and protein levels in the SGC-7901 cells and SGC-7901/DDP cells was determined by qPCR and Western blot .The expression of integrin β1 in the SGC-7901/DDP cells was silenced by antisense oligodeoxynucleotide .The cell viability was detected by the CCK-8 assay, the cell apoptosis were ana-lyzed by flow cytometry, and the protein levels of integrin β1, Bcl-2/Bax, cleaved caspase-3/caspase-3, cytochrome C ( Cyt-C) and p-AKT/AKT were determined by Western blot .RESULTS:The expression of integrin β1 at both mRNA and protein levels was significantly upregulated in SGC-7901/DDP cells.The expression of integrin β1 was increased in SGC-7901 cells treated with chemotherapeutic agents such as cisplatin , paclitaxel and 5-fluorouracil .Knockdown of integrin β1 induced apoptosis of SGC-7901/DDP cells with an increased sensitivity to the chemotherapeutic agents .Meanwhile , knock-down of integrin β1 downregulated the protein levels of Bcl-2/Bax, p-AKTSer473 and p-AKTThr308 , while promoted the release of Cyt-C and upregulated the protein level of cleaved caspase-3.CONCLUSION:Knockdown of integrin β1 increases the sensitivity of SGC-7901/DDP cells to the chemotherapeutic agents , and promotes the cell apoptosis via mitochondrial apop-tosis pathway .The mechanism may be related to the attenuation of AKT pathway by inhibiting phosphorylations of AKT at Ser473 and Thr308.
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<p><b>OBJECTIVE</b>To explore the effects of acupuncture at Baihui (GV 20) and Zusanli (ST 36) on the peripheral serum expression of microRNA 124 (miRNA 124), laminin and integrin β1 in rats with cerebral ischemia reperfusion injury (CIRI).</p><p><b>METHODS</b>Seventy-two healthy male Sprague-Dawley rats were randomized into a model group, an acupuncture group, and a sham-operated group using a random digits table, with 24 rats per group. Each group was further randomly divided into 1-, 3-, 5-, and 7-day subgroups based on the reperfusion time according to a random digits table, with 6 rats in each subgroup. In the model and acupuncture groups, CIRI was induced using the thread occlusion method. Electroacupuncture stimulation was applied daily to GV 20 and left ST 36 for 20 min at the indicated time points after successful operations. Serum was sampled for detecting laminin and integrin β1 protein via enzyme-linked immunosorbent assay, and serum miRNA 124 was examined using quantitative polymerase chain reaction.</p><p><b>RESULTS</b>The serum level of miRNA 124 in the cerebral ischemia rats increased significantly, and the peak expression of miRNA 124 in both the model and acupuncture groups occurred at 3 days. The expression of miRNA 124 in the acupuncture group was higher than in the model group at the same time point (5.96±0.01 vs. 3.11±0.04, P <0.05). Laminin expression in serum from the cerebral ischemia group was higher than that in the sham-operated group. Compared with the model group, the level of laminin in the serum of the acupuncture group was significantly lower at each time point, especially at the 3-day, and 7-day time points (589.12±3.57 vs. 793.05±5.28, and 600.53±3.05 vs. 899.06±5.74, P <0.05). The level of integrin β1 in the serum from the acupuncture group was lower than that in the model group particularly at the 3-day and 7-day time points (208.66±0.95 vs. 280.83±1.77, and 212.36±0.95 vs. 316.77±2.42, P <0.05). Additionally, the model group and the acupuncture group showed dual peaks of integrin β1 and laminin expression at 3-day and 7-day.</p><p><b>CONCLUSIONS</b>Acupuncture at GV 20 and ST 36 in rats alleviated CIRI and was associated with upregulated expression of miRNA 124 and with downregulated expression of integrin β1 and laminin in peripheral serum. These changes may represent one of the mechanisms underlying acupuncture's attenuation of CIRI.</p>
Subject(s)
Animals , Male , Acupuncture Points , Acupuncture Therapy , Methods , Brain Ischemia , Blood , Genetics , Therapeutics , Gene Expression Regulation , Integrin beta1 , Blood , Genetics , Laminin , Blood , MicroRNAs , Blood , Genetics , Rats, Sprague-Dawley , Reperfusion Injury , Blood , Genetics , TherapeuticsABSTRACT
Objective: To probe into the relationship between integrin β1 expression in human cervical carcinoma HCE1 multicellular spheroids (HCE1/MCS) and their invasion into human umbilical vein endothelium cells (HUVEC), so as to assess the inhibitory effect of anti-integrin β1 monoclonal antibody P5D2 on the invasion of HCE1/MCS into HUVEC. Methods: A model of HUVEC monolayer invaded by HCE1/MCS was established (in brief, the HCE1 invading model). Morphology changes of the HCE1 invading model were observed under inverted microscope and analyzed by Motic Med System after P5D2 treatment. The expressions of β1 integrin on HCE1 monolayer cells, HCE1/MCS, HVUEC monolayer cells, and P5D2-treated HCE1 invading models were measured by immunocytochemistry SABC assay. Results: A HCE1 invading model was successfully established. The HCE1/MCS proliferated rapidly after culture, and on the 7th day the invading area of HCE1/MCS was 40.42 folds larger than the original HCE1/MCS. Invading areas of P5D2-treated HCE1/MCS were significantly smaller than that of the control group after 1, 4, 7 day (P<0.05, or P<0.01), with the invading area after 7 days reduced by (84.68±0.08) % compared with the control group. Integrin β1 expression in HCE1/MCS was significantly higher than that in HCE1 monolayer cells (P<0.01), and the expression was negative in HUVEC. Integrin β1 expression in P5D2-treated HCE1/MCS was significantly lower than that the in untreated HCE1/MCS (P<0.01). Conclusion: Upregulated expression of integrin β1 in HCE1/MCS and HCE1 invading model may be associated with their enhanced adhesion and invasion abilities. Anti-integrin β1 monoclonal antibody P5D2 can partially block the invasion of HCE1/MCS into HUVEC.
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Organ-specific tumor cell adhesion to extracellular matrix (ECM) components and cell migration into host organs often involve integrin-mediated cellular processes.Direct integrin-mediated cell adhesion to ECM components in the space of Disse appears to be required for the successful liver metastatic formation of colon cancer.In the present study,human colon cancer HT-29 cells were transfected by liposome with integrin-β1 antisense oligodeoxynucleotide (ASODN).The integrin-β1 gene expression in HT-29 cells was significantly down-regulated.The migration of HT-29 cells was assayed using transwell cell culture chambers in vitro.The number of migrating HT-29 cells in experimental group was far less than that in control group (P＜0.05).The models of hepatic metastasis in nude mice were established by the intrasplenic injection of transfected HT-29 cells.Thirty days later,the nude mice were killed and the average number of hepatic metastases (4.00±0.93 per mouse),average volume (10.10±6.50 mm3 per mouse),average weight (0.0440±0.0008 g per mouse) in experimental group were remarkably reduced as compared with those in control group (P＜0.05).Integrin-β1 expression in the hepatic metastasis was studied by immunohistochemistry (SP).Positive cell percentage of hepatic metastases in experimental group was markedly decreased as compared with that in control group (P＜0.05).It was concluded that integrin-β1 may take part in hepatic metastasis,and down-regulation of integrin-β1 expression may play a key role in decreasing migration and hepatic metastasis of human colon carcinoma cells (HT-29).
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Objective To investigate the expression of integrin β1 and VEGF in gastric carcinoma and evaluate its correlation with invasion and metastasis.Methods Immunohistochemistry techniques were used to study the expression of integrin β1 and VEGF in 63 gastric carcinoma specimens.We analyzed the variance with histological grade and stage and classification of clinical date.Results No significant relationship was observed among age,sex and histological classification of the expression of integrin β1 in the gastric cancer group (P>0.05).With the depth of invasion and lymphatic metastasis increasing,integrin β1 expression improved.The positive rate of integrin β1 among tubular adenocarcinoma,papillary carcinoma,mucinous carcinoma and undifferentiated carcinoma was 70.00 %,55.56 %,57.14 % and 64.86 %.No significant relationship was observed in it.Integrin β1 in cases with lymphnode metastasis was significantly higher than those without lymph node metastasis (P<0.05).No significant relationship was observed among VEGF expression and age,sex,histological classification and depth of invasion (P>0.05).Significant relationship was observed among VEGF expression and lymphatic metastasis.VECF in cases with lymph node metastasis was significantly higher than those without lymph node metastasis (P<0.05).Conclusion Immunohistochemistry techniques indicated that integrin β1 participated in the procession of invasion and metastasis.The expression of integrin β1 and VEGF are important factor,forecasting the invasion and metastasis.VEGF is an important factor for lymphnade metastasis of gastric carcinoma.VEGF in cases with lymphnode metastasis was significantly higher than those without lymphnode metastasis(P<0.05).The usage of integrin β1 and VEGF will improve the rightness in forecasting the invasion and lymph node metastasis of gastric carcinoma.