Study on the relationship between long non-coding RNA DHRS4-AS1 and disease-free survival in patients with osteosarcoma and its effect on osteosarcoma cell proliferation and migration in vitro / 肿瘤研究与临床

Objective:To investigate the relationship between long non-coding RNA (lncRNA) DHRS4-AS1 and disease-free survival in osteosarcoma patients and the mechanisms of its effect on proliferation and migration of osteosarcoma cells in vitro.Methods:The data of DHRS4-AS1 transcriptome levels and survival status of osteosarcoma patients in GEPIA database were collected since the database was established, and the patients were divided into high DHRS4-AS1 expression group and low DHRS4-AS1 expression group based on the median DHRS4-AS1 transcriptome level, with 59 cases in each group, and the Kaplan-Meier method was used to analyze the disease-free survival of the two groups. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of DHRS4-AS1 in osteosarcoma cell lines MG-63, HOS, 143B, U-2OS, Saos2 and normal osteoblast cell line hFOB1.19, and the osteosarcoma cell line with the lowest DHRS4-AS1 expression level was selected for subsequent experiments. The plasmid carrying DHRS4-AS1 sequence and the plasmid carrying negative control sequence were transfected into the selected osteosarcoma cells as DHRS4-AS1 group and control group. CCK-8 method was used to detect the proliferation of each group of cells, and the absorbance value was used as the cell proliferation ability; cell scratch assay was used to detect the migration of each group of cells. The bioinformatics website starBase V2.0 was used to predict the target genes of DHRS4-AS1, and the dual luciferase reporter gene assay was used to verify the targeting relationship between DHRS4-AS1 and the target genes. The expression levels of target genes and downstream genes of osteosarcoma cells in control group and DHRS4-AS1 group were detected by qRT-PCR and Western blotting.Results:Survival analysis showed that the disease-free survival of osteosarcoma patients in the high DHRS4-AS1 expression group in GEPIA database was superior to that of the low DHRS4-AS1 expression group ( P < 0.001). Compared with normal osteoblastic hFOB1.19 cells, the expression level of DHRS4-AS1 was low in all osteosarcoma cells (all P < 0.01), with the lowest expression level of DHRS4-AS1 in U-2OS cells ( P < 0.001). Cell proliferation ability was reduced in U-2OS cells of the DHRS4-AS1 group after 1, 2, 3 and 4 d of culture compared with the control group (all P < 0.05). The migration rate of U-2OS cells in the DHRS4-AS1 group was lower than that in the control group [(31±6)% vs. (63±4)%, t = 4.38, P = 0.005]. starBase V2.0 website predicted that DHRS4-AS1 complementarily bound to miRNA-411-3p (miR-411-3p); dual luciferase reporter gene assay showed that miR-411-3p overexpression reduced the luciferase activity of the wild-type DHRS4-AS1 reporter gene ( P < 0.001), but had no effect on the luciferase activity of the mutant DHRS4-AS1 reporter gene ( P > 0.05). qRT-PCR showed that the relative expression of miR-411-3p in U-2OS cells of the DHRS4-AS1 group was low (0.22±0.06 vs. 1.06±0.23, t = 3.55, P = 0.012) and the relative expression of metastasis suppressor MTSS1 mRNA was high (5.58±1.03 vs. 1.06±0.22, t = 4.28, P = 0.005) compared with the control group; Western blotting showed that MTSS1 expression was elevated, and the expression levels of cell proliferation phenotype proteins CDK3 and cyclin C and cell migration phenotype proteins ZEB2 and KLF8 were low. Conclusions:Osteosarcoma patients with high expression of lncRNA DHRS4-AS1 have better disease-free survival, and its expression is low in osteosarcoma cell lines. DHRS4-AS1 may promote MTSS1 gene expression and inhibit cell proliferation and migration by targeting and down-regulating miR-411-3p expression in osteosarcoma cells.

Expression of intergenic long non-coding RNA RP11-488L18.10 in cholangiocarcinoma and its association with prognosis / 临床肝胆病杂志

ObjectiveTo screen out the key intergenic long non-coding RNAs (lncRNAs) in cholangiocarcinoma (CCA) and their association with patient prognosis. MethodsCCA expression and clinical data were obtained from The Cancer Genome Atlas (TCGA). High-throughput gene expression data in GSE107943 dataset were obtained from the Gene Expression Omnibus (GEO). R software was used to analyze differentially expressed genes and determine significantly differentially expressed lncRNAs between tumor and normal samples, and the key lncRNAs were screened out. The receiver operating characteristic (ROC) curve was used to analyze the value of gene expression in the diagnosis of CCA; the Kaplan-Meier survival curves were used to analyze prognosis; Gene Set Enrichment Analysis (GSEA) was used to investigate significantly enriched pathways; quantitative real-time PCR was used to verify gene expression in clinical samples. ResultsA total of 451 upregulated lncRNAs and 154 downregulated lncRNAs in both TCGA and GSE107943 dataset were screened out. The lncRNA RP11-488L18.10 in TCGA and GSE107943 dataset was highly abundant in tumor tissue. Based on TCGA and GSE107943 dataset, the ROC curve analysis of the association between lncRNA RP11-488L18.10 expression and CCA showed that the expression of lncRNA RP11-488L18.10 had an area under the ROC curve of 1 in TCGA (P＜0.000 1) and 0.9469 in GSE107943 (P＜0.000 1). In TCGA, the high expression of lncRNA RP11-488L18.10 significantly predicted overall survival rate (P=0.016) and recurrence-free survival rate (P=0.017) in patients with CCA; in GSE107943, the high expression of lncRNA RP11-488L18.10 significantly predicted overall survival rate (P=0.023) and recurrence-free survival rate (P=0.005) in patients with CCA. LncRNA RP11-488L18.10 was significantly positively correlated with MCM2. Compared with the adjacent tissue, CCA tissue had high expression of lncRNA RP11-488L18.10 (P=0.010) and MCM2 (P=0.023). ConclusionLncRNA RP11-488L18.10 is closely associated with the development and prognosis of CCA and thus may become a target for the diagnosis and treatment of CCA.

Regulatory effect of long non-coding RNAs on liver fibrosis / 临床肝胆病杂志

A large number of studies in recent years have shown that long non-coding RNAs (lncRNAs) play an important regulatory role in the progression of liver fibrosis. This article briefly describes the definition, classification, and biological functions of lncRNAs and summarizes recent reports on the regulatory role of lncRNAs in liver fibrosis by acting as competitive endogenous RNA, including downregulated maternally expressed gene 3, growth arrest-specific transcript 5, and long intergenic non-coding RNA-p21 and upregulated lung adenocarcinoma-associated transcript 1, lncRNA-activated by transforming growth factor beta, plasmacytoma variant translocation 1, homeobox transcript antisense RNA, lncRNA-H19, and small nuclear RNA host gene 7, so as to provide insights into the diagnosis of liver fibrosis, the screening of therapeutic targets, and the development of clinical treatment regimens for the reversal of liver fibrosis.

Cardiovascular RNA interference therapy: the broadening tool and target spectrum.

Understanding of the roles of noncoding RNAs (ncRNAs) within complex organisms has fundamentally changed. It is increasingly possible to use ncRNAs as diagnostic and therapeutic tools in medicine. Regarding disease pathogenesis, it has become evident that confinement to the analysis of protein-coding regions of the human genome is insufficient because ncRNA variants have been associated with important human diseases. Thus, inclusion of noncoding genomic elements in pathogenetic studies and their consideration as therapeutic targets is warranted. We consider aspects of the evolutionary and discovery history of ncRNAs, as far as they are relevant for the identification and selection of ncRNAs with likely therapeutic potential. Novel therapeutic strategies are based on ncRNAs, and we discuss here RNA interference as a highly versatile tool for gene silencing. RNA interference-mediating RNAs are small, but only parts of a far larger spectrum encompassing ncRNAs up to many kilobasepairs in size. We discuss therapeutic options in cardiovascular medicine offered by ncRNAs and key issues to be solved before clinical translation. Convergence of multiple technical advances is highlighted as a prerequisite for the translational progress achieved in recent years. Regarding safety, we review properties of RNA therapeutics, which may immunologically distinguish them from their endogenous counterparts, all of which underwent sophisticated evolutionary adaptation to specific biological contexts. Although our understanding of the noncoding human genome is only fragmentary to date, it is already feasible to develop RNA interference against a rapidly broadening spectrum of therapeutic targets and to translate this to the clinical setting under certain restrictions.

Identification of differentially expressed long non-coding RNAs in human ovarian cancer cells with different metastatic potentials.

OBJECTIVE: To identify differentially expressed long non-coding RNAs (lncRNAs) involved in the metastasis of epithelial ovarian cancer. METHODS: An in vitro invasion assay was performed to validate the invasive capability of SKOV3 and SKOV3.ip1 cell lines. Total RNA was then extracted, and microarray analysis was performed. Moreover, nine lncRNAs were selected for validation using RT-qPCR. RESULTS: Compared with the SKOV3 cells, the SKOV3.ip1 cells significantly improved in the in vitro invasive activity. Of the 4,956 lncRNAs detected in the microarray, 583 and 578 lncRNAs were upregulated and downregulated, respectively, in SKOV3.ip1 cells, compared with the parental SKOV3 cells. Seven of the analyzed lncRNAs (MALAT1, H19, UCA1, CCAT1, LOC645249, LOC100128881, and LOC100292680) confirmed the deregulation found by microarray analysis. CONCLUSION: LncRNAs clusters were differentially expressed in ovarian cancer cells with varying metastatic potentials. This result indicates that some lncRNAs might exert a partial or key role in epithelial ovarian cancer metastasis. Further studies should be conducted to determine the roles of these lncRNAs in ovarian cancer metastasis.