HOTTIP Expression Research Articles

<i>HOTTIP</i> Dependent R-Loop Formation Regulates CTCF Boundary Activity and TAD Integrity in Leukemia

HOTTIP lncRNA is highly expressed in acute myeloid leukemia patients carrying MLL rearrangement or NPM1 mutation to facilitate the formation of HOXA topologically associated domains (TADs) and drive aberrant transcription. However, the mechanism through which HOTTIP accesses CTCF chromatin boundaries and regulates CTCF-mediated leukemic genome topology remains unknown. Here, we show that HOTTIP directly interacts and regulates a fraction of CTCF-binding sites (CBSs) including key canonical beta-catenin target loci in the AML genome by forming R-loops that reinforce the CTCF boundary and facilitate formation of TADs to drives gene transcription. Either deleting CBSs or targeting RNaseH to eliminate R-loop in CBSs of beta-catenin TAD resulted in impaired CTCF boundary activity, inhibited promoter/enhancer interactomes, reduced beta-catenin target expression, and mitigated leukemogenesis in PDX mouse models with aberrant HOTTIP expression. Thus, HOTTIP-mediated R-loop formation directly reinforces CTCF chromatin boundary activity and TAD integrity to drive oncogene transcription and pathogenesis of leukemia.

Non-coding RNAs repressive role in post-transcriptional processing of RUNX2 during the acquisition of the osteogenic phenotype of periodontal ligament mesenchymal stem cells

Mesenchymal stem cells are candidates for therapeutic strategies in periodontal repair due to their osteogenic potential. In this study, we identified epigenetic markers during osteogenic differentiation, taking advantage of the individual pattern of mesenchymal cells of the periodontal ligament with high (h-PDLCs) and low (l-PDLCs) osteogenic capacity. We found that the involvement of non-coding RNAs in the regulation of the RUNX2 gene is strongly associated with high osteogenic potential. Moreover, we evaluated miRs and genes that encode enzymes to process miRs and their biogenesis. Our data show the high expression of the XPO5 gene, and miRs 7 and 22 observed in the l-PDLCs might be involved in acquiring osteogenic potential, suppressing RUNX2 gene expression. Further, an inversely proportional correlation between lncRNAs (HOTAIR and HOTTIP) and RUNX2 gene expression was observed in both l- and h-PDLCs, and it was also related to the distinct osteogenic phenotypes. Thus, our results indicate the low expression of XPO5 in h-PDLC might be the limiting point for blocking the miRs biogenesis, allowing the high gene expression of RUNX2. In accordance, the low expression of miRs, HOTAIR, and HOTTIP could be a prerequisite for increased osteogenic potential in h-PDLCs. These results will help us to better understand the underlying mechanisms of osteogenesis, considering the heterogeneity in the osteogenic potential of PDLCs that might be related to a distinct transcriptional profile of lncRNAs and the biogenesis machinery.

HOTTIP polymorphism may affect gastric cancer susceptibility by altering HOTTIP expression.

Background: Non-coding RNA polymorphisms can affect disease risk and prognosis by influencing gene expression. Here, we first investigated the association between single nucleotide polymorphisms (SNPs) of long non-coding RNA (lncRNA) HOTTIP and gastric cancer risk/prognosis. Methods: A total of five HOTTIP SNPs among 627 gastric cancer cases and 935 controls were tested by Kompetitive Allele Specific PCR (KASP) assay. The functional SNPs underwent eQTL analysis and the expression of HOTTIP was assessed by quantitative RT-PCR. Results: The rs2067087 and rs3807598 SNPs of HOTTIP increased susceptibility to gastric cancer (rs2067087: dominant model, P=0.008, odds ratio (OR) = 1.35; rs3807598: recessive model, P=0.037, OR = 1.29). Both HOTTIP rs2067087 and rs3807598 could affect the expression of mature lncRNA (P=0.003 and P=0.032, respectively). Conclusion: The rs2067087 and rs3807598 SNPs of HOTTIP are associated with gastric cancer risk, possibly by affecting the expression of mature HOTTIP.

Association Between rs1859168/HOTTIP Expression Level and Colorectal Cancer and Adenomatous Polyposis Risk in Egyptians.

LncRNA HOTTIP is a new lncRNA that is strictly linked to the susceptibility, growth, propagation, and prognosis of several human cancers together with colorectal cancer. lncRNA HOTTIP rs1859168 may confer colorectal cancer susceptibility through regulating its gene expression level. To elucidate its role in colorectal cancer risk, we genotyped rs1859168 A>C and measured serum HOTTIP expression level in colorectal cancer, adenomatous polyposis patients and controls by real-time polymerase chain reaction. The results displayed that rs1859168 A>C single-nucleotide polymorphism is a risk factor for colorectal cancer among adenomatous polyposis patients and controls, AC versus CC genotypes [adjusted odds ratio (OR) = 2.256, 95% confidence interval (CI) = 1.316-3.868, P = 0.003] when compared with controls and (adjusted OR = 9.521, 95% CI = 3.330-27.217, P < 0.0001) when compared with adenomatous polyposis. Serum HOTTIP was upregulated in the colorectal cancer group when compared with adenomatous polyposis or controls [median (interquartile range) = 3.64 (2.46-5.02) (P < 0.0001)]. A significant difference in serum HOTTIP was found to be associated with different rs1859168 genotypes. rs1859168 A>C and higher serum HOTTIP were significantly associated with distant metastasis, lymph nodes metastasis, and grade III of colorectal cancer. Both rs8159168 and high HOTTIP confer increased risk for colorectal cancer development. [Figure: see text].

LncRNA HOTTIP Participates in Cisplatin Resistance of Tumor Cells by Regulating miR-137 Expression in Pancreatic Cancer.

AimThis study aimed to investigate the effect of HOTTIP and miR-137 on cisplatin resistance of pancreatic cancer cells, and study the mechanism of the effect of HOTTIP on the resistance to cisplatin in pancreatic cancer cells, so as to provide new targets for clinical treatment of pancreatic cancer.MethodsPancreatic cancer cells were induced to be resistant to cisplatin by gradually increasing cisplatin concentration at a low concentration gradient in vitro. The changes of HOTTIP and miR-137 were detected, and the effects of HOTTIP and miR-137 on cisplatin efficacy of pancreatic cancer cisplatin-resistant cells were analyzed to explore the mechanism of HOTTIP on cisplatin resistance of pancreatic cancer cells.ResultsAfter inducing cisplatin resistance in pancreatic cancer cells, the expression level of HOTTIP in pancreatic cancer cells further increased and miR-137 decreased. Silencing HOTTIP or over-expression of miR-137 can increase the sensitivity of pancreatic cancer cisplatin-resistant cells to cisplatin, inhibit the proliferation of pancreatic cancer cells, and promote apoptosis. And we found HOTTIP can target to inhibit miR-137 expression. Rescue experiments showed that regulating miR-137 cannot affect the expression of HOTTIP, miR-137 is a downstream target of HOTTIP, and down-regulation of miR-137 expression can obviously hinder the cisplatin sensitization effect of silencing HOTTIP on cisplatin-resistant pancreatic cancer cells.ConclusionSilencing HOTTIP reverses cisplatin resistance of pancreatic cancer cells by promoting miR-137 expression.

Hottip Lncrna Reinforces CTCF Defined Chromatin Boundaries and Drives Wnt Target Gene Expression in AML Leukemogenesis

We reported recently that HOXA locus associated lncRNA, HOTTIP, is highly expressed in AML patients carrying MLL rearrangement and NPM1c+ mutations. The expression of HOTTIP positively correlates with posterior HOXA gene expression and poor patient survival. We further demonstrated that HOTTIP acts as an epigenetic regulator to define oncogenic HOXA topologically associated domain (TAD) and drive HOXA associated leukemic transcription program. However, it remains unclear whether and how HOTTIP lncRNA is involved in remodeling leukemic genome to facilitate AML leukemogenesis. Here, we showed that HOTTIP regulates a fraction of CTCF binding sites (CBSs) in the AML genome by directly interacting with CTCF and its binding motifs. We carried out CTCF ChIP-seq and HOTTIP ChIRP (chromatin isolation by RNA purification)-seq comparing WT and HOTTIP knockout (KO) MOLM13 cells. KO of HOTTIP in MLL-rearranged MOLM13 AML cells specifically impaired CTCF binding sites that were co-occupied by HOTTIP lncRNA, whereas loss of HOTTIP did not affect global CTCF binding. These target genes include posterior HOXA genes and Wnt target genes such as C-MYC, EVI1, AXIN, and TWIST1. Furthermore, we found that HOTTIP interacts with its putative target sites by formation of DNA: RNA hybridization structure triple helix and R-loop in vivo and in vitro. We then carried out DRIP (DNA-RNA immunoprecipitation)-seq and DRIPc(DNA-RNA immunoprecipitation followed by cDNA conversion)-Seq, which utilize a sequence independent but structure-specific S9.6 antibody for DRIP to capture global R-loops, by comparing WT and HOTTIP KO MOLM13 cells. The obtained DRIP-seq and DRIPc-seq data were then incorporated and integrated with the HOTTIP ChIRP-seq and CTCF ChIP-seq data to explore global collaboration between R-loop and HOTTIP associated CTCF binding sites. We found that HOTTIP interacts with CTCF binding motif that defines the TADs and the promoters of the HOTTIP target genes by formation of R-loop or triple helix structure. Loss of HOTTIP disrupted the R-loop formation at promoters and enhancers of the HOTTIP target genes to inhibit their expression. In MLL-rearranged AML genome, in addition to the HOXA locus, CTCF forms leukemic specific TADs that protect aberrant Wnt target genes. Depletion of HOTTIP lncRNA impaired CTCF defined TADs in the Wnt target gene loci and reduced Wnt target gene expression. In contrast, overexpression of Hottip lncRNA (Hottip-Tg) in the mice bone marrow hematopoietic compartment perturbs hematopoietic stem cell (HSC) self-renewal and differentiation leading to AML like disease by reinforcing CTCF defined TADs, enhancing chromatin accessibility within TADs, and upregulating gene transcription in the Wnt target loci. Finally, when we treated HOTTIP expressed primary patient AML cells carrying MLL-rearrangement and their derived PDX mouse model with a canonical Wnt inhibitor, ICG-001, ICG-001 inhibited AML LSC self-renewal in in vitro by LTC-IC assays and in vivo leukemogenesis in the PDX mouse models with an aberrant HOTTIP lncRNA expression, but not in HOTTIP negative/low non-MLL AML samples. Thus, HOTTIP lncRNA and CTCF cooperate to specifically reinforce CTCF defined WNT target locus TADs and drive Wnt target gene expression in the HOTTIP expressed AML. Disclosures No relevant conflicts of interest to declare.

P1.12-01 CD81-HOTTIP Regulating Loop Enhances Myc-Mediated Chemoresistance in Small Cell Lung Cancer via ERK Signaling Pathway

The majority of small cell lung cancer (SCLC) patients who are treated with platinum-based chemotherapy will eventually develops into drug resistance. Currently, there are no effective durable therapies for patients with resistant recurrent SCLC. High expression of CD81 is associated with SCLC chemoresistance and poor clinical outcome. Here, our goal is to study the underlying mechanism and define a novel signaling pathway controlled by CD81 -modulating SCLC chemotherapy resistance. The expression of CD81, HOTTIP, and MYCBP were examined in human SCLC tissues, patient-derived xenograft (PDX) models, cell lines derived xenograft tumor model, and SCLC cell lines. The correlations between CD81 and HOTTIP were also confirmed by RNA immunoprecipitation and luciferase reporter assay. Functional analysis of HOTTIP, MYCBP, and ERK signaling was investigated in SCLC cell lines and xenografts. Knockdown CD81 enhanced cell responsiveness to chemotherapeutic drugs in SCLC and decreased the expression of HOTTIP, as well as inhibited the intra-nuclear translocation of MYCBP/MYC complex. Conversely, overexpression of CD81 decreased the chemotherapeutic reactivity and increased the expression of HOTTIP and promoting nuclear translocation of MYCBP/MYC complex. CD81 upregulation of HOTTIP requires the transcription factor TCF4. Further analysis demonstrated that CD81-mediated activation of ERK signaling pathway and inhibition of β-catenin degradation leads to promotion of β-catenin transfer to the nucleus. Nuclear β-catenin enhances transcriptional activity of TCF4 and then promotes the transcription of HOTTIP. Synergistically, HOTTIP could bind and increase the nucleus transduction of the MYCBP, form a HOTTIP/MYCBP/MYC complex in nucleus, resulting in increased MYC levels in nucleus. Nuclear MYC further mediates chemoresistance and promotes CD81 expression, forming a positive feedback regulatory pathway in SCLC. In addition, in vivo experiments showed that inhibitors that target the CD81-HOTTIP feedback regulatory loop combined with chemotherapeutic drugs can reverse the chemotherapeutic resistance of SCLC. Our findings suggest a novel feedback regulatory pathway dominated by CD81 and HOTTIP controlling SCLC chemotherapeutic resistance, which could serve as a new therapeutic targets for this devastating disease.

692P - Expression of long noncoding RNA and clinical outcomes of pancreatic cancer patients who received adjuvant chemotherapy by S-1 or GEM after curative resection

BackgroundLong noncoding RNAs (lncRNAs) have recently been known to play various important roles in maintaining molecular regulations. Significant amounts of lncRNAs HOTTIP, PVT1 and H19 are well known to be expressed in pancreatic cancer cells, however their association with efficacy of adjuvant chemotherapy for pancreatic cancer have not been fully investigated. The aim of this study was to evaluate expression of HOTTIP, PVT1 and H19 in the pancreatic cancer cells, and to clarify the predictive roles of those lncRNAs in pancreatic cancer patients who received adjuvant chemotherapy by S-1, an oral fluoropyrimidine or gemcitabine (GEM) after curative resection. MethodsPancreatic cancer patients who underwent curative resection followed by adjuvant chemotherapy by S-1 or GEM since 2005 to 2017 at Kanagawa Cancer Centre were enrolled in this study. In situ hybridization (RNA scope®) to detect each three lncRNAs was performed using tissue microarrays prepared from formalin-fixed and paraffin-embedded tissues of resected specimens. The relations between the expression of HOTTIP, PVT1 and H19 and clinicopathological characteristics were statistically analyzed. ResultsWe analyzed 204 eligible pancreatic cancer patients. 114 patients received adjuvant chemotherapy by S-1, and 90 patients by GEM. The patients with higher expression of H19 were significantly associated with shorter relapse free survival (RFS) regardless of adjuvant chemotherapy regimens (p<0.01). Contrary, the patients with higher expression of PVT1 were significantly associated with longer RFS when they were received adjuvant chemotherapy with S-1 (p=0.048), but not with GEM (p=0.65). The patients with higher expression of HOTTIP were significantly associated with longer overall survival (OS) in the GEM group (p<0.01), and not in the S-1 group (p=0.38). ConclusionsThe expression levels of PVT1 and HOTTIP in pancreatic cancer cells were associated with the efficacy of adjuvant chemotherapy for pancreatic cancer in the present study. The higher expression of H19 may serve as a significant predictive/prognostic marker for patients who received adjuvant chemotherapy by S-1 or GEM after curative resection. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Salinomycin reduces epithelial-mesenchymal transition-mediated multidrug resistance by modifying long noncoding RNA HOTTIP expression in gastric cancer cells.

Chemotherapy is the main treatment for advanced gastric cancer. However, the emergence of multidrug resistance (MDR) has become a major obstacle in chemotherapy in many tumors, including gastric cancer. Epithelial-mesenchymal transition (EMT), which is considered an important process in cancer development, also contributes toward tumor MDR. Salinomycin, an EMT blocker, shows broad-spectrum antitumor and chemosensitization properties. Here, we hypothesized that salinomycin could reverse the MDR of SGC7901/cisplatin (CDDP) gastric cancer cell by inhibiting EMT and further explored its possible underlying mechanisms. Our results indicated higher 50% inhibiting concentration (IC50) and stronger migration capacity in SGC7901/CDDP than in SGC7901 cells, whereas salinomycin could reduce the IC50 (50% inhibition of the concentration of chemodrugs after 4 μmol/l salinomycin treatment) and migration capacity in SGC7901/CDDP cells. At the molecular level, we found that the expression of E-cadherin, ZO-1 decreased, whereas the expression of N-cadherin, Vimentin, ZEB-1, and Twist increased in SGC7901/CDDP cells, and that salinomycin potently blocked the EMT by enhancing the expression of E-cadherin, ZO-1 and reducing the expression of N-cadherin, Vimentin, ZEB-1, and Twist in the above MDR cells. In addition, we also found that long noncoding RNA HOTTIP, an oncogenic regulator, was upregulated in SGC7901/CDDP cells, whereas its downregulation could markedly attenuate the EMT, thereby reversing the MDR. Furthermore, our data showed that the salinomycin-elicited MDR-reversion effect was associated closely with suppression of EMT through inhibition of the expression of long noncoding RNA HOTTIP. Collectively, our findings suggest a new underlying mechanism and applicable therapeutic regimen for MDR gastric cancer.

Expression of HOXA terminal transcript antisense RNA in hepatocellular carcinoma tissues and its effect on proliferation, invasion and migration of hepatocellular carcinoma HepG2 cells

Objective To investigate the expression of HOXA terminal transcript antisense RNA (HOTTIP) in hepatocellular carcinoma (HCC) tissues and to explore its effect on proliferation, invasion and migration in HepG2 cells. Methods A total of 60 cases with HCC tissues undergoing excision surgery and 60 cases of corresponding paracancerous tissues from January 2012 to June 2018 in Dandong First Hospital of Liaoning Province were collected. The expressions of HOTTIP in HCC tissues and paracancerous tissues were detected by using real-time quantitative polymerase chain reaction (RT-qPCR), and the relationship between the expression and clinicopathological features was analyzed. HepG2 cell line with high expression of HOTTIP constructed by cell transfer technique was treated as the experimental group, and the empty plasmid pcDNA3.1-NC was treated as the control group. The effect of HOTTIP on the proliferation of HepG2 cells was detected by using CCK-8 method, and the effect of HOTTIP on invasion and migration of HepG2 cells was detected by using Transwell assay. Results The expression of HOTTIP mRNA in HCC tissues was higher than that in paracancerous tissues, and there was no statistically significant difference (1.9±0.6 vs. 0.9±0.7, t = 6.069, P 0.05). The results of RT-qPCR showed that the expression of HOTTIP mRNA in HepG2 cells was increased after transfection of overexpressed HOTTIP and the differences was statistically significant compared with the control group (63±6 vs. 13±9, t = 9.129, P < 0.01). The results of CCK-8 method showed that the proliferation activity of cells was enhanced after the overexpression of HOTTIP in HepG2 cells (24 h, 36 h, 72 h at 490 nm absorbance was 1.497 ± 0.017 vs. 0.826±0.006, 2.002±0.025 vs. 1.211±0.020, 3.257±0.042 vs. 1.772±0.021), and the differences were statistically significant (t values were 5.321, 7.349, 8.793, all P < 0.01). In Transwell invasion assay, the number of cells transferred into the basement membrane in the experimental group was more than that in the control group (101±9 vs. 41±11), and the difference was statistically significant (t = 6.839, P < 0.01). In Transwell migration assay, the number of cells transferred into the basement membrane in the experimental group was more than that in the control group (112±9 vs. 53±11), and the difference was statistically significant (t = 7.105, P < 0.01). Conclusion The expression of HOTTIP in HCC tissues is up-regulated, and the overexpression of HOTTIP can promote the proliferation, invasion and migration of HCC cells. Key words: Liver neoplasms; HOXA terminal transcript antisense RNA; Cell proliferation; Neoplasm invasiveness; Neoplasm metastasis

Long Non-coding RNA HOTTIP Promotes CCL3 Expression and Induces Cartilage Degradation by Sponging miR-455-3p.

Long non-coding RNAs (lncRNAs) play pivotal roles in diseases such as osteoarthritis (OA). However, knowledge of the biological roles of lncRNAs is limited in OA. We aimed to explore the biological function and molecular mechanism of HOTTIP in chondrogenesis and cartilage degradation. We used the human mesenchymal stem cell (hMSC) model of chondrogenesis, in parallel with, tissue biopsies from normal and OA cartilage to detect HOTTIP, CCL3, and miR-455-3p expression in vitro. Biological interactions between HOTTIP and miR-455-3p were determined by RNA silencing and overexpression in vitro. We evaluated the effect of HOTTIP on chondrogenesis and degeneration, and its regulation of miR-455-3p via competing endogenous RNA (ceRNA). Our in vitro ceRNA findings were further confirmed within animal models in vivo. Mechanisms of ceRNAs were determined by bioinformatic analysis, a luciferase reporter system, RNA pull-down, and RNA immunoprecipitation (RIP) assays. We found reduced miR-455-3p expression and significantly upregulated lncRNA HOTTIP and CCL3 expression in OA cartilage tissues and chondrocytes. The expression of HOTTIP and CCL3 was increased in chondrocytes treated with interleukin-1β (IL-1β) in vitro. Knockdown of HOTTIP promoted cartilage-specific gene expression and suppressed CCL3. Conversely, HOTTIP overexpression reduced cartilage-specific genes and increased CCL3. Notably, HOTTIP negatively regulated miR-455-3p and increased CCL3 levels in human primary chondrocytes. Mechanistic investigations indicated that HOTTIP functioned as ceRNA for miR-455-3p enhanced CCL3 expression. Taken together, the ceRNA regulatory network of HOTTIP/miR-455-3p/CCL3 plays a critical role in OA pathogenesis and suggests HOTTIP is a potential target in OA therapy.

Long noncoding RNA HOTTIP is associated with male infertility and promotes testicular embryonal carcinoma cell proliferation.

BackgroundIt has been proposed that lncRNAs, widely transcribed from genomes, play pivotal regulatory roles in a variety of biological processes, but their function in regulating spermatogenesis in human males is rarely reported.MethodsQRT‐PCR was adopted to detect HOTTIP expression level in testicular tissues from hypospermatogenesis (Hypo) patients or controls. The proliferation levels of NT2 and 293T were measured via CCK‐8 and EdU detection. Meanwhile, luciferase reporter gene assay and bioinformatics analysis were carried out to identify a target of HOTTIP. Additionally, the underlying mechanism of HOTTIP’s function was investigated using western blotting and RIP analysis.ResultsThe research results manifested that the expression of HOTTIP in testicular tissues from Hypo patients was prominently reduced in comparison with that in control testicular tissues. Interestingly, it was noted that HOTTIP exhibited a high expression in testicular embryonal carcinoma cell line NT2 compared with that in normal control cell line 293T. It was denoted in cell function evaluation that cell proliferation was impeded by downregulated HOTTIP but evidently stimulated by overexpressed HOTTIP. Moreover, HOTTIP was capable of positively modulating HOXA13 expression via the competitive binding to miR‐128‐3p.ConclusionTherefore, HOTTIP acting as ceRNAs to promote testicular embryonal carcinoma cell proliferation.

HOTTIP Predicts Poor Survival in Gastric Cancer Patients and Contributes to Cisplatin Resistance by Sponging miR-216a

Background: Gastric cancer (GC) is a huge burden worldwide and cisplatin resistance is the major obstruction in the effective chemotherapy of this disease. Previous studies have revealed that HOTTIP was involved in the pathology of GC and related to the poor overall survival of patients. However, the functional role of HOTTIP in GC chemoresistance remains obscure. Methods: Quantitative real-time PCR was used to detect HOTTIP expression in tissues of GC patients received cisplatin-based chemotherapy and GC cells. The chemoresistance effects of HOTTIP were assessed by cell viability, apoptosis and autophagy assays. The relationships among HOTTIP, miR-216a and Bcl-2 were confirmed by luciferase reporter and western blot assay. Findings: HOTTIP was remarkably upregulated in tissues of GC patients who were treated with gastrectomy and cisplatin chemotherapy, especially those with recurrent tumor. Meanwhile, HOTTIP was increased in the cisplatin-resistant cell, SGC7901/DDP, than in its parental cell, SGC7901. Functional assays demonstrated that HOTTIP expression could promote cisplatin-resistance and inhibit apoptosis and autophagy in GC cells. Mechanistic investigations revealed that HOTTIP might regulate functions of GC cells by sponging miR-216a. MiR-216a overexpression could decrease Bcl-2 expression, enhance Beclin1 expression and active autophagy. Interpretation: HOTTIP is closely associated with the recurrence of GC patients. And HOTTIP expression confers the cisplatin resistance via regulating miR-216a/BCL-2/Beclin1/autophagy pathways, which provides a novel strategy to overcome the resistance to chemotherapy in GC. Funding: The study was sponsored by Shandong Key Research and Development Program (2016GSF201122), Natural Science Foundation of Shandong Province (ZR2017MH044), Jinan Science and Technology Development Plan (201805084, 201805003), Shandong Medical and Health Technology Development Project (2018WSB20002). Declaration of Interest: The authors declare that they have no potential conflicts of interest. Ethical Approval: All patients provided their written informed consents and the Ethic committee of the Qilu Hospital of Shandong University approved this study.

Long noncoding RNA HOTTIP is a significant indicator of ovarian cancer prognosis and enhances cell proliferation and invasion.

Long noncoding RNAs (LncRNAs) are involved in the occurrence and progression of human tumors including ovarian cancer (OC). Long noncoding RNA HOTTIP has been found to be involved in several human tumors development. However, the role of HOTTIP in OC remains large unknown. In the present study, our results observed that lncRNA HOTTIP expression levels were notably higher in ovarian cancer tissue samples compared to adjacent normal tissue samples. Increased lncRNA HOTTIP expression levels were significantly associated with advanced FIGO stage and lymph node metastasis of ovarian cancer patients. Survival plots analysis results showed high lncRNA HOTTIP expression levels in ovarian cancer patients showed a poor prognosis compared to patients with low lncRNA HOTTIP expression levels. Function assays showed that lncRNA HOTTIP knockdown in ovarian cancer cells decreased cell proliferation and cell invasion capacities. Furthermore, we demonstrated that inhibition of lncRNA HOTTIP suppressed Wnt/β-catenin signaling by downregulating β-catenin expression. Thus, these results suggest that aberrant HOTTIP expression level could serve as a promising biomarker for monitoring ovarian cancer and potential target of ovarian cancer treatment.

Clinical significance of long non-coding RNA HOTTIP in early-stage non-small-cell lung cancer

BackgroundHOTTIP, a long non-coding RNA located in the HOXA cluster, plays a role in the patterning of tissues with mesodermal components, including the lung. Overexpression of HOXA genes, including HOTTIP, has been associated with a more aggressive phenotype in several cancers. However, the prognostic impact of HOTTIP has not yet been explored in non-small-cell lung cancer (NSCLC). We have correlated HOTTIP expression with time to relapse (TTR) and overall survival (OS) in early-stage NSCLC patients.MethodsNinety-nine early-stage NSCLC patients who underwent surgical resection in our center from June 2007 to November 2013 were included in the study. Mean age was 66; 77.8% were males; 73.7% had stage I disease; and 55.5% had adenocarcinoma. A validation data set comprised stage I-II patients from The Cancer Genome Atlas (TCGA) Research Network.ResultsHOTTIP was expressed in all tumor samples and was overexpressed in squamous cell carcinoma (p = 0.007) and in smokers (p = 0.018). Patients with high levels of HOTTIP had shorter TTR (78.3 vs 58 months; p = 0.048) and shorter OS (81.2 vs 61 months; p = 0.023) than those with low levels. In the multivariate analysis, HOTTIP emerged as an independent prognostic marker for TTR (OR: 2.05, 95%CI: 1–4.2; p = 0.05), and for OS (OR: 2.31, 95%CI: 1.04–5.1; p = 0.04). HOTTIP was validated as a prognostic marker for OS in the TCGA adenocarcinoma cohort (p = 0.025). Moreover, we identified a 1203-mRNA and a 61-miRNA signature that correlated with HOTTIP expression.ConclusionsThe lncRNA HOTTIP can be considered a prognostic biomarker in early-stage NSCLC.

Diagnostic Value of Plasma Long Non-coding RNA HOTTIP as a Non-invasive Biomarker for Colorectal Cancer (A Case- Control Study).

Long non-coding RNAs (lncRNAs), associated with various cancers including colorectal cancer (CRC), could be collected from body fluids easily. Our aims were to determine the expression level of HOTTIP lncRNA in plasma samples of healthy individuals and CRC patients as well as their relationship with clinico-pathological characteristics of patients. First, total RNA was extracted from the plasma samples of 100 subjects including 50 patients and 50 age and sex matched healthy persons. Then, gene expression was measured using real-time PCR technique. The sensitivity and specificity of HOTTIP dysregulation in CRC and healthy individual’s plasma was measured by receiver operating characteristic (ROC) analysis. As compared with healthy controls, HOTTIP lncRNA was over expressed in a statistically significant manner in plasma samples of patients (P=0.001). Significant relationship between HOTTIP expression and positive family history of CRC was observed, too (P=0.04). The ROC curve analysis showed an AUC value of 0.775, a specificity of 82%, a sensitivity of 76%, with a cut off value equal to 2.40 (P =0.001). HOTTIP transcript can be proposed as a new biomarker for early diagnosis due to the increased expression in plasma samples of patients with CRC and the relatively high sensitivity and specificity.

Long noncoding RNA HOTTIP expression predicts tumor recurrence in hepatocellular carcinoma patients following liver transplantation.

The long noncoding RNA HOTTIP has recently been described as a biomarker of poor prognosis in patients with hepatocellular carcinoma (HCC). In the present study, we evaluated the clinical significance of HOTTIP expression in predicting the rate of tumor recurrence in HCC patients after liver transplantation (LT). We examined the expression pattern and single nucleotide polymorphism (SNP) genotype of HOTTIP in HCC samples from 155 patients underwent LT, and its correlation with clinical parameters and patient prognosis was analyzed. HOTTIP was suppressed using siRNA to explore the role HOTTIP plays in tumor progression. The expression level of HOTTIP in cancer tissues was higher than in adjacent noncancerous tissues. Multivariate analyses revealed that HOTTIP expression was an independent prognostic factor for tumor recurrence and lower overall survival times in HCC patients after LT. Patients who beyond the Milan criteria and exhibit decreased HOTTIP expression experienced longer recurrence-free survival and overall survival. HOTTIP rs2071265 is associated with an earlier recurrence in HCC patients. Moreover, the suppression of HOTTIP in liver cancer cell lines reduced cell invasion rates and increased chemosensitivity. In summary, the high expression level of HOTTIP in HCC could serve as a candidate biomarker for predicting poor prognosis in HCC patients underwent liver transplant therapy. Furthermore, HOTTIP might be a potential therapeutic target.

Activation of Hottip lncrna Perturbs HSC Function Leading to AML like Disease in Mice

Several HOX loci associated long noncoding RNAs (lncRNAs) have been shown to regulate transcription of HOX genes through influencing epigenetic landscape. Especially, the posterior HOXA domain associated lncRNA HOTTIP acts as an epigenetic regulator that recruits WDR5/MLL complex to coordinate active chromatin modifications and HOXA genes expression in the development of animal digits. Despite HOX genes, especially HOXA genes, are highly expressed in many acute myeloid leukemia (AML) patients, it remains largely unknown whether and how HOTTIP lncRNA regulates hematopoietic stem cell (HSC) function and contributes to leukemogenesis.We showed previously that disruption of the CTCF boundary located between HOXA7 and HOXA9 genes (CBS7/9) resulted in reduced lncRNA HOTTIP and HOXA genes expression in MLL rearranged AML suggesting that HOTTIP may play a role in ectopic expression of the posterior HOXA gene. We employed a pooled CRISPR-Cas9 KO library to specifically screen lncRNAs in four HOX gene loci and identify HOTTIP as acritical regulator in controlling oncogenic HOX chromatin signature and associated gene expression patterns in AML by collaborating with posterior HOXA chromatin boundary. HOTTIP is upregulated in AML patients with MLL-rearrangement or NPM1 mutation. AML patients with high HOTTIP expression exhibits a significantly shortened survival compared to low HOTTIP expressing patients. To test whether HOTTIP acts to coordinate posterior chromatin domain and HOXA genes activation in AML, we manipulated HOTTIP lncRNA expression levels in the MLL-AF9 rearranged MOLM13 by loss-of-function KO and gain-of function rescue, as well as carried out genome wide chromatin and transcriptomic analysis to intterrogate the role of HOTTIP in control of AML specific posterior HOXA chromatin domain. We found that knock-out of HOTTIP lncRNA led to a loss of active chromatin structure and invasion of repressive H3K27me3 mark over the posterior HOXA domain. HOTTIP KO attenuated progression of AML in the transplanted AML mouse model resembling the effect of CBS7/9 boundary disruption, while transcriptional activation of HOTTIP lncRNA in the CBS7/9 boundary-disrupted AML cells restored HOXA locus chromatin signature and gene expression as well as reversed the CBS7/9-mediated anti-leukemic effects.To further determine the role of HOTTIP lncRNA in regulating HSC function and leukemogenesis, we generated transgenic mice that expresses Hottip lncRNA under the control of the hematopoietic specific Vav1 enhancer and promoter. The Hottip transgenic (Tg) mice exhibited increased WBC and neutrophil counts and developed splenomegaly indicating that enforced expression of Hottip lncRNA resulted in perturbation of hematopoiesis. Furthermore, overexpression of Hottip lncRNA in mice bone marrow hematopoietic compartment strongly perturbed hematopoietic stem and progenitor cell (HSC/HPC) function by altering self-renewal and differentiation property of HSC/HPCs through affecting homeotic gene associated oncogenic transcription program. Approximately 20% of Hottip lncRNA transgenic mice developed abnormal hematopoietic phenotypes resembling AML-like disease. RNA-seq and ATAC-seq analysis indicated that overexpression of Hottip enhanced promoter chromatin accessibility and stimulates transcription of genes and pathways involved in HSC function and leukemogenesis, including WNT signaling, hematopoietic cell lineage, cell cycle, Hoxa9, Hoxa13, and Meis1, Runx1, and Twist1 genes. Thus, Hottip lncRNA overexpression acts as an oncogenic event to promote HSC self-renewal and HPC proliferation by reprograming leukemic associated chromatin signature and transcription programs. DisclosuresNo relevant conflicts of interest to declare.

HOTTIP participates in mammary cancer by promoting cell proliferation via PI3K/AKT pathway.

To investigate the role of HOTTIP in the development of mammary cancer and its underlying mechanism. 70 mammary cancer tissues and paracancerous tissues surgically resected from mammary cancer patients were enrolled in this study. HOTTIP expressions in these mammary cancer tissues and paracancerous tissues were detected by qRT-PCR (quantitative real-time polymerase chain reaction). The relationship between HOTTIP expression, prognosis, tumor size, and stage of mammary cancer patients was analyzed. Subsequently, we constructed lentivirus of HOTTIP. Proliferation, apoptosis, cell cycle, and invasion of mammary cancer cells transfected with HOTTIP lentivirus were detected by CCK-8 (cell counting kit-8), colony formation, flow cytometry, and transwell assay, respectively. The effect of overexpressed HOTTIP on PI3K/AKT pathway was detected by Western blot. HOTTIP was overexpressed in mammary cancer tissues than that of paracancerous tissues. HOTTIP expression was negatively correlated with the prognosis of mammary cancer. Overexpressed HOTTIP remarkably promoted cell cycle, and increased expressions of CyclineD1 and PCNA. Meanwhile, overexpressed HOTTIP inhibited cell apoptosis, whereas promoted proliferation and colony formation abilities. Western blot results demonstrated that overexpressed HOTTIP promotes proliferation of mammary cancer cells via PI3K/AKT pathway. HOTTIP remarkably promotes proliferative and invasive abilities, but inhibits cell apoptosis of mammary cancer cells via PI3K/AKT pathway.

Long non-coding RNA HOTTIP is upregulated in renal cell carcinoma and regulates cell growth and apoptosis by epigenetically silencing of LATS2.

Renal cell carcinoma (RCC) is one of the most aggressive malignancies with increasing incidence worldwide and is characterized by dismal prognosis owing to a lack of early detection and prognostic biomarkers for this fatal disease. Accumulating studies demonstrated that abnormally expressed long non-coding RNAs (lncRNAs) are involved in tumorigenesis and progression. Specifically, HOTTIP is upregulated and exerts oncogenic properties in some cancers. However, its clinical significance, biological functions and molecular mechanisms in RCC have not been studied. In the current study, RT-qPCR was performed to quantify the relative expression of HOTTIP in RCC tissues and cells. Additionally, we explored its clinical value using Fisher's exact test. Moreover, cell growth and apoptosis altered by HOTTIP was evaluated in vitro and in vivo. Mechanistically, RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP) analysis was used to determine its molecular mechanism in cell growth and apoptosis. As a result, upregulated HOTTIP is closely associated with unfavorable phenotypes in RCC patients. The mechanistic investigations showed that HOTTIP could bind to enhancer of zeste homolog 2 (EZH2) and lysine specific demethylase 1 (LSD1), thereby repressing LATS2 expression. Collectively, our study illustrates how HOTTIP plays an oncogenic role in RCC and may offer a potential therapeutic target for treating this fatal disease.