Tumor Formation In Nude Mice Research Articles

An in vitro and in vivo study of the role of long non-coding RNA-HOST2 in the proliferation, migration, and invasion of human glioma cells.

Gliomas are the most commonly occurring primary malignant brain tumors in the central nervous system of adults. They are rarely curable and the prognosis for high grade gliomas is generally poor. Recently, long non-coding RNA (lncRNA) human ovarian cancer-specific transcript 2 (HOST2) has been reported to be expressed at high levels in human ovarian cancer, involving tumorigenesis. However, little is still known about whether and how HOST2 regulates glioma development and progression. Therefore, this study aims to investigate the role of HOST2 in human glioma cells. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was used to determine the expression of lncRNA HOST2, let-7b, and PBX3 in human glioma cells. Cultured human glioma cells were treated with siRNA (si)-lncRNA HOST2, let-7b mimic, si-lncRNA HOST2 + let-7b inhibitor, and si-PBX3. Parameters including cell viability, colony formation, cell migration, and cell invasion were detected by cell counting kit-8 assay, colony formation assay, scratch test, and Transwell assay respectively to determine the effects of down-regulated HOST2 on glioma cells. Tumor formation in nude mice was evaluated by subcutaneous tumor formation experiment. Results showed that HOST2 and PBX3 were highly expressed in glioma tissue whereas let-7b was expressed at much lower levels. In response to treatment with si-lncRNA HOST2, si-PBX3, and let-7b mimic, glioma cell lines exhibited decreased cell viability, suppressed cell migration, invasion, and reduced colony formation of glioma cells. This was accompanied by an attenuated tumor formation with smaller volume and weight in nude mice, suggesting that down-regulated HOST2 could inhibit the tumorigenicity of glioma cells. Lastly, we found that lncRNA HOST2 was highly expressed in glioma tissues and its down-regulation could inhibit the growth and invasion of glioma cells. © 2018 IUBMB Life, 71(1):93-104, 2019.

Increased resistin suggests poor prognosis and promotes development of lung adenocarcinoma.

Resistin is considered to be a risk factor for several types of cancer, but its functions are controversial and not well studied in lung cancer. The present study is aimed to investigate the expression of resistin in lung adenocarcinoma tissues, in order to evaluate its association with the clinicopathological characteristics of cancer patients and to investigate the effects of resistin in lung adenocarcinoma cells. A total of 70 pairs of lung adenocarcinoma tissues and normal tissues were collected and immunohistochemistry was performed to examine resistin expression. Resistin overexpressed cells were established by plasmid transfection in A549 or H1975 cells. Alterations in cell proliferation, apoptosis, migration and invasion were analyzed in vitro. A nude mouse tumorigenicity assay was used to test the effect of resistin in vivo. High expression of resistin was predominantly observed in lung adenocarcinoma tissues but not in adjacent normal lung tissues. Resistin expression was significantly associated with increased tumor size, clinical stage as well as lymph node metastasis while negatively associated with progression-free survival (PFS) and overall survival (OS). Expression of resistin was an independent risk factor for PFS and OS. Overexpression of resistin promoted significant proliferation, migration and invasion, while also inhibited apoptosis in vitro. Resistin also promoted tumor formation in nude mice. The potential molecular mechanism was also investigated by in vitro experiments. In conclusion, the present study revealed that a high level of resistin expression in lung adenocarcinoma tissues is associated with poor clinicopathological status and survival. Resistin, which promotes the development of lung adenocarcinoma in vitro and in vivo may be a novel target for lung adenocarcinoma.

METTL3 promotes ovarian carcinoma growth and invasion through the regulation of AXL translation and epithelial to mesenchymal transition

ObjectiveAs the most prevalent internal modification in mammalian messenger RNA, N6‑methyladenosine (m6A) plays an important role in posttranscriptional gene regulation. METTL3 is a key component of the m6A methyltransferase complex and has recently been shown to play important roles in cancer development and progression. The current study was aimed to explore the function and underlying mechanism of METTL3 in ovarian cancer. MethodsMETTL3 expression was assessed by immunohistochemistry in 162 ovarian carcinoma patients. Stable cell lines with METTL3 gene overexpression or knockdown were established to investigate the function of METTL3 in ovarian cancer in vitro and in vivo. ResultsMETTL3 was frequently upregulated in ovarian carcinoma and that a high level of METTL3 was significantly associated with tumor grade (P = 0.001), pT status (P = 0.002), pN/pM status (P < 0.001), FIGO stage (P < 0.001), and overall survival rate (P < 0.001). Stable overexpression of METTL3 in the OVCAR3 and COV504 cell lines significantly increased cellular proliferation, focus formation, motility, invasion, and tumor formation in nude mice. Silencing METTL3 expression in the SKOV3 and HO-8910 cell lines with short hairpin RNA effectively inhibited its oncogenic function. Further study found that METTL3 promoted epithelial-mesenchymal transition (EMT) by upregulating the receptor tyrosine kinase AXL. ConclusionOur findings suggest that METTL3 plays very important oncogenic roles in ovarian carcinoma development and/or aggressiveness by stimulating AXL translation and EMT and that METTL3 may serve as a novel prognostic and/or therapeutic target of interest in ovarian cancer.

Integrative analysis of gene expression profile and DNA methylation profile of long-term cultivated porcine bone marrow mesenchymal stem cells

To integrate the result of whole genome expression data and whole genome promoter CpG island methylation data, to screen the epigenetic modulated differentially expressed genes from transformed porcine bone marrow mesenchymal stem cells (BMSCs) after long-term cultivation. Bone marrow from 6 landrace pigs, 3-month-old about 50 kg weight, was aspirated from the medullary cavity of the proximal tibia. The BMSCs were isolated, and purified by Ficoll density gradient centrifugation combined with adherent culture method. The transfor mation of BMSCs was tested by several methods including cell morphology observation, karyotype analysis, clone forming in soft agarose, serum requirement assay, and tumor forming in mice. The Agilent Pig 4x44k Gene Expression Microarray was used to investigate the differentially expressed mRNA. The methylated genes expression profile was performed using customized pig methylation chip. The gene expression and DNA methylation profiles were integrated to find out the epigenetic modulated differentially expressed genes, and to complete the bioinformatic analysis. BMSCs showed a change in appearance, from the initial spindle shape to a more flatted morphology then to small contact shape. After additional passages, BMSCs gradually acquired recovery of proliferating capacity and transformation properties such as anchorage-independent growth, chromosomal abnormality, and tumor formation in nude mice. The gene chip analysis demonstrated that 257 genes were upregulated and 315 genes were downregulated during long-term cultures as well as multiple signal pathways transduction involved, such as cell cycle, ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton, pathways in cancer, and P53. The analysis from methylation chip of coding genes suggested epigenetic regulation was involved in BMSCs spontaneous transformation and play a important role on it; 962 genes were hypermethylated and 1219 genes were hypomethylated, which were involved in the biological process of cellular metabolic, structure, and tumor generation. The combined analysis of genes regulated by methylation in the transformation process of BMSCs found that the methylation changes of the 35 genes were contrary to the direction of expression change (correlation coefficient r=-0.686, P=0.000); in which the methylation level of 21 genes promoter regions were increased while the gene expression decreased, and the methylation level of the 14 genes promoter regions decreased and the gene expression increased. At the same time, KEGG enrichment analysis revealed multiple genes regulated by methylation, involved in stem cell differentiation and multiple cell signaling pathways. Among the 14 down-regulated genes, many of them have the role of regulating the interaction of tumor and immunization, and the change of the methylation status of the CDKN3 promoter region may be closely related to the cell oncology. The results deepen our understanding of the crucial role of coding genes methylation modification in BMSCs transformation, and may provide new approach to establish safe criteria for BMSCs clinical applications and transformation prevention.

PDSS2 Deficiency Induces Hepatocarcinogenesis by Decreasing Mitochondrial Respiration and Reprogramming Glucose Metabolism.

Glucose metabolic reprogramming from oxidative phosphorylation to glycolysis is one of the hallmarks of cancer development. Coenzyme Q10 (CoQ10) is essential for electron transport in the mitochondrial respiratory chain and for antioxidant defense. Here, we investigated the role of a key factor in CoQ10 synthesis, prenyldiphosphate synthase subunit 2 (PDSS2), in hepatocellular carcinoma (HCC) tumorigenesis. PDSS2 was frequently downregulated in HCC tissues and was significantly associated with poorer HCC prognosis (P = 0.027). PDSS2 downregulation was a prognostic factor independent of T status and stage (P = 0.028). Downregulation of CoQ10 was significantly correlated with downregulation of PDSS2 in HCC tumor tissues (R = 0.414; P < 0.001). Of the six different splicing isoforms of PDSS2, the five variants other than full-length PDSS2 showed loss of function in HCC. Reintroduction of full-length PDSS2 into HCC cells increased CoQ10 and mitochondrial electron transport complex I activity and subsequently induced a metabolic shift from aerobic glycolysis to mitochondrial respiration in cells. Reintroduction of PDSS2 also inhibited foci formation, colony formation in soft agar, and tumor formation in nude mice. Knockdown of PDSS2 induced chromosomal instability in the MIHA immortalized human liver cell line. Furthermore, knockdown of PDSS2 in MIHA induced malignant transformation. Overall, our findings indicate that PDSS2 deficiency might be a novel driving factor in HCC development.Significance: Downregulation of PDSS2 is a driving factor in hepatocellular carcinoma tumorigenesis. Cancer Res; 78(16); 4471-81. ©2018 AACR.

RETRACTED: LINC01116 targets miR-520a-3p and affects IL6R to promote the proliferation and migration of osteosarcoma cells through the Jak-stat signaling pathway

ObjectiveThe purpose of this study was to find out the important lncRNA-miRNA-mRNA axis and pathway in osteosarcoma (OS) through bioinformatics analysis and verify the biological functions of lncRNA/miRNA/mRNA in OS through in vitro and in vivo assays. MethodsThe differential expression mRNAs and lncRNAs were identified through microarray analysis, and the altered pathways were identified by GSEA. The Pearson Coefficient was used to analyze the correlations between mRNAs and lncRNAs. Kaplan-Meier survival analysis was preformed using patient information in GEO database. Their target miRNAs were predicted by Targetscan and miRanda database and confirmed by dual luciferase reporter assay. QRT-PCR were utilized to detected the relative expressions of mRNAs, miRNAs and lncRNAs. The expressions of IL6R protein and pathway related proteins were detected by western blot. OS cell viability, migration and apoptosis were determined through MTT assay, Transwell assay and flow cytometry. Tumor formation in nude mice verified the influence of LINC01116 in vivo. ResultsThe Jak-stat signaling pathway was activated in OS tissues. LINC01116 expression was positively correlated with IL6R expression. MiR-520a-3p targeted the 3’-UTR of LINC01116 and IL6R. Lower expression levels of miR-520a-3p significantly correlated with shorter survival of patients. LINC01116 and IL6R were up-regulated while miR-520a-3p was down-regulated in OS. LINC01116 and IL6R promoted the viability and migration of OS cells, while miR-520a-3p acted as a tumor suppressor. MiR-520a-3p inhibitor could rescue the suppressive effects of si-LINC011116 and si-IL6R on OS development. The Jak-stat signaling pathway related proteins were also down-regulated by miR-520a-3p. Down-regulation of LINC01116 inhibited the tumor growth in nude mice. ConclusionLINC01116 up-regulated IL6R in OS through targeting miR-520a-3p, thus activating the Jak-stat signaling pathway and promoting the progression of OS.

Abstract 4472: Downregulation of POTEG predicts poor prognosis in esophageal squamous cell carcinoma

Abstract POTE ankyrin domain family, member G (POTEG) belongs to POTE family. The POTE gene family encodes very closely related proteins that are highly expressed in prostate, ovary, testis, and placenta. Recent studies indicated that the POTE proteins have a pro-apoptotic function. To examine whether POTEG also underwent apoptosis in ESCC cancer cells and the functions of POTEG in the development of esophageal squamous cell carcinoma (ESCC), we determined the location and expression of POTEG in ESCC cancer cells. Clinical association studies determined that POTEG downregulation was associated with poor clinical outcomes. Ectopic re-expression of POTEG effectively suppressed the tumorigenesis and metastasis of ESCC cells in vitro and in vivo, including the inhibition of cell growth rate, foci formation, soft agar colony formation, migration, invasion and tumor formation in nude mice. Molecular analyses revealed that POTEG downregulated CDKs, leading to subsequent inhibition of Rb phosphorylation, and consequently arrested Cell Cycle at G1/S Checkpoint. Prospectively, POTEG can reduce the level of anti-apoptotic proteins, while promote the level of proapoptotic proteins and inducing apoptosis. On the other side, POTEG inhibit metastasis by reducing epithelial-mesenchymal transition in ESCCs. Taken together, our results revealed that POTEG has a pivotal function in ESCC pathogenesis, with possible use as a biomarker and intervention point for new therapeutic strategies. Citation Format: Yan Li. Downregulation of POTEG predicts poor prognosis in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4472.

Upregulation of the Kank1 gene inhibits human lung cancer progression invitro and invivo.

Kank1, an important member of the Kank gene family, plays an important role in the development of many malignant tumors. As a brand new tumor-suppressor gene, the expression of Kank1 gene has been revealed to be downregulated or absent in many malignant tumors. However, there is no study concerning the specific role of Kank1 in the development and progression of lung cancer. In the present study, we found that the Kankl gene was significantly downregulated in human lung cancer and lung cancer cells. When the Kankl gene was upregulated, we found that the proliferation of lung cancer cells was significantly inhibited, the cells were arrested in the G0/G1phase, the apoptosis of cells was observed, the expression of the caspase family of genes was altered with mainly the activation of caspase-3and-9, and Bcl-2/Bax ratio was significantly imbalanced. In addition, we also found that upregulation of the Kankl gene resulted in the inhibition of tumor cell invasion and metastasis. Concurrently, invivo nude mice experiments also confirmed that upregulation of Kank1 expression led to reduced tumor formation in nude mice and significantly reduced tumor volume. These results revealed that upregulation of the Kankl gene inhibited the progression of lung cancer both invitro and invivo, and its mechanism was closely related to cell apoptosis as well as tumor invasion and metastasis.

Effect of A20 gene induced silencing on the biological behaviors of human nasopharyngeal carcinoma cell

Objective: To observe the effect of knockdown A20 gene expression on the proliferation, invasion and metastasis of human nasopharyngeal carcinoma cell in vivo and in vivo. Methods: Human nasopharyngeal carcinoma cell 5-8F-H3 was transfected with A20-specific shRNA Tet-on inducible plasmid vectors, and A20 silenced cells were screened by Puromycin. Quantitative RT-PCR and Western blot analysis were used to detect the mRNA level and protein of A20. The cell proliferation was detected by cell counting kit-8 (CCK8) and plate colony formation assays. The cell cycle and apoptosis were measured by flow cytometry. And the ability of cell invasion was measured using Boyden chamber assay in vivo. Subcutaneous tumor formation and liver metastasis in vivo were examined with whole-body fluorescence imaging system to observe the influence of silencing A20 gene expression in nude mice. Results: The stable A20 inducible silencing cells line 5-8F-H3/A20-shRNA was established successfully. Down-regulation of A20 mRNA and protein expression were observed in 5-8F-H3/A20-shRNA cells treated with DOX(both P<0.01). The results of CCK-8 assay (F=18.542, P=0.003), clone formation experiment (F=40.080, P<0.001) and flow cytometry analysis (F=7.398, P=0.024) in vivo showed that the cell proliferation of 5-8F-H3 was remarkably inhibited by down-regulation of A20 gene expression. The results of Boyden chamber assay showed that A20 gene silencing could inhibit the cell invasion ability (F=26.157, P<0.001). Silencing of A20 inhibited tumorigenesis and metastasis via subcutaneous tumor formation and liver metastasis experiments in nude mice. Conclusion: A20 gene is closely related to the malignant biological behaviors of nasopharyngeal carcinoma, and it may serve as a potential molecular target for the treatment of nasopharyngeal carcinoma.

MicroRNA-183 induces epithelial-mesenchymal transition and promotes endometrial cancer cell migration and invasion in by targeting CPEB1.

The aim of this study is to evaluate the ability of microRNA-183 (miR-183) to influence epithelial-mesenchymal transition (EMT) and cell proliferation, migration, invasion, and apoptosis in endometrial cancer (EC) by targeting cytoplasmic polyadenylation element binding protein 1(CPEB1). EC tissues with matched nonmalignant tissues were collected from 208 EC patients. Ishikawa and RL95-2 cells were selected for cell experiments in vitro and each kind of cells were grouped into blank, negative control (NC), miR-183 mimic, miR-183 inhibitor, CPEB1 overexpression, and miR-183 mimic + CPEB1 overexpression groups. Expressions of miR-183, CPEB1, E-cadherin, and Vimentin were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Cell viability, colony formation ability, migration, invasion, and apoptosis were assessed by MTT assay, clone formation assay, scratch test, Transwell assay, and flow cytometry. In vivo tumorigenesis of Ishikawa cells was evaluated by tumor formation in nude mice. The miR-183 expression was higher, but the CPEB1 expression was lower in EC tissues than in adjacent nonmalignant tissues. CPEB1 was confirmed as the target of miR-183 by dual-luciferase reporter assay. The miR-183 mimic group had increased cell viability, colony formation ability, cell invasion and migration, tumor volume and weight in nude mice, but decreased cell apoptosis when compared with the blank group. The expression of E-cadherin was down-regulate, but expression of Vimentin was up-regulate in the miR-183 mimic group in comparison with the blank group. In terms of a comparison between the blank group and CPEB1 overexpression group, the CPEB1 overexpression group had suppressed cell viability, colony formation ability, cell invasion and migration, tumor volume and weight, but increased cell apoptosis. The expression of E-cadherin was up-regulated, but the expression of Vimentin was down-regulated in the CPEB1 overexpression group in comparison with the blank group. The miR-183 mimic + CPEB1 overexpression group had higher miR-183 expression than the blank group. These findings indicate that miR-183 induces EMT, inhibits apoptosis, and promotes cell proliferation, migration, invasion, and in vivo tumorigenesis in EC by targeting CPEB1.

Hsa_circ_0046264 up-regulated BRCA2 to suppress lung cancer through targeting hsa-miR-1245

ObjectiveLung cancer had been leading mounts of deaths worldwide. Advances in genes microarray had helped human further understand genes and identify novel circular RNAs. This study aimed at investigating the biological functions and molecular mechanisms of hsa_circ_0046264 in lung cancer which may be helpful in lung cancer early diagnosis and clinical treatment.MethodsGene microarray data screened the differential gene of hsa_circ_0046264 and its downstream genes were found by bioinformatics analysis and verified by luciferase reporter assay. QRT-PCR and Western blot was used to detect the RNA and protein levels respectively. RNase R digestion confirmed the existences of circular RNA. Cell viability, invasion and apoptosis were determined by MTT assay, flow cytometry and DNA damage assay. Tumor formation in nude mice and immunohistochemistry proved the functions of hsa_circ_0046264 in vivo.ResultsHsa_circ_0046264 and BRCA2 were down-regulated in lung cancer tissues while miR-1245 was up-regulated. Hsa_circ_0046264 induced apoptosis but inhibited proliferation and invasion of lung cancer cells through targeting miR-1245 to up-regulate BRCA2. Hsa_circ_0046264 inhibited the tumor growth in vivo.ConclusionHsa_circ_0046264 was a tumor suppressor in lung cancer. Overexpression of hsa_circ_0046264 could up-regulate BRCA2 expression through down-regulating of miR-1245.

Gemcitabine and cisplatin for treatment of lung cancer in vitro and vivo.

To evaluate the antitumor activity of gemcitabine (GEM), cisplatin (DDP) as well as the combination of these two agents in lung cancer cells and mice. The cell viability was evaluated by the CCK-8 assay. Cell apoptosis was measured by flow cytometry assay and Hoechst staining. The protein expression of VEGF, VEGFR2, Ang II, AT1R, and ACE2 was examined by Western blotting. The effect of GEM and DDP on tumor growth and survival time was also measured in lung cancer mice in vivo. The results revealed that alone or combined administration of GEM and DDP could inhibit the growth, induce apoptosis and apoptotic body formation of A549 cells compared with control cells, with the most significance detected in a combination of GEM and DDP administration. It is indicated that combined administration of GEM and DDP could delay the progress of tumor formation in nude mice. The cell apoptosis- and angiogenesis-related proteins expressions were decreased both in A549 cells and lung cancer mice. GEM plus DDP can be an option for patients with lung cancer treatment. However, further prospective evaluation and randomized trials are to provide more accurate information through clinical trials.

Total Saponins of Rubus Parvifolius L. Exhibited Anti-Leukemia Effect in vivo through STAT3 and eIF4E Signaling Pathways.

To investigate the anti-leukemia effect of total saponins of Rubus parvifolius L. (TSRP) on K562 cell xenografts in nude mice and the mechanisms of action. The K562 cell xenografts in nude mice were established, and then randomly divided into 5 groups, the control group, the cytosine arabinoside group(Ara-c) and 3 TSRP groups (20, 40 and 100 mg/kg). The tumor volume and mass of each group of nude mice were measured and the anti-tumor rates of TSRP were calculated subsequently. The apoptosis status of tumor cells was detected by hematoxylin-eosin (HE) and terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining analysis. Finally, the activities of apoptosis related signaling of signal transducer and activator of transcription 3 (STAT3), eukaryotic initiation factor 4E (eIF4E) and B-cell lymphoma-2 (bcl-2) were determined with immunohistochemistry tests. Subcutaneous injection of K562 cells induced tumor formation in nude mice, and the TSRP treated group showed a signifificant inhibitory effect on tumor formation. The nude mice treated with TSRP showed a signifificant decrease in tumor growth rate and tumor weight in comparison to the control group (all P<0.05). The HE staining and TUNEL assay showed that TSRP induced cell death by apoptosis. The immunohistochemical assay showed down-regulation of the bcl-2 gene in the TSRP treated cells. The phosphorylation levels of eIF4E and STAT3 were decreased obviously after the treatment of TSRP. TSRP had an excellent tumor-suppressing effect on K562 cells in the nude mice xenograft model, suggesting that TSPR can be developed as a promising anti-chronic myeloide leukemia drug.

Abstract B24: Role of Dermcidin in growth and survival of melanoma tumor cells

Abstract Dermcidin (DCD) is a candidate oncogene localized at 12q13.1 which is normally expressed in the skin sweat gland and brain and overexpressed in various tumor cells. We have established the role of DCD in melanoma growth and survival by silencing DCD via shRNA expression in human melanoma G361 cell line. Biologic and biochemical assays showed that DCD gene knockdown decreased significantly melanoma cell growth in culture and tumor formation in NUDE mice. A DNA microarray-based study identified a total of 162 upregulated and 212 downregulated genes, in a G-361 control clone (pLKO) and clones expressing shRNA to DCD mRNA (IBC-I). We found, notably, genes involved in the nucleosome organization and signaling pathway to APRIL/BAFF receptor. Additionally, we examined DCD expression by immunohistochemistry in TMA (tissue microarray) of 101 patients and found that DCD-negative tumor had a 65.54% survival rate and DCD-positive tumor 43.33% survival rate in 5 years. We found an association with BRAF V600E and ERBB4 mutation in DCD-positive tumor with most frequent E43K mutation. Finally, we have screened clones resistant to Vemurafenib after long-term treatment in vitro and in xenografts in nude mice. Overall, these studies provide further support for DCD as oncogene to melanomas. Financial support: FAPESP, CNPq and CAPES. Citation Format: Jennifer N. Montoya, Marcela N. Perez-Sosa, Beatriz A. Sangiuliano, Jose E. Belizario. Role of Dermcidin in growth and survival of melanoma tumor cells [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B24.

Chronic Microcystin-LR Exposure Induces Hepatocarcinogenesis via Increased Gankyrin in Vitro and in Vivo

Background/Aims: Our recent study indicated that the serum microcystin-LR (MC-LR) level is positively linked to the risk of human hepatocellular carcinoma (HCC). Gankyrin is over-expressed in cancers and mediates oncogenesis; however, whether MC-LR induces tumor formation and the role of gankyrin in this process is unclear. Methods: We induced malignant transformation of L02 liver cells via 35 passages with exposure to 1, 10, or 100 nM MC-LR. Wound healing, plate and soft agar colony counts, and nude mice tumor formation were used to evaluate the tumorigenic phenotype of MC-LR-treated cells. Silencing gankyrin was used to confirm its function. We established a 35-week MC-LR exposure rat model by twice weekly intraperitoneal injection with 10 μg/kg body weight. In addition, 96 HCC patients were tested for tumor tissue gankyrin expression and serum MC-LR levels. Results: Chronic low-dose MC-LR exposure increased proliferation, mobility, clone and tumor formation abilities of L02 cells as a result of gankyrin activation, while silencing gankyrin inhibited the carcinogenic phenotype of MC-LR-treated cells. MC-LR also induced neoplastic liver lesions in Sprague-Dawley rats due to up-regulated gankyrin. Furthermore, a trend of increased gankyrin was observed in humans exposed to MC-LR. Conclusion: These results suggest that MC-LR induces hepatocarcinogenesis in vitro and in vivo by increasing gankyrin levels, providing new insight into MC-LR carcinogenicity studies.

Nicotine-enhanced stemness and epithelial-mesenchymal transition of human umbilical cord mesenchymal stem cells promote tumor formation and growth in nude mice.

Cigarette smoking is a well-known risk factor in the development and progression of malignant diseases. Nicotine, the major constituent in cigarette smoke, has also shown negative effects on stem cells. Mesenchymal stem cells (MSCs) have been widely demonstrated to migrate into tumors and play key roles in cancer progression. However, the mechanisms by which nicotine impacts MSCs and tumorigenesis of lung cancer are still undetermined. In this study we investigated the effects of nicotine on human umbilical cord mesenchymal stem cells (hUC-MSCs) and the impacts of nicotine-treated hUC-MSCs on tumor formation and progression. We found that nicotine has a toxic effect on hUC-MSCs and changes the morphology, inhibits proliferation and promotes apoptosis of hUC-MSCs in a dose-dependent manner. Nicotine-treated hUC-MSCs produce higher level of IL-6. Moreover, nicotine promotes migration, stemness and epithelial-mesenchymal transition (EMT) of hUC-MSCs by inhibiting E-cadherin expression and upregulating mesenchymal markers such as N-cadherin and Vimentin, leading to the induction of stem cell markers Sox2, Nanog, Sall4, Oct4 and CD44. Migration and proliferation of non-small cell lung cancer A549 cells and breast cancer MCF-7 cells are promoted after their coculture with nicotine-treated hUC-MSCs in a cell-cell contact-independent manner. Furthermore, nicotine-treated hUC-MSCs promote tumor formation and growth of A549 cells in nude mice. These studies demonstrated that the enhanced stemness and EMT of hUC-MSCs induced by nicotine are critical for the development of tobacco-related cancers.

Overexpression of MUC13, a Poor Prognostic Predictor, Promotes Cell Growth by Activating Wnt Signaling in Hepatocellular Carcinoma

Recently RNA sequencing revealed high mucin 13 (MUC13) expression in hepatocellular carcinoma (HCC) tissues. To understand the clinicopathologic significance of MUC13 in HCC, quantitative PCR and immunohistochemistry were used to detect its expression in paired tumor tissues and nontumor tissues. The oncoprotein role of MUC13 was determined by invitro and invivo assays. Overexpression of MUC13 was detected in 74 of 168 primary HCC cases (44%) and was significantly associated with tumor size (P=0.027), stage (P=0.006), encapsulation (P=0.044), venous invasion (P=0.024), and poor outcome (P=0.004). Functional studies demonstrated MUC13 had strong oncogenic activity by promoting cell growth, colony formation, cell migration, and tumor formation in nude mice. The pro-oncogenic effect of MUC13 were effectively inhibited by RNA interference. MUC13 promoted cellular G1/S phase transition by activating Wnt signaling. Mechanistically, MUC13 bound to β-catenin and increased its phosphorylation at Ser552 and Ser675 sites, which subsequently promoted nuclear translocation of β-catenin and up-regulation of its downstream target genes Axin2, c-Myc, and CyclinD1. Knockdown of AKT with shRNA in MUC13-overexpressing cells nullified the elevated phosphorylation of β-catenin by MUC13. In clinical HCC samples, nuclear translocation of β-catenin was significantly associated with MUC13 overexpression (P=0.001). Overexpression of MUC13 plays a critical role in the development and progression of HCC by activating Wnt signaling.

Calcium-binding protein 39 promotes hepatocellular carcinoma growth and metastasis by activating extracellular signal-regulated kinase signaling pathway.

Taken together, our findings suggested that CAB39 played very important oncogenic roles in HCC pathogenesis and progression by activating the ERK signaling pathway. Better understanding of CAB39 may lead to its clinical application as a biomarker for a prognosis predictor and a novel therapeutic target. (Hepatology 2017;66:1529-1545).

MiR-26b regulates invasion and migration of lung cancer cells through targeting hENT1 depending on RhoA/ROCK-1 pathway

To investigate the effect of miR-26b on the invasion and migration of lung cancer cell and to explore its mechanism. Methods: qPCR was used to detect the expression of miR-26b in lung cancer. Luciferase reporter gene was used to detect interaction between miR-26b and hENT1. Transwell assay was used to detect invasion ability after treatment of miR-26b mimics. Scratch assay was used to detect migration ability after treatment of miR-26b mimics. The expressions of hENT1, ROCK-1 and RhoA were detected by Western blot. The changes of cytoskeleton after miR-26b mimics treatment with phalloidin were observed. The effect of miR-26b mimics on the tumor size and volume of lung cancer was determined by subcutaneous tumor formation in nude mice. Results: MiR-26b expression was significantly reduced in lung cancer. With the progress of lung cancer, the expression of miR-26b was reduced. With the progress in differentiation of lung cancer, the expression of miR-26b was decreased. Decrease of miR-26b was associated with lung cancer lymph node metastasis. HENT1 was the direct target of miR-26b; miR-26b regulated the invasion and migration ability of human lung carcinoma A549 cells. MiR-26b regulated the expression of hENT1, ROCK-1 and RhoA. After the treatment with miR-26b mimics, the F-actin staining was significantly reduced, whereas the formation of wrinkles and the formation of pseudopodia were significantly reduced. Subcutaneous tumor formation in nude mice showed that miR-26b mimics treatment significantly reduced the tumor size and mass. Conclusion: MiR-26b plays a role in tumor suppression in lung cancer. miR-26b can regulate the invasion and migration ability of lung carcinoma A549 cells by targeting hENT1 depending on the RhoA/ROCK-1 pathway.

Overexpression of SIRT6 attenuates the tumorigenicity of hepatocellular carcinoma cells.

ObjectiveThis study aimed to explore the effects of overexpression of sirtuin 6 (SIRT6) on the tumorigenicity of hepatocellular carcinoma (HCC) cellsMethodsStable SIRT6-overexpressed HCC cell lines were established by transfecting SIRT6 plasmid. Soft agar assay and tumor xenograft assay in nude mice were applied. Flow cytometry was employed to detect cell cycle distribution. Western blotting analysis was used to detect the expression of proteins.ResultsOverexpression of SIRT6 attenuated HepG2 and HCCLM3 cells proliferation, colony formation in vitro and tumor formation in nude mice, and resulted in the G1 phase cell cycle arrest. Overexpression of SIRT6 reduced the expression of cyclin D1 and p-ERK proteins in both HepG2 and HCCLM3 cells.ConclusionOverexpression of SIRT6 attenuates the tumorigenicity of HCC cells.