Effects of miR-515-5p on the proliferation and metastasis of ovarian cancer cell A2780 by regulating the expression of chondroitin sulfate proteoglycan 4 and its mechanism / 解剖学报

Objective To investigate the targeted regulation of miR-515-5p on chondroitin sulfate proteoglycan 4 (CSPG4) and its effect on the proliferation and metastasis of ovarian cancer cell A2780. Methods The target genes of miR-515-5p were predicted by bioinformatics tools. Real-time PCR and Western blotting were used to detect the expression of miR-515-5p and CSPG4 in 65 cases ovarian cancer tissues and adjacent tissues. Ovarian cancer cells A2780 were divided into miR-NC group, miR-515-5p group, si-NC group, si-CSPG4 group, miR-515-5p + pcDNA group, miR-515-5p + pcDNA-CSPG4 group. MTT assay was used to detect cell proliferation. Transell was applied to detect cell migration and invasion, and Western blotting was selected to detect cyclinD1, P21, matrix metalloproteinase (MMP)-2 and MMP-9 protein expression. The double luciferase reporter gene experiment and Western blotting confirmed the regulation effect of miR-515-5p on CSPG4 gene. Results Bioinformatics analysis showed that CSPG4 was one of the potential target genes of miR-515-5p. Compared with the adjacent tissues, the expression of miR-515-5p was lower in ovarian cancer tissues, and the expression of CSPG4 was higher (P< 0. 05). Compared with the miR-NC group, the expression of cyclinD1, MMP-2 and MMP-9 protein was lower in A2780 cells of miR-515-5p group, the expression of P21 protein was higher, the cell viability was lower, and the number of cell migration and invasion was lower (P<0. 05). Compared with the si-NC group, the expression of cyclinD1, MMP-2 and MMP-9 proteins were lower in A2780 cells in si-CSPG4 group, P21 protein was higher, cell viability was lower, and the number of cell migration and invasion was lower (P<0. 05). Compared with the miR-515-5p + pcDNA group, the expression of cyclinD1, MMP-2 and MMP-9 proteins was higher in A2780 cells of miR-515-5p + pcDNA-CSPG4 group, the expression of P21 protein was lower, and the cell viability was higher, the number of cell migration and invasion was higher (P<0. 05). MiR-515-5p targeted and negatively regulated CSPG4 expression. Conclusion MiR-515-5p could inhibit the proliferation and metastasis of ovarian cancer cell A2780 by targeting CSPG4.

Screening key factors from bone marrow mesenchymal stem cells for alleviating silicosis in mice / 中国职业医学

OBJECTIVE: To explore the mechanism of bone marrow mesenchymal stem cells(BMSCs) in alleviating silica-induced lung injury in mice. METHODS: Ten specific pathogen free healthy male C57 BL/6 mice were selected for isolating BMSCs and bone marrow macrophages(BMDMs). Transwell chamber was used, BMDMs were inoculated onto the upper chamber and BMSCs in the lower chamber. We divided them into sequencing control group and silica(SiO_2) exposure group. All cells were pre-stimulated with 50 μg/L mass concentration lipopolysaccharide for 4 hours. In the SiO_(2 ) group, 250 mg/L mass concentration SiO_2 was added to the upper chamber of transwell and cultured for 16 hours. Total RNA was extracted from the BMSCs collected from the lower chamber. HiSeq/MiSeq high-throughput sequencing technology was used to detect the BMSCs RNA paired-end sequencing. Transcriptome sequencing data was obtained and bioinformatics analysis was performed. Another 12 specific pathogen free healthy male C57 BL/6 mice were randomly divided into control group and experimental group. All mice received one intra-tracheal injection of 20.0 μL(250 g/L mass concentration) of silica dust suspension. After 6 hours, the mice in the control group was given 500.0 μL of 0.9% sodium chloride solution and mice in the experimental group was given 500.0 μL of BMSCs suspension(cell density 1×10~9/L) by tail vein infusion.Mice were sacrificed 12 hours later. The relative mRNA expression of interleukin(IL)-1 Ra, IL-10, tumor necrosis factor stimulating gene 6(TSG-6) and prostaglandin E2(PGE2) in lung tissues of mice were measured by quantitative real-time PCR(q-PCR). Meanwhile, BMDMs and BMSCs transwell co-culture models were established. The cells were divided into 5 groups: BMSCs group, BMSCs+BMDMs group, BMSCs+BMDMs+ lipopolysaccharide(LPS) group, 50 mg/L SiO_2 group, and 100 mg/L SiO_2 group. After 16 hours of corresponding SiO_2 stimulation, BMSCs of each group were collected and the relative mRNA expression levels of IL-1 Ra, IL-10, TSG-6, and PGE2 in the cells were detected by q-PCR. RESULTS: Compared with sequencing control group, BMSCs co-cultured with SiO_2 had 19 genes up-regulated and 21 genes down-regulated, including 10 genes up-regulated for more than 2.0-fold. The relative mRNA expression of IL-1 Ra, IL-10, PGE2 and TSG-6 in the lung tissue of mice increased in the experimental group than that of the control group(all P<0.05). The relative mRNA expression of TSG-6 increased by 37.5 times higher than that of the control group. Compared with the BMSCs+BMDM+LPS group, the level of TSG-6 mRNA relative expression increased in both the 50 mg/L SiO_2 group and the 100 mg/L SiO_2 group(all P<0.05). CONCLUSION: TSG-6 could be the key factor of BMSCs that can attenuate silica-induced lung injury.

Effect of dihydromyricetin on the proliferation and migration ability in choriocarcinoma JEG-3 and JAR cells / 解剖学报

Objective To investigate the effect of dihydromyricetin (DM Y) on proliferation and migration in choriocarcinoma JEG-3 and JAR cells. Methods JEG-3 and JAR cells were treated with different concentrations (0 mg/L, 20 mg/L, 40 mg/L, 60 mg/L, 80 mg/L) of DMY for 24 hours and 48 hours, and the proliferation was analyzed by methylthio tetrazole (MTT) assay. The effect of DMY on migration was detected by wound healing (after 24 hours) and Transwcll assay (after treated JEG-3 for 36 hours and JAR for 24 hours). The expression of matrix metalloproteinase 2 (MMP-2) at mRNA and protein levels with different concentrations of DMY(0 mg/L, 40 mg/L, 60 mg/L, 80 mg/L) were detected by Real-time PCR (after 12 hours, 24 hours, 36 hours, 48 hours) and Western blotting (after treated 36 hours) respectively. Results The proliferation of JEG-3 and JAR ceils was inhibited significantly (/><0. 05) , after DMY treatment for 24 hours and 48 hours.DMY inhibited the migration of JEG-3 and JAR cells significantly (P<0. 05) with a dose-dependent manner. After JEG-3 and JAR Cells treated with DMY for 36 hours and 48 hours, the expression of MMP-2 mRNA decreased significantly (P<0. 05) , there were no significantly changes in DMY treatment with 12 hours and 24 hours. The expression level of MMP-2 protein was inhibited significantly after treatment with DMY for 36 hours (P<0. 05). Conclusion DMY might inhibit the proliferation in choriocarcinoma JEG-3 and JAR cells with a dose-dependent manner. The invasion and migration were inhibited by DMY through downregulation of MMP-2.

Inhibition of invasion and metastasis of human tonguesquamous cell carcinoma Tca8113 cells by Src kinase inhibitor PP2 / 西安交通大学学报(医学版)

Objective: To investigate the inhibitory effects of Src kinase inhibitor PP2 on migration and invasion of Tca8113 cells. Methods: Tca8113 cells were cultured for 24 h with 5 mol/L, 10 mol/L and 20 micron mol/L of Src kinase inhibitor PP2. The effects of PP2 on the invasion and migration of Tca8113 cells were measured with Transwell chamber and scratch method, respectively. Results: After the treatment with PP2 for 24 h, the expression of p-Src in 5, 10, 20 μmol/L of Src kinase inhibitor PP2 treatment groups was significantly lower than that of the non-drug treatment group (all P<0.05). The number of Tca8113 cells in the non-drug treatment group and the 5, 10, and 20 μmol/L of Src kinase inhibitor PP2 treatment groups was (232.76±28.65), (186.53±21.34), (129.18±17.96), and (37.82±12.41), respectively; the number of migratory cells was (259.38±25.27), (193.45±20.18), (143.24±18.04), and (32.94±14.39), respectively, the cell migration rate was (11.51±0.84)%, (8.06±0.51)%, (5.13±0.57)%, and (3.18±0.12)%, respectively; the overall difference was statistically significant (F=73.852, 85.687, 48.157, all P=0.000). It had a negative correlation with PP2 dose. Conclusion: Src kinase inhibitor PP2 can effectively inhibit the invasion and migration of Tca8113 cells in the concentration-dependent manner, and it may have certain clinical value in the treatment of human tongue squamous cell carcinoma.

Biological features of tumor associated fibroblasts and effect of Transwell co-culture on proliferation and metastasis of gastric cancer cells / 中国药理学通报

; Aim To culture primary human gastric cancer associated fibroblasts (CAFs) and normal fibroblasts (NFs), and to explore the biological characteristics and their effects on gastric cancer cells. Methods After isolation and culture of CAFs and NFs, growth curve was drawn by MTT. The a-smooth muscle actin (ot-SMA) and Vimentin were detected by Immunofluorescence, Western blot and qRT-PCR. MGC-803 cells were co-cultured with CAFs and NFs in Transwell suspension mode. The migration and invasion ability of gastric cancer cells was detected by Transwell. The proliferation activity and AMD3100 on CAFs-gastric cancer co-culture system were compared by MTT. The acidic property, lactic acid and ROS contents of co-culture system were determined by PH meter, lactic acid and DCFH-DA method. Results The morphology of CAFs, NFs cells were in long spindle or flat star shape. The proliferation ability and overlapping growth phenomenon of CAFs were higher than those of NFs. The expression of ct-SMA and Vimentin cells was positive in CAFs, but low or negative in NFs cells. The activity of gastric cancer in low density co-culture group > medium density group > high density group, the PH value of CAFs co-culture system decreased, the content of lactic acid and ROS was high, and only CAFs low density co-culture group had significant effect on promoting cancer. Conclusions The co-culture of gastric cancer cells with CAFs and NFs is greatly affected by the proportion. Low density co-culture can significantly improve the proliferation and metastasis ability of gastric cancer cells. High density co-culture may in turn inhibit the growth and metastasis of cancer cells, which may be related to the content of lactic acid and ROS.

Effect of oxygen glucose deprivation-reperfusion in astrocytes overexpressing endothelin-1 on the proliferation of neural stem/progenitor cells / 器官移植

Objective To investigate the effect of oxygen glucose deprivation-reperfusion (OGD-R) in astrocytes overexpressing endothelin (ET)-1 on the proliferation of neural stem/progenitor cells (NSPCs). Methods OGD-R models of negative control astrocytes (C6-Mock) and astrocytes over-expressing ET-1 (C6-ET-1) were constructed. Transwell co-culture system of astrocytes and NSPCs was established. Morphologic observation and identification of the astrocytes and primary NSPCs were performed. The cells were divided into four groups: C6-Mock+NSPCs, OGD-R+C6-Mock+NSPCs, C6-ET-1+NSPCs and OGD-R+C6-ET-1+NSPCs groups and co-cultured for 0, 24, 48 and 72 h respectively. The diameter of neurosphere was measured in each group. Results In the C6-Mock and C6-ET-1 cells, type Ⅰ astrocytes in fibrous morphology were observed. Glial fibrillary acidic protein (GFAP) was expressed in the cytoplasm of these two types of cells. Primary NSPCs were positive for nestin staining. After co-culture for 48 and 72 h, the neurosphere diameter in the OGD-R+C6-Mock+NSPCs group was significantly greater than that in the C6-Mock+NSPCs group. The neurosphere diameter in the OGD-R+C6-ET-1+NSPCs group was considerably greater than that in the C6-ET-1+NSPCs group. The neurosphere diameter in the OGD-R+C6-ET-1+NSPCs group was significantly greater compared with that in the OGD-R+C6-Mock+NSPCs group (all P<0.05). Conclusions OGD-R astrocytes can promote the proliferation of NSPCs. ET-1 over-expression further accelerates the proliferation of NSPCs.

A Randomised Approach for Enumerating Migrated Cells in a Transwell Migration Assay

@#The transwell migration assay is commonly used for assessing cell migration. It involves the enumeration of cells that have migrated across a pore-containing membrane. We describe a randomised approach to quantifying migrated cells and compare it to a conventional full cell count. We used ATP as a chemoattractant and automatic cell quantification performed on all fields (Full count; FC) or 10 randomly selected fields (Randomised count; RC). The two methods were compared by evaluating standard deviations (SD), coefficient of variation (CV) and using the Bland-Altman analysis. The dispersion of data is higher with the RC approach (3.77-6.66% CV for control; 3.89-4.48% CV for ATP-treated wells) compared to FC (0.27-0.46% CV for control; 0.05-0.09% CV for ATP-treated wells), but are acceptable considering that the number of migrated cells are in the thousands. Both methods verified that an ATP migration assay for BV2 microglia was established, demonstrating that the RC approach is reliable and comparable to a full count.

MiR-26b regulates invasion and migration of lung cancer cells through targeting hENTI depending on RhoA/ROCK-I pathway / 中南大学学报(医学版)

Objective:To investigate the effect of miR-26b on the invasion and migration of lung cancer cell and to explore its mechanism.Methods:qPCR was used to detect the expression of miR-26b in lung cancer.Luciferase reporter gene was used to detect interaction between miR-26b and hENT1.Transwell assay was used to detect invasion ability after treatment of miR-26b mimics.Scratch assay was used to detect migration ability after treatment of miR-26b mimics.The expressions of hENT1,ROCK-I and RhoA were detected by Western blot.The changes of cytoskeleton after miR-26b mimics treatment with phalloidin were observed.The effect of miR-26b mimics on the tumor size and volume of lung cancer was determined by subcutaneous tumor formation in nude mice.Results:MiR-26b expression was significantly reduced in lung cancer.With the progress of lung cancer,the expression of miR-26b was reduced.With the progress in differentiation of lung cancer,the expression of miR-26b was decreased.Decrease of miR-26b was associated with lung cancer lymph node metastasis.HENT1 was the direct target of miR-26b;miR-26b regulated the invasion and migration ability of human lung carcinoma A549 cells.MiR-26b regulated the expression of hENT1,ROCK-1 and RhoA.After the treatment with miR-26b mimics,the F-actin staining was significantly reduced,whereas the formation of wrinkles and the formation of pseudopodia were significantly reduced.Subcutaneous tumor formation in nude mice showed that miR-26b mimics treatment significantly reduced the tumor size and mass.Conclusion:MiR-26b plays a role in tumor suppression in lung cancer.miR-26b can regulate the invasion and migration ability of lung carcinoma A549 cells by targeting hENT1 depending on the RhoA/ROCK-1 pathway.

Long non-coding RNA H19 promoted lung cancer cells migration and invasion through miR-107 / 中国免疫学杂志

Objective:To investigate the effect and the related mechanism of H19 on the invasion and migration ability of pancreatic cancer cells.Methods: The expression of H19 in tissues of pancreatic carcinoma and non-carcinoma adjacent tissues of pancreatic carcinoma were detected.qPCR was used to detect the expression of H19 in different pancreatic cancer cells.The migration and invasion ability of pancreatic cancer cells was detected after silencing H19 using wound healing assays and Transwell matrigel invasion assays.Dual-Luciferase Reporter Assay System was used to detect miR-107regulating H19.The expression of miR-107 in tissues of pancreatic carcinoma and non-carcinoma adjacent tissues of pancreatic carcinoma were detected.The regulation of miR-107 on the migration and invasion ability of pancreatic cancer cells were detected after silencing H19 using wound healing assays and Transwell matrigel invasion assays.Subcutaneous tumor was used to detect the size and volume of the tumor after inject the tumor cells.Immunohistochemistry was used to detect the expression of Ki67 and PCNA.Results: Compared with non-carcinoma adjacent tissues of pancreatic carcinoma,the expression of H19 of tissues of pancreatic carcinoma was significantly increased.The expression of H19 in pancreatic cancer cell A549 was highest.Silencing H19 could inhibit the migration of human pancreatic cancer A549 cells.miR-107 was the direct target of H19.Compared with non-carcinoma adjacent tissues of pancreatic carcinoma,the expression of H19 of tissues of pancreatic carcinoma was significantly decreased.After silencing H19,inhibiting the expression of miR107 could inhibit the migration of human pancreatic cancer A549 cells.Compared with the group of H19-siRNA,H19-siRNA+miR-107-inhibitor group mice tumor volume and weight were significantly bigger.Immunohistochemistry showed that compared with H19-siRNA group,the expression Ki67 and PCNA expression in H19-siRNA+miR-107-inhibitor group increased.Conclusion: H19 plays the role of tumor promotor factor in pancreatic cancer.H19 can affect the invasion and migration ability of pancreatic carcinoma cells by regulating miR-107.

Effect of Knocking Down ski on Migration of Astrocytes in Rats / 中国康复理论与实践

Objective To investigate the effect of ski gene in migration process of astrocytes in rats. Methods Astrocytes were obtained from rats' cerebral cortex and cultured in vitro. siRNA targeting ski gene and negative control sequences were prepared. The ski-siRNA group, siRNA negative control group and untreated group were set in this experiment. The specific siRNA targeting ski gene was transfected into astrocytes with Lipofectamine?RNAiMAX Reagent. Then the ski protein levels were determined with Western blotting. After transfec-tion, the changes in migration of astrocytes were measured with wound scratch assay and Transwell migration assay. Results Western blot-ting showed that the expression of ski protein was significantly lower in the ski-siRNA group than in the siRNA negative control group and untreated group (F=132.957, P47.197, P69.187, P<0.001). Conclusion Ski knocked down by siRNA could inhibit the migration ability of astrocytes. It is a reminding that ski may take part in the migration process of astrocytes, and moreover, ski may play an important role in the formation of glial scar.

Mechanism of miR-34a on invasion and migration ability of human lung carcinoma by Snail induced EMT / 中国免疫学杂志

Objective:To investigate the exression of miR-34a on lung cancer and normal lung tissues,and the effect and mechanism of miR-34a in lung cancer cell invasion and migration.Methods: qPCR was used to detect the expression of miR-34a on lung cancer.miR-34a-mimic and miR-34a-inhibitor were used to overexpress and knockdown miR-34a.qPCR was used to detect the effectiveness.Western blot was used to detect the expression of Snail after induced with miR-34a-mimic and miR-34a-inhibitor.Luciferase reporter gene was used to detect interaction between miR-34a and Snail.Transwell invasion assay was used to detect invasion ability after induced with miR-34a-mimic and miR-34a-inhibitor.Scratch assay was used to detect migration ability after induced with miR-34a-mimic and miR-34a-inhibitor.The expression of E-cadherin,Vimentin and Twist were detected by Western blot.Results: miR-34a expression was significantly reduced in lung cancer.With the stage of lung cancer progression,the expression of miR-34a reduced.With the differentiation of lung cancer progression,the expression of miR-34a decreased.Decreasing of miR-34a was associated with lung cancer lymph node metastasis.miR-34a-mimic and miR-34a-inhibitor could overexpress and knockdown miR-34a.miR-34a could regulate expression of Snail.Snail was the direct target of miR-34a;miR-34a could regulate the invasion ability of human lung carcinoma H1650 cells;miR-34a could regulate the migration of human lung carcinoma H1650 cells;miR-34a could regulate the expression of E-cadherin,Vimentin and Twist.Conclusion: miR-34a plays the role of tumor suppressor factor in lung cancer.miR-34a can regulate the invasion and migration ability of lung carcinoma H1650 cells by Snail induced EMT.

Effects of miR-125b on proliferation and invasion of hepatocarcinoma cells by ART4 protein / 中国免疫学杂志

Objective:To investigate the effect of miR-125 b on ART4 protein and its effect on proliferation and invasion of hep-atocellular carcinoma cells .Methods:The expression of miR-125 b in hepatocellular carcinoma and hepatocellular cells was detected by qPCR.The expression of miR-125b and ART4 was detected by qPCR after overexpression of miR-125b.The expression of miR-125b and ART4 was detected by double luciferase assay .The effect of miR-125 b on the proliferation of hepatoma cells was detected by MTT assay.The effect of miR-125b on the invasion of hepatoma cells was detected by Transwell invasion assay .MTT assay was used to detect the effect of miR-125 b on the proliferation of hepatoma cells after overexpression of ART 4.Transwell invasion assay was used to detect the effect of miR-125 b on the invasion of hepatoma cells after overexpression of ART 4.Results: The expression of miR-125 b in hepatoma cells was low,and overexpression of miR-125b could inhibit the expression of ART4 protein.Overexpression of miR-125b could inhibit the proliferation and invasion of hepatoma cells .Overexpression of ART4 could reverse the proliferation and invasion of hepatoma cells by miR-125b.Conclusion:Expression of miR-125b in hepatocellular carcinoma was down-regulated.Meanwhile,miR-125 b can regulate the expression of ART 4 and affect the proliferation and invasion of hepatoma cells .

Effects of miR-143 on invasion and migration of B cell lymphoma cells through regulating expression of PAN3 protein / 中国免疫学杂志

Objective:To investigate the effect of miR-143 on the invasion and migration of B cell lymphoma cells .Methods:The expression of miR-143 in normal bone marrow and lymphoma was detected by qPCR .The expression levels of miR-143 in different cell lines were examined by qPCR .qPCR was used to detect the ability of miR-143 on PAN3.The relationship between miR-143 and PAN3 was detected by double luciferase assay .The effect of miR-143 expression on the migration ability of B cell lymphoma E 6-1 cells was examined by scratch test .The effect of miR-143 expression on the invasion ability of B cell lymphoma E 6-1 cells was exa mined by transwell test.Results:Compared with normal bone marrow ,miR-143 was down-regulated in B-cell lymphoma.Double luciferase assay showed that miR-143 could regulate the expression of PAN 3.Overexpression of miR-143 ,E6-1 cells significantly reduced the ability to attack and migrate.Conclusion:miR-143 can regulate the migration and invasion of B cell lymphoma cells by regulating the expression of PAN3.

Effects of BCYRN1 on miR-503 on migration and invasion of lung cancer by Notch1 pathway / 中国免疫学杂志

Objective:To investigate the mechanism of BCYRN1 regulating miR-503 through Notch1 signaling pathway in the migration and invasion of lung cancer. Methods:The expression of BCYRN1 and miR-503 in different lung cancer cell lines were detected by qPCR. Immunofluorescence and qPCR were used to detect the transfection efficiency of lentivirus BCYRN1 + siRNA transfected lung cancer cells. Double luciferase reporter gene was used to detect the interaction between BCYRN1 and miR-503. Transwell invasion test and scratch test were used to detect the invasion and migration of lung cancer cells after silencing BCYRN1. Western blot was used to detect the expression of Notch1 signaling pathway silence BCYRN1. The effect of silencing BCYRN1 on lung cancer cells in nude mice was measured by subcutaneous tumor formation in nude mice. Results:The expression level of BCYRN1 was the highest in lung cancer cell H1299,and the expression level of miR-503 was relatively high. Immunofluorescence and mRNA levels demonstrated that BCYRN1 + siRNA lentivirus could be effectively transfected into H1299 cells;BCYRN19 binds specifically to the 3′-UTR of miR-503;silencing BCYRN1 could inhibit the invasion and migration of lung cancer H1299 cells;the expression of Notch1 pathway protein was down-regulated after silencing BCYRN1. Compared with NC group,tumor volume and weight of BCYRN1-siRNA group were significantly decreased. Conclusion:BCYRN1 can target the regulation of miR-503 through Notch1 signaling pathway in the invasion and migration of lung cancer H1299 cells.

Effects of miR-210 on Mex3 B protein on migration and proliferation of lung cancer cells / 中国免疫学杂志

Objective:To investigate the effect of miR-210 on the migration and proliferation of lung cancer cells. Methods:The expression of miR-210 in normal lung tissues and lung cancer tissues were detected by qPCR. The expression of Mex3B in lung cancer tissues and adjacent tissues were detected by qPCR. qPCR was used to detect the expression of miR-210 in different lung cancer cell lines(A549,H1299,H1650 and H358). The effect of miR-210 on the transcription of Mex3B was detected by double luciferase reporter gene system. The effect of miR-210 expression on the activity of lung cancer cells was detected by cell viability assay. The effect of miR-210 on the proliferation of A549 cells were detected by plate cloning assay. Transwell invasion assay were used to detect the effect of miR-210 on the invasion ability of lung cancer cell line A549. Results:Compared with adjacent tissues,the expression of miR-210 were significantly decreased in lung cancer tissues,Mex3B were lower in lung cancer. Double luciferase reporter gene system results showed that miR-210 can directly regulate the transcriptional activity of Mex3B. The invasion and proliferation ability of lung cancer cell line were significantly reduced after inhibition of miR-210. Conclusion:miR-210 can target regulate the expression of Mex3B, and affects the invasion and proliferation of lung cancer cells.

c-Myc promoted migration and invasion of malignant melanoma B16-F10 cells through regulating PIK3 R3 / 中国免疫学杂志

Objective:To investigate the effect and the related mechanism of c-Myc on the proliferation,invasion and migration ability of malignant melanoma B16-F10 cells. Methods:We detected the expression of PIK3R3 in malignant melanoma and normal tis-sues. Efficiency of gene silencing of c-Myc and PIK3R3 was determined by qPCR and Western blot. We detected the proliferate ability of B16-F10 cells after silencing c-Myc and PIK3R3 using EdU assay. We detected the migration and invasion ability of B16-F10 cells after silencing c-Myc and PIK3R3 using wound healing assays and Transwell matrigel invasion assays. The expression of miR-199a after silencing c-Myc and PIK3R3 using qPCR. The expression of c-Myc and PIK3R3 was detected by qPCR after transfecting miR-199a mimics or miR-199a inhibitor. Dual-luciferase reporter assay system was used to detect miR-199a regulating c-Myc and PIK3R3. Results:Compared normal skin tissue,expression of PIK3R3 was significantly increased in malignant melanoma tissue;after silencing c-Myc and PIK3R3 gene,the proliferation,invasion and metastasis of melanoma cell line B16-F10 were significantly reduced;expression of miR-199a upregulated after silencing c-Myc and PIK3R3 genes, expression of c-Myc and PIK3R3 decreased after transfecting miR-199a mimics, expression of c-Myc and PIK3R3 upregulated after transfecting miR-199a inhibitor, dual luciferase reporter system test results revealed miR-199a can directly regulate c-Myc and PIK3R3 transcription activity. Conclusion: miR199a regulated the expression of c-Myc,then promote proliferation,migration and invasion in malignant melanoma cells.

Effects of BCYRN1 on miR-503 on migration and invasion of lung cancer by Notch1 pathway / 中国免疫学杂志

Objective:To investigate the mechanism of BCYRN1 regulating miR-503 through Notch1 signaling pathway in the migration and invasion of lung cancer. Methods:The expression of BCYRN1 and miR-503 in different lung cancer cell lines were detected by qPCR. Immunofluorescence and qPCR were used to detect the transfection efficiency of lentivirus BCYRN1 + siRNA transfected lung cancer cells. Double luciferase reporter gene was used to detect the interaction between BCYRN1 and miR-503. Transwell invasion test and scratch test were used to detect the invasion and migration of lung cancer cells after silencing BCYRN1. Western blot was used to detect the expression of Notch1 signaling pathway silence BCYRN1. The effect of silencing BCYRN1 on lung cancer cells in nude mice was measured by subcutaneous tumor formation in nude mice. Results:The expression level of BCYRN1 was the highest in lung cancer cell H1299,and the expression level of miR-503 was relatively high. Immunofluorescence and mRNA levels demonstrated that BCYRN1 + siRNA lentivirus could be effectively transfected into H1299 cells;BCYRN19 binds specifically to the 3′-UTR of miR-503;silencing BCYRN1 could inhibit the invasion and migration of lung cancer H1299 cells;the expression of Notch1 pathway protein was down-regulated after silencing BCYRN1. Compared with NC group,tumor volume and weight of BCYRN1-siRNA group were significantly decreased. Conclusion:BCYRN1 can target the regulation of miR-503 through Notch1 signaling pathway in the invasion and migration of lung cancer H1299 cells.

Effects of miR-210 on Mex3 B protein on migration and proliferation of lung cancer cells / 中国免疫学杂志

Objective:To investigate the effect of miR-210 on the migration and proliferation of lung cancer cells. Methods:The expression of miR-210 in normal lung tissues and lung cancer tissues were detected by qPCR. The expression of Mex3B in lung cancer tissues and adjacent tissues were detected by qPCR. qPCR was used to detect the expression of miR-210 in different lung cancer cell lines(A549,H1299,H1650 and H358). The effect of miR-210 on the transcription of Mex3B was detected by double luciferase reporter gene system. The effect of miR-210 expression on the activity of lung cancer cells was detected by cell viability assay. The effect of miR-210 on the proliferation of A549 cells were detected by plate cloning assay. Transwell invasion assay were used to detect the effect of miR-210 on the invasion ability of lung cancer cell line A549. Results:Compared with adjacent tissues,the expression of miR-210 were significantly decreased in lung cancer tissues,Mex3B were lower in lung cancer. Double luciferase reporter gene system results showed that miR-210 can directly regulate the transcriptional activity of Mex3B. The invasion and proliferation ability of lung cancer cell line were significantly reduced after inhibition of miR-210. Conclusion:miR-210 can target regulate the expression of Mex3B, and affects the invasion and proliferation of lung cancer cells.

Relaxation of Danhong Injection in vascular smooth muscle cells in co-culture system and study on its mechanism / 中草药

Objective: In this study, we examined whether Danhong Injection (DHI) exerted a relaxation effect in vascular smooth muscle cells (A7r5) in co-culture system by human umbilical vein endothelial cells (Ea.Hy926) and studied its mechanisms. Methods: First of all, Western blotting assay was carried out to quantify the protein expression of MLC and P-MLC in A7r5 cells. Secondly, calcium assay kit was used to detect the changes in intracellular calcium while the changes of CaM mRNA expression were subsequently detected by RT-PCR. MTT assay was used to detect the effect of DHI in proliferation of A7r5 cell, which is induced by PDGF-BB. Finally, the co-culture system of EA.Hy926 and A7r5 cells was constructed by a Transwell. After the treatment of Hy926 cells with DHI for co-culture system, the protein expression of MLC and P-MLC in A7r5 cells in co-culture system was also quantified by Western blotting. Cell-based ELISA was used to observe the cAMP generation in A7r5 cells. Results: DHI (10 μL/mL) contributed to the decrease of the ratio of P-MLC/MLC (P < 0.05) and CaM mRNA expression (P < 0.01) in A7r5 cells, which are cultured individually, but had no significant effect in intracellular calcium concentration and the expression of MLC proteins. In co-culture system, DHI (10 μL/mL) could still decrease the ratio of P-MLC/MLC (P < 0.05) and increase the secretion of cAMP (P < 0.01) in A7r5 cells. In addition, DHI contributed to suppress the proliferation of A7r5 cells which were induced by the PDGF-BB (P < 0.01). Conclusion: DHI can directly relax vascular smooth muscle cells by reducing the ratio of P-MLC/MLC and the expression of CaM mRNA, meanwhile indirectly relaxing the vascular smooth muscle cells in co-culture system by promoting the secretion of cAMP which is increased by vasodilatory factors in endothelial cells.

Effects of 5-aza-2′deoxycytidine on RECK gene expression and tumor invasion in salivary adenoid cystic carcinoma

Reversion-inducing cysteine-rich protein with kazal motifs (RECK), a novel tumor suppressor gene that negatively regulates matrix metalloproteinases (MMPs), is expressed in various normal human tissues but downregulated in several types of human tumors. The molecular mechanism for this downregulation and its biological significance in salivary adenoid cystic carcinoma (SACC) are unclear. In the present study, we investigated the effects of a DNA methyltransferase (DNMT) inhibitor, 5-aza-2′deoxycytidine (5-aza-dC), on the methylation status of the RECK gene and tumor invasion in SACC cell lines. Methylation-specific PCR (MSP), Western blot analysis, and quantitative real-time PCR were used to investigate the methylation status of the RECK gene and expression of RECK mRNA and protein in SACC cell lines. The invasive ability of SACC cells was examined by the Transwell migration assay. Promoter methylation was only found in the ACC-M cell line. Treatment of ACC-M cells with 5-aza-dC partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression of mRNA and protein, and 5-aza-dC significantly suppressed ACC-M cell invasive ability. Our findings showed that 5-aza-dC inhibited cancer cell invasion through the reversal of RECK gene hypermethylation, which might be a promising chemotherapy approach in SACC treatment.