Changes In Biological Behavior Research Articles

MicroRNA-326 inhibits cell proliferative capacity and invasiveness through inhibiting the expression of ETS1 in nasopharyngeal carcinoma.

This study aims to detect microRNA-326 expression in nasopharyngeal carcinoma (NPC) tissues and cell lines and to explore the potential mechanism of microRNA-326 inhibiting the proliferative capacity and invasiveness of NPC cells. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine microRNA-326 expression in 40 cases of NPC tissue samples and cell lines. Meanwhile, microRNA-326 mimics were transfected into NPC cells to up-regulate microRNA-326 level. Next, the influence of microRNA-326 mimics on the proliferation and invasiveness of NPC cells was observed by Cell Counting Kit-8 (CCK-8) assay and transwell assay, respectively. Bioinformatics analysis was applied to search for target genes which may have direct effects on microRNA-326. An EST1 luciferase reporter vector containing microRNA-326 binding site was constructed and the binding relation between ETS1 and microRNA-326 was detected by Dual-Luciferase reporting assay. Lastly, the underlying mechanism of microRNA-326 and ETS1 in NPC was further verified via cell reverse experiments. Compared with control group, microRNA-326 expression was found remarkably decreased both in NPC tissue specimens and cell lines. The low expression of microRNA-326 could predict poor prognosis of patients with NPC. In vitro cell experiments revealed that overexpression of microRNA-326 remarkably inhibited the proliferative capacity and invasiveness of NPC cells. Bioinformatics analysis and Dual-Luciferase assay results suggested that microRNA-326 may bind to ETS1. Cell reverse assay indicated that inhibiting ETS1 expression partially reversed the changes in cell biological behavior induced by the down-regulation of microRNA-326 in CNE1 and 5-8F cell lines. Overexpression of microRNA-326 in NPC cells could remarkably inhibit the proliferative capacity and invasiveness of NPC cells. In addition, microRNA-326 may participate in the development of NPC by inhibiting ETS1 expression.

Long non-coding RNA TTN-AS1 promotes breast cancer cell migration and invasion via sponging miR-140-5p.

Breast cancer (BC) is one of the most common fatal cancers. Recent studies have identified the vital role of long non-coding RNAs (lncRNAs) in the development and progression of BC. In this investigation, lncRNA TTN-AS1 was studied to identify its function in the metastasis of BC. TTN-AS1 expression of tissues was detected by real-time quantitative polymerase chain reaction (RT-qPCR) in 56 BC patients. Wound healing assay and transwell assay were used to observe the biological behavior changes of BC cells through gain or loss of TTN-AS1. In addition, luciferase assays and RNA immunoprecipitation (RIP) assay were performed to discover the potential targets of TTN-AS1 in BC cells. TTN-AS1 expression level in BC samples was higher than that of adjacent tissue. Besides, the ability of cell migration and invasion of BC cells was inhibited after TTN-AS1 was silenced, while cell migration and cell invasion of BC cells were promoted after TTN-AS1 was overexpressed. In addition, miR-140-5p was upregulated after silencing of TTN-AS1 in BC cells, while miR-140-5p was downregulated after overexpression of TTN-AS1 in BC cells. Furthermore, luciferase assays and RIP assay showed that miR-140-5p was a direct target of TTN-AS1 in BC. Our study uncovered a new oncogene in BC and suggests that TTN-AS1 enhances BC cell migration and invasion via sponging miR-140-5p, which provides a novel therapeutic target for BC patients.

Circ-0003998 promotes cell proliferative ability and invasiveness by binding to miR-197-3p in osteosarcoma.

The aim of this study was to investigate the level of circ-0003998 in osteosarcoma tissues and cell lines, and to analyze its relation with prognosis of patients, as well as its effect on biological behaviors of osteosarcoma cells. In addition, the potential mechanism of circ-0003998 in promoting osteosarcoma cell proliferation and invasion was explored. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to examine circ-0003998 expression in 60 clinical osteosarcoma tissues and cell lines. The association between circ-0003998 expression and the overall survival rate of patients was explored. After shRNA-circ-0003998 was constructed to down-regulate circ-0003998 expression in osteosarcoma cell lines, the proliferation of osteosarcoma cells was observed through the Cell Counting Kit-8 (CCK-8) and colony formation assay. Meanwhile, cell invasiveness was detected by the transwell invasion assay. Bioinformatics was used to search for microRNAs (miRNAs) that contained the direct effect on circ-0003998. Subsequently, the luciferase reporter vector of circ-0003998 or Krüppel-like factor 10 (KLF10) containing miR-197-3p binding site was constructed. Then, the binding of circ-0003998 or KLF10 to miR-197 was detected using the Dual-Luciferase assay. Furthermore, the function recovery experiment was designed to validate the biological function of circ-0003998 and miR-197 in osteosarcoma. Compared to normal control tissues and cells, the expression of circ-0003998 was significantly up-regulated in both osteosarcoma tissue samples and cell lines. Highly-expressed circ-0003998 was significantly associated with poor overall survival of patients with osteosarcoma. In vitro experiments revealed that the down-regulation of circ-0003998 significantly inhibited the proliferative ability and invasiveness of osteosarcoma cells. Bioinformatics analysis and Dual-Luciferase reporter gene assay indicated that circ-0003998 might bind to miR-197-3p in MG-63, and Saos-2 cell lines. Meanwhile, the functional recovery experiment demonstrated that inhibiting miR-197-3p expression could partially restore the changes in cellular biological behaviors induced by circ-0003998 down-regulation in MG-63 and Saos-2 cells. In addition, miR-197-3p was remarkably down-regulated in osteosarcoma tissues, while KLF10 was up-regulated. However, KLF10 was significantly up-regulated after the knockdown of miR-197-3p in osteosarcoma cells. Circ-0003998 plays a vital role in promoting the development of osteosarcoma, whose high expression can predict poor clinical prognosis. Circ-0003998 is highly expressed in osteosarcoma tissues and cell lines. The down-regulation of its level can significantly inhibit the proliferative ability and invasiveness of osteosarcoma cells. Meanwhile, circ-0003998 up-regulates the expression of KLF10 by binding to miR-197-3p, thereby promoting osteosarcoma cell growth and invasion, and accelerating the progression of osteosarcoma.

Long non-coding RNA ROR1-AS1 induces tumorigenesis of colorectal cancer by affecting Wnt/β-catenin signaling pathway.

Recent studies have discovered that long noncoding RNAs (lncRNAs) play an important role in malignant tumors. In this research, lncRNA ROR1-AS1 was selected to identify how it affects the development of colorectal cancer (CRC). ROR1-AS1 expression was detected by RT-qPCR in CRC tissue samples. ROR1-AS1 expression level and patients’ overall survival time were analyzed. Functional experiments were conducted to identify the changes of biological behaviors in CRC cells after knockdown of ROR1-AS1. Moreover, we also explored the underlying mechanism. Detection of ROR1-AS1 expression level in patients’ tissues showed that ROR1-AS1 was higher in CRC tissues than that in adjacent ones. ROR1-AS1 expression was negatively associated with patients’ overall survival time. Cell growth ability was inhibited due to knockdown of ROR1-AS1 in vitro. Moreover, cell migration and invasion were repressed after ROR1-AS1 knockdown. Furthermore, due to knockdown of ROR1-AS1, the targeted proteins in Wnt/β-catenin signaling pathway were suppressed. These results suggest that ROR1-AS1 could enhance cell metastasis and proliferation via inducing Wnt/β-catenin signaling pathway, which might offer a potential therapeutic target in CRC.

LncRNA CASC15 promotes migration and invasion in prostate cancer via targeting miR-200a-3p.

Prostate cancer (PC) is one of the most ordinary malignant cancers. Recent researches have proved that long noncoding RNAs (lncRNAs) act as an important role in cancers. Our study aims to explore the function of lncRNA CASC15 in the tumor metastasis of PC. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was utilized to detect CASC15 expression in 50 PC patients. Besides, the wound healing assay and transwell assay were performed to identify the biological behavior changes of PC cells after CASC15 was silenced in PC cells. In addition, the potential mechanism was also explored using the luciferase assay. CASC15 expression level was significantly higher in PC tissues and cell lines. Results of wound healing assay and transwell assay revealed that cell migrated ability and invaded ability were suppressed via silence of CASC15 in PC cells. Furthermore, the expression of miR-200a-3p was upregulated via silence of CASC15 in PC cells. Luciferase assay showed that miR-200a-3p was a direct target of CASC15 in PC. In addition, miR-200a-3p expression was negatively correlated with CASC15 expression in PC tissues. Rescue experiments also revealed that the inhibition of PC migration and invasion by silence of CASC15 could be reversed through knockdown of miR-200a-3p. Our study uncovers that CASC15 could enhance cell migration and invasion of PC cells by sponging miR-200a-3p, which might be applied as a novel therapeutic target for PC patients.

LINC00511 promotes the progression of non-small cell lung cancer through downregulating LATS2 and KLF2 by binding to EZH2 and LSD1.

Lung cancer is a malignant tumor with extremely high morbidity and mortality. Recent studies have identified the vital role of LINC00511 (lncRNAs) in the development and progression of non-small cell lung cancer (NSCLC). In this research, we aim to explore the biological function of LINC00511 in the development and metastasis of NSCLC. LINC00511 expression in 57 paired NSCLC patients' tissues and matched normal tissues were detected by Real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation assay, colony formation assay and transwell assay were conducted to observe the biological behavior changes of NSCLC cells through the influence of LINC00511. In addition, dual-luciferase reporter gene assay, RNA immunoprecipitation assay (RIP) and, chromatin immunoprecipitation (ChIP) were performed to discover the potential targets of LINC00511 in NSCLC cells. LINC00511 was highly expressed in NSCLC tissues and cell lines compared with controls. LINC00511 expression was positively correlated with tumor size, tumor stage, lymph node metastasis and distant metastasis, but negatively correlated with overall survival (OS) of NSCLC patients. Receiver Operating Characteristic (ROC) curves suggested that LINC00511 could be an effective indicator to distinguish NSCLC patients from normal people. Cell counting kit-8 (CCK-8), flow cytometry and transwell assay showed that knockdown of LINC00511 in A549 cells decreased viability, accelerated apoptosis and inhibited invasive and migratory abilities. Overexpression of LINC00511 in PC9 cells obtained the opposite biological effects. Chromatin fractionation predicted that LINC00511 was mainly distributed in the nucleus. RIP and ChIP assay showed that LINC00551 directly bound to lysine-specific demethylase 1 (LSD1) and enhancer of zeste homolog 2 (EZH2). It inhibited expressions of LATS2 and KLF2 by binding to their promoter regions. LINC00511 is upregulated in NSCLC tissues and cell lines. It is closely related to tumor size, tumor stage, lymph node metastasis and, distant metastasis of NSCLC patients. Knockdown of LINC00511 attenuates proliferative, migratory and invasive capacities, but induces apoptosis of NSCLC cells. LATS2 and KLF2 are target genes of LINC00511, which are regulated by LINC00511 through binding to EZH2 and LSD1, thus influencing the progression of NSCLC.

ROR1-AS1 promotes tumorigenesis of colorectal cancer via targeting Wnt/β-catenin.

Recent studies have discovered that long noncoding RNAs (lncRNAs) play an important role in malignant tumors. In this research, lncRNA ROR1-AS1 was selected to identify how it affected the development of colorectal cancer (CRC). ROR1-AS1 expression was detected by Real-time quantitative polymerase chain reaction (RT-qPCR) in CRC tissue samples. ROR1-AS1 expression level and patients' overall survival time were analyzed. Functional experiments were conducted to identify the changes of biological behaviors in CRC cells after knockdown of ROR1-AS1. Moreover, we also explored the underlying mechanism. Detection of ROR1-AS1 expression level in patients' tissues showed that ROR1-AS1 was higher in CRC tissues than that in adjacent ones. ROR1-AS1 expression was negatively associated with patients' overall survival time. Cell growth ability was inhibited due to knockdown of ROR1-AS1 in vitro. Moreover, cell migration and invasion abilities were repressed after ROR1-AS1 was knockdown. Furthermore, due to the knockdown of ROR1-AS1, the targeted proteins in Wnt/β-catenin signaling pathway were suppressed. These results suggested that ROR1-AS1 could enhance cell metastasis and proliferation via inducing Wnt/β-catenin signaling pathway, which might offer a potential therapeutic target in CRC.

F factor plasmid-mediated Epstein-Barr virus genome introduction establishes an EBV positive NPC cell model.

BackgroundMost Epstein-Barr virus (EBV)-positive cells lose the EBV episomes upon prolonged propagation. PurposeThe purposes of this study were to establish a simple cell model for nasopharyngeal carcinoma (NPC) research by introducing a plasmid with the EBV genome into NPC cells and then to investigate the resulting changes in malignant biological behaviour and NPC-associated signalling pathways. MethodsHONE1 NPC cells were transfected with F-factor plasmids including the EBV genome (HONE1-EBV cells). Then cell proliferation, migration, cell cycle distribution and apoptosis were evaluated in vitro by using CCK8, transwell and flow cytometry assays respectively. EBV-encoded proteins and cell signal tranducting proteins were detected by western blot assays. EBV-encoded RNAs were detected by in situ hybridization. EBV particles were assayed by transmission electron microscope (TEM). The morphology of cells were detected by immunofluorescence assays for alpha-tubulin. ResultsLatent membrane protein 1 (LMP1), latent membrane protein 2A (LMP2A), Epstein-Barr nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) were successfully expressed in HONE1-EBV cells. No EBV particles were founded by TEM. Introduction of the EBV genome significantly promoted proliferation, cell cycle progression and migration and inhibited apoptosis in HONE1 cells. Immunofluorescence assays showed that the morphology of HONE1-EBV cells changed into spindle. Furthermore, EBV genome introduction significantly inhibited the JAK/STAT signalling pathway, while it activated the PI3K-AKT and NF-κB signalling pathways in HONE1 cells. ConclusionThese findings suggest that F-factor plasmid-mediated EBV genome introduction was successful in constructing an EBV positive cell model, which showed deteriorated biological behavior and activated NPC-associated signalling pathways. This model can serve as a good tool for studying EBV in NPC, but the subtle differences in cancer-associated pathways must be considered.

MiR-1261/circ-PTPRZ1/PAK1 pathway regulates glioma cell growth and invasion.

Glioma is the most common primary brain tumor in adults, with high malignancy and poor prognosis. According to the research in these years, the relationship between circular RNAs (circRNAs) and glioma development is abnormally close. Studies on circRNAs in glioma cells revealed that miR-1261 had almost completely matched binding site on the circ-PTPRZ1 sequence, and dual-luciferase reporter gene assay confirmed that circ-PTPRZ1 was a target gene of miR-1261. MiR-1261 inhibited circ-PTPRZ1 expression in glioma cells, while circ-PTPRZ1 did not affect miR-1261 expression. At the same time, circ-PTPRZ1 could promote p-PAK1 expression, while miR-1261 suppressed the activation of PAK1 by regulating the expression of circ-PTPRZ1. Biological behaviors of glioma cells were detected, circ-PTPRZ1 enhanced cell proliferation and invasion, and inhibited cell apoptosis; miR-1261 had the opposite effects, and could terminate the above effects of circ-PTPRZ1. When co-transfected with PAK1 siRNAs and circ-PTPRZ1 over-expression vector, the changes of above biological behaviors were not obvious. Therefore, in glioma cells, the expression of circ-PTPRZ1/PAK1 is regulated by miR-1261, which affects the proliferation, apoptosis, and invasion. This finding provides another powerful evidence for the role of circRNAs in glioma.

Long non-coding RNA LINP1 induces tumorigenesis of Wilms' tumor by affecting Wnt/β-catenin signaling pathway.

Recent studies have discovered that long non-coding RNAs (lncRNAs) play an important role in the development of malignant tumors. The aim of this work was to investigate the exact role of lncRNA LINP1 in the development of Wilms' tumor and to explore the possible underlying mechanism. The expression of lncRNA in non-homologous end joining pathway 1 (LINP1) in tissue samples of Wilms' tumor was detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). The relationship between the expression of lung cancer associated transcript 1 (LUCAT1) and patients' overall survival time was analyzed. Subsequent functional experiments were conducted to identify the changes in biological behaviors of Wilms' tumor cells after the gain or loss of LINP1. Moreover, the underlying mechanism of LINP1 function was explored. QRT-PCR results showed that LINP1 expression level in Wilms' tumor tissues was significantly higher than that of adjacent tissues. LINP1 expression was negatively associated with the overall survival time of patients with Wilms' tumor. Cell growth ability was markedly inhibited and promoted after down-regulation and overexpression of LINP1 in vitro, respectively. Moreover, after the loss and gain of LINP1 in vitro, cell migration and invasion abilities were remarkably repressed and promoted, respectively. Furthermore, the loss of LINP1 in vitro could significantly decrease the expressions of targeted proteins in the Wnt/β-catenin signaling pathway. However, the expressions of targeted proteins in the Wnt/β-catenin signaling pathway were remarkably up-regulated after over-expression of LINP1. LINP1 could enhance cell metastasis and proliferation via inducing the Wnt/β-catenin signaling pathway. Our findings might provide a new prospect for the diagnosis and therapy of Wilms' tumor.

Construction and effects of functionalized graphene oxide nonparticles-mediated RNA interference of hypoxia-inducible factor-1α gene

Objective To explore the effects of graphene oxide (GO)-polyethylene glycol (PEG)-folic acid (FA)-pyrenemethylamine hydrochloride (PyNH2)-mediated RNA interference (RNAi) of hypoxia-inducible factor-1α (HIF-1α) on the biological behaviors of human pancreatic cancer Patu8988 cells. Methods GO-PEG-FA-PyNH2 and RNAi targeting HIF-1α gene (GO-PEG-FA-PyNH2-HIF-1α-RNAi) was constructed. The expressions of HIF-1α and glucose transporter 1 (Glut-1) in Patu8988 cells were determined after knockdown of HIF-1α by RNAi. The invasive ability, the proliferation and the cell cycle of Patu8988 cells were investigated. The effect of HIF-1α knockdown on the uptake of 18F-fluorodeoxyglucose (FDG) in Patu8988 cells was also detected. Comparison of data was conducted by one-way analysis of variance and least significant difference t test. Results The GO-PEG-FA-PyNH2 was successfully constructed, and no cytotoxicity was found. Under the hypoxia or normoxia state, the mRNA and protein levels of HIF-1α and mRNA level of Glut-1 in cells transfected with GO-PEG-FA-PyNH2-HIF-1α-RNAi (study group) were lower than those in cells transfected with GO-PEG-FA-PyNH2(negative group) and cells transfected with Opti-minimal essential medium (Opti-MEM, control group; F=30.25-32.58, t=3.66-5.81, all P<0.05); the numbers of migrated cells in the study group were much lower than those in the negative group and the control group (F=38.63 and 41.35, t=20.51-35.25, all P<0.01); the cell proliferation in the study group was significantly lower than that in the negative group and the control group (F=35.19 and 38.11, t=15.11-22.19, all P<0.05). The proportions of G0/G1 cells in the study group were higher than those in the negative group and the control group (F=34.60 and 34.83, t=11.55-34.56, all P<0.05); the 18F-FDG uptake in the study group was lower than that in the negative group and control group (F=29.85 and 31.69, t=3.35-4.35, all P<0.05). Conclusion GO-PEG-FA-PyNH2-mediated HIF-1α RNAi inhibits the expression of HIF-1α in pancreatic cancer cells, leading to changes in related biological behaviors. Key words: Pancreatic neoplasms; Tumor cells, cultured; RNA, small interfering; Hypoxia-inducible factor 1, alpha subunit; Graphene oxide; Polyethylene glycols; Folic acid; 1-Pyrenemethylamine

Circ-ZNF264 Promotes the Growth of Glioma Cells by Upregulating the Expression of miR-4493 Target Gene Apelin.

Glioma is the most common malignant tumor in the brain and nervous system, with high recurrence and high mortality rate. Recent researches have shown that circular RNAs (circRNAs) play key roles in the genesis and progress of glioma. Detection of circRNAs in glioma cells revealed that the expression of circ-ZNF264 was upregulated. At the same time, the expression of miR-4493 was downregulated in glioma cells and had multiple binding sites on the circ-ZNF264 sequence. Dual luciferase reporter gene assay confirmed that miR-4493 could bind to circ-ZNF264 and apelin specifically. MiR-4493 expression was not changed, but its target gene apelin expression could be significantly upregulated by circ-ZNF264. MiR-4493 could inhibit the expression of circ-ZNF264 and apelin. Biological behaviors of glioma cells were detected; circ-ZNF264 promoted cell proliferation and invasion and inhibited apoptosis. MiR-4493 had the opposite effects and could terminate the above effects of circ-ZNF264. When the expression of apelin was downregulated and that of circ-ZNF264 was upregulated, the changes of the above biological behaviors were not obvious. Therefore, in glioma cells, circ-ZNF264 can inhibit the function of miR-4493 and then upregulate its target gene apelin expression, thus regulating glioma cell proliferation, apoptosis, and invasion. This finding provides more evidence for the role of circRNAs in glioma.

High-pressure artificial pneumothorax promotes invasion and metastasis of oesophageal cancer cells.

This study aimed to investigate the viability, apoptosis, invasion and metastasis of oesophageal cancer cells in a simulated artificial pneumothorax model and to explore its potential mechanism of action. Oesophageal cancer cells were subjected to a simulated thoracoscopic CO2 pneumothorax environment with different pressures and exposure times (low-pressure group: 8 mmHg 1 h or 8 mmHg 4 h; high-pressure group: 12 mmHg 1 h). Cell viability, apoptosis, invasive capacity and mRNA expression of adhesion- and metastasis-related molecules in each group were detected. To explore in greater detail the potential reasons for the changes in biological behaviour under the high-pressure CO2 environment, we designed 3 additional experimental groups: (i) high-pressure group, (ii) hypoxia group and (iii) pH decrease group. An miRNA microarray analysis was performed by comparing 2 paired samples of cells from the high-pressure group and the control group. Treatment with high-pressure CO2 pneumothorax significantly increased the cell viability (P < 0.001) and the cell invasion (P < 0.001). Significantly higher expression of adhesive- and metastasis-related molecules was also observed. Further experiments indicated that the high-pressure CO2 pneumothorax might increase cell invasion and metastasis through the high pressure and decreased pH. The miRNA microarray analysis results suggested that several potential pathways related to cancer development: the RhoA pathway, the PI3K-Akt signalling pathway and the MAPK signalling pathway. The application of high-pressure CO2 pneumothorax promoted the invasion and metastasis of oesophageal cancer cells through high pressure and decreased pH. This process might be related to several signalling pathways.

Long noncoding RNA SNHG14 promotes breast cancer cell proliferation and invasion via sponging miR-193a-3p.

Breast cancer (BC) is one of the most ordinary fatal cancers. Recent studies have identified the vital role of long noncoding RNAs (lncRNAs) in the development and progression of BC. In this research, lncRNA SNHG14 was studied to identify how it functioned in the development and metastasis of BC. SNHG14 expression of tissues was detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) in 50 paired patients with BC. And cell proliferation assay, colony formation assay, and transwell assay were enrolled to observe the biological behavior changes of BC cells through gain or loss of SNHG14. In addition, luciferase assays and RNA immunoprecipitation assay (RIP) were performed to discover the potential targets of SNHG14 in BC cells. SNHG14 expression level of BC samples was higher than that of adjacent ones. Besides, cell growth ability and cell invaded ability of BC cells were inhibited after SNHG14 was silenced, while cell growth ability and cell invaded ability of BC cells were promoted after SNHG14 was overexpressed. In addition, miR-193a-3p was upregulated after silence of SNHG14 in BC cells, while miR-193a-3p was downregulated after overexpression of SNHG14 in BC cells. Furthermore, luciferase assays and RNA immunoprecipitation assay (RIP) showed that miR-193a-3p was a direct target of SNHG14 in BC. Our study uncovers a new oncogene in BC and suggests that SNHG14 could enhance BC cell proliferation and invasion via sponging miR-193a-3p, which provided a novel therapeutic target for BC patients.

Non-tight junction-related function of claudin-7 in interacting with integrinβ1 to suppress colorectal cancer cell proliferation and migration.

PurposeWe conducted a preliminarily exploration of the role and possible mechanism of the non-tight junction-related function of claudin-7 in the occurrence and development of colorectal cancer.MethodsWe selected the colorectal cancer cell line HCT116, constructed a stably transfected claudin-7 knockdown cell line via RNAi and lentiviral infection, and determined the claudin-7 knockdown efficiency. We assessed the biological behavior changes (cell viability, apoptosis, and migration) in the stably transfected HCT116 cells and observed structural changes in the tight junction by transmission electron microscopy. We used a subcutaneous tumor formation model to assess the tumorigenicity of HCT116 cells after claudin-7 knockdown. We assessed the expression and localization of integrinβ1 in the stably transfected cell line by immunofluorescence staining and investigated the interaction between integrinβ1 and claudin-7 by co-immunoprecipitation.ResultsAfter the knockdown of claudin-7 the expression, the viability and migration ability of HCT116 cells increased and apoptosis decreased. Transmission electron microscopy indicated that the intercellular tight junction structure did not change substantially. Furthermore, the tumor growth in nude mice was enhanced. Immunofluorescence staining showed that integrinβ1 and claudin-7 were co-expressed and co-localized on the cell membrane, and immunoprecipitation suggested that claudin-7 interacts with integrinβ1.ConclusionClaudin-7 may inhibit the proliferation and migration of tumor cells by interacting with integrinβ1, subsequently participating in the development of colorectal cancer.

Lyophilization as an alternative method for preservation of some continuous cell cultures

This work is a trial to provide lyophilization as a simple method for preservation of continuous cell lines instead of the freezing process in liquid nitrogen. Comparative evaluation of lyophilized African green monkey kidney (VERO), baby hamster kidney (BHK) and Madin Darby bovine kidney (MDBK) cell lines and those preserved by freezing in liquid nitrogen was carried out. Such evaluation on 6 months intervals revealed that both of lyophilized cell cultures showed delayed cell adhesion to the culture surface extending to 2-3 days post culturing while propagated and frozen cells adhered to the culture surface within few hours. However there were an increased number of adhered viable cells in case of loading of cells using trehalose and sucrose on days post culturing. In addition cultured lyophilized cells which loaded by trehalose or sucrose exhibited abnormal shape (showing cell rounding) in comparison to the other cultures. Also cell dispersing of confluent sheets of cultured lyophilized cells were found to take longer time (one hour) than that required to other cultures (few minutes) even on using EDTA and incubation at 37 °C. Further studies are needed to investigate the biological behavior or cell changes which may be occurred through the lyophilization process in addition to study the susceptibility of such cells to virus infection. Depending on these facts it could be said that lyophilization is not valid for VERO, BHK and MDBK which usually used for preparation of virus vaccine in stationary or roller systems. It also could be suggested that preservation of cell lines by lyophilization may be of value in cell culturing suspension system.

Zoledronate inhibits fibroblasts' proliferation and activation via targeting TGF-β signaling pathway.

BackgroundPrevious preclinical and clinical studies have demonstrated that zoledronate might inhibit neointimal hyperplasia at least partly by inhibiting the proliferation, adhesion and migration of vascular smooth muscle cells (VSMCs). However, whether zoledronate influences fibroblasts’ proliferation and activation, which also play a key role in neointimal hyperplasia and vascular remodeling, remains largely unknown. In the present study, the effect of zoledronate on fibroblasts was investigated and the underlying molecular mechanisms were examined.MethodsAfter treatment with zoledronate, changes in biological behaviors, including the morphology, proliferation, cell-cycle distribution and migration of fibroblasts (NIH3T3 cells), were observed. The expression of α-SMA, TGF-β1 and TGF-β2 and the level of Smad2/3 phosphorylation in cultured fibroblasts were examined by Western blot. In vivo expression of α-SMA and TGF-β1 was assessed by immunohistochemical staining.ResultsIt was shown that the typical fibroblast cell morphology was altered after zoledronate exposure. Cultured fibroblasts treated with zoledronate displayed dose-dependent inhibition of cell proliferation due to cell-cycle arrest in the S phase. Cell migration activities were also dose dependently suppressed by zoledronate treatment. Expression of α-SMA in cultured fibroblasts was significantly reduced by zoledronate treatment. Further analysis showed decreased expression of TGF-β1 and α-SMA by periadventitial delivery of zoledronate in the rat carotid balloon-injury model. The expression of TGF-β1 and TGF-β2 and the phosphorylation of Smad2/3 in cultured fibroblasts were significantly inhibited by zoledronate treatment.ConclusionOur findings demonstrated that zoledronate can inhibit the proliferation, migration and activation of fibroblasts via the TGF-β signaling pathway and revealed a novel mechanism of zoledronate action against neointimal hyperplasia.

Fasudil inhibits proliferation and migration of Hep-2 laryngeal carcinoma cells.

BackgroundRho-kinase signal pathway is a new target for cancer therapy. Fasudil, a selective Rho-kinase inhibitor, is found to exert antitumor effects on several types of cancer, but whether fasudil has antitumor effects on laryngeal carcinoma is still unknown. The aim of this study was to determine the effects of fasudil on laryngeal carcinoma and explore the underlying molecular mechanisms in this process.MethodsAfter treatment with fasudil, changes in biological behaviors, including the growth, proliferation, clone formation, apoptosis, and migration of human laryngeal carcinoma cells (Hep-2 cells) were observed. The influences on apoptotic protease activity factor-1 (APAF-1)-mediated apoptosis pathway and the activities of matrix metalloproteinases (MMP-2 and MMP-9) were measured by Western blotting and gelatin zymography assay.ResultsHalf-maximal inhibitory concentration of fasudil to Hep-2 cells was ~3.40×103 µM (95% CI: 2.53–4.66×103 µM). Moreover, fasudil treatment significantly decreased the ability of growth, proliferation, clone formation, and migration of Hep-2 cells, while remarkably increased the apoptosis rate. Furthermore, the expressions of APAF-1, caspase-9, and caspase-3 significantly increased in fasudil treatment group. Meanwhile, fasudil led to a remarkable decrease in the expressions and activities of MMP-2 and MMP-9.ConclusionOur findings first demonstrate that fasudil not only inhibits the proliferation of laryngeal carcinoma cells through activating APAF-1-mediated apoptosis pathway, but also prevents migration by inhibiting the activities of MMP-2 and MMP-9. Therefore, fasudil is an attractive antitumor drug candidate for the treatment of laryngeal carcinoma.

Acquired tumor cell resistance to sunitinib by increased invasion and epithelial-mesenchymal transition in LL/2 murine lung cancer.

ObjectiveThis study aims to investigate biological behavior changes in a murine lung cancer cell characterized by acquired resistance to sunitinib, a potent inhibitor of multiple-targeted receptor tyrosine kinase.MethodsA lung cancer cell line resistant to sunitinib (LL/2-R) was developed from its parental cell line (LL/2-P). Differences in biological characteristics and associated molecular profiles between these two cells were compared in vitro and in vivo.ResultsLL/2-R cells showed an approximately 5-fold higher IC50 of sunitinib than LL/2-P cells and exhibited a reduced growth inhibition following sunitinib treatment compared with LL/2-P. In LL/2-R cells and tumors, increased migration, invasion and metastasis were observed, along with upregulation of MMP-2 and MMP-9. We also analyzed the molecular profiles involved in EMT, and found that E-cadherin was downregulated in LL/2-R tumors, and vimentin was upregulated in LL/2-R cells and tumors, along with β-catenin translocating to the nuclei in LL/2-R cells. Furthermore, transcriptional factors mediated EMT, snail and twist, and the secretion of TGFβ1 also increased in LL/2-R cells and tumors.ConclusionsWe established a sunitinib-resistant lung cancer cell line and confirmed its drug-resistance to sunitinib in vivo. Our results implied that increased invasion and EMT may associate with the acquisition of resistant phenotype to sunitinib in cancer cells.

Hypoxia-induced tumor biological behavior changes and application of radiotracers for PET imaging of tumor hypoxia

Hypoxia exists in a wide range of advanced solid tumors and is often associated with poor prognosis. Hypoxia can enhance tumor invasion, promote tumor angiogenesis, impact genomic stability and change the tumor microenvironment by activating expression of relevant genes through hypoxia-induced factors (HIFs). Hypoxia provides favorable conditions for tumor metastasis and relapse, and it is involved in all aspects of tumor development. The study of hypoxia in tumors may provide useful information for pre-clinical evaluation, individualized treatment and clinical prognosis. Positron emission tomography (PET) is a non-invasive imaging method, which can be used for assessing and quantifying tumor hypoxic microenvironment. This article reviews the mechanisms of hypoxia-induced tumor biological behavior changes and the research progress of the application of radiotracers for PET imaging of tumor hypoxia. Key words: Cell hypoxia, neoplasms; Hypoxia-inducible factor 1, alpha subunit; Radioactive tracers