Number Of Transmembrane Cells Research Articles

EGFR Inhibitor CL-387785 Suppresses the Progression of Lung Adenocarcinoma.

This study aimed to explore the influence of the irreversible EGFR inhibitor CL-387785 on invasion, metastasis, and radiation sensitization of non-small cell lung cancer cells. The proliferation inhibitory rate at different time points was detected by MTT assay. The apoptosis of H1975 cells treated with CL-387785 was detected using flow cytometry. The invasion and migration of H1975 cells treated with CL-387785 were determined by Transwell assay and wound healing assay. The survival fraction (SF) of H1975 cells cultured with CL- 387785 under X-ray (0, 2, 4, 6, 8, and 10 Gy) was detected by cloning formation experiment, and the sensitization ratio (SER) was calculated by clicking the multi-target model to fit the cell survival curve. CL-387785 restrained H1975 cell proliferation in a concentration- and time-dependent manner. CL-387785 promoted H1975 cell apoptosis and reduced cell migration distance and the number of transmembrane cells. The SF treated by different concentrations of CL-387785 (10, 25, 50, and 100 nM) was all below 0 nM. The radiation SER of CL-387785 (10, 25, 50 and 100 nM) were 1.17, 1.39, 2.88, and 3.64, respectively. The invasion and metastasis of H1975 cells were restrained by irreversible EGFR inhibitor CL-387785. CL-387785 also exhibited the effect of radiotherapy sensitization.

Effect of Kaempferol on the Biological Behavior of Human Colon Cancer via Regulating MMP1, MMP2, and MMP9

Kaempferol is a kind of flavonoid, which has a significant anticancer effect. MMPs were discovered with the function of cleaving the extracellular matrix. We utilized bioinformatics to analyze the association and bonding mode between the traditional Chinese medicine (TCM) monomer composition (i.e., kaempferol) and the target proteins. The purpose of our research was to verify the effect of kaempferol on the biological behavior of human colon cancer cells HCT116 and HT29 and the expression of matrix metalloproteinase (MMP) 1, 2, and 9 genes. We detected the changes in the biological behavior of colon cancer cells treated with kaempferol by CCK-8, wound healing assay, transwell migration/invasion assay, and flow cytometry. Meanwhile， we detected the expression difference of the target gene by qRT-PCR and western blot. Compared with the two control groups, the cell viability of the kaempferol group decreased, the rate of cell migration and the number of transmembrane cells in the kaempferol group decreased significantly, and the early apoptosis rate increased, the number of cells in the G1 phase increased and in the S phase decreased. The results of qRT-PCR and western blot showed that the expression of target genes MMP1, 2, and 9 in the kaempferol group was lower than that in the two control groups. Kaempferol can significantly inhibit the proliferation, invasion, and migration ability of colon cancer cells; induce their apoptosis; and block the cell cycle. Meanwhile, the expression of MMP1, 2, and 9 genes was downregulated, which verified the results of bioinformatic analysis.

The effect and mechanism of exosomes derived from human amniotic epithelial cells on the proliferation and migration of HaCaT in high glucose environment

Objective: To investigate the effect and mechanism of exosomes derived from human amniotic epithelial cells (hAEC-Exos) on the proliferation and migration of HaCaT in high glucose environment. Methods: The experimental research method was adopted. The amniotic membrane tissue was collected from 10 healthy pregnant women at full term delivery in the Department of Obstetrics and Gynecology of Fujian Medical University Union Hospital from January to June 2019, and the primary human amniotic epithelial cells (hAECs) were isolated. The growth status and morphological changes of the primary hAECs on the 2nd, 4th, and 7th day of culture were observed, and the expressions of the cells surface markers of CD73, CD90, CD29, CD34, and human leukocyte antigen DR (HLA-DR). The 2nd to 4th passages of hAECs were used in the following experiments. The hAEC-Exos were separated by ultracentrifugation method. The HaCaT and hAEC-Exos were co-cultured for 3 h, and the uptake of hAEC-Exos by HaCaT was observed by inverted fluorescence microscopy. The HaCaT were divided into phosphate buffer solution (PBS) group and hAEC-Exos group or dimethyl sulfoxide (DMSO)+PBS group, DMSO+hAEC-Exos group, and LY294002+hAEC-Exos group, which were dealt correspondingly, with 3 wells in each group. Cell counting kit 8 (CCK-8) method was used to detect cell proliferation activity after 0 (immediately), 12, 24, 36, 48, and 60 h of culture. The scratch test was conducted to detect the scratch healing at 0, 24, 48, and 72 h after the scratch, and the scratch healing rate was calculated, respectively. The Transwell experiment was conducted to detect the number of transmembrane cells after 48 h of culture. The Western blotting was used to detect the protein expressions of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), protein kinase B (Akt), and phosphorylated Akt (p-Akt) related to phosphatidylinositol 3-kinase-Akt-mTOR (PI3K-Akt-mTOR) pathway after 24 h of culture. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and independent sample t test. Results: Most of the primary hAECs were oval and uniform in size on the 2nd day of culture. The hAECs were arranged in a typical cobblestone-like monolayer on the 4th and 7th day of culture. The primary hAECs highly expressed CD73, CD90, and CD29 of mesenchymal stem cell related surface markers, and were with no or low expressions of CD34 and HLA-DR of hematopoietic stem cell related surface markers. After 3 h of culture, hAEC-Exos were successfully endocytosed by HaCaT into the cytoplasm and gathered around the nucleus. After 12, 24, 36, 48, and 60 h of culture, the proliferation activity of HaCaT in hAEC-Exos group was significantly higher than that in PBS group (t=3.691, 10.861, 12.121, 10.531, 14.931, P<0.01). At 24, 48, and 72 h after scratch, the scratch healing rates of HaCaT in PBS group were significantly lower than those in hAEC-Exos group (t=3.342, 6.427, 5.485, P<0.05 or P<0.01). After 48 h of culture, the number of transmembrane HaCaT in hAEC-Exos group was significantly more than that in PBS group (t=5.385, P<0.01). After 24 h of culture, the protein expressions of p-mTOR and p-Akt in HaCaT of hAEC-Exos group were significantly higher than those in PBS group (t=4.240, 5.586, P<0.01), while the protein expressions of mTOR and Akt in HaCaT of the two groups were similar (P>0.05). After 24 h of culture, the protein expressions of p-mTOR and p-Akt in HaCaT of DMSO+hAEC-Exos group were significantly higher than those in DMSO+PBS group (t=6.155, 8.338, P<0.01) and LY294002+hAEC-Exos group (t=5.030, 3.960, P<0.01), while the protein expressions of mTOR and Akt in HaCaT of the three groups were similar (P>0.05). The proliferation activity of HaCaT in DMSO+hAEC-Exos group at 12, 24, 36, 48, and 60 h of culture was 0.78±0.05, 1.23±0.07, 1.60±0.09, 1.86±0.09, and 2.03±0.08, which was significantly higher than 0.46±0.04, 0.69±0.07, 0.98±0.08, 1.16±0.08, and 1.26±0.11 in DMSO+PBS group (t=4.376, 7.398, 8.488, 9.766, 10.730, P<0.01). The proliferation activity of HaCaT in DMSO+hAEC-Exos group at 24, 36, 48, and 60 h was significantly higher than 0.96±0.09, 1.20±0.08, 1.39±0.08, and 1.55±0.10 in LY294002+hAEC-Exos group (t=3.639, 5.447, 6.605, 6.693, P<0.05 or P<0.01). The scratch healing rates of HaCaT in DMSO+hAEC-Exos group at 24, 48, and 72 h after scratch were significantly higher than those in DMSO+PBS group (t=4.003, 6.349, 7.714, P<0.01) and LY294002+hAEC-Exos group (t=3.805, 4.676, 4.067, P<0.05 or P<0.01). After 48 h of culture, the number of transmembrane HaCaT in DMSO+hAEC-Exos group was significantly more than that in DMSO+PBS group and LY294002+hAEC-Exos group, respectively (t=7.464, 1.232, P<0.01). Conclusions: PI3K-Akt-mTOR pathway can promote the proliferation and migration of HaCaT in high glucose environment by mediating hAEC-Exos.

Activation of the ERK1/2 signaling pathway enhances proliferation and apoptosis of trophoblast in preeclampsia rats.

The aim of this study was to investigate the influences of the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway on preeclampsia rats as well as the proliferation and apoptosis of trophoblasts. A proper number of conceived rats were applied to prepare the preeclampsia model (group P). Meanwhile, others were enrolled as control group (group C). The differences in placental structure between the two groups were compared via hematoxylin-eosin (HE) staining. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were compared between the two groups as well. In addition, T25 cell lines were divided into three groups, including Control group, hypoxia/reoxygenation (H/R) group and H/R + Staurosporine group (an activator of the ERK1/2 signaling pathway). The protein expression of phosphorylated (p)-ERK1/2 in the aforementioned model groups and cells was detected through Western blotting. Cell apoptosis rate was determined by a flow cytometer. Moreover, methyl thiazolyl tetrazolium was utilized to measure the proliferative capacity of trophoblasts in the three groups. Transwell chamber assay was adopted to count the transmembrane cells. The cells in group P were arranged disorderly. Group P had remarkably lower SOD activity but higher MDA content than group C (p<0.05). The protein expression levels of ERK1/2 and p-ERK1/2 in the placenta of group C were evidently higher than those of group P (p<0.05). Besides, the protein expression levels of ERK1/2 and p-ERK1/2 were markedly up-regulated in Control group when compared with H/R + Staurosporine group, with the lowest in H/R group (p<0.05). The proliferative capacity of cells was gradually enhanced in the three groups with the increase of culture time. Cell proliferation was the strongest in Control group, followed by H/R + Staurosporine group and H/R group (p<0.05). The apoptosis and death rates of cells were the highest in H/R group, followed by H/R + Staurosporine group and Control group (p<0.05). However, the number of transmembrane cells was the largest in Control group, followed by H/R + Staurosporine group and H/R group (p<0.05). The ERK1/2 signaling pathway is associated with preeclampsia in rats, whose activation can enhance cell proliferation and weaken cell apoptosis.

Effect of miR-146a on biological behavior of esophageal squamous cell carcinoma cells and its mechanism

Objective: To investigate the effects and mechanisms of miR-146a on the proliferation, invasion, migration, apoptosis and cell cycle of esophageal squamous cell carcinoma cells. Methods: The expressions of miR-146a in 3 esophageal squamous carcinoma cell lines (Eca109, KYSE140, KYSE150) were detected by real-time quantitative polymerase chain reaction (RT-qPCR). MiR-146a mimics was transfected into Eca109 to up-regulate the expression of miR-146a. Effects of miR-146a on cell proliferation, invasion and migration were evaluated by cell counting kit 8 (CCK-8), Transwell assay and wound-healing assay, respectively. The cell apoptosis and cycle were assessed by flow cytometry (FCM). Finally, relevant bioinformatics techniques were used to predict the target gene of miR-146a. Dual luciferase reporter assay was used to identify the interaction of 3' terminal untranslated region (3' UTR) of miR-146a and its target gene, interleukin-1 receptor-associated kinase (IRAK1). RT-qPCR and western blot were used to detect the mRNA and protein expressions of IRAK1, respectively. Results: The relative expressions of miR-146a in 3 esophageal squamous cell carcinoma cells (Eca109, KYSE140, KYSE150) were 0.36±0.05, 0.16±0.06 and 0.09±0.02, respectively, all of which were significantly lower than 1±0.05 of normal esophageal epithelial cells (HEEC) (P<0.01). The model of esophageal squamous cell carcinoma cells was constructed by transfecting miR-146a mimics into Eca109 cells. The results showed that the ability of absorbance value, the number of transmembrane cells (52±18), the reduced scratch distances at 48 hours and 72 hours after transfection [(25.29±0.77) μm, (30.66±0.91) μm] were significantly lower than those of the negative control group and blank control group (all P<0.01). The early apoptosis rate was (6.13±0.91)%, higher than (2.50±0.68)% of the negative control group (P<0.01) and (1.70±0.20)% of blank control group (P<0.01). The percentage of cells in G(1) phase [(44.74±6.76)%] was decreased while the G(2)/M phase [(41.88±2.88)%] was increased when compared with the negative control group and the blank control group (P<0.05 and P<0.01, respectively). The results of dual luciferase reporter gene assay showed that luciferase activity in the group co-transfected with IRAK1-wild-type and miR-146a mimics was significantly lower than that in the control groups (P<0.01). The results of RT-qPCR and western blot showed that the mRNA and protein expressions of IRAK1 in the co-transfected group were 1.02±0.28 and 1.00±0.05, respectively, both lower than those in the negative control group and the blank control group (P<0.01). Conclusions: The expressions of miR-146a are decreased in the esophageal squamous cell lines, which plays a role as tumor suppressor gene. MiR-146a can inhibit the proliferation, invasion and migration of esophageal squamous cell cells, promote apoptosis, and block the cell cycle at G(2)/M stage. MiR-146a may mediate the malignant biological behavior of esophageal cancer cells through the regulation of IRAK1.

Study on the expression of non-coding microRNA-376b-3p in serum exosomes of patients with malignant glioma and the mechanism of anti-angiogenesis

Objective: To screen the different microRNAs in the serum exosomes of patients with malignant glioma, to explore the effect of non-coding microRNA-376b-3p (miR-376b-3p) on the proliferation, invasion and tumor vasculogenic mimicry of glioma cells, and to verify its targeting effect on HOXD10. Methods: HiSeq/MiSeq high-throughput sequencing was used to screen the different microRNA expression profiles, target genes and action pathways in the serum exosomes of patients with malignant glioma. Samples were used to evaluate the expression of candidate microRNAs in serum exosomes of high-grade gliomas. The effects of miR-376b-3p on the proliferation, invasion and angiogenesis of glioma cells were detected by MTT assay, Transwell migration assay and Matrigel vasculogenic mimicry assay. The mRNA and protein expression of HOXD10 were detected to evaluate the regulatory effect of miR-376b-3p on it. Results: There were 144 different expression microRNAs in the serum exosomes between malignant glioma and the normal control. Focal adhesion and tumor protein polysaccharides were involved in the regulation of glioma enriched by KEGG(Kyoto Encyclopedia of Genes and Genomes). MiR-376b-3p was down regulated in malignant glioma, and AUC of malignant glioma was 0.85 (P<0.01). MTT test showed that the proliferation ability of miR-376b-3p inhibitor group was higher than that of the control group, and that of miR-376b-3p mimic group was lower than that of the control group. Transwell migration test showed that the number of transmembrane cells in miR-376b-3p inhibitor group was higher than that in NC inhibitor group, and the number of transmembrane cells in miR-376b-3p mimic group was lower than that in NC mimic group. The number of tubes of vasculogenic mimicry in miR-376b-3p mimic group was lower than that in NC mimic group. MiR-376b-3p inhibitor decreased the expression level of HOXD10 mRNA and protein, and miR-376b-3p mimic increased the expression level of HOXD10 mRNA and protein. Conclusions: MiR-376b-3p is down-regulated in the serum exosomes of malignant glioma patients. The up-regulated miR-376b-3p can reduce the proliferation and invasion of glioma cells, inhibit the formation of vasculogenic mimicry, and increase the expression of HOXD10, which is expected to inhibit the formation of two forms of angiogenesis at the same time. MiR-376b-3p may be a new therapeutic target of anti-angiogenesis for malignant glioma.

Study on the mechanism of llinc00052 regulating Wnt pathway affecting invasion and metastasis of gastric cancer

Objective To explore the mechanism of llinc00052 regulating Wnt pathway affecting invasion and metastasis of gastric cancer. Methods SGC-7901 cells were selected from gastric cancer cell lines, and siRNAs related to INC0005 and had invasion and metastasis effects in gastric cancer cells were screened by binding of IncRNA expression profiles to qRT-PCR.Ilinc00052 and miRNA expression changes were studied by in vitro cell transfection experiments.Through molecular experiments, the expression of llinc00052 and the effect on Wnt/β-catenin expression were investigated to explore whether llinc00052 could affect the invasion and metastasis of gastric cancer cells by regulating miRNAs affecting Wnt/β-catenin signaling pathway. Results Transwell chamber test showed that the number of transmembrane cells in the untransfected plasmid group was (134.10±4.29), and the number of transmembrane cells in the overexpressed llinc000522 plasmid group was (169.24±6.99)(t=8.956, P=0.001). The scratch test showed that the migration distance in the llinc000522 overexpression transfection plasmid group was significantly higher than that in the no-load plasmid transfection group(r=0.907, P<0.01). The clone formation rate of llinc000522 overexpressed transfected plasmid group was significantly higher than that of the empty plasmid group[(92.75±6.32)% vs.(73.34±9.14)%](t=5.998, P<0.05). Compared with the transfection of blank plasmid, the expressions of Wnt1, Wnt3a, Wnt2 and β-catenin mRNA in the llinc000522 overexpression transfection group were significantly up-regulated(P<0.05), while the miRNA transfection group had no significant effect on the expression.The expressions of Wnt1, Wnt3a, Wnt2, and β-catenin mRNA were significantly increased [(1.82±0.11), (1.52±0.15), (1.42±0.21), (1.71±0.19)](P<0.05), but their expressions were still lower than those of the genes transfected with llinc000522 alone.Compared with the blank plasmid transfection, the expressions of Wnt1, Wnt3a, Wnt2 and β-catenin protein in the llinc000522 overexpression transfection group were significantly up-regulated(all P<0.05), while the miRNA transfection group had no significant effect on its expression.The protein expressions of Wnt1, Wnt3a, Wnt2 and β-catenin were also significantly increased[(1.53±0.09), (1.4±0.21), (1.33±0.07), (1.47±0.19)](P<0.05), but their expressions were still lower than those of the gene transfected with llinc000522 alone. Conclusion In gastric cancer cells, llinc00052 can affect the invasion and metastasis of gastric cancer by regulating the level of miRNA and affecting the Wnt/β-catenin pathway. Key words: Stomach neoplasms; Neoplasm lnvasiveness; Neoplasm metastasis; Wnt proteins; Long-chain non-coding RNA; Plasmids; Transfection; Clone cells

Effect of down-regulation of fatty acid synthase expression on proliferation, migration and invasion of bladder carcinoma UMUC3 cell lines

To investigate the effects of fatty acid synthase (FASN) on proliferation, migration and invasion of bladder cancer UMUC3 cell lines and possible mechanism. The expression levels of FASN protein in 30 cases of bladder cancer and 15 cases of normal bladder tissues were detected by Immunohistochemistry. FASN siRNA and nonsense siRNA were transfected into UMUC3 cell lines by lipofectamine 2000 respectively, and the stable siFASN and siControl cell lines were successfully obtained after screening and identification for several times. The siFASN cell lines were set as the experimental group, while the siControl cell lines were set as the control group. The expressions of FASN protein and mRNA in the experimental group and the control group were detected by Western blot and real-time quantitative PCR (RT-PCR) respectively. Cell proliferation activities in two groups were detected by MTT assay and cell invasion and migration in two groups were detected by cell scratch test and Transwell invasive assays respectively. FASN protein was overexpressed in bladder cancer tissues, and it was closely correlated with pathological stage and grade (P＜0.05). Compared with the siControl group, the expressions of FASN mRNA and protein in the siFASN group cell lines were decreased significantly (P＜0.05). The cell proliferation ability, the migration ability and the number of transmembrane cells of siFASN group cell lines were reduced significantly (P＜0.05). The FASN overexpression may play an essential role in the development and progression of bladder cancer. Down-regulation of FASN expression can inhibit the proliferation, migration and invasion of bladder cancer cells, and inhibition of FASN expression is expected to be a new treatment for bladder cancer.

Inhibitory effects of plasmid small interfering RNA targeting signal transducer and activator of transcription‑3 in C6 glioma cells.

The current study investigated the effect of plasmid small interfering RNA(psiRNA)‑mediated silencing of signal transducer and activator of transcription‑3 (STAT3) on the invasion, apoptosis, and expression levels of cyclinD1, caspase‑3 and B‑cell lymphoma‑2(Bcl‑2) in C6 glioma cells. Cell invasion was determined using a Transwell assay, while the apoptosis rate and cell cycle distribution of cells were assessed using AnnexinV‑FITC/PI double staining. The expression levels of cyclinD1, caspase‑3 and Bcl‑2 proteins were measured by western blotting. Transfection with psiRNA‑STAT3 was observed to significantly decrease the number of transmembrane cells compared with the control groups (P<0.05). In addition, the proportion of cells at G0/G1 phase was increased in the psiRNA‑STAT3 group compared with the controls. Western blotting indicated that psiRNA‑STAT3 decreased the expression of cyclinD1, caspase‑3 and Bcl‑2 proteins. Taken together, psiRNA‑STAT3 inhibited the migration and invasive abilities, and induced the apoptosis of C6 glioma cells, possibly through regulation of the expression of cyclinD1, caspase‑3 and Bcl‑2 proteins.

Effects of human leucocyte antigen-G expression on invasion and proliferation of chorionic trophoblastic cell line JEG-3

Objective: To investigate the effects of human leukocyte-associated antigen-G (HLA-G) expression in silencing trophoblast cell line JEG-3 under normal and hypoxic conditions on invasion and proliferation of JEG-3 cells. Methods: Inhibition of HLA-G expression in JEG-3 cells by transfection of small interfering RNA (siRNA),the transfected JEG-3 cells were divided into 4 groups: normoxia control group, hypoxia control group, normoxia inhibition group and hypoxia inhibition group. The levels of HLA-G mRNA and protein in 4 groups of cells were detected by real-time quantitive PCR and western blot. The proliferation activity and invasion ability of 4 groups of cells were determined by methylthiazolyl tetrazolium (MTT) assay and invasion assay. Results: (1) Real-time quantitive PCR technology showed: the level of HLA-G mRNA in the hypoxic inhibition group (0.220±0.050) was significantly different (P<0.05), when compared with that in the hypoxic control group (0.630±0.030) and normoxic inhibition group (0.400±0.020). (2) Western blot analysis showed: the expression level of HLA-G protein in the hypoxic inhibition group was 0.260±0.010, statistically different from that in the hypoxic control group (0.850±0.100) and the normoxic inhibition group (0.560±0.020; P<0.05).(3) MTT showed: proliferative activity of JEG-3 cells in the normoxic inhibition group was 0.490±0.070, the ability of cell proliferation was reduced. When compared with that in the normoxic control group (0.850±0.050), the differences was statistically significant (P<0.05). The proliferative activity of JEG-3 cells in the hypoxic inhibition group (0.330±0.070) was lower than that in the normoxic inhibition group (0.490±0.070), and there was a significant difference (P<0.05). (4) Invasion assay showed: compared with the normoxic control group (98±7), the invasive ability of JEG-3 cells in the normoxic inhibition group (73±7) was weakened, and the difference was statistically significant (P<0.05). The number of transmembrane cells (52±11) of JEG-3 cells in the hypoxic inhibition group was lower than that in the hypoxic control group (72±7), and the difference was statistically significant (P<0.05). Compared with the normoxic inhibition group, the invasion ability of JEG-3 cells in the hypoxic inhibition group decreased, and the difference was statistically significant (P<0.05). Conclusion: Under hypoxia, using siRNA technology to down-regulate the expression of HLA-G may affect the proliferation and invasion ability of trophoblast cells, which may be involved in the occurrence of hypertensive disorder of pregnancy.

The expression and clinical significance of Rho-associated Kinase 1 in glioma tissues

Objective To explore the expression and significance of Rho-associated kinase 1 (ROCK1) in glioma. Methods Western blotting was used to detect the protein expression levels of ROCK1 in glioma tissues. Product-Limit (Kaplan-Meier) was used to analyze the relationship between ROCK1 and clinical features of glioma patients. Small interfering RNA (siRNA) interference was applied to inhibit the expression of ROCK1 in glioblastoma cells. The migration of U251 cells was measured by Transwell assay. The proliferation of U251 cells was examined by cell counting kit-8 (CCK-8) assay. Results Western blotting results showed that ROCK1 protein was highly expressed in glioma tissues. The expression of ROCK1 was not significantly correlated with gender, age, and tumor size (P>0.05), but significantly with World health organization (WHO) classification (P<0.05). Kaplan-Meier analysis showed that the median of ROCK1 high expression group was 13.00±2.04, and that of ROCK1 low expression group was 25.00±6.98. Patients with high expression of ROCK1 had a poor prognosis (P<0.01). Transwell assay results showed that there was a 45%-50% reduction in number of transmembrane cells in si-ROCK1 group, indicating that ROCK1 promotes cancer cell invasion. Transwell migration assay results showed the number of transmembrane cells in the si-ROCK1 group was reduced by 45%-50%, indicating that ROCK1 promotes tumor cell invasion. CCK-8 assay results showed that the number of clonal cells in the si-ROCK1 group was reduced by about 56 compared to the control group, indicating that ROCK1 promotes glioma cell proliferation. Conclusion ROCK1 is highly expressed in glioma patients and is associated with the development, progression, and metastasis of glioma. Key words: Rho-associated protein kinase 1; Glioma; Invasion; Proliferation

Long non-coding RNA RP11-486P11.1 regulates the role of metastasis-associated in colon cancer-1 in invasion and metastasis of lung adenocarcinoma

Objective To observe the regulatory effects of long non-coding RNA (lncRNA) RP11-486P11.1 on metastasis-associated in colon cancer-1 (MACC1) and its role in invasion and metastasis of lung adenocarcinoma. Methods A total of 180 lung cancer patients were enrolled in the lung tissue samples, and 3 samples were selected for lncRNA expression profiling to screen for lncRNA with significant differences in lung adenocarcinoma. The relative expression levels of non-coding RNA RP11-486P11.1 in lung cancer tissues and adjacent normal tissues, lung cancer cell lines H1229, A549 abd NL9980, and human bronchial epithelioid cells HBE were detected by real-time fluorescent quantitative polymerase chain reaction. RP11-486P11.1 lentiviral expression plasmid was transfected into A549 cells. Transwell chamber was used to detect cell invasive ability, and scratch assay was used to detect cell migrative ability. Results The results of microarray showed that 45 lncRNAs in adenocarcinoma showed high expression to varying degrees, and 31 showed low expression, RP11-486P11.1 had statistically significant difference in chip results (P<0.05), and the expression level in each group was lower than that in normal tissues. The expression of lncRNA RP11-486P11.1 in lung cancer tissues was significantly lower than that in adjacent tissues (P<0.05). The expression in lung cancer cell lines H1229, A549 and NL9980 was significantly lower than that om HBE cells. Transfection of RP11- 486P11.1 lentiviral expression plasmid could increase the expression level of RP11-486P11.1 in A549 cells. Transwell chamber results showed the number of transmembrane cells in the plasmid transfection group was 16.6±2.15, which was significantly reduced as compared with the lentiviral empty vector group (37.7±2.4) and the normal control group (39.2±3.5). The results of the scratch test showed that the distance in the normal control group and the plasmid transfection group was (896±23) and (294±18) μm respectively (P<0.01). Conclusion The low expression of lncRNA RP11-486P11.1 in lung cancer cells and tissues plays an important role in the invasion and migration of lung cancer. Key words: Lung cancer; Long non-coding RNA; RP11-486P11.1; Invasion; Migration

Inhibitory effect of docetaxel combined with oncolytic adenovirus carrying short hairpin RNA targeting special AT rich sequence binding protein-1 on proliferation and invasion of breast cancer MDA-MB-231 cells

Objective To investigate the effect of docetaxel and oncolytic adenovirus carrying short hairpin RNA (shRNA) targeting special AT rich sequence binding protein-1 (SATB1) (ZD55-SATB1) on the proliferation and invasion of breast cancer MDA-MB-231 cells. Methods MDA-MB-231 cells were divided to 5 groups and treated with phosphate buffer (PBS), docetaxel (DOC, 1×10-6 mol/L), ZD55-enhanced green fluorescent protein (EGFP) (10 multiplicity of infection, MOI), ZD55-SATB1 (10 MOI), ZD55-SATB1 plus DOC (10 MOI + 1×10-6 mol/L) respectively. The viability of MDA-MB-231 cells treated with DOC (1×10-9, 1×10-8, 1×10-7, 1×10-6, 1×10-5 mol/L) alone and ZD55-SATB1 (0, 0.1, 1.0, 10.0, 100.0 MOI) alone for 96 h was assessed by cell counting kit-8 (CCK-8) assay. And the viability of MDA-MB-231 cells of 5 groups with 48-h treatment was assessed by CCK-8 assay. Cell invasion and migration capacities of MDA-MB-231 cells were detected by Transwell assay and Wound-Healing Assay respectively. The expression of E1A, SATB1 and invasion associated protein matrix metalloproteinase (MMP)-2, Vimentin, DE-cadherinF in MDA-MB-231 cells was detected by Western blotting. Results CCK-8 assay showed that DZD55-SATB1F (10 MOI) plus DOC (1×10-6 mol/L) (35.16±2.17)%, compared with corresponding dose ZD55-SATB1 (10 MOI) (58.26±2.02)% (P=0.015), ZD55-EGFP (10 MOI) (71.25±3.49)% (P=0.023), DOC (1×10-6 mol/L) (50.16±3.17)% (P=0.018), could significantly inhibit the viability of MDA-MB-231 cells after 48 h. Wound-healing assay showed that ZD55-SATB1 plus DOC could significantly inhibit the ability of cell migration as compared with ZD55-SATB1 group, ZD55-EGFP group or DOC group. Transwell assay showed the number of transmembrane cells in combination group (356.2±101.5), produced obvious inhibition of cell invasion and migration capacity of the MDA-MB-231 cells as compared with ZD55-SATB1 group (576.1±89.4) (P=0.035), ZD55-EGFP group (935.2±201.3) (P=0.027) and DOC group (878.4±178.7) (P=0.016), and the difference was significant. Western blotting showed that the adenovirus ZD55-SATB1 and ZD55-EGFP were efficient replication in MDA-MB-231 cells and DOC had no effect on adenovirus replication. In addition, SATB1 expression in the ZD55-SATB1 plus DOC group was lower than PBS group, DOC group, ZD55-SATB1 group and ZD55-EGFP group. Western blotting analysis also confirmed that ZD55-SATB1 plus DOC could increase the expression level of E-cadherin, concomitant with down-regulating the protein expression of MMP-2, Vimentin (P=0.000). Conclusion Oncolytic adenovirus carrying shRNA targeting SATB1 gene could inhibit the growth and invasion of MDA-MB-231 cells. The combined use of ZD55-SATB1 with DOC could significantly inhibit the proliferation and invasion of MDA-MB-231 cells. And the combination of the treatment of oncolytic adenovirus and DOC is better than DOC or adenovirus alone. Key words: Special AT rich sequence binding protein-1; Breast cancer; Docetaxel; Invasion; Migration

Effects of secreted frizzled-related proteins 2 and β-catenin on proliferation, migration and invasion of colorectal cancer cells

Objective To observe the effects of secreted frizzled-related proteins 2 (sFRP2) and β-catenin on proliferation, migration and invasion of colorectal cancer cells. Methods The pcDNA3.1 plasmid containing sFRP2 gene sequence was constructed to overexpress sFRP2 in HCT116 by transfection. The expression level of sFRP2 in HCT116 cells after transfection was detected by Western blotting. Cell counting kit-8 (CCK-8) assay was used to detect the effect of sFRP2 upregulation on the proliferation of colorectal cancer cells; Wound-healing assay was used to detect the effect of sFRP2 up-regulation on the migration of colorectal cancer cells; Transwell assay was used to detect the effect of sFRP2 up-regulation on the invasion ability of colorectal cancer cells. Results After plasmid transfection, the expression of sFRP2 and β-catenin significantly increased. The ratio of sFRP2 protein to β-actin was 0.62±0.03 vs. 0.21±0.02 (t=25.430, P=0.001), and the ratio of β-catenin to β-actin was 0.32±0.02 vs. 0.17±0.01 (t=15.000, P=0.001), and the difference was statistically significant. The absorbance values at 12, 24, 36, 48 and 60 h in sFRP2 transfection group, control group and empty plasmid group were measured to be 0.213±0.015 vs. 0.215±0.018 vs. 0.218±0.012, 0.345±0.053 vs. 0.351±0.041 vs. 0.362±0.038, 0.382±0.058 vs. 0.413±0.051 vs. 0.433±0.048, 0.441±0.079 vs. 0.503±0.033 vs. 0.534±0.053, and 0.482±0.034 vs. 0.594±0.045 vs. 0.628±0.062. As compared with the control group and the empty plasmid group, the cell proliferation rate in sFRP2 transfection group was significantly decreased (12 h: t=0.016, P=0.988 and t=0.041, P=0.970; 24 h: t=0.155, P=0.884 and t=0.452, P=0.675; 36 h: t=0.695, P=0.525 and t=1.173, P=0.306; 48 h: t=1.254, P=0.278 and t=1.693, P=0.166; 60 h: t=3.440, P=0.026 and t=3.576, P=0.023). The difference in the distances between the both sides of scratches at 0 h and 48 h in the blank group was used as control. The distance in the blank group, empty plasmid group, and sFRP2 transfection group was 1.00±0.00, 0.91±0.06, and 0.61±0.04, respectively. The distance in sFRP2 transfection group was farther than blank group and empty plasmid group, and the migration speed was significantly slower (t=16.890, P=0.001 and t=7.206, P=0.002); The number of transmembrane cells in the sFRP2 transfection group was (75.69±7.19) cells, significantly less than that in the control group [(195.39±8.68) cells, t=25.459, P=0.001], indicating the invasive ability of the cells was significantly decreased in the sFRP2 transfection group. Conclusion The up regulation of sFRP2 significantly inhibited the proliferation, migration and invasion of colorectal cancer cell line HCT116. Key words: Colorectal cancer; Secreted Frizzled-related proteins 2; β-catenin; Proliferation; Migration; Invasion

Expression of HPA in astrocytomas and its effect on invasiveness of tumor cells

Objective: To observe the expression of heparanase (HPA) in human astrocytomas tissue and its effect on invasiveness of tumor cells. Methods: Immunohistochemical Elivision™plus two step method and Realtime-PCR method were used to detect the expression of HPA protein and mRNA in 75 cases of astrocytoma tissues and 40 cases of normal brain tissues. Silencing HPA expression in astrocytoma U87 cells by siRNA interference technique, and adopting Realtime PCR, Western blot, Transwell chamber experiment, the expression and cell invasiveness change of tumor cells HPA, vascular endothelial growth factor (VEGF), matrix metalloproteinase -9 (MMP.9) were detected. Results: The expression of HPA protein was not found in normal brain tissues, HPA positive expression rate in astrocytoma tissues was 78.67% (59/75), and the positive expression rate of HPA in astrocytoma tissues was significantly higher than that in normal brain tissues, and its expression level of mRNA was significantly higher than that in normal brain tissues, the difference of which was statistically significant (χ2=67.8, P<0.05). The positive expression rate of HPA in grade II to IV group of astrocytoma increased gradually, the difference of which was statistically significant (χ2=131.5, P<0.05); the positive expression rate of HPA in metastasis group was significantly higher than that in non-metastasis group (χ2=89.3, P<0.05). SiRNA can effectively inhibit the expression of HPA in astrocytoma U87 cells, and down regulate the expression of VEGF, MMP-9, at the same time the number of transmembrane cells decreased significantly, and the difference was statistically significant (P<0.05). Conclusion: HPA is highly expressed in astrocytomas tissues, and with the increase of malignancy of tumor, the expression increases gradually. The cell invasiveness decreased significantly after siRNA silenced HPA, and HPA might be a potential target for the treatment of astrocytomas.

Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133.

The present study aimed to isolate and identify the properties of the cluster of differentiation (CD)133+ subset in human gallbladder cancer cells. The CD133+ and CD133− subpopulations of the GBC-SD cell line were separated using immunomagnetic separation, and the biological features of the two subpopulations were analyzed in vitro and in vivo. In particular, the present study aimed to determine whether the two subpopulations were resistant to anti-tumor reagents and to identify the underlying molecular mechanisms involved. Following cell sorting of GBC-SD cells using immunomagnetic beads, 90.2±2% of cells were identified as CD133+. Immunofluorescence confirmed that CD133 was expressed at higher levels in the Cd133+ group compared with the CD133− group. The proliferation of the CD133+ group was significantly increased compared with the CD133− group in vitro and in vivo. Following treatment with fluorouracil or gemcitabine, cells in the CD133+ group exhibited a decreased sensitivity to these drugs. The number of transmembrane cells was significantly increased in the CD133+ group compared with the CD133− group. In addition, the expression levels of ATP binding cassette subfamily G member 2, CD44, C-X-C motif chemokine receptor 4 (CXCR4), phosphorylated-protein kinase B (Akt) and CD133 in the CD133+ group were significantly increased, compared with those in the CD133− group. In CD133+ GBC-SD cells, stromal cell-derived factor 1α (SDF-1α) or treatment with AMD3100, an inhibitor of CXCR4, promotes or suppresses the SDF-1α/CXCR4 axis, respectively, resulting in increased or decreased CD133 expression through the Akt signaling pathway. Inhibition of the Akt signaling pathway resulted in decreased CD133 expression in GBC-SD cells. Immunomagnetic beads were successfully used for isolation of the CD133+ subset from GBC-SD cells. Furthermore, the CD133+ subset revealed an increased potential for tumor formation, cell proliferation, invasion and resistance to chemotherapeutic agents with expression of stem cell-associated genes. Therefore, in GBC-SD cells, the CXCR4/Akt/CD133 signaling pathways may be activated.

Bcl-2 associated athanogene 3 affects the epithelial-mesenchymal transition in human cervical cancer

Objective: To investigate the expression of Bcl-2 associated athanogene 3 (BAG3) in cervical cancer tissues and cells and its role in epithelial mesenchymal transition (EMT) of cervical cancer. Methods: (1) Cervical cancer samples were collected from September 2015 to March 2017 in the Qilu Hospital of Shandong University and Shangdong Provincial Hospital. While, 50 normal tissues were collected from August 2015 to March 2017 in the Dezhou Municiple Hospital, which were obtained from patients with uterine myoma underwent hysterectomy and patients with cervical biopsy. Reverse transcription (RT)-PCR and western blot were used to detect the expression of BAG3 mRNA and protein, and their clinical significances were analyzed. (2) The expression of BAG3 mRNA and protein was detected using RT-PCR and western blot method in HeLa and SiHa cell lines and normal cervical epithelial cells. The experiment was divided into two groups, BAG3 small interfering RNA transfected group (si-BAG3) and the control group transfected with small interfering RNA (siRNA). Cell counting kit 8 (CCK-8) analysis was used to detect cell proliferation of two groups. Wound-healing and transwell assay were used to detect the migration and invasion ability of HeLa and SiHa cells. The xenograft model of cervical cancer in nude mice was used to observe the effect of BAG3 on tumor xenografts and the tumor-related biomarkers were tested by western blot. Results: (1) The expression levels of BAG3 mRNA and protein in cervical carcinoma tissues were 1.20±0.15 and 1.10±0.16, which were significantly higher than that in normal cervical tissue, 0.23±0.04 and 0.29±0.03 (both P<0.01). The results showed that the expression levels of BAG3 mRNA and protein were significantly correlated with cervical carcinoma staging and lymph node metastasis (P<0.05).However, its expression was not correlated with the patient's age, pathological grade, and diameter of tumor (all P>0.05). (2) Compared with normal cervical epithelial cells, the expression of BAG3 mRNA and protein levels in HeLa and SiHa cells were significantly increased (P<0.01), the expression levels of BAG3 mRNA and protein in HeLa and SiHa cells transfected with si-BAG3 were significantly lower than that in control group (all P<0.01). After post-transfected 72 hours, A value of HeLa and SiHa with transfection were significantly lower than those in control group [(0.88±0.08) vs (1.22±0.13), (0.92±0.09) vs (1.35±0.12); both P<0.01]. After post-transfected 24 hours, the migration level of HeLa and SiHa cells with transfection were significantly lower than those in the control group [(20.1±2.1)% vs (58.6±5.6)%, and (21.1±2.1)% vs (61.7±5.4)%; both P<0.01]. The transmembrane cell number in HeLa and SiHa cells with transfection were 76±11 and 71±8, which were significantly less than those in control group (131±12 and 129±14; both P<0.01). After the inoculation into nude mice, tumor formation time of HeLa and SiHa cells with transfection were (9.5±0.5) and (10.5±1.3) days, respectively, which were significantly longer than those in control group [(4.5±0.5) and (5.2±1.1) days; both P<0.05]. Compared with those in the control group, the expression level of Slug, N-cadherin and matrix metalloproteinase-2 (MMP-2) protein in HeLa and SiHa cells with transfected in tumor tissues were significantly decreased (all P<0.01), while the expression level of E-cadherin protein was significantly increased (P<0.01). Conclusion: BAG3 could be involved in the proliferation, migration and invasion of cervical cancer cells by affecting cervical cancer EMT, and BAG3 may be an effective target for the treatment of cervical cancer.

Effect of plumbagin on epithelial-mesenchymal transition and underlying mechanisms in human tongue squamous cell carcinoma cells

Objective: To study the effect of plumbagin on epithelial-mesenchymal transition (EMT) and underlying mechanisms in human tongue squamous cell carcinoma (TSCC) cells. Methods: Methyl thiazolyl tetrazolium assay was apllied to examine the proliferation inhibition effect and half maximal inhibitory concentration (IC(50)) of plumbagin (0.1, 1.0, 5.0, 10.0, 20.0 μmol/L) in 12, 24, 48 h in TSCC cells. Transwell assay was used to count the number of transmembrane cells and scratch test was performed to examine cells mobility. The flow cytometry was applied to measure intracellular reactive oxygen species (ROS) level in control group, plumbagin group (1.0 μmol/L, 24 h) and glutathione (GSH)+plumbagin group. The expression of E-cadherin, vimentin, Slug, p38 mitogen activated protein kinases (p38MAPK) and phospho-p38MAPK (p-p38MAPK) proteins were determined by Western blotting. The expression of E-cadherin, vimentin and Slug were detected by Western blotting in control group, plumbagin group, activator combined group (p38MAPK activator+plumbagin) and inhibitor combined group (p38MAPK inhibitor+plumbagin). Results: After the treatment of plumbagin for 12, 24, and 48 h, the IC(50) of TSCC cells were 10.3, 3.1, 1.5 μmol/L. After treated by 1.0 μmol/L plumbagin for 24 h, the number of transmembrane cells were significantly reduced ([50±13], P<0.05) in comparison to control group (204±6), as well as the cells mobility ([18.2±2.3]%, P<0.05) in comparison to control group ([49.3±1.2]%). Compared to control group (2.32±0.52), the ROS level was increased in plumbagin group (902.20±10.69), while compared to plumbagin group, the ROS level was reduced in GSH combined group (2.18±0.15). In comparison to control group, the expression of E-cadherin was up-regulated (P<0.05), and vimentin, Slug, p-p38MAPK/p38MAPK were down-regulated in plumbagin group (P<0.05). In comparison to plumbagin group, the expression of E-cadherin was down-regulated (P<0.05), and vimentin, Slug, p-p38MAPK/p38MAPK were up-regulated in GSH combined group (P<0.05). Treatment of cells with p38MAPK activator could decrease the expression of E-cadherin significantly (P<0.05) and increase the expression of vimentin (P<0.05) and Slug (P<0.05) in comparison to plumbagin group. Treatment of cells with p38MAPK inhibitor could increase the expression of E-cadherin significantly (P<0.05) and decrease the expression of vimentin (P<0.05) and Slug (P<0.05) in comparison to plumbagin group. Conclusions: Plumbagin inhibits EMT via ROS/p38MAPK-mediated pathway in human TSCC cells.

Role of SDF-1/CXCR4 axis in the invasion and migration of human choriocarcinoma cells and its significance in preeclampsia

Objective To investigate the effects of stromal cell-derived factor 1 (SDF-1) and an CXC chemokine receptor 4 (CXCR4) antagonist (AMD3100) on the invasion and migration capabilities of the huaman choriocarcinoma cell line JAR for further elucidating the role of SDF-1/CXCR4 axis in the pathogenesis of preeclampsia. Methods JAR cells were divided into four groups: SDF-1 group (treated with 50 ng/ml of SDF-1), SDF-1+ AM3100 mixed group (first treated with 100 ng/ml of AMD3100 for 2 hours and then treated with 50 ng/ml of SDF-1), AMD3100 group (treated with 100 ng/ml of AMD3100) and blank control group (without any treatment). RT-PCR was performed to detect the expression of CXCR4 at mRNA level in JAR cells. Western blot assay was used to measure the expression of CXCR4 and p-AKT at protein level. MTT assay was used to analyze the effects of different concentrations of SDF-1 (10, 30, 50 and 100 ng/ml) on the proliferation of JAR cells at different time points (0, 24, 48, 72 h). Transwell invasion assay and wound-healing assay were used to test the changes in invasion and migration capabilities of JAR cells after different treatments. Results (1) Results of the RT-PCR showed that the expression of CXCR4 at mRNA level in JAR cells was increased in the SDF-1 group (1.839±0.083) as compared with that in the blank control group (1.372±0.086), AMD3100 group (0.694±0.045) or SDF-1+ AM3100 mixed group (0.703±0.093). Moreover, the differences between the SDF-1 group and the other three groups were statistically significant (F=30.67, P<0.05). Compared with the blank control group, the expression of CXCR4 at mRNA level in JAR cells was decreased in the AMD3100 group (P<0.01). (2) Results of the Western blot assay showed that the expression of CXCR4 and p-AKT at protein level in JAR cells were enhanced in the SDF-1 group as compared with that in the blank control group, AMD3100 group or SDF-1+ AM3100 mixed group. Compared with the blank control group, the expression of CXCR4 and p-AKT at protein level in JAR cells were inhibited in the AMD3100 group. (3) Results of the MTT assay showed that SDF-1, especially at the concentration of 50 ng/ml, could enhance the proliferation of JAR cells (P<0.05) and its best effect on proliferation was seen at 48 h. (4) Results of the Transwell invasion assay showed that the number of transmembrane cells in the SDF-1 group (70.49±2.42) was more than that in the blank control group (54.36±2.26), AMD3100 group (21.68±8.31), or SDF-1+ AMD3100 mixed group (28.18±4.61). The differences between the SDF-1 group and the other three groups were statistically significant (F=116.26, P<0.01). Compared with the blank control group, the number of transmembrane cells was reduced in the AMD3100 group (P<0.05). (5) Results of the wound-healing assay showed that the relative migration distance was increased in the SDF-1 group (1.162±0.034) as compared with that in the blank control group (0.823±0.101), AMD3100 group (0.160±0.047), or SDF-1+ AMD3100 mixed group (0.183±0.064). The differences between the SDF-1 group and the other three groups were statistically significant (F=30.500, P<0.05). Compared with the blank control group, the relative migration distance was decreased in the AMD3100 group (P<0.01). Conclusion The invasion and migration of huaman choriocarcinoma JAR cells can be enhanced by SDF-1, but inhibited by AMD3100. This study indicates that the blocked biological axis of SDF-1/CXCR4 may play an important role in the pathogenesis of preeclampsia through inducing abnormal activation of PI3K/AKT pathway, which results in inhibited invasion and migration of trophoblast cells and placenta abnormality. Key words: SDF-1/CXCR4 axis; Invasion; Migration; PI3K/AKT pathway; Trophoblast cell; Preeclampsia

Effects of TRAM-34 on Proliferation and Invasion of Leukemia Cell Line HL-60

To investigate the effects of KCa3.1 channel inhibitor TRAM-34 on the proliferation and invasion of leukemia cell line HL-60. HL-60 cells at logarithmic growth phase exposed to TRAM-34 at the final concentration of 25, 50, 75 and 100 nmol/L were used as experimental group. The HL-60 cells of control group was cultured in 10% fetal bovine serum-RPMI 1640. The proliferation inhibition rate of TRAM-34 on HL-60 cells was detected by adding MTT solution after 24, 48 and 72 h culture. The cell apoptotic rate and cell cycle distribution of HL-60 cells treated with TRAM-34 were evaluated by flow cytometry with Annexin V-FITC/propidium iodide(PI) double staining or PI single staining. The number of transmembrane cells was detected by Transwell at 24 and 48 h after treatment with TRAM-34. The effect of TRAM-34 on CDK6, P53 and MMP-2 mRNA level was detected by real-time quantitative PCR. Compared with the control group (0 nmol/L), the inhibition rate, apoptosis rate, G0/G1 phase cell proportion and P53 mRNA level all increased, but the percentages of cells in S phase, cell number penetrating the membrane and mRNA levels of CDK6 and MMP-2 in the TRAM-34-treated group decreased (P<0.05) except for 24 h proliferation rate of TRAM-34 at low concentration (25 nmol/L). The effect of TRAM-34 on the above indices was enhanced with the increase of concentration and prolongation of time, and the differences were statistically significant (P<0.05). TRAM-34 can inhibit the proliferation and invasion of HL-60 cells, and can induce cell apoptosis and G0/G1 arrest. The time and concentration of TRAM-34 have effect on the malignant behavior of HL-60 cells.