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In recent years, more and more attention has been paid to the integration of nano-material graphene and field effect transistor to construct biosensor for detecting biomolecules.This is mainly due to the following advantages of graphene field effect transistor inculding high sensitivity and specificity, rapid analysis, label-free detection, cheap price, miniaturization and integration.This review summarizes the structure and working principle of graphene field effect transistor biosensor, the preparation and functionalization of graphene, and the application of graphene field effect transistor in medical detections.
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Objective To explore the expression of tiny RNA-25 (microRNA-25, miR-25) in the plasma、tissues of triple-negative breast cancer(TNBC) patients and cell lines, to investigate the potential molecular mechanisms of miR-25 on migration and invasion of TNBC. Methods Real-time fluorescent quantitative PCR was used to detect the expression of miR-25 in the plasma of TNBC patients. Linked omics web platform was used to analyse miR-25 level in samples of TNBC and non-TNBC. Real-time fluorescent quantitative PCR was also used to detect the miR-25 level in TNBC cell lines. The wound healing and transwell assay was applied to assess the effects on migration and invasion of TNBC cell lines which transfected with miR-25 inhibitor or the negative control. The luciferase reporter assay was used to validate the relationship between miR-25 and the sphingosine-1-phosphate phosphatase 1 (SGPP1) in HEK293T cell. The wound healing and transwell assay was used to detect the migration and invasion ability of TNBC cell lines when cotransfected with pCMV6-SGPP1 and miR-25. Furthermore, Western blot was performed to detect the SGPP1 level in TNBC cell lines. Results The expression of miR-25 was significantly elevated in the plasma of 86 TNBC patients compared with the healthy controls (P value was 0.031). LinkedOmics web platform analysis showed that miR-25 expression was significantly higher in TNBC samples than in non-TNBC samples with Luminal A or Luminal B (P value was<0.001 and 0.006). The level of miR-25 was also elevated in TNBC cell lines HS578T, HCC1806, MDA-MB-231 and BT549(P value was 0.006, 0.01, 0.029 and 0.046). The MDA-MB-231 and HS578T cells which transfected with miR-25 inhibitor exhibited a significant slower wound healing rate than control (P value was 0.035 and 0.001). At the same time, when transfected with miR-25 inhibitor, MDA-MB-231 and HS578T both exhibited a decreased invasion ability compared with the control group(P value was 0.002 and 0.001). LinkedOmics web platform analysis showed that sphingosine-1-phosphate phosphatase 1 (SGPP1) gene level was negatively correlated with miR-25 in the tissues of TNBC patients (P value was 0.037). The luciferase reporter assay validated that SGPP1 was a directed target of miR-25. The western blot assay indicated that the SGPP1 level was increased in MDA-MB-231 and HS578T after transfection with miR-25 inhibitor. Over-expression of SGPP1 could abrogate the positive effects of miR-25 on migration and invasion when pCMV6-SGPP1 was cotransfected with miR-25 (P value was all 0.002). Conclusions MiR-25 was elevated in both plasma and tissues of TNBC patients and also increased in TNBC cell lines. Transfection of MDA-MB-231 and HS578T cells with miR-25 inhibitor resulted in reduced migration and invasion. Moreover, SGPP1 was identified as a novel target of miR-25. The ability of miR-25 to promote TNBC cell migration and invasion is attributable to its effect on SGPP1 suppression.
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Objective To evaluate the effect of Annexin-A1 mimetic peptide Ac2-26 on activation of astrocytes in rats.Methods The primarily cultured astrocytes from the cortex of fetal Sprague-Dawley rats after 4 passages were divined into 4 groups (n =24 each) using a random number table method:control group (C group),LPS group,LPS+scramble peptide group (LPS+Src group) and LPS+Ac2-26 group.LPS was added to LPS group with the final concentration of 1 mg/ml.LPS at the final concentration of 1 mg/ml and scramble peptide at the final concentration of 3.3 mmol/L were added to LPS+Src group.LPS at the final concentration of 1 mg/ml and Ac2-26 at the final concentration of 3.3 mmol/L were added to LPS+Ac2-26 group.After 24-h incubation,the cell survival rate was measured by CCK-8 assay,the migration was determined by Transwell assay,the concentrations of tumor necrosis factor-alpha (TNF-α),interleukin-1 beta (IL-1β),monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 a (MIP-1a) in the supernatant were measured (by enzyme-linked immunosorbent assay),and the expression of glial fibrillary acidic protein (GFAP),extracellular signal-regulated kinase (ERK),phosphorylated ERK (p-ERK),c-Jun N-terminal kinase (JNK),phosphorylated JNK (p-JNK),p38 mitogen-activated protein kinase (p38MAPK),and phosphorylated p38MAPK (p-p38MAPK) in astrocytes was detected by Western blot.Results Compared with group C,the expression of GFAP was significantly up-regulated,and the cell mobility,concentrations of TNF-α,IL-1β,MCP-1 and MIP-1α in the supernatant,p-ERK/ERK ratio,p-JNK/JNK ratio and p-p38MAPK/p38MAPK ratio were increased (P＜0.05),and no siguificant change was found in the cell survival rate in group LPS (P＞0.05).Compared with group LPS,the expression of GFAP was significantly down-regulated,and the cell mobility,concentrations of TNF-α,IL-1β,MCP-1 and MIP-1α in the supernatant,p-JNK/JNK ratio and p-p38MAPK/p38MAPK ratio were decreased in group LPS+Ac2-26 (P＜0.05),and no significant change was found in the parameters mentioned above in group LPS+Src (P＞0.05).Conclusion Ac2-26 can inhibit activation of astrocytes and produces anti-inflammatory effect in rats.
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Objective@#To evaluate the effect of Annexin-A1 mimetic peptide Ac2-26 on activation of astrocytes in rats.@*Methods@#The primarily cultured astrocytes from the cortex of fetal Sprague-Dawley rats after 4 passages were divined into 4 groups (n=24 each) using a random number table method: control group (C group), LPS group, LPS+ scramble peptide group (LPS+ Src group) and LPS+ Ac2-26 group.LPS was added to LPS group with the final concentration of 1 mg/ml.LPS at the final concentration of 1 mg/ml and scramble peptide at the final concentration of 3.3 mmol/L were added to LPS+ Src group.LPS at the final concentration of 1 mg/ml and Ac2-26 at the final concentration of 3.3 mmol/L were added to LPS+ Ac2-26 group.After 24-h incubation, the cell survival rate was measured by CCK-8 assay, the migration was determined by Transwell assay, the concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1a (MIP-1a) in the supernatant were measured (by enzyme-linked immunosorbent assay), and the expression of glial fibrillary acidic protein (GFAP), extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), p38 mitogen-activated protein kinase (p38MAPK), and phosphorylated p38MAPK (p-p38MAPK) in astrocytes was detected by Western blot.@*Results@#Compared with group C, the expression of GFAP was significantly up-regulated, and the cell mobility, concentrations of TNF-α, IL-1β, MCP-1 and MIP-1α in the supernatant, p-ERK/ERK ratio, p-JNK/JNK ratio and p-p38MAPK/p38MAPK ratio were increased (P<0.05), and no significant change was found in the cell survival rate in group LPS (P>0.05). Compared with group LPS, the expression of GFAP was significantly down-regulated, and the cell mobility, concentrations of TNF-α, IL-1β, MCP-1 and MIP-1α in the supernatant, p-JNK/JNK ratio and p-p38MAPK/p38MAPK ratio were decreased in group LPS+ Ac2-26 (P<0.05), and no significant change was found in the parameters mentioned above in group LPS+ Src (P>0.05).@*Conclusion@#Ac2-26 can inhibit activation of astrocytes and produces anti-inflammatory effect in rats.
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Objective To investigate the role of OMA1 in acute kidney injury (AKI) induced by lipopolysaccharide (LPS).Methods OMA1 wild-type and knocked out mice (8 week old) were injected with 10 mg/kg body weight of LPS.The model was confirmed by testing mouse serum creatinine and blood urea nitrogen.The apoptosis in mouse kidney cortex was examined by TUNEL staining and cleaved caspase 3.In vitro,in humam kidney proximal tubular cells (HK2) were knocked down OMA1 by transfecting OMA1 shRNA,with the scramble shRNA being used as negative control of transfection.HK2 cells were cultured with 5 μg/ml of LPS for 24 hours to induce apoptosis.DAPI staining of cells and caspase-3 activity were applied to test apoptosis.The images of mitochondria in cells were obtained by transfection of mito-green plasmid and OMA1 shRNA.Western blotting was used to exam the OMA1 and Cytochrome C expressions.Resudts Compared with OMA1 KO mice,LPS induced more severe AKI of WT mice with higher Scr [(97.2±26.5) μmol/L vs (53.0±17.7) μmol/L,P ＜ 0.05] and BUN [(43.3± 13.7) mmol/L vs (29.7±7.7) mmol/L,P ＜ 0.05].Moreover,there were more apoptosis cells in kidney cortex in WT mice than in OMA1 KO mice [(75.4± 26.1)/ram2 vs (38.3± 14.4)/mm2,P＜ 0.05].About 46％ of OMA1 expressions in HK2 cells were inhibited by OMA1 shRNA transfection (P ＜ 0.05).Further,OMA1 shRNA cells with LPS stimulation had decreased mitochondria fragmentation [(29.8±10.9)％ vs (43.2±6.8)％,P ＜ 0.05],Cytochrome C release [(37.0±12.3)％ vs (76.0±26.2)％,P ＜ 0.05],and cell apoptosis [(13.2±3.9)％ vs (25.0±7.1)％,P ＜ 0.05] as compared with control cells.Conclusion Knockdown of OMA1 alleviated septic AKI through inhibition of cell apoptosis,mitochondria fragmentation,and Cytochrome C release.
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Objective To investigate the effects of Annexin-A1 ( Anxa1 ) gene silencing induced by siRNA on the growth and migration of microglial BV-2 cells and its possible mechanisms.Methods A synthesized siRNA duplex targeting Anxa1 gene was transfected into BV-2 cells.The efficiency of siRNA-in-duced Anxa1 gene silencing was evaluated on both mRNA and protein levels by using reverse-transcription PCR and Western blot assay.MTT assay was performed to measure the proliferation of BV-2 cells with si-lenced expression of Anxa1 gene.Flow cytometry with Annexin V-FITC/PI double staining was used to de-tect the apoptosis rate of BV-2 cells.Transwell chambers were used to analyze the effects of siRNA-induced Anxa1 gene silencing on the migration of BV-2 cells.Western blot assay was performed to detect the expres-sion of signaling proteins related to cell cycle and migration.Results Compared with the siRNA negative control ( siRNA-NC) group, the inhibitory rates of siRNA-induced Anxa1 gene silencing on the proliferation of BV-2 cells were significantly increased at the time points of 24 h, 48 h and 72 h after intervention [(16.9 ±2.1)%, (23.1±3.6)%and (42.4±1.7)%vs (1.35±0.5)%, (2.06±0.7)% and (8.65±0.9)%, P<0.05 ].The apoptosis rate of BV-2 cells transfected with Anxa1 siRNA was (18.4±2.1)%, which was significantly elevated as compared with that of the siRNA-NC group (5.2±0.3)%and control group (4.3±0.2)%.Cell migration of the Anxa1 siRNA transfected BV-2 cells was inhibited remarkably at 48 h as com-pared with that of the siRNA-NC group (28.7±5.2 vs 173.4±11.4, P<0.01).Moreover, the suppressed expression of Cyclin D1 protein and activation of p38 and JNK signaling pathways were induced by silenced expression of Anxa1 gene in BV-2 cells.Conclusion The growth and migration of BV-2 cells were signifi-cantly inhibited by silencing the expression of Anxa1 gene with siRNA, the possible mechanisms might be associated with the suppressed expression of Cyclin D1protein and the activation of p38 and JNK signaling pathways.
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OBJECTIVE@#To investigate the association between-1304T/G polymorphism in the promoter of MKK4 gene and the susceptibility in sporadic nasopharyngeal carcinoma.@*METHOD@#MKK4-1304T/G genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 90 NPC cases and 30 healthy controls.@*RESULT@#The number of nasopharyngeal carcinoma patients carrying with TG+GG genotype was much higher than those of controls (82.2% vs 66.7%, χ² =10.076, P < 0.05). Analysis showed that compared with the-1304TT genotype, -1304TG heterozygous reduced risk of nasopharyngeal carcinoma 0.56 fold (95% CI = 0.164-1.178, P < 0.01) and-1304GG lower 0.58 fold (95% CI = 0.126-1.381, P < 0.01), TG+ GG genotype variation risk of nasopharyngeal carcinoma decreased 0.72 fold (95% CI = 0.105-0.753, P < 0.01).@*CONCLUSION@#MKK4 gene-1304TG genotype can reduce risk of nasopharyngeal carcinoma, and it may be an independent protection factor in sporadic nasopharyngeal carcinoma.
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Humanos , Carcinoma , Predisposición Genética a la Enfermedad , Genotipo , Heterocigoto , MAP Quinasa Quinasa 4 , Genética , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Genética , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Regiones Promotoras GenéticasRESUMEN
Objective To investigate the expression level of microRNA-145 in breast cancer cell lines andtissues and its impact on breast cancer invasion and metastasis.Methods MiR-145 expression was detected by FQ-PCR in 5 breast cancer cell lines ( HBL-100, MCF-7, MDA-MB-231, MDA-MB-468 and SK-BR-3)and in breast cancer tissue and paraneoplastic tissues (n=39).The miR-145 expression plasmid ( Psif-miR-145 ) and negative control plasmid were transfected into SK-BR-3 using lipofectamine, respectively.The characteristics of invasion and migration of the transfected SK-BR-3 cells were examined by scratch test and transwell assay.The target genes of miR-145 were predicted by bioinformatics and the ANGPT2 gene were verified as miR-145 target by the dual-luciferase reporter assay.The expression levels of ANGPT2 protein was examined by western blot after pSIF-miR-145 transfection by lipofectamine in breast cancer cell line SK-BR-3.Results FQ-PCR result indicated that miR-145 expression level waslower in breast cancer tissue (45.93 ±22.02)than paraneoplastic tissue [ (182.04 ±56.92), U value was 7, P<0.01].MiR-145 expression level was lower in breast cancer cell lines than normal breast cells.miR-145expression in 4 breast cancer cell lines was 0.51 ±0.05, 0.07 ±0.01, 0.36 ±0.04 and 0.04 ±0.01, respectively.Compare with normal breast cell, miR-145 was lower expressed in all 4 breast cancer cell lines (t value separately was 15.93, 308.17, 25.02, 201.30;P<0.05).Lower expression of miR-145 was observed in the highly invasive breast cancer cells (MDA-MB-231, MDA-MB-468 and SK-BR-3), compared with weakly invasive breast cancer cell (MCF-7) (t value separately was14.18, 3.78, 15.20;P<0.05). Wound healing assay shows that overexpression of miR-145 in SK-BR-3 significantly reducedthe motility as compared with control group (P <0.01).The cell invasion assay indicated the numbers of miR-145 overexpressed SK-BR-3 cells, which invased to lower chamber, was 137 ±37, the numbers of invased cells was 617 ±80 when the negative control was applied. Over-expression of miR-145 could repress the expression levelsof ANGPT2 protein;miR-145 could repress the activity of luciferase reporter carrying a 3′-untranslated region of ANGPT2 mutated the predicted binding site, the activity of luciferase was reversed. Conclusions MiR-145 depressed in breast cancer cell lines and breast cancer tissues.MiR-145 maybe plays an important role in breast cancer invasion and migration by directly target ANGPT2.
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Objective:To investigate the expression of MKK 4 protein in the nasopharyngeal carcinoma and its clinical significance.Methods:Immunohistochemical methods were employed to analyze MKK 4 positive expression intensity and positive cells in freshly collected nasopharyngeal carcinoma of both 90nasopharyngeal carcinoma cases and 20 chronic nasopharyngitis control.The clinical pathological characteristic were analyzed.Results:The data obtained by MKK4 immunohistochemistry showed that the MKK 4 positive rate was higher in control group than in the NPC group (95.5%vs 75.6%,P0.05 ) . Conclusion:Positive rate of MKK4 protein in nasopharyngeal carcinoma tissues is lower than in chronic nasopharyngitis.MKK4 protein expressions in nasopharyngeal carcinoma tissues negatively correlated with lymph node metastasis ,clincal stage ,invasive depth ,and TTP (Time to progression),but not with age,gender,location and tumor volume.
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Objective To investigate the expression of microRNA-21(miR-21)in breast cancer cell lines and serum of patients with breast cancer and the impact on the invasion and migration of breast cancer cells.Methods From Jan 2013 to Feb 2014, miR-21 expression were determined by fluorescent quantity polymerase chain reaction (FQ-PCR) in 4 breast cell lines (HBL-100, MCF-7, MDA-MB-231 and MDA-MB-468) and in serum from breast cancer patients ( n =56 ) , breast benign disease patients ( n =39 ) andhealth controls ( n =45 ) . The characteristics of cell invasion and migration were examined by transwellinvasion and migration assay afterbreast cancer cell line MDA-MB-231 were transfectedwith miR-21 inhibitor or negative control by lipofectamin.The t test was used to analysis the normal distribution data. Results FQ-PCR results showed that the relative expression of miR-21 in the normal breast epithelial cell line HBL-100 was 1.01 ±0.04, in the breast cancer cell line MCF-7, MDA-MB-231 and MDA-MB-468 were 1.99 ±0.11,4.02 ±0.38 and 3.73 ±0.79 respectively.Compared with the normal controls, miR-21 were highly expressed in the three breast cancer cell lines, the difference was statistically significant (t=9.01, 9.20 and 4.55, respectively, P<0.01); and the miR-21 was highly expressed in invasive and metastatic breast cancer cell lines (MDA-MB-231 and MDA-MB-468),compared with weakly invasive breast cancer cell line MCF-7, the difference was statistically significant ( t values were 6.14 and 2.91, P<0. 05), suggesting that miR-21 is highly expressed in breast cancer cells, and is closely related to the invasion and metastasis.The relative expression of miR-21 in serum of breast cancer was 2.63 (1.57-4.59), in benign breast disease group was 1.34 (1.01-1.78), in healthy control group was 0.81 (0.52-1.59), the miR-21 expression in the serum of breast cancer patients was significantly higher than in patients with benign lesions and normal control group (U values were 208 and 279, P<0.01), whereas no significant difference in serum in patients with benign lesions and normal control group, the miR-21 expression in the serum of breast cancer patients with lymph node metastasis (U=95 , P=0.19) was 3.55 (2.44-5.26), significantly higher than those without lymph node metastasis [2.11(1.59-3.25), U=216,P=0.021]. The results of invasion and migration assay showed that cells treated with miR-21 inhibitor invasion was:44 ±18, the number of cell migration was:98 ±22, while the negative control treated cells after invasion was:133 ±44, migration cell number:255 ±35;miR-21 inhibitor treatment compared with the negative control, cell invasion and migration was also significantly decreased( t values were 5.46 and 9.08, P<0. 01) .The cell invasion and migration assay indicated the numbers of MDA-MB-231 cells, which invaded or migrated to lower chamber, were 44 ±18 and 98 ±22 respectively after miR-21 inhibitor was applied, The numbers of invaded or migrated cells were 133 ±44 and 255 ±35 when the negative control was applied.The ability of cell invasion and migration was decreased significantly in the inhibitor group compared with the negative group(tvalue separately was 5.46, 9.08, P<0.01).The capacity of breast cancer cell invasion and migrationwas significantly decreased after transfection ofmiR-21 inhibitor.Conclusions MiR-21 is highly expressed in breast cancer cell lines and breast cancer patients′serum.Altered expression of miR-21 maybeplays an important role in breast cancer invasion and migration.MiR-21 may serve as new biomarker to early detectionand prognosis estimation of breast cancer.
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Objective To investigate the expression levels of serum visfatin ,leptin and other indexes and their correlation and clinical significance in the patients with type 2 diabetes mellitus(T2DM ) .Methods Eighty-two age-matched and gender-matched cases of T2DM (T2DM group)and 71 cases of healthy subjects(health control group)were tested serum visfatin ,leptin ,insulin ,lipid and glycemia levels .Results The serum visfatin ,leptin ,triglycerides(TG) ,total cholesterol(TC) ,high-density lipoprotein choles-terol(HDL-C) and low density lipoprotein cholesterol(LDC-C) ,fasting plasma glucose ,fasting insulin(FINS) ,homeostasis model assessment of insulin resistance(OMA-IR) ,2 h FPG ,2 h FINS had statistically significant differences between the T 2DM group and the health control group(P< 0 .05);the stepwise regression analysis showed that visfatin were positively correlated with BMI , FINS ,HOMA-IR ,TC ,TG ,LDL-C (P<0 .05)and negatively correlated with HDL-C(P<0 .05);leptin were positively correlated with BMI ,FINS ,HOMA-IR(P<0 .05) .Conclusion Serum visfatin and leptin in the T2DM patients are hither than those in the healthy subjects and closely related with blood lipid ,blood glucose and insulin ,which may become the new diagnostic indexes of T2DM .
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Objective To apply nitric oxide(NO) electrochemical microsensor in the real time detection of NO released from RAW 264 .3 cells infected by E .coli ,and to explore the application value of this NO microsensor in the research area of infection im‐munity against bacterium .Methods Taking NO microsensor to detect NO released from RAW 264 .3 cells respectively stimulated by E .coli of different densities and of 1 × 107 mL -1 for different time .Results The level of NO released from RAW 264 .3 cells was enhanced obviously when incubated with E .coli as compared with that of normal cells and the extent of incersase depended on the density of E .coli (P<0 .01) .The released level of NO increased gradually from the beginning and reached its peal at the time of 12 h then decreased slowly when incubated with E .coli of 1 × 107 mL -1 .Conclusion The electrochemical microsensor was applied in the real time detection of NO released from macrophages activated by E .coli successfully .
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Objective To explore the performance of the novel nitric oxide(NO) electrochemical microsensor based on Au nano-particles(nano-Au) modified glass-fiber .Methods With columnar glass-fiber as the substrate material ,the in-situ chemical seed-growth technology was adopted to fabricate one kind of electrochemical microsensor based on gold nanoparticles pole electrodes and the amperometric response method was used to investigate the various properties of the NO microsensor .Results This microsensor had high sensitivity response to NO ,its linear range was 7 .2 nmol/L -11 .7μmol/L(r=0 .998) with a detection limit of 3 .6 nmol/L .Conclusion A novel electrochemical microsensor based on nano-Au pole electrode was fabricated .The NO microsensor posseses high sensitivity ,wide linear range ,good reproducibility and excellent anti-interference ability .
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Background and purpose: Plumbagin is the main active components of traditional Chinese medicine of plumbago zeylanica. The present studies show that plumbagin has a killing effect on tumor cells. This study aimed to investigate the function and primary mechanism of plumbagin on invasion and metastasis of human liver cancer SK-hep-1 cells. Methods:With the treatment of plumbagin in vitro, cell proliferation and adhesion of SK-hep-1 cells were detected by MTS staining, cell cycle of SK-hep-1 cells were detected by lfow cytometry, the self-renewal and propagation abilities of SK-Hep-1 cells were conducted by colony formation assay , invasion in cells were performed using transwell invasion assay, and the p21 and MMP-2/9 mRNA levels were detected by real-time RT-PCR. Results:With the treatment of plumbagin, SK-Hep-1 cells proliferation was decreased with plumbagin concentration-dependency and the IC50 value of plumbagin in SK-Hep-1 cells was 22.04 mmol/L. The colony formation ability of SK-Hep-1 cells was decreased and the percentage of cells in G0/G1 phase was increased in a dose-dependent manner, as compared to control. The cell adhesion and invasion abilities were decreased. The real-time RT-PCR showed that p21 mRNA expression was increased and the MMP-2/9 mRNA was decreased. Conclusion:Plumbagin could suppress the proliferation and invasiveness of human liver cancer SK-hep-1 cells in vitro, and these effects may be by up-regulation of p21 and down-regulation of MMP-2 and MMP-9.
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ObjectiveTo study the expression of microRNA-21 ( miR-21 )in serum of patient with diffuse large B cell lymphoma (DLBCL) and DLBCL cell lines and validate the significance of miR-21 in early diagnosis,genotyping and prognosis estimates of DLBCL.MethodsmiR-21 expression were detected by fluorescent quantity polymerase chain reaction (FQ-PCR)in 9 lymphoma cell lines (OCI-Ly1,OCI-Ly3,OCI-Ly4,OCI-Ly7,OCI-Ly8,OCI-Ly10,OCI-Ly18,OCI-Ly19 and HBL),the serum from DLBCL patients (n =62) and health controls (n =50 ).Kaplan-Meier survival analysis was carried out during the relapsefree survival period of DLBCL patients to explore the relationship between the prognosis and microRNA expression level.ResultsReal time FQ-PCR result indicated that miR-21 expression was higher in DLBCL cell lines than that in normal B cells (BC).miR-21 expression in normal B cell and 9 DLBCL cell lines separately were 1.04 ± 0.02,2.30 ± 0.35,237.97 ± 56.19,5.27 ± 0.83,3.40 ± 0.30,11.22 ± 2.70,133.55 ± 16.78,6.63 ±0.24,4.91 ±0.37 and 81.59 ±6.64.Compared with BC,the expression of miR-21 were higher in all 9 DLBCL cell lines ( t =7.3,13.7,21.0,6.2,8.8,13.6,6.5,39.5,18.1 ;P ＜ 0.01 ).miR-21 expression segregates with specific molecular subgroups of DLBCL The expression was higher in the ABC type cell lines (OCI-Ly3,OCI-Ly10,HBL) than GCB type cell lines (OCI-Ly1,OCI-Ly4,OCI-Ly7,OCI-Ly8,OCI-Ly18,OCI-Ly19;t =11.18,P ＜ 0.01 ).Consistent with the cell line models,miR-21 expression levels were higher in serum from DLBCL patients [21.38 (10.26-45.21 )] than from controls [1.87 ( 1.05-3.97 ),U =168,P =0.000],and the levels were higher in DLBCL cases with an ABC-type [28.68 ( 14.92-98.44 )] than those in GCB-type [18.30 ( 7.32-33.46 ),U =336,P =0.043].MiR-21 expression levels were different in sera from different clinical stage DLBCL patients.The miR-21 level in serum of patients with subgroup ABC and subgroup GCB in stage Ⅰ and Ⅱ were 47.49( 25.65-295.41 ) and 24.74( 16.08-50.38) respectively and in stage Ⅲ and Ⅳ were 16.66 ( 5.35-44.30 ) and 11.96 ( 4.10-21.05) respectively.The levels were higher in DLBCL cases withⅠ -Ⅱ stage than those with Ⅲ-Ⅳ stage (U =62,P =0.013 in GCB type; U =53,P =0.014 in ABC type).Moreover,compare with relapse-free survival in DLBCL patients,high miR-21 expression was associated with well prognosis ( U =259,P =0.035).ConclusionsMiR-21 is high expression in DLBCL cell lines and DLBCL patients serum.miR-21 level in sera from DLBCL patients is associated with clinical stage,molecular subgroup and prognosis estimates.MiR-21 may serve as a new biomarker to early detection,genotyping and prognosis estimates of DLBCL.
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Objective To study the expression of microRNA-301 in pancreatic carcinoma andvalidate the significance of miR-301 in invasion and metastasis of pancreatic carcinoma.Methods miR-301 expression were detected by FQ-PCR in 5 pancreatic cancer eell lines(PANC-1,PaCa-2,AsPC-1,Hs766T.BxPC-3).Further immunohistochemistry in pancreatic cancer tissue microarrays was detected miR-301 expression,which contained 60 pancreatic cancer specimens along with 10 normal adjacent tissues and 10 normal pancreas tissues.After high expression of miR-301 in pancreatic carcinoma being confirmed.the clinical significance of high expression of miR-301 in invasion and metastasis of pancreatic carcinoma were studed.Pancreatic cancer cell lines(PANC-1.PaCa-2)were transfected by 100 nmoml/L miR-301 inhibitor(anti-miR-301)or negative eontrol(Anti-miR~(TM) Negative Control#1).COX-2 and MMP-2 protein expression in pancreatic cancer cell lines were detected by WB.and cell migration assays were performed using transwell technology.Results FQ-PCR resuhs indicated that miR-301 expression was higher in pancreatic cancer cell lines than normal pancreatic cells.The relative level of miR-301 in 5 pancreatic cancer cell lines(PANC-1,PaCa-2,AsPC-1,Hs-766T,BxPC-3)and normal pancreatic cell were 33.09± 4.21,30.76±3.18,47.57±3.56,20.20 ±1.21,76.75±13.51 and 1.00±0.08 respectively.The miR-301 level in all 5 pancreatic cancer cells were significantly higher than those of normal pancreatic cell(t=8.86,9.53,6.39,6.77,11.18,P<0.01).Immunohistochemistry results also showed miR-301 expression was higher in pancreatic carcinoma tissues than those in the cancer adjacent tissues and normal pancreatic tissues.The relative levels of miR-301 in pancreatic carcinoma tissues.normal adjacent tissues and normal pancreas tissues were 0.88±0.09,0.22±0.04 and 0.14±0.05 respectively.The miR-301 levels in pancreatic carcinoma tissues were significantly higher than those of normal adjacent tissues and normal pancreatic tissues(t=15.1,10.6,P<0.01).There was no significant difference between normal adjacent tissues and normal pancreas tissues(t=1.32,P=0.22).After miR-301 inhibitor was introduced into pancreatic cancer cells PANC-1 and PaCa-2.miR-301 levels were reduced while the protein levels of COX-2 and MMP-2.which were invasion and metastasis related factors,were down-regulated.The cell migration assay indicated the numbers of PANC-1 and PaCa-2 cells,which migrated to lower chamber.were 587±27 and 363±13 respectively after miR-301 inhibitor was applied.The numbers of migrated cells were 1091 4-15.737±44 when the netative control was applied.The cell invasion ability was decreased significantly in the inhibitor group compared with the negative group(t=7.89,7.56,P<0.01).Conclusions miR-301 is highly expressed in pancreatic cancer cell lines and pancreatic cancer tissues.Inhibition of miR-301 expression can effectively supress the invasion of pancreatic cancer cells.miR-301 may serve as a new biomarker for early detection of pancreatic cancer and molecular target for early treatment of pancreatic cancer.