Integrative analysis and validation of dysregulated long non-coding RNAs in colon cancer.

It is an increasing evidence that long non-coding RNAs (lncRNAs) are involved in tumour initiation and progression. Here, we analysed RNA-sequencing data from the Cancer Genome Atlas (TCGA) datasets. Totally, 1176lncRNAs, 245miRNAs and 2081mRNAs were identified to be differentially expressed (DE) in colon cancer tissues compared with normal tissues. CASC21, a novel lncRNA located in 8q24.21 locus, was significantly overexpressed in 30 colon cancer tissues compared with matched normal tissues by qRT-PCR assay. CASC21 tended to higher expression as the increase of the tumour-node-metastasis (TNM) classification. Functionally, CASC21 promoted cell proliferation by regulating cell cycle and enhanced tumour metastasis by epithelial-mesenchymal transition (EMT) in colon cancer. Mechanism study indicated that CASC21 might be involved in activating WNT/ß-catenin pathway in colon cancer. In addition, we also built a competing endogenous RNA (ceRNNA) network by bioinformatic analysis using TCGA datasets. Together, our results not only provide novel lncRNAs as potential candidates for further study but also prove that CASC21 is an oncogenic regulator through activating WNT/ß-catenin signalling in colon cancer.

LINC00365 promotes colorectal cancer cell progression through the Wnt/ß-catenin signaling pathway.

In the past decade, substantial evidence established that long noncoding RNAs are serious about mediating the evolution of malignancies. In previous studies, LINC00365, which has not been reported in colorectal cancer (CRC), was selected using the bioinformatics analysis in GSE109454 and GSE41655 data sets. However, the function and mechanism of LINC00365 are still obscure. In our study, LINC00365 was found upregulated in CRC specimens and intimately connected with the prognosis of patients with CRC. In addition, LINC00365 overexpression enhances the cell abilities of proliferation, migration, and invasion in vitro. Meanwhile, mechanistic studies showed that LINC00365 might involve in CRC cell progression by mediating the Wnt/ß-catenin pathway. Furthermore, LINC00365 upregulation increased CDK1 protein expression. In conclusion, this study suggests that LINC00365 acts as a vital part in facilitating CRC progression and might play as a therapeutic target for patients with CRC.

Construction and analysis of dysregulated lncRNA-associated ceRNA network in colorectal cancer.

Colorectal cancer (CRC) is one of the most frequently diagnosed digestive system cancer. The aim of the present study was to investigate the interactions among messenger RNAs (mRNAs), microRNAs (miRNAs), and long noncoding RNAs (lncRNAs) in CRC to reveal the mechanisms of CRC. Differentially expressed genes (DEGs) were identified from public gene expression data sets. One thousand eighty-one common dysregulated mRNAs in two data sets were identified. Gene function analysis and protein-protein interaction network analysis indicated that these DEGs might play important roles in CRC. LINC00365 was selected through coding- noncoding network analysis and its expression was validated upregulated in 22 paired clinical samples and four CRC cell lines. A competing endogenous RNA network composed of 70 miRNAs, nine mRNAs, and LINC00365 was constructed. Eight of nine mRNAs were validated upregulated in The Cancer Genome Atlas data set. Our results suggested that LINC00365 was an oncogene in CRC and it could regulate the expression of several mRNAs through sponging miRNAs.

Values of circulating GPC-3 mRNA and alpha-fetoprotein in detecting patients with hepatocellular carcinoma.

BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is poor and its early diagnosis is of the utmost importance. This study aimed to investigate the values of glypican-3 (GPC-3) expression in the liver and sera and its gene transcription for diagnosis and monitoring of metastasis of HCC. METHODS: Liver GPC-3 was analyzed in HCC tissues from 36 patients by immunohistochemistry and Western blotting. GPC-3 mRNA from circulating peripheral blood mononuclear cells from 123 HCC patients or 246 patients with other diseases or 36 HCC tissues was amplified by RT-PCR, quantitative real-time PCR, and confirmed by DNA sequencing. Circulating GPC-3 level was detected by ELISA. RESULTS: The increasing expression of GPC-3 was observed from non-cancerous to cancerous tissues, with brown granule-like staining localized in tumor parts of atypical hyperplasia and HCC formation. The positive rate of GPC-3 was 80.6% in HCC, 41.7% in their paracancerous tissues, and none in distal cancerous tissues (P<0.001), with no significant difference in differentiation grade and tumor number except for size (Z=2.941, P=0.003). Serum GPC-3 was detected only in HCC (52.8%) and significant difference was found between GPC-3 and tumor size (X2=6.318, P=0.012) or HBV infection (X2=23.362, P<0.001). Circulating GPC-3 mRNA was detected in 70.7% of HCC tissues, with relation to TNM stage, periportal cancerous embolus, and extra-hepatic metastasis (P<0.001). The combination of circulating GPC-3, GPC-3 mRNA and alpha-fetoprotein is of complementary value for HCC diagnosis (94.3%). CONCLUSION: Both GPC-3 overexpression and GPC-3 mRNA abnormality could be used as markers for the diagnosis of HCC and monitoring its metastasis.

Oncofetal antigen glypican-3 as a promising early diagnostic marker for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by a multi-cause, multi-stage and multi-focus process of tumor progression. Its prognosis is poor and early diagnosis is of utmost importance. This study was undertaken to investigate the dynamic expression of oncofetal antigen glypican-3 (GPC-3) and GPC-3 mRNA in hepatocarcinogenesis and to explore their early diagnostic value for HCC. METHODS: A hepatoma model was induced in male Sprague-Dawley rats with 0.05% 2-fluorenylacetamide and confirmed by hematoxylin and eosin staining and gamma-glutamyltransferase (GGT) expression. Total RNA was purified and transcribed into cDNA by reverse transcription. Fragments of the GPC-3 gene were amplified by nested RT-PCR, and confirmed by sequencing. GPC-3 was analyzed by immunohistochemistry, Western blotting or ELISA. RESULTS: Positive GPC-3 expression showed as brown granule-like staining localized in the cytoplasm. Histological examination of hepatocytes revealed three morphological stages of granule-like degeneration, atypical hyperplasia (precancerous), and cancer formation, with a progressive increase of liver total RNA and GGT expression. The incidence of liver GPC-3 mRNA and GPC-3, and serum GPC-3 was 100%, 100% and 77.8% in the HCC group, 100%, 100%, and 66.7% in the precancerous group, 83.3%, 83.3%, and 38.9% in the degeneration group, and no expression in the liver or blood of the control group, respectively. There was a positive correlation between liver GPC-3 mRNA and total RNA level (r=0.475, P<0.05) or liver GPC-3 (r=1.0, P<0.001) or serum GPC-3 (r=0.994, P<0.001). CONCLUSION: Abnormal oncofetal antigen GPC-3 and GPC-3 mRNA expression in hepatocarcinogenesis may be promising molecular markers for early diagnosis of HCC.

[Expression features of glypican-3 and its diagnostic and differential values in hepatocellular carcinoma].

OBJECTIVE: To investigate the expression features of glypican-3 (GPC-3) and its diagnostic and differential values in hepatocellular carcinoma (HCC). METHODS: Rat hepatoma models were made and the dynamic expression features of GPC-3 protein and its gene were investigated by Western blotting and RT-PCR respectively. Liver specimens from 36 HCC patients were collected by self-control method and the expression and clinicopathological features of GPC-3 were analyzed by immunohistochemistry. Serum GPC-3 levels were quantitatively detected by ELISA and its efficiency for HCC diagnosis was evaluated in patients with liver diseases. RESULTS: The incidence of GPC-3 was 0% in control, 83.3% in degeneration, 100% in precancerosis and 100% in canceration during dynamic formation of rat hepatoma, respectively. The positive GPC-3 was brown granule- like staining localized in membrane and cytoplasm in human HCC. CONCLUSIONS: The GPC-3 positive rates were 80.6% in HCC, 41.7% in surrounding tissues and none in distal tissues (P < 0.01), respectively. No positive relationship presented between GPC-3 and differentiation grade or the number of tumor except of tumor size (Z = 2.941, P < 0.01). The incidence of serum GPC-3 was 52.8% in HCC patients except of one patient with cirrhosis. No significant differences were found between GPC-3 and sex, age, AFP, tumor number, Child classification or extrahepatic metastasis except of tumor size (χ² = 6.318, P < 0.05) and HBV infection (χ² = 23.362, P < 0.01). Combined detection of GPC-3 and AFP could rise up diagnosis of HCC. GPC-3 expression closely associated with HCC and might be useful for early diagnosis of HCC.

Suppression of human hepatoma (HepG2) cell growth by nuclear factor-kappaB/p65 specific siRNA.

Nuclear factor-kappaB (NF-κB) is a transcription factor and antagonist of apoptosis during liver regeneration and closely related to the formation and development of hepatocellular carcinoma. In the present study, we investigated the effect of small interference RNA (siRNA)-mediated inhibition of NF-κB on growth of human hepatoma (HepG2) cells. Our data indicated that the expression of NF-κB/p65 mRNA was significantly higher in the HepG2 cells than that in the normal liver (LO2) cells before transfection, and the expression of NF-κB/p65 in the HepG2 cells with NF-κB/p65 siRNA (100 nMol/L) transfection at 72 h was reduced at the levels of mRNA (93%) and protein (62%) using real-time reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting. Interestingly, the apoptosis index of the HepG2 cells increased up to 85%, detected by Annexin V-fluorescein isothiocyanate, suggesting that NF-κB is overexpressed in hepatoma cells and can be inhibited by NF-κB/p65 siRNA through the apoptotic mechanism. Thus, we conclude that NF-κB is a potential molecular target for HCC gene therapy.

[Dynamic alterations of VEGF and intervention of its expression on effect of hepatocyte malignant transformation].

OBJECTIVE: To investigate the influences of VEGF expression through the intervention of thalidomide in malignant transformation of hepatocytes. METHODS: Hepatoma model was induced with 2-fluorenyl-acetamide (2-FAA, 0.05%) in male SD rats. And thalidomide was administered intragastrically to block the progress of hepatoma. Some rats were sacrificed at a fortnightly interval. Morphological changes were observed by pathological examinations (HE staining). The VEGF expressions in rat liver tissues were detected by ELISA and immunohistochemistry respectively. RESULTS: Hepatocytes in rats fed with 2-FAA showed vacuole-like denaturations at an early stage, dysplastic nodules appeared at a middle stage and finally progressed to tubercles of cancerous nest. All were the manifestations of highly differentiated hepatocellular carcinoma (HCC). An increasing tendency of hepatic VEGF protein was found for normal liver to precancerous to cancerous tissues during the development of hepatoma. The VEGF levels in hepatoma were significantly higher than those in normal ones. Thalidomide repressed the morphologic change of hepatic cells. And the VEGF level of thalidomide group was lower than those in 2-FAA group. CONCLUSION: Thalidomide can inhibit the hepatic VEGF expression and arrest the development of rat hepatoma.

PLGA nanoparticle-based docetaxel/LY294002 drug delivery system enhances antitumor activities against gastric cancer.

Docetaxel (TXT) is acknowledged as one of the most important chemotherapy agents for gastric cancer (GC). PI3K/AKT signaling is frequently activated in GC, and its inhibitor LY294002 exerts potent antitumor effects. However, the hydrophobicity of TXT and the poor solubility and low bioavailability of LY294002 limit their clinical application. To overcome these shortcomings, we developed poly(lactic acid/glycolic) (PLGA) nanoparticles loaded with TXT and LY294002. PLGA facilitated the accumulation of TXT and LY294002 at the tumor sites. The in vitro functional results showed that PLGA(TXT+LY294002) exhibited controlled-release and resulted in a markedly reduced proliferative capacity and an elevated apoptosis rate. An in vivo orthotopic GC mouse model and xenograft mouse model confirmed the anticancer superiority and tumor-targeting feature of PLGA(TXT+LY294002). Histological analysis indicated that PLGA(TXT+LY294002) was biocompatible and had no toxicity to major organs. Characterized by the combined slow release of TXT and LY294002, this novel PLGA-based TXT/LY294002 drug delivery system provides controlled release and tumor targeting and is safe, shedding light on the future of targeted therapy against GC.

KCNQ1OT1/miR-217/ZEB1 feedback loop facilitates cell migration and epithelial-mesenchymal transition in colorectal cancer.

Long noncoding RNAs are widely acknowledged as a group of regulatory factors in various diseases, especially in cancers. KCNQ1 overlapping transcript 1 (KCNQ1OT1) has been reported as oncogene in human cancers. However, the role of KCNQ1OT1 in colorectal cancer (CRC) has not been fully explained. Based on the database analysis, KCNQ1OT1 was highly expressed in CRC samples and predicted the poor prognosis for CRC patients. Functional experiments revealed that KCNQ1OT1 knockdown negatively affected the proliferation, migration and epithelial-mesenchymal transition (EMT) in CRC cells. Moreover, we identified the cytoplasmic localization of KCNQ1OT1 in CRC cells, indicating the post-transcriptional regulation of KCNQ1OT1 on gene expression. Mechanism experiments including RNA Immunoprecipitation (RIP) assay and dual luciferase reporter assays verified that KCNQ1OT1 acted as a competing endogenous RNA (ceRNA) in CRC by sponging microRNA-217 (miR-217) to up-regulate the expression of zinc finger E-box binding homeobox 1 (ZEB1). Further mechanism investigation revealed that ZEB1 enhanced the transcription activity of KCNQ1OT1 by acting as a transcription activator. Finally, rescue assays were designed to demonstrate the effect of KCNQ1OT1-miR-217-ZEB1 feedback loop on proliferation, migration, and EMT of CRC cells. In brief, our research findings revealed that ZEB1-induced upregulation of KCNQ1OT1 improved the proliferation, migration and EMT formation of CRC cells via regulation of miR-217/ZEB1 axis.

Gambogic acid regulates the migration and invasion of colorectal cancer via microRNA-21-mediated activation of phosphatase and tensin homolog.

Gambogic acid (GA) has been reported to inhibit cancer cell proliferation and migration and enhance apoptosis. Several signaling pathways were identified to be involved in GA function, including PI3K/Akt, caspase-3 apoptosis and TNF-α/NF-κB. However, to the best of our knowledge, the association between miRNA and GA has not been explored. The present study initially demonstrated that GA could inhibit HT-29 cancer cell proliferation using an MTT assay. In addition, a Transwell assay and a wound-healing assay respectively indicated that GA inhibited HT-29 cancer cell invasion and migration, which was also confirmed by the increased MMP-9 protein expression. Furthermore, GA induced the apoptosis of HT-29 cancer cells in an Annexin V and PI double staining assay. Moreover, treatment with GA significantly decreased miR-21 expression in these cells. Additionally, western blot analysis demonstrated that GA treatment enhanced the activation of phosphatase and tensin homolog (PTEN) along with the suppression of PI3K and p-Akt. Furthermore, miR-21 mimics reversed all the aforementioned activities of GA, which indicated that miR-21 was the effector of GA and blocked PI3K/Akt signaling pathway via enhancing PTEN activity. In summary, GA induced HT-29 cancer cell apoptosis via decreasing miR-21 expression and blocking PI3K/Akt, which may be a useful novel insight for future CRC treatment.

Characteristics of hepatic igf-ii expression and monitored levels of circulating igf-ii mRNA in metastasis of hepatocellular carcinoma.

The prognosis of hepatocellular carcinoma (HCC) remains dismal. Insulin-like growth factor II (IGF-II), a fetal growth factor, is highly expressed during HCC development. We examined serum IGF-II levels and circulating IGF-II messenger RNA (mRNA) expression and analyzed the clinicopathologic characteristics in patients with liver diseases. The higher IGF-II level in the serum of patients with HCC could be correlated with hepatitis B virus infection but not with patient sex, age, tumor size, or α-fetoprotein (AFP) level. Total RNAs were extracted from liver tissues or peripheral blood mononuclear cells, and IGF-II complementary DNA (cDNA) and AFP cDNA were synthesized through random primers and reverse transcriptase; gene fragments were amplified by nested polymerase chain reaction and confirmed by sequencing. The incidence of the hepatic IGF-II gene was 100% in HCC, 54.3% in paracancerous tissues, and none in noncancerous tissues. The incidence rates for circulating IGF-II and AFP genes were 34.3% and 52.7%, respectively, and for both, 61.6% in patients with HCC. They were 100% in cases with extrahepatic metastasis. The IGF-II abnormality associates with HCC, and circulating IGF-II and IGF-II mRNA are useful molecular markers for HCC differential diagnosis and hematogenous metastasis.