Xenograft Tumor Formation Assay Research Articles

Establishment and characterization of a novel cell line (SCCOHT-CH-1) and PDX models derived from Chinese patients of small cell ovarian carcinoma of the hypercalcemic type

Small cell carcinoma of the ovary hypercalcemic type (SCCOHT) is a rare and aggressive malignancy that poses a significant clinical challenge due to its grim prognosis. Unfortunately, only three SCCOHT cell lines are currently available for scientific research. In this study, we have successfully established a novel SCCOHT cell line from a recurrent lesion of a SCCOHT patient, named SCCOHT-CH-1. We comprehensively characterized the novel cell line by employing techniques such as morphological observation, CCK-8 assay, Transwell assay, clone formation assay, short tandem repeat sequence (STR) analysis, karyotype analysis, immunohistochemical staining, western blot assay, and xenograft tumor formation assay. SCCOHT-CH-1 cells were small circular and had a unique STR profile. The population-doubling time of SCCOHT-CH-1 was 33.02 h. The cell line showed potential migratory and invasive ability. Compared with another SCCOHT cell line COV434, SCCOHT-CH-1 exhibited higher expression of AKT, VIM, and CCND1. At the same time, SCCOHT-CH-1 has the ability of tumorigenesis in vivo. We also successfully constructed three patient-derived xenograft (PDX) models of SCCOHT, which were pathologically diagnosed to be consistent with the primary tumor, accompanied by loss of SAMRCA4 protein expression. The establishment of SCCOHT-CH-1 cell line and PDX models from Chinese people represent a pivotal step toward unraveling the molecular mechanism of SCCOHT and fostering the development of targeted interventions to tackle this challenging malignancy.

Circular RNA circLRCH3 Inhibits Proliferation, Migration, and Invasion of Colorectal Cancer Cells Through miRNA-223/LPP Axis.

PurposeColorectal cancer (CRC) is one of the most common carcinomas worldwide with a high mortality rate. Numerous studies suggest that circular RNA (circRNA) plays a crucial role in the progression of various carcinomas, including CRC. The present work focused on exploring the role and underlying molecular mechanism of action of the circRNA circLRCH3 in CRC.MethodsReal-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was conducted to detect expression levels of circLRCH3, miR-233, and lipoma preferred partner (LPP). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure the proliferation of CRC cells and the transwell assay was used to evaluate cell migration and invasion capacity. A flow cytometry assay was used to analyze the effect of circLRCH3 on the distribution of the cell cycle and apoptosis of CRC cells. The expression of LPP was analyzed using Western blotting or an RT-qPCR assay. The relationship between miR-223 and circLRCH3, and that between miR-223 and LPP, was predicted and examined using bioinformatics analysis and luciferase reporter gene experiments. A xenograft tumor formation assay was also performed.ResultsWe found that the expression level of circLRCH3 was downregulated in CRC cells and negatively correlated with miR-223. The overexpression of circLRCH3 or silencing of miR-223 inhibited the growth, invasion, and migration of CRC cells, but promoted their apoptosis. In contrast, overexpression of miR-223 and depletion of LPP severally abrogated the tumor suppressive roles of circLRCH3 and miR-223 knockdown in CRC cells in vitro. The xenograft experiments in nude mice also proved the antitumor effect of circLRCH3.ConclusionThese results suggested that the circLRCH3/miR-223/LPP axis likely plays a critical role in CRC.

Circular RNA circ_UBAP2 facilitates the progression of osteosarcoma by regulating microRNA miR-637/high-mobility group box (HMGB) 2 axis

ABSTRACT Circular RNA circ_UBAP2 has been reported to be closely associated with various tumors. The present work focused on exploring the roles of circ_UBAP2 and its molecular mechanism in osteosarcoma (OS). Circ_UBAP2, miR-637, and high-mobility group box (HMGB) 2 levels in OS cells and tissues were detected by quantitative real-time polymerase chain reaction. The relationship between miR-637 and circ_UBAP2, as well as between miR-637 and HMGB2, was predicted and examined through bioinformatics analysis and luciferase reporter gene experiments. Moreover, OS cell growth, invasion, migration, and apoptosis were detected using the cell counting kit-8 (CCK-8), Transwell and flow cytometry assays, respectively. HMGB2 protein levels were measured using Western blotting. Xenograft tumor formation assay was also performed. Circ_UBAP2 showed high expression levels in OS tissues and cells, which was directly proportional to metastasis and clinical stage of OS. The overexpression of circ_UBAP2 enhanced the growth, invasion, and migration of OS cells, but suppressed their apoptosis. In contrast, circ_UBAP2 silencing had opposite effects. Furthermore, miR-637 served as a downstream target of circ_UBAP2, which played opposite roles to circ_UBAP2 in OS. More importantly, HMGB2 served as miR-637ʹs downstream target. The xenograft experiments in nude mice also proved that knockdown of circ_UBAP2 could increase miR-637 expression, but decrease HMGB2 expression, thus alleviating OS progression. Mechanistically, circ_UBAP2 exerts a cancer-promoting effect on OS by downregulating miR-637 and upregulating the expression of HMGB2. Circ_UBAP2 plays a promoting role in OS, and the circ_UBAP2/miR-637/HMGB2 axis is involved in OS progression.

LncRNA PVT1 promotes gemcitabine resistance of pancreatic cancer via activating Wnt/\u03b2-catenin and autophagy pathway through modulating the miR-619-5p/Pygo2 and miR-619-5p/ATG14 axes

BackgroundPancreatic cancer is one of the most lethal malignancies and has an extremely poor diagnosis and prognosis. The development of resistance to gemcitabine is still a major challenge. The long noncoding RNA PVT1 was reported to be involved in carcinogenesis and chemoresistance; however, the mechanism by which PVT1 regulates the sensitivity of pancreatic cancer to gemcitabine remains poorly understood.MethodsThe viability of pancreatic cancer cells was assessed by MTT assay in vitro and xenograft tumor formation assay in vivo. The expression levels of PVT1 and miR-619-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Western blotting analysis and qRT-PCR were performed to assess the protein and mRNA levels of Pygo2 and ATG14, respectively. Autophagy was explored via autophagic flux detection under confocal microscopy and autophagic vacuole investigation under transmission electron microscopy (TEM). The functional role and mechanism of PVT1 were further investigated by gain- and loss-of-function assays in vitro.ResultsIn the present study, we demonstrated that PVT1 was up-regulated in gemcitabine-resistant pancreatic cancer cell lines. Gain- and loss-of-function assays revealed that PVT1 impaired sensitivity to gemcitabine in vitro and in vivo. We further found that PVT1 up-regulated the expression of both Pygo2 and ATG14 and thus regulated Wnt/β-catenin signaling and autophagic activity to overcome gemcitabine resistance through sponging miR-619-5p. Moreover, we discovered three TCF/LEF binding elements (TBEs) in the promoter region of PVT1, and activation of Wnt/β-catenin signaling mediated by the up-regulation of Pygo2 increased PVT1 expression by direct binding to the TBE region. Furthermore, PVT1 was discovered to interact with ATG14, thus promoting assembly of the autophagy specific complex I (PtdIns3K-C1) and ATG14-dependent class III PtdIns3K activity.ConclusionsThese data indicate that PVT1 plays a critical role in the sensitivity of pancreatic cancer to gemcitabine and highlight its potential as a valuable target for pancreatic cancer therapy.

PDLIM2 acts as a cancer suppressor gene in non-small cell lung cancer via the down regulation of NF-κB signaling

PDZ and LIM domain containing protein 2 (PDLIM2) has been identified as a vital tumor-associated gene that is aberrantly expressed in various types of tumors. Yet, the involvement of PDLIM2 in non-small cell lung cancer (NSCLC) is currently undetermined. The design of the current study was to evaluate whether PDLIM2 plays a role in NSCLC. We found that PDLIM2 expression was commonly decreased in NSCLC tissues. Moreover, low expression of PDLIM2 was also detected in NSCLC cell lines and demethylation treatment restored PDLIM2 expression. The re-expression of PDLIM2 impeded the proliferative, colony-forming, and invasive capabilities of NCLCL cells. In contrast, depletion of PDLIM2 markedly enhanced the malignant behaviors of NSCLC cells. Notably, PDLIM2 overexpression downregulated the expression of nuclear factor (NF)-κB p65 subunit and repressed NF-κB transcription reporter activity in NSCLC cells. The overexpression of p65 significantly reversed PDLIM2-mediated antitumor effects in NSCLC cells. Additionally, the Xenograft tumor formation assay revealed that the overexpression of PDLIM2 markedly restricted the tumor growth of NSCLC in vivo. Overall, our study confirms that PDLIM2 acts as a tumor-inhibitor in NSCLC through the inactivation of NF-κB, suggesting PDLIM2 as a candidate therapeutic target for NSCLC.

Knock-down of the TIM/TIPIN complex promotes apoptosis in melanoma cells.

The Timeless (TIM) and it's interacting partner TIPIN protein complex is well known for its role in replication checkpoints and normal DNA replication processes. Recent studies revealed the involvement of TIM and TIPIN in human malignancies; however, no evidence is available regarding the expression of the TIM/TIPIN protein complex or its potential role in melanoma. Therefore, we investigated the role of this complex in melanoma.To assess the role of the TIM/TIPIN complex in melanoma, we analyzed TIM/TIPIN expression data from the publicly accessible TCGA online database, Western blot analysis, and RT-qPCR in a panel of melanoma cell lines. Lentivirus-mediated TIM/TIPIN knockdown in A375 melanoma cells was used to examine proliferation, colony formation, and apoptosis. A xenograft tumor formation assay was also performed.The TIM/TIPIN complex is frequently overexpressed in melanoma cells compared to normal melanocytes. We also discovered that the overexpression of TIM and TIPIN was significantly associated with poorer prognosis of melanoma patients. Furthermore, we observed that shRNA-mediated knockdown of TIM and TIPIN reduced cell viability and proliferation due to the induction of apoptosis and increased levels of γH2AX, a marker of DNA damage. In a xenograft tumor nude mouse model, shRNA-knockdown of TIM/TIPIN significantly reduced tumor growth.Our results suggest that the TIM/TIPIN complex plays an important role in tumorigenesis of melanoma, which might reveal novel approaches for the development of new melanoma therapies. Our studies also provide a beginning structural basis for understanding the assembly of the TIM/TIPIN complex. Further mechanistic investigations are needed to determine the complex’s potential as a biomarker of melanoma susceptibility. Targeting TIM/TIPIN might be a potential therapeutic strategy against melanoma.

MiR-320/ELF3 axis inhibits the progression of breast cancer via the PI3K/AKT pathway.

There is increasing evidence demonstrating that disorders affecting microRNAs (miRs) influence tumorigenesis and progression, which results in a poor prognosis in patients with breast cancer (BC). In the present study, the precise molecular mechanism underlying the role of miR-320 in the progression of BC was investigated. Reverse transcription-quantitative PCR was conducted to determine mRNA expression, and western blot analysis was used to test protein levels. An MTT assay was conducted to detect cell viability and Transwell assays were used to analyze cell migration and invasion abilities. Furthermore, E74-like factor 3 (ELF3) protein density was tested via immunohistochemistry. Tumor volume was detected by xenograft tumor formation assay. The current results indicated that miR-320 expression was downregulated in BC tissues and cells, and was associated with a poor prognosis of patients with BC. Overexpression of miR-320 inhibited cell proliferation, migration and invasion via inhibition of the epithelial-mesenchymal transition and the PI3K/AKT signaling pathway in BC cells. Furthermore, it was revealed that the tumor size and weight were smaller in nude mice that had been transfected to overexpress miR-320. The luciferase reporter assay demonstrated the direct binding of miR-320 to the 3′ untranslated region of ELF3 mRNA, which may further downregulate ELF3. Overall, the present results provided evidence that miR-320 may be a tumor suppressor in BC, and that the miR-320/ELF3 axis regulated tumor progression via the PI3K/AKT signaling pathway, which may represent a novel treatment strategy for BC.

Knock-Down of the TIM/TIPIN Complex Promotes Apoptosis in Melanoma Cells

Background: The Timeless (TIM) and its interacting partner TIPIN protein complex is well known for its role in replication checkpoint and normal DNA replication processes. TIM appears to be an essential component of the DNA damage response. Recent studies revealed the involvement of TIM and TIPIN in human malignancies; however, no evidence is available regarding TIM/TIPIN protein complex expression and its potential role in melanoma. Therefore, we investigated the role of TIM as well as TIPIN in melanoma. Methods: To assess the role of TIM/TIPIN in melanoma, we analyzed TIM/TIPIN expression data from the publicly accessible TCGA online database, Western blot analysis, and RT-qPCR in a panel of melanoma cell lines. Lentivirus-mediated TIM/TIPIN knockdown in A375 melanoma cells was used to examine cell proliferation, colony formation, and apoptosis. A xenograft tumor formation assay was performed using shTIM, shTIPIN, and shMock cells. Findings: The TIM/TIPIN complex was frequently overexpressed in melanoma cells compared to normal melanocytes. We also discovered that the overexpression of TIM and TIPIN was significantly associated with poorer prognosis of melanoma patients. Furthermore, we observed that shRNA-mediated knockdown of TIM and TIPIN reduced cell viability and proliferation due to the induction of apoptosis and increased levels of γH2AX, a marker of DNA damage. In a xenograft tumor nude mouse model, shRNA-knockdown of TIM/TIPIN significantly reduced tumor growth. Interpretation: Our results suggest that TIM and TIPIN play an important role in tumorigenesis of melanoma, which might reveal novel approaches for the development of new melanoma therapies. Further mechanistic investigations are needed to determine this complex’s potential as a biomarker of melanoma susceptibility. Targeting TIM/TIPIN might be a potential therapeutic strategy against melanoma. Funding Statement: This work was supported by the Hormel Foundation (Z. Dong). Declaration of Interests: All authors have agreed to publish this manuscript and declare no competing interests. Ethics Approval Statement: All animal studies were performed following the guidelines approved by the University of Minnesota Institutional Animal Care and Use Committee (Protocol ID: 1803-35739A).

MiR-16 inhibits hepatocellular carcinoma progression by targeting FEAT through NF-κB signaling pathway.

MicroRNA-16 (miR-16) expression has been proved to take part in the initiation and development of several cancers, including hepatocellular carcinoma (HCC). However, its role and its molecular mechanism in HCC cells remain unclear. Our study aimed to elucidate miR-16 probable role and potential mechanism in HCC cells. MiR-16 expression in HCC was measured by Real Time-Polymerase Chain Reaction (RT-PCR). MiR-16 mimic or inhibitor was applied to increase or decrease miR-16 expression in Huh7 cells separately. The cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). The invaded cells and migrated cells were detected by the transwell assay. The epithelial-mesenchymal transition (EMT) and the nuclear factor-κB (NF-κB) were performed using Western blot. The tumor growth was measured via xenograft tumor formation assay. Moreover, bioinformatical methods and luciferase reporter assay were carried out to confirm the miR-16 target gene. MiR-16 expression was downregulated in HCC tissues and cells. Furthermore, the increasing miR-16 expression was suppressed, whereas the decreasing miR-16 expression promoted cell proliferation, invasion, and migration in Huh7 cells. Moreover, miR-16 targeted FEAT in regulating HCC progression. FEAT was associated with a poor prognosis of HCC patients. Especially, miR-16 suppressed EMT and NF-κB pathway in HCC and inhibited the tumor growth in vivo. We stated that miR-16 suppressed HCC cell progression by targeting FEAT and inhibiting EMT and NF-κB pathway. MiR-16 may be clinically utilized as a factor for the clinical diagnosis and prognosis of HCC.

MiR-15 suppressed the progression of bladder cancer by targeting BMI1 oncogene via PI3K/AKT signaling pathway.

To investigate the role of microRNA-15 (miR-15) in the progression of bladder cancer (BC) cell and its underlying mechanism. Human BC specimens were collected from BC patients during operations. BC cell lines (T24, BIU87, and HT1376) and normal uroepithelial cell lines SV-HUV-1 were cultured. The abilities of cell proliferation and invasion were detected by Methyl thiazolyl tetrazolium (MTT) and transwell assay, respectively. Additionally, the relevant mRNA and protein expressions were measured by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), Western blot and immunohistochemistry, respectively. Furthermore, the luciferase reporter assay was used to verify the target gene of miR-15. Besides, Xenograft tumor formation assay was performed to confirm the effect of miR-15 on tumor growth. A low expression of miR-15 was detected by qRT-PCR, whereas the high expression of B cell-specific Moloney murine leukemia virus integration site 1 (BMI1) was detected by immunocytochemical assay in BC tissues. Moreover, miR-15 expression and BMI1 expression were significantly associated with the overall survival of BC patients. MTT and transwell assay results stated that the up-regulation of miR-15 inhibited BC cell proliferation, migration, and invasion. BMI-1 was verified as a direct target of miR-15 in BC using Luciferase reporter assay. Besides, miR-15 regulated epithelial-mesenchymal transition (EMT)-related makers, protein kinase B (AKT), and the phosphorylation of AKT protein levels in BC using the Western blot assay. Xenograft tumor formation assay indicated that the over-expression of miR-15 inhibited the tumor growth. We stated that miR-15 suppressed BC cell progression by targeting BMI1 through the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, which provided a potential target for BC treatment.

MicroRNA-361-5p suppresses the tumorigenesis of hepatocellular carcinoma through targeting WT1 and suppressing WNT/β-cadherin pathway.

MicroRNA-361-5p (miR-361-5p) has been found to be involved in the pathogenesis of several human cancers. However, the specific role of miR-361-5p is still unclear in hepatocellular carcinoma (HCC). Therefore, this study was designed to elucidate the function of miR-361-5p in HCC. The expression levels of miR-361-5p and Wilms' tumor-1 (WT1) were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assay. Moreover, the function of miR-361-5p was examined through Cell Counting Kit-8 (CCK-8) and transwell assays. The protein expressions were examined via Western blot analysis and immunocytochemical assay. Tumor growth of HCC was observed via xenograft tumor formation assay. The relationship between miR-361-5p and WT1 was verified by the Dual-Luciferase assay. Downregulation of miR-361-5p was identified in HCC, which predicted a worse prognosis in HCC patients. Furthermore, it was found that miR-361-5p suppressed cell proliferation, migration, and invasion in HCC by inhibiting WT1. MiR-361-5p also inhibited tumor growth of HCC. Besides that, miR-361-5p suppressed EMT and negatively activated the WNT/β-cadherin pathway in HCC. MiR-361-5p suppressed tumorigenesis of HCC by targeting WT1 and inactivating the WNT/β-cadherin pathway.

MicroRNA-23b suppresses cervical cancer biological progression by directly targeting six1 and affecting epithelial-to-mesenchymal transition and AKT/mTOR signaling pathway.

To elucidate the effects and mechanism of microRNA-23b (miR-23b) in cervical cancer (CC) progression. Fifty-six pairs of CC tissue samples and matched para-carcinoma tissue samples were collected. Meanwhile, human normal cervical epithelial cell and CC cell lines were cultured. The abilities of cell proliferation and migration were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays and transwell assays. The correlation between sine oculis homeobox 1 (six1) and miR-23b was clarified by dual-luciferase reporter assay. The relative protein and mRNA expression were detected by quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC) and Western blot. In addition, Xenograft tumor formation assay was performed in this study. MiR-23b was remarkably down-regulated in CC and the low miR-23b expressions were associated with the poor prognosis and worse OS of CC patients. Additionally, the functional assays demonstrated that miR-23b overexpression obviously repressed CC cell proliferation, invasion and migration abilities through the regulation of the AKT/mTOR pathway and the epithelial-to-mesenchymal transition (EMT) progress. Moreover, the luciferase reporter assay indicated that six1 was one functional target for miR-23b in CC cells, indicating that the inhibitory functions of miR-23b in CC cells were partially regulated by six1. Moreover, miR-23b restoration could prominently repress tumor growth in vivo. MiR-23b suppressed CC progression via directly targeting six1 and affecting AKT/mTOR signaling pathway as well as EMT progress. Therefore, miR-23b/six1 may be promising biomarkers for CC diagnosis and therapy.

MicroRNA-191-5p promotes the development of osteosarcoma via targeting EGR1 and activating the PI3K/AKT signaling pathway.

MicroRNA-191 (miR-191) has been reported to be abnormally expressed in human cancers and other diseases. The function of miR-191 was contradictory in different cancers. In the present study, we confirmed the specific function of miR-191-5p in osteosarcoma (OS). The effects of miR-191-5p on cellular behaviors of OS cells were investigated through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and transwell assay. The quantitative Real-time-polymerase chain reaction (qRT-PCR) was applied to examine the expressions of miR-191-5p and early growth response gene 1 (EGR1). Western blot and immunocytochemical assay were used to detect the protein expression of EGR1. The binding relationship between miR-191-5p and EGR1 was confirmed by Dual-Luciferase reporter gene assay. Xenograft tumor formation assay was conducted to examine the in vivo effect of miR-191-5p on tumor growth of OS. MiR-191-5p was upregulated in OS tissues, which was related to poor prognosis of OS patients. Moreover, miR-191-5p promoted cell proliferation, migration and invasion by regulating EGR1 in OS. Furthermore, EGR1 was downregulated in OS tissues, which was associated with poor prognosis of OS patients. MiR-191-5p was found to promote epithelial-mesenchymal transition (EMT) and PI3K/AKT pathway, thus promoting the development of OS. MiR-191-5p promoted the development of OS via targeting EGR1 and positively regulated the PI3K/AKT signaling pathway.

MicroRNA‑485 inhibits the malignant behaviors of retinoblastoma by directly targeting Wnt3a.

Deregulation of microRNAs (miRNAs) has been widely reported in retinoblastoma (RB), and the aberrantly expressed miRNAs may serve as crucial epigenetic regulators in the occurrence and development of RB. Therefore, the identification of dysregulated miRNAs in RB may be useful for the development of effective targets for the therapy patients with this disease. miRNA (miR)‑485‑5p (miR‑485) is deregulated in multiple human cancer types and serves crucial roles in their progression and development. However, the expression pattern of miR‑485 and its role in RB have not been well investigated. In the present study, expression levels of miR‑485 in RB tissues and cell lines were measured using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). The effects of miR‑485 overexpression on RB cell proliferation, apoptosis, migration and invasion were examined using Cell Counting Kit‑8 assay, flow cytometric analysis and in vitro migration and invasion assays, respectively. Xenograft tumor formation assay was utilized to determine the influence of miR‑485 on RB tumor growth in vivo. The mechanism responsible for the tumor‑suppressing roles of miR‑485 in RB progression was determined through a series of experiments, including bioinformatics prediction, luciferase reporter assay, RT‑qPCR, western blot analysis and rescue experiments. Herein, a marked downregulation of miR‑485 expression in human RB tissues and cell lines was observed. miR‑485 overexpression suppressed RB cell proliferation, induced cell apoptosis, attenuated cell migration and cell invasion in vitro, and restrained the growth of RB cells in vivo. Additionally, Wnt3a was revealed to be a direct target gene of miR‑485 in RB cells. Wnt3a was upregulated in human RB tissues, and its upregulation was inversely associated with miR‑485. Furthermore, the tumor suppressive roles of Wnt3a silencing were similar to those of miR‑485 overexpression in RB cells. In addition, restoration of Wnt3a expression partially reversed the tumor suppressor action of miR‑485 in RB cells. However, miR‑485 upregulation directly targeted Wnt3a to inhibit activation of the Wnt/β‑catenin signaling pathway in RB cells both in vitro and in vivo. Notably, these results demonstrated that the tumor‑suppressive roles of miR‑485 were at least partially mediated by Wnt3a in RB cells. Therefore, miR‑485 is a potential therapeutic target for treating patients with RB.

MiR-598 Suppresses Invasion and Migration by Negative Regulation of Derlin-1 and Epithelial-Mesenchymal Transition in Non-Small Cell Lung Cancer

Background/Aims: MicroRNAs regulate a wide range of biological processes of non-small cell lung cancer (NSCLC). Although miR-598 has been reported to act as a suppressor in osteosarcoma and colorectal cancer, the physiological function of miR-598 in NSCLC remains unknown. In this study, the role of miR-598 in NSCLC was investigated. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to estimate the expression of miR-598 and Derlin-1 (DERL1) in both NSCLC tissues and cell lines. Immunohistochemistry (IHC) analyzed the association between the miR-598 expression and epithelial-mesenchymal transition (EMT) hallmark genes (E-cadherin, Vimentin) by staining the tumors representative of the high- and low-expression groups. The effect of miR-598 and DERL1 on invasion and migration was determined in vitro using transwell and wound-healing assays. The molecular mechanism underlying the relevance between miR-598 and DERL1 was elucidated by luciferase assay and Western blot. Western blot assessed the expression levels of EMT hallmark genes in cell lines. Xenograft tumor formation assay was conducted as an in vivo experiment. Results: In this study, a relatively low level of miR-598 and high DERL1 expressions were found in NSCLC specimens and cell lines. IHC results established a positive correlation between the miR-598 expression and E-cadherin and a negative with Vimentin. DERL1 was verified as a direct target of miR-598 by luciferase assay. In vitro, the over-expression of miR-598 negatively regulated DERL1 and EMT for the suppression of invasion and migration. In vivo, the over-expression of miR-598 could inhibit tumor cell metastasis in NSCLC. Conclusions: These findings for the first time revealed that miR-598, as a tumor suppressor, negatively regulate DERL1 and EMT to suppress the invasion and migration in NSCLC, thereby putatively serving as a novel therapeutic target for NSCLC clinical treatment.

SNHG6 Promotes Tumor Growth via Repression of P21 in Colorectal Cancer

Background/Aims: SNHG6 (Small Nucleolar RNA Host Gene 6) is a novel non-coding RNA (ncRNA) and its cellular function is largely unknown. Methods: Cell Counting Kit-8 (CCK-8) cell growth assay, colony formation and flow cytometry were used to determine colorectal cancer cell proliferation, cell cycle progression and apoptosis in vitro. The xenograft tumor formation assay in nude mice was established to evaluate tumor growth in vivo. RNA immunopreciptation (RIP) analysis was performed to examine whether SNHG6 could bind to EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit), and chromatin immunoprecipitation (ChIP) assay was conducted to examine whether SNHG6 could repress p21 transcription by recruiting EZH2 to the p21 promoter. Results: Here we found that SNHG6 was upregulated and its expression levels were positively correlated with advanced tumor stage in colorectal cancer. Survival analysis suggested that higher expression of SNHG6 predicted poor prognosis in patients with colorectal cancer. Functional studies indicated that SNHG6 could promote cell proliferation via a direct suppression of p21 expression in colorectal cancer cells. Moreover, SNHG6 repressed p21 transcription through recruiting EZH2 to the p21 promoter in colorectal cancer cells. Conclusion: Taken together, our study demonstrates that SNHG6 promotes tumor growth via repression of p21 in colorectal cancer, which may provide a promising target for novel anticancer therapeutics.

Proliferin-related protein overexpression in SGC-7901 gastric cancer cells inhibits in vitro cell growth and tumorigenesis in nude mice

As reported in the literature, the worldwide 5-year overall survival rate for patients with gastric cancer receiving surgical treatment in the progressive stage is less than 25%. Therefore, there is an urgent need for the development of novel therapeutic strategies. Our preliminary studies demonstrated that proliferin-related protein (PRP) inhibits the proliferation of TM3 Leydig testicular cells. To evaluate whether PRP has antitumor effects invitro and invivo, we stably expressed PRP in SGC-7901 gastric carcinoma cells. PRP inhibited the proliferation and cell cycle progression of SCG-7901 cells, as determined by cell growth and cell cycle assays. Transwell experiments demonstrated that PRP inhibited the cell migration and invasion of SCG-7901 cells. Western blotting demonstrated that PRP-overexpressing cells had upregulated matrix metalloproteinase 9 (MMP-9) and downregulated tissue inhibitor of metalloproteinases-1 (TIMP-1). In a xenograft tumor formation assay using nude mice, tumors formed by PRP-overexpressing cells had significantly lower weights than those formed by control cells, and the tumor inhibitory rate reached 71.9%. We demonstrated for the first time that PRP inhibits gastric carcinoma cell proliferation, motility, and tumorigenicity invivo, suggesting that PRP may become an important target for the development of gastric cancer gene therapy.

Epstein-Barr Virus-Encoded LMP2A Induces an Epithelial–Mesenchymal Transition and Increases the Number of Side Population Stem-like Cancer Cells in Nasopharyngeal Carcinoma

It has been recently reported that a side population of cells in nasopharyngeal carcinoma (NPC) displayed characteristics of stem-like cancer cells. However, the molecular mechanisms underlying the modulation of such stem-like cell populations in NPC remain unclear. Epstein-Barr virus was the first identified human tumor virus to be associated with various malignancies, most notably NPC. LMP2A, the Epstein-Barr virus encoded latent protein, has been reported to play roles in oncogenic processes. We report by immunostaining in our current study that LMP2A is overexpressed in 57.6% of the nasopharyngeal carcinoma tumors sampled and is mainly localized at the tumor invasive front. We found also in NPC cells that the exogenous expression of LMP2A greatly increases their invasive/migratory ability, induces epithelial–mesenchymal transition (EMT)-like cellular marker alterations, and stimulates stem cell side populations and the expression of stem cell markers. In addition, LMP2A enhances the transforming ability of cancer cells in both colony formation and soft agar assays, as well as the self-renewal ability of stem-like cancer cells in a spherical culture assay. Additionally, LMP2A increases the number of cancer initiating cells in a xenograft tumor formation assay. More importantly, the endogenous expression of LMP2A positively correlates with the expression of ABCG2 in NPC samples. Finally, we demonstrate that Akt inhibitor (V) greatly decreases the size of the stem cell side populations in LMP2A-expressing cells. Taken together, our data indicate that LMP2A induces EMT and stem-like cell self-renewal in NPC, suggesting a novel mechanism by which Epstein-Barr virus induces the initiation, metastasis and recurrence of NPC.