Introducing graphitic carbon nitride nanosheets as supersandwich-type assembly on porous electrode for ultrasensitive electrochemiluminescence immunosensing.

Biomarkers in blood or tissue provide essential information for clinical screening and early disease diagnosis. However, increasing the sensitivity of detecting biomarkers remains a major challenge in a wide variety of electrochemical immunoassays. Herein, we present an electrochemiluminescence (ECL) immunosensing strategy with 1: Nn amplification ratio (target-to-signal probe) for biomarkers detection on a porous gold electrode. The high porosity of the electrode surface provides enough bonding sites for capturing the target biomolecules and thus many DNA labels can be introduced. On the basis of this concept, a great number of graphitic carbon nitride (g-C3N4) nanosheets are employed to create a supersandwich-type assembly on a porous electrode via the DNA hybridization process. Furthermore, compared with the traditional sandwich immunoassay (the ratio of target-to-signal probe is 1 : 1), the supersandwich construction can introduce a large number of signal probes, thus resulting in a highly improved sensitivity. The proposed ECL immunosensor exhibits an excellent performance in a concentration range from 0.01 fg mL-1 to 1 µg mL-1 with an ultralow detection limit of 0.001 fg mL-1 (S/N = 3) and excellent selectivity. This sensing strategy could be developed into a real-time assay for the disease-related molecular targets, with many practical applications in biotechnology and life science.

HMGB3 promotes growth and migration in colorectal cancer by regulating WNT/ß-catenin pathway.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths and a major health problem. High mobility group box 3 (HMGB3), a member of the high-mobility group box (HMGB) family, was reported to be over-expressed in gastric carcinoma and bladder cancer. However, the function of HMGB3 in CRC remains unclear. Here, we found that HMGB3 was up-regulated in CRC at both mRNA and protein levels. qRT-PCR results showed that high expression of HMGB3 had positive correlation with serosal invasion, lymph metastasis, and tumor-node-metastasis (TNM) stage in CRC patient. Functional experiments showed that HMGB3 can promote CRC cells proliferation and migration in vitro. Moreover, we found HMGB3 can active WNT/ß-catenin pathway to increase the expression level of c-Myc and MMP7. These results may be the reason for HMGB3 oncogene role in CRC. In summary, our data indicated that HMGB3 may serve as an oncoprotein and could be used as a potential prognostic marker in CRC.

Long non-coding RNA CASC11 interacts with hnRNP-K and activates the WNT/ß-catenin pathway to promote growth and metastasis in colorectal cancer.

The abnormal expression of many long non-coding RNAs (lncRNAs) has been reported in the progression of various tumors, and these lncRNAs can be useful as diagnostic indicators and anti-tumor targets. Therefore, it is important to identify lncRNAs that can be used for the clinical prevention and treatment of colorectal cancer (CRC). Here, we report that cancer susceptibility candidate 11 (CASC11) was upregulated in CRC tissues; increased CASC11 expression in CRC was associated with tumor size, serosal invasion, lymph metastasis, and the tumor-node-metastasis (TNM) stage. Functional experiments showed that CASC11 can promote CRC cell proliferation and metastasis in vitro and in vivo. Furthermore, CASC11 can target heterogeneous ribonucleoprotein K (hnRNP-K) to activate WNT/ß-catenin signaling in CRC cells. In addition, we found that c-Myc directly bound to the promoter regions of CASC11 and increased promoter histone acetylation to enhance CASC11 expression. Together, our findings indicate that the novel lncRNA CASC11 may serve as a candidate diagnostic biomarker and a promising therapeutic target for CRC.

Loss of TINCR expression promotes proliferation, metastasis through activating EpCAM cleavage in colorectal cancer.

Long non-coding RNAs (lncRNAs) are involved in kinds of human diseases, including colorectal cancer (CRC). TINCR, a 3.7 kb long non coding RNA, was associated with cell differentiation in keratinocyte and gastric cancer cells. However, little is known about the role of TINCR in regulation CRC progression. Here, we showed that lncRNA TINCR was associated with CRC proliferation and metastasis. TINCR was statistically downregulated in CRC tissues and metastatic CRC cell lines compared with their counterparts. TINCR was reversely correlated with CRC progression and promoted tumor cells growth, metastasis in vivo and in vitro. While overexpression of TINCR had opposite effect. In addition, we also found that TINCR specifically bound to EpCAM through RNA IP and RNA pull down assays. Loss of TINCR promoted hydrolysis of EpCAM and then released EpICD, subsequently, activated the Wnt/ß-catenin pathway. Further studies shown that c-Myc repressed the expression of TINCR through repressing sp1 transcriptive activity, which established a positive feedback loop controlling c-Myc and TINCR expression. These findings elucidate that loss of TINCR expression promotes proliferation and metastasis in CRC and it could be considered as a potential cancer suppressor gene.

IFN-γ-mediated IRF1/miR-29b feedback loop suppresses colorectal cancer cell growth and metastasis by repressing IGF1.

To investigate the clinicopathological significance and underlying mechanism of microRNA-29b (miR-29b) in colorectal cancer (CRC), the role of miR-29b was investigated using in vivo and in vitro assays. Luciferase reporter assays were conducted to determine the association between miR-29b and the insulin-like growth factor 1 (IGF1) 3' untranslated region (3'UTR). Chromatin immunoprecipitation (ChIP) assays were employed to assess the direct binding of interferon regulatory factor 1 (IRF1) to miR-29b. We found that interferon (IFN)-γ could induce miR-29b by recruiting IRF1 to binding sites in the miR-29b promoter. A low level of miR-29b was significantly associated with an aggressive phenotype. MiR-29b inhibited CRC cell growth and invasion. IGF1, an activator of PI3K/Akt signaling, was confirmed as a novel target of miR-29b. Moreover, miR-29b increased IRF1 expression, and the inhibition of miR-29b suppressed IFN-γ-induced apoptosis. We elucidated the potential signaling pathway, IFN-γ/IRF1/miR-29b/IGF1, and its implication for CRC tumorigenesis. A positive feedback loop between IRF1 and miR-29b may contribute to the sensitivity of CRC cells to IFN-γ. Targeting miR-29b may provide a strategy for blocking CRC growth and metastasis.

Revealing the anti-tumor effect of artificial miRNA p-27-5p on human breast carcinoma cell line T-47D.

microRNAs (miRNAs) cause mRNA degradation or translation suppression of their target genes. Previous studies have found direct involvement of miRNAs in cancer initiation and progression. Artificial miRNAs, designed to target single or multiple genes of interest, provide a new therapeutic strategy for cancer. This study investigates the anti-tumor effect of a novel artificial miRNA, miR P-27-5p, on breast cancer. In this study, we reveal that miR P-27-5p downregulates the differential gene expressions associated with the protein modification process and regulation of cell cycle in T-47D cells. Introduction of this novel artificial miRNA, miR P-27-5p, into breast cell lines inhibits cell proliferation and induces the first "gap" phase (G1) cell cycle arrest in cancer cell lines but does not affect normal breast cells. We further show that miR P-27-5p targets the 3'-untranslated mRNA region (3'-UTR) of cyclin-dependent kinase 4 (CDK4) and reduces both the mRNA and protein level of CDK4, which in turn, interferes with phosphorylation of the retinoblastoma protein (RB1). Overall, our data suggest that the effects of miR p-27-5p on cell proliferation and G1 cell cycle arrest are through the downregulation of CDK4 and the suppression of RB1 phosphorylation. This study opens avenues for future therapies targeting breast cancer.

Prediction of human miRNAs using tissue-selective motifs in 3' UTRs.

MicroRNAs (miRNAs) play an important role in posttranscriptional regulation of genes. We developed a method to predict human miRNAs without requiring cross-species conservation. We first identified lowly/moderately expressed tissue-selective genes using EST data and then identified overrepresented motifs of seven nucleotides in the 3' UTRs of these genes. Using these motifs as potential target sites of miRNAs, we recovered more than two-thirds of the known human miRNAs. We then used those motifs that did not match any known human miRNA seed region to infer novel miRNAs. We predicted 36 new human miRNA genes with 44 mature forms and 4 novel alternative mature forms of 2 known miRNA genes when a stringent criterion was used and many more novel miRNAs when a less stringent criterion was used. We tested the expression of 11 predicted miRNAs in three human cell lines and found 5 of them expressed in all three cell lines and 1 expressed in one cell line. We selected 2 of them, P-2 and P-27-5p, to do functional validation, using their mimics and inhibitors and using both luciferase assay and Western blotting. These experiments provided strong evidence that both P-2 and P-27-5p are novel miRNAs and that CREB3L3, which encodes cAMP-responsive element binding protein 3-like 3, is a target gene of P-2, whereas LAMB3, which encodes laminin beta3, is a target gene of P-27-5p.