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.

Evaluation of the Regulatory Role of Circadian Rhythm Related Long Non-Coding RNAs in ADHD Etiogenesis.

OBJECTIVE: ADHD is associated with increased sleep problems and circadian rhythm disturbances. This study aimed to examine ADHD patients and healthy controls in terms of chronotypic features and expression levels of CLOCK, PER1, lncRNA HULC, lncRNA UCA1. METHOD: Eighty-three children were included (43 ADHD). Conner's Parent Rating Scale-Revised Short Form, Childhood Chronotype Questionnaire, Children's Sleep Disorders Scale were administered. Gene expression levels were studied from peripheral blood. RESULTS: Evening chronotype, sleep initiation/maintenance disorder, sleep-wake transition disorder, excessive sleepiness disorder were higher in the ADHD group compared to the controls in the scales reported by the parents. Expression levels of all examined genes were statistically significantly higher in the ADHD group. There was no significant relationship between genes and sleep parameters in the ADHD group. CONCLUSION: Our study provides the first evidence that lncRNA HULC and lncRNA UCA1 might have a role in the etiology of ADHD.

Long noncoding RNA HULC contributes to paclitaxel resistance in ovarian cancer via miR-137/ITGB8 axis.

Long noncoding RNA (lncRNA) highly upregulated in liver cancer (HULC) has been reported to be implicated in chemoresistance. However, the potential mechanism of HULC in paclitaxel (PTX)-resistant ovarian cancer (OC) remains undefined. The expression of RNAs and proteins was measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot assay. The PTX resistance and apoptotic rate were assessed via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. Furthermore, the interaction between miR-137 and HULC or integrin beta-8 (ITGB8) was predicted by miRcode and starBase v2.0 and then verified by dual luciferase reporter and RNA pull-down assays. In addition, the xenograft mice model was established to explore the effects of HULC in vivo. HULC was significantly upregulated and miR-137 was downregulated in PTX-resistant OC tissues and cells. Also, the HULC depletion suppressed tumor growth and PTX resistance in PTX-treated mice. miR-137 was verified as a target of HULC and directly targeted ITGB8. And HULC knockdown downregulated ITGB8 expression by targeting miR-137. miR-137 inhibitor or ITGB8 overexpression mitigated the suppressive impacts of HULC knockdown on PTX resistance. Collectively, HULC modulated ITGB8 expression to promote PTX resistance of OC by sponging miR-137.

Long non-coding RNA HULC stimulates the epithelial-mesenchymal transition process and vasculogenic mimicry in human glioblastoma.

BACKGROUND: Long non-coding RNA (lncRNA) HULC (highly upregulated in liver cancer) is considered as an oncogenic factor for various malignant tumors. This study aimed to reveal the role of lncRNA HULC in the malignant behavior of glioblastoma (GBM) by exploring its effects on the epithelial-mesenchymal transition (EMT) and vasculogenic mimicry (VM) of human GBM. MATERIALS AND METHODS: The contents of VM in 27 GBM samples were assessed by immunohistochemistry-histology and their association with progress-free survival (PFS) was analyzed. Human GBM SHG44 and U87 cells were manipulated to establish stable lncRNA HULC overexpressing and silencing cells by lentivirus-based technology. The effects of altered lncRNA HULC on vasculogenic tubular formation, invasion, and EMT process in GBM cells were tested in vitro and the growth of implanted GBM tumors and their EMT process were examined in vivo. RESULTS: The numbers of VM were positively associated with disease progression, but negatively with PFS periods of GBM patients. Compared with the control vec cells, lncRNA HULC overexpression significantly increased the tubular formation, invasion, and EMT process of both SHG44 and U87 cells, accompanied by promoting the growth of implanted GBM tumors and EMT process in mice. LncRNA HULC silencing had opposite effects on the tubular formation, invasion, and EMT process as well as tumor growth of GBM cells. CONCLUSION: LncRNA HULC stimulates the EMT process and VM in human GBM, and may be a therapeutic target for intervention of GBM.

Expression of lncRNA HULC in bladder cancer tissues and its effect on the malignant biological behaviors of 5637 cells / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the expression of long non-coding RNA (lncRNA) HULC in bladder cancer tissues and its relationship with the clinicopathological features of patients, as well as the effect of silencing HULC on the proliferation, apoptosis, migration and invasion of bladder cancer 5637 cells. Methods: A total of 102 pairs of cancer tissue and adjacent normal tissue samples from bladder cancer patients who underwent surgical resection in Zhengzhou People’s Hospital from June 2014 to December 2017 were selected, as well as bladder cancer 5637 cell line and human normal bladder epithelial SV-HUC-1 cell line. The expression of HULC in bladder cancer tissues and cells was detected by qPCR, and the correlation between HULC and clinicopathological features of bladder cancer patients was analyzed. The effect of HULC on prognosis was evaluated by Kaplan-Meier survival curve. si-HUL and si-NC plasmids were transfected into 5637 cells by siRNA interference technology, and the effects of silencing HULC on proliferation, apoptosis, migration and invasion of 5637 cells were determined by CCK-8, Flow cytometry, Wound-healing assay and Transwell method, respectively. Results: The expression of HULC in bladder cancer tissues was significantly higher than that in normal tissues (P<0.05), and its expression level was correlated with tumor grade, tumor stage and lymph node metastasis (P<0.05). The OS and PFS of patients with high HULC expression were significantly lower than those with low expression (all P<0.05). The expression level of HULC in 5637 cells was significantly higher than that in SV-HUC-1 cells (P<0.01). After silencing HULC, the proliferation, migration and invasion of 5637 cells were significantly decreased (P<0.01), and the apoptosis rate was significantly increased (P<0.01). Conclusion: lncRNA HULC is highly expressed in bladder cancer tissues and 5637 cells. Silencing HULC expression can inhibit the proliferation, migration and invasion but promote apoptosis of bladder cancer cells.

LncRNA PTCSC3 and lncRNA HULC Negatively Affect Each Other to Regulate Cancer Cell Invasion and Migration in Gastric Cancer.

BACKGROUND: LncRNA PTCSC3 (PTCSC3) inhibits thyroid cancer cervical carcinoma and glioma, while its roles in gastric cancer are unknown. Studies have reported that HULC could serve as a potential biomarker for the diagnosis and prognosis of gastric cancer (GC). Our study aimed to investigate the potential interaction between PTCSC3 and HULC in gastric cancer. METHODS: This study enrolled 77 gastric cancer patients at the First Affiliated Hospital of Anhui Medical University from January 2016 to January 2018. RT-qPCR was performed to analyze gene expression levels. Cell transfections were carried out to evaluate gene interactions. Transwell assays and wound healing assays were used to analyze the effects of transfection on cell invasion and migration. Western blotting was also used to illustrate the possibility that lncRNA PTCSC3 and lncRNA HULC negatively affected each other through WNT signal path. RESULTS: We showed that PTCSC3 was downregulated in tumor tissues of gastric cancer patients in comparison to that in adjacent healthy tissues, and an inverse correlation between the expression levels of PTCSC3 and AJCC stage was observed. LncRNA HULC (HULC) was upregulated in tumor and inversely correlated with PTCSC3 in tumor tissues. Overexpression of PTCSC3 mediated the inhibition of HULC, while overexpression of HULC also mediated the inhibition of PTCSC3. PTCSC3 inhibited, while HULC promoted invasion and migration of gastric cancer cells. In addition, overexpression of HULC attenuated the effects of overexpression of PTCSC3. However, overexpression of PTCSC3 showed no significant effects on cell proliferation. We also found that PTCSC3/HULC affected each other to regulate cell invasion and migration through the Wnt/ß-catenin signaling. CONCLUSION: Therefore, overexpression of PTCSC3 inhibited the invasion and migration of gastric cancer cells, and the function of PTCSC3 is associated with HULC.

Long noncoding RNA HULC in acute ischemic stroke: Association with disease risk, severity, and recurrence-free survival and relation with IL-6, ICAM1, miR-9, and miR-195.

BACKGROUND: This study aimed to evaluate the clinical role of long noncoding RNA (lncRNA) HULC in acute ischemic stroke (AIS). METHODS: LncRNA HULC in plasma samples from 215 first episode AIS patients and 215 age/gender-matched non-AIS controls was detected by reverse transcriptional-quantitative polymerase chain reaction (RT-qPCR). Then, in AIS patients, interleukin-6 and intercellular adhesion molecule 1 (ICAM1), as well as microRNA (miR) target of lncRNA HUCL (miR-9 and miR-195), were detected by enzyme-linked immunosorbent assay and RT-qPCR, respectively. Disease severity was assessed by National Institution of Health stroke scale (NIHSS) score. AIS recurrence or death was recorded, and recurrence-free survival (RFS) was calculated. RESULTS: LncRNA HULC was increased in AIS patients compared to non-AIS controls (P < .001), and receiver operating characteristic curve showed that it was correlated with increased AIS risk (area under curve: 0.876, 95% confidence interval: 0.843-0.908). Meanwhile, lncRNA HULC was positively correlated with NIHSS score (P < .001, r = .456), interleukin-6 (P < .001, r = .275) and ICAM1 (P < .001, r = .383), whereas negatively correlated with miR-9 (P < .001, r = -.438) but not miR-195 (P = .205, r = -.087) in AIS patients. Additionally, miR-9 was negatively correlated with NIHSS score (P < .001, r = -.335), interleukin-6 (P = .001, r = -.231), and ICAM1 (P < .001, r = -.280), while miR-195 was only negatively associated with NIHSS score (P = .041, r = -.139) in AIS patients. Moreover, lncRNA HULC high expression predicted worse RFS (P = .013) in AIS patients. CONCLUSION: LncRNA HULC is correlated with higher AIS risk, increased disease severity and worse prognosis in AIS patients. Meanwhile, it associates with higher IL-6, elevated ICAM1, and lower miR-9 AIS patients.

Quantitative Proteomics Analysis Indicates That Upregulation of lncRNA HULC Promotes Pathogenesis of Glioblastoma Cells.

PURPOSE: Glioblastoma (GBM) is an aggressive central nervous system (CNS) cancer and a serious threat to human health. The long noncoding RNA (lncRNA) HULC has been implicated in GBM, but the molecular mechanism is uncertain. This study used quantitative proteomic analysis for global identification of HULC-regulated proteins in glioblastoma cells and identification of potential biomarkers. MATERIALS AND METHODS: qRT-PCR was used to determine the expression of HULC in U87 cells stably transfected with HULC or an empty vector (control). The CCK-8 assay, transwell assay, and wound-scratch assay were used to measure cell proliferation, invasion, and migration. Quantitative proteomics using Tandem Mass Tag (TMT) labeling, high-performance liquid chromatography (HPLC) fractionation, and liquid chromatography-mass spectrometry (LC-MS/MS) analysis were used to identify differentially expressed proteins (DEPs). Screened proteins were validated by parallel reaction monitoring (PRM) and Western blotting. RESULTS: Overexpression of HULC led to increased cell proliferation, invasion, and migration. HULC overexpression also led to significant upregulation of 37 proteins and downregulation of 78 proteins. Bioinformatics analysis indicated these proteins had roles in cellular component, biological process, and molecular function. PRM results of 8 of these proteins (PTK2, TNC, ITGAV, LASP1, MAPK14, ITGA1, GNA13, RRAS) were consistent with the LC-MS/MS and Western blotting results. CONCLUSION: The results of present study suggest that lncRNA HULC promotes GBM cell proliferation, invasion, and migration by regulating RRAS expression, suggesting that RRAS may be a potential biomarker or therapeutic target for this cancer.

Long Non-Coding RNA HULC Enhances Tumor Growth in Orthotopic Xenograft Model of Glioblastoma / 中山大学学报(医学科学版)

@#【Objective】To investigate the mechanism of action of long non-coding RNA highly up-regulated in liver cancer（LncRNA HULC）on the growth of glioblastoma U87 cells in vitro and in vivo.【Methods】The cultured glioblastoma U87 cells were divided into four groups：overexpression group（HULC-over）and its vector control group（VEC），silent expression group（HULC- siRNA）and its negative control group（NC）.Quantitative real- time polymerase chain reaction PCR（qRT-PCR）was used to verify the expression levels of HULC. CCK8 proliferation assay and colony formation assay were adopted to monitor the proliferation of glioblastoma U87 cells. Flow cytometry was utilized to detect the apoptosis of glioblastoma U87 cells. By injecting U87 cells，we divided the orthotopic xenograft mouse model into HULC- over group（n=10），VEC group（n=10），HULC-siRNA group（n=10）and NC group（n=10）accordingly. The survival of the mice in each group was observed. The expression of Ki67 was analyzed by immunohistochemistry. 【Results】 The expression level of HULC was significantly higher in HULC-over group than that in VEC group and significantly lower in HULC-siR NA group than that in NC group（P < 0.01）. The cell proliferation ability was significantly increased in HULC-over group compared with that in VEC group and significantly decreased in HULC- siRNA group compared with that in NC group（P < 0.01 on days2，3and4）. The colony formation rates in VEC group，HULC-over group，NC group and HULC-siRNA group were，respectively，（34.47 ± 1.56）% ，（95.4 ± 2.74）% ，（23.83 ± 0.92）% and （10.23 ± 0.61）% ，which revealed that overexpression of HULC elevated the colony formation rate and silencing expression of HULC reduced the colony formation rate（P < 0.01）. The early apoptosis rates in VEC group，HULC- over group，NC group and HULC- siRNA group were，respectively，（3.55±0.56）% ，（0.09±0.01）% ，（2.89±0.67）% ，and（7.13±0.14）% ，which showed that overexpression of HULC elevated the early apoptosis rate and silencing expression of HULC reduced the early apoptosis rate （P <0.01）. The survival curve of nude mouse indicated shorter survival time in HULC-over group than that in VEC group and longer survival time in HULC-siRNA group than that in NC group（P < 0.05）. Ki67 protein expression was up-regulated in the HULC-over group compared with that in VEC group and down-regulated in the HULC-siRNA group compared with that in NC group（P < 0.05）.【Conclusion】LncRNA HULC can enhance the growth of glioblastoma U87 cells in vitro and in vivo.

Long Non-Coding RNA HULC Promotes the Development of Breast Cancer Through Regulating LYPD1 Expression by Sponging miR-6754-5p.

INTRODUCTION: Long non-coding RNAs (lncRNAs) were found to regulate many biological processes including cancer development, immunology and other diseases. LncRNA HULC was found to be oncogenes in many cancer progression. However, the role of HULC in the regulation of breast cancer remains unclear. METHODS: The expression of HULC and miR-6754-5p was examined by RT-PCR. Through knockdown of HULC, we found that the proliferation abilities coupled with migration and invasion abilities were significantly decreased. And also, we verified that overexpression of miR-6754-5p significantly decreased the proliferation ability of breast cancer cells. RESULTS: In this study, we found that lncRNA HULC was overexpressed in breast cancer tissues and cell lines compared to normal healthy breast tissues and normal breast cell line. Moreover, the high expression of HULC was associated with metastasis and malignancy of breast cancers. Mechanically, we found that HULC can bind to miR-6754-5p directly through complementary base pairing. Furthermore, we found that HULC regulates the expression of LYPD1 through sponging miR-6754-5p. Moreover, overexpression of LYPD1 can rescue the migration and invasion abilities of breast cancer cells decreased by knockdown of HULC or overexpression of miR-6754-5p. CONCLUSION: Our study showed the role of HULC in promoting breast cancer development and explained the detailed molecular mechanisms.

METase/lncRNA HULC/FoxM1 reduced cisplatin resistance in gastric cancer by suppressing autophagy.

BACKGROUND: Autophagy plays an important role in regulating cisplatin (CDDP) resistance in gastric cancer cells. However, the underlying mechanism of methioninase (METase) in the regulation of autophagy and CDDP resistance of gastric cancer cells is still not clear. MATERIALS AND METHODS: Western blot was used to detect the levels of autophagy-related proteins, multidrug-resistant 1 (MDR-1), and FoxM1 protein. LncRNA HULC was detected by qRT-PCR. Cell viability was detected using CCK-8 assay. The interaction between lncRNA HULC and FoxM1 was confirmed by RNA pull-down and RIP assay. RESULTS: Lentiviral vector carrying METase (LV-METase) suppressed autophagy and CDDP resistance of drug-resistant gastric cancer cells. LncRNA HULC was significantly downregulated in drug-resistant gastric cancer cells transfected with LV-METase. Besides, we found that lncRNA HULC interacted with FoxM1. In addition, METase suppressed autophagy to reduce CDDP resistance of drug-resistant gastric cancer cells through regulating HULC/FoxM1, and interfering HULC suppressed autophagy to reduce CDDP resistance of drug-resistant gastric cancer cells through regulating FoxM1. Finally, interfering HULC inhibited tumor growth in vivo. CONCLUSION: METase suppressed autophagy to reduce CDDP resistance of drug-resistant gastric cancer cells through regulating HULC/FoxM1 pathway.

[LncRNA HULC promots HCC growth by downregulating miR-29].

Objective: To explore the effects of lncRNA HULC on hepatocellular carcinoma (HCC) growth by down-regulating miR-29. Methods: The expression levels of HULC and miR-29 in HCC tissues and cells were detected by real-time quantitative PCR (RT-qPCR), and the correlation analysis was performed. After HCC cells were transfected with HULC overexpressed plasmid or siRNA, the expressions of miR-29 and its target gene SETDB1 were determinate by RT-qPCR. According to the bioinformatic prediction of the miR-29 binding site in the HULC sequence, the report gene plasmids were constructed. The HCC cells were co-transfected with miR-29 mimics or miR-29 inhibitor, and the HULC targeted regulation of miR-29 was verified by dual luciferase reporter assay. The effect of miR-29 on the HULC-mediated proliferation in HCC cells was detected by cell count kit 8 (CCK-8) experiment. Expression of tumor proliferation antigen Ki-67 was detected by RT-qPCR.The Hep3B cells were inoculated in mice and miR-29 mimics and miR-29 negative control (NC) further injected into the lesions. The tumor volume was observed, and the expressions of tumor proliferation antigen ki-67 in tumor tissues were detected by immunohistochemical staining. Results: The expression of HULC was significantly up-regulated while the expression of miR-29 was significantly down-regulated in HCC tissues and cells (P<0.01). The level of HULC was negatively correlated with miR-29 in tumor tissues (r=-0.754, P<0.01) and HCC cells (r=-0.865, P<0.05). The in vitro experiments showed that, compared with the blank control group, the expression of miR-29 in HULC overexpressed Huh7 cells was significantly reduced, while the mRNA level of miR-29 target gene SETDB1 was increased (P<0.01). The expression of miR-29 was significantly increased in HULC deleted Hep3B cells, while the mRNA expression of SETDB1 was decreased (P<0.01). Double luciferase reporter gene assay showed that miR-29 mimics significantly inhibited the luciferase activity of Hep3B cells transfected with HULC wide type (psi-HULC-WT) plasmid but had no effect on Hep3B cells transfected with mutant plasmid (psi-HULC-Mut). However, the miR-29 inhibitor antagonized the inhibitory effect of miR-29 mimics on luciferase activity of psi-HULC-WT (P<0.01). Cell proliferation experiments showed that, compared with the control group, the proliferation ability of miR-29 mimics overexpressed Huh7 cells was significantly reduced.After 24, 48 and 72 hours of treatment, the proliferation rates of Huh7 cells in the HULC overexpressed group were (43.87±3.82)%, (83.45±7.46)% and (123.34±8.67)%, respectively, significantly higher than (13.45±1.77)%, (23.54±1.37)% and (38.21±2.09)% of control group (P<0.05). After treatment for 48 and 72 hours, the proliferation rates of miR-29 mimics transfected Huh7 cells were (57.10±1.94)% and (73.76±3.46)%, respectively, significantly lower than (83.45±7.46)% and (123.34±8.67)% of control group (P<0.05). After treatment for 48 and 72 hours, the proliferation rates of Huh7 cells transfected with miR-29 mimics and miR-29 inhibitor group were (76.45±3.24)% and (89.37±4.37)%, respectively, significant higher than (57.10%±1.94)% and (73.76±3.46)% of the control group (P<0.05). After 48 h transfection, the expression of Ki-67 in Huh7 transfected with miR-29 mimics was significantly inhibited compared with the control group (P<0.01). However, the expression of Ki-67 mRNA was increased in Huh7 cells transfected with miR-29 inhibitor (P<0.01). The results of in vivo experiments showed that the tumor volumes of the control group, miR-29 mimics group and miR-29 mimics + miR-29 inhibitors group were (504.0±19.6) mm(3), (310.0±24.3) mm(3) and (483.7±21.2) mm(3), respectively. Injection of miR-29 mimics reduced while miR-29 inhibitor promoted tumorigenesis ability of Huh7 in nude mice (P<0.01). The immunohistochemical staining showed that the average optical density values of Ki-67 protein in tumor tissues of the control group, miR-29 mimics group and miR-29 analogue+ miR-29 inhibitor group were 0.65±0.08, 0.36±0.07 and 0.56±0.06, respectively. The expression level of Ki-67 protein in miR-29 mimics group was significantly reduced (P<0.01) while increased in the miR-29 mimics+ miR-29 inhibitor group (P<0.01). Conclusion: LncRNA HULC promotes HCC growth by down-regulating miR-29.

Clinical important dysregulation of long non-coding RNA CCHE1 and HULC in carcinogenesis of cervical cancer.

Genetic analysis has revealed that the lncRNAs acted in the critical pathways including cell growth, cell differentiation, and tumorigenesis. The purpose of the present survey was to evaluate the lncRNA-CCHE1 and lncRNA-HULC expression variations in subjects with cervical malignancy. In our case-control analysis planned on 100 formalin-fixed paraffin-embedded (FFPE) samples comprising 50 paraffin blocks of cervical cancerous and 50 paraffin blocks of non-tumors samples. We have calculated the lncRNA-CCHE1 and lncRNA-HULC gene expression alterations using a quantitative RT-PCR approach. Our results declared that the lncRNA-CCHE1 expression was considerably raised in tumorous matched to the non-tumorous samples (P = 0.001). Moreover, the lncRNA-HULC expression was not considerably modified between tumorous samples matched to the non-tumorous samples (P = 0.060). In extension, lncRNA-CCHE1 expression variation was correlated with the histological type (P = 0.001), tumor size (P = 0.001) and FIGO stage (P = 0.001). Our data advised that the lncRNA-CCHE1 gene may influence cervical malignancy progression, held as a prognostic marker and useful curative target. However, the character of lncRNA-HULC in carcinogenesis of cervical malignancy requires further analyses.

Genome-wide identification of a competing endogenous RNA network in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is the second widespread liver tumor with relatively poor survival. Increasing evidence in recent studies showed long noncoding RNAs (lncRNAs) exert a crucial impact on the development and progression of CCA based on the mechanism of competing endogenous RNAs (ceRNAs). However, functional roles and regulatory mechanisms of lncRNA-regulated ceRNA in CCA, are only partially understood. The expression profile of messenger RNAs (mRNAs), lncRNAs, and microRNAs (miRNAs) downloaded from The Cancer Genome Atlas were comprehensively investigated. Differential expression of these three types of RNA between CCA and corresponding precancerous tissues were screened out for further analysis. On the basis of interactive information generated from miRDB, miRTarBase, TargetScan, and miRcode public databases, we then constructed an mRNA-miRNA-lncRNA regulatory network. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses were conducted to identify the biological function of the ceRNA network involved in CCA. As a result, 2883 mRNAs, 136 miRNAs, and 993 lncRNAs were screened out as differentially expressed RNAs in CCA. In addition, a ceRNA network in CCA was constructed, composing of 50 up and 27 downregulated lncRNAs, 14 up and 7 downregulated miRNAs, 29 up and 25 downregulated mRNAs. Finally, gene set enrichment and pathway analysis indicated our CCA-specific ceRNA network was related with cancer-related pathway and molecular function. In conclusion, our research identified a novel lncRNA-related ceRNA network in CCA, which might act as a potential therapeutic target for patients with CCA.

Long non-coding RNA highly up-regulated in liver cancer protects tumor necrosis factor-alpha-induced inflammatory injury by down-regulation of microRNA-101 in ATDC5 cells.

BACKGROUND: Osteoarthritis (OA) is a familiar joint degenerative disease. Long non-coding RNAs (lncRNAs) play vital roles in the pathogenesis of OA. Nevertheless, the regulatory impacts of lncRNA highly up-regulated in liver cancer (lncRNA-HULC) on OA remain dimness. The study tried to probe the protective effect of HULC on ATDC5 cells against tumor necrosis factor-alpha (TNF-α)-induced inflammatory injury. METHODS: Relative expression levels of pro-inflammatory cytokines (IL-6, IL-8 and MCP-1) and HULC in OA cartilage tissues and normal cartilage tissues were determined by RT-qPCR. TNF-α induced inflammatory injury model in ATDC5 cells was constructed, and the biological functions of HULC overexpression or suppression in TNF-α-injured ATDC5 cells were assessed. The relevancy between miR-101 and HULC was investigated by utilizing bioinformatics prediction, luciferase reporter assay, RNA pull-down and immunoprecipitation. MiR-101 mimic and inhibitor were transfected into ATDC5 cells, and its regulatory effect on TNF-α-injured ATDC5 cells was examined. Further, NF-κB and MAPK signaling pathways were finally detected by western blot. RESULTS: Enhancement of IL-6, IL-8 and MCP-1 were observated in OA cartilage tissues, but repression of HULC was discovered in OA cartilage tissues. HULC expression was decreased by TNF-α treatment, and overexpressed HULC significantly relieved TNF-α-induced ATDC5 cells injury. Additionally, miR-101 was mutual repressed with HULC, and overexpressed miR-101 reversed the protective effect of HULC in TNF-α-injured ATDC5 cells. Besides, HULC blocked NF-κB and MAPK pathways via repression of miR-101. CONCLUSIONS: The discoveries testified that HULC protected ATDC5 cells against TNF-α-induced inflammatory injury by repression of miR-101.

Inhibition of lncRNA HULC improves hepatic fibrosis and hepatocyte apoptosis by inhibiting the MAPK signaling pathway in rats with nonalcoholic fatty liver disease.

This study is conducted to investigate the role of long noncoding RNA highly upregulated in liver cancer (lncRNA HULC) on hepatic fibrosis and hepatocyte apoptosis by inhibiting the mitogen-activated protein kinase (MAPK) signaling pathway in rats with nonalcoholic fatty liver disease (NAFLD). The successfully modeled rats were injected with HULC siRNA or small interfering RNA (siRNA) negative control into the tail vein. The expression of HULC in liver tissues was detected by reverse transcription quantitative polymerase reaction chain. The role of HULC in pathological state and liver function-related indexes of liver lipid deposition, the degree of hepatic fibrosis and hepatocyte apoptosis in rats with NAFLD were also investigated through a series of experiments. Increased expression of HULC was found in liver tissue of NAFLD rats. Inhibition of HULC improved the pathological state and liver function-related indexes of liver lipid deposition, improved the degree of hepatic fibrosis, reduced hepatocyte apoptosis, and inhibited the MAPK signaling pathway in the liver tissue of NAFLD rats. The inhibition of p38 and JNK improved the pathological state of liver lipid deposition and liver function to some extent, improved the degree of hepatic fibrosis, and reduced the apoptosis of hepatocytes in NAFLD rats. Collectively, this present study provides evidence that inhibition of lncRNA HULC improves hepatic fibrosis and decrease hepatocyte apoptosis in rats with NAFLD by inhibiting the MAPK signaling pathway.

LncRNA HULC promots HCC growth by downregulating miR-29 / 中华肿瘤杂志

Objective@#To explore the effects of lncRNA HULC on hepatocellular carcinoma (HCC) growth by down-regulating miR-29.@*Methods@#The expression levels of HULC and miR-29 in HCC tissues and cells were detected by real-time quantitative PCR (RT-qPCR), and the correlation analysis was performed. After HCC cells were transfected with HULC overexpressed plasmid or siRNA, the expressions of miR-29 and its target gene SETDB1 were determinate by RT-qPCR. According to the bioinformatic prediction of the miR-29 binding site in the HULC sequence, the report gene plasmids were constructed. The HCC cells were co-transfected with miR-29 mimics or miR-29 inhibitor, and the HULC targeted regulation of miR-29 was verified by dual luciferase reporter assay. The effect of miR-29 on the HULC-mediated proliferation in HCC cells was detected by cell count kit 8 (CCK-8) experiment. Expression of tumor proliferation antigen Ki-67 was detected by RT-qPCR.The Hep3B cells were inoculated in mice and miR-29 mimics and miR-29 negative control (NC) further injected into the lesions. The tumor volume was observed, and the expressions of tumor proliferation antigen ki-67 in tumor tissues were detected by immunohistochemical staining.@*Results@#The expression of HULC was significantly up-regulated while the expression of miR-29 was significantly down-regulated in HCC tissues and cells (P<0.01). The level of HULC was negatively correlated with miR-29 in tumor tissues (r=-0.754, P<0.01) and HCC cells (r=-0.865, P<0.05). The in vitro experiments showed that, compared with the blank control group, the expression of miR-29 in HULC overexpressed Huh7 cells was significantly reduced, while the mRNA level of miR-29 target gene SETDB1 was increased (P<0.01). The expression of miR-29 was significantly increased in HULC deleted Hep3B cells, while the mRNA expression of SETDB1 was decreased (P<0.01). Double luciferase reporter gene assay showed that miR-29 mimics significantly inhibited the luciferase activity of Hep3B cells transfected with HULC wide type (psi-HULC-WT) plasmid but had no effect on Hep3B cells transfected with mutant plasmid (psi-HULC-Mut). However, the miR-29 inhibitor antagonized the inhibitory effect of miR-29 mimics on luciferase activity of psi-HULC-WT (P<0.01). Cell proliferation experiments showed that, compared with the control group, the proliferation ability of miR-29 mimics overexpressed Huh7 cells was significantly reduced.After 24, 48 and 72 hours of treatment, the proliferation rates of Huh7 cells in the HULC overexpressed group were (43.87±3.82)%, (83.45±7.46)% and (123.34±8.67)%, respectively, significantly higher than (13.45±1.77)%, (23.54±1.37)% and (38.21±2.09)% of control group (P<0.05). After treatment for 48 and 72 hours, the proliferation rates of miR-29 mimics transfected Huh7 cells were (57.10±1.94)% and (73.76±3.46)%, respectively, significantly lower than (83.45±7.46)% and (123.34±8.67)% of control group (P<0.05). After treatment for 48 and 72 hours, the proliferation rates of Huh7 cells transfected with miR-29 mimics and miR-29 inhibitor group were (76.45±3.24)% and (89.37±4.37)%, respectively, significant higher than (57.10%±1.94)% and (73.76±3.46)% of the control group (P<0.05). After 48 h transfection, the expression of Ki-67 in Huh7 transfected with miR-29 mimics was significantly inhibited compared with the control group (P<0.01). However, the expression of Ki-67 mRNA was increased in Huh7 cells transfected with miR-29 inhibitor (P<0.01). The results of in vivo experiments showed that the tumor volumes of the control group, miR-29 mimics group and miR-29 mimics + miR-29 inhibitors group were (504.0±19.6) mm3, (310.0±24.3) mm3 and (483.7±21.2) mm3, respectively. Injection of miR-29 mimics reduced while miR-29 inhibitor promoted tumorigenesis ability of Huh7 in nude mice (P<0.01). The immunohistochemical staining showed that the average optical density values of Ki-67 protein in tumor tissues of the control group, miR-29 mimics group and miR-29 analogue+ miR-29 inhibitor group were 0.65±0.08, 0.36±0.07 and 0.56±0.06, respectively. The expression level of Ki-67 protein in miR-29 mimics group was significantly reduced (P<0.01) while increased in the miR-29 mimics+ miR-29 inhibitor group (P<0.01).@*Conclusion@#LncRNA HULC promotes HCC growth by down-regulating miR-29.

Effects of Silencing Expression of Long Non-Coding RNA HULC on Proliferation and Apoptosis of Human Glioblastoma Cell SHG44 / 中山大学学报(医学科学版)

@#【Objective】 To explore the effects of long- chain non- coding RNA highly up- reglated in liver cancer（LncRNA HULC）silencing expression on proliferation and apoptosis of human glioblastoma cell line SHG44.【Methods】 Quantitative Real- time Polymerase Chain Reaction （qRT- PCR） was used to verify the expression level of HULC in HULC silent expression group （HULC- siRNA）and negative control group （NC group）. CCK8 proliferation assay and plate colony formation assay were used to detect the proliferation of glioblastoma cells. Cell cycle and apoptosis assays were used to detect the cell cycle distribution and apoptosis of glioblastoma cells. 【Results】 qRT- PCR confirmed that HULC- siRNA group had significantly lower expression of HULC than NC group （P=0.003）. CCK8 proliferation experiment showed that the proliferation rate of HULC-siRNA group was significantly lower than that of NC group on the second，third and fourth days of experiment（Day 2，P=0.003；Day 3，P=0.005；Day 4，P=0.009）. Plate colony formation assay showed the cloning rates in the NC and HULC-siRNA groups were（34.11 ± 1.24）% and（14.44 ± 0.87）%，and it showed that the cell clone formation rate was clearly decreased after silenced expression of HULC （P<0.001）. The cell cycle assay showed that the numbers of cells in NC group and HULC-siRNA group were G1 phase（36.89 ± 4.09，51.74± 0.68）and S phase（46.95 ± 2.49，36.89 ± 2.13），and it showed that the cell cycle was blocked in G1/S phase after silencing HULC expression （G1 phase，P=0.023，S phase，P=0.038）. Apoptosis experiment showed that the early apoptotic rates of NC group and HULC-siRNA group were（2.57 ± 0.22）% and（7.063 ± 0.71）%，and it showed that the early apoptotic rate of the cells was significantly increased after silencing HULC expression （P=0.004）. 【Conclusion】 Silencing of LncRNA HULC can inhibit the proliferation of glioblastoma cells and promote their apoptosis.

Pro-Angiogenic Role of LncRNA HULC in Microvascular Endothelial Cells via Sequestrating miR-124.

BACKGROUND/AIMS: HULC is a multifunctional lncRNA that has pro-angiogenic function in various cancers. The present study was designed to see the role of lncRNA HULC in normal endothelial cells angiogenesis. METHODS: Cell viability, apoptosis, migration, tube formation and expression levels of angiogenesis-related proteins were respectively assessed in human microvascular endothelial HMEC-1 cells after lncRNA HULC was silenced by shRNA transfection. Cross-regulation between lncRNA HULC and miR-124, and between miR-124 and MCL-1 were detected by qRT-PCR, sequence analysis, and luciferase reporter assay. RESULTS: Silence of lncRNA HULC significantly reduced viability, migration, tube formation and protein levels of VEGF, VEGFR2, CD144 and eNOS in HMEC-1 cells. Meanwhile, silence of lncRNA HULC induced apoptosis in HMEC-1 cells, as Bcl-2 was down-regulated, Bax was up-regulated, and caspase-3 and -9 were cleaved. miR-124 expression was negatively regulated by lncRNA HULC, and HULC worked as a molecular sponge for miR-124, in having miR-124 exhausted. Besides, MCL-1 was a target gene of miR-124. Rescue assay results showed that the effects of lncRNA HULC silence on HMEC-1 cells growth, migration and angiogenesis were abolished by miR-124 suppression. Similarly, the effects of miR-124 on HMEC-1 cells were abolished by MCL-1 overexpression. Furthermore, MCL-1 activated PI3K/AKT and JAK/STAT signaling pathways. CONCLUSION: These findings suggest a pro-angiogenic role of lncRNA HULC in endothelial cells. The pro-angiogenic actions of lncRNA HULC may be through sponging miR-124, preventing MCL-1 from degradation by miR-124.

RETRACTED: Long non-coding RNA HULC promotes UVB-induced injury by up-regulation of BNIP3 in keratinocytes.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Concerns were raised about the background pattern of the Western Blots from Figures 4 and 5. Given the comments of Dr Elisabeth Bik regarding this article "This paper belongs to a set of over 400 papers (as per February 2020) that share very similar Western blots with tadpole-like shaped bands, the same background pattern, and striking similarities in title structures, paper layout, bar graph design, and - in a subset - flow cytometry panels", the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.