HOTAIR/miR-1277-5p/FBN2 signaling axis is involved in recurrent spontaneous abortion by regulating the growth, migration, and invasion of HTR-8/SVneo cells.

OBJECTIVE: This study aimed to explore the specific pathways by which HOX transcript antisense intergenic RNA (HOTAIR) contributes to the pathogenesis of unexplained recurrent spontaneous abortion (URSA). METHODS: Real-time quantitative PCR (RT-qPCR) was employed to assess the differential expression levels of HOTAIR in chorionic villi tissues from URSA patients and women with voluntarily terminated pregnancies. HTR-8/SVneo served as a cellular model. Knockdown and overexpression of HOTAIR in the cells were achieved through siRNA transfection and pcDNA3.1 transfection, respectively. Cell viability, migration, and invasion were evaluated using cell counting kit-8 (CCK-8), scratch, and Transwell assays, respectively. The interaction among the HOTAIR/miR-1277-5p/fibrillin 2 (FBN2) axis was predicted through bioinformatics analysis and confirmed through in vitro experiments. Furthermore, the regulatory effects of the HOTAIR/miR-1277-5p/FBN2 signaling axis on cellular behaviors were validated in HTR-8/SVneo cells. RESULTS: We found that HOTAIR was downregulated in chorionic villi tissues from URSA patients. Overexpression of HOTAIR significantly enhanced the viability, migration, and invasion of HTR-8/SVneo cells, while knockdown of HOTAIR had the opposite effects. We further confirmed the regulatory effect of the HOTAIR/miR-1277-5p/FBN2 signaling axis in URSA. Specifically, HOTAIR and FBN2 were found to reduce the risk of URSA by enhancing cell viability, migration, and invasion, whereas miR-1277-5p exerted the opposite effects. CONCLUSION: HOTAIR promotes URSA development by targeting inhibition of miR-1277-5p/FBN2 axis.

LncRNA-HOTAIR promotes endothelial cell pyroptosis by regulating the miR-22/NLRP3 axis in hyperuricaemia.

Long non-coding RNA (lncRNA) plays an important role in the renal inflammatory response caused by hyperuricaemia. However, the underlying molecular mechanisms through which lncRNA is involved in endothelial injury induced by hyperuricaemia remain unclear. In this study, we investigated the regulatory role of lncRNA-HOTAIR in high concentration of uric acid (HUA)-induced renal injury. We established hyperuricaemia mouse model and an in vitro uric acid (UA)-induced human umbilical vein endothelial cell (HUVEC) injury model. In HUA-treated HUVECs and hyperuricaemia mice, we observed increased HOTAIR and decreased miR-22 expression. The expression of pyroptosis-associated protein (NLRP3, Caspase-1, GSDMD-N, GSDMD-FL) was increased. The release of LDH, IL-1ß and IL-18 in cell supernatants and the sera of model mice was also increased. The proliferation of HUVECs stimulated by HUA was significantly inhibited, and the number of TUNEL-positive cells in hyperuricaemia mouse kidney was increased. Bioinformatics analysis and luciferase reporter and RIP assays confirmed that HOTAIR promoted NLRP3 inflammasome activation by competitively binding miR-22. In gain- or loss-of-function experiments, we found that HOTAIR and NLRP3 overexpression or miR-22 knock down activated the NLRP3 inflammasome and promoted pyroptosis in HUA-treated HUVECs, while NLRP3 and HOTAIR knockdown or a miR-22 mimic exerted the opposite effects. Furthermore, in vivo experiments validated that HOTAIR knockdown alleviated renal inflammation in hyperuricaemia mice. In conclusion, we demonstrated that in hyperuricaemia, lncRNA-HOTAIR promotes endothelial cell pyroptosis by competitively binding miR-22 to regulate NLRP3 expression.

Long non-coding RNA HOTAIR knockdown alleviates gouty arthritis through miR-20b upregulation and NLRP3 downregulation.

This study aimed to determine the mechanism underlying the regulation of gout by the HOX transcript antisense RNA (HOTAIR) long non-coding RNA (lncRNA). The expression levels of HOTAIR, miR-20b, and Nlrp3 were estimated by qRT-PCR and western blotting. The methylation level of HOTAIR was detected by methylation-specific PCR. The recruitment of DNA methyltransferase 1 (DNMT1) to the lncRNA HOTAIR promoter was confirmed by a ChIP assay. RNA immunoprecipitation and RNA pull-down assays were used to confirm the interaction between HOTAIR and miR-20b. LncRNA HOTAIR and Nlrp3 expression was upregulated, and that of miR-20b was downregulated in synovial fluid mononuclear cells (SFMCs) collected from patients with gouty arthritis and monosodium urate (MSU)-stimulated THP-1 cells. Interleukin (IL)-1ß level increased substantially upon stimulation by MSU crystals. The methylation percentage of HOTAIR was reduced in SFMCs from patients with gouty arthritis and MSU-stimulated THP-1 cells. DNMT1 expression was downregulated in MSU-stimulated THP-1 cells, and DNMT1 knockdown increased lncRNA HOTAIR expression. In addition, the interaction of HOTAIR with miR-20b was confirmed. HOTAIR knockdown suppressed Nlrp3 expression and the secretion of inflammatory cytokines via miR-20b regulation. Finally, in vivo experiments showed that HOTAIR knockdown alleviated ankle swelling in a mouse model of gouty arthritis. These findings suggest that lncRNA HOTAIR knockdown suppresses inflammatory cytokine secretion by upregulating miR-20b and downregulating NLRP3, thereby alleviating ankle swelling in gouty arthritis.

Analysis of clinical important of LncRNA-HOTAIR gene variations and ovarian cancer susceptibility.

LncRNAs are of functional long non-coding RNAs, which have been shown to be involved in critical pathways in cancer development. LncRNA-HOTAIR gene overexpression has been reported in several cancers. The aim of this study was to evaluate the associations between two variants of lncRNA-HOTAIR (rs1899663 G>T and rs4759314 A>G) gene polymorphisms and the risk of ovarian cancer (OC) susceptibility. We performed a case and control analysis on two hundred individuals consisting of 100 cases with OC and 100 women cancer-free in East Azerbaijan of Iranian population. To evaluate the association between two SNPs of lncRNA-HOTAIR with the risk of OC susceptibility used the polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) method. We revealed that two SNPs in the lncRNA-HOTAIR gene were significantly associated with the risk of OC. The dominant model of rs4759314 in lncRNA-HOTAIR (AA vs. GA/GG) showed a significantly increased risk with an OR of 10.036 (CI 2.253-44.712, P = 0.000); the recessive model of rs1899663 (TT vs. GT/GG) revealed a significantly increased risk with OR of 0.910 (CI 0.856-0.968; P = 0.002). In addition, our findings demonstrated that the 4759314G (OR 13.500; CI 3.146-57.940; P = 0.000) and 1899663T (OR 3.273; CI 1.433-7.475; P = 0.003) alleles are increased the risk of OC susceptibility. Our findings provide evidence that the specific genetic variants in lncRNA-HOTAIR gene may affect OC susceptibility in an Iranian population.

Overexpression of LncRNA-HOTAIR promotes chemoresistance in acute leukemia cells.

Chemotherapy treatment of acute myeloid leukemia (AML) can be compromised due to the multidrug resistance (MDR) of leukemia cells. HOTAIR, a long noncoding RNA (LncRNA), is involved in MDR development of various solid tumors. However, whether it functions in MDR development of leukemia remains unclear. In this study, expressions of HOTAIR in leukemia cell line K562/A02 and bone marrow samples from 10 patients with refractory and relapsed AML were detected by qRT-PCR. The apoptosis, proliferation, and susceptibility of K562/A02 cells to Adriamycin (ADR) were analyzed by flow cytometry and CCK8 assay, respectively. The expression of cell cycle regulator P21 and Notch1 in the K562/A02 cells was examined by qRT-PCR. The accumulation of total AKT and the phosphorylated AKT (pAKTS473) were detected by western blotting. We found that the expression of HOTAIR in drug-resistant cells and patient samples was increased. Inhibition of HOTAIR expression could suppress the proliferation, increase the apoptosis, and promote the doxorubicin sensitivity of K562/A02 cells. Moreover, inhibiting expression of HOTAIR could attenuate the expression of P21 and Notch1 and inhibit the phosphorylation of AKT in drug-resistant cells. In conclusion, our results demonstrated that LncRNA-HOTAIR is involved in MDR development of leukemia cells by regulating the expression of P21 and the AKT/Notch1 signaling pathway.

lncRNA HOTAIR promotes malignant biological behaviors of breast cancer SKBR3 cells through miR-519d-3p/CCND1 axis / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To explore the effect of lncRNA HOTAIR/miR-519d-3p/cyclin D1 (CCND1) axis on the proliferation and metastasis of breast cancer cells and its underlying mechanism. Methods: A total of 50 pairs of breast cancer tissues and corresponding para-cancer tissues resected from breast cancer patients in the Department of Breast Surgery, the Third Hospital of Nanchang from March 2017 to February 2019 were collected for this study. The expression level of HOTAIR in breast cancer tissues and paired paracancer tissues was detected by qPCR, in addition, the expressions of HOTAIR and miR-519d-3p in normal breast epithelial cells and breast cancer cell lines were also detected. Breast cancer SKBR3 cells were divided into NC group (without any treatment), si-HOTAIR group, mir-519d-3p mimics group, miR-519d-3p mimic+pcHOTAIR group, miR-519d-3p mimic+pcCCND1 group, and si-HOTAIR+ pcCCND1 group. The proliferation ability of SKBR3 cells was detected by CCK-8. Invasion and migration of SKBR3 cells were detected by Transwell. The expression levels of E-cadherin, N-cadherin, Vimentin and CCND1 in SKBR3 cells were detected by Western blotting. The targeting relationship between HOTAIR and miR-519d-3p, miR-519d-3p and CCND1 was detected by Dualluciferase reporter gene system. Results: HOTAIR was highly expressed in breast cancer tissues and cell lines, with the highest expression in SKBR3 cells. HOTAIR knockdown significantly inhibited the proliferation, invasion and migration of SKBR3 cells, as well as increased the expression level of E-cadherin and decreased the expression levels of N-cadherin and Vimentin. Dual-luciferase reporter gene assay showed that HOTAIR targetedly down-regulated the expression of miR-519d-3p, and miR-519d-3p targetedly downregulated the expression of CCND1. Further studies showed that knockout of HOTAIR inhibited the EMT, proliferation, invasion and migration of SKBR3 cells through enhancing the inhibitory effect of miR-519d-3p on CCND1 expression (all P<0.05). Conclusion: HOTAIR knockdown inhibits proliferation and metastasis of SKBR3 cells by regulating the axis of miR-519d-3p/CCND1.

Identification and molecular analysis of a lncRNA-HOTAIR transcript from secondary hair follicle of cashmere goat reveal integrated regulatory network with the expression regulated potentially by its promoter methylation.

The HOTAIR transcript is transcribed from the antisense strand within the HOXC gene cluster, and it is thought to play a role in regulating the inductive capacity of dermal papilla cells during the reconstruction of hair-follicle. In the current investigation, we firstly isolated and characterized a lncRNA-HOTAIR transcript from the secondary hair follicle of cashmere goat. Also, we analyzed its transcriptional pattern and methylation level of HOTAIR gene promoter in secondary hair follicle of cashmere goat during anagen and telogen stages. Nucleotide composition analysis indicated that the contents of Adenine (A) and Thymine (T) are higher than that of Guanine (G) and Cytosine (C) in lncRNA-HOTAIR transcript of cashmere goat with the highest frequency distribution of AG nucleotide pair (8.06%). The regulatory network analysis showed a directly or indirectly complex regulatory relationships between lncRNA-HOTAIR of cashmere goat and its potential target molecules: miRNAs, mRNAs and proteins. Also, we showed that lncRNA-HOTAIR was properly transcribed at both anagen and telogen stages of secondary hair follicle of cashmere goat with the anagen being significantly higher than telogen in its expression, which suggest that lncRNA-HOTAIR transcript might be involved in the reconstruction of secondary hair follicle with the formation and growth of cashmere fiber. Taken together with methylation analysis of HOTAIR gene promoter, our data suggest that the promoter methylation of HOTAIR gene most likely is involved in its transcriptional suppression in secondary hair follicle of cashmere goat.

The action mechanism of lncRNA-HOTAIR on the drug resistance of non-small cell lung cancer by regulating Wnt signaling pathway.

The action mechanism of long non-coding ribonucleic acid-homeobox transcript antisense ribonucleic acid (lncRNA-HOTAIR) in the regulation of the Wnt signaling pathway on the drug resistance of non-small cell lung cancer was investigated. Forty eight patients with non-small cell lung cancer, who were treated with cisplatin (DDP) as neoadjuvant chemotherapy, were selected from the specimen bank of the Department of Pathology of Peking Union Medical College Hospital. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the messenger RNA (mRNA) level of lncRNA-HOTAIR in cancer and cancer-adjacent tissues. The correlation curve of the expression of lncRNA-HOTAIR with the overall survival (OS) was plotted using the Kaplan-Meier method. NCI-H1299 DDP-resistant cell lines were constructed, and the half maximal inhibitory concentration (IC50) value was measured. The expression of lnc-HOTAIR in NCI-H1299/DDP cells was detected by the target interference of small interfering RNA (siRNA). The effect of si-HOTAIR on cell resistance was detected by Cell Counting Kit-8 (CCK-8). Western blot analysis was used to detect the effects of si-HOTAIR on multidrug resistance proteins, multidrug resistance-associated protein 1 (MRP1) and multidrug resistance 1 (MDR1), and Wnt signaling pathways, Wnt3a, adenomatous polyposis coli (APC) and ß-catenin. The mRNA level of lncRNA-HOTAIR in cancer tissues was significantly higher than that in cancer-adjacent tissues (P<0.05), and the high expression of lncRNA-HOTAIR indicated that the OS of patients was shortened (P<0.05). The IC50 of NCI-H1299/DDP cells inhibiting DDP was 127.82 µM, which was significantly higher than that of parental NCI-H1299 cells (IC50=8.40 µM) (P<0.05). si-HOTAIR interference significantly decreased the sensitivity of cells to DDP, the IC50 of cells was decreased from 131.85 to 44.34 µM (P<0.05), the expression levels of MRP1 and MDR1 were significantly decreased, and the activation of Wnt signaling pathway was significantly inhibited (P<0.05). Thus, lncRNA-HOTAIR plays an important role in the occurrence and development of non-small cell lung cancer, and it may be an important factor in the clinical prognosis of patients with non-small cell lung cancer.

Influence of long chain non-coding RNA (lncRNA)-HOTAIR on migration and invasion ability of endometrial cancer / 临床与实验病理学杂志

Purpose To investigate the influence of long chain non-coding RNA (lncRNA)-HOTAIR on endometrial cancer cell proliferation,invasion,metastasis and other biological behaviour.Methods 20 cases of endometrial carcinoma tissue specimen,20 cases of hyperplasia tissue sample and 10 cases of normal tissue specimen were collected.Difference expression of lncRNA-HOTAIR in normal endometrium,hyperplasia endometrium tissue and endometrial carcinoma tissue at all periods were detected with RT-PCR assay.HOTAIR-siRNA transfection into Ishikawa cells was utilized with Lipofectamine 2000.MTT experiment was used to detected the proliferation ability of cells in all groups.Transwell chamber experiment was used to test the migration and invasion ability of cells in all groups.Results The gene expression level of of lncRNA-HOTAIR in endometrial carcinoma group at all stages was prominently increased compared with normal endometrium group (P ＜ 0.05).The expression level of lncRNA-HOTAIR in simple hyperplasia endometrium group and atypical hyperplasia endometrial group was not significantly different (P ＞ 0.05).Cell proliferation,invasion ability and migration ability of HOTAIR-siRNA targeting suppression group were lower than the blank control group and the negative control group significantly (P ＜ 0.05).Conclusion lncRNA-HOTAIR may involved in occurrence and development of endometrial cancer,which may play an important role in the aggression and metastasis of endometrial cancer.