LncRNA LINC00924 upregulates NDRG2 to inhibit epithelial-mesenchymal transition via sponging miR-6755-5p in hepatitis B virus-related hepatocellular carcinoma.

Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is a life-threatening cancer. Long noncoding RNAs participate in HBV-related HCC progression. Based on the bioinformatics analysis, LINC00924 downregulation is positively related to unfavorable outcomes in patients with HBV-related HCC. Herein, we detected the biological functions and regulatory system of LINC00924 in HCC. The LINC00924 downregulation in HBV-related HCC tissues and cells was revealed by reverse transcription-quantitative polymerase chain reaction. Functionally, as Transwell assays and western blotting indicated, LINC00924 elevation inhibited HCC cell invasion and epithelial-mesenchymal transition (EMT). The binding site between LINC00924 and miR-6755-5p was determined by luciferase reporter assays. miR-6755-5p was confirmed to target NDRG2. miR-6755-5p upregulation decreased NDRG2 messenger RNA (mRNA) and protein levels. The mRNA and protein levels of NDRG2 were downregulated in tissues and cells. NDRG2 knockdown attenuated the inhibition induced by LINC00924 overexpression on invasion and EMT of HCC cells. In summary, LINC00924 increases NDRG2 expression to inhibit EMT by targeting miR-6755-5p in HBV-related HCC.

LncRNA H19-Derived miR-675-5p Accelerates the Invasion of Extravillous Trophoblast Cells by Inhibiting GATA2 and Subsequently Activating Matrix Metalloproteinases.

The invasion of extravillous trophoblast (EVT) cells into the maternal decidua, which plays a crucial role in the establishment of a successful pregnancy, is highly orchestrated by a complex array of regulatory mechanisms. Non-coding RNAs (ncRNAs) that fine-tune gene expression at epigenetic, transcriptional, and post-transcriptional levels are involved in the regulatory mechanisms of EVT cell invasion. However, little is known about the characteristic features of EVT-associated ncRNAs. To elucidate the gene expression profiles of both coding and non-coding transcripts (i.e., mRNAs, long non-coding RNAs (lncRNAs), and microRNAs (miRNAs)) expressed in EVT cells, we performed RNA sequencing analysis of EVT cells isolated from first-trimester placentae. RNA sequencing analysis demonstrated that the lncRNA H19 and its derived miRNA miR-675-5p were enriched in EVT cells. Although miR-675-5p acts as a placental/trophoblast growth suppressor, there is little information on the involvement of miR-675-5p in trophoblast cell invasion. Next, we evaluated a possible role of miR-675-5p in EVT cell invasion using the EVT cell lines HTR-8/SVneo and HChEpC1b; overexpression of miR-675-5p significantly promoted the invasion of both EVT cell lines. The transcription factor gene GATA2 was shown to be a target of miR-675-5p; moreover, small interfering RNA-mediated GATA2 knockdown significantly promoted cell invasion. Furthermore, we identified MMP13 and MMP14 as downstream effectors of miR-675-5p/GATA2-dependent EVT cell invasion. These findings suggest that miR-675-5p-mediated GATA2 inhibition accelerates EVT cell invasion by upregulating matrix metalloproteinases.

Huaier Extract Inhibits Breast Cancer Progression Through a LncRNA-H19/MiR-675-5p Pathway.

BACKGROUND/AIMS: Increasing evidence indicates that Huaier extract has promising therapeutic effects against cancer. However, the mechanisms that underlie its anti-tumor effects remain unclear. In recent years, various studies have shown that long noncoding RNAs (lncRNAs) play a critical role in the regulation of cancer development and progression. Here, we explored the role of lncRNAs in Huaier-induced tumor suppression. METHODS: Microarray profiling was performed to identify the candidate lncRNAs affected by Huaier extract. Quantitative realtime PCR (qPCR) was used to evaluate the transfection efficiency and the influence of Huaier extract on H19 expression. The effect of Huaier extract on the cell viability was examined by MTT. Moreover, the rates of apoptotic cells were detected using flow-cytometric analysis. Western blot analysis was applied to show the protein levels of CBL. RESULTS: Microarray data derived from Huaier-treated breast cancer cells identified H19 as a potential target. Huaier extract reduced the expression of H19. The over-expression of H19 inhibited the cytotoxic effects of Huaier extract; in contrast, reduced H19 expression enhanced the function of Huaier extract. MiR-675-5p was identified as a mature product of H19. Moreover, Huaier extract reduced the miR-675-5p expression. Upregulating miR-675-5p reversed the inhibitory effects of Huaier extract, whereas downregulating miR-675-5p sensitized breast cancer cells to the effect of Huaier extract. In addition, Huaier extract increased the expression of CBL protein, a direct target of miR-675-5p. CONCLUSION: Collectively, the data demonstrate that Huaier extract reduces viability and induces apoptosis in breast cancer cells via H19-miR-675-5p-CBL axis regulation.

Hypoxia-inducible factor 1Α may regulate the commitment of mesenchymal stromal cells toward angio-osteogenesis by mirna-675-5P.

BACKGROUND AIMS: During bone formation, angiogenesis and osteogenesis are regulated by hypoxia, which is able to induce blood vessel formation, as well as recruit and differentiate human mesenchymal stromal cells (hMSCs). The molecular mechanisms involved in HIF-1α response and hMSC differentiation during bone formation are still unclear. This study aimed to investigate the synergistic role of hypoxia and hypoxia-mimetic microRNA miR-675-5p in angiogenesis response and osteo-chondroblast commitment of hMSCs. METHODS: By using a suitable in vitro cell model of hMSCs (maintained in hypoxia or normoxia), the role of HIF-1α and miR-675-5p in angiogenesis and osteogenesis coupling was investigated, using fluorescence-activated cell sorting (FACS), gene expression and protein analysis. RESULTS: Hypoxia induced miR-675-5p expression and a hypoxia-angiogenic response, as demonstrated by increase in vascular endothelial growth factor messenger RNA and protein release. MiR-675-5p overexpression in normoxia promoted the down-regulation of MSC markers and the up-regulation of osteoblast and chondroblast markers, as demonstrated by FACS and protein analysis. Moreover, miR-675-5p depletion in a low-oxygen condition partially abolished the hypoxic response, including angiogenesis, and in particular restored the MSC phenotype, demonstrated by cytofluorimetric analysis. In addition, current preliminary data suggest that the expression of miR-675-5p during hypoxia plays an additive role in sustaining Wnt/ß-catenin pathways and the related commitment of hMSCs during bone ossification. DISCUSSION: MiR-675-5p may trigger complex molecular mechanisms that promote hMSC osteoblastic differentiation through a dual strategy: increasing HIF-1α response and activating Wnt/ß-catenin signaling.

LMP1 Induces p53 Protein Expression via the H19/miR-675-5p Axis.

Epstein-Barr virus (EBV), a ubiquitous oncogenic herpesvirus, infects more than 90% of the adult population worldwide. The long noncoding RNA H19 is downregulated in EBV-positive gastric cancer (EBVaGC) and nasopharyngeal cancer (NPC). In this study, we found that loss of H19 is caused by hypermethylation status of the H19 promoter in EBV-positive GC and NPC cell lines. Furthermore, latent membrane protein 1 (LMP1), encoded by EBV, induced H19 promoter hypermethylation and deregulated the expression of H19 by upregulating DNMT1 expression. Transwell assays showed that H19 promoted cell migration. Furthermore, H19 promoted cell proliferation and inhibited apoptosis in CCK-8 and flow cytometry assays, respectively. p53, a well-known tumor suppressor, was upregulated in EBVaGC and NPC cell lines. miR-675-5p derived from H19 inhibited p53 protein expression by targeting the 3' untranslated region of the gene. Overall, we found that LMP1 induced p53 protein expression via the H19/miR-675-5p axis in EBVaGC and NPC. LMP1 induced H19 promoter hypermethylation, which repressed the expression of H19 and miR-675-5p and caused p53 protein overexpression in EBVaGC and NPC cells. IMPORTANCE Epstein-Barr virus (EBV) is the first virus to be known to have direct association with human cancer and to be considered as an important DNA tumor virus. The EBV life cycle consists of both latent and lytic modes of infection in B lymphocytes and epithelial cells. The persistence of EBV genomes in malignant cells promoted cell growth. p53, acting as a critical gatekeeper tumor suppressor, is involved in multiple virus-mediated tumorigeneses. Overexpression of p53 inhibits the ability of BZLF1 (EBV-encoded immediate early gene) to disrupt viral latency. In our study, we found LMP1 induces H19 promoter hypermethylation, which represses the expression of H19 and miR-675-5p and results in p53 protein overexpression in EBVaGC and NPC cells. These observations suggest a new mechanism of aberrant expression of p53 by LMP1, which facilitates EBV latency.

The EGFR-P38 MAPK axis up-regulates PD-L1 through miR-675-5p and down-regulates HLA-ABC via hexokinase-2 in hepatocellular carcinoma cells.

BACKGROUND: Immunotherapy has been shown to be a promising strategy against human cancers. A better understanding of the immune regulation in hepatocellular carcinoma (HCC) could help the development of immunotherapy against HCC. The epidermal growth factor receptor (EGFR) signaling is frequently activated in HCC and plays important roles in tumorigenesis. However, its role in HCC immunity is still largely unknown. This study aimed to investigate the impact of EGFR signaling on programmed death-ligand 1 (PD-L1) and human leukocyte antigen class-I (HLA-I) expression in HCC cells and its underlying mechanisms. METHODS: The expression of phosphorylated EGFR (p-EGFR), PD-L1, and HLA-I (HLA-ABC) in HCC specimens was detected by immunohistochemistry, and their correlations were analyzed. PD-L1 and HLA-ABC expression in EGFR-activated HCC cells were detected by quantitative real-time PCR, Western blotting, and flow cytometry, and T cell-mediated lysis was performed to test the immunosuppressive effects of PD-L1 and HLA-ABC alterations in HCC cells. Furthermore, the underlying mechanisms of EGFR activation-induced PD-L1 up-regulation and HLA-ABC down-regulation were explored by animal experiments, luciferase reporter assay, and gene gain- and loss-of-function studies. RESULTS: p-EGFR was positively correlated with PD-L1 and negatively correlated with HLA-ABC expression in HCCs. EGFR activation by its ligand EGF up-regulated PD-L1 and down-regulated HLA-ABC in HCC cells, which was functionally important and could be abolished by the EGFR inhibitor, gefitinib, both in vitro and in vivo. Mechanistically, enhanced P38 mitogen-activated protein kinase (MAPK) activation down-regulated microRNA-675-5p (miR-675-5p) and up-regulated glycolysis-related enzyme hexokinase 2 (HK2); miR-675-5p down-regulation enhanced the stability of PD-L1 mRNA probably via the 3'-untranslated region (3'-UTR) of PD-L1 and thereby caused PD-L1 accumulation, and HK2 up-regulation enhanced aerobic glycolysis and mediated a decrease in HLA-ABC. CONCLUSIONS: The EGFR-P38 MAPK axis could up-regulate PD-L1 through miR-675-5p and down-regulate HLA-ABC via HK2 in HCC cells. Our study reveals a novel signaling network that may cause immune suppression in HCC and suggests that EGFR signaling can be targeted for HCC immunotherapy.

Overexpression of lncRNA H19 leads to reduced proliferation in TSCC cells through miR-675-5p/GPR55.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is a highly malignant tumor and oral disease. We intended to identify the function and mechanism of lncRNA H19 in TSCC. METHODS: Twenty-two TSCC samples were obtained, and expression levels of lncRNA H19 were measured by quantitative real time PCR (qRT-PCR). After experimentally upregulating expression of lncRNA H19 in TSCC cells, proliferation, invasion and migration were assessed. Further, dual-luciferase reporter gene assays were used to examine the relationship between lncRNA H19 and GPR55. RESULTS: LncRNA H19 was expressed at lower levels in TSCC tissues than in normal tissues. When lncRNA H19 was overexpressed, miR-675-5p expression levels were also upregulated, leading to reduced proliferation, invasion and migration of CAL27 and SCC9 cells. Dual-luciferase reporter gene assays revealed that miR-675-5p binds to GPR55, which might promote growth in TSCCs. CONCLUSIONS: The lncRNA H19/miR-675-5p/GPR55 axis might inhibit cell proliferation, invasion and migration in TSCC.

Therapeutic effect of psoralen on muscle atrophy induced by tumor necrosis factor-α.

OBJECTIVES: To observe and determine the effect and mechanism of psoralen on tumor necrosis factor-α (TNF-α)-induced muscle atrophy. MATERIALS AND METHODS: Three sets of C2C12 cells, including blank control, TNF-α (10 or 20 ng/ml) treatment and a TNF-α (10 or 20 ng/ml) plus psoralen (80 µM) administration were investigated. Cell viability was assessed using Cell Counting Kit-8 (CCK-8) assay. Western blot analysis was used to detect protein expression of atrophic markers. Flowcytometry was used to observe the effect of psoralen on apoptosis. A quantitative real-time PCR (qRT-PCR) assay was performed to detect the mRNA level of miR-675-5P. RESULTS: TNF-α (1, 10, 20 and 100 ng/ml) treatment inhibited C2C12 myoblast viability (P<0.001), while 24 hr of psoralen administration increased the viability, and lowered TNF-α cytotoxicity (P<0.001). MURF1, MAFbx, TRIM62 and GDF15 expressions were significantly increased in TNF-α (10 ng/ml or 20 ng/ml)-treated group (P<0.001), and psoralen could significantly decrease the expression of these proteins (P<0.001). Apoptotic rate of C2C12 myoblasts was increased after TNF-α (10 ng/ml and 20 ng/ml) treatment, and was significantly decreased after psoralen treatment (P<0.001). miR-675-5P was increased in TNF-α-treated C2C12 myoblasts compared to control group, and it was significantly decreased after psoralen treatment. CONCLUSION: Psoralen could reduce TNF-α-induced cytotoxicity, atrophy and apoptosis in C2C12 myoblasts. The therapeutic effect of psoralen may be achieved by down-regulating miR-675-5P.

H19/miR-675-5p Targeting SFN Enhances the Invasion and Metastasis of Nasalpharyngeal Cancer Cells.

AIMS: The aim is to study the role of miR-675-5p coded by long non-coding RNA H19 in the development of Nasopharyngeal Cancer (NPC) and whether miR-675-5p regulates the invasion and metastasis of NPC through targeting SFN (14-3-3σ). The study further validated the relationship between H19, miR-675-5p and SFN in NPC and their relationship with the invasion and metastasis of NPC. METHODS: Western blot was used to detect the expression of 14-3-3σ protein in immortalized normal nasopharyngeal epithelial cells NP69 and different metastatic potential NPC cells, 6-10B and 5-8F. At the same time, to find out the relationship between 14-3-3σ protein and the expression of H19 and miR-675-5p, the expression of H19 and miR-675-5p in normal nasopharynx epithelial cells NP69 and varied nasopharyngeal carcinoma cells 6-10B and 5-8F were quantified by real-time PCR. MiR-675-5p mimic and inhibitor were transfected into NPC 6-10B to over-express and down-express miR-675-5p; miR-675-5p mimic negative control and inhibitor negative control were transfected into NPC 6-10B as control groups. The effect of over-expression and down-expression by miR-675-5p on the expression of 14-3-3σ protein was detected by Western blotting. The 3'-UTR segments of SFN, containing miR-675-5p binding sites were amplified by PCR and the luciferase activity in the transfected cells was assayed to detect whether SFN is the direct target of miR-675-5p. Transwell and scratch assays were used to verify the changes in NPC invasion and metastasis ability of mimics and inhibitors transfected with miR-675-5p. RESULTS: The expression of 14-3-3σ protein in normal nasopharynx epithelial cells NP69 is significantly higher than in varied nasopharyngeal carcinoma cells, 6-10B and 5-8F (P<0.05), and the 14-3-3σ protein levels in low-metastatic nasopharyngeal carcinoma cell 6-10B is higher than in high-metastatic nasopharyngeal carcinoma cell 5-8F. The expression of H19 and miR-675-5p are significantly higher in NPC cells than in NP69 cell (P<0.05). The expression of H19 and miR-675-5p in high-Metastatic nasopharyngeal carcinoma cell 5-8F was higher than in low-Metastatic nasopharyngeal carcinoma cell 6-10B. The expression of 14-3-3σ protein in miR-675-5p mimic cells was significantly lower than in mimic NC (negative control) group and blank control group. However, compared with the blank control group, mimic NC showed no significant difference in 14-3-3σ protein between the two groups. The miR-675-5p inhibitor group was significantly higher than the inhibitor NC group and the blank control group (p<0.05), but there was no significant difference in the expression of 14-3-3σ protein in the inhibitor NC group and the blank control group (p>0.05). Dual-luciferase reporter assay system shows the 3'-UTR segments of SFN containing miR-675-5p binding sites. SFN was the target gene of miR-675-5p. CONCLUSION: 14-3-3σ is downregulated in NPC and is involved in the development of NPC. H19 and miR- 675-5p are upregulated in NPC, which is related to the development of NPC. The over-expression of miR- 675-5p inhibits the expression of 14-3-3σ protein. SFN is the target gene of miR-675-5p. MiR-675-5p targets SFN, downregulates its protein expression and promotes the invasion and metastasis of NPC.

Co-expression profiling of plasma miRNAs and long noncoding RNAs in gastric cancer patients.

PURPOSE: The recent researches indicate that differential non-coding RNAs expression signatures could be associated with the pathogenesis of gastric cancer (GC). However, there are few studies focused on lncRNA-miRNAs co-expression profiling in GC patients. Therefore, in the present study the expression of H19 and MEG3 and their related miRNAs including miR-148a-3p, miR-181a-5p, miR-675-5p and miR-141-3p were determined in the plasma samples of GC patients and controls. MATERIALS AND METHODS: This case-control study included 62 GC patients and 40 age- sex matched controls. The non-coding RNA levels were assessed by real-time PCR. Further, using in silico analysis, we identified shared targets of studied miRNAs and performed GC-associated pathway enrichment analysis. RESULTS: Our results showed that the H19 level was significantly (P = 0.008) elevated and MEG3 expression was significantly (P = 0.002) down-regulated in GC patients compared to healthy participants. Furthermore, it was revealed that the miR-675-5p level was increased, while miR-141-3p plasma levels were significantly reduced in GC patients (P < 0.05). We did not observe a significant difference for miR-148a-3p (P = 0.682) and miR-181a-5p (P = 0.098) expression between groups. In addition, the expression levels of H19, MEG3 and miR-148a-3p were associated with some clinicopathological features of patients (P < 0.05). ROC analysis revealed that a combination of H19, MEG3 and miR-675-5p levels able to discriminate controls and GC subjects with 88.87% sensitivity and 85% specificity (AUC, 0.927; 0.85-0.96 CI, P < 0.0001). CONCLUSION: The results of current study demonstrated that combination of H19, MEG3 and miR-675-5p expression levels could provide a potential diagnostic panel for GC.

Cancer stem cells from peritumoral tissue of glioblastoma multiforme: the possible missing link between tumor development and progression.

In glioblastoma multiforme (GBM), cancer stem cells (CSCs) are thought to be responsible for gliomagenesis, resistance to treatment and recurrence. Unfortunately, the prognosis for GBM remains poor and recurrence frequently occurs in the peritumoral tissue within 2 cm from the tumor edge. In this area, a population of CSCs has been demonstrated which may recapitulate the tumor after surgical resection. In the present study, we aimed to characterize CSCs derived from both peritumoral tissue (PCSCs) and GBM (GCSCs) in order to deepen their significance in GBM development and progression. The stemness of PCSC/GCSC pairs obtained from four human GBM surgical specimens was investigated by comparing the expression of specific stem cell markers such as Nestin, Musashi-1 and SOX2. In addition, the growth rate, the ultrastructural features and the expression of other molecules such as c-Met, pMet and MAP kinases, involved in cell migration/invasion, maintenance of tumor stemness and/or resistance to treatments were evaluated. Since it has been recently demonstrated the involvement of the long non-coding RNAs (lncRNAs) in the progression of gliomas, the expression of H19 lncRNA, as well as of one of its two mature products miR-675-5p was evaluated in neurospheres. Our results show significant differences between GCSCs and PCSCs in terms of proliferation, ultrastructural peculiarities and, at a lower extent, stemness profile. These differences might be important in view of their potential role as a therapeutic target.

miR-675-5p enhances tumorigenesis and metastasis of esophageal squamous cell carcinoma by targeting REPS2.

Recently H19 has been demonstrated to be up-regulated in esophageal squamous cell carcinoma (ESCC) and shown to be the precursor of miR-675 that encodes miR-675-5p conservatively. miR-675 is overexpressed in many human cancers; however, the function of miR-675-5p is largely unknown in ESCC. In this study, we found that miR-675-5p expression was significantly increased in ESCC tissues and cell lines and related with ESCC progression and poor prognosis. We also showed here that down-regulation of miR-675-5p in ESCC cells dramatically induced cell G1 arrest and reduced cell proliferation, colony formation, migration and invasion in vitro as well as tumorigenesis and tumor metastasis in vivo. We subsequently identified that REPS2 was a target gene of miR-675-5p. We found that inhibition of miR-675-5p up-regulated the expression of REPS2, inhibited RalBP1/RAC1/CDC42 signaling pathway. Inversely, interference of REPS2 abrogated the effect induced by miR-675-5p inhibition, which resembled the function of miR-675-5p up-regulation. Taken together, our findings suggested that miR-675-5p might play an oncogenic role in ESCC through RalBP1/RAC1/CDC42 signaling pathway by inhibiting REPS2 and might serve as a valuable prognostic biomarker and therapeutic target for ESCC patients.