Mouse Xenograft Assay Research Articles

Methylation-Mediated Silencing of MicroRNA-211 Decreases the Sensitivity of Melanoma Cells to Cisplatin.

BackgroundMalignant melanoma is recalcitrant to most existing chemotherapies, and aberrant expression of miR-211 plays prominent roles in progression of melanoma. However, the trigger mechanism of aberrant miR-211 expression in melanoma is still elusive.Material/MethodsWe used qRT-PCR to test miR-211 expression. Cell viability assay and mouse xenograft assay were performed to examine the role of miR-211 on the sensitivity of melanoma cells to cisplatin. The epigenetic modification of miR-211 promoter was assess by DNA methylation analysis and DAC treatment.ResultsIn this study, decreased miR-211 expression was detected. Bisulfite sequencing PCR showed that DNA hypermethylation contributed to the downregulation of miR-211 in melanoma tissues. In melanoma cells, overexpressed 211 could enhance the anticancer effect of cisplatin and restoration of miR-211 rendered susceptibility to cisplatin in cisplatin-resistant cells. And the same result was showed in vivo by mouse xenograft assay. What is more, DAC treatment could increase miR-211 expression and EZH2 expression was increased in cisplatin-resistant cells. MiR-211 could be transcriptionally repressed by EZH2 mediated promoter methylation.ConclusionsTaken together, our findings revealed that epigenetic modification of miR-211 governed melanoma cell chemosensitivity and were involved in the progression of tumorigenesis.

Systematic Cancer-Testis Expression Analysis Identifies CDCA5 As a Potential Therapeutic Target in Esophageal Squamous Cell Carcinoma

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies with poor prognosis. Recently, the discovery of the immunogenic proteins, cancer-testis antigens, have been vigorously pursued as targets for therapeutic cancer vaccines. Methods: A systematic screening strategy was adopted to screen cancer-testis genes (CTGs) in ESCC by integrating multiple public databases and RNA expression microarray data from 119 ESCC subjects. For our identified novel CTG, independent cohort with 118 ESCC patients were recruited to validate the protein level by immunohistochemistry. Furthermore, functional assays were used to determine the underlying mechanisms in vitro and in vivo. Findings: 21 genes were recognized as CTGs, in particular, CDCA5 was aberrantly upregulated in 99.16% (118/119) of ESCC specimens and higher CDCA5 mRNA expression showed significant association with poor ESCC prognosis (HR = 1.85, 95%CI: 1.14-3.01, P = 0.013). Subsequently, immunohistochemical staining further confirmed that positive CDCA5 protein expression was associated with advanced TNM stage and shorter overall survival rate (45.59% vs 28.00% for CDCA5-/+ subjects, P = 1.86×10-3). Mechanistic exploration revealed that the activation of CDCA5 is associated with gain of H3K27ac. Silencing of CDCA5 significantly suppressed proliferation, invasion, migration and apoptosis resistance in ESCC cells. What's more, mouse xenograft assay showed that repressed CDCA5 inhibited tumor formation in vivo (P<0.01). Further investigations indicated that CDCA5 knockdown led to arrest in G2/M phase and expression changes of factors with functions in the cell cycle. Interpretation: CDCA5 contributed to ESCC progression and might serve as an attractive target for ESCC immunotherapy. Funding Statement: This work was supported by the Natural Science Foundation of Jiangsu Province (No. BK20181083 and BK20181496), Jiangsu Top Expert Program in Six Professions (No. WSW-007), Major Program of Science and Technology Foundation of Jiangsu Province (No. BE2016790 and BE2018746), Jiangsu Medical Young Talent Project (No. QNRC2016566), the Program of Jiangsu Medical Innovation Team (No. CXTDA2017006) and Jiangsu Province 333 Talents Project (No. BRA2017545). Declaration of Interests: The authors declare no competing financial interest. Ethics Approval Statement: This study was approved by the medical ethics committees of the First Affiliated Hospital of Nanjing Medical University.

Overexpression of CDC42SE1 in A431 Cells Reduced Cell Proliferation by Inhibiting the Akt Pathway

Cell division cycle 42 (CDC42), a small Rho GTPase, plays a critical role in many cellular processes, including cell proliferation and survival. CDC42 interacts with the CRIB (Cdc42- and Rac-interactive binding) domain of CDC42SE1, a small effector protein of 9 kDa. We found that the expression of CDC42SE1 was reduced in human skin cancer samples relative to matched perilesional control. Exogenous expression of CDC42SE1 but not CDC42SE1H38A (mutation within CRIB domain) in A431 cells (A431SE1, A431SE1-H38A) reduced cell proliferation. Antibody microarray analysis of A431Ctrl and A431SE1 lysate suggested that reduced A431SE1 cells proliferation was due to inhibition of Akt pathway, which was confirmed by the reduced P-Akt and P-mTOR levels in A431SE1 cells compared to A431Ctrl cells. This suggests that CDC42SE1 modulates the CDC42-mediated Akt pathway by competing with other effector proteins to bind CDC42. A431SE1 cells formed smaller colonies in soft agar compared to A431Ctrl and A431SE1-H38A cells. These findings correlate with nude mice xenograft assays, where A431SE1 cells formed tumors with significantly-reduced volume compared to the tumors formed by A431Ctrl cells. Our results suggest that CDC42SE1 is downregulated in skin cancer to promote tumorigenesis, and thus CDC42SE1 might be an important marker of skin cancer progression.

Citron kinase (CIT-K) promotes aggressiveness and tumorigenesis of breast cancer cells in vitro and in vivo: preliminary study of the underlying mechanism

ObjectivesCitron kinase (CIT-K), as a key Rho effector, functions to maintain proper structure of the midbody during cell mitosis. This study assessed CIT-K expression and its role in breast cancer cells.MethodsParaffin-embedded breast cancer and para-tumor tissues from 43 invasive breast cancer patients and 33 normal mammary glands were collected for immunohistochemistry. CIT-K expression knockdown was achieved using lentivirus carrying CIT-K shRNA in a wide range of breast cancer cell lines. Cells were then subjected to Western blot, qRT-PCR, cell proliferation, colony formation, transwell, and flow cytometric assays. The tumorigenicity of CIT-K knocked-down breast cancer cells was assessed using the nude mouse xenograft assay. Microarray analysis was performed to elucidate the underlying gene regulation after CIT-K silencing.ResultsCIT-K protein was overexpressed in breast cancer tissues, which is associated with advanced tumor stage, HER-2 expression and Ki-67 expression, whereas knockdown of CIT-K expression reduced breast cancer cell proliferation and colony formation, but promoted tumor cell apoptosis and cell-cycle arrest. Knockdown of CIT-K expression also inhibited breast cancer cell migration and invasion capacity. Moreover, CIT-K knockdown suppressed the tumorigenicity of breast cancer cells in nude mice. Molecularly, the expression of a variety of signaling genes, such as cyclin D1, EGFR, JAK1, TGF-α, PTK2, RAF1, RALB, SOS1, mTOR, and PTGS2, were altered after CIT-K knockdown.ConclusionsThis study demonstrated that CIT-K is associated with aggressive breast cancer behavior and targeting CIT-K may be a novel strategy for the future control of breast cancer.

FOXF2 inhibits proliferation, migration, and invasion of Hela cells by regulating Wnt signaling pathway.

This article was aimed to study the FOXF2 effects on cervical cancer. Tumor tissues and adjacent tissues of 41 cervical cancer patients were collected. Human endometrial epithelial cells (hEEC) and Hela cells were cultured. FOXF2 expression vector and its empty vector were transfected into Hela cells, and named as pcDNA 3.1-FOXF2 group and Vector group, respectively. Hela cells without any treatment were set as Blank group. qRT-PCR was used to detect mRNA expression. Nude mouse xenograft assay was performed to test Hela cells proliferation ability in vivo. FOXF2 and β-catenin positive cell numbers were detected by immunohistochemistry. Protein expression was analyzed by Western blot. Cells migration and invasion were conducted by Transwell. Tumor tissues and Hela cells FOXF2 expression were lower than that in adjacent tissues and hEEC (P<0.01). Low FOXF2 expression predicted poor outcomes of cervical cancer patients. Compared with Blank group and Vector group, Hela cells of pcDNA 3.1-FOXF2 group were with higher FOXF2 expression, lower OD495 value, migrated and invaded cells, higher E-cadherin expression, lower Vimentin and Snail expression, smaller tumor volume in nude mice, lower c-Myc, CyclinDl, MMP9, Lgr5, and nuclear β-catenin expression (all P<0.01). FOXF2 inhibits Hela cells proliferation, migration, and invasion through regulating Wnt signaling pathway.

GINS2 promotes cell proliferation and inhibits cell apoptosis in thyroid cancer by regulating CITED2 and LOXL2.

To explore the mechanisms of GINS2 on cell proliferation and apoptosis in thyroid cancer (TC) cells. Expressions of GINS2 were inhibited in K1 and SW579 cells using gene interference technology. The abilities of proliferation and apoptosis, and cell cycle were determined by MTT assay and flow cytometric assay. The downstream molecules of GINS2 were searched by microarray and bioinformatics and validated by qRT-PCR and western blotting. In the in vivo study, the tumor growth was compared and the whole-body fluorescent imaging was analyzed. After GINS2 was interfered, cell proliferation was significantly inhibited (P < 0.01) and apoptosis rate increased (P < 0.01) in both K1 and SW579 cells. Cell cycle changed significantly in K1 cells, but not in SW579 cells. With bioinformatics upstream analysis, TGF-β1 was found as the most significantly upstream regulator. Expressions of TGF-β1 and its downstream target molecules CITED2 and LOXL2 were validated and found downregulated significantly in mRNA and protein levels (P < 0.05). The results of the nude mouse xenograft assay suggested that the volume and weight of tumor in ones infected with shGINS2 were statistically smaller than controls (P < 0.05). GINS2 plays an important role in cell proliferation and apoptosis of thyroid cancer by regulating the expressions of CITED2 and LOXL2, which may be a potential biomarker for diagnosis or prognosis and a drug target for therapy.

Oncogenic Activity of Wrap53 in Human Colorectal Cancer In Vitro and in Nude Mouse Xenografts.

BackgroundWD40-encoding RNA antisense to p53 (Wrap53) has been implicated in cancer development. However, the role of Wrap53 remains unknown in colorectal cancer. The aim of this study was to elucidate the function of Wrap53 in colorectal cancer tumorigenesis and development.Material/MethodsThis study analyzed Wrap53 expression in colorectal cancer tissue specimens using The Cancer Genome Atlas data and tumor cell lines and assessed the effects of Wrap53 knockdown on regulation of cancer cell malignant phenotypes in vitro and in nude mouse xenografts.ResultsWrap53 expression was upregulated in colorectal cancer tissue specimens and cell lines. Knockdown of Wrap53 expression induced colorectal cancer cell line apoptosis and cell cycle arrest in the G1 phase, but reduced tumor cell line proliferation and invasion in vitro. Knockdown of Wrap53 in a nude mouse xenograft assay inhibited tumor cell line xenograft formation and growth.ConclusionsWrap53 is likely a potential oncogene or possesses oncogenic activity in colorectal cancer, promoting colorectal tumorigenesis. Targeting Wrap53 expression may represent a novel strategy for the control of colorectal cancer.

The relationship between lncRNA PCGEM1 and STAT3 during the occurrence and development of endometrial carcinoma

We investigated the effects of lncRNA PCGEM1 on the tumorigenesis and development of endometrial carcinoma (EC) and its possible molecular mechanism. We found that PCGEM1 expression was significantly higher in EC tissues than in normal endometrial tissues via quantitative reverse transcription (qRT)-PCR. The upregulation of PCGEM1 promoted the proliferation, migration, and invasive ability of EC cells while inhibiting apoptosis. The silencing of PCGEM1 had the opposite effects. Nude mouse xenograft assay showed that PCGEM1 overexpression could promote tumor growth in vivo. Western blotting and immunohistochemistry showed that PCGEM1 also upregulated STAT3 expression, which affected the expression of B-cell lymphoma-2, survivin, vascular endothelial growth factor A, and matrix metalloproteinase-2. Furthermore, the dual-luciferase reporter assay confirmed that miR-129-5p could bind directly to both PCGEM1 and STAT3. In addition, qRT-PCR showed that overexpression of PCGEM1 caused a decrease in miR-129-5p expression, and silencing of PCGEM1 produced the opposite result. In the PCGEM1-overexpressing cells, overexpression of miR-129-5p reduced the expression of STAT3 at both mRNA and protein levels. Thus, we confirmed that PCGEM1 could upregulate the expression of STAT3 by acting as a competing endogenous RNA for miR-129-5p, thereby affecting the occurrence and development of EC.

CircFBLIM1 act as a ceRNA to promote hepatocellular cancer progression by sponging miR-346

BackgroudAccumulating evidences indicate that circular RNAs (circRNAs), a class of non-coding RNAs, play important roles in tumorigenesis. However, the function of circRNAs in hepatocellular cancer (HCC) is largely unknown.MethodsWe performed circRNA microarrays to identify circRNAs that are aberrantly expressed in HCC tissues. Expression levels of a significantly upregulated circRNA, circFBLIM1, was detected by quantitative real-time PCR (qRT-PCR) in HCC cell lines and tissues. Then, we examined the functions of circFBLIM1 in HCC by cell proliferation, apoptosis, invasion and mouse xenograft assay. In addition, luciferase assay and RNA immunoprecipitation (RIP) assay were used to explore the miRNA sponge function of circFBLIM1 in HCC.ResultsMicroarray analysis and qRT-PCR verified a circRNA termed circFBLIM1 that was upregulated in HCC tissues and cell lines. Knockdown of circFBLIM1 inhibited proliferation, invasion and promoted apoptosis in HCC. Via luciferase reporter assays, circFBLIM1 and FBLIM1 were observed to directly bind to miR-346. Subsequent experiments showed that circFBLIM1 and FBLIM1 regulated the expression of each other by sponging miR-346.ConclusionsTaken together, we conclude that circFBLIM1 may function as a competing endogenous RNA (ceRNA) to regulate FBLIM1 expression through sponging miR-346 to exert regulatory functions in HCC. circFBLIM1 may be a diagnostic biomarker and potential target for HCC therapy.

The oncogenic potentials and diagnostic significance of long non-coding RNA LINC00310 in breast cancer.

Recent studies have revealed that long non‐coding RNAs (lncRNAs) are involved in different physiological processes and human diseases. However, to date, the function and overall clinical significance of the vast majority of lncRNAs in breast cancer remain largely unexplored. Here, we focused on LINC00310 by interrogating the breast invasive carcinoma data set of the Cancer Genome Atlas (TCGA). The results showed that LINC00310 was increased as breast cancer progressed, and the deregulation of LINC00310 was significantly associated with patients’ survival. Experiments with knockout (KO) approach by CRISPR/Cas9 system and the subsequent rescue experiments revealed that LINC00310 promoted cell proliferation by regulating c‐Myc expression in vitro. Nude mouse xenograft assay demonstrated that LINC00310 KO significantly suppressed tumour growth in vivo. Furthermore, we found that serum LINC00310 expression was significantly up‐regulated in patients with breast cancer, and receiver operating characteristic (ROC) curve analysis indicated that LINC00310 had a powerful capability of distinguishing patients with breast cancer from healthy individuals (the area under curve 0.828). Taken together, these results provide a more intuitive approach to explore the clinical relevance and functional roles of lncRNAs. As a result, lncRNAs, such as LINC00310, may be used in clinical applications as circulating markers for breast cancer.

Phytochemical naphtho[1,2-b] furan-4,5‑dione induced topoisomerase II-mediated DNA damage response in human non-small-cell lung cancer

BackgroundPhytochemical naphtho[1,2-b] furan-4,5‑dione (NFD) presenting in Avicennia marina exert anti-cancer effects, but little is known regarding about DNA damage-mediated apoptosis in non-small-cell lung carcinoma (NSCLC). PurposeTo examine whether NFD-induced apoptosis of NSCLC cells is correlated with the induction of DNA damage, and to investigate its underlying mechanism. Study designThe anti-proliferative effects of NFD were assessed by MTS Assay Kit FACS assay, and in vivo nude mice xenograft assay. The DNA damage related proteins, the Bcl-2 family and pro-apoptotic factors were examined by immunofluorescence assay, q-PCR, and western blotting. The activity of NF-κB p65 in nuclear extracts was detected using a colorimetric DNA-binding ELISA assay. The inhibitory activity of topoisomerase II (TOPO II) was evaluated by molecular docking and TOPO II catalytic assay. ResultsNFD exerted selective cytotoxicity against NSCLC H1299, H1437 and A549 cells rather than normal lung-embryonated cells MRC-5. Remarkably, we found that NFD activated the hull marker and modulator of DNA damage repairs such as γ-H2AX, ATM, ATR, CHK1, and CHK2 probably caused by the accumulation of intracellular reactive oxygen species (ROS) and inhibition of TOPO II activity. Furthermore, the suppression of transcription factor NF-κB by NFD resulted in significantly decreased levels of pro-survival proteins including Bcl-2 family Bcl-2, Bcl-xL and Mcl-1 and the endogenous inhibitors of apoptosis XIAP and survivin in H1299 cells. Moreover, the nude mice xenograft assay further validated the suppression of H1299 growth by NFD, which is the first report for evaluating the anti-cancer effect of NFD in vivo. ConclusionThese findings provide a novel mechanism indicating the inhibition of TOPO II activity and NF-κB signaling by NFD, leading to DNA damage and apoptosis of NSCLC tumor cells.

Neoisoliquiritigenin Inhibits Tumor Progression by Targeting GRP78-β- catenin Signaling in Breast Cancer.

Breast cancer mortality has been stable or decreasing in the world, its incidence and recurrence rates have sharply risen worldwide in the recent years. To investigate the clinicopathological significance and potential function of GRP78 in the development and progression of breast cancer. To explore the effects of neoisoliquiritigenin (NISL) in breast cancer and the underlying mechanism. GRP78 was detected by immunohistochemistry (IHC) using breast cancer tissue microarrays (TMAs), and the association between GRP78 levels and clinicopathological factors and prognosis was analyzed. The functional effects of GRP78 on breast cancer were validated by an MTT assay, foci formation assay, Matrigel invasion assay and mouse xenograft assay. The effects of NISL were tested by an MTT assay, apoptosis assay and mouse xenograft assay. A LigandFit algorithm, ATPase activity assay, western blot and IHC assay were used to discover the underlying mechanism of the effects of NSIL. GRP78 was highly expressed in breast cancer cell lines and tissues. In addition, high expression of GRP78 was correlated to poor outcomes and distant metastasis. Functional experiments showed that GRP78 promoted breast cancer proliferation and invasion in vitro and in vivo. NISL inhibited cell proliferation and induced cell apoptosis in breast cancer by directly binding to GRP78 to regulate the β-catenin pathway. Taken together, these results highlighted the significance of GRP78 in breast cancer development and suggested NISL as a natural candidate to inhibit breast cancer by targeting GRP78 and β-catenin signaling.

20(S)-Protopanaxadiol-Induced Apoptosis in MCF-7 Breast Cancer Cell Line through the Inhibition of PI3K/AKT/mTOR Signaling Pathway.

20(S)-Protopanaxadiol (PPD) is one of the major active metabolites of ginseng. It has been reported that 20(S)-PPD shows a broad spectrum of antitumor effects. Our research study aims were to investigate whether apoptosis of human breast cancer MCF-7 cells could be induced by 20(S)-PPD by targeting the Phosphatidylinositol 3-kinase/Protein kinase B/Mammalian target of rapamycin (PI3K/AKT/mTOR) signal pathway in vitro and in vivo. Cell cycle analysis was performed by Propidium Iodide (PI) staining. To overexpress and knock down the expression of mTOR, pcDNA3.1-mTOR and mTOR small interfering RNA (siRNA) transient transfection assays were used, respectively. Cell viability and apoptosis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-test and Annexin V /PI double-staining after transfection. The antitumor effect in vivo was determined by the nude mice xenograft assay. After 24 h of incubation, treatment with 20(S)-PPD could upregulate phosphorylated-Phosphatase and tensin homologue deleted on chromosome 10 (p-PTEN) expression and downregulate PI3K/AKT/mTOR-pathway protein expression. Moreover, G0/G1 cell cycle arrest in MCF-7 cells could be induced by 20(S)-PPD treatment at high concentrations. Furthermore, overexpression or knockdown of mTOR could inhibit or promote the apoptotic effects of 20(S)-PPD. In addition, tumor volumes were partially reduced by 20(S)-PPD at 100 mg/kg in a MCF-7 xenograft model. Immunohistochemical staining indicated a close relationship between the inhibition of tumor growth and the PI3K/AKT/mTOR signal pathway. PI3K/AKT/mTOR pathway-mediated apoptosis may be one of the potential mechanisms of 20(S)-PPD treatment.

MiR-182-5p Promotes Growth in Oral Squamous Cell Carcinoma by Inhibiting CAMK2N1

Background/Aims: Emerging evidence suggests that the propagation of oral squamous cell carcinoma (OSCC) is influenced by the abnormal expression of microRNAs (miRNAs). This study aimed to characterize the involvement of miR-182-5p in OSCC by targeting the calcium/ calmodulin-dependent protein kinase II inhibitor CAMK2N1. Methods: miR-182-5p expression was quantified in OSCC tissues and cell lines with reverse transcription polymerase chain reaction (RT-PCR). Cell colony formation, Cell Counting Kit-8 (CCK-8), Ki-67, and nude mouse xenograft assays were used to characterize the role of miR-182-5p in the proliferation of OSCC. A miR-182-5p target gene was identified with western blotting, RT-PCR, and luciferase activity assays. OSCC patient survival based on CAMK2N1 expression was also analyzed. Results: miR-182-5p was up-regulated in in vitro cell lines and in vivo clinical OSCC samples. CCK-8, colony formation, and Ki-67 assays revealed that miR-182-5p promoted the growth and proliferation of OSCC cells. miR-182-5p directly targeted CAMK2N1, as evidenced by luciferase assays and target prediction algorithms. CAMK2N1 operated as a tumor suppressor gene in patients with OSCC. Down-regulating miR-182-5p expression in the CAL-27 cell line restored CAMK2N1-mediated OSCC cell proliferation. miR-182-5p expression inhibited the activation of AKT, ERK1/2, and NF-κB. Mice injected with CAL-27 cells transfected with miR-182-5p-inhibitor demonstrated a significant increase in tumor size and weight and increased CAMK2N1 mRNA and protein expression compared with the miR-negative control group. Conclusion: The miR-182-5p-CAMK2N1 pathway can be potentially targeted to regulate the proliferation of OSCC cells.

Retracted Article: TRIM22 functions as an oncogene in gliomas through regulating the Wnt/β-catenin signaling pathway.

The tripartite motif-containing (TRIM) family is a group of proteins that are implicated in a plethora of pathological conditions. TRIM22 has been found to be involved in various cancers; however, the role of TRIM22 in gliomas has not been reported. The present study aimed to evaluate the expression pattern of TRIM22 and its function in gliomas. TRIM22 expressions in glioma tissues and cell lines were measured by RT-PCR and western blot analysis. To knockdown TRIM22 by small hairpin RNAs (shTRIM22), the U118 cells were transfected with pLKO.1-shTRIM22 plasmid or pLKO.1 plasmid. Cell proliferation was measured using CCK-8 assay. Transwell assays were performed to evaluate the migration and invasion. The epithelial–mesenchymal transition (EMT) was assessed by detecting the expressions of E-cadherin, N-cadherin and vimentin with western blot analysis. A xenograft mouse model was established to evaluate the effect of TRIM22 silencing on tumor growth in vivo. The expressions of β-catenin, cyclin D1, and c-Myc were analyzed by western blot analysis. TRIM22 was significantly overexpressed in glioma tissues and cell lines. In vitro studies demonstrated that TRIM22 knockdown inhibited cell proliferation, migration, and invasion. Additionally, TRIM22 silencing increased the expressions of E-cadherin, and decreased the expressions of N-cadherin and vimentin. Nude mouse xenograft assay showed that TRIM22 silencing inhibited tumor growth in vivo. Furthermore, silencing of TRIM22 inhibited the activation of the Wnt/β-catenin pathway. Treatment with LiCl, an activator of the Wnt/β-catenin pathway, attenuated the effects of shTRIM22 on U118 cells. Silencing of TRIM22 inhibited proliferation, migration and invasion, as well as repressing the EMT process in glioma cells. The Wnt/β-catenin pathway was involved in the effect of TRIM22.

LncRNA PCGEM1 Induces Ovarian Carcinoma Tumorigenesis and Progression Through RhoA Pathway

Background/Aims: Prostate cancer gene expression marker 1 (PCGEM1) is a long noncoding RNA (lncRNA) and is well known as a promoter in prostate cancer and osteoarthritis synoviocytes. However, the role PCGEM1 plays in epithelial ovarian cancer is unknown. Methods: PCGEM1 expression was examined in epithelial ovarian cancer and normal ovarian tissues using reverse transcription–PCR. Ovarian cancer cell phenotypes and genotypes were examined after PCGEM1 overexpression or downregulation in vitro; besides, the effects of PCGEM1 overexpression was also examined in vivo. Results: PCGEM1 expression level was higher in epithelial ovarian cancer tissues than in normal ovarian tissues and was positively associated with differentiation (Well vs. Mod/Poor). Upregulation of PCGEM1 induced cancer cell proliferation, migration, and invasion, but decreased cell apoptosis through upregulating RhoA, YAP (Yes-associated protein), MMP2 (matrix metalloproteinase 2), Bcl-xL, and P70S6K expression; while PCGEM1 downregulation had the opposite effect. The nude mouse xenograft assay demonstrated that PCGEM1 overexpression promoted tumor growth. Furthermore, silencing RhoA expression reversed the effect of PCGEM1 and significantly inhibited RhoA, YAP, MMP2, Bcl-xL, and P70S6K protein expression. Conclusion: In conclusion, we suggest that PCGEM1 may be an inducer in epithelial ovarian cancer tumorigenesis and progression by upregulating RhoA and the subsequent expression of YAP, P70S6K, MMP2, and Bcl-xL.

Cysteine Dioxygenase 1 Mediates Erastin-Induced Ferroptosis in Human Gastric Cancer Cells

BACKGROUND: Ferroptosis is a recently discovered form of iron-dependent nonapoptotic cell death. It is characterized by loss of the activity of the lipid repair enzyme, glutathione peroxidase 4 (GPX4), and accumulation of lethal reactive lipid oxygen species. However, we still know relatively little about ferroptosis and its molecular mechanism in gastric cancer (GC) cells. Here, we demonstrate that erastin, a classic inducer of ferroptosis, induces this form of cell death in GC cells and that cysteine dioxygenase 1 (CDO1) plays an important role in this process. METHODS: We performed quantitative real-time polymerase chain reaction, Western blotting, cell viability assay, reactive oxygen species (ROS) assay, glutathione assay, lipid peroxidation assay, RNAi and gene transfection, immunofluorescent staining, dual-luciferase reporter assay, transmission electron microscopy, and chromatin immunoprecipitation assay to study the regulation of ferroptosis in GC cells. Mouse xenograft assay was used to figure out the mechanism in vivo. RESULTS: Silencing CDO1 inhibited erastin-induced ferroptosis in GC cells both in vitro and in vivo. Suppression of CDO1 restored cellular GSH levels, prevented ROS generation, and reduced malondialdehyde, one of the end products of lipid peroxidation. In addition, silencing COO1 maintained mitochondrial morphologic stability in erastin-treated cells. Mechanistically, c-Myb transcriptionally regulated CDO1, and inhibition of CDO1 expression upregulated GPX4 expression. CONCLUSIONS: Our findings give a better understanding of ferroptosis and its molecular mechanism in GC cells, gaining insight into ferroptosis-mediated cancer treatment.

CircGFRA1 and GFRA1 act as ceRNAs in triple negative breast cancer by regulating miR-34a

BackgroudAccumulating evidences indicate that circular RNAs (circRNAs), a class of non-coding RNAs, play important roles in tumorigenesis. However, the function of circRNAs in triple negative breast cancer (TNBC) is largely unknown.MethodsWe performed circRNA microarrays to identify circRNAs that are aberrantly expressed in TNBC cell lines. Expression levels of a significantly upregulated circRNA, circGFRA1, was detected by quantitative real-time PCR (qRT-PCR) in TNBC cell lines and tissues. Kaplan-Meier survival analysis was used to explore the significance of circGFRA1 in clinical prognosis. Then, we examined the functions of circGFRA1 in TNBC by cell proliferation, apoptosis and mouse xenograft assay. In addition, luciferase assay was used to explore the miRNA sponge function of circGFRA1 in TNBC.ResultsMicroarray analysis and qRT-PCR verified a circRNA termed circGFRA1 that was upregulated in TNBC. Kaplan-Meier survival analysis showed that upregulated circGFRA1 was correlated with poorer survival. Knockdown of circGFRA1 inhibited proliferation and promoted apoptosis in TNBC. Via luciferase reporter assays, circGFRA1 and GFRA1 was observed to directly bind to miR-34a. Subsequent experiments showed that circGFRA1 and GFRA1 regulated the expression of each other by sponging miR-34a.ConclusionsTaken together, we conclude that circGFRA1 may function as a competing endogenous RNA (ceRNA) to regulate GFRA1 expression through sponging miR-34a to exert regulatory functions in TNBC. circGFRA1 may be a diagnostic biomarker and potential target for TNBC therapy.

MiR-150 Inhibits Cell Growth In Vitro and In Vivo by Restraining the RAB11A/WNT/β-Catenin Pathway in Thyroid Cancer.

BackgroundEmerging evidence has shown that downregulation or upregulation of microRNAs (miRNAs) plays an important role in the development and progression of thyroid cancer (TC). However, the potential role of miR-150 and its biological function in TC remains largely unclear.Material/MethodsReal-time polymerase chain reaction (RT-qPCR) was employed to detect the expression level of miR-150 and RAB11A in human TC tissue and human normal thyroid tissue. MTT assay, colony formation assay, flow cytometry cell cycle, and apoptosis assay were used to investigate the role of miR-150 and RAB11A on the malignant phenotypes in vitro. Nude mouse xenograft assay and western blot assay was used to verify the function of miR-150 in vivo. Western blot assay and immunofluorescence assay were used to detect the activation of WNT/β-catenin pathway mediated by miR-150 and RAB11A. EGFP reporter assay, RT-qPCR assay, and western blot assay were used to validate the regulation relationship.ResultsThis study demonstrated that miR-150 expression in human TC tissues was markedly downregulated. Moreover, overexpression of miR-150 markedly inhibited cell proliferation via inducing the cell cycle arrest and promoting cell apoptosis by directly targeting RAB11A in vitro and suppressing tumor growth in vivo. However, overexpression of RAB11A promoted cell malignant phenotypes. In addition, miR-150 restrained the RAB11A mediated WNT/β-catenin activation in TC cells.ConclusionsmiR-150 may function as a suppressor gene in TC cells by inhibiting the RAB11A/WNT/β-catenin pathway.

TGF‐β1 secreted by Tregs in lymph nodes promotes breast cancer malignancy via up‐regulation of IL‐17RB

Lymph node (LN) metastasis is commonly associated with systemic distant organ metastasis in human breast cancer and is an important prognostic predictor for survival of breast cancer patients. However, whether tumor‐draining LNs (TDLNs) play a significant role in modulating the malignancy of cancer cells for distant metastasis remains controversial. Using a syngeneic mouse mammary tumor model, we found that breast tumor cells derived from TDLN have higher malignancy and removal of TDLNs significantly reduced distant metastasis. Up‐regulation of oncogenic Il‐17rb in cancer cells derived from TDLNs contributes to their malignancy. TGF‐β1 secreted from regulatory T cells (Tregs) in the TDLNs mediated the up‐regulation of Il‐17rb through downstream Smad2/3/4 signaling. These phenotypes can be abolished by TGF‐β1 neutralization or depletion of Tregs. Consistently, clinical data showed that the up‐regulation of IL‐17RB in cancer cells from LN metastases correlated with the increased prevalence of Tregs as well as the aggressive growth of tumors in mouse xenograft assay. Together, these results indicate that Tregs in TDLNs play an important role in modulating the malignancy of breast cancer cells for distant metastasis. Blocking IL‐17RB expression could therefore be a potential approach to curb the process.