Circulating lncRNA SNHG11 as a novel biomarker for early diagnosis and prognosis of colorectal cancer

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer mortality worldwide. Emerging evidence indicates that tumour cells release substantial amounts of RNA into the bloodstream, in which RNA strongly resists RNases and is present at sufficient levels for quantitative analyses. Our study aimed to discover blood-based markers for the early detection of CRC and to ascertain their efficiency in discriminating healthy controls, patients with polyps and adenomas and cancer patients. We first analysed and screened ZFAS1, SNHG11, LINC00909 and LINC00654 in a bioinformatics database and then collected clinical plasma samples for preliminary small-scale analysis and further large-scale verification. We then explored the mechanism of dominant lncRNA SNHG11 expression in CRC by in vitro and in vivo assays. The combination of ZFAS1, SNHG11, LINC00909 and LINC00654 showed high diagnostic performance for CRC (AUC: 0.937), especially early-stage disease (AUC: 0.935). Plasma levels of the four candidate lncRNAs were significantly reduced in postoperative samples compared to preoperative samples. A panel including these four lncRNAs performed well in distinguishing patient groups with different stages of colon disease, and SNHG11 exhibited the greatest diagnostic ability to identify precancerous lesions and early-stage tumour formation. Mechanistically, high SNHG11 expression promotes proliferation and metastasis by targeting the Hippo pathway. Taken together, the data indicate that SNHG11 may be a novel therapeutic target for the treatment of CRC and a potential biomarker for the early detection of CRC.     

Restoration of the ATG5-dependent autophagy sensitizes DU145 prostate cancer cells to chemotherapeutic drugs

Autophagy serves an important role in cancer cell survival and drug resistance. In the present study, the prostate cancer DU145 cell line was used, which lacks autophagy related 5 (ATG5) expression and is defective in induction of ATG5-dependent autophagy. The aim of the study was to examine the effects of the restoration of autophagy on cell proliferation and migration, and to assess the cytotoxicity caused by chemotherapeutic drugs, using microscopic, wound-healing, western blot and apoptotic assays. The restoration of the autophagic activity in DU145 cells by the overexpression of ATG5 enhanced the cell proliferation and migration rates. Notably, restoration of the ATG5-dependent autophagy in DU145 cells significantly increased the cytotoxic effects of the chemotherapeutic drugs, docetaxel and valproic acid, and the endoplasmic reticulum stress inducers, brefeldin A, tunicamycin and thapsigargin. The present study provides a novel perspective on the role of ATG5-dependent autophagy in drug resistance and chemotherapy.     

Pleotropic role of RNA binding protein CELF2 in autophagy induction

We previously reported that ionizing radiation (IR) mediates cell death through the induction of CUGBP elav‐like family member 2 (CELF2), a tumor suppressor. CELF2 is an RNA binding protein that modulates mRNA stability and translation. Since IR induces autophagy, we hypothesized that CELF2 regulates autophagy‐mediated colorectal cancer (CRC) cell death. For clinical relevance, we determined CELF2 levels in The Cancer Genome Atlas (TCGA). Role of CELF2 in radiation response was carried out in CRC cell lines by immunoblotting, immunofluorescence, autophagic vacuole analyses, RNA stability assay, quantitative polymerase chain reaction and electron microscopy. In vivo studies were performed in a xenograft tumor model. TCGA analyses demonstrated that compared to normal tissue, CELF2 is expressed at significantly lower levels in CRC, and is associated with better overall 5‐year survival in patients receiving radiation. Mechanistically, CELF2 increased levels of critical components of the autophagy cascade including Beclin‐1, ATG5, and ATG12 by modulating mRNA stability. CELF2 also increased autophagic flux in CRC. IR significantly induced autophagy in CRC which correlates with increased levels of CELF2 and autophagy associated proteins. Silencing CELF2 with siRNA, mitigated IR induced autophagy. Moreover, knockdown of CELF2 in vivo conferred tumor resistance to IR. These studies elucidate an unrecognized role for CELF2 in inducing autophagy and potentiating the effects of radiotherapy in CRC.     

LncRNA SNHG5 affects cell proliferation,metastasis and migration of colorectal cancer through regulating miR-132-3p/CREB5

We aimed at the effects of long non-coding RNA (lncRNA) SNHG5 on proliferation, metastasis and migration of colorectal cancer (CRC) cells. We also investigated regulatory relationships among miR-132-3p, SNHG5 and CREB5 and their roles in CRC. 25 pairs of samples containing CRC tissues and matched para-tumor tissues were obtained to examine SNHG5, miR-132-3p and CREB5 expression by qRT-PCR or Western blot. The targeted relationship between miR-132-3p and SNHG5 or CREB5 was confirmed by dual luciferase report assay as well as RNA pull down assay. The expression of SNHG5, miR-132-3p and CREB5 in CRC cells were regulated by cell transfection. CRC cellular proliferation was assayed by CCK-8 and meanwhile flow cytometry was adopted to observe apoptosis. Metastasis and migration of CRC cells were determined respectively by means of Transwell assay and scratch test. The effects of SNHG5 on CRC were researched in vivo, too. SNHG5 or CREB5 was up-regulated in CRC tissues and cells, whereas miR-132-3p was down-regulated. Overexpression of SNHG5 and CREB5 resulted in the enhancement of proliferation, metastasis, migration and the inhibition of apoptosis in CRC cells, while miR-132-3p led to the opposite result. LncRNA SNHG5 promoted proliferation, migration and metastasis of CRC cells but inhibited apoptosis by modulating miR-132-3p/CERB5.     

miR‐20a inhibits hypoxia‐induced autophagy by targeting ATG5/FIP200 in colorectal cancer

Autophagy is a highly conserved lysosome‐mediated protective cellular process in which cytosolic components, including damaged organelles and long‐lived proteins, are cleared. Many studies have shown that autophagy was upregulated in hypoxic regions. However, the precise molecular mechanism of hypoxia‐induced autophagy in colorectal cancer (CRC) is still elusive. In this study, we found that miR‐20a was significantly downregulated under hypoxia in colon cancer cells, and overexpression of miR‐20a alleviated hypoxia‐induced autophagy. Moreover, miR‐20a inhibits the hypoxia‐induced autophagic flux by targeting multiple key regulators of autophagy, including ATG5 and FIP200. Furthermore, by dual‐luciferase assay we demonstrated that miR‐20a directly targeted the 3′‐untranslated region of ATG5 and FIP200, regulating their messenger RNA and protein levels. In addition, reintroduction of exogenous ATG5 or FIP200 partially reversed miR‐20a‐mediated autophagy inhibition under hypoxia. A negative correlation between miR‐20a and its target genes is observed in the hypoxic region of colon cancer tissues. Taken together, our findings suggest that hypoxia‐mediated autophagy was regulated by miR‐20a/ATG5/FI200 signaling pathway in CRC. miR‐20a‐mediated autophagy defect that might play an important role in hypoxia‐induced autophagy during colorectal tumorigenesis.     

miR-425 regulates lipophagy via SIRT1 to promote sorafenib resistance in liver cancer

Liver cancer is one of the most malignant cancer, with poor outcomes and a high incidence rate, and current treatment approaches to prevent tumor progression and development remain unsatisfactory. Therefore, it is urgent to explore novel methods to inhibit tumor growth and metastasis. Autophagy is a highly conserved process associated with metastasis and drug resistance. Lipids are selectively recognized and degraded via autophagy; thus, autophagy is a crucial process to maintain tumor self-protection. MicroRNA (miR)-425 is a tumor-associated gene involved in liver cancer development that can induce cell proliferation and drug resistance. Using Cell Counting Kit-8 assays, western blot analysis and immunofluorescence assays, the present study revealed that inhibition of miR-425 promoted lipophagy by mediating the autophagy process, which in turn helps to promote sorafenib resistance. Using a bioinformatics website, it was revealed that autophagy promoted lipophagy by targeting silent information regulator 2 homolog 1 (SIRT1). The results of luciferase reporter assays supported this finding, and rescue experiments provided additional evidence. Overall, the current results suggested that inhibition of miR-425 expression increased SIRT1 expression to promote lipophagy, leading to the inhibition of liver cancer cell proliferation.     

Modulation of pediatric brain tumor autophagy and chemosensitivity

Brain and spinal tumors are the second most common malignancies in childhood after leukemia, and they remain the leading cause of death from childhood cancer. Autophagy is a catabolic cellular process that is thought to regulate chemosensitivity, however its role in pediatric tumors is unknown. Here we present studies in pediatric medulloblastoma cell lines (DAOY, ONS76) and atypical teratoid/rhabdoid tumor cell lines (BT-16, BT-12) to test this role. Autophagy was inhibited using siRNA against autophagy-related genes ATG12 and ATG7 or pharmacologically induced or inhibited using rapamycin and chloroquine to test the effect of autophagy on chemosensitivity. Autophagic flux was measured using Western blot analysis of LC3-II and p62 and cell viability was determined using MTS assays and clonogenic growth. We found that when pediatric brain tumor cells under starvation stress, exposed to known autophagy inducers such as rapamycin, or treated with current chemotherapeutics (lomustine, cisplatin), all stimulate autophagy. Silencing ATG12 and ATG7 or exposure to a known autophagy inhibitor, chloroquine, could inhibit this autophagy increase; however, the effect of autophagy on tumor cell killing was small. These results may have clinical relevance in the future planning of therapeutic regimens for pediatric brain tumors.     

Autophagyinhibition by chloroquine sensitizes HT-29 colorectal cancer cells to concurrent chemoradiation Schonewolf CA,Mehta M,Schiff D,Wu H,Haffty BG,Karantza V,Jabbour SK简

AIM: To investigate whether the inhibition of autophagy by chloroquine (CQ) sensitizes rectal tumors to radiation therapy (RT) or concurrent chemoradiation (chemoRT).METHODS: In vitro, HCT-116 and HT-29 colorectal cancer (CRC) cell lines were treated as following: (1) PBS; (2) CQ; (3) 5-fluorouracil (5-FU); (4) RT; (5) CQ and RT; (6) 5-FU and RT; (7) CQ and 5-FU; and (8) 5-FU and CQ and RT. Each group was then exposed to various doses of radiation (0-8 Gy) depending on the experiment. Cell viability and proliferative capacity were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays. Clonogenic survival curves were constructed and compared across treatment groups. Autophagy status was determined by assessing the LC3-II to LC3-I ratio on western blot analysis, autophagosome formation on electron microscopy and identification of a perinuclear punctate pattern with GFP-labeled LC3 on fluorescence microscopy. Cell cycle arrest and cell death were evaluated by FACS and Annexin V analysis. All experiments were performed in triplicate and statistical analysis was performed by the student’s t test to compare means between treatment groups.RESULTS: RT (2-8 Gy) induced autophagy in HCT-116 and HT-29 CRC cell lines at 4 and 6 h post-radiation, respectively, as measured by increasing LC3-II to LC3-I ratio on western blot. Additionally, electron microscopy demonstrated autophagy induction in HT-29 cells 24 h following irradiation at a dose of 8 Gy. Drug treatment with 5-FU (25 μmol/L) induced autophagy and the combination of 5-FU and RT demonstrated synergism in autophagy induction. CQ (10 μmol/L) alone and in combination with RT effectively inhibited autophagy and sensitized both HCT-116 and HT-29 cells to treatment with radiation (8 Gy; P < 0.001 and 0.00001, respectively). Significant decrease in clonogenic survival was seen only in the HT-29 cell line, when CQ was combined with RT at doses of 2 and 8 Gy (P < 0.5 and P = 0.05, respectively). There were no differences in cell cycle progression or Annexin V staining upon CQ addition to RT.CONCLUSION: Autophagy inhibition by CQ increases CRC cell sensitivity to concurrent treatment with 5-FU and RT in vitro, suggesting that addition of CQ to chemoRT improves CRC treatment response.     

Zebrafish xenograft model for studying the function of lncRNA SNHG4 in the proliferation and migration of colorectal cancer

BackgroundThe zebrafish xenograft model has become a reliable in vivo model for human cancer research. Compared to a mouse model, the zebrafish xenograft has many advantages, including optical transparency, intuitive in vivo observation, and speed. Long noncoding RNAs (lncRNAs) have been identified as crucial regulatory factors in the progression of colorectal cancer (CRC). The biological function of lncRNA small nucleolar RNA host gene 4 (SNHG4) in CRC is still unclear.MethodsWe analyzed the expression of SNHG4 in CRC patient samples by the Gene Expression Profiling Interactive Analysis (GEPIA) software. The quantitative real time-polymerase chain reaction (qRT-PCR) was used to verify in CRC cell lines. The colony formation assay was used to study the cell proliferation, and we used the transwell assay to detect the migration ability. Then the zebrafish xenograft models were used to confirm these roles of SNHG4 in vivo. Moreover, we detected epithelial mesenchymal transition (EMT) related genes by qRT-PCR.ResultsWe found the expression of SNHG4 was upregulated in CRC patient samples by analyzing GEPIA software, which was also verified in CRC cell lines. We also found that silencing SNHG4 inhibited the proliferation and migration of CRC cells, and its roles were verified in zebrafish xenografts in vivo. Further, we found that the expression of E-cadherin was significantly upregulated and N-cadherin was downregulated when knocking-down SNHG4 in CRC cells.ConclusionsOur findings demonstrated that SNHG4 played oncogenic roles in CRC, which could be a potential target for treatment of CRC patients, and the results strongly revealed that zebrafish xenograft could be used for functional research of lncRNAs in human cancer.     

MTDH基因和ATG7基因在结直肠癌中的表达及临床意义

目的探讨异黏蛋白(MTDH)基因和自噬相关基因7 (ATG7)在结直肠癌(CRC)中的表达及临床意义。方法选取66例CRC患者的CRC组织及癌旁组织,比较CRC组织及癌旁组织中的MTDH mRNA和ATG7mRNA的相对表达量;比较不同临床特征CRC患者的CRC组织/癌旁组织MTDH mRNA、ATG7 mRNA的相对表达量;采用受试者工作特征(ROC)曲线分析CRC组织/癌旁组织中MTODH mRNA和ATG7 mRNA对结直肠癌的诊断价值,并分析MTDH mRNA与ATG7 mRNA的相关性。结果 CRC组织中MTDH mRNA和ATG7 mRNA相对表达量显著高于癌旁组织(P <0.01);TNM分期为Ⅲ~Ⅳ和有肝转移的CRC患者中,CRC组织/癌旁组织中的MTDH mRNA和ATG7 mRNA相对表达量均显著高于Ⅰ~Ⅱ期和无肝转移的CRC患者,低分化程度的CRC患者中CRC组织/癌旁组织中的MTDH mRNA和ATG7 mRNA相对表达量最高(P <0.01)。ROC曲线分析结果 CRC组织/癌旁组织中MTDH mRNA、ATG7 mRNA的AUC面积分别是0.934、0.900,诊断敏感度分别为90.91%、89.39%,特异度分别为86.37%、84.35%。Pearson分析结果表明CRC中MTDH的表达与ATG7的表达呈正相关(r=0.372,P=0.002)。结论 CRC组织中MTDH mRNA、ATG7 mRNA的表达高于癌旁组织。CRC组织中MTDH mRNA、ATG7 mRNA与TNM分期、分化程度、伴肝转移相关。CRC组织中MTDH mRNA、ATG7 mRNA具有诊断价值且两者具有正相关。     

Activation of autophagy in mesenchymal stem cells provides tumor stromal support

Recent studies have implicated multipotential mesenchymal stem cells (MSCs) as an aid to breast cancer cell proliferation and metastasis, partly as a result of the MSCs secretome. As the tumor gets beyond 2 mm in diameter, the stromal cells could undergo starvation due to the lack of sufficient nutrients in solid tumor microenvironment. In this study, we investigated the survival mechanisms used by stressed stromal cells in breast cancers. We used serum-deprived mesenchymal stem cells (SD-MSCs) and MCF-7 breast cancer cells as model system with a hypothesis that stromal cells in the nutrient-deprived core utilize survival mechanisms for supporting surrounding cells. We tested this hypothesis using in vivo tumor xenografts in immunodeficient mice, which indicated that SD-MSCs supported MCF-7 tumor growth by protection from apoptosis. Histochemical assays showed that SD-MSCs-injected tumors exhibited higher cellularity, decreased apoptosis and decreased differentiation. Beclin-1 staining indicated autophagic areas surrounded by actively proliferating cells. Furthermore, in vitro studies demonstrate that SD-MSCs survive using autophagy and secrete paracrine factors that support tumor cells following nutrient/serum deprivation. Western blot and immunocytochemistry analysis of SD-MSCs demonstrated upregulation and perinuclear relocation of autophagy key regulators such as beclin-1, ATG10, ATG12, MAP-LC3 and lysosomes. Electron microscopic analysis detected a time-dependent increase in autophagosome formation and HDAC6 activity assays indicated the upregulation of autophagy. Taken together, these data suggest that under nutrient-deprived conditions that can occur in solid tumors, stromal cells utilize autophagy for survival and also secrete anti-apoptotic factors that can facilitate solid tumor survival and growth.     

The Influence of Tumor Microenvironment on ATG4D Gene Expression in Colorectal Cancer Patients

Despite great progress in research on the subject, the involvement of autophagy in colorectal cancer (CRC) pathogenesis (initiation, progression, metastasis) remains obscure and controversial. Autophagy is a catabolic process, fundamental to cell viability and connected with degradation/recycling of proteins and organelles. In this study, we aimed at investigating the relative expression level of mRNA via Real-Time PCR of 16 chosen genes belonging to Atg8 mammalian orthologs and their conjugation system, comprising GABARAP, GABARAPL1, GABARAPL2, MAP1LC3A, MAP1LC3B, MAP1LC3C, ATG3, ATG7, ATG10, ATG4A, ATG4B, ATG4C, ATG4D, and three genes encoding proteins building the multimeric ATG16L1 complex, namely ATG5, ATG12, and ATG16L1, in 73 colorectal tumors and paired adjacent normal colon mucosa. Our study demonstrated the relative downregulation of all examined genes in CRC tissues in comparison to adjacent noncancerous mucosa, with the highest rate of expression in both tumor and non-tumor tissues observed for GAPARBPL2 and the lowest for MAP1LC3C. Moreover, in patients with advanced-stage tumors and high values of regional lymph nodes, statistically significant downregulation of ATG4D expression in adjacent normal cells was observed. Our study confirms the role of autophagy genes as cancer suppressors in colorectal carcinogenesis. Furthermore, in regard to the ATG4D gene, we observed the influence of tumor microenvironments on gene expression in adjacent colon mucosa.