Qntrolling the LncRNA HULC-Tregs-PD-1 axis inhibits immune escape in the tumor microenvironment.

Background: Immune escape remains a major challenge in the treatment of malignant tumors. Here, we studied the mechanisms underlying immune escape in the tumor microenvironment and identified a potential therapeutic target. Methods: Pathological specimens from patients with liver cancer, soft tissue sarcoma, and liver metastasis of colon cancer were subjected to immunohistochemistry analysis to detect the expression of programmed death-1 (PD-1) in the tumor microenvironment (TME). Additionally, the expression of regulatory T cells (Tregs) and long non-coding RNAs (lncRNAs), such as highly upregulated in liver cancer (HULC) was evaluated by fluorescence in situ hybridization, and the relationship between HULC, Treg cells, and PD-1 was determined. The animals were divided into H22 hepatic carcinoma and S180 sarcoma groups. Each group was divided into Foxp3-/-C57BL/6J and C57BL/6J mice. Thereafter, mice were inoculated with 0.1 ml S180 sarcoma cells or 0.1 ml H22 hepatoma cells, at a concentration of 1 × 107/ml. The number of splenic CD4+CD25+Foxp3+ T cells was detected by flow cytometry, and serum interleukin-10 (IL-10) and transforming growth factor ß1 (TGF-ß1) levels were detected using a Luminex liquid suspension chip. Expression of PD-1, fork head box P3 (Foxp3), and HULC in the TME, were analyzed and the therapeutic effect of inhibiting the lncRNA HULC-Treg-PD-1 axis in malignant tumors was determined. Results: High expression of lncRNA HULC promotes the proliferation of Treg cells and increases PD-1 expression in the tumor microenvironment. The HULC-Treg-PD-1 axis plays an immunosuppressive role and promotes the proliferation of malignant tumors. Knocking out the Foxp3 gene can affect the HULC-Treg-PD-1 axis and reduce PD-1, IL-10, and TGF-ß1 expression to control the growth of malignant tumors. Conclusion: The lncRNA HULC-Treg-PD-1 axis promotes the growth of malignant tumors. This axis could be modulated to reduce PD-1, IL-10, and TGF-ß1 expression and the subsequent immune escape. The inhibition of immune escape in the tumor microenvironment can be achieved by controlling the LncRNA HULC-Treg-PD-1 axis.

Association between multiple sclerosis and cancer risk: A two-sample Mendelian randomization study.

Multiple Sclerosis (MS) is an immune-related disease and the relationship between MS and cancer has raised attention. Previous studies of the relationship between MS and cancer have reached conflicting conclusions. In this study, the two-sample MR method is used to investigate whether MS has a causal correlation with cancers and offer scientific evidence for cancer prevention. Single nucleotide polymorphisms (SNPs) related to MS were obtained from the genome-wide association study (GWAS) based on International Multiple Sclerosis Genetics Consortium (IMSGC) and SNPs related to 15 types of cancers were obtained from the GWASs based on UK Biobank. Inverse variance weighted (IVW) method was mainly used to assess causal effects. Sensitivity analyses were conducted with Cochran's Q-test, MR Egger intercept, leave-one-out test, and MR Steiger method. IVW analysis showed that MS was only associated with a marginal increased risk of cervical cancer (OR 1.0004, 95% CI 1.0002-1.0007, p = 0.0003). Sensitivity analyses showed that the results of MR analysis were robust and found no heterogeneity, no pleiotropy, and no reverse causation. In conclusion, this study finds no causal relationship between MS and 15 types of cancers except cervical cancer.

Global expression profiling of metabolic pathway-related lncRNAs in human gastric cancer and the identification of RP11-555H23.1 as a new diagnostic biomarker.

BACKGROUND: Long noncoding RNAs (lncRNAs) have roles in regulating metabolism; however, the global expression profile of metabolic pathway-associated lncRNAs in gastric cancer is unknown. The purpose of our study was to examine metabolic pathway-related lncRNAs in gastric cancer and their possible diagnostic values. METHODS: Differential expression patterns of metabolic pathway-related lncRNAs between gastric cancer and paired nontumor tissues were detected using metabolic pathway-associated lncRNA microarrays. The expression of RP11-555H23.1, one representative metabolic pathway-associated lncRNA, was validated using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The associations between RP11-55H23.1 expression and the clinicopathological features of gastric cancer patients were analyzed. A receiver operating characteristic (ROC) curve was further established. RESULTS: A total of 114 differentially expressed metabolic pathway-associated lncRNAs (fold change >2, P < 0.05) between cancer and nontumor tissues were found (GEO No. GSE96856). Among them, TUG1, RP11-555H23.1, RP1-257I20.13, UGP2, GCSHP3, and XLOC_000889 lncRNAs were downregulated more than sixfold in gastric cancer tissues. In contrast, RP11-605F14.2, TBC1D3P5, BC130595, LINC00475, RP11-19P22.6, BC080653, XLOC_004923, AFAP1-AS1, EPB49, and RP11-296I10.3 lncRNAs were upregulated more than sixfold in gastric cancer tissues. We further demonstrated that RP11-555H23.1 expression was significantly correlated with TNM stage (P = 0.038). The area under the ROC curve (AUC) was 0.65, and the specificity and sensitivity were 62% and 81%, respectively. CONCLUSIONS: Metabolic pathway-associated lncRNAs play an important role in the occurrence of gastric cancer, and metabolic pathway-associated lncRNAs, such as RP11-555H23.1, may represent novel biomarkers of gastric cancer.

The Study on Polymorphism of TrxR and Nrf2/HO-1 Signaling Pathway in Kaschin-Beck Disease.

Kaschin-Beck disease (KBD) is an endemic, chronic, and degenerative osteoarthropathy, which seriously impairs the quality of patients' life. We detected the expression of TrxR by ELISA and found that TrxR was lower in KBD than in normal control group significantly (P < 0.001); this result indicated that TrxR must be related to KBD. We retrieved cSNPs in NCBI SNP database and used three bioinformatics programmers, including SIFT, PolyPhen, and SNP3d, to help select the researched nsSNP. Then, we used PCR-RFLP to analyze the relationship between the SNP site rs5746841 in TrxR2 gene and susceptibility of KBD and detected the expression of Nrf2 and HO-1 by western blot. The results showed that the genotype of rs5746841 in 93 normal controls and 103 KBD subjects were C/C totally, but A/A and A/C were not found, which indicated preliminarily that there was no correlation between rs5746841 in TrxR2 gene and susceptibility of KBD. The expression of TrxR was lower in KBD than in normal control group significantly, while the expressions of Nrf2 and HO-1 were higher in KBD than in normal control group. These results indicated that the low expression of selenoprotein TrxR may be a candidate factor of KBD, which related to Nrf2/HO-1 signaling pathway.

Identification and functional annotation of metabolism-associated lncRNAs and their related protein-coding genes in gastric cancer.

BACKGROUND: Long noncoding RNAs (lncRNAs) play important roles in carcinogenesis. However, the roles of metabolism-associated lncRNAs in cancers are still unclear. METHODS: A microarray of metabolism-associated lncRNAs was used to detect their expression patterns between gastric cancer and paired nontumorous tissues. Its results and gastric cancer differential gene expression data from public databases were used to screen the metabolic pathway-associated lncRNAs. A metabolic network with microRNAs (miRNAs), lncRNAs, and protein-coding genes was further constructed. Finally, the expression of TOPORS antisense RNA 1 (TOPORS-AS1), a screened highly expressed lncRNA and its associated protein-coding gene, NADH: ubiquinone oxidoreductase subunit B6 (NDUFB6), were verified by reverse transcription polymerase chain reaction. RESULTS: A total of eight upregulated and one downregulated lncRNAs and 25 upregulated and 20 downregulated protein-coding genes were found to be involved in metabolism in gastric cancer. Within the lncRNAs-miRNAs-mRNAs metabolic network, 78 miRNA-target links, 546 positive coexpression relationships, and 191 protein-protein interactions were found. The expression of TOPORS-AS1 and its associated gene, NDUFB6 in gastric cancer tissues was significantly lower than that in adjacent nontumor tissues. Moreover, NDUFB6 expression was associated with the invasion and distal metastasis of gastric cancer. CONCLUSIONS: The metabolism-associated lncRNAs play important roles in the occurrence of gastric cancer.

Screening differential circular RNA expression profiles reveals hsa_circ_0004018 is associated with hepatocellular carcinoma.

Circular RNAs (circRNAs) have been emerged as an indispensable part of endogenous RNA network. However, the expression significance of circRNAs in hepatocellular carcinoma (HCC) is rarely revealed. The aim of this study was to determine the circRNA expression profile in HCC, and to investigate their clinical significances and relevant mechanisms for cancer progression. The global circRNA expression profile in HCC was measured by circRNA microarray. Levels of one representative circRNAs, hsa_circ_0004018, were confirmed by real-time reverse transcription-polymerase chain reaction. The expression levels of hsa_circ_0004018 in HCC were significantly lower compared with para-tumorous tissue (P<0.001). Our data further showed that lower expression of hsa_circ_0004018 was correlated with serum alpha-fetoprotein (AFP) level, tumor diameters, differentiation, Barcelona Clinic Liver Cancer stage and Tumor-node-metastasis stage. More importantly, we detected liver tissues from chronic hepatitis, cirrhosis and HCC patients; and found that hsa_circ_0004018 harbored HCC-stage-specific expression features in diverse chronic liver diseases (P<0.001). The area under receiver operating characteristic curve was up to 0.848 (95% CI=0.803-0.894, P<0.001). The sensitivity and specificity were 0.716 and 0.815, respectively. Finally, hsa_circ_0004018 might be involved in cancer-related pathways via interactions with miRNAs.

Circular RNA 0000096 affects cell growth and migration in gastric cancer.

BACKGROUND: Circular RNAs (circRNAs) are a class of non-coding RNAs broadly expressed in cells of various species. Their role in cancers, especially in gastric cancer, is poorly understood. METHODS: Circular RNA 0000096 (hsa_circ_0000096) levels in 101 paired gastric cancer tissues and adjacent non-tumorous tissues from patients with gastric cancer were detected by real-time quantitative reverse transcription-polymerase chain reaction. A receiver operating characteristic curve was generated to evaluate the diagnostic value of hsa_circ_0000096. RNA interference was used to manipulate the expression of hsa_circ_0000096. Its biological effects were evaluated by flow cytometry, real-time cell analysis, a wound scratch assay, western blot analysis and xenograft models. RESULTS: Hsa_circ_0000096 was found to be significantly downregulated in gastric cancer tissues and gastric cancer cell lines compared with paired adjacent non-tumorous tissues and normal gastric epithelial cells (P<0.001). Moreover, knockdown of hsa_circ_0000096 significantly inhibited cell proliferation and migration in vitro and in vivo. The results of both immunohistochemical and western blot analyses showed that the protein levels of cyclin D1, cyclin-dependent kinase 6 (CDK6), matrix metalloproteinase-2 and MMP-9 were significantly reduced in vitro and in vivo. A gastric cancer xenograft nude mouse model indicated that Ki67 and VEGF were reduced in a dose-dependent manner following knockdown of hsa_circ_0000096. However, the expression of E-cadherin increased. CONCLUSIONS: Hsa_circ_0000096 may be used as a potential novel biomarker for gastric cancer. It affects gastric cancer cell growth and migration by regulating cyclin D1, CDK6, MMP-2 and MMP-9.

Clinical significance of the long noncoding RNA RP11-19P22.6-001 in gastric cancer.

BACKGROUND: Long non-coding RNAs (lncRNAs) play roles in carcinogenesis; however, the significance of most lncRNAs in gastric cancer is unclear. OBJECTIVE: To explore the diagnostic value of the long noncoding RNA RP11-19P22.6-001 in gastric cancer. METHODS: RP11-19P22.6-001 levels in gastric cancer tissues and paired non-tumor tissues were analyzed by quantitative reverse transcription-polymerase chain reaction. Since RP11-19P22.6-001 acts in cis to regulate nitric oxide synthase 2 (NOS2), we also analyzed NOS2 expression and its correlation with gastric cancer. Finally, to analyze the potential diagnostic values of RP11-19P22.6-001, a receiver operating characteristic (ROC) curve was constructed. RESULTS: RP11-19P22.6-001 expression was significantly downregulated in 70.91% (78/110) of gastric cancer tissues compared to that of adjacent normal tissues. However, NOS2 was upregulated in 75.45% (83/110) of gastric cancer tissues. RP11-19P22.6-001 expression levels were associated with invasion, lymph node metastasis, and TNM stage. The areas under the ROC curves (AUC) were 0.662 and 0.671 for RP11-19P22.6-001 and NOS2, respectively. More importantly, the combined use of these parameters increased the AUC to 0.704. CONCLUSIONS: RP11-19P22.6-001 and NOS2 may be new biomarkers for the diagnosis and prognosis of gastric cancer. The combined use of lncRNAs and their target genes may be a promising method to increase the diagnostic value of lncRNAs in cancer.

Effects of sodium selenite on c-Jun N-terminal kinase signalling pathway induced by oxidative stress in human chondrocytes and c-Jun N-terminal kinase expression in patients with Kashin-Beck disease, an endemic osteoarthritis.

The c-Jun N-terminal kinases (JNK) are members of the mitogen-activated protein kinase family and are activated by environmental stress. Se plays an important role in the biological pathways by forming selenoprotein. Selenoproteins have been shown to exhibit a variety of biological functions including antioxidant functions and maintaining cellular redox balance, and compromise of such important proteins would lead to oxidative stress and apoptosis. We examined the expression levels of JNK in Kashin-Beck disease (KBD) patients, tested the potential protective effects of sodium selenite on tert-butyl hydroperoxide (tBHP)-induced oxidative injury and apoptosis in human chondrocytes as well as its underlying mechanism in this study. We produced an oxidative damage model induced by tBHP in C28/I2 human chondrocytes to test the essential anti-apoptosis effects of Se in vitro. The results indicated that the expression level of phosphorylated JNK was significantly increased in KBD patients. Cell apoptosis was increased and molecule expressions of the JNK signalling pathway were activated in the tBHP-injured chondrocytes. Na2SeO3 protected against tBHP-induced oxidative stress and apoptosis in cells by increasing cell viability, reducing reactive oxygen species generation, increasing Glutathione peroxidase (GPx) activity and down-regulating the JNK pathway. These results demonstrate that apoptosis induced by tBHP in chondrocytes might be mediated via up-regulation of the JNK pathway; Na2SeO3 has an effect of anti-apoptosis by down-regulating the JNK signalling pathway.

Molecular mechanisms of long noncoding RNAs on gastric cancer.

Long noncoding RNAs (lncRNAs) are non-protein coding transcripts longer than 200 nucleotides. Aberrant expression of lncRNAs has been found associated with gastric cancer, one of the most malignant tumors. By complementary base pairing with mRNAs or forming complexes with RNA binding proteins (RBPs), some lncRNAs including GHET1, MALAT1, and TINCR may mediate mRNA stability and splicing. Other lncRNAs, such as BC032469, GAPLINC, and HOTAIR, participate in the competing endogenous RNA (ceRNA) network. Under certain circumstances, ANRIL, GACAT3, H19, MEG3, and TUSC7 exhibit their biological roles by associating with microRNAs (miRNAs). By recruiting histone-modifying complexes, ANRIL, FENDRR, H19, HOTAIR, MALAT1, and PVT1 may inhibit the transcription of target genes in cis or trans. Through these mechanisms, lncRNAs form RNA-dsDNA triplex. CCAT1, GAPLINC, GAS5, H19, MEG3, and TUSC7 play oncogenic or tumor suppressor roles by correlated with tumor suppressor P53 or onco-protein c-Myc, respectively. In conclusion, interaction with DNA, RNA and proteins is involved in lncRNAs' participation in gastric tumorigenesis and development.

[Circular RNAs and their roles in tumorigenesis].

Recently, with the development of RNA research techniques, a wide variety of circular RNAs (circRNAs) have been discovered and some of them are confirmed to have crucial biological functions. CircRNAs arise from exons (i.e. exonic circRNAs) or introns (i.e. intronic circRNAs). Acting as microRNA sponges or combining with proteins, circRNAs participate in the regulation of gene expression and influence the activity of some proteins. In addition, some circRNAs even encode proteins. More importantly, several circRNAs play a key role in the occurrence and progression of some tumors, including stomach, liver, colon, breast, cervical, and ovarian cancers. Therefore, circRNAs may be a novel type of diagnostic marker and therapeutic target of cancers.

Using circular RNA as a novel type of biomarker in the screening of gastric cancer.

BACKGROUND: Circular RNAs (circRNAs), a class of endogenous RNAs, have emerged as an enigmatic class of RNAs. Little is known about their value in the diagnosis of cancers. METHODS: The targeted circRNA of this study was selected using two circRNA databases: CircBase (http://circbase.org/) and circ2Traits (http://gyanxet-beta.com/circdb/). Divergent primers, rather than commonly used convergent primers, for the circRNA were designed. The circRNA levels in 101 paired gastric cancer tissues and adjacent nontumorous tissues from surgical gastric cancer patients and 36 paired plasma samples from preoperative and postoperative gastric cancer patients were analyzed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The specificity of the amplified products was measured by melting curve analysis and DNA sequencing. To observe the stability of circRNA, three randomly selected samples of gastric cancer tissues were stored at room temperature, 4°C and -20°C, and then, their circRNA levels were analyzed. To verify the reproducibility of qRT-PCR, circRNA levels were detected in a set of specimens (n=15) in two independent experiments with an interval of one day. Then, the correlation of their Ct values was determined. The relationships between circRNA expression levels and clinicopathological factors of patients with gastric cancer were further analyzed by one-way analysis of variance. A receiver operating characteristic (ROC) curve was established to evaluate the diagnostic value. RESULTS: Hsa_circ_002059, a typical circular RNA, was first found to be significantly downregulated in gastric cancer tissues compared with paired adjacent nontumorous tissues (p<0.001). Its levels in plasma collected from postoperative gastric cancer patients were found significantly different from those from preoperative gastric cancer patients. The area under the ROC curve was 0.73. Importantly, we further found that lower expression levels were significantly correlated with distal metastasis (P=0.036), TNM stage (P=0.042), gender (P=0.002) and age (P=0.022). The stability of circRNAs and the reproducibility of the qRT-PCR method for detecting circRNA levels were determined. CONCLUSION: These results suggested that circRNAs are highly stable in mammalian cells and that one specific circRNA, hsa_circ_002059, may be a potential novel and stable biomarker for the diagnosis of gastric carcinoma.

SAMD4B, a novel SAM-containing protein, inhibits AP-1-, p53- and p21-mediated transcriptional activity.

The sterile alpha motif (SAM) is a putative protein interaction domain involved in a wide variety of biological processes. Here we report the identification and characterization of a novel gene, SAMD4B, which encodes a putative protein of 694 amino acids with a SAM domain. Northern blot and RT-PCR analysis showed that SAMD4B is widely expressed in human embryonic and adult tissues. Transcriptional activity assays show SAMD4B suppresses transcriptional activity of L8G5-luciferase. Over-expression of SAMD4B in mammalian cells inhibited the transcriptional activities of activator protein-1 (AP-1), p53 and p21, and the inhibitory effects can be relieved by siRNA. Deletion analysis indicates that the SAM domain is the main region for transcriptional suppression. The results suggest that SAMD4B is a widely expressed gene involved in AP-1-, p53-and p21-mediated transcriptional signaling activity.

ZNF424, a novel human KRAB/C2H2 zinc finger protein, suppresses NFAT and p21 pathway.

Zinc finger-containing transcription factors are the largest single family of transcriptional regulators in mammals, which play an essential role in cell differentiation, cell proliferation, apoptosis, and neoplastic transformation. Here we have cloned a novel KRAB-related zinc finger gene, ZNF424, encoding a protein of 555aa. ZNF424 gene consisted of 4 exons and 3 introns, and mapped to chromosome 19p13.3. ZNF424 gene was ubiquitously expressed in human embryo tissues by Northern blot analysis. ZNF424 is conserved across species in evolution. Using a GFP-labeled ZNF424 protein, we demonstrate that ZNF424 localizes mostly in the nucleus. Transcriptional activity assays shows ZNF424 suppresses transcriptional activity of L8G5-luciferase. Overexpression of ZNF424 in HEK- 293 cells inhibited the transcriptional activity of NFAT and p21, which may be silenced by siRNA. The results suggest that ZNF424 protein may act as a transcriptional repressor that suppresses NFAT and p21 pathway to mediate cellular functions.