Protein Expression Levels Of Vimentin Research Articles

Alisol A attenuates malignant phenotypes of colorectal cancer cells by inactivating PI3K/Akt signaling.

Despite the advancement in the diagnosis and therapeutic strategies for colorectal cancer, the outcomes of patients with colorectal cancer remain unsatisfactory. Alisol A is a natural constituent of Alismatis rhizoma (zexie) and has demonstrated anti-cancer properties; however, the function of Alisol A in colorectal cancer is still unknown. In the present study, the effect of Alisol A on colorectal cancer progression was investigated. MTT and colony formation assays showed that treatment with Alisol A repressed colorectal cancer cell proliferation in a dose-dependent manner. Similarly, western blot analysis demonstrated that Alisol A upregulated E-cadherin protein expression levels, but downregulated N-cadherin and Vimentin protein expression levels in colorectal cancer cells. In addition, the number of cells in G0/G1 phase was enhanced, while that of S phase was reduced in Alisol A-treated colorectal cancer cells. Apoptosis and pyroptosis of colorectal cancer cells were stimulated following treatment with Alisol A. Alisol A suppressed the migration ability of colorectal cancer cells in a dose-dependent manner. Moreover, Alisol A increased the chemotherapeutic sensitivity of colorectal cancer cells to cisplatin. Mechanically, western blot analysis confirmed that Alisol A repressed the phosphorylation levels of PI3K, Akt and mTOR in colorectal cancer cells. The Akt activator, SC79 reversed the effect of Alisol A on colorectal cancer cell proliferation and apoptosis. In conclusion, Alisol A induced an inhibitory effect on colorectal cancer progression by inactivating PI3K/Akt signaling.

TFEB and TFE3 Depletion Increases Cancer Stem Cell Characteristics and EMT in Triple‐Negative Breast Cancer

Breast cancer is currently the second leading cause of death for women worldwide. One subtype of breast cancer with poor prognosis is triple‐negative breast cancer (TNBC), which is characterized by the lack of expression of estrogen, progesterone, and human epidermal growth factor 2 (HER2) receptors. TNBC is difficult to treat due to its drug resistance and aggressiveness. Tumor invasiveness is driven by epithelial‐mesenchymal transition (EMT), which allows cells to acquire mobility to metastasize. EMT is also relevant to maintaining cancer stem cell (CSC) characteristics. Drug resistance is driven by CSCs, which are slow to divide, thus surviving chemotherapies, the standard of treatment. Thus, new treatments that inhibit CSCs and/or EMT in TNBC are highly desirable. TFEB and TFE3, two transcription factors of the MiT‐TFE family that operate as a complex, are involved in regulating lysosome biogenesis and autophagy. Our preliminary data and other studies have shown that TFEB and TFE3 regulate pluripotency and EMT in various in vitro models such as gastric and pancreatic cancer, but this regulation is poorly understood in TNBC. To develop new targeted therapies for TNBC, this study aimed to investigate the effects of depleting TFEB and TFE3 on CSC abundance and EMT. CSC renewal capacity was assessed via mammosphere assays and flow cytometry using CD44 and ALDH1A. The MDA MB 231 TFEBKO cells grew the highest number and largest mammospheres compared to the control, TFE3 knock‐down, and double knock‐out cell lines. TFEBKO and TFEBKOTFE3sh showed increases in CD44 expression and ALDH1A activity. While ALDH1A positivity was 1.16% in controls, TFEBKO and TFEBKOTFE3sh significantly increased the population to 22.6% and 21.9%, respectively. Western blotting and a wound healing assay were used to evaluate EMT marker expression and migration, respectively. Quantification revealed that TFEBKO had a two‐fold increase in protein expression levels of EMT markers Vimentin, Snail, and Slug, while TFE3sh showed higher protein expression levels of Vimentin and Slug. TFEBKOTFE3sh showed higher protein expression levels of Slug. The TFEBKO cell line had the highest rate of migration when compared to the control. In conclusion, the data showed that knockout of TFEB and combined knockout of TFEB and TFE3 resulted in an increase in stem cell character and EMT, which could correlate to increased tumor metastasis and poorer prognosis. These results are promising to investigate how depletion or inhibition of TFEB and TFE3 can affect TNBC tumor cell stem cell character and EMT.

Sufentanil inhibits the proliferation and epithelial mesenchymal transition of lung cancer cells through Wnt/beta-catenin signaling pathway

ABSTRACT Lung cancer is the most common malignancy and leading cause of cancer-related death. Sufentanil is a commonly used opioid anesthetic in clinics. This study aimed to explore the effects of sufentanil on the malignant behavior of lung cancer cells. H460 and H1299 lung cancer cell lines were selected for in vitro experiments. The MTT assay was conducted to detect cell viability. Proliferation ability was determined by colony formation and EdU assays. Transwell assays were performed to measure migration and invasion abilities. Western blotting was used to detect the expression of related proteins. LiCl was used to activate the Wnt/β-catenin signaling pathway. Sufentanil decreased the proliferation, migration, and invasion of H460 and H1299 cells. The protein expression levels of vimentin, N-cadherin, β-catenin, c-Myc, and MMP2 were downregulated, while those of E-cadherin and ZO-1 were upregulated after sufentanil treatment. LiCl treatment reversed the effects of sufentanil on H460 and H1299 cells. Sufentanil inhibited the proliferation, migration, invasion, and epithelial–mesenchymal transition of lung cancer cells by regulating the Wnt/β-catenin signaling pathway.

Effects of simvastatin on pulmonary fibrosis and endothelial - mesenchymal transition in the pulmonary fibrosis tissue of rats

Objective: To investigate the effects of simvastatin (SIM) on pulmonary fibrosis and the expression of VE-cadherin(VE-cad),vimentin(VIM) and alpha-smooth muscle actin(α-SMA)in the pulmonary fibrosis tissue of rats. Methods: Sixty healthy male SD rats were randomly divided into control group(group A), bleomycin group(group B), 5 mg SIM group (group C) and 10 mg SIM group (group D),15 rats in each group. The model of rat pulmonary fibrosis was established by itraperitoneal injection of bleomycin(5 mg/kg). Since the first day of modeling, the rats of group C and D were treated with simvastatin suspension 5 mg/(kg·d) and 10 mg/(kg·d) by intragastric administration everyday, and the rats of group A and B were treated with equal volume of saline 10 ml/(kg·d) everyday. Five rats of each group were sacrificed randomly at the 7th, 14th and 28th day. Masson staining was used to observe the morphological changes of lung tissue in rats. The degree of fibrosis in lung tissues of each group was evaluated by the content of hydroxyproline (HYP) . The microvessel density (MVD) was analyzed by immunohistochemistry,The expressions of protein and mRNA of VE-cad, VIM and α-SMA were determined by immunohistochemistry and RT-PCR. Results: ①Compared with group A, the levels of HYP and MVD, the mRNA and protein expression levels of VIM and α-SMA in lung tissues of groups B, C and D were increased significantly at the 7th, 14th and 28th day(all P＜0.05), which reached highest level at the 28th day. However, the mRNA and protein expression levels of VE-CAD were decreased significantly at the corresponding time (P＜0.05), which reached lowest level at 28th day. ②Compared with group B, the levels of HYP and MVD, the mRNA and protein expression levels of VIM and α-SMA in groups C and D were decreased at the 7th, 14th and 28th day (all P＜0.05), which were decreased more obviously in group D at the 28th day. However, the mRNA and protein expression levels of VE-CAD were increased at the corresponding time (all P＜0.05), which were increased more obviously in group D at the 28th day. Conclusion: Simvastatin can reduce the degree of pulmonary fibrosis in rats through inhibiting the process of EnMT, which can enhance the expression of VE-cad and reduce the expression of VIM and α-SMA.

MicroRNA-1298-5p inhibits the tumorigenesis of breast cancer by targeting E2F1.

Studies performed in the last two decades have identified microRNA (miR)-1298-5p to display tumor-suppressive functions in several types of malignancy. In addition, the regulatory role of E2F transcription factor 1 (E2F1) has been reported in multiple types of cancer, including breast cancer (BC). However, whether miR-1298-5p participates in BC progression and whether a regulatory association exists between miR-1298-5p and E2F1 remains to be explored. The present study aimed to determine the role of miR-1298-5p and its interaction with E2F1 in BC. The expression of miR-1298-5p and E2F1 was examined by reverse transcription-quantitative PCR and western blot assays. The viability and proliferative capacity of BC cells were evaluated by Cell Counting Kit-8 and 5-bromo-2′-deoxyuridine assays, respectively. The apoptotic rate was assessed by the caspase-3 activity assay and flow cytometry; the protein expression levels of vimentin and E-cadherin were evaluated by western blotting. In addition, the adhesive and migratory abilities of BC cells were determined by conducting cell adhesion and wound healing assay, respectively. The target relationship between miR-1298-5p and E2F1 was validated by the luciferase reporter assay. The results of the present study revealed that the levels of miR-1298-5p were downregulated in BC tissues and cells compared with those in normal breast tissues and cells, respectively. In addition, miR-1298-5p was demonstrated to inhibit the proliferation, adhesion and migration of BC cells and to promote BC cell apoptosis. E2F1 was verified as a target gene of miR-1298-5p using the luciferase reporter assay. Additionally, E2F1 exhibited an opposite expression pattern compared with that of miR-1298-5p in BC tissues. Furthermore, the downregulation of miR-1298-5p in BC cells was reversed by silencing E2F1. Overall, the results of the present study suggested that miR-1298-5p repressed BC cell proliferation, adhesion and migration, and enhanced BC cell apoptosis by downregulating E2F1.

Long non-coding RNA DUXAP8 promotes tumorigenesis by regulating IGF1R via miR-9-3p in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide with a low 5-year survival rate. Long non-coding RNA (lncRNA) double homeobox A pseudogene 8 (DUXAP8) is an oncogene and a potential biomarker in various tumors, such as ovarian, colorectal and non-small-cell lung cancer. However, the function and molecular mechanism underlying DUXAP8 in HCC progression is not completely understood. The expression of DUXAP8, microRNA (miR)-9-3p and insulin-like growth factor 1 receptor (IGF1R) in HCC tissues and cells was detected via reverse transcription-quantitative PCR. The expression levels of IGF1R and epithelial-mesenchymal transition-associated proteins (Snail, Slug, E-cadherin, N-cadherin and vimentin) were assessed via western blotting. The effects of DUXAP8, miR-9-3p and IGF1R on proliferation, migration and invasion were examined by conducting Cell Counting Kit-8 and Transwell assays, respectively. The interaction between miR-9-3p and DUXAP8 or IGF1R was predicted using StarBase or TargetScan, and further assessed using dual luciferase reporter and RNA immunoprecipitation assays. DUXAP8 and IGF1R were upregulated and miR-9-3p was downregulated in HCC tissues and cells compared with adjacent healthy tissues and a normal liver cell line, respectively. miR-9-3p overexpression decreased the protein expression level of IGF1R, and miR-9-3p knockdown enhanced the protein expression level of IGF1R in HCC cells compared with the corresponding control groups. Moreover, compared with the corresponding control groups, DUXAP8 knockdown and miR-9-3p overexpression increased E-cadherin protein expression levels, and decreased Snail, Slug, N-cadherin and vimentin protein expression levels. However, miR-9-3p inhibitor and IGF1R overexpression reversed DUXAP8 knockdown- and miR-9-3p overexpression-induced effects, respectively. In addition, compared with the corresponding control groups, DUXAP8 knockdown and miR-9-3p overexpression suppressed proliferation, migration and invasion, which was reversed by miR-9-3p inhibitor and IGF1R overexpression, respectively. Moreover, miR-9-3p as the target of DUXAP8 and IGF1R as the target of miR-9-3p were verified in HCC cells. lncRNA DUXAP8 contributed to HCC tumorigenesis via the miR-9-3p/IGF1R axis, providing a novel therapeutic approach for HCC diagnosis and treatment.

Lentivirus-mediated silencing of CNTN1 enhances gefitinib sensitivity by reversing epithelial-mesenchymal transition in lung adenocarcinoma A549 cells.

Contactin-1 (CNTN1), a neuronal cell adhesion molecular, functions in nervous system development and has been associated with carcinogenesis and tumor progression. To investigate the role of CNTN1 in gefitinib resistance in lung adenocarcinoma, lentivirus-mediated short hairpin (sh)RNA was used to silence CNTN1 and its physiological function was analyzed in the A549 cell line. A cell cytotoxicity assay revealed that CNTN1 knockdown enhanced gefitinib sensitivity in the A549 cells. In addition, CNTN1 knockdown, together with gefitinib treatment, resulted in a significant inhibition of colony formation and migration, and promotion of apoptosis. Furthermore, CNTN1 knockdown also reversed the epithelial-mesenchymal transition (EMT) phenotype by increasing E-cadherin protein expression level, and decreasing N-cadherin and vimentin protein expression levels. The PI3K/Akt signaling pathway was also association with the effects of CNTN1 on EMT progression and gefitinib resistance in the A549 cells. Collectively, knockdown of CNTN1 reversed the EMT phenotype and enhanced gefitinib sensitivity in the A549 cells by inhibiting the activation of the PI3K/Akt signaling pathway. These results suggested that CNTN1 may represent a potential therapeutic target for reserving EGFR-tyrosine kinase inhibitor resistance in non-small cell lung cancer.

Linc‑ROR facilitates hepatocellular carcinoma resistance to doxorubicin by regulating TWIST1‑mediated epithelial‑mesenchymal transition.

Long non-coding RNAs are associated with cancer progression. Long intergenic non-protein coding RNA (linc)-regulator of reprogramming (ROR) enhances tumor development in hepatocellular carcinoma (HCC). However, the effect of chemoresistance and its underlying mechanisms in HCC are not completely understood. The present study aimed to identify the effect of ROR on sensitivity to doxorubicin (DOX) in HCC cells. In the present study, Cell Counting Kit-8 and EdU assays were performed to assess cell viability and proliferation, respectively. In addition, E-cadherin and vimentin protein expression levels were assessed via western blotting and immunofluorescence. The results of the present study demonstrated that HCC cells with high linc-ROR expression levels were more resistant to DOX, and linc-ROR knockdown increased HCC cell DOX sensitivity compared with the control group. The results indicated that compared with the NC siRNA group, linc-ROR knockdown notably suppressed epithelial-mesenchymal transition by downregulating twist family bHLH transcription factor 1 (TWIST1) expression. TWIST1 knockdown displayed a similar effect on HCC cell DOX sensitivity to linc-ROR knockdown. Moreover, linc-ROR knockdown-induced HCC cell DOX sensitivity was inhibited by TWIST1 overexpression. The present study provided evidence that linc-ROR promoted HCC resistance to DOX by inducing EMT via interacting with TWIST1. Therefore, linc-ROR might serve as a therapeutic target for reducing DOX resistance in HCC.

ROS‑mediated hypomethylation of PRDX5 promotes STAT3 binding and activates the Nrf2 signaling pathway in NSCLC.

Deoxyribonucleic acid (DNA) epigenetic modification has been linked to specific sequences of CpG islands and plays roles in the progression of lung cancer. In this study, it was found that peroxiredoxin-5 (PRDX5) was highly expressed in non-small cell lung cancer (NSCLC) tissues; however, its specific regulatory mechanisms and functions in NSCLC remain unknown. The present study therefore explored the regulatory mechanism of PRDX5 under conditions of oxidative stress (OS) in NSCLC. The results revealed that 79 of 121 NSCLC patients exhibited demethylation in the PRDX5 promoter region, which was related to the tumor, node and metastasis (TNM) stage (P=0.027). PRDX5 messenger ribonucleic acid (mRNA) expression positively correlated with the demethylation status of the promoter region. The results of bisulfite sequencing polymerase chain reaction (BSP) revealed lower demethylation frequencies in H1299 cells treated with 0 µM H2O2, but maximum demethylation following treatment with 100 µM H2O2. Using chromatin immunoprecipitation (ChIP) and luciferase detection assays, the effective binding of STAT3 to the transcriptional binding sites of the PRDX5 promoter region was confirmed (2 sites confirmed: Site 1, -444 to -434 bp; and site 4, -1,417 to -1,407 bp). STAT3 knockdown significantly decreased the protein expression of PRDX5, while the overexpression of STAT3 significantly increased the protein levels of PRDX5. When PRDX5 was overexpressed in lung cancer cells under conditions of OS, the levels of the epithelial-mesenchymal transition (EMT) biomarkers, E-cadherin and vimentin, were significantly decreased and increased, respectively. By contrast, PRDX5 knockdown resulted in significantly increased E-cadherin and decreased vimentin protein expression levels. Ultimately, when PRDX5-small interfering RNA (siRNA) or pcDNA3.1-PRDX5expression vector were constructed and transfected into H1299 cells pre-treated with 100 µM H2O2, the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway was inhibited or activated. All these results suggested that the reactive oxygen species (ROS)-mediated hypomethylation of PRDX5 enhanced STAT3 binding affinity with the promoter region, and resulted in the promotion of cell migration and invasion, as well as in the activation of the Nrf2 signaling pathway in NSCLC. The demethylation status of the PRDX5 promoter may thus be used as an epigenetic biomarker in NSCLC. STAT3/PRDX5 signaling may also prove to be a potential strategy for the treatment of this type of cancer.

LncRNA RPPH1 promotes non-small cell lung cancer progression through the miR-326/WNT2B axis.

Long non-coding RNAs (lncRNAs) serve important regulatory roles in human tumors. The aim of the present study was to examine the role of ribonuclease P RNA component H1 (RPPH1) in non-small cell lung cancer (NSCLC). RPPH1 expression was assessed in datasets from The Cancer Genome Atlas, as well as lung cancer cell lines and patients with NSCLC. RPPH1 was significantly upregulated in NSCLC cell lines, compared with a normal lung epithelial cell line. Moreover, high RPPH1 expression was associated with poor overall survival and disease progression. RPPH1 was knocked down in A549 and H1299 cells using short hairpin (sh) RNA constructs, and the expressions of target genes and proteins were determined by reverse transcription-quantitative PCR and western blotting. Cell invasion potential was also determined using Transwell Matrigel assays. Compared with the negative control, RPPH1 silencing significantly reduced the number of invading cells, increased E-cadherin expression and reduced vimentin protein expression. Cell resistance to cisplatin/cis-diamminedichloridoplatinum (CDDP) was also evaluated using Cell Counting Kit-8 and colony formation assays. RPPH1 overexpression increased the resistance of A549 and H1299 cells to CDDP. Moreover, the potential interactions between RPPH1, microRNA (miR)-326 and Wnt family member 2B (WNT2B) were investigated using luciferase reporter assays and co-transfection experiments. MiR-326 expression was directly inhibited by RPPH1. In A549 cells co-transfected with shRPPH1 and miR-326 inhibitor, the invading cell number significantly increased compared with cells transfected with shRPPH1 alone. In addition, E-cadherin expression levels were reduced, and vimentin was upregulated. MiR-326 overexpression partially reduced the resistance of A549 cells to CDDP induced by RPPH1 overexpression. WNT2B expression was directly suppressed using miR-326. A549 cells co-transfected with a miR-326 mimic and a WNT2B overexpression vector demonstrated increased invasion potential, reduced E-cadherin and increased vimentin protein expression levels, compared with cells transfected with the mimic alone. miR-326 overexpression reduced CDDP resistance in A549 cells. However, co-transfection with WNT2B partially enhanced CDDP resistance, compared with the mimic alone. In conclusion, RPPH1 promoted NSCLC progression and lung cancer cell resistance to CDDP through miR-326 and WNT2B.

Circular RNA hsa_circ_0096157 contributes to cisplatin resistance by proliferation, cell cycle progression, and suppressing apoptosis of non-small-cell lung carcinoma cells.

Circular RNAs (circRNAs) play a major role in cancer development and chemotherapy resistance. This study aimed to characterize circRNA profiles associated with Cisplatin (diamminedichloroplatinum, DDP) resistance of non-small-cell lung carcinoma (NSCLC) cells. The half-maximal inhibitory concentration (IC50) of A549 and A549/DDP cells was determined using CCK-8 assay. Further, circRNA profiles and differentially expressed genes in A549 and A549/DDP cells were characterized by deep sequencing and cell proliferation was measured using MTS assay. Cell cycle progression was analyzed using flow cytometry. Apoptosis experiment was performed by TUNEL assay and flow cytometry. Cell migration and invasion were assessed using the Transwell system. Finally, signalling protein levels related to cell cycle progression and migration were measured by western blot. CCK-8 assay showed that A549/DDP cells obtained strong DDP resistance. Further deep sequencing results showed that 689 circRNAs and 87 circRNAs were significantly upregulated and downregulated in A549/DDP cells compared to A549 cells, respectively. Moreover, the circRNA hsa_circ_0096157 with the highest expression level in A549/DPP cells was further analyzed for its potential mechanism of DDP resistance in A549/DDP. With or without DDP treatment, hsa_circ_0096157 knockdown inhibited proliferation, migration, invasion and cell cycle progression but promoted apoptosis of A549/DDP cells. In addition, the western blot results also showed that hsa_circ_0096157 knockdown in A549/DDP cells increased P21 and E-cadherin but decreased CDK4, Cyclin D1, Bcl-2, N-cadherin, and Vimentin protein expression levels, indicating that cell cycle progression might be inhibited by increased P21 protein level to inhibit the expression of CDK4-cyclin D1 complex and decreased Bcl-2 protein level; and migration and invasion were suppressed by the increased E-cadherin and decreased N-cadherin and Vimentin expression levels. In contrast, hsa_circ_0096157 overexpression in A549 cells caused the opposite cellular and molecular alterations. DDP resistance in NSCLC cells was associated with significant circRNA profile alterations. Moreover, increased hsa_circ_0096157 expression contributed to DDP resistance in NSCLC cells by promoting cell proliferation, migration, invasion and cell cycle progression and inhibiting apoptosis.

Silence of cytoskeleton-associated protein 2 represses cell proliferation and migration and promotes apoptosis in liver cancer cell lines.

To investigate the roles of cytoskeleton-associated protein 2 (CKAP2) in proliferation, apoptosis, and migration in liver cancer cells and the potential mechanisms. Human normal hepatocyte L02 and liver cancer cell lines HepG2, Huh7, and SMMC-7721 were cultured. The CKAP2 expression was detected by real-time PCR and Western blotting. HepG2 cells were randomly divided into a control group, a negative control (NC) group, and a CKAP2 silencing (siCKAP2) group. CCK-8 and BrdU assays were used to evaluate cell viability and proliferation, respectively. Transwell assay was employed to determine cell migration and invasion. The protein levels of cleaved-caspase 3, Bax, E-cadherin, N-cadherin, Vimentin, phosphorylated Janus kinase 2 (p-JAK2), and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) were determined by Western blotting. Compared with normal hepatocyte L02, CKAP2 was highly expressed in liver cancer cell lines HepG2, Huh7, and SMMC-7721 (all P<0.05). Compared with the NC group, cell viability and proliferation rate of the siCKAP2 group were decreased (both P<0.05). The apoptotic rate, protein expression of cleaved-caspase 3 and Bax in the siCKAP2 group were significantly higher than those in the NC group (all P<0.05). Compared with the NC group, cell migration and invasion rates of the siCKAP2 group were significantly attenuated (both P<0.05). Compared with the NC group, E-cadherin protein expression in siCKAP2 group was increased, while protein expression levels of Vimentin, N-cadherin, p-JAK2, and p-STAT3 were decreased (all P<0.05). CKAP2 gene silence inhibits proliferation, migration, and invasion, and promotes apoptosis in liver cancer cells, while JAK2/STAT3 signaling pathway may be involved in these processes.

Circular RNA hsa_circ_0012673 facilitates lung cancer cell proliferation and invasion via miR-320a/LIMK18521 axis.

Lung cancer is one of the most malignant tumors with high morbidity and mortality in the world. The incidence and mortality of lung cancer were increased per year in many countries over the past 50 years. The increasing studies had shown that circular RNA (circRNA) was involved in the progression of lung cancer. Therefore, it was significant to seek the molecular mechanism of circ_0012673 in lung cancer. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to estimate the expression levels of circ_0012673, miR-320a and LIM domain kinase 1 (LIMK1) in lung cancer tissues and cells. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT), flow cytometry and transwell assays were recruited to evaluate proliferation, apoptosis and mobility of lung cancer cells, respectively. The relative protein expression levels of Vimentin, N-cadherin, E-cadherin and LIMK1 were determined with Western blot assay. The relationships among circ_0012673, miR-320a and LIMK1 were analyzed by starBase database, dual-luciferase reporter assay, and Pearson's correlation. Circ_0012673 was overexpressed in lung cancer tissues and cell lines. Loss-of-functional experiment confirmed that knockdown of circ_0012673 constrained proliferation, motility and Epithelial-Mesenchymal Transition (EMT), but induced apoptosis by targeting miR-320a. Furthermore, LIMK1 was a target of miR-320a in lung cancer cells. Elevated LIMK1 could abolish the overexpression of miR-320a induced effects on lung cancer cells. Mechanistically, circ_0012673 contributed to lung cancer progression through mediating miR-320a /LIMK1 pathway. Circ_0012673 was a tumor-promoter in lung cancer via acting as competing endogenous RNA to regulate LIMK1 expression by binding miR-320a.

Silencing Smad4 attenuates sensitivity of colorectal cancer cells to cetuximab by promoting epithelial‑mesenchymal transition.

The aberrant expression of tumor suppressor Smad4 often occurs in colorectal cancer (CRC), and this phenomenon is believed to be associated with drug resistance. The present study aimed to investigate the effects of Smad4 on the sensitivity of CRC cells to cetuximab, and the possible mechanism underlying such an effect. A total of 629 colorectal adenocarcinoma cases were downloaded from The Cancer Genome Atlas (TCGA) database, and a Smad4 mutation rate of ~21% was demonstrated among the cases. Low expression of Smad4 was present in CRC tissues analyzed by TCGA and in four CRC cell lines, as determined by reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis. Cell Counting kit-8 (CCK-8) was used to measure the effects of different concentrations of cetuximab on SW480 cell viability at 24 and 48 h. The results demonstrated that treatment of SW480 cells with 20 µg/ml cetuximab for 48 h markedly reduced cell viability. In addition, plasmids were transfected into SW480 cells to induce Smad4 silencing or overexpression. Silencing Smad4 attenuated the sensitivity of SW480 CRC cells to cetuximab; this effect was reflected in increased cell viability and slightly increased migration and invasion, as determined by CCK-8, wound scratch and Transwell analyses. RT-qPCR and western blotting was performed to assess the expression levels of apoptosis- and epithelial-mesenchymal transition (EMT)-related genes. Silencing Smad4 partly reversed the effects of cetuximab on the mRNA and protein expression levels of vimentin, Bax/Bcl-2 and E-cadherin. However, Smad4 overexpression enhanced SW480 cell sensitivity to cetuximab. In conclusion, Smad4 may serve a vital role in the sensitivity of CRC cells to chemotherapeutic drugs by promoting EMT.

MicroRNA-34a suppresses the invasion and migration of colorectal cancer cells by enhancing EGR1 and inhibiting vimentin.

MicroRNAs (miRNAs/miRs) are small non-coding RNAs that serve a post-transcriptional regulatory role in eukaryotes. Previous studies have demonstrated that the expression of miR-34a in colorectal cancer (CRC) tissues is decreased compared with that in normal colorectal tissues. However, the role of miR-34a in the invasion and metastasis of CRC remains unclear. In the present study, the levels of miR-34a expression were measured in various CRC cell lines. The cells were transfected with miR-34a mimics or inhibitors in order to assess the proliferation rate, and the colony forming, invasive and migratory abilities. Furthermore, the protein expression levels of vimentin and early growth response protein 1 (EGR1) were examined by western blot analysis. The results revealed that the expression of miR-34a was low in SW620, RKO, LoVo and Caco-2 cell lines and high in the SW480 and SW1116 cell lines. The migration, invasion and proliferation levels of SW480 cells were facilitated by decreasing the expression of miR-34a. Transient transfection with miR-34a mimics in SW620 cells caused a notable decrease in cell migration, invasion and proliferation levels compared with the control group, and a downregulation of vimentin and upregulation of EGR1 protein expression. The present study demonstrated that miR-34a was deregulated in a highly invasive CRC cell lines, and that it may attenuate the migratory, invasive and proliferative capabilities of CRC cells by enhancing the expression of EGR1 and inhibiting that of vimentin. The results of the present study represent important progress towards understanding the mechanisms of CRC recurrence and metastasis.

Functional analysis of protein disulfide isomeraseP5 in glioblastoma cells as a novel anticancer target.

P5, which is a member of the protein disulfide isomerase family, possesses isomerase and chaperone activity invitro; however, the physiological functions of this enzyme in cells remain unclear. To understand the important roles of P5 in cancer cells, the present study examined its expression on the surface of normal and cancer cell lines by flow cytometry using an affinity‑purified anti‑P5 antibody labeled with 6‑(fluorescein‑5‑carboxamido) hexanoic acid succinimidyl ester. P5 expression was increased on the surface of various cancer cell lines, including leukemia cells, and glioblastoma, breast, colon, ovarian and uterine cervical cancer cells, compared with normal cells. However, P5 was constantly expressed within both normal and cancer cell lysates, and its total expression levels were not significantly different between the cells. P5 knockdown in glioblastoma cells by small interfering RNA affected Bip promoter activation during cancer cell growth, and significantly inhibited cancer cell growth and migration. Immunoprecipitation using an anti‑P5 antibody in cancer and normal cells demonstrated that vimentin was bound to P5, predominantly in U251 glioblastoma cells. P5 knockdown in glioblastoma cells did not affect the protein expression levels of vimentin; however, it did affect the expression of numerous epithelial‑mesenchymal transition markers, including Snail and Slug. These results suggested that P5 may serve an important role in cancer cell growth, and may be considered an attractive and potent target for the treatment of glioblastoma.

Pectolinarigenin inhibits non‑small cell lung cancer progression by regulating the PTEN/PI3K/AKT signaling pathway.

Lung cancer is the principal cause of cancer-associated mortality. Pectolinarigenin (Pec) reportedly has effective antitumor activity against certain cancer types. Phosphatase and tensin homolog (PTEN) is a well-known tumor suppressor and serves a vital role in cancer progression. However, the effect of Pec on non-small cell lung cancer (NSCLC) cell proliferation and metastasis, and the underlying mechanism, has not yet been elucidated. In the present study, it was demonstrated that Pec inhibited the proliferation of A549 and Calu-3 cells in dose- and time-dependent manners. The apoptosis rate significantly increased with increasing doses of Pec. Apoptosis-associated protein expression was additionally altered by Pec exposure. Pec was able to suppress the metastasis of NSCLC cells; it upregulated the mRNA and protein expression levels of E-cadherin, and downregulated the mRNA and protein expression levels of vimentin. Additionally, Pec was able to activate PTEN and subsequently downregulate the PI3K/protein kinase B (AKT) signaling pathway. In summary, Pec was able to inhibit cell proliferation, promote apoptosis and suppress metastasis in NSCLC cells through the PTEN/PI3K/AKT signaling pathway, indicating that Pec is a potential agent for NSCLC therapy.

Silymarin Attenuates Invasion and Migration through the Regulation of Epithelial-mesenchymal Transition in Huh7 Cells

Hepatocellular carcinoma (HCC), a major type of hepatoma, is associated with high recurrence and mortality because of its uncontrolled metastatic feature. Silymarin is a polyphenolic flavonoid from Silybum marianun (milk thistle) and exhibits anti-carcinogenic activity through modulation of the epithelial-mesenchymal transition (EMT) in several cancer cells. In this study, the inhibitory mechanism of silymarin against migration and invasion was investigated in the Huh7 HCC cell line. Wound healing and in vitro invasion assays were conducted to examine the effects of silymarin on migration and invasion. Western blot analysis was also applied to evaluate the inhibitory effects of silymarin on the EMT-related genes and their upstream signaling molecules. Silymarin inhibited the migratory and invasive activities of Huh7 cells. In addition, silymarin attenuated the protein expression levels of vimentin and matrix metalloproteinase (MMP)-9 as well as their transcription factors, Snail, and nuclear factor (NF)-κB, while the expression of E-cadherin was increased by the silymarin treatment. Among the upstream signaling molecules, the phosphorylation of Akt was inhibited by the silymarin treatment, which was confirmed by the selective inhibitor, LY294002. Consequently, silymarin inhibited the invasive and migratory activities in Huh7 cells through the modulation of EMT-related gene expression by the PI3K/Akt signaling pathway, which may have potential as a chemopreventive agent against HCC metastasis.

AngII induces HepG2 cells to activate epithelial-mesenchymal transition.

The present study aimed to determine whether HepG2 can induce epithelial-mesenchymal transition (EMT) via angiotensin II (AngII) simulation. The expression levels of EMT markers vimentin and E-cadherin in cancer tissues and adjacent tissues of patients with hepatocellular carcinoma (HCC) were detected by immunohistochemistry. In addition, HepG2 cells were stimulated with AngII, and the gene and protein expression levels of vimentin and E-cadherin were measured by reverse transcription-quantitative polymerase chain reaction and western blot analyses, respectively, whereas cell migration and invasion were assessed using Transwell assays. The AngII inhibitor Ang1-7 and the Ang1-7 inhibitor A779 were added to the system to further evaluate AngII-induced EMT. Compared with that in normal tissue, the expression level of vimentin in HCC tissue was increased, whereas that of E-cadherin was decreased. EMT occurred 48 h following AngII stimulation. The transcription level of E-cadherin in HepG2 cells was decreased, whereas that of vimentin was increased. In addition, the migration and invasion abilities of the cells were increased simultaneously. Ang1-7 partly inhibited AngII-induced EMT. When stimulated at an appropriate time, HepG2 cells have the ability to undergo EMT.

Effects of obatoclax combined with gemcitabine on the biological activity of pancreatic cancer cells under hypoxic conditions.

The present study aimed to investigate the effects of obatoclax (OBX) combined with gemcitabine (GEM) treatment on the proliferation, migration, invasion and epithelial‑mesenchymal transition (EMT) related proteins of pancreatic cancer cell line BxPC‑3 under hypoxic conditions. Protein expression levels of hypoxia‑inducible factor 1α (HIF‑1α) in BxPC‑3 pancreatic cancer cells under normoxic and hypoxic conditions were detected by western blotting. Cells were divided into four groups: Normoxia group, hypoxia group, OBX group and OBX + GEM group. The proliferation activity of BxPC‑3 cells was detected by Cell Counting kit‑8. The migratory and invasive abilities of BxPC‑3 cells were detected by the scratch test and Matrigel assay, respectively. The protein expression levels of vimentin, E‑cadherin and p53 in BxPC‑3 cells were also detected by western blotting. HIF‑1α expression under hypoxic conditions was significantly increased compared with expression under normoxic conditions. Under hypoxic conditions, OBX treatment reduced cell activity, decreased cell migration and invasion, promoted the expression of E‑cadherin and p53. In the OBX + GEM group, BxPC‑3 cell activity decreased significantly, cell migration and invasion decreased significantly, the expression of vimentin was significantly reduced and the expression of E‑cadherin and p53 further increased. In conclusion, the present results demonstrated that under hypoxic conditions, OBX combined with a small dose of GEM may be able to inhibit the growth, migration and invasion of pancreatic cancer cells, possibly via inhibition of EMT process. These results may provide a promising strategy for pancreatic cancer therapy.