Silencing MTA1 by RNAi Reverses Adhesion,Migration and Invasiveness of Cervical Cancer Cells （SiHa） via Altered Expression of p53, and E-cadherin/β-catenin Complex

It has been reported that metastasis-associated gene 1 (Mta1) is overexpressed in many malignant tumors with high metastatic potential. In addition, some studies indicated that MTA1 participated in invasion, metastasis, and survival of cancer cells by regulating cell migration, adhesion and proliferation. But the role of MTA1 is unclear in vitro in the development of cervical cancer cells. This study investigated whether and how MTA1 mediated cell proliferation, migration, invasion and adhesion in cervical cancer. MTA1 expression level was detected by Western blot in two cervical cancer cell lines of different invasion potentials. The effects of MTA1 expression on SiHa cell apoptosis, cycle, proliferation, migration, invasion and adhesion were tested by flow cytometry, MTT, wound-healing assay, Transwell assay and adhesion assay, respectively. The expression levels of p53, E-cadherin, and β-catenin activity were evaluated in untreated and treated cells. The results showed that MTA1 protein expression was significantly higher in SiHa than in HeLa, which was correlated well with the potential of migration and invasion in both cell lines. Furthermore, the cell invasion, migration and adhesion capabilities were decreased after inhibition of MTA1 expression mediated by Mta1-siRNA transfection in SiHa. However, no significant differences were found in cell apoptosis, cycle, and proliferation. In addition, E-cadherin and p53 protein levels were significantly up-regulated, while β-catenin was significantly down-regulated in SiHa transfected with the siRNA. These results demonstrated that MTA1 played an important role in the migration and invasion of cervical cancer cells. It was speculated that the decreased migration and invasion capability by inhibiting the MTA1 expression in the SiHa cell line may be mediated through the altered expression of p53, and E-cadherin/β-catenin complex. MTA1 could serve as a potential therapeutic target in cervical cancer.     

Effect of siRNA targeting MTA1 on metastasis malignant phenotype of ovarian cancer A2780 cells

Ovarian cancer is the fifth lethal gynecologic malignancy. Metastasis-associated gene 1 (MTA1) is overexpressed in many malignant tumors with high metastatic potential. This study investigated whether down-regulation of MTA1 expression by RNAi in A2780 ovarian cancer cells could affect proliferation, anoikis, migration, invasion and adhesion of the cells and to research the potential for MTA1 gene therapy of ovarian cancer. After transfection with effective Mta1 gene siRNA, the effects on proliferation, anoikis, migration, invasion and adhesion of A2780 cells were tested by MTT assay, flow cytometry, wound-healing assay, Transwell assay and adhesion assay. Expression levels of PTEN, beta 1 integrin, MMP-9, phosphor-AKT (Ser473), and total AKT activity were evaluated in control and transfected cells. The results showed that inhibition of MTA1 mediated by Mta1-siRNA transfection decreased the cell invasion, migration and adhesion, and induced the increased cell anoikis, but no significant difference was found in proliferation of A2780 cancer cells. In addition, beta 1 integrin, MMP-9, and phosphor-AKT protein levels were significantly down-regulated, while PTEN was significantly up-regulated. These results demonstrated that MTA1 played an important role in the cell metastasis in ovarian cancer. MTA1 could serve as another novel potential therapeutic target in ovarian cancer.     

miR-200c inhibits metastasis of breast cancer cells by targeting HMGB1

miR-200c has been shown to regulate the epithelial-mesenchymal transition （EMT） by inhibiting ZEB1 and ZEB2 expression in breast cancer cells. This study further examined the role of miR-200c in the invasion and metastasis of breast cancer that goes beyond the regulation on ZEB1 and ZEB2 expression. In this study, the bioinformatics software （miRanda） was used to predict the target gene of miR-200c and Renilla luciferase assay to verify the result. The metastatic breast cancer cells MDA-MB-231 were cultured and transfected with the miR-200c mimic or inhibitor. The expressions of miR-200c and HMGB 1 were detected by RT-PCR and Western blotting, respectively. Transwell assay and wound healing assay were employed to examine the invasive and migrating ability of transfected cells. Target prediction and Renilla luciferase analysis revealed that HMGB1 was a putative target gene of＇miR-200c. After transfection of MDA-MB-231 cells with the miR-200c mimic or inhibitor, the expression of miR-200c was significantly increased or decreased when compared with cells transfected with the miR-200c mimic NC or inhibitor NC. Moreover, the expression of HMGB1 was reversely correlated with that of miR-200c in transfected cells. Tranwell assay showed that the number of invasive cells was significantly reduced in miR-200c mimic group when compared with miR-200c inhibitor group. It was also found that the migrating ability of cells transfected with miR-200c mimics was much lower than that of cells transfected with miR-200c inhibitors. It was suggested that miR-200c can suppress the invasion and migration of breast cancer cells by regulating the expression of HMGB1. miR-200c and HMGB1 may become useful biomarkers for progression of breast cancer and targets of gene therapy.     

Inhibitory effects of microRNA-34a on cell migration and invasion of invasive urothelial bladder carcinoma by targeting notch1

MicroRNAs (miRNAs or miRs) are a class of short, non-coding RNAs that participate in various oncological processes. This study aims to explore the roles of microRNA-34a (miR-34a) in invasive urothelial bladder carcinoma. miR-34a was transfected into bladder cancer cell lines 253J and J82. The miR-34a expression levels in tissues and cells were detected by using qRT-PCR. The Notch1 expression was detected by qRT-PCR and Western blotting. Cell migratory and invasive abilities were measured by Transwell chamber assay. Bioinformatics and luciferase assay were performed to predict and analyze the binding sites between miRNA-34a and Notch1. It was found that there was aberrant expression of miR-34a in bladder cancer tissues. Moreover, we revealed that ectopic expression of miR-34a suppressed cell migration and invasion, while forced expression of Notch1 increased cell migratory and invasive abilities. Finally, we observed that miR-34a transfection significantly down-regulated luciferase activity and reduced the mRNA and protein levels of Notch1. Our study concluded that microRNA-34a antagonizes Notch1 and inhibits cell migration and invasion of bladder cancer cells, which indicates the tumor-suppressive function of microRNA-34a in bladder cancer.     

Vascular Endothelial Growth Factor165-regulated Nasopharyngeal Carcinoma Cell Lines Invasion and Migration Involve Expression and Activation of Matrix Metalloproteinase-2

The effect of vascular endothelial growth factor （VEGF） overexpression on matrix metalloproteinase-2 （MMP-2） in nasopharyngeal carcinoma （NPC） cells in vitro and the possible mechanism involved were investigated, and the correlation between the expression of VEGF and MMP-2 in NPC evaluated. The NPC cells were transfected with PAd-trackVEGF165 plasmid. The expression levels of VEGF and MMP-2 mRNA and protein in NPC cells were detected by semi-quantitative RT-PCR and Western blot respectively. It was found that the expression of VEGF and MMP-2 mRNA and protein was significantly increased in NPC cells after transfection of VEGF 165. It was concluded that the expression of VEGF was correlated to the in vitro invasion of NPC cells, and the induction of MMP-2 by VEGF was a key process of NPC cell invasion.     

MicroRNA-34a inhibits human brain glioma cell growth by down-regulation of notch1

The effects of microRNA-34a (miR-34a)-regulated Notch1 gene on the proliferation and apoptosis of the human glioma cell line U87 were investigated in this study. The U87 cells were divided into miR-34a mimics, negative control, mock transfection and blank control groups in terms of different treatments. In miR-34a mimics group, human U87 glioma cells were transfected with miR-34a mimics by using lipofectamine 2000. The cells transfected with nonsense microRNA were set up as negative control group. Those treated with lipofectamine 2000 only were designated to the mock tranfection group. In the blank control group, the cells were cultured routinely and no treatment was given. The expression of miR-34a and Notch1 was detected by using real-time RT-PCR. Western blotting was employed to monitor the change in Notch1 protein. Cell proliferation and apoptosis were measured by CCK-8 and flow cytometry. The results showed that the proliferative ability of U87 cells was significantly reduced and the apoptotic cells increased in miR-34a mimics group relative to control groups. The expression of miR-34a was significantly up-regulated in mimics group as compared with control groups (P<0.05). Furthermore, Notch1 protein levels were significantly decreased in miR-34a mimics group when compared with control groups (P<0.05), but the mRNA expression of Notch1 showed no significant difference among these groups. It was concluded that miR-34a may suppress the proliferation and induce apoptosis of U87 cells by decreasing the expression of target gene Notch1, suggesting that miR-34a may become a promising gene therapeutic target for brain glioma.     

Effect and mechanism of tacrolimus on melanogenesis on A375 human melanoma cells

Background Topical tacrolimus has been used for vitiligo as a common treatment option for more than ten years while the underlying mechanism is still uncertain.The aim of this study was to investigate the direct effects of tacrolimus on the melanogenesis and migration on human A375 melanoma cells.The expression of c-KIT mRNA and protein of human A375 cells were also investigated.Methods The cultured A375 human melanoma cells were randomly assigned to control and tacrolimus treatment groups （10,102,103and 104 nmol/L）.The cell proliferation was measured with Cell Counting Kit-8 assays.Melanin content was measured with NaOH method.Transwell migration assay was used to measure cell migration.The expression of c-KIT mRNA and protein were measured with real-time fluorescence quantitative polymerase chain reaction and immunohistochemistry respectively.Results The cell proliferation of the 103 and 104 nmol/L tacrolimus groups were significantly lower （0.666±0.062 and 0.496±0.038） as compared with the control （0.841±0.110,P 〈0.05）.The mean melanin content in all groups treated with different concentration of tacrolimus （10,102,103,104 nmol/L） increased compared with the control group （P 〈0.05）.Dosedependent increase in cell migration were seen in all tacrolimus-treated groups （P 〈0.01）.The expression of c-KIT mRNA level in A375 cells exposed to tacrolimus （103and 104 nmol/L） had significantly increased by 3.03-fold and 3.19-fold respectively compared with the control （P 〈0.05）.Conclusions Although tacrolimus had no effects on cell proliferation on A375 human melanoma cells,it could increase the melanin content and cell migration.The expression of c-KIT mRNA and protein increased dose-dependently in tacrolimus-treated groups as compared with the control.Our study demonstrated that tacrolimus could enhance the melanogenesis and cell migration on A375 cells.     

Ezrin promotes invasion and migration of the MG63 osteosarcoma cell

Background Evidence shows that ezrin plays an important role in the development of some human malignancies.But the mechanism by which ezrin may affect tumor cell invasion and metastasis remains unclear.Methods In this study,the expression of ezrin was verified in osteosarcoma （OS） cells and tissues by comparison with normal bone cells and tissues using Western blotting.OS-MG63 were transfected with pcDNA3.1-ezrin or pGenesil-1/ shRNA-ezrin and the stably transfected cells were selected with G418 to yield the ezrin cell line.The OS-MG63 tumor cells were delivered by tail vein to female BALB/c to develop pulmonary metastasis model in vivo.Ezrin was identified as a direct target of miR-183 via a luciferase reporter carrying the 3＇-untranslated region of ezrin.Migration assays and invasion assays were done with the transwells.Signaling pathway was studied by Western blotting and/or inhibitor.Results Ectopic overexpression of ezrin in OS cell line MG63 promoted tumor cell invasion and migration.Consistent with this,knockdown of ezrin inhibited tumor cell invasion and migration.Similar results were obtained in the experimental metastasis model in vivo.We identified ezrin as a direct target of miR-183.What is more,ectopic expression of ezrin could induce the expression of N-cadherin and enhance the activity of extracellular signal-regulated kinase （ERK） signaling.Conclusion Collectively,these results suggest that ezrin as a direct target of miR-183 promotes the aggressiveness of OS via increased N-cadherin and activating ERK signaling.     

Expressions of miR-22 and miR-135a in acute pancreatitis

This study examined the expressions of miR-22 and miR-135a in rats with acute edematous pancreatitis （AEP） and their target genes in order to shed light on the involvement of miR-22 and miR-135a in the pathogenesis of acute pancreatitis （AP）. The in vivo model of AEP was established by introperitoneal injection of L-arginine （150 mg/kg） in rats. The miRNA microarray analysis was used to detect the differential expression of miRNAs in pancreatic tissue in AEP and normal rats. The in vitro AEP model was established by inducing the rat pancreatic acinar cell line （AR42J） with 50 ng/mL re- combinant rat TNF-ct. Real-time quantitative RT-PCR was employed to detect the expression of miR-22 and miR-135a in AR42J cells. Lentiviruses carrying the miRNA mimic and anti-miRNA oligonucleotide （AMO） of miR-22 and miR-135a were transfected into the AR42J cells. The AR42J cells transfected with vehicle served as control. Western blotting was used to measure the expression of activated cas- pase3 and flow cytometry analysis to detect the apoptosis of AR42J cells. Targets of miR-22 and miR-135a were predicted by using TargetScan, miRanda, and TarBase. Luciferase reporter assay and quantitative real-time RT-PCR were performed to confirm whether ErbB3 and Ptk2 were the target gene of miR-22 and miR-135a, respectively. The results showed that the expression levels of miR-22 and miR-135a were obviously increased in AEP group compared with the control group in in-vivo and in-vitro models. The expression levels of miR-22 and miR-135a were elevated conspicuously and the expression levels of their target genes were reduced significantly in AR42J cells transfected with Ienti- viruses carrying the miRNA mimic. The apoptosis rate was much higher in the TNF-ct-induced cells than in non-treated cells. The AR42J cells transfected with miRNA AMOs expressed lower level of miR-22 and miR-135a and had lower apoptosis rate, but the expression levels of ErbB3 and Ptk2 were increased obviously. It was concluded that the expression levels of miR-22 and miR-135a were elevated in AEP. Up-regulating the expression of miR-22 and miR-135a may promote the apoptosis of pancreatic acinar cells by repressing ErbB3 and Ptk2 expression in AEP.     

Immunohistochemical Study of HLA-DR Antigen in Endometrial Tis-sue of Patients with Endometriosis

In order to evaluate the expression of HLA-DR antigen in glandular cells in eutopic and ectopic endometrium in patients with endometriosis, 19 infertile patients with endometriosis were analyzed immunohistochemically by labelled streptavidin biotin (LSAB) method. Nineteen infertile patients without endometriosis were studied as controls. The results showed that the expression of HLA-DR antigen in the glandular cells in both eutopic and ectopic endometrium was increased significantly as compared with that in the controls (P＜0.01). It is likely that aberrant expression of HLADR antigen in endometriotic tissue is involved in abormal immunogenesis of endometriosis.     

Effect of leptin on cytotrophoblast proliferation and invasion

The effects of leptin on cytotrophoblast proliferation and invasion activity were investigated. Immunohistochemistry was used to determine the placental expression of leptin in first-trimester pregnancy. By using RT-PCR and quantitative real-time PCR, the expression of leptin in cytotrophoblast and the effect of leptin on cytotrophoblast secretion were detected. The potential of cell proliferation, invasiveness and migration was assessed by MTT, Transwell invasion assay and migration assay respectively when the cytotrophoblast was cultured with different concentrations of leptin. The results showed that： （1） Leptin was distributed diffusely around cell membrane, in cytoplasma, and on nuclear mem- brane of cytotrophoblast; （2） Leptin mRNA was expressed in cytotrophoblast. Ten ng/mL leptin could promote the secretion of cytotrophoblast significantly （P〈0.01）; （3） After culture with different concentrations of leptin for 24 h or longer, the proliferation of cytotrophoblast was inhibited, while in 24 h leptin could promote cytotrophoblast invasion and migration. Leptin at a concentration of 500 ng/mL could promote cytotrophoblast invasiveness and migration significantly as compared with controls （P〈0.05）. It was suggested that leptin could inhibit cytotrophoblast proliferation, and promote cytotro- phoblast invasion and migration activity.     

Effect of siRNA Targeting Survivin Gene on the Biological Behavior of Hepatocellular Carcinoma

To study the influence of siRNA targeting survivin gene on the biological behavior of hepatocellular carcinoma (HCC), one pair of 21bp reverse repeated motifs of survivin target sequence with 9 spacers were synthesized and inserted into plasmid psilencer2.1 to generate siRNA eukaryotic expression vector. After stable transfection into HepG2 cells, the biological behaviors of the survivin siRNA transfected HCC cells were observed. After the recombinant plasmid Psilence( )-survivin was successfully constructed, survivin mRNA and protein expression inhibition ratio reached 73% and 75% respectively compared to control groups. Transfected cells with survivin siRNA demonstrated significantly inhibited cell growth and increased apoptosis. Subsequent study in nude mouse model demonstrated lower succeeding rate in cells transfected with survivin siRNA and slow growth rate. The results elucidated the siRNA targeting survivin gene could specially suppress its expression in HepG2 cells and inhibit tumor cells growth both in vivo and in vitro. This provides a theoretical basis to turn the drug resistance in tumor cells.     

Inhibition of Invasion and Up-regulation of E-cadherin Expression in Human Malignant Melanoma Cell Line A375 by（-）-Epigallocatechin-3-gallate

The inhibitory effects of （-）-epigallocatechin-3-gallate （EGCG） on the invasion of human malignant melanoma cell line A375 and the possible molecular mechanisms of this effect were investiaged. A375 cells were pretreated with 20 μg/mL EGCG for 24, 48 and 72 h respectively and the E-cadherin expression was detected by Western blot analysis. A375 cells were also pretreated with different concentrations of EGCG （1, 5, 10 and 20 μg/mL） for 72 h and the expression of E-cadherin was measured by RT-PCR. The adhesion and invasion of A375 cells were tested by cell-matrigel adhesion assay and matrigel invasion assay respectively. The results showed that EGCG could significantly up-regulate the expression of E-cadherin time-and concentration-dependently （both P〈0.05）. Statistical analysis showed that A375 cells invasion was inhibited by EGCG and correlated with the up-regulation of E-cadherin expression. It was suggested that EGCG strongly inhibited invasion of A375 cells, and the inhibition mechanism was possibly associated with the up-regulation of E-cadherin expression.     

The Effect of Targeted Magnetic Nanopaticles on Hepatoma and the Expression of bcl-2/bax Protein

The effect of targeted magnetic nanoparticles on hepatoma and the underlying mechanism were examined. Nude mice transplanted with a human hepatoma cell line （HepG2 cells） were randomized into 5 groups, including： （1） group A, receiving normal saline, （2） group B, receiving 5-fluorouracil （5-Fu）, （3） group C, receiving magnetic nanoparticles containing 5-Fu, （4） group D, consisting of treatment with magnetic nanoparticles containing 5-Fu and inside magnetic field and （5） group E, receiving pure magnetic nanoparticles and inside magnetic field. Morphological features of transplanted tumors in mice in each group were observed under transmission electron microscope （TEM）. The expression of bcl-2/bax protein was immunohistochemically detected by SABC method. The results showed that a large number of apoptotic tumor cells were found in group B and group D under TEM. The expression of bcl-2 protein was significantly decreased and the expression of bax protein increased significantly in both group B and D as compared with those in group A, C and E （P〈0.01 for all）. The decrease in bcl-2 and the increase in bax were more in group D as compared with group B （P〈0.01）. It is concluded that the targeted magnetic nanoparticles containing 5-Fu can improve the chemotherapeutic effect of 5-Fu by decreasing bcl-2 expression, increasing bax expres- sion and inducing apoptosis of the liver cancer cells.     

Vascular endothelial growth inhibitor (VEGI) affects the invasion, apoptotics and vascularisation in breast cancer cell line MDA-MB-231

Background: Breast cancer is one of the most common malignant female diseases worldwide. It is a significant threat to every woman’s health. Vascular endothelial growth inhibitor (VEGI) is known to be abundant in endothelial cells. According to previous literature overexpression of VEGI has been shown to inhibit tumor neovascularisation and progression in cellular and animal models, but there has been limited research on the significance of VEGI in breast. Methods: In our study, cell lines MDA-MB-231were first constructed in which VEGI is lentivirus-mediatedly over-expressed. The effects of VEGI over-expression on MDA-MB-231 cells were investigated both in vitro and in vivo. The expression of VEGI in the MDA-MB-231 cells after infection of lentivirus was analyzed using real-time PCR and Western blot. The effect of biological characteristics of MDA-MB-231 cells was assessed by growth, invasion, adhesion, and migration assay with subcutaneous tumor-bearing nude mice models. Then the growth curves of the subcutaneous tumors were studied. Expressions of VEGI, CD31 and CD34 in the tumors were analyzed by immunohistochemistry and apoptosis was detected by flow cytometry and immunohistochemistry. Results: Infection of MDA-MB-231 cells within the lentivirus resulted in an approximately a 1000-fold increase in the expression of VEGI. As can be seen in the invasion, adhesion and migration assay - the over-expression of VEGI can inhibit the ability of MDA-MB-231 cells during migration, adhesion and invasion. The volume of the subcutaneous tumor in over-expression group was distinctly and significantly less than that of the controlled groups. Immunohistochemistry analysis of the tumor biopsies clearly showed the expression of VEGI in the over-expression group increased while CD31 and CD34 decreased significantly. In vitro and in vivo, the early apoptosis rate and the apoptosis index were increased within the VEGI over-expression group as compared with the control group. Conclusions: Taken together, recombinant lentivirus that were successfully constructed, demonstrated up-regulated VEGI gene expression in breast cancer cells. In vitro, lentivirus-mediated over-expression of VEGI weakened the ability of the breast cancer cell migration, adhesion and invasion. Over-expression of VEGI diminished the tumorigenicity capacity of breast cancer cells in vivo. Up-regulation of VEGI gene expression however inhibited breast cancer MDA-MB-231 cell in the early apoptosis In vitro and In vivo.     

Vascular Endothelial Growth Factorl65-regulated Nasopharyngeal Carcinoma Cell Lines Invasion and Migration Involve Expression and Activation of Matrix Metalloproteinase-2

The effect of vascular endothelial growth factor (VEGF) overexpression on matrix met-alloproteinase-2 (MMP-2) in nasopharyngeal carcinoma (NPC) cells in vitro and the possible mechanism involved were investigated, and the correlation between the expression of VEGF and MMP-2 in NPC evaluated. The NPC cells were transfected with PAd-trackVEGF165 plasmid. The expression levels of VEGF and MMP-2 mRNA and protein in NPC cells were detected by semi-quantitative RT-PCR and Western blot respectively. It was found that the expression of VEGF and MMP-2 mRNA and protein was significantly increased in NPC cells after transfection of VEGF165. It was concluded that the expression of VEGF was correlated to the in vitro invasion of NPC cells, and the induction of MMP-2 by VEGF was a key process of NPC cell invasion.     

Vascular endothelial growth inhibitor affects the invasion, apoptosis and vascularisation in breast cancer cell line MDA-MB-231

Background Breast cancer is one of the most common malignant female diseases worldwide.It is a significant threat to every woman＇s health.Vascular endothelial growth inhibitor （VEGI） is known to be abundant in endothelial cells.According to previous literature,overexpression of VEGI has been shown to inhibit tumor neovascularisation and progression in cellular and animal models,but there has been limited research on the significance of VEGI in the breast cancer.Methods In our study,cell lines MDA-MB-231 were first constructed in which VEGI mediated by lentivirus over-expressed.The effects of VEGI over-expression on MDA-MB-231 cells were investigated both in vitro and in vivo.The expression of VEGI in the MDA-MB-231 cells after infection of lentivirus was analyzed using real-time PCR and Western blotting.The effect of the biological characteristics of MDA-MB-231 cells was assessed by growth,invasion,adhesion,and migration assay with subcutaneous tumor-bearing nude mice models.Then the growth curves of the subcutaneous tumors were studied.Expressions of VEGI,CD31 and CD34 in the tumors were analyzed by immunohistochemistry and apoptosis was detected by flow cytometry and immunohistochemistry.Results Infection of MDA-MB-231 cells within the lentivirus resulted in approximately a 1 000-fold increase in the expression of VEGI.As can be seen in the invasion,adhesion and migration assay,the over-expression of VEGI can inhibit the ability of MDA-MB-231 cells during migration,adhesion and invasion.The volume of the subcutaneous tumor in the over-expression group was distinctly and significantly less than that of the control groups.Immunohistochemistry analysis of the tumor biopsies cleady showed the expression of VEGI in the over-expression group increased while CD31 and CD34 decreased significantly.In vitro and in vivo,the early apoptosis rate and the apoptosis index were increased within the VEGI over-expression group as compared with the control group.Conclusions Taken together,recombinant lentivirus that were successfully constructed,demonstrated up-regulated VEGI gene expression in breast cancer cells.Lentivirus-mediated over-expression of VEGI weakened the ability of the breast cancer cell migration,adhesion and invasion.Over-expression of VEGI diminished the tumorigenic capacity of breast cancer cells in vivo.Up-regulation of VEGI gene expression however inhibited breast cancer MDA-MB-231 cell in the early apoptosis.