Plate Clone Formation Research Articles

MiR-744-5p Inhibits Multiple Myeloma Proliferation, Epithelial Mesenchymal Transformation and Glycolysis by Targeting SOX12/Wnt/β-Catenin Signaling.

PurposeThis study investigated the function and molecular mechanisms of miR-744-5p in multiple myeloma (MM).MethodsmiR-744-5p and SRY-related high-mobility-group box 12 (SOX12) expression in clinical tissues and MM cells was monitored by quantitative real-time polymerase chain reactions and Western blot. miR-744-5p expression in MM cells was regulated by transfection. Cell proliferation was researched by cell counting kit-8 assay and plate clone formation experiment. Transwell experiment was utilized for migration and invasion detection. Glycolysis test was conducted for the detection of glucose uptake and lactate production of MM cells. The relationship between miR-744-5p and SOX12 was determined by dual-luciferase reporter gene assay and RNA pull-down experiment. In vivo experiment was conducted using nude mice.ResultsmiR-744-5p expression was reduced in MM patients (P<0.01). Low miR-744-5p expression was associated with lower 60-month survival in MM patients (P=0.0402). miR-744-5p overexpression inhibited MM cells proliferation, invasion, migration, glucose uptake, lactate production, and epithelial mesenchymal transformation (EMT) (P<0.01). miR-744-5p directly inhibited SOX12 expression. miR-744-5p silencing promoted MM cells proliferation, invasion, migration, glucose uptake, lactate production, and EMT by elevating SOX12 (P<0.01). miR-744-5p inhibited the growth of MM xenograft tumors in vivo (P<0.001).ConclusionmiR-744-5p inhibits MM cells proliferation, invasion, migration, EMT, and glycolysis by targeting SOX12/Wnt/β-catenin.

Aberrantly high activation of a FoxM1\u2013STMN1 axis contributes to progression and tumorigenesis in FoxM1-driven cancers

Fork-head box protein M1 (FoxM1) is a transcriptional factor which plays critical roles in cancer development and progression. However, the general regulatory mechanism of FoxM1 is still limited. STMN1 is a microtubule-binding protein which can inhibit the assembly of microtubule dimer or promote depolymerization of microtubules. It was reported as a major responsive factor of paclitaxel resistance for clinical chemotherapy of tumor patients. But the function of abnormally high level of STMN1 and its regulation mechanism in cancer cells remain unclear. In this study, we used public database and tissue microarrays to analyze the expression pattern of FoxM1 and STMN1 and found a strong positive correlation between FoxM1 and STMN1 in multiple types of cancer. Lentivirus-mediated FoxM1/STMN1-knockdown cell lines were established to study the function of FoxM1/STMN1 by performing cell viability assay, plate clone formation assay, soft agar assay in vitro and xenograft mouse model in vivo. Our results showed that FoxM1 promotes cell proliferation by upregulating STMN1. Further ChIP assay showed that FoxM1 upregulates STMN1 in a transcriptional level. Prognostic analysis showed that a high level of FoxM1 and STMN1 is related to poor prognosis in solid tumors. Moreover, a high co-expression of FoxM1 and STMN1 has a more significant correlation with poor prognosis. Our findings suggest that a general FoxM1-STMN1 axis contributes to cell proliferation and tumorigenesis in hepatocellular carcinoma, gastric cancer and colorectal cancer. The combination of FoxM1 and STMN1 can be a more precise biomarker for prognostic prediction.

Effect of Ras-guanine nucleotide release factor 1-mediated H-Ras/ERK signaling pathway on glioma

ObjectiveTo investigate the function of Ras-guanine nucleotide release factor 1 (Ras-GRF1) in glioma through mediating H-Ras/ERK signaling pathway. MethodsRas-GRF1, H-Ras, K-Ras and N-Ras expressions in glioma and normal brain tissues were detected via Immunohistochemistry. Glioma cells (U87 cells, U251 cells and primary human glioma cells) were transfected with Ras-GRF1 siRNA, H-Ras siRNA and/or Ras-GRF1 lentivirus activation particles. Then, the following aspects were evaluated: cell proliferation by MTT assay, clonogenic ability by the plate clone formation experiment, cell migration and invasion by Wound-healing and Transwell assays, and cell apoptosis by Annexin-V-FITC/PI staining. The protein expressions were measured by Western blotting. Subcutaneous and orthotopic mouse models of glioma were conducted to determine the role of Ras-GRF1 in glioma tumorigenesis. ResultsRas-GRF1, H-Ras, K-Ras and N-Ras expressions were upregulated in the glioma tissues, which were correlated with the WHO grade of glioma. Besides, Ras-GRF1 expression was positively related to H-Ras expression. Ras-GRF1 siRNA could reduce the expression of H-Ras and p-ERK/ERK in glioma cell. H-Ras siRNA inhibited the proliferation, clone formation, migration and invasion, and enhance the apoptosis of glioma cells, which, however, were reversed by Ras-GRF1 lentivirus activation particles. In vivo experiments also revealed that Ras-GRF1 shRNA reduced the volume and weight of the tumors in the nude mice, with down-regulations of H-Ras and p-ERK/ERK. ConclusionRas-GRF1 was upregulated in glioma tissues and correlated with its malignancy and prognosis. Silencing Ras-GRF1, through mediating H-Ras/ERK pathway, may suppress the growth and metastasis of glioma.

Study on Effect of Vitexicarpin on the Biological Behavior of Colorectal Cancer Cells by Circ_0000419/MicroRNA-224

To investigate the effect and molecular mechanism of vitexicarpin on the biological behavior of colorectal cancer cells is the objective of the study. The human epithelial cell line Caco-2 cells were divided into control group, low, middle and high dose groups of vitexicarpin, plasmid cloning DNA-circ_0000419 group, plasmid cloning DNA group, anti-microRNA-224 group, anti-microRNA-224-negative control group, vitexicarpin+small interfering-circ_0000419 group and vitexicarpin+microRNA-224 group. Cell activity was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. Colony formation number was detected by plate clone formation experiment. Apoptosis was detected by flow cytometry. Scratch test was used to detect the migration distance of cells. Transwell method was used to detect the number of invasive cells; protein expression was detected by western blot; the expression levels of circ_0000419 and microRNA-224 were detected by real-time fluorescence quantitative polymerase chain reaction; dual-luciferase reporter assay was used to detect the targeting relationship between circ_0000419 and microRNA-224. After treatment with low, middle and high doses of vitexicarpin, the viability of Caco- 2 cells decreased, the number of colony formation decreased, the apoptosis rate increased, the migration distance decreased, the number of invasive cells decreased, the expression level of E-cadherin increased, the expression level of N-cadherin decreased, the expression level of circ_0000419 increased and the expression level of microRNA-224 decreased (p<0.05). Overexpression of circ_0000419 or inhibition of miR-224 expression decreased Caco-2 cell viability, decreased colony formation, increased apoptosis rate, decreased cell migration distance, decreased invasive cell number, increased expression of E-cadherin and decreased expression of N-cadherin (p<0.05). Interference with circ_0000419 or overexpression of microRNA-224 reverses the effects of vitexicarpin on proliferation, apoptosis, migration and invasion of Caco-2 cells; circ_0000419 targets the regulation of microRNA-224 expression. Vitexicarpin can inhibit the malignant biological behavior of colorectal cancer cells by circ_0000419/microRNA-224.

The long non-coding RNA SNHG4/microRNA-let-7e/KDM3A/p21 pathway is involved in the development of non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a foremost cause of malignancy-associated mortality globally. Recent studies have emphasized long non-coding RNAs (lncRNAs) as important biomarkers with diagnostic and therapeutic potential in regard to NSCLC. This study aimed to elucidate the functional role of lncRNA small nucleolar RNA host gene 4 (SNHG4) in NSCLC. Initially, 50 paired cancerous and noncancerous tissues were obtained from NSCLC patients. Human NSCLC H1299 cells were assayed to evaluate viability, colony formation, invasion, migration, cycle arrest, and apoptosis via Cell Counting Kit-8 (CCK-8), plate clone formation, and transwell invasion assays, as well as a scratch test and flow cytometry. A dual-luciferase reporter gene assay was used to examine lncRNA SNHG4 binding with miR-let-7e and miR-let-7e binding with lysine demethylase 3A (KDM3A). H1299 cells were xenografted into nude mice. lncRNAs SNHG4 and KDM3A were both upregulated in NSCLC tissues. The knockdown of lncRNA SNHG4 or KDM3A inhibited H1299 cell viability, colony formation, invasion, migration, and cycle progression while inducing apoptosis. lncRNA SNHG4 was found to bind to miR-let-7e that negatively targeted KDM3A. KDM3A inhibited p53-K372me1, thus reducing p21 expression. The NSCLC development was inhibited by downregulating lncRNA SNHG4 in nude mice. Taken together, the key findings of the current study demonstrate a novel lncRNA SNHG4/let-7e/KDM3A/p21 axis in NSCLC, highlighting a promising therapeutic target for NSCLC.

MicroRNA-198 inhibits metastasis of thyroid cancer by targeting H3F3A.

This study was designed to investigate the role of microRNA-198 in thyroid cancer (TCa) progression. Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to examine microRNA-198 and H3F3A levels in tumor tissue specimens and paracancerous ones collected from 50 patients with TCa, and the interplay between microRNA-198 or H3F3A and some clinical indicators or prognosis of TCa patients was analyzed as well. MicroRNA-198 and H3F3A overexpression models were constructed using lentivirus in TCa cell lines TPC-1 and BHP2-7, and the impacts of microRNA-198 on TCa cell functions were evaluated by using cell counting kit-8 (CCK-8), plate clone formation, and transwell assays. Finally, recovery investigations were conducted to explore the underlying mechanisms as well as the interaction between microRNA-198 and H3F3A. QRT-PCR indicated that in tumor tissues of TCa patients, microRNA-198 showed a remarkably lower expression than in adjacent normal tissue samples. Compared with patients with high expression of microRNA-198, those with microRNA-198 low expression had more advanced tumor stage, larger tumor size, higher lymph node metastasis rate, and lower overall survival rate. Meanwhile, the results of research on H3F3A were just opposite to the above observations on microRNA-198. In in vitro cell experiments, overexpression of microRNA-198 significantly weakened the proliferation and migration ability of thyroid tumor cells. Besides, Luciferase reporter gene experiment revealed that H3F3A was a specific target gene for microRNA-198. Moreover, qRT-PCR indicated that H3F3A and microRNA-198 were negatively correlated in thyroid carcinoma tissues. In addition, compared with NC group, overexpression of H3F3A markedly enhanced the migration and proliferative capacity of TCa cells. Lastly, recovery experiment revealed a mutual regulation between microRNA-198 and H3F3A, the two of which may together participate in the malignant progression of TCa. MicroRNA-198 is remarkably reduced in TCa and inhibits malignant progression of TCa by regulating H3F3A. Meanwhile, microRNA-198 is remarkably associated with pathological stage, tumor size, lymph node metastasis, and poor prognosis of TCa.

Long-term 1800MHz electromagnetic radiation did not induce Balb/c-3T3 cells malignant transformation

ABSTRACT There is an increased public concern about potential health hazards of exposure to electromagnetic radiation (EMR). To declare the carcinogenic effects of 1800 MHz EMR. In this study, Balb/c-3T3 cells were exposed to 1800 MHz EMR for 80 days. The cells were harvested for cell proliferation detection, cell cycle assay, plate clone, and soft agar formation assay, transwell assay, and mRNA microarray detection. 1800 MHz EMR promoted Balb/c-3T3 proliferation. No clones were observed in both plate clone and soft agar clone formation assay. The percentage of cells in S phase in Balb/c-3T3 cells of 80d Expo was obviously higher than the percetage in 80d Sham cells. 80d Expo Balb/c-3T3 cells had stronger migration ability than Sham cells. The mRNA microarray results indicated that cell cycle, cell division, and DNA replication were the main biological processes the significant genes enriched, with higher expression of RPs and Mcms. 1800 MHz EMR promoted Balb/c-3T3 cells proliferation and migration. The mRNA microarray results indicated that cell cycle, cell division, and DNA replication were the main biological processes the significant genes enriched.

Gallic acid from Terminalia chebula inhibited the growth of esophageal carcinoma cells by suppressing the Hippo signal pathway.

Objective(s):To explore the molecular mechanism of gallic acid (GA) from Terminalia chebula in suppressing the growth of esophageal carcinoma (EC).Materials and Methods:Human EC cells (EC9706 and KYSE450) were treated with different concentrations of GA (10, 20, and 40 μg/ml), which were subjected to 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, plate clone formation assay, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining, and Western blotting. EC mice were divided into Model, 0.3% GA, and 1% GA groups to observe the tumor volume and the expressions of YAP, TAZ, Ki-67, and Caspase-3 in tumor tissues. Results:GA decreased cell viability and colony formation of EC9706 and KYSE450 cells, which was more obvious as the concentration increased. In addition, GA promoted cell apoptosis in a concentration-dependent manner with the up-regulation of pro-apoptotic proteins (Bax, cleaved caspase-3, and cleaved caspase-9) and nuclear YAP/TAZ, as well as the down-regulation of anti-apoptotic protein Bcl-2 and the levels of p-YAP and p-TAZ. Moreover, GA decreased the growth of xenograft tumor in vivo, with the reduction in the tumor volume and the reduction of YAP and TAZ expressions in the tumor tissues. In addition, Ki-67 expression in GA groups was lower than those in the Model group, with the increase in caspase-3 expression in the tumor tissues. Changes aforementioned were obviously shown in the 0.3% GA group. Conclusion:GA blocked the activity of the Hippo pathway to suppress cell proliferation of EC and facilitate cell apoptosis, which is expected to be a novel strategy for treatment of EC.

ERCC6L promotes the progression of hepatocellular carcinoma through activating PI3K/AKT and NF-\u03baB signaling pathway

BackgroundExcision Repair Cross-Complementation group 6-like (ERCC6L) has been shown to exhibit carcinogenic effect in several malignant tumors. However, the function and molecular mechanism of the ERCC6L in hepatocellular carcinoma (HCC) have not been investigated extensively.MethodsImmunohistochemistry analyses were used to detect ERCC6L expression in a HCC tissue microarray, and the Chi-square test was used to assess the correlation between ERCC6L expression and patients’ clinicopathological features. shRNA was used to down-regulation ERCC6L expression in HCC cell lines. MTT assay, plate clone formation assay, flow cytometry, caspase 3/7 activity and migration assays were performed to evaluate the impact of ERCC6L on HCC cells in vitro. Nude mice xenograft models were used to assess the role of ERCC6L in vivo. The regulatory of mechanism of PI3K/AKT pathway was evaluated by western blotting.ResultsERCC6L was highly expressed in HCC tissue compared with tumor adjacent tissues in 90 paired samples. ERCC6L expression positively correlated with gender, tumor encapsulation, and pathological stage. Patients with low ERCC6L expression had significantly longer OS than those with high ERCC6L expression. Knockdown of ERCC6L expression significantly inhibited proliferation, invasion and metastasis in vitro and tumor growth in vivo, and it promoted cell cycle arrest and apoptosis. Mechanistic analyses revealed that PI3K/AKT and NF-κB signaling pathway were inhibited by silencing ERCC6L.ConclusionThese results demonstrate that ERCC6L plays a critical role in HCC progression, and thereby might be a potential therapeutic target for HCC patients.

&lt;p&gt;Mechanism of Sanguinarine in Inhibiting Macrophages to Promote Metastasis and Proliferation of Lung Cancer via Modulating the Exosomes in A549 Cells&lt;/p&gt;

ObjectiveSanguinarine (SNG) is a benzophenanthridine alkaloid obtained from the roots of Sanguinaria canadensis and has an anticancer effect. The aim of this study was to explore the mechanism of SNG in inhibiting macrophages via regulating the exosomes derived from lung carcinoma cells to reduce metastasis and proliferation of lung carcinoma.MethodsHuman lung cancer cells (A549 cells) were treated with 4μM of SNG. Exosomes of A549 cells were extracted from A549 cells supernatant, and THP-1 cells were cultured with exosomes. Then, the supernatant of THP-1 cells was collected and cultured with A549 cells. Cell proliferation was measured via plate clone formation and CCK-8 assays. Migration was assessed by using Transwell assay and scratch test. Cellular invasion was detected by Transwell assay. Apoptosis was determined using flow cytometry. Moreover, the protein expressions of GAPDH, P65 and P-P65 in THP-1 cells were measured by Western blot. Levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and chemotactic cytokines ligand 2 (CCL-2) extracted from THP-1 cells were determined by reverse transcription-polymerase chain reaction (RT-PCR).ResultsCompared to the control group, exosomes could activate THP-1 cells, and the invasion, migration, and proliferation of A549 cells were consequently enhanced. Exosomes could increase the protein expression of p-p65 and the RNA expression levels of TNF-α, IL-6, and CCL-2 in THP-1 cells. Compared with the exosome group, SNG-treated exosomes inhibited THP-1 cells so that the invasion, proliferation, and migration of A549 cells were attenuated and apoptosis was promoted. In THP-1 cells, SNG-treated exosomes inhibited P-P65 expression and the RNA expression levels of TNF-α, IL-6, and CCL-2.ConclusionExosomes treated by SNG inhibited THP-1 cells so that the invasion, proliferation, and migration of A549 cells were inhibited, and the apoptosis was promoted. The mechanism is possibly associated with the inhibition of NF-κB pathway in THP-1 cells.

LncRNA PVT1 Regulates TRPS1 Expression in Breast Cancer by Sponging miR-543.

BackgroundBreast cancer is the most common female malignancy with high invasion and metastasis abilities. Studies have shown that long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 gene (PVT1) is an oncogene and is positively correlated with progression and metastasis of breast tumors. However, the detailed mechanism of PVT1 in breast cancer tumorigenesis is not fully understood.MethodsReal-time polymerase quantitative chain reaction (RT-qPCR) was performed to identify the expression levels of PVT1, miR-543 and trichorhinophalangeal syndrome-1 gene (TRPS1) in breast cancer tissues and cells. Cell proliferation was measured by plate clone formation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay. Apoptosis and motility of MCF-7 and MDA-MB-436 cells were assessed with flow cytometry assay and transwell migration and invasion analyses, respectively. In addition, a model was established to probe the function of PVT1 silencing in vivo. The target relationship among PVT1, miR-543 or TRPS1 was confirmed by dual-luciferase reporter analysis, RNA immunoprecipitation (RIP) and RNA pull down assays. The protein expression level of TRPS1 was evaluated with Western blot assay.ResultsPVT1 expression was upregulated in breast cancer tissues and cell lines. In addition, PVT1 silencing inhibited breast cancer cell growth and motility, while increased apoptosis. Meanwhile, the effects of PVT1 or miR-543 could be reversed by introducing overexpressed plasmid of miR-543 or TRPS1 in breast cancer cell lines, respectively.ConclusionKnockdown of PVT1 repressed breast cancer cell growth and motility, and induced apoptosis in vitro and reduced tumor volume and weight in vivo. Mechanically, the overexpression of PVT1 enhanced TRPS1 level by negatively targeted miR-543 in breast cancer.

High APOBEC1 Complementation Factor Expression Positively Modulates the Proliferation, Invasion, and Migration of Endometrial Cancer Cells Through Regulating P53/P21 Signaling Pathway.

Background: APOBEC1 complementation factor (A1CF) is a component of the apolipoprotein-B messenger RNA editing complex that participates in various cellular processes and acts as an oncogene in many cancers. In this study, it was aimed to investigate the roles of A1CF and its potential mechanism in endometrial cancer (EC). Materials and Methods: Gene expression prolife was downloaded from The Cancer Genome Atlas database. Then Kaplan-Meier and Cox regression analyses were conducted to assess the prognostic value of A1CF in EC. Cell Counting Kit-8, plate clone formation, and transwell assays were used to estimate the functions of A1CF on the proliferation, invasion, and migration of EC cell. The gene set enrichment analysis was used to analyze the pathway that is enriched by A1CF, whereas quantitative real-time polymerase chain reaction and Western blot analyses were utilized to detect the mRNA and protein expression involved. Results: It was detected that the upregulated A1CF was enriched in P53/P21 signaling pathway and tightly associated with patients' age, stage, and death. Besides, high A1CF expression led to a shorter overall survival of patients and predicted a poor prognosis in EC. The overexpression of A1CF promoted the proliferation, invasion, and migration of EC cells, whereas the depletion of A1CF suppressed these processes. Moreover, P21 and P53 were reduced whereas cyclin D1 and proliferating cell nuclear antigen were induced along with the increasing of A1CF. However, the effects of silencing A1CF on these protein expressions were on the contrary. Conclusion: A1CF was highly expressed and closely related to the prognosis and progression of EC through the regulation of P53/P21 signaling pathway, providing a possible new therapy target site for EC.

Nobiletin inhibits viability of human renal carcinoma cells via the JAK2/STAT3 and PI3K/Akt pathway

Nobiletin, a flavonoid found chiefly in oranges and lemons, has exhibited potential anti-proliferative and pro-apoptotic activities in various types of cancers. However, the inhibitory effect and mechanisms of nobiletin on renal cancer cells are unclear. CCK8 and plate clone formation assay were used to determine the effect of nobiletin on the proliferation of renal cancer cells, while scratch healing test was used to assay its effect on migration ability. The effect of nobiletin on the invasion of renal cancer cells was determined using Trans well chamber assay. Flow cytometry was used to determine the effect of nobiletin on apoptosis of renal cancer cells, while Western blotting assay was used to determine its effect on the expressions of JAK2/STAT3 and PI3K/Akt pathway proteins, and apoptosis-related proteins. Nobiletin inhibited the proliferation of renal carcinoma cells in a time- and dose-dependent manner. It inhibited the migration and invasion of renal cancer cells, and promoted their apoptosis. Western blot results showed that nobiletin inhibited the phosphorylations of JAK2, STAT3, PI3K, and Akt, and promoted the expressions of apoptosis-related proteins. Nobiletin inhibits the proliferation, invasion and migration of renal cell carcinoma by inhibiting JAK2/STAT3 and PI3K/Akt pathways, and promotes their apoptosis. These findings provide a new experimental basis for the application of nobiletin in the treatment of renal cancer.

Atorvastatin Enhances Effects of Radiotherapy on Prostate Cancer Cells and Xenograft Tumor Mice Through Triggering Interaction Between Bcl-2 and MSH2.

BackgroundProstate cancer (PCa) is considered to be the 4th most common cancer in males in the world. This study aimed to explore effects of atorvastatin on colony formation of PCa cells and radio-resistance of xenograft tumor models.Material/MethodsPCa cell lines, including PC3, DU145, and Lncap, were treated with irradiation (4 Gy) and/or atorvastatin (6 μg/mL). Cells were divided into tumor cell group, irradiation treatment group (IR group) and irradiation+atorvastatin treatment group (IR-AS group). Xenograft tumor mouse model was established. Plate clone formation assay (multi-target/single-hit model) was conducted to evaluate colony formation. Flow cytometry analysis was employed to detect apoptosis. Interaction between Bcl-2 and MSH2 was evaluated with immuno-fluorescence assay.ResultsAccording to the plate colony formation assay and multi-target/single-hit model, IR-treatment significantly suppressed colony formation in PCa cells (including PC3, DU145, and Lncap cells) compared to no-IR treated cells (P<0.05). Atorvastatin remarkably enhanced inhibitive effects of irradiation on colony formation of PCa cells (P<0.05), however, the IR+AS group demonstrated no effects on apoptosis, comparing to IR group (P>0.05). Atorvastatin administration (IR+AS group) significantly reduced tumor size of IR-treated PCa cells-induced xenograft tumor mice (P<0.05). Bcl-2 interacted with MSH2 both in tumor tissues of xenograft tumor mice.ConclusionsAtorvastatin administration inhibited colony formation in PCa cells and enhanced effects of radiotherapy on tumor growth of xenograft tumor mice, which might be associated with interaction between Bcl-2 and MSH2 molecule.

Integrin α5 silencing inhibits proliferation, invasion and metastasis of human liver cancer Bel-7404 cells in vitro

To analyze the association of integrinα5 (ITGA5) with grading of liver cancer and the overall patient survival and investigate the effects of integrin α5 (ITGA5) silencing on the proliferation, invasion and migration abilities of human liver cancer Bel-7404 cells. UALCAN was used to analyze the expression of ITGA5 in liver cancer tissues and normal tissues, and expression in different grades of liver cancer tissues. GEPIA was used to analyze the relationship between ITGA5 expression and the survival of liver cancer patients through.The ITGA5 shRNA lentiviral vector was used to infect Bel-7404 cells to establish a cell line with stable ITGA5 silencing verified by Western blotting. Plate clone formation assay and Transwell assay were used to detect the proliferation, invasion and migration of Bel-7404 cells. The correlation between ITGA5 and PI3K in liver cancer tissues and control tissues was analyzed using Oncomine cancer specimen database. The expression of ITGA5 was significantly higher in liver cancer than in normal tissues (P &lt; 0.05). The expression of ITGA5 was significantly lower in grade 1 than in grade 2 liver cancer, and also lower in grade 2 than in grade 3 liver cancer (P &lt; 0.05). The patients with high ITGA5 expression had a significantly lower overall survival rate than those with low ITGA5 expression (P &lt; 0.05). Plate clone formation assay showed that the clone formation rate was significantly lowered in Bel-7404 cells with ITGA5 silencing compared with the blank and negative control cells (P &lt; 0.05). ITGA5 silencing significantly attenuated the migration of Bel-7404 cells as shown by Transwell assay (P &lt; 0.05). ITGA5 and PI3K were both highly expressed and showed a positive correlation in liver cancer tissues (P &lt; 0.05). ITGA5 is closely related to the progression of liver cancer and the patients' prognosis. ITGA5 silencing inhibits the proliferation, invasion and migration of liver cancer cells. ITGA5 promotes the liver cancer growth and metastasis possibly by regulating the PI3K signaling pathway.

Loss-of-function mutations in KEAP1 drive lung cancer progression via KEAP1/NRF2 pathway activation

Background and purposeTargeted therapy and immunotherapy have led to dramatic change in the treatment of lung cancer, however, the overall 5-year survival rate of lung cancer patients is still suboptimal. It is important to exploit new potential of molecularly targeted therapies. High-frequency somatic mutations in KEAP1/NRF2 (27.9%) have been identified in lung squamous cell carcinoma. In this research, we explored the role of KEAP1 somatic mutations in the development of LSCC and whether a nuclear factor erythroid 2-related factor 2(NRF2) inhibitor be potential to target lung cancer carrying KEAP1/NRF2 mutations.MethodsLung cancer cell lines A549 and H460 with loss-of-function mutations in KEAP1 stably transfected with wild-type (WT) KEAP1 or somatic mutations in KEAP1 were used to investigate the functions of somatic mutations in KEAP1. Flow cytometry, plate clone formation experiments, and scratch tests were used to examine reactive oxygen species, proliferation, and migration of these cell lines.ResultsThe expression of NRF2 and its target genes increased, and tumor cell proliferation, migration, and tumor growth were accelerated in A549 and H460 cells stably transfected with KEAP1 mutants compared to control cells with a loss-of-function KEAP1 mutation and stably transfected with WT KEAP1 in both in vitro and in vivo studies. The proliferation of A549 cell line trasfected with the R320Q KEAP1 mutant was inhibited more apparent than that of the A549 cell line trasfected with WT KEAP1 after treatment with NRF2 inhibitor ML385.ConclusionSomatic mutations of KEAP1 identified from patients with LSCC likely promote tumorigenesis mediated by activation of the KEAP1/NRF2 antioxidant stress response pathway. NRF2 inhibition with ML385 could inhibit the proliferation of tumor cells with KEAP1 mutation.27fX3rkGbRpRrWDtKhtxqBVideo abstract

Six-transmembrane epithelial antigen of the prostate 1 expression promotes ovarian cancer metastasis by aiding progression of epithelial-to-mesenchymal transition.

Ovarian cancer is a severe malignant tumour of the female genital organs. Six-transmembrane epithelial antigen of the prostate 1 (STEAP1) expression is correlated with the occurrence and progression of multiple cancers. Here, we assessed STEAP1 expression in ovarian cancer and explored the relationship between STEAP1 and ovarian cancer progression. We used immunohistochemistry and public databases to test STEAP1 expression in normal human ovarian tissues, benign ovarian tumours, and ovarian cancer. The expression of STEAP1 and epithelial-to-mesenchymal transition (EMT)-related genes was analysed using immunocytochemistry, quantitative reverse transcription polymerase chain reaction, and western blotting in ovarian cancer cell lines. Lentivirus was used to knockdown and overexpress STEAP1. Invasion, migration, growth, clonogenicity, and apoptosis were assessed using transwell assay, growth curve, plate clone formation assay, and flow cytometry. We used a tumour xenograft to verify the relationship between STEAP1 and in vivo ovarian cancer cell growth. Matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) activities were examined using Matrix metalloproteinase zymography assay. STEAP1 was highly expressed in the human ovarian cancer tissues and a highly invasive ovarian cancer cell line. Overexpression of STEAP1 was related to poor prognosis in ovarian cancer patients. Down-regulation of STEAP1 suppressed the invasion, migration, proliferation, clonogenicity, EMT progression in human ovarian cancer cells and xenograft tumour growth in vivo, but it enhanced apoptosis. In human ovarian cancer, the STEAP1 gene is highly expressed, and its function is correlated with human ovarian cancer cell metastasis and growth. STEAP1 may be a possible target for suppressing ovarian cancer metastasis.

Downregulation of MicroRNA-551b Correlates With Dissemination of Human Oral Squamous Cell Carcinoma

Altered expression of microRNAs contributes to invasion and metastasis of many human cancers; however, the importance of microRNAs in head and neck cancers remains to be elucidated. In this study, we examined whether altered microRNA (miR)-551b expression correlated with invasive phenotypes of human oral squamous cell carcinoma (OSCC) invivo and invitro. Real-time polymerase chain reaction was used to detect the expression level of miR-551b in 71 OSCC tissues with lymph node metastasis and 50 nonmetastatic OSCC tissues. We also constructed miR-551b mimic-transfected cell lines HN4 and HN12. The effects of overexpressing miR-551b on the proliferation, migration, and invasion of OSCC cells were examined using Cell Counting Kit 8 (Dojindo, Kumamoto, Japan), plate clone formation, wound healing, and Transwell invasion experiments (Corning, Corning, NY).The association between clinical pathologic parameters and the expression level of miR-551b was analyzed using Kaplan-Meier survival analysis. The expression of miR-551b measured 0.33±0.11 in the 71 OSCC tissues with lymph node metastasis versus 0.54±0.06 in the 50 tissues with non-lymph node metastasis (P=.021).Regarding OSCC patients, the expression of miR-551b negatively correlated with patients' overall survival (P=.035). The ectopic expression of miR-551b inhibited the invasion and migration of OSCC cells. This is the first report showing that reduced miR-551b expression may be an event leading to OSCC invasion and metastasis.

Targeting PIK3CG in Combination with Paclitaxel as a Potential Therapeutic Regimen in Claudin-Low Breast Cancer.

PurposeMolecular targeting is a powerful approach for aggressive claudin-low breast cancer (CLBC). Overexpression of PI3K catalytic subunit gamma (PIK3CG) in human CLBC is offering a promising opportunity for targeted therapies. We utilized a specific inhibitor of PIK3CG combined with paclitaxel (PTX) to treat CLBC cells in vitro and in vivo.Patients and MethodsThe tumor cells growth and apoptosis in vitro were analyzed by CCK8, plate clone formation assay, tumorsphere assay, Hoechst staining and flow cytometry. The invasion and metastasis ability of tumor cells in vitro were investigated by wound healing and transwell experiments. Critical gene expression levels were checked by qRT-PCR and Western blot. Xenograft models with CLBC cell lines in SCID mice were established to investigate the effect of combined drugs in vivo.ResultsWe identified that PIK3CG was a potential therapeutic target for CLBC patients. Targeting PIK3CG potentiated CLBC cells growth inhibition in 2D and 3D cultures by PTX. Inhibition of PIK3CG activation could enhance CLBC cells apoptosis and migration suppression induced by PTX. Manipulating autophagy was a validated approach for the use of PIK3CG inhibitor. Using CLBC xenograft mice model, we found that CLBC tumors in vivo could be well treated by combined drugs of PIK3CG inhibitor and PTX.ConclusionWe demonstrated that PIK3CG was a potential target for the therapy of CLBC and inhibition of PIK3CG activation could reinforce the therapeutic effect of this aggressive disease by PTX. The combined use of PIK3CG inhibitor and PTX might be a potential regimen for treating this subtype of breast cancer.

High Expression of CARM1 Inhibits Lung Cancer Progression by Targeting TP53 by Regulating CTNNB1.

To explore the role of CARM1 in lung cancer (LC) and its relationship with TP53 and CTNNB1. Lung cells H1299 and PC14 were randomly divided into six groups: ov-H1299, si-H1299, ov-PC14, si-PC14, Con-H1299, and Con-PC14. Transwell assay, plate clone formation assay, and flow cytometry were used to determine the migration, clone formation capacity, and apoptosis situation of LC cells in the six groups, respectively. Western blot assay was used to determine the protein expression of CARM1, TP53, and CTNNB1 in the six groups. CHIP assay was applied to analyze the combined characteristics of JUN and TP53 promoter. Co-immunoprecipitation was used to analyze the interaction between TP53 and CARM1/CTNNB1. Cox proportional hazard regression model was used to analyze the relevance between the expression of CARM1 and clinicopathological information of the patient. Kaplan-Meier plot was used to determine the relevance between CARM1 and patient survival. High expression of CARM1 inhibits the migration and proliferation of LC cells and promoted the apoptosis of LC cell. Overexpression of CARM1 promotes the expression of CARM1 and TP53, while decreases CTNNB1 expression. CARM1 supplementation of H1299 cells induced JUN aggregation on the TP53 promoter. TP53 and CARM1 protein/TP53 and CTNNB1 protein in H1299 cells were immunoprecipitated together. High expression of CARM1was negatively correlated with the degree of tumor metastasis. The survival period of patients with high expression CARM1 was greater than that of low expression. Overexpression of CARM1 may inhibit the progression of LC by targeting TP53 via regulation CTNNB1.