HOXA Transcript At The Distal Tip Knockdown Research Articles

Long non-coding RNA HOTTIP exerts an oncogenic function by regulating HOXA13 in nasopharyngeal carcinoma

BackgroundThe long non-coding RNA HOXA transcript at the distal tip (HOTTIP) and homeobox A13 (HOXA13) have been identified as oncogenes that play a pivotal role in tumorigenesis. However, their specific mechanisms of action in nasopharyngeal carcinoma (NPC) progression remain unclear.Methods and resultsIn the present study, RT-qPCR was employed to quantify RNA expression in NPC cells and tissues. Flow cytometry, MTT, CCK8 and colony formation assays were utilized to assess cell apoptosis and proliferation. Transwell assay was conducted to evaluate migration and invasion while Western blotting was performed for protein expression analysis. Our findings revealed that the expression of HOTTIP was significantly upregulated in NPC cell lines. Inhibition of HOTTIP could induce apoptosis and suppress proliferation, clonogenicity, invasion and metastasis in NPC cells. Knockdown of HOTTIP led to downregulation of HOXA13 expression, which subsequently inhibited the proliferation and metastasis in NPC cells. The inhibitory effects on cell proliferation and metastasis caused by HOTTIP silencing were rescued by HOXA13 overexpression. Additionally, there was a significant positive correlation between HOTTIP and HOXA13, which were found to be elevated in NPC tissues compared to normal tissues.ConclusionsWe have determined that LncRNA HOTTIP facilitates tumorigenesis by modulating the expression of HOXA13 in NPC cells. Targeting HOTTIP/HOXA13 may be a promising therapeutic strategy for NPC.

LncRNA HOTTIP PROMOTES OVARIAN CANCER CELL INVASION AND METASTASIS BY STABILIZING HIF-1α IN THE ANOXIC CELLULAR MICROENVIRONMENT.

The high recurrence rate and low survival rate of ovarian cancer (OC) patients are closely related to an anoxic environment. We aim to study the mechanism of long non-coding RNA (lncRNA) HOXA transcript at the distal tip (HOTTIP) on hypoxia ovarian cancer cells (OCC) and its mechanism was investigated. Knockdown and overexpression of HOTTIP in human OCC (SKOV-3, OVCAR3) were performed. The expression levels of HOTTIP and HIF-1α were monitored by qRT-PCR and WB. Transwell was conducted to validate the cell migration and invasion. ELISA was performed to calculate VEGF concentration in cells. Cell viability was monitored by CCK-8. Cell apoptosis and cycle were tested by flow cytometry. RNA pull-down was used to analyze the interaction between HIF-1α and HOTTIP. HOTTIP was highly expressed in OCC. After HOTTIP knockdown, HIF-1α expression and VEGF concentration in OCC were decreased. Cell migration, invasion, and cell viability were decreased. Cell apoptosis rate and G0/G1 phase cells were increased. RNA pull-down indicated a direct interaction between HIF-1α and HOTTIP. HOTTIP formed a positive feedback loop with HIF-1α to promote the development and metastasis of hypoxia ovarian cancer. This study provided theoretical support for the development of new OC treatment strategies.

HOTTIP downregulation reduces neuronal damage and microglial activation in Parkinson's disease cell and mouse models.

HOXA transcript at the distal tip (HOTTIP), a newly identified long noncoding RNA, has been shown to exhibit anti-inflammatory effects and inhibit oxygen-glucose deprivation-induced neuronal apoptosis. However, its role in Parkinson's disease (PD) remains unclear. 1-Methyl-4-phenylpyridium (MPP+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were used to establish PD models in SH-SY5Y and BV2 cells and in C57BL/6 male mice, respectively. In vitro, after HOTTIP knockdown by sh-HOTTIP transfection, HOTTIP and FOXO1 overexpression promoted SH-SY5Y apoptosis, BV2 microglial activation, proinflammatory cytokine expression, and nuclear factor kappa-B and NACHT, LRR and PYD domains-containing protein 3 inflammasome activation. Overexpression of miR-615-3p inhibited MPP+-induced neuronal apoptosis and microglial inflammation and ameliorated HOTTIP- and FOXO1-mediated nerve injury and inflammation. In vivo, HOTTIP knockdown alleviated motor dysfunction in PD mice and reduced neuronal apoptosis and microglial activation in the substantia nigra. These findings suggest that inhibition of HOTTIP mitigates neuronal apoptosis and microglial activation in PD models by modulating miR-615-3p/FOXO1. This study was approved by the Ethics Review Committee of the Affiliated Hospital of Qingdao University, China (approval No. UDX-2018-042) in June 2018.

Knockdown of lncRNA HOTTIP Inhibits Retinoblastoma Progression by Modulating the miR-101-3p/STC1 Axis.

Objective:Retinoblastoma (RB) is a frequent eye cancer in children. Long non-coding RNA (LncRNA) HOXA transcript at the distal tip (HOTTIP) is aberrantly expressed in cancer tissues. This study explores the underlying mechanism of lncRNA HOTTIP in RB.Methods:HOTTIP expression in normal retinal cells and RB cell lines was detected using qRT-PCR. The proliferation of RB cells was measured using CCK-8 and EdU assays, and apoptosis was detected using flow cytometry and Western blotting after the transfection of si-HOTTIP into Y79 cells and pc-HOTTIP into HXO-RB-44 cells. The target relationships between HOTTIP and miR-101-3p, and miR-101-3p and STC1 were predicted by bioinformatics website and verified using dual-luciferase reporter gene assay. The binding of HOTTIP and miR-101-3p was verified using RNA pull-down assay. STC1 mRNA and protein in RB cells were measured using qRT-PCR and Western blotting. Moreover, si-HOTTIP and in-miR-101-3p/in-NC, and si-HOTTIP and pc-STC1/pcDNA were co-transfected into Y79 cells respectively to evaluate cell proliferation and apoptosis. Xenograft study was conducted, and Ki67-positive expression was detected using immunohistochemical staining.Results:HOTTIP expression was promoted in RB tissues and cells. Downregulation of HOTTIP inhibited proliferation and promoted apoptosis of Y79 cells, while upregulation of HOTTIP promoted proliferation and inhibited apoptosis of HXO-RB-44 cells. There were target relationships between HOTTIP and miR-101-3p, and miR-101-3p and STC1. Inhibition of miR-101-3p or overexpression of STC1 reversed the effect of si-HOTTIP on the proliferation and apoptosis of RB cells. Xenograft study showed that knockdown of HOTTIP suppressed the growth of RB in vitro.Conclusion:It could be concluded that HOTTIP sponged miR-101-3p to upregulate STC1 expression, thereby promoting RB cell proliferation and inhibiting apoptosis.

LncRNA HOTTIP facilitates the stemness of breast cancer via regulation of miR-148a-3p/WNT1 pathway.

Emerging evidence suggests that dysregulation of long non‐coding RNA (lncRNA) plays a key role in tumorigenesis. The lncRNA, HOXA transcript at the distal tip (HOTTIP), has been reported to be up‐regulated in multiple cancers, including breast cancer, and is involved in various biological processes, including the maintenance of stemness. However, the biological function and underlying modulatory mechanism of HOTTIP in breast cancer stem cells (BCSCs) remains unknown. In this study, we found that HOTTIP was markedly up‐regulated in BCSCs and had a positive correlation with breast cancer progression. Functional studies revealed that overexpression of HOTTIP markedly promoted cell clonogenicity, increased the expression of the stem cell markers, OCT4 and SOX2, and decreased the expression of the differentiation markers, CK14 and CK18, in breast cancer cells. Knockdown of HOTTIP inhibited the CSC‐like properties of BCSCs. Consistently, depletion of HOTTIP suppressed tumour growth in a humanized model of breast cancer. Mechanistic studies demonstrated that HOTTIP directly binds to miR‐148a‐3p and inhibits the mediation of WNT1, which leads to inactivation of the Wnt/β‐catenin signalling pathway. Our study is the first to report that HOTTIP regulates the CSC‐like properties of BCSCs by as a molecular sponge for miR‐148a‐3p to increase WNT1 expression, offering a new target for breast cancer therapy.

HOTTIP knockdown inhibits cell proliferation and migration via regulating miR-490-3p/HMGB1 axis and PI3K-AKT signaling pathway in ox-LDL-induced VSMCs

AimsAtherosclerosis (AS) is a common cardiovascular disease with complicated pathogenesis. Long non-coding RNAs (lncRNAs) have been reported to be associated with AS progression. We aimed to explore the role and underlying mechanism of HOXA transcript at the distal tip (HOTTIP) in AS. Materials and methodsQuantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of HOTTIP, miR-490-3p and high mobility group B 1 (HMGB1) in AS patients' sera and oxidized low-density lipoprotein (ox-LDL) induced human aortic vascular smooth muscle cells (HA-VSMCs). Cell Counting Kit-8 (CCK-8) assay and transwell assay were conducted to evaluate the proliferation and migration of HA-VSMCs, respectively. Western blot assay was carried out to determine the levels of proliferating cell nuclear antigen (PCNA), matrix metalloprotein 2 (MMP2), MMP9 and HMGB1. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to verify the targeting association between HOTTIP and miR-490-3p, as well as miR-490-3p and HMGB1. Key findingsHOTTIP and HMGB1 were upregulated and miR-490-3p was downregulated in the sera of AS patients and ox-LDL-stimulated HA-VSMCs. HOTTIP knockdown suppressed ox-LDL induced proliferation and migration in HA-VSMCs. MiR-490-3p was identified as a target of HOTTIP and HOTTIP overexpression abolished the inhibition on cell proliferation and migration mediated by miR-490-3p in ox-LDL-induced HA-VSMCs. Moreover, miR-490-3p inhibition promoted cell proliferation and migration by directly targeting HMGB1 in ox-LDL-induced HA-VSMCs. Besides, HOTTIP knockdown repressed the activation of PI3K-AKT signaling pathway. SignificanceHOTTIP knockdown suppressed cell proliferation and migration by regulating miR-490-3p/HMGB1 axis and PI3K-AKT pathway in ox-LDL-induced HA-VSMCs.

Downregulation of lncRNA HOTTIP Suppresses the Proliferation, Migration, and Invasion of Oral Tongue Squamous Cell Carcinoma by Regulation of HMGA2-Mediated Wnt/β-Catenin Pathway.

Background: Oral tongue squamous cell carcinoma (OTSCC) is a common type of oral tumor. LncRNAs (long noncoding RNAs) and miRNAs (microRNAs) were identified as regulators in many human cancers. This study aims to explore the molecular basis of HOXA transcript at the distal tip (HOTTIP) in regulating OTSCC progression. Materials and Methods: The expression of HOTTIP, miR-124-3p, and high-mobility group AT-hook 2 (HMGA2) was detected by quantitative real-time polymerase chain reaction. Next, the proliferation was evaluated by 3-(4,5-dimethylthiazole-2-y1)-2,5-biphenyl tetrazolium bromide (MTT) assay. The migration and invasion were assessed by transwell assay. Furthermore, dual-luciferase reporter assay was performed to confirm the combination between HOTTIP and miR-124-3p, miR-124-3p, and HMGA2. Protein levels of HMGA2, β-catenin, c-Myc, and E-cadherin were examined by Western blot. The nude mice model was employed to test the tumor growth in vivo. Results: HOTTIP was upregulated in OTSCC tissues and cells, and was highly expressed in positive lymph node metastasis and late-stage OTSCC patients. Silencing HOTTIP impeded proliferation, migration, and invasion of OTSCC cells. Moreover, HOTTIP knockdown inhibited proliferation, migration, and invasion of OTSCC cells by targeting miR-124-3p. Besides, miR-124-3p targeted HMGA2 to block proliferation, migration, and invasion. HMGA2 could rescue the inhibitory effects of HOTTIP interference on proliferation, migration, and invasion. In addition, HMGA2 overexpression reversed the downregulation of β-catenin and c-Myc protein levels and upregulation of E-cadherin level affected by HOTTIP silencing. Finally, HOTTIP silencing repressed tumor growth and resulted in a great rise on miR-124-3p and E-cadherin expression and a distinct fall on HMGA2, β-catenin, and c-Myc protein levels. Conclusions: HOTTIP knockdown restrained proliferation, migration, and invasion of OTSCC cells by miR-124-3p/HMGA2 axis through Wnt/β-catenin pathway.

The Long Non-coding RNA HOTTIP Is Highly Expressed in Colorectal Cancer and Enhances Cell Proliferation and Invasion

Long non-coding RNAs (lncRNAs) are associated with a spectrum of biological processes such as gene regulation on transcriptional and post-transcriptional levels. The HOXA transcript at the distal tip (HOTTIP) lncRNA plays an important role in carcinogenesis; however, the underlying role of HOTTIP in colorectal carcinoma (CRC) remains unknown. The aim of the present study was to evaluate the expression and function of HOTTIP in CRC. In the present study, we analyzed HOTTIP expression levels of CRC patients in tumor and adjacent normal tissue by real-time quantitative PCR. Knockdown of HOTTIP by RNA interference was performed to explore its roles in cell proliferation, migration, and invasion. Our results found that HOTTIP was upregulated in human primary CRC tissues. Knockdown of HOTTIP inhibited CRC cell proliferation, migration, and invasion. Above all, knockdown of HOTTIP could represent a rational therapeutic strategy for CRC.

The long noncoding RNA HOTTIP promotes breast cancer cell migration, invasiveness, and epithelial-mesenchymal transition via the Wnt-β-catenin signaling pathway.

Long noncoding RNA HOTTIP (HOXA transcript at the distal tip) has recently been reported to have a role in the proliferation of various cancer cells, yet its role in cell migration, invasiveness, and the EMT (epithelial-mesenchymal transition) in breast cancer and the potential mechanisms remain unknown. Breast cancer cell lines MDA-MB-231 and MDA-MB-468 were transfected with shRNA (short hairpin RNA) that specifically targeting HOTTIP. We observed a remarkable decrease in migration and invasiveness in these two breast cancer cell lines after knock-down of HOTTIP by shHOTTIP. We also demonstrated that the EMT of these two breast cell lines was suppressed after HOTTIP knock-down, as evidenced by increased E-cadherin levels, and decreased levels of N-cadherin, Snail, and Twist. Moreover, HOTTIP silencing also suppressed tumor metastasis in nude mice in vivo. In addition, we found that the expression of β-catenin was significantly decreased in breast cancer cells after knock-down of HOTTIP. In a further rescue experiment using overexpression of β-catenin, the rates of cell migration, invasiveness, and EMT of HOTTIP-silenced breast cancer cells were promoted, disclosing a potential role of the Wnt-β-catenin signaling pathway in this process. Overall, we discovered the positive regulatory function of HOTTIP in the migration, invasiveness, and EMT of breast cancer cells, via regulating the Wnt-β-catenin pathway.

Long noncoding RNA HOTTIP mediates SRF expression through sponging miR-150 in hepatic stellate cells.

HOXA transcript at the distal tip (HOTTIP) has been shown to be up‐regulated in a variety of cancers and is identified as an oncogenic long noncoding RNA. However, the biological role of HOTTIP in liver fibrosis is unclear. Here, we reported that HOTTIP was specifically overexpressed in activated hepatic stellate cells (HSCs). HOTTIP knockdown suppressed the activation and proliferation of HSCs. Luciferase reporter assay showed that HOTTIP and serum response factor (SRF) were targets of miR‐150. RNA binding protein immunoprecipitation assay indicated the interaction between miR‐150 and HOTTIP. Further study revealed that HOTTIP increased SRF expression as a competing endogenous RNA for miR‐150, thus prompting HSC activation. Taken together, we provide a novel HOTTIP‐miR‐150‐SRF signalling cascade in liver fibrosis.

Knockdown of HOXA transcript at the distal tip suppresses the growth and invasion and induces apoptosis of oral tongue squamous carcinoma cells.

BackgroundOral tongue squamous cell carcinoma (OTSCC) is an aggressive cancer which has high mortality rates. HOXA transcript at the distal tip (HOTTIP) is a lncRNA that can be used as a prognostic marker in multiple carcinomas. The expression of HOTTIP is found to be elevated in OTSCC tissues, and such elevation is correlated with poor prognosis. However, its functional role in regulating the growth and metastasis of OTSCC cells remains elusive and requires further investigation.MethodsHOTTIP-silenced OTSCC cells were established by inhibiting HOTTIP expression via its exclusive shRNA. Whether HOTTIP knockdown affected the aggressive tumor behaviors of OTSCC cells was investigated in vitro and in vivo.ResultsWe found that HOTTIP shRNA restrained the cell proliferation and arrested the cell cycle at G1 phase in TSCCA and TCA8113 cells. The expression levels of cyclins B, D1, and E were downregulated in HOTTIP-silenced cells. HOTTIP silencing suppressed the growth of xenograft tumors. Moreover, the silencing of HOTTIP triggered apoptosis in TSCCA and TCA8113 cells and altered the expression of a group of apoptosis-related molecules: downregulated Bcl-2, upregulated Bax, and enhanced the cleavage of caspase 3 and PARP. Knockdown of HOTTIP also suppressed the migration, invasion, and epithelial–mesenchymal transition (EMT) of both TSCCA and TCA8113 cell lines.ConclusionOur findings suggest that HOTTIP is required by the OTSCC cells to maintain their growth and metastasis in vitro. It may serve as a promising potential candidate for OTSCC therapy.

Long non-coding RNA HOTTIP promotes prostate cancer cells proliferation and migration by sponging miR-216a-5p.

Long non-coding RNAs (lncRNAs) are a class of ncRNAs with >200 nts in length that regulate gene expression. The HOXA transcript at the distal tip (HOTTIP) lncRNA plays an important role in carcinogenesis, however, the underlying role of HOTTIP in prostate cancer (PCa) remains unknown. The aim of the present study was to evaluate the expression and function of HOTTIP in PCa. In the present study, we analyzed HOTTIP expression levels of 86 PCa patients in tumor and adjacent normal tissue by real-time quantitative PCR (qPCR). Knockdown or overexpression of HOTTIP was performed to explore its roles in cell proliferation, migration, invasion, and cell cycle. Furthermore, bioinformatics online programs predicted and luciferase reporter assay were used to validate the association of HOTTIP and miR-216a-5p in PCa cells. Our results found that HOTTIP was up-regulated in human primary PCa tissues with lymph node metastasis. Knockdown of HOTTIP inhibited PCa cell proliferation, migration, and invasion. Overexpression of HOTTIP promoted cell proliferation, migration, and invasion of PCa cells. Bioinformatics online programs predicted that HOTTIP sponge miR-216a-5p at 3′-UTR with complementary binding sites, which was validated using luciferase reporter assay. HOTTIP could negatively regulate the expression of miR-216a-5p in PCa cells. Above all, the knockdown of HOTTIP could represent a rational therapeutic strategy for PCa.

Long non-coding RNA HOTTIP promotes renal cell carcinoma progression through the regulation of the miR-615/IGF-2 pathway.

Emerging evidence has indicated that long non‑coding RNA (lncRNA) HOXA transcript at the distal tip (HOTTIP) regulates cell growth, differentiation, apoptosis and cancer progression. However, the expression and function of HOTTIP in the progression of renal cell carcinoma(RCC) remain largely unknown. In this study, we investigated the role of the lncRNA HOTTIP in RCC. The expression levels of HOTTIP in RCC tissues and cell lines were determined by RT‑qPCR. The association between HOTTIP expression and clinicopathological characteristics and prognosis was analyzed in patients with RCC from the TCGA database. Loss‑of‑ function assays were designed and conducted to verify the oncogenic function of HOTTIP in RCC progression. Luciferase assay was performed to explore the mechanisms of the miRNA‑lncRNA sponge. The results revealed that HOTTIP expression was upregulated in RCC. An increased HOTTIP expression in RCC was associated with a larger tumor size and a higher clinical stage, lymph node metastasis and vascular invasion. Additionally, patients RCC with a high HOTTIP expression had a significantly shorter overall survival(OS) and disease‑free survival(DFS). HOTTIP knockdown significantly inhibited cell proliferation, migration and invasion, and increased the apoptosis of RCC cells invitro. Mechanistic analyses revealed that HOTTIP functioned as a competing endogenous RNA (ceRNA) for hsa‑miR‑615‑3p, and led to the derepression of its endogenous target, insulin‑like growth factor-2(IGF‑2), which is a protein hormone that exerts a stimulatory effect on tumor cell growth. miR‑615 inhibition reversed the suppressive effects of HOTTIP knockdown on RCC cell progression. HOTTIP regulated IGF‑2 expression in a miR‑615‑dependent manner in RCC cells. In addition, IGF‑2 expression was significantly upregulated in the RCC specimens and a positive association between the expression of HOTTIP and IGF‑2 in RCC tissues was detected. The effect of HOTTIP was abolished by the siRNA‑mediated silencing of IGF-2 in RCC cells. On the whole, this study demonstrates, for the first time, at least to the best of our knowledge, that the HOTTIP/miR‑615/IGF‑2 axis plays an important role in RCC progression and potentially contributes to the improvement of RCC diagnosis and therapy.

HOTTIP and HOXA13 are oncogenes associated with gastric cancer progression.

A long non-coding RNA named HOTTIP (HOXA transcript at the distal tip) coordinates the activation of various 5'HOXA genes which encode master regulators of development through targeting the WDR5/MLL complex. HOTTIP acts as an oncogene in several types of cancers, whereas its biological function in gastric cancer has never been studied. In the present study, we investigated the role of HOTTIP in gastric cancer. We found that HOTTIP was upregulated in gastric cancer cell lines. Knockdown of HOTTIP in gastric cancer cells inhibited cell proliferation, migration and invasion. Moreover, downregulation of HOTTIP led to decreased expression of homeobox protein Hox-A13 (HOXA13) in gastric cancer cell lines. HOXA13 was involved in HOTTIP‑induced malignant phenotypes of gastric cancer cells. Our data showed that the levels of HOTTIP and HOXA13 were both markedly upregulated in gastric cancer tissues compared with their counterparts in non-tumorous tissues. Furthermore, the expression levels of HOTTIP and HOXA13 were both higher in gastric cancer which was poorly differentiated, at advanced TNM stages and exhibited lymph node-metastasis. Spearman analyses indicated that HOTTIP and HOXA13 had a highly positive correlation both in non-tumor mucosae and cancer lesions. Collectively, these findings suggest that HOTTIP and HOXA13 play important roles in gastric cancer progression and provide a new insight into therapeutic treatment for the disease.

The long noncoding RNA HOXA transcript at the distal tip promotes colorectal cancer growth partially via silencing of p21 expression.

Accumulating evidence strongly suggests that dysregulation of long noncoding RNAs (lncRNAs) is associated with human carcinogenesis. The lncRNA HOXA transcript at the distal tip (HOTTIP) is involved in the development of several cancers. However, the biological role of HOTTIP in colorectal cancer (CRC) has not yet been discussed. Here, we report that HOTTIP acts as a functional oncogene in the pathogenesis of CRC. In this study, quantitative polymerase chain reaction (qPCR) was performed to detect the expression of HOTTIP in 48 pairs of colorectal cancer samples. We found that overexpression of HOTTIP is correlated with an advanced pathological stage and a larger tumor size. Moreover, functional analyses revealed that the knockdown of HOTTIP expression by small interfering RNA (siRNA) or small hairpin RNA (shRNA) could inhibit cell proliferation and induce cell apoptosis. More importantly, we observed that HOTTIP knockdown induced a marked increase in the number of cells in the G0/G1 phase and a reduction in the number of cells in the S phase in both DLD-1 cells and SW480 cells. An in vivo experiment also revealed that the knockdown of HOTTIP inhibited tumor growth. Western blot and immunohistochemistry analyses indicated that HOTTIP potentially contributed to CRC cell growth partially through the silencing of p21 expression. Collectively, our results suggest that HOTTIP is involved in the progression of CRC and may provide evidence for HOTTIP being a target for therapy of this disease.