Downregulation of Roundabout guidance receptor 2 suppresses hepatocellular carcinoma progression by interacting with Y-box binding protein 1.

Roundabout guidance receptor 2 (Robo2) is closely related to malignant tumors such as pancreatic cancer and liver fibrosis, but there is no relevant research on the role of Robo2 in HCC. The study will further explore the function and mechanism of Robo2 and its downstream target genes in HCC. Firstly, Robo2 protein levels in human HCC tissues and paired adjacent normal liver tissues were detected. Then we established HepG2 and Huh7 hepatoma cell lines with knock-down Robo2 by transfection with lentiviral vectors, and examined the occurrence of EMT, proliferation and apoptosis abilities in HCC cells by western blot, flow cytometry, wound healing assay and TUNEL staining. Then we verified the interaction between Robo2 and its target gene by Co-IP and immunofluorescence co-staining, and further explored the mechanism of Robo2 and YB-1 by rescue study. The protein expression level of Robo2 in HCC was considerably higher than that in the normal liver tissues. After successfully constructing hepatoma cells with knock-down Robo2, it was confirmed that down-regulated Robo2 suppressed EMT and proliferation of hepatoma cells, and accelerated the cell apoptosis. High-throughput sequencing and validation experiments verified that YB-1 was the downstream target gene of Robo2, and over-expression of YB-1 could reverse the apoptosis induced by Robo2 down-regulation and its inhibitory effect on EMT and proliferation. Robo2 deficiency inhibits EMT and proliferation of hepatoma cells and augments the cell apoptosis by regulating YB-1, thus inhibits the occurrence of HCC and provides a new strategy for the treatment of HCC.

Effects of Let-7c on the processing of hepatitis B virus associated liver diseases.

BACKGROUND: The most common type of cancer of the digestive system is hepatocellular carcinoma. In China, many patients harbour HBV. The lin28B/Let-7c/MYC axis is associated with the occurrence of many cancers. Therefore, we aimed to illuminate the function of the lin28B/Let-7c/MYC axis in hepatocellular carcinoma. We aimed to evaluate the critical involvement of lin28B and Let-7c in the carcinogenesis of human hepatocellular carcinoma (B-HCC). METHODS: Data from the GEO database were used to analyse differentially expressed genes and IRGs. A protein - protein interaction (PPI) network and Venn diagram were generated to analyse relationships. Real-time RT-PCR, Western blotting, and cell counting kit-8 assays were used to examine the association of lin28B, Let-7c, and MYC with cell proliferation. RESULTS: A total of 2552 functionally annotated differentially expressed RNAs were analysed in HBV patients from the GSE135860 database. In addition, 46 let-7c target genes were screened in HBV patients, and the interactions were analysed through PPI network analysis. The results confirmed that Let-7c and its target genes play a key role in HBV-related diseases. Next, we discovered a gradual decrease in Let-7c expression during the progression from HBV-associated chronic hepatitis (B-CH) and HBV-associated liver cirrhosis (B-LC) to B-HCC. We found evidence for a negative association between lin28B expression and Let-7c expression. The expression of MYC was obviously upregulated when Let-7c was inhibited. CONCLUSION: Our results highlight that Let-7c and lin28B participate in the carcinogenesis of HBV-associated diseases through the lin28B/Let-7c/MYC axis.

Cyto-biological effects of microRNA-424-5p on human colorectal cancer cells.

MicroRNA (miR)-424-5p is overexpressed in colorectal cancer (CRC); however, its role, clinical significance and underlying molecular mechanism have remained to be fully elucidated. The aim of the present study was to investigate the roles of miR-424-5p in CRC and the underlying mechanisms. It was demonstrated that miR-424-5p is overexpressed in CRC, based on bioinformatics analysis using The Cancer Genome Atlas TCGA and analysis of tissue samples from patients with CRC from The First Hospital of Hebei Medical University, and the expression of miR-424-5p was associated with the depth of invasion and Dukes' staging. In CRC cells, the oncogenic roles of miR-424-5p were also verified by Cell Counting Kit-8, wound healing and Transwell assays. To identify target genes, all transcripts were compared between miR-424-5p mimic-transfected SW480 cells and mimic control cells by transcriptome sequencing. Subsequently, the differentially expressed genes (DEGs) were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The DEGs were revealed to be significantly enriched in the GO terms 'serine hydrolase activity,' 'serine-type peptidase activity' and 'serine-type endopeptidase activity'. KEGG signaling pathway analysis indicated that the DEGs were significantly enriched in 'endocytosis', 'regulation of actin cytoskeleton', 'Wnt signaling pathway' and 'ubiquitin-mediated proteolysis signaling pathway'. These results suggested that miR-424-5p is a potential target in the treatment of CRC.

TRIM67 inhibits tumor proliferation and metastasis by mediating MAPK11 in Colorectal Cancer.

Purpose: We recently reported that tripartite motif-containing 67 (TRIM67) activates p53 to suppress colorectal cancer (CRC). However, the function and mechanism of TRIM67 in the inhibition of CRC cell proliferation and metastasis remains to be further elucidated. Methods: We detected the expression of TRIM67 in CRC tissues compared with normal tissues and confirmed its relationship with clinicopathological features. DNA methylation of TRIM67 was analyzed to determine its significantly hypermethylated sites in CRC tissues. CCK-8, colony formation, transwell migration, and Matrigel invasion assays were performed to evaluate the effects of TRIM67 on cell proliferation and metastasis in CRC cells. RNA sequencing of TRIM67 and TRIM67 rescue experiments were performed to reveal its mechanisms in CRC cell proliferation and metastasis. Results: TRIM67 expression was significantly downregulated in CRC tissues and its expression was associated with clinical stage, invasive depth, tumor size, lymph node metastasis, and Dukes' stage. Three methylation sites were significantly hypermethylated and negatively correlated with TRIM67 expression in CRC tissues. TRIM67 suppressed proliferation, migration, and invasion in CRC cells. RNA sequencing revealed that protein mitogen-activated protein kinase 11 (MAPK11) was a potential downstream negative regulatory gene of TRIM67. Reversing MAPK11 expression could rescue the effects of TRIM67 on the proliferation and metastasis of CRC cells. Conclusion: TRIM67 inhibited cell proliferation and metastasis by mediating MAPK11 in CRC, and may be a potential target to inhibit CRC metastasis.

Thapsigargin promotes colorectal cancer cell migration through upregulation of lncRNA MALAT1.

Colorectal cancer (CRC) is the third most common tumor in the world; however, the role and mechanism of endoplasmic reticulum (ER) stress in CRC metastasis remains largely unclear. Metastasis­associated lung adenocarcinoma transcript 1 (MALAT1) is a long non­coding RNA (lncRNA), which has previously been associated with CRC metastasis. It has been suggested that ER stress pathways regulate lncRNA expression; however, the effect of ER stress on MALAT1 expression in cancer is unknown. The present study aimed to investigate the relationship between ER stress pathways, MALAT1 expression and cell migration in CRC cells. ER stress was induced by thapsigargin (TG); low dose TG induced the migration of HT29 and HCT116 cells, but not SW1116 and SW620 cells. This effect was associated with increased expression levels of MALAT1, as the knockdown of MALAT1 prevented TG­induced cell migration. TG­induced MALAT1 expression was associated with inositol­requiring enzyme 1 (IRE1) expression and activation of the protein kinase R (PKR)­like ER kinase (PERK) signaling pathway. X­box­binding protein 1 (XBP1) and activating transcription factor 4 (ATF4) binding sites were predicted to be located in the MALAT1 gene promoter regions and the expression of MALAT1 was positively associated with XBP1 and ATF4 expression levels in CRC tissue samples. Thus, these findings indicated that ER stress may promote the migration of CRC cells and contribute to the progression of CRC through the activation of the IRE1/XBP1 and PERK/eIF2α/ATF4 signaling pathways. In conclusion, to the best of our knowledge, this study is the first report that lncRNA MALAT1 expression is regulated by the IRE1/XBP1 pathway in CRC.

ITGB4 is a novel prognostic factor in colon cancer.

Integrin ß4 (ITGB4) has been reported to be involved in carcinomas. Currently, ITGB4 has been characterized in colon cancer, however, its clinical significance is not very clear. In the present study, we utilized the large public datasets from NCBI Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases and collected clinical samples in our center to investigate the transcriptional expressions of ITGB4 in colon cancer, and then explored the associations of ITGB4 with clinicopathological features and overall survival. The statistical analyses suggested that ITGB4 mRNA expressions were up-regulated significantly in colon cancer. High ITGB4 expression was observed to be associated with elder onset age, proximal tumor location, and high microsatellite instability (MSH) status. Further, Kaplan-Meier curves and univariate analysis demonstrated high ITGB4 expression was significantly associated with unfavorable overall survival in colon cancer (HR=1.292, 95%CI=1.084-1.540, P=0.004). And significant association was also found after adjusting the confounding factors including age, gender, and stage (adjusted HR=1.254, 95%CI=1.050-1.497, P=0.012). The annotation of ITGB4 co-expressed genes suggested the pathways including cell growth, positive regulation of cell migration, and apoptotic signaling might be involved in the potential mechanisms of ITGB4 in colon cancer development. The molecular regulation mechanism of ITGB4 ectopic expression in colon cancer was also explored and the results indicated that ITGB4 might be up-regulated by the transcription factor FOSL1 (FOS like 1, AP-1 Transcription Factor Subunit) and its promoter hypomethylation. Our results revealed that ITGB4 might be a therapeutic target and prognosis marker for individual therapy of colon cancer.

Identification and characterization of loop7 motif and its role in regulating biological function of human APOBEC3G through molecular modeling and biological assay.

Human APOBEC3G (hA3G) is a cytidine deaminase which inhibits HIV-1 replication. The HIV-1 accessory protein viral infectivity factor (Vif) counteracts with hA3G by targeting it for proteasomal degradation. In this work, we constructed and optimized molecular models of the hA3G dimer and the hA3G-Vif complex. The molecular modeling study revealed that the loop7 motif of hA3G appears on the interfaces of both the hA3G-Vif complex and the hA3G dimer. Biochemical analysis provided evidence suggesting that binding of Vif to hA3G results in steric blocking of hA3G dimerization, implying that monomeric hA3G serves as a substrate for Vif-mediated degradation. Furthermore, we presented evidence for the important roles of the loop7 motif, especially the central residues within the region, in hA3G dimerization, hA3G--Vif interaction, Vif-mediated hA3G degradation as well as subcellular localization of hA3G. This work highlights a multiple-task interface formed by loop7 motif, which regulates biological function of hA3G, thus providing the feasibility of the strategy of blocking Vif-mediated A3G degradation by targeting the putative site around loop7.

Biological effects and clinical characteristics of microRNA-106a in human colorectal cancer.

MicroRNAs serve important roles in various diseases, particularly cancer. microRNA-106a (miR-106a) exhibits abnormal expression and oncogenic activity in carcinogenesis. The clinical significance of the abnormal expression of miR-106a in colorectal cancer is poorly understood. In the present study, miR-106a expression from colorectal cancer tissues was quantified using the reverse transcription-quantitative polymerase chain reaction. The overexpression or knockdown of miR-106a was performed by transfection with microRNA mimic or inhibitor in human colorectal carcinoma HCT116 cells. The overexpression of miR-106a promoted viability and inhibited apoptosis in colorectal cancer cells. The association between miR-106a expression and clinicopathological factors was analyzed, and it was identified that miR-106a exhibited significantly increased expression in adenocarcinoma tissues compared with in mucinous carcinoma tissues, and the expression of miR-106a was identified to be associated with the depth of invasion and differentiation. The expression of miR-106a in plasma was also determined and it was identified that increased expression of miR-106a, as a characteristic of patients with colorectal cancer, may be distinguished from that of other patients by digitization of the areas under the receiver operating characteristic curves. These data suggested that miR-106a is a potential biomarker in the diagnosis of colorectal carcinoma. However, the underlying molecular mechanism of miR-106a-promoted viability and inhibition of apoptosis requires further investigation.

Tumor-suppressing roles of miR-214 and miR-218 in breast cancer.

MicroRNAs (miRNAs) are small non-coding RNAs that are key post-transcriptional regulators of gene expression. MicroRNA-214 (miR-214) and microRNA-218 (miR-218) have shown the function of tumor suppressors in various types of human cancers. However, the biological functions of miR-214 and miR-218 in breast cancer have not been elucidated completely. The present study evaluated the expression and biological function of miR-214 and miR-218 in human breast cancer. Our results revealed that the expression of miR-214 and miR-218 were significantly decreased in breast cancer tissues compared with adjacent tissues. The aberrant expression of miR-214 and miR-218 were negatively associated with Ki-67, and the miR-218 expression was positively associated with progesterone receptor (PR) in breast cancer tissues. In vitro, the cell proliferation and migration were decreased, cell apoptosis was induced, and cell cycle was also disturbed in miR-214 or miR-218 overexpressed breast cancer cells. Our results demonstrated that miR-214 and miR-218 function as tumor suppressors in breast cancer, and may become biomarkers and potential therapeutic targets in breast cancer.

Functional analysis of the two cytidine deaminase domains in APOBEC3G.

Human APOBEC3G (hA3G), a cytidine deaminase with two cytidine deaminase domains (CDs), has been identified as an anti-HIV-1 host factor. Although the two CDs of hA3G have been extensively characterized, there is still debate on the role of the CDs in the biological function of hA3G. In this work, we constructed three hA3G mutants CD1-1, CD2-2 and CD2-1, which contain duplicate CD1 domain, duplicate CD2 domain and position switched CD domain respectively, and investigated the effect of CD domain replacement or switch upon virion encapsidation, Vif-mediated degradation, deamination and antiviral activity of hA3G. The results showed that the two CD domains were functionally equivalent in virion encapsidation and the interaction with HIV-1 Vif of hA3G, whereas CD domain switch or replacement greatly affected the sensitivity to Vif induced degradation, editing and antiviral activity of hA3G. Although the CD2 domain was shown to possess the deamination activity, CD2-2 incorporated efficiently into HIV-1 was unable to mutate viral cDNA, suggesting that CD1 also involved in the enzymatic function. Interestingly, CD2-1 retained considerable deamination activity with a different sequence preference. Taken together, our results suggest that CD domain may play a structural role in virion encapsidation and Vif-mediated degradation of hA3G, and coordination of the two CD domains is required for its editing and antiviral activity.