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Objective To investigate the regulation effect of berberine(BBR) on abnormal proliferation of mesangial cells(MCs) induced by high glucose(HG) and its possible mechanism. Methods MCs were cultured in vitro. The experiment was divided into control group, HG model group and BBR different concentration gradient action group(30, 60, 90 μmol/L). CCK-8 method was used to detect cell viability; high content system imaging method was used to detect cell proliferation; laser confocal method was used to detect extracellular matrix(ECM) deposition protein collagen Ⅳ(Col-Ⅳ) and fibronectin(FN) expression; Western blot was used to detect extracellular signal-regulated kinase(ERK),p-ERK, Phosphorylated mitogen-activated kinase 1(p-MSK1), IκB, p-IκB, p65, p-p65 protein expression. Results The results of CCK-8 method showed that BBR(30,60,90 μmol/L) could regulate the proliferation of MCs; high-content system imaging results showed that BBR(30,60,90 μmol/L)could inhibit the abnormal proliferation of MCs induced by HG; the results of laser confocal experiments showed BBR(90 μmol/L) could reduce the expression of ECM deposition proteins Col-Ⅳ, FN; Western blot results showed that BBR(30,60,90 μmol/L) could reduce the expression of p-ERK, p-MSK1, p-IκB,p-p65 protein. Conclusion BBR may regulate the ERK/NF-κB signaling pathway of MCs and improve the abnormal proliferation of MCs induced by HG.

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Objective To investigate the effect and underlying mechanisms of different culture conditions including growth status of parent culture and cell density on U251 cell proliferation and apoptosis. Methods Cell proliferation assay was applied to determine the growth of U251 cells under different culture conditions including growth status of parent culture and cell reseeding density. Western blot analysis was used to test the expressions of p-ERK1/2,cleaved-caspase3,cleaved-caspase8,p27,cyclin D1,p-PLK,p-FPAK. Caspase 3 activity assay was used to test cell apoptosis. Flow cytometry was performed for cell cycle distribution and proliferation. Results Re-plating of plateau phase U251 cells at high density resulted in increased proliferation. Re-plating of exponentially growing cells at high density resulted in limited proliferation accompanied by cell detachment. EH cells showed activated cleaved-Caspase 3 and cleaved-Caspase 8 as well as increased activity of Caspase 3, suggesting EH cells underwent apoptosis. Moreover, EH cells were accumulated in G2 phase by flow cytometry. Cyclin D1 and p-PLK1 were down-regulated while p27 expression was up-regulated in EH cells measured by Western blot. Increasing FAK expression did not rescue cells from apoptosis. Functional p27 expression prevented cells from undergoing apoptosis and kept cell proliferation. Conclusion The growth status of parent culture and cell density have impacts on U251 cell proliferation and apoptosis. Functional p27 expression plays an important role in the proliferation and survive of glioma.

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Objective To establish a stable human trophoblast cell line HTR-8/SVneo with overexpression of HOTAIR and to verify the expression of HOTAIR. Methods PCR was used to amplify the full length sequence of HOTAIR gene and clone it into LV5 vector. LV5-HOTAIR vector was constructed. The recombinant plasmid was identified by double enzyme digestion and verified by sequencing. The constructed overexpressed HOTAIR lentivirus vector was infected with HTR-8/SVneo cells,and the cell lines with stable expression of HOTAIR were screened by purinomycin,and the level of GFP expression was observed by fluorescence microscope. qRT-PCR verified the expression of HOTAIR. Results The bands obtained by recombinant plasmid digestion were consistent with the expectation,and the sequences of overexpressed HOTAIR lentivirus vectors were consistent with the target sequences.Under fluorescence microscope,the cells in the overexpressed HOTAIR group showed green fluorescence expression. qRT-PCR results showed that the HOTAIR expression in HTR-8/Svneo stable cell lines with overexpression of HOTAIR was 206. 3 times higher than that in the Control group( P<0. 05) and 232. 8 times higher than that in the NC group( P<0. 05). Conclusion The stable HOTAIR-overexpressed HTR-8/SVneo cells were successfully established,and the expression level of HOTAIR mRNA significantly increased in the stable HOTAIR-overexpressed HTR-8/SVneo cell lines.

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Objective To investigate the effect and mechanism of Ghrelin receptor antagonist(D-Lys3)-GHRP-6 on adipose inflammation and oxidative stress in high fat diet(HFD)-induced obese mice. Methods 4-week old male C57 BL/6 J mice were randomly divided into the normal diet(RD), high-fat diet(HFD) and HFD+(D-Lys3)-GhRP-6 groups. After 16 weeks of feeding, the body weight, body fat rate and body fat composition of mice in each group were analyzed, and the blood and adipose tissue of mice in each group were collected. Mouse monocyte chemokine 1(MCP-1), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α) and IL-10 were detected by ELISA. HE staining was used to assess adipocyte diameter and lipid droplet level. Immunofluorescence staining and flow cytometry were used to analyze the number and phenotype of SVF macrophages in adipose tissue. The activities of total superoxide dismutase(SOD), malondialdehyde(MDA), catalase(CAT), xanthine oxidase(XO), glutathione peroxidase(GPx) and XDH in serum of each group were detected. Results Compared with RD group, mice body weight, body fat rate, epididymal white adipose tissue(eWAT), subcutaneous white adipose tissue(sWAT), interscapular brown adipose tissue(iBAT), diameter of fat cells and the fat droplet of iBAT all significantly increased in HFD group. However, the above indexes in HFD+(D-Lys3)-GHRP-6 group were significantly lower than those in HFD group(P<0.05). Compared with HFD group, serum IL-6, TNF-α, MCP-1, MDA, XO and XDH significantly reduced, while the activity of IL-10, SOD, CAT and GPx increased in HFD+(D-Lys3)-GHRP-6 group(P<0.05). In addition, the number of coronary structure(CLS) of eWAT tissue, the total number of macrophages in SVF and the M1 phenotype of macrophages markedly decreased while the M2 phenotype increased in the HFD+(D-Lys3)-GHRP-6 group(P<0.05). Conclusion (D-Lys3)-GhRP-6 may alleviate HFD-induced obesity and adipose inflammation by inhibiting the recruitment of pro-inflammatory macrophages, accelerating their M1-to-M2-phenotype conversion and maintaining the balance of oxidative stress.

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Objective To investigate the effects of MiR-34 a-5 p overexpression on malignant proliferation, invasion and tumor formation of hepatocellular carcinoma HepG2 cells. Methods The relationship between MiR-34 a-5 p and MET was detected by luciferase assay. MET pcDNA vector was constructed to overexpress MET, miR-34 a-5 p and PCDNA-MET were transfected into HepG2 alone or in combination, and the cells were randomly divided into 4 groups: Control group, mimic group, MET group and mimic+MET group for subsequent experiments. Cell proliferation was detected by clone formation method. Flow cytometry was used to detect apoptosis. Transwell detected cell invasion. Protein expression levels of Ki67, PCNA, Bax, Bcl-2 and Caspase-3 were detected by Western blot. Nude mice were randomly divided into Control group and mimic group to detect tumor volume change and tumor weight at 30 d, immunohistochemistry was used to detect Ki67 positive expression rate and TUNEL staining cell apoptosis. Results There was a direct targeting relationship between MiR-34 a-5 p and MET. Compared with the Control group, the cell clone formation rate of mimic group significantly reduced(P<0.05), the levels of Ki67 and PCNA protein significantly reduced(P<0.05), the apoptosis rate significantly increased(P<0.05), the ratio of Bax/Bcl-2 and cleaved caspase-3/caspase-3 significantly increased(P<0.05), and the number of invaded cells significantly reduced(P<0.05). In the MET group, the cell clonal formation rate significantly increased(P<0.05), the levels of Ki67 and PCNA significantly increased(P<0.05), the apoptosis rate significantly decreased(P<0.05), the Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios significantly decreased(P<0.05), and the number of invaded cells significantly increased(P<0.05). Compared with the MET group, mimic+MET group showed significantly lower cell clone formation rate(P<0.05), significantly lower Ki67 and PCNA protein levels(P<0.05), significantly higher apoptosis rate(P<0.05), significantly higher Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios(P<0.05), and significantly lower cell invasion number(P<0.05). In the tumor formation experiments of nude mice, compared with the Control group, the tumor volume of the group mimic significantly decreased at day 25 and 30(P<0.05), the weight of tumor tissue significantly decreased(P<0.05), the number of Ki67 positive cells significantly decreased(P<0.05), and the apoptosis rate of tumor tissue significantly increased(P<0.05). Conclusion miR-34 a-5 p overexpression inhibits MET, inhibits HepG2 proliferation, invasion and promotes cell apoptosis, and inhibits tumor growth of xenograft tumor in nude mice with HCC.

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Objective To investigate the effect of SOAT1 on proliferation, migration and invasion of esophageal cancer cells by down-regulating the expression of SOAT1 using RNA interference. Methods Firstly, the relative expression levels of SOAT1 protein and mRNA in esophageal cancer tissues and cell lines were detected using western blot and qRT-PCR assay, respectively. Next, after that SOAT1 was down-regulated in KYSE30 cells by RNA interference technique, the proliferation and apoptosis were detected by Cell Counting Kit-8 and flow cytometry respectively, and the expression of apoptosis-related proteins(cleaved PARP and cleaved caspase-3) were detected by western blot, and the cell migration and invasion were detected by scratch and transwell assay. Results The expression of SOAT1 protein and mRNA in esophageal cancer tissues was higher than that in adjacent normal tissues(allP<0.01), and the expression of SOAT1 protein and mRNA in esophageal cancer cell lines was also higher than that in normal esophageal epithelial cells(F=85.72,P<0.000 1). Knock-down of SOAT1 expression could weaken the proliferation ability of KYSE30 cells(t=10.73,P=0.000 4) and increase the percentage of apoptosis, and also weaken the migration(t=9.923,P=0.000 6) and invasion(t=3.357,P=0.028 4) ability of KYSE30 cells. Conclusion Knock-down of SOAT1 expression can suppress the proliferation and metastasis of esophageal carcinoma cells.

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Objective To investigate the interaction of JAK2/STAT3 pathway and transient receptor potential channel 6(TRPC6) signaling path way in Podocyte induced by Angiotensin Ⅱ(Ang Ⅱ). Methods Podocyte in mice was treated by the Ang Ⅱ, Ang Ⅱ+chlorine mitotane, Ang Ⅱ+AG490, and Ang Ⅱ+SKF96365. The expression levels of TRPC6, JAK2/STAT3 protein and mRNA were detected by Western blot and qRT-PCR. The secretion of tumor necrosis factor α(TNF-α) and interleukin 6(IL-6) was detected by ELISA. Results The expression of JAK2/STAT3, TRPC6、TNF-α and IL-6 in Ang Ⅱ injury group was higher than those in control group(P<0.05). The expression of JAK2/STAT3, TRPC6、TNF-α and IL-6 in Ang Ⅱ+chlorine mitotane group was lower than those in Ang Ⅱ injury group(P<0.05). The expression of JAK2/STAT3, TRPC6、TNF-α and IL-6 in Ang Ⅱ+AG490 group was lower than those in Ang Ⅱ injury group(P<0.05). The expression of JAK2/STAT3, TRPC6、TNF-α and IL-6 in Ang Ⅱ+SKF96365 group was lower than those in Ang Ⅱ injury group(P<0.05). Conclusion Ang Ⅱ can activate JAK2/STAT3 pathway by TRPC6, and the expression of TRPC6 can be cut by the inhibition of JAK2/STAT3. There is a positive feedback mechanism between JAK2/STAT3 and TRPC6.

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Objective To explore the effects of different concentrations of sevoflurane on the apoptosis, self-renewal and neural differentiation of human embryonic stem cells(hESCs). Methods hESCs were treated with 2.1%(low-dose) or 4.1%(high-dose) sevoflurane for 6 hours. Alkaline phosphatase staining, quantitative real-time PCR, Western blot and immunofluorescence were used to detect the levels of markers of apoptosis, self-renewal and neural cells. Meanwhile, the total and phosphorylated proteins of SMAD and ERK were also examined. Results Compared with the no treatment control cells, there was no significant change in the apoptosis, undifferentiated state and neural differentiation of hESCs in the low-dose group cells. However, the apoptosis of hESCs was induced by the treatment of high-dose sevoflurane. Consistently, the protein level of CASPASE3(P<0.05), an apoptotic marker, increased. At the same time, many hESC colonies partially differentiated, characterized by the downregulation of self-renewal markersOCT4,SOX2,NANOGandPRDM14 and the upregulation of differentiation associated genesGATA4 andGATA6(P<0.01). Moreover, the high-dose sevoflurane inhibited the efficiency of neural differentiation of hESCs, shown by the lower expression levels of neural cell markersTUJ1,ASCL1 andMAP2(P<0.05) when compared with control and low-dose group cells. In addition, the treatment of high-dose sevoflurane reduced the phosphorylation level of ERK in hESCs(P<0.05), suggesting that the effect of high-dose sevoflurane may be related to the ERK signaling. Conclusion High concentration of sevoflurane has a certain inhibitory effect on the self-renewal and neural differentiation of hESCs. The downstream mechanism is partially associated with the decreased activity of ERK signal. Our study will provide useful clues for the safe application of sevoflurane in the future.

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Objective To study the inhibitory effect of forsythoside A(FA) on the inflammation induced by ischemia/reperfusion in PC12 cells. Methods Oxygen glucose deprivation/reoxygenation(OGD/R) model was established in PC12 cells, and PC12 cells were treated with different concentrations of FA(1.25, 2.5 and 5 μmol/L) to determine inflammatory factors(IL-1β, TNF-α and IL-6) level, Western blot was used to determine the expression of toll-like receptor-4(BTLR4);nuclear factor kappa(NF-κB) pathway-related proteins, and the mechanism was verified using lentiviral activation particles. Results FA could significantly inhibit the release of inflammatory factors, inhibit the protein expression of TLR4 and NF-κB p65, and also inhibit the expression of NF-κB p65 in the nucleus. TLR4 and NF-κB lentiviral activation particles were used to verify that FA regulated NF-κB p65 nuclear transfer through TLR4, thereby inhibiting the release of inflammatory factors and reducing inflammation. Conclusion FA can inhibit the expression of inflammatory factors by inhibition of NF-κB p65 nuclear translocation regulated by TLR4, so as to reduce the inflammatory response induced by ischemia-reperfusion and show brain protection effects.

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Objective To investigate the role and mechanism of microRNA(miR)-205 in promoting the differentiation of human adipose-derived stem cells(hASCs) cell lines to chondrocytes by regulating the expression of core-binding factor α-1(Cbfα-1).Methods Lenti-miR-205 and lenti-control lentivirus solution were transfected into the hASCs cells by lentivirus transfection method, which were named miR-205 overexpression group and miR-NC group. The untreated cells were taken as the control groups. The expression of miR-205 gene was detected by RT-qPCR and cell proliferation was detected by MTT colorimetry. The induced differentiation of the third generation hASCs cells after transfection was observed by toluidine blue staining, immunocytochemistry and immunofluorescence staining. The expressions of Cbfα-1, Smad3, TGF-β1, ColⅡ mRNA and protein were detected by RT-qPCR and Western blot.Results The relative expression leve of miR-205 gene in miR-205 overexpression group was higher than that in control group and miR-NC group(P<0.001). The absorbance(A) value of MTT test of the miR-205 overexpression group was higher than that of the control group and the miR-NC group(P<0.05). After 2 weeks of induction,the toluidine blue staining showed that the miR-205 overexpression group stained strongly positively in dark blue staining,while the control group and miR-NC group were weakly light blue. Immunocytochemical staining showed that the cells and extracellular matrix of miR-205 overexpression group were strongly positive brown-yellow staining,while those of the control group and miR-NC group were weakly positive yellowish staining.Immunofluorescence staining showed that the red fluorescence intensity of miR-205 overexpression group was stronger,and the red fluorescence intensity of control group and miR-NC group was dim. The toluidine blue staining in the control group and miR-NC group was weak light blue. The relative expression levels of Cbfα-1 mRNA and protein in the miR-205 overexpression group were lower than those in the control group and miR-NC group,and the relative expression levels of Smad3,TGF-β1,ColⅡ mRNA and protein in the miR-205 overexpression group were higher than those in the control group and miR-NC group( P<0. 05). Conclusion MiR-205 overexpression can promote hASCs cell proliferation and differentiation towards chondrocytes,which may play a regulatory role by inhibiting Cbfα-1 and activating TGF-β1/Smad3 signaling pathway.

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Objective To observe the effect of silencing prostate transmembrane protein, androgen induced 1(PMEPA1) on proliferation and migration of HepG2 hepatocellular carcinoma(HCC) cells. Methods qRT-PCR, immunohistochemistry, and Western blot were used to detect the mRNA and protein expression of PMEPA1 in HepG2 HCC cells. Besides, the relationship between the expression of PMEPA1 in liver cancer and its clinicopathological features was analyzed. Additionally, siRNA was employed to interfere with the expression of PMEPA1 in HepG2 HCC cells. MTT and Transwell methods were adopted to detect the proliferation and migration of HepG2 HCC cells. Results The expression of PMEPA1 protein in liver cancer was higher than that in normal liver, which was correlated with tumour size(P=0.036 6) and lymph node metastasis(P=0.035 8). Meanwhile, it had no correlation with gender, age, AFP level, vascular infiltration, histological grade, and TNM stage of HCC patients. Moreover, the PMEPA1 protein and mRNA expression of PMEPA1-siRNA HepG2 HCC cells was down-regulated(P<0.01), and the proliferation and migration of transfected cells in siPMEPA1 both decreased(P<0.01). Conclusion The expression of PMEPA1 may promote the proliferation and migration of hepatocellular carcinoma cells.

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Objective To compare different effects of sodium butyrate, Hemin and DMSO on erythroid differentiation of K562 cells.Methods K562 cells were treated with 1.5 mmol/L sodium butyrate, 20 μmol/L Hemin and 2% DMSO for 120 h and the medium of K562 was changed every 24 h. The changes of cell counts,cell cycle,the positive rate of TMB and the expression of CD235 a were detected. Results Proliferation of K562 was inhibited in G0/G1 from 48 h to 72 h and G2/M phase from 96 h to 120 h by sodium butyrate,and the positive rate of TMB was( 22. 02 ± 2. 27) % at 120 h. Hemin had no statistical significance on the proliferation and cell cycle of K562,and the positive rate of TMB was( 90. 83 ± 2. 69) % at 120 h. The proportion of G0/G1 of K562 treated with DMSO increased,but there was no statistical significance on cell proliferation after 72 h culture,and the positive rate of TMB was( 1. 84 ± 0. 48) %,which was not significantly different from that of the control group( 2. 89 ± 0. 18) %.The level of CD235 a of K562 exposed to sodium butyrate and Hemin improved compared with control group and DMSO group after cultured 120 h and there was no statistical significance between two groups. However,there was no statistical significance between the expression of CD235 a of the DMSO group and the control group. Conclusion According to the comparison the changes of proliferation,cell cycle,positive rate of TMB and expression of CD235 a of K562 after erythorid induction,it is found that Hemin is a more effective reagent than DMSO and sodium butyrate in inducing erythroid differentiation of K562.

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Objective To investigate the molecular mechanisms of advanced glycation end products(AGEs)-induced macrophage M1 polarization. Methods Primary M0 macrophages were isolated from mice bone marrow. Specific inhibitors of Toll-like receptor 4(TLR4) and receptors for AGEs(RAGE), namely TAK-242 and FPS-ZM1 were used to pre-treat the macrophages. Then the macrophages were exposed to AGEs at 2.5, 5 and 10 μmol/L respectively. Flow cytometry was used to detect intracellular reactive oxygen species(ROS) levels; immumofluorescence staining was used to observe the expression of M1 macrophage phenotype hall marker inducible nitric oxide synthase(iNOS); ELISA was used to determine the concentrations of inflammatory cytokines secreted from macrophages; Western blot was used to evaluate relative expression levels of cytosol and nuclear proteins. Results AGEs treatment induced elevation of intracellular ROS levels, interleukin 6(IL6), IL12 and tumor necrosis factor α(TNFα) concentrations, relative expression levels of cytosol TLR4, phosphorylated signal transducers and activators of transcription 1(p-STAT1) and nuclear STAT1 expressions in macrophages in a concentration dependent manner(allP<0.001). Pre-treatment of TAK242 significantly reduced iNOS expression, inflammatory cytokine concentrations, p-STAT1 expression and STAT1 nuclear translocation in AGEs-treated macrophages in a concentration dependent manner(allP<0.001) without affecting TLR4 expression and intracellular ROS levels. Pre-treatment of FPS-ZM1 significantly reduced iNOS expression, intracellular ROS levels(P<0.001), inflammatory cytokine concentrations(allP<0.001), TLR4 and p-STAT1 expressions(allP<0.001) and STAT1 nuclear translocation(P<0.001) in AGEs-treated macrophages. Conclusion AGEs can induce macrophage M1 polarization via RAGE/ROS/TLR4/STAT1 signaling pathway.

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Objective To establish a prognostic model for renal clear cell carcinoma based on differentially expressed autophagy related genes in TCGA database. Methods The RNA sequencing data for 537 cases of renal clear cell carcinoma and 72 cases of normal tissues were downloaded from the TCGA database for extracting the expression profiles of autophagy related genes. A comparison between carcinoma and normal tissue was conducted to obtain a list of differentially expressed genes, in which 489 samples of renal clear cell carcinoma with paired clinical information were randomized 7 ∶3 to training group(n=344) and validation group(n=145). For the training group, the univariate and multivariate Cox regression analyses were used to construct the prognostic model, which was then validated by the validation group. Results Total 36 differentially expressed autophagy-related genes were screened out. The univariate and multivariate Cox regression analyses screened out 8 differentially expressed autophagy-related genes(CX3 CL1,PRKCQ,RAB,VMP1,IFNG,EIF4 EBP1,ATG16 L2and BID) and relevant risk factors, and the risk score was calculated for each patient. With the median risk score as the cutoff value, the patients in the training group and validation group were divided into low risk group and high risk group. The Kaplan-Meier and log-rank analyses showed that the overall survival rate of patients with high risk scores was lower than that of patients with low risk scores(P<0.05). The results obtained in the validation group were consistent(P<0.05). In addition, the multivariate regression analysis revealed that the prognostic model was an independent risk factor for predicting the prognosis of renal clear cell carcinoma(P<0.05). Conclusion The differentially expressed autophagy-related genes are potential diagnostic and prognostic markers in patients with renal clear cell carcinoma, which may support individualized postoperative treatment theoretically.

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Objective To explore the effect of the competitive binding of LncCCAT1 to miR-583 p on the stem-like characteristics of MCF-7 cells by regulating the expression of MAPK13. Methods Breast cancer tumor stem cells were isolated from MCF-7 cells and the expressions of CCAT1 and miR-583 p were detected by q-PCR in Adherent cells, suspension Spheres and Re-adherent cells. Expression of miR-583 p was measured after transfection of shRNA-CCAT1-2 and miR-583 p inhibitor into MCF-7 cells alone or in combination. The luciferase reporting experiment verified the targeting relationship between miR-583 p and CCAT1. The cells were divided into four groups: Control, shRNA-CCAT1-2, miR-583 p inhibitor and shRNA-CCAT1-2+miR-583 p inhibitor. ALDEFLUOR analysis was used to detect ALDH activity. The expression levels of SOX2, NANOG and ALDH1 were detected by Western blot. The pellet diameter was measured by pellet formation experiment. The lentivirus LV-MAPK13 was constructed and infected with MCF-7 cells to overexpress MAPK13. The MAPK13 level was detected by q-PCR, and the cells were divided into four groups: Control, shRNA-CCAT1-2, LV-MAPK13 and shRNA-CCAT1-2+LV-MAPK13 groups. The protein Western blot was used to detect the protein expression levels of MAPK13, SOX2, NANOG and ALDH1. Results Results showed that compared with Adherent group, CCAT1 expression levels increased(P<0.05)while miR-583 p expression levels decreased(P<0.05)in Spheres group. There was a direct targeting relationship between miR-583 p and CCAT1. Compared with the control group, the ALDH activity of shRNA-CCAT1-2 group decreased, the protein levels of SOX2, NANOG and ALDH1 decreased(P<0.05), and the ball diameter decreased(P<0.05). In the miR-583 p inhibitor group, ALDH activity increased, SOX2, NANOG, and ALDH1 protein levels increased(P<0.05), and ball diameter increased(P<0.05). Compared with the miR-583 p inhibitor group, the ALDH activity, SOX2, NANOG and ALDH1 protein levels and ball diameter of the shRNA-CCAT1-2+miR-583 p inhibitor group decreased(P<0.05). There was a direct targeting relationship between miR-583 p and MAPK13. Compared with Adherent group, MAPK13 levels of Spheres group increased(P<0.01). Compared with Control group, MAPK13 level of LV-MAPK13 group increased(P<0.001). Compared with the control group, MAPK13, SOX2, NANOG, and ALDH1 protein levels in shRNA-CCAT1-2 group decreased(P<0.05), while those in LV-MAPK13 group increased(P<0.05). Compared with LV-MAPK13 group, the protein levels of MAPK13, SOX2, NANOG and ALDH1 in shRNA-CCAT1-2+LV-MAPK13 group decreased(P<0.05). Conclusion LncCCAT1 competitively binds to miR-583 p to inhibit the stem cell like characteristics of MCF-7 cells by regulating the expression of MAPK13.

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Objective To observe the expression of miR-214-3 p in gastric cancer tissues, explore its effect on the metastatic ability of gastric cancer cells, and explore the mechanism of its role. Methods The expression of miR-214-3 p in 41 pairs of gastric cancer and adjacent normal tissues and its relationship with tumor clinicopathological characteristics were detected by Real-time quantitative PCR; gastric cancer cell MGC-803 was inoculated in culture dishs, with 3 replicate wells in each group. The experiment was divided into 3 groups: miR-214-3 p up-regulated group(transfected miR-214-3 p mimic), miR-214-3 p down-regulated group(transfected miR-214-3 p inhibitor), and a NC group was set respectively; Real-time quantitative PCR was used to detect the expression levels of p53 in each group, and the Transwell experiment was used to detect the migration and invasion ability of MGC-803 cells after transfection with miR-214-3 p inhibitor. Bioinformatics analysis and luciferase activity determination determine whether p53 is the target gene of miR-214-3 p. Results Compared with adjacent tissues, miR-214-3 p was up-regulated in gastric cancer tissues and was associated with tumor lymph node metastasis; compared with the NC group, the expression of p53 in the miR-214-3 p up-regulated group decreased, and the expression of p53 increased in the miR-214-3 p down-regulated group; the results of Transwell experiment showed that compared with the NC group, cells of the miR-214-3 p down-regulated group had lower migration and invasion capabilities; bioinformatics analysis and luciferase activity measurement results showed that miR-214-3 p inhibited p53 expression by targeting its 3′UTR. Conclusion miR-214-3 p may play an important role in gastric cancer development and progression, and miR-214-3 p can be used as a potential targeted biomark for gastric cancer.

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Objective To observe the effect of MYDGF on the angiogenesis of chorioallantoic membrane(CAM) in chickens. Methods Fresh eggs were incubated for 7 days. The normally developed chicken embryos were randomly divided into blank control group, PBS group, low-dose MYDGF group, medium-dose MYDGF group and high-dose MYDGF group, with 10 eggs in each group. 30 μl samples were added to each group by air chamber(MYDGF solution dissolved in PBS with concentrations of 10 ng/ml, 100 ng/ml and 500 ng/ml respectively in the low and medium and high dose MYDGF group). After 2 days, the vascular growth of CAM was recorded by taking photos. The computer software IPP 6.0 was used to analyze the angiogenic area(VA) and CAM area and calculate the ratio of the two(VA/CAM). HUVECs were cultured in vitro. The effect of MYDGF on the proliferation of HUVECs was detected by CCK-8 assay. The effect of MYDGF on the tube-forming function of HUVECs was detected by tube-forming experiment. Results There were( 20. 80 ± 2. 49) VN in the PBS group,( 27. 60 ± 3. 17) VN in the medium-dose MYDGF group,and( 28. 60 ± 3. 27) VN in the high-dose MYDGF group. The differences between the medium-dose MYDGF group and the high-dose MYDGF group were statistically significant compared with the PBS group( P<0. 05). VA/CAM values were respectively( 23. 53 ± 1. 96) % in the PBS group,( 27. 87 ±3. 10) % in the medium-dose MYDGF group,and( 29. 26 ± 2. 73) % in the high-dose MYDGF group. The differences between the medium-dose MYDGF group and the high-dose MYDGF group were statistically significant compared with the PBS group( P<0. 05). Compared with the control group,MYDGF( 100 ng/ml) could promote the proliferation of HUVECs( P<0. 05) and tubulization( P<0. 05),and the difference was statistically significant.Conclusion MYDGF can promote angiogenesis in chorioallantoic membrane of chicken embryo. MYDGF can enhance the proliferation and tubulization of HUVECs.

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Objective To verify the mRNA that inhibits the replication and expression of hepatitis B virus(HBV) in hepatocytes. Methods The peroxisome proliferator activated receptor(PPAR) α(PPARα), nuclear factor B(NFIB) and interleukin-8(IL-8) genes were transferred into HepG2.2.15 and HBV whole genome 1.3 ploid HepG2(HBV1.3 P-HepG2) cell models by lentiviral mediation. 48 hours after transfection, mRNA expression and HBVDNA replication level were detected by qPCR with blank control group(NC) as control. The expression of HBsAg and HBeAg in the cell supernatant was detected by chemiluminescence, and the expression of HBsAg in the cells was detected by immunofluorescence. Results In HepG2.2.15 and HBV1.3 P-HepG2 cell models, PPARα could promote the replication of HBVDNA and the expression of HBsAg, and the expression of HBVDNA was 1.46 and 1.27 times higher than that of NC group(P<0.05). NFIB could inhibit the replication of HBVDNA and the expression of HBsAg, and the expression of HBVDNA was 0.76 and 0.55 times higher than that of NC group(P<0.05). IL-8 could promote the replication of HBVDNA and the expression of HBsAg, and the expression of HBVDNA was 1.54 times and 1.62 times higher than that of NC group(P<0.05). Conclusion PPARα and IL-8 in hepatocytes can promote the replication and expression of HBV, while NFIB can inhibit the replication and expression of HBV, which can provide laboratory basis for follow-up research.

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Objective To investigate the effect of lysophosphatidic acid(LPA) on the expressions of CCAAT/enhancer binding protein β(C/EBPβ) and cyclooxygenase-2(COX-2) and proliferation in hepatocellular carcinoma cells, and further determine the possible effect of sinomenine hydrochloride(SIN), an anti-inflammatory drug, on it. Methods Human hepatocellular carcinoma Huh7 cell line was employed. The mRNA and protein expression levels of genes were detected by Real Time PCR and western blot, respectively. Cell proliferation was detected by MTT assay. Results After Huh7 cells were treated with 10 μmol/L of LPA for 0～6 h, the protein and mRNA expression levels of C/EBPβ and COX-2 were up-regulated in a time-dependent manner. After treatment with different concentrations of LPA(0～10 μmol/L) for 6 h, the protein and mRNA expression levels of C/EBPβ and COX-2 were increased in a concentration-dependent manner. SIN suppressed the increase of C/EBPβ and COX-2 and cell proliferation induced by LPA in a concentration-dependent manner. Conclusion LPA promotes the proliferation of human hepatocellular carcinoma Huh7 cells by inducing the expression levels of C/EBPβ-COX-2 pathway. SIN can inhibit LPA-induced cell proliferation through downregulating C/EBPβ-COX-2 pathway.

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Objective To investigate the effect and mechanism of glucose transporter 1(Glut1) gene silencing on the proliferation, differentiation and apoptosis of human colorectal cancer HT-29 cells. Methods Glut1 interference sequence(siRNA) was transfected into human colon cancer HT-29 cells as shGlut1 group, non-interfering sequence was transferred to HT-29 cells as NC group, HT-29 cells as Blank group, and normal tissue cells adjacent to cancer as Control Group. qRT-PCR and Western blot were used to detect the expression levels ofGlut1, TGF-β1, PI3 K, AKT, PTEN, mTORmRNA and corresponding proteins in each group of cells, and to detect the Bcl-2/Bax ratio, p-PI3 K, p-S 473-AKT, p-S 389-S6 K1, p-T 70-4 EBP1, CleavedCaspase-3, Cleaved-PARP protein expression; MTT method was used to detect changes in cell proliferation in each group, flow cytometry was used to detect cell cycle and apoptosis, clone formation experiments was used to detect tumor cell proliferation and tumor formation abilityin vitro. Results The immunofluorescence results showed that the transfection efficiency of shGlut1 group was higher, which met the experimental standards. Compared with the Control group, the expression ofGlut1, TGF-β1, PI3 K, AKT, mTOR, Bcl-2 mRNA and protein were up-regulated in colon cancer tissues, while PTEN, Bax mRNA and protein expression were down-regulated, p-PI3 K, p-S 473-AKT, p-S 389-S6 K1 and p-T 70-4 EBP1 protein expression was up-regulated, Cleavedcaspase-3, Cleaved-PARP protein expression was down-regulated,(allP<0.05); compared with the Blank group and NC group, the expression of Glut 1, TGF-β1, PI3 K, AKT, mTOR, Bcl-2 mRNA and protein was down-regulated in the shGlut 1 group, and the expression of PTEN, Bax mRNA and protein was up-regulated, p-PI3 K, p-S 473-AKT, The expression of p-S 389-S6 K1 and p-T 70-4 EBP1 was down-regulated, the expression of Cleavedcaspase-3, Cleaved-PARP protein was up-regulated, the proliferation rate was down-regulated, the G0/G1phase was more and the S phase was less, the apoptotic rate was up-regulated, and the clone formation was fine, the cell growth was slower(allP<0.05). Conclusion Glut 1 gene silencing can inhibit the proliferation and differentiation of colorectal cancer cells and promote their apoptosis. It is considered to inhibit the TGF-β/PI3 K-AKT-mTOR signaling pathway.

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Objective To explore effects of tetrandrine(TET) on growth, movement of tongue squamous cell SCC9 and tumorigenesis in nude mice and its mechanism. Methods Human tongue squamous cells SCC9 were culturedin vitro. They were divided into blank group, low-dose TET group(TET 2.5 μmol/L), medium-dose TET group(TET 5μmol/L) and high-dose TET group(TET 10 μmol/L). And these groups were pretreated with corresponding doses of TET. Cell growth was detected by colony formation assay. The apoptosis was detected by flow cytometry. Cell invasion was detected by Transwell. The expression levels of Ki67, Caspase-3, vascular endothelial cell growth factor(VEGF), PI3 K, p-PI3 K, AKT, p-AKT, mTOR and p-mTOR proteins were detected by Western blot. A total of 60 nude mice were selected. 0.002% 4-nitroquinoline-1-oxide natural drinking water was applied to feed them for 36 weeks to establish animal models of tongue squamous cell carcinoma. They were given low-dose TET[12.5 mg/(kg·d)], medium-dose TET [25 mg/(kg·d)]and high-dose TET [50 mg/(kg·d)] for gavage treatment. 29 d later, tumor weight was detected. The expression of Ki67, caspase-3 and VEGF in tongue squamous tissues was detected by immumohistochemical staining. Results Compared with those in blank group, colony formation rate of tongue squamous cells, number of invasive cells per unit area, expression of p-PI3 K, p-AKT and p-mTOR protein were significantly decreased after treated with TET. The above indexes were the highest in high-dose TET group, followed by medium-dose TET group and low-dose TET group. Compared with those in blank group, there was no significant difference in expression levels of PI3 K, AKT and mTOR protein among the other three groups. Compared with those in blank group, expression level of Caspase-3 protein was significantly increased after treated with TET. The above index was the highest in high-dose TET group, followed bymedium-dose TET group and low-dose TET group.In vivoexperiments of mice showed that compared with those in blank group, tumor mass of nude mice, expression level and positive rate of Ki67 protein and VEGF were significantly decreased after gavage treatment with TET(P<0.05), and the higher the gavage concentration, the lower the above indexes. Compared with those in blank group, expression level and positive rate of Caspase-3 protein in nude mice were significantly increased after gavage treatment with TET(P<0.05), and the higher the gavage concentration, the higher the above indexes. Conclusion TET can inhibit growth and movement of tongue squamous cells by inhibiting PI3 K/AKT/mTOR signaling pathway, and promote apoptosis of tongue squamous cells, which shows concentration-dependence within certain concentration range.

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Objective To investigate the relationship between the expression of uteroglobin-related protein 1(UGRP1) in thyroid cells and the properties, functions and the mutation of BRAFV600Egene in patients of thyroid nodules.Methods According to the results of cytological examination, 153 cases of aspirated thyroid tissues from 131 patients with thyroid nodules as the chief complaints undergone thyroid fine needle aspiration cytology(FNAC) examinations, were divided into thyroid nodules group(47 cases), Hashimoto′s thyroiditis group(38 cases) and thyroid carcinoma group(46 cases). Thyroid tissues adjacent to benign nodules were classified as normal thyroid group( 22 cases). The expressions of UGRP1 in thyroid cells obtained by FNAC were detected by immunohistochemistry,and the expressions of UGRP1 in the four groups were analyzed,as well as the thyroid functions,ultrasonic characteristics,the relationship between UGRP1 expression and BRAFV600Egene mutation in the positive and negative UGRP1 expression groups. Results The positive UGRP1 expression rates in Hashimoto' s thyroiditis group,thyroid nodules group and thyroid carcinoma group were 71. 1%,4. 3% and 2. 2% respectively. UGRP1 was negatively expressed in normal thyroid group. There was significant difference between the four groups( P<0. 05). There was no significant difference in TT3,TT4 and TSH between the positive and negative UGRP1 expression groups. There was a correlation between UGRP1 expression and BRAFV600Egene mutation( P = 0. 023,C =0. 414). Conclusion In thyroid nodules,Hashimoto's thyroiditis was highly positive expression for UGRP1. The positive expression of UGRP1 may be one of the reference indexes to distinguish Hashimoto's thyroiditis from normal thyroid and thyroid carcinoma.

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Objective To investigate whether in the onset time of sudden sensorineural hearing loss(SSHL) is seasonal. Methods The clinical data of 348 patients with SSHL in the past 10 years were collected. According to sex, age, unilateral/bilateral, accompanying symptoms and risk factors, they were divided into different subgroups. The seasonal variability in the onset of patients in general group and each subgroup was identified by circular distribution statistics. Results The overall onset of SSHL had no significant seasonal variability, but showed a seasonal trend of more in spring and autumn and least in summer. The male group had significant seasonal variability(P<0.05), and the incidence was the highest in autumn. There was significant seasonal variability in hypertension group and hyperglycemia group(P<0.05), and the incidence was concentrated in cold months. There was no significant seasonal variability in the other subgroups. Conclusion The onset of SSHL has seasonal trend of more in spring and autumn and the least in summer. The onset of SSHL in male group, hypertension group and hyperglycemia group has significant seasonal variability.

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Objective To investigate the clinical, endoscopic and pathological features of early esophageal squamous cell carcinoma with epithelial keratosis. Methods Patients with early esophageal squamous cell carcinoma confirmed by pathology after endoscopic submucosal dissection were collected. The patients were divided into two groups according to the manifestation of leukoplakia under endoscopy. The clinical, endoscopic and pathological data were collected and analyzed. Results Among the 263 patients with esophageal squamous cell carcinoma, 90 cases were complicated with epithelial keratosis. In patients with keratosis, the proportion of keratosis in patients over 60 years old was much higher than that in patients under 60 years old. Patients over 60 years old had a higher proportion of esophageal keratosis(81.11%vs66.47%,P=0.013) compared with non-epithelial keratosis group. Moreover, esophageal keratosis was more likely to occur in women(40.00%vs21.97%,P=0.002). In addition, more keratosis patients had a history of smoking than non-keratosis patients(35.56%vs23.70%,P=0.042). In patients with keratosis, there were more leukoplakia than those with scattered lesions, with larger lesions(80.77%vs48.44%,P=0.005) and deeper infiltration(M1: 3.85%vs20.31%; M2: 57.69%vs71.88%; M3: 26.92%vs4.69%; SM: 11.54%vs3.13%,P<0.001). Conclusion For elderly patients, women and people with a history of smoking, early esophageal squamous cell carcinoma is more likely to be complicated with esophageal keratosis. Clinicians should thoroughly observe the red area which is not completely covered by the white spots of keratosis. For the lesions with severe keratosis, we should be aware that the depth of invasion may be relatively deep.

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To establish a method for the isolation, culture and identification of primary fibroblasts from salivary gland of neonatal rat. The salivary gland of neonatal SD rat was digested by 0.125% trypsin. The proliferation of fibroblast cells was detected by high content cell imaging system. Vimentin was detected by cell immunofluorescence and Western blot to identify fibroblasts. RT-PCR detected the expression level of vimentin mRNA, CD90 labeled fibroblasts to identify cell purity. The primary cells obtained from isolation were bright and spherical, with a clear outline, they were distributed separately or aggregated into cell clusters. Most of the cells were attached to the wall bottom after 90 min-culture; part of them began to stretch at this point. Cell monolayer was formed after 5 to 6 days. Cells presented as spindle-shaped or polygon-shaped after subculture. Positive vimentin expressing cells were identified as fibroblasts. The percentage of fibroblast reached to around 87% in passage 3. After 24 hours of culture in DMEM/F12 medium containing 5% FBS, the proliferation of cells could be detected. Trypsin digestion is an effective method for the isolation of fibroblasts from the salivary gland of neonatal rat.

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158 patients with fistula chronic apical inflammation requiring root canal therapy were randomly divided into the experimental group and the control group. In the experimental group, iRoot SP sealant was used for Single-cone obturation technique. In the control group, the root canals were filled with iRoot SP sealant by continuous wave warm vertical condensation. The time of root canal filling, the incidence of postoperative pain at 48 hours and the clinical and X-ray examination results at 1 year were recorded. The root filling time of the experimental group and control group was( 86. 6 ± 12. 1) s and( 138. 6 ± 11. 2) s respectively( P<0. 05). The incidence of pain 48 h after operation in the two groups was 6. 25% and 16. 67%,respectively( P<0. 05). One year after the operation,the effective rates of the two groups were 89. 47% and 92. 00% respectively. Single-cone obturation technique with iRoot SP paste has less postoperative pain response,shorter operation time and more reliable clinical efficacy in the treatment of fistula chronic periapical inflammation. Single-cone obturation technique with iRoot SP paste can be used in the treatment of fistula chronic periapical inflammation.

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To investigate the effects of osteogenic inducer(OI) sustained-release system carried by poly(lactide-co-glycolide)(PLGA) gel on the bone remodeling of tooth extraction sockets. After tooth extraction, 27 New Zealand white rabbits were randomly divided into 3 groups: extraction sockets filled with OI sustained-release system(PLGA+OI group), extraction sockets filled with PLGA gel(PLGA group), and extraction sockets without any treatment(control group). After 2, 4, 8 weeks of tooth extraction, 3 rabbits of each group were killed and the samples of their mandibular alveolar bone were taken for radiological and histological observation. The BMD of the PLGA+OI group at 2, 4, 8 weeks was significantly higher than that of the PLGA group and control group. The variation of ABW and ABH of PLGA+OI group at 8 weeks was significantly lower than that of the PLGA group and control group(P<0.001). Histological results showed that new bone formation of the PLGA+OI group was quicker than that of the other groups. OI sustained-release system can effectively promote the bone remolding process of tooth extraction sockets and reduce the resorption of ABW and ABH, which can be a promising biomaterial for alveolar bone preservation in extraction sockets.

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MC3 T3-E1 cells were cultured and treated with different concentrations of strontium and icariin. On the 1 st, 4 th, 7 th and 11 th day, cell morphology was observed and cell number was calculated under fluorescent microscope. CCK-8 method was also applied to analyze the effect of strontium and icariin on proliferation of MC3 T3-E1 cells. Alkaline phosphatase(ALP) activity detection(on 4 th and 7 th day) and Alizarin red staining(on 21 th day) were performed to detect the effect of strontium and icariin on osteogenic differentiation of MC3 T3-E1 cells. The results showed that compared with other groups, group S1 I2(80 mg/L Sr+7×10-2mg/L ICA) had more MC3 T3-E1 cells and exhibited higher cell proliferation activity. Furthermore, ALP activity was the highest in S1 I2 group and more red-stained mineralization nodules were observed in group S1 I2. These suggest that the combination of strontium and icariin can promote proliferation and osteogenic differentiation of MC3 T3-E1 cells under adequate concentrations.

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241 patients were included in this study. Each patient had a BI-RADS 4 breast lesion, and each breast lesion was examined by conventional ultrasound, color doppler ultrasound and ARFI. The resistance index, shear wave velocity(SWV, SWVlesionsand SWVglands) and their ratios(SWVlesions/SWVglands) were recorded. The diagnostic performance of each quantitative parameter was evaluated by the subject operation characteristic curve, and then the classification algorithm was used for classification analysis to construct the prediction model. Among the 241 breast masses, 140 cases were malignant and 101 cases were benign. In terms of quantitative indicators RI, SWVlesionsand SWVlesions/SWVglands, RI and SWVlesionswere included in the classification algorithm, and the depth of the prediction model included two branches(SWVlesions>or ≤3.795 m/s; if SWVlesions≤3.795 m/s, then RI≤0.620 or 0.6200.790, and if SWVlesions>3.795 m/s then RI≤0.710 or>0.710). The classification algorithm led to an AUC of 0.938, a sensitivity of 98.6%, and a specificity of 57.4%. The prediction model of ultrasonic quantitative parameters can significantly improve the accuracy in the diagnosis of benign and malignant BI-RADS 4 breast lesions, and can avoid the puncture biopsy of 24% breast lesions.

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The data of 218 patients with tinnitus, including specialist otolaryngology examination, hearing measurement, tinnitus matching, tinnitus handicap inventory(THI), VAS annoyance, and tinnitus multielement integration sound therapy(T-MIST) were retrospectively analyzed. The patients were divided into 4 groups according to age: 18～30 y, 31～44 y, 45～60 y and 61～70 y. The effects of clinical characteristics, hearing level, matching tinnitus pitch and loudness, tinnitus handicap inventory(THI), visual assessment scale(VAS) annoyance, and residual inhibition(RI) were compared among the four groups. The results showed that there was no statistically significant difference in the prognosis of the patients treated by voice therapy in the groups. However, the age was correlated with annoyance, THI and tinnitus loudness(P<0.05). The hearing loss, tinnitus loudness, THI and VAS annoyance increased with the increase of the age(P<0.05). Therefore, the age had no effect on the prognosis of acoustic therapy. Compared with the younger patients, the elderly were more easily affected by the psychological disorders because of tinnitus. Account for most of the elderly tinnitus patients with hearing loss, hearing rehabilitation should give priority to this age group.