Objective To investigate the molecular characteristics of immune response signaling molecules induced by transfection of coxsackievirus B2 ( CVB2 ) structural proteins into epithelial cells. Methods Recombinant eukaryotic expression plasmids containing the coding regions of CVB2 structural proteins VP1-VP4 were constructed and then transfected into 16HBE cells. Culture supernatants and cell ly-sates of the transfected 16HBE cells were collected. Expression of signaling molecules involved in innate im-mune responses in transfected 16HBE cells at mRNA level was detected by RT-Q-PCR. The proliferation of T cells co-cultured with culture supernatants and cell lysates of the transfected 16HBE cells was analyzed by ELISPOT. Results Expression of innate immunity-related signaling molecules such as TGF-β-activated ki-nase ( TAK) , NF-κB-inducing kinase ( NIK) , IκB kinase α ( IKKα) and IFN-β at mRNA level was up-regulated in 16HBE cells transfected with CVB2 structural proteins VP1-VP4. Both culture supernatants and cell lysates of the transfected 16HBE cells enhanced the proliferation of T cells. Conclusions CVB2 struc-tural proteins VP1-VP4 could enhance the expression of innate immunity-related signaling molecules to var-ying degrees and promote the activation of adaptive immunity.

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.

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.

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.

Objective To investigate the significance and mechanism of intracerebroventricular injection viper venom nerve growth factor (Vngf) in rat neural plasticity after cerebral ischemia reperfusion injury.Methods Ninety Wistar male rats were randomly assigned into Vngf-25 U group (n = 18), Vngf-50 U group (n = 18), Vngf-100 U group (n = 18), ischemia reperfusion group (n = 18) and sham operated group.The expression of candidate plasticity-related gene 15(cpg-15) Mrna and nuclear factor of kappa B ( NF-Κb ) Mrna in rat brain tissues which were collection at 2,7,14 days after surgery were evaluated by the real time PCR.Results The expression of cpg-15 Mrna and NF-Κb Mrna began to increase after surgery( the F value of cpg-15:70.43, 34.11, 31.89, the F value of NF-Κb: 27.47, 34.56, 31.89,P＜0.01).At the same time, expression of cpg-15 Mrna and NF-Κb Mrna in the Vngf groups was significantly different from the I/R group and the sham operated group (the F value of cpg-15:48.18, 55.93, 78.43, the F value of NF-Κb: 45.92, 55.72, 50.49, P ＜0.01).The more Vngf were injected, the more cpg-15 Mrna and NF-Κb Mrna were expressed in Vngf groups.Conclusions The Vngf could accelerate neural plasticity and restore neurofunctional defect through up-regulated the expression of cpg-15 and NF-Κb.

Objective To investigate the dynamic changes of cardiomyocyte apoptosis and Caspase-12 activation after coronary microembolization (CME) in rats. Methods The CME models were produced by injection of 42 μm microspheres (3000/0.1 ml) into the left ventricle during clampinduced ascending aorta occlusion for 10 seconds in adult male Sprague-Dawley rats (CME group).The sham-operation group was injected with saline instead (S group). The survivors were randomly divided into five groups: 3 h, 6 h, 12 h, 24 h and 4 weeks (n=10, each), respectively. In addition,10 rats were designed as normal control group. Cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The expressions of Caspase-3, 12 and procaspase-3 and 12 were detected with Western-blot analysis. The activity of Caspase-12 was determined with fluorometric assay kit. Results (1)Compared with the shamoperation group and normal control group, the apoptosis rates of cardiomyocytes in CME group were significantly increased at each time point respectively (all P＜0.05). Apoptotic cardiomyocytes were found mainly in the border zones and infarct foci. The apoptosis rates of cardiomyocytes at 3 h, 6 h,12 h, 24 h and 4 weeks after CME were (1.76±0.68)%, (3.17±1.26)%, (1.34±0.12)%,(1.07±0.65)% and (0.30±0.13)%, respectively. The apoptosis rates of cardiomyocytes increased at 3 h after CME, peaked at 6 h after CME (all P＜0.05), and then gradually decreased with lowest value at 4 weeks (all P＜0.01). (2)Compared with sham-operation group and normal control group,the relative activation level of Caspase-3 and 12 in CME group increased remarkably (all P＜0.05).The time courses of Caspase-3 and 12 expressions corresponded well to those of cardiomyocyte apoptosis after CME. Conclusions The amount of cardiomyocytes apoptosis is significantly increased after CME. Caspase-12 may be involved in the apoptosis of cardiomyocyte after CME.

Objective To investigate the levels of oxidative stress in liver tissues of hepatocelluar carcinoma(HCC)patients after transcatheter arterial chemotherapy(TAC).Methods Immunohistochemistry streptavidin biotinylated peroxidase(S-P)method was used to detect the cellular levels of 8-hydroxy-2'-deoxyguanosine(8-OHdG),p53 and p21~(waf1/cip1).Eighty-nine HCC patients were divided into TAC group(39 cases)and Non-TAC group(50 cases).15 Non-HCC liver tissues served as controls.Result 8-OHdG level was higher in Non-TAC group than that in TAC group in tumor tissues (F=9.516,P＜0.05),with that being the lowest in control group(F=9.516,P＜0.01);8-OHdG levels in cancer tissues were significantly higher than that in tumor surrounding tissues in both TAC group (t=7.101,P＜0.001)and Non-TAC group(t=8.020,P＜0.001),there was no significant difference of 8-OHdG levels between para-tumor tissues and controls.The levels of 8-OHdG between tumor and its surrounding tissues in TAC group(r=0.651,P＜0.001)and non-TAC group(r=0.493,P＜0.01)was in positive correlation.The difference of p53 levels in cancer tissues in TAC group and Non-TAC group were not statistically significant and p53 was not detected in para-tumor tissues.The difference of p21~(waf1/cip1) levels among TAC group,Non-TAC group and controls was statistically significant,the levels of p21~(waf1/cip1) in normal group was the highest(F=13.459,P＜0.001),followed by that in TAC and Non-TAC group in cancer tissues(TAC vs.Non-TAC group,P＜0.01);p21~(waf1/cip1) expression in normal controls was significantly higher than that in both TAC and Non-TAC group in para-tumor tissues(F=16.613,P＜0.001).The correlation of p21 ~(waf1/cip1) levels between tumor and its surrounding tissues was significant in non-TAC group(r=0.872,P＜0.001).Conclusions Oxidative stress levels in HCC tumor tissues were higher than in para-tumor tissues and non-HCC liver tissues.Cancer cells probably survive chemotherapy by fortifying oxidative stress repair mechanism.

Objective To investigate the cerebral ischemia/reperfusion protection mechanism of viper venom nerve growth factor(vNGF) by the change of expression of growth associated protein-43 (GAP-43) and neurological function.Methods 45 adult male Wistar rats (weight 220～280 g) were divided randomly into 3 groups: sham group(S, n=9), balanced salt solution group (BSS, n=9) and venom nerve growth factor group (vNGF, n=27). Each group was observed for 7 days. vNGF group was divided into 25 U, 50 U and 100 U subgroups respectively. The following indexes in 3 groups were observed respectively: neurologic deficits and the expression of GAP-43 (immunohistochemistry method).Results Neurological function: The scores of neurological function was 0 in S group. The neurological deficits score was lower at the same time in vNGF group than that in BSS group (P<0.05). Immunohistochemistry: GAP-43 expressed in both BSS group and vNGF group. The expression of GAP-43 in vNGF group increased in 25 U, and to maximum in 100 U. The expression of GAP-43 in BSS group was significantly lower than in vNGF group (P<0.05). Conclusion vNGF can effectively enhance and prolong the expression of GAP-43, increase the survival rats of nerve cells, and has the protection effect on nerve cells after cerebral ischemia injured.

Objective To observe the changes of cardiac rhythms in a swine model of adult asphyxia! cardiac arrest. Method Sixteen Pigs were aphyxiated by endotracheal tube clamping until 8 min after loss of aortic pulsations. Resuscitation was then provided and swinds were assigned to received 0.045 mg/kg epinephrine intravenously after 3 min of basic life support. The animals with restoration of spontaneous circulation within 20 min from CPR were defined as successfully resuscitated, while the rest were identified as unresuscitation. Electrocardiogram ( EGG) were monitored from the start of asphyxia to the start of the CPR. Results When loss of pulsations occurred, 2 of 16 animals had ventricular fibrillation; 10 pigs exhibited pulseless electrical activity, and 4 pigs had asystole. During the 8 min after the loss of aortic pulsations, pulseless electrical activity converted to VF in 7 pigs. Immidiatedly prior to resuscitation, VF occurred in 9 pigs, asystole in 4 pigs, and PEA in 3 pigs. Conclusions Most of animals in this swine model of asphyxial cardiac arrest presented PEA, but most of them converted to VF especially late in the asphyxial process.

Objective To develop rats model for human dilated cardiomyopathy(DCM).Methods Male Sprague-Dawley rats were administered adriamycin intraperitoneally 2.8 mg/kg? week)for 11 weeks,and then observed for 2 weeks.Plasma levels of brain natriuretic peptide(BNP)were studied by ELISA;left ventricular end diastolic diameter(LVEDD),left ventricular end systolic diameter(LVESD)and left ventricular ejection fraction(LVEF)were measured by echocardiogram;and morphology of the hearts and pathological lesions of cardiac muscle tissues were observed.Results(1)The levels of BNP of the DCM group were higher than those of the normal group(P