KCNQ1OT1 Knockdown Research Articles

Knockdown of KCNQ1OT1 Alleviates the Activation of NLRP3 Inflammasome Through miR-17-5p/TXNIP Axis in Retinal Müller Cells

Purpose Diabetic retinopathy (DR) is one of the most severe and common complications caused by diabetic mellites. Inhibiting NLRP3 inflammasome activation displays a crucial therapeutic value in DR. Studies have shown that KCNQ1OT1 plays a critical role in regulating NLRP3 inflammasome activation and participates in the pathogenesis of diabetic complications. The present study aims to explore the role, and the potential mechanism of KCNQ1OT1 in regulating the activation of NLRP3 inflammasome in DR. Methods qRT-PCR was used to detect the expression of KCNQ1OT1, miR-17-5p, TXNIP, NLRP3, ASC, caspase-1 and IL-1β. Western blot was performed to detect the expression of NLRP3, ASC, caspase-1, IL-1β and TXNIP. Immunohistochemistry and immunostaining were performed to detect the expression of caspase-1. The levels of the inflammatory cytokine IL-1β were determined by ELISA assay. FISH was used to detect the subcellular localisation of KCNQ1OT1. Bioinformatic analysis, luciferase reporter assay and in vitro studies were performed to elucidate the mechanism of KCNQ1OT1-mediated dysfunction. Results The expression of KCNQ1OT1 and the activation of NLRP3 inflammasome were increased in experimental DR models. KCNQ1OT1 knockdown alleviated NLRP3 inflammasome-associated molecules expression. In addition, KCNQ1OT1 was found to be localized mainly in the cytoplasm of Müller cells and facilitated TXNIP expression by acting as a miR-17-5p sponge. KCNQ1OT1 promoted the activation of NLRP3 inflammasome through miR-17-5p/TXNIP axis. Conclusions In conclusion, it was found in this study that KCNQ1OT1 promoted the activation of NLRP3 inflammasome both in vitro and in vivo, which was mediated by miR-17-5p/TXNIP axis. KCNQ1OT1 might be an effective interference target for the prevention and treatment of DR.

Imprinted lncRNA KCNQ1OT1 regulates CDKN1C expression through promoter binding and chromatin folding in pigs

Long noncoding RNAs (lncRNAs) are implicated in a number of regulatory functions in eukaryotic genomes. In humans, KCNQ1OT1 is a 91 kb imprinted lncRNA that inhibits multiple surrounding genes in cis. Among them, CDKN1C is closely related to KCNQ1OT1 and is involved in multiple epigenetic disorders. Here, we found that pigs also had a relatively conserved paternal allele expressing KCNQ1OT1 and had a shorter 5′ end (∼27 kb) compared to human KCNQ1OT1. Knockdown of KCNQ1OT1 using antisense oligonucleotides (ASO) showed that upregulation of CDKN1C expression in pigs. However, porcine KCNQ1OT1 did not affect the DNA methylation status of the CpG islands in the promoters of KCNQ1OT1 and CDKN1C. Inhibition of DNA methyltransferase using Decitabine treatment resulted in a significant increase in both KCNQ1OT1 and CDKN1C expression, suggesting that the regulation between KCNQ1OT1 and CDKN1C may not be dependent on RNA interference. Further use of chromosome conformation capture and reverse transcription-associated trap detection in the region where CDKN1C was located revealed that KCNQ1OT1 bound to the CDKN1C promoter and affected chromosome folding. Phenotypically, inhibition of KCNQ1OT1 at the cumulus-oocyte complex promoted cumulus cell transformation, and to upregulated the expression of ALPL at the early stage of osteogenic differentiation of porcine bone marrow mesenchymal stem cells. Our results confirm that the expression of KCNQ1OT1 imprinting in pigs as well as porcine KCNQ1OT1 regulates the expression of CDKN1C through direct promoter binding and chromatin folding alteration. And this regulatory mechanism played an important role in cell differentiation.

Long noncoding RNA KCNQ1OT1 aggravates cerebral infarction by regulating PTBT1/SIRT1 via miR-16-5p.

Cerebral infarction (CI) is one of the leading causes of disability and death. LncRNAs are key factors in CI progression. Herein, we studied the function of long noncoding RNA KCNQ1OT1 in CI patient plasma samples and in CI models. Quantitative real-time PCR and Western blotting tested gene and protein expressions. The interactions of KCNQ1OT1/PTBP1 and miR-16-5p were analyzed using dual-luciferase reporter and RNA immunoprecipitation assays; MTT assays measured cell viability. Cell migration and angiogenesis were tested by wound healing and tube formation assays. Pathological changes were analyzed by triphenyltetrazolium chloride and routine staining. We found that KCNQ1OT1 and PTBP1 were overexpressed and miR-16-5p was downregulated in CI patient plasma and in oxygen-glucose deprived (OGD) induced mouse brain microvascular endothelial (bEnd.3) cells. KCNQ1OT1 knockdown suppressed pro-inflammatory cytokine production and stimulated angiogenic responses in OGD-bEnd.3 cells. KCNQ1OT1 upregulated PTBP1 by sponging miR-16-5p. PTBP1 overexpression or miR-16-5p inhibition attenuated the effects of KCNQ1OT1 knockdown. PTBP1 silencing protected against OGD-bEnd.3 cell injury by enhancing SIRT1. KCNQ1OT1 silencing or miR-16-5p overexpression also alleviated ischemic injury in a mice middle cerebral artery occlusion model. Thus, KCNQ1OT1 silencing alleviates CI by regulating the miR-16-5p/PTBP1/SIRT1 pathway, providing a theoretical basis for novel therapeutic strategies targeting CI.

From Phenomenon to Essence: A Newly Involved lncRNA Kcnq1ot1 Protective Mechanism of Bone Marrow Mesenchymal Stromal Cells in Liver Cirrhosis.

Bone marrow mesenchymal stromal cells (BMSCs) have a protective effect against liver cirrhosis. Long noncoding RNAs (lncRNAs) play crucial roles in the progression of liver cirrhosis. Therefore, it is aimed to clarify the lncRNA Kcnq1ot1 involved protective mechanism of BMSCs in liver cirrhosis. This study found that BMSCs treatment attenuates CCl4 -induced liver cirrhosis in mice. Additionally, the expression of lncRNA Kcnq1ot1 is upregulated in human and mouse liver cirrhosis tissues, in addition to TGF-β1-treated LX2 cells and JS1 cells. The expression of Kcnq1ot1 in liver cirrhosis is reversed with BMSCs treatment. The knockdown of Kcnq1ot1 alleviated liver cirrhosis both in vivo and in vitro. Fluorescence in situ hybridization (FISH) confirms that Kcnq1ot1 is mainly distributed in the cytoplasm of JS1 cells. It is predicted that miR-374-3p can directly bind with lncRNA Kcnq1ot1 and Fstl1, which is verified via luciferase activity assay. The inhibition of miR-374-3p or the overexpression of Fstl1 can attenuate the effect of Kcnq1ot1 knockdown. In addition, the transcription factor Creb3l1 is upregulated during JS1 cells activation. Moreover, Creb3l1 can directly bind to the Kcnq1ot1 promoter and positively regulate its transcription. In conclusion, BMSCs alleviate liver cirrhosis by modulating the Creb3l1/lncRNA Kcnq1ot1/miR-374-3p/Fstl1 signaling pathway.

Retraction: Knockdown of long non-coding RNA KCNQ1OT1 restrained glioma cells' malignancy by activating miR-370/CCNE2 axis.

[This retracts the article DOI: 10.3389/fncel.2017.00084.].

Long non-coding RNA KCNQ1OT1 alleviates postmenopausal osteoporosis by modulating miR-421-3p/mTOR axis

The prevention and treatment of postmenopausal osteoporosis (PMOP) is a significant public health issue, and non-coding RNAs are of vital importance in this process. In this study, we find that the long non-coding RNA potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (lncRNA KCNQ1OT1) can alleviate the ovariectomy-induced (OVX) PMOP in vivo. We determined that over-expression of KCNQ1OT1 could enhance functions of MC3T3-E1 cells, whereas an opposite trend was observed when KCNQ1OT1 was knocked down. Subsequently, miR-421-3p targeting KCNQ1OT1 was detected through a database search, and RNA fluorescent in situ hybridization, RNA immunoprecipitation, dual luciferase reporter assays all verified this relationship. Notably, KCNQ1OT1 stifled the miR-421-3p expression. The inhibition of proliferation, migration, and osteogenic differentiation caused by KCNQ1OT1 knock-down were reversed by an miR-421-3p inhibitor, further confirming the above findings. We verified that miR-421-3p specifically targeted the mammalian target of rapamycin (mTOR), and miR-421-3p inhibitor could reverse the negative effects of small interfering RNA of mTOR (si-mTOR) on MC3T3-E1 cells. Finally, osteoblasts isolated and cultured from OVX mice model and control mice also confirmed the observed trend. In combination, results mentioned above reveal that KCNQ1OT1 regulates MC3T3-E1 cell functions by regulating the miR-421-3p/mTOR axis.

The involvement and therapeutic potential of lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 pathway in arsenic trioxide-induced cardiotoxicity

Background/AimsArsenic trioxide (ATO) is the first-line therapeutic drug for acute promyelocytic leukemia. However, the cardiotoxicity of ATO limits its clinical application. This study aims to explore the long noncoding RNA (lncRNA) involved molecular mechanism in ATO-induced cardiotoxicity and to identify available prevention strategies.MethodsATO was administered to mice or primary cultured mouse cardiomyocytes. Small interfering RNA targeting lncRNA Kcnq1ot1 (si-Kcnq1ot1) was used to knockdown lncRNA Kcnq1ot1. MiR-34a-5p mimic and antisense morpholino oligonucleotide targeting miR-34a-5p (AMO-34a-5p) were used to upregulate and downregulate the expression of miR-34a-5p, respectively. TUNEL staining was conducted to detect cell DNA damage. Flow cytometry assay was used to detect cell apoptosis. Western blot was conducted to detect Bcl-2, Bax and Sirt1 protein expression. Real-time PCR was used to detect lncRNA Kcnq1ot1, miR-34a-5p, and Sirt1 mRNA expression. Dual-luciferase reporter assay was performed to validate the predicted binding site.ResultsATO induced apoptosis in cardiomyocytes both in vivo and in vitro. Simultaneously, the expression of lncRNA Kcnq1ot1 and Sirt1 was downregulated, and miR-34a-5p was upregulated. MiR-34a-5p has binding sites with lncRNA Kcnq1ot1 and Sirt1. Knockdown of lncRNA Kcnq1ot1 induced apoptosis of cardiomyocytes, with increased miR-34a-5p and decreased Sirt1 expression. Inhibition of miR-34a-5p attenuated si-Kcnq1ot1-induced apoptosis in cardiomyocytes. Therefore, the lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 signaling pathway is involved in ATO-induced cardiotoxicity. Propranolol alleviated ATO-induced apoptosis in cardiomyocytes both in vivo and in vitro, which was related to the lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 signaling pathway.ConclusionThe lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 pathway is involved in ATO-induced cardiotoxicity. Propranolol can attenuate ATO-induced cardiotoxicity at least partially through the lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 pathway. Combined administration with propranolol may be a new strategy for alleviating the cardiotoxicity of ATO.

LncRNA KCNQ1OT1 enhances the radioresistance of lung squamous cell carcinoma by targeting the miR-491-5p/TPX2-RNF2 axis.

Lung cancer, especially lung squamous cell carcinoma (LUSC), is one of the most common malignant tumors worldwide. Currently, radiosensitization research is a vital direction for the improvement of LUSC therapy. Long non-coding RNAs (lncRNAs) can be novel biomarkers due to their multiple functions in cancers. However, the function and mechanism of lncRNA KCNQ1OT1 in the radioresistance of LUSC remain to be elucidated. The clonogenic assay was employed to determine the radioresistance of SK-MES-1R and NCI-H226R cells. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot were conducted for the detection of gene expression. Cell proliferation was determined by the methyl thiazolyl tetrazolium (MTT) assay, colony formation assay, and 5-ethynyl-2'-deoxyuridine (EdU) staining, and cell apoptosis was assessed by flow cytometry. The relationships between genes were also evaluated by applying the luciferase reporter and radioimmunoprecipitation (RIP) assays. Radioresistant LUSC cells (SK-MES-1R and NCI-H226R) had strong resistance to X-ray irradiation, and lncRNA KCNQ1OT1 was highly expressed in SK-MES-1R and NCI-H226R cells. Moreover, knockdown of lncRNA KCNQ1OT1 prominently suppressed proliferation, attenuated radioresistance, and accelerated the apoptosis of SK-MES-1R and NCI-H226R cells. More importantly, we verified that miR-491-5p was a regulatory target of lncRNA KCNQ1OT1, and Xenopus kinesin-like protein 2 (TPX2) and RING finger protein 2 (RNF2) were the target genes of miR-491-5p. The rescue experiment results also demonstrated that miR-491-5p was involved in the inhibition of cell proliferation and the downregulation of TPX2 and RNF2 expression mediated by lncRNA KCNQ1OT1 knockdown in SK-MES-1R and NCI-H226R cells. LncRNA KCNQ1OT1 was associated with the radioresistance of radioresistant LUSC cells, and the lncRNA KCNQ1OT1/miR-491-5p/TPX2-RNF2 axis might be used as a therapeutic target to enhance the radiosensitivity of radioresistant LUSC cells.

LncRNA KCNQ1OT1 promotes the metastasis of ovarian cancer by increasing the methylation of EIF2B5 promoter

BackgroundLong non-coding RNAs (lncRNAs) have emerged as regulators of human malignancies, including ovarian cancer (OC). LncRNA KCNQ1OT1 could promote OC progression, and EIF2B5 was associated with development of several tumors. This project was aimed to explore the role of lncRNA KCNQ1OT1 in OC development, as well as the involving action mechanism.MethodsReverse transcription quantitative polymerase chain reaction (RT-qPCR) or Western blotting was employed to determine the expression levels of KCNQ1OT1 and EIF2B5. OC cell proliferation was evaluated by MTT and colony formation assays, and wound healing and Transwell assays were implemented to monitor cell migration and invasion, respectively. The methylation status of EIF2B5 promoter was examined by MS-PCR, to clarify whether the expression of EIF2B5 was decreased. The binding activity of KCNQ1OT1 to methyltransferases DNMT1, DNMT3A and DNMT3B was determined by dual luciferase reporter assay or RIP assay, to explore the potential of KCNQ1OT1 alters the expression of its downstream gene. ChIP assay was carried out to verify the combination between EIF2B5 promoter and above three methyltransferases.ResultsExpression of lncRNA KCNQ1OT1 was increased in OC tissues and cells. EIF2B5 expression was downregulated in OC, which was inversely correlated with KCNQ1OT1. Knockdown of KCNQ1OT1 inhibited OC cell proliferation and metastasis. KCNQ1OT1 could downregulate EIF2B5 expression by recruiting DNA methyltransferases into EIF2B5 promoter. Furthermore, interference of EIF2B5 expression rescued KCNQ1OT1 depletion-induced inhibitory impact on OC cell proliferation and metastasis.ConclusionOur findings evidenced that lncRNA KCNQ1OT1 aggravated ovarian cancer metastasis by decreasing EIF2B5 expression level, and provided a novel therapeutic strategy for OC.

Salvianolic acid B activates chondrocytes autophagy and reduces chondrocyte apoptosis in obese mice via the KCNQ1OT1/miR-128-3p/SIRT1 signaling pathways

PurposeSalvianolic acid B (Sal B) possesses strong anti-inflammatory and antioxidant activity. This study aims to explore the underlying mechanism of Sal B to improve the obesity-related osteoarthritis (OA).MethodsC57BL/6 J male mice were fed with a normal control diet (NCD), a high fat diet (HFD), or HFD with Sal B (25 mg/kg), and mouse body weights and osteoarticular inflammatory factor levels were examined. Mouse chondrogenic cell line ATDC5 were transfected with lncRNA KCNQ1 overlapping transcript 1 small hairpin RNA (KCNQ1OT1 shRNA), miR-128-3p mimic or Sirtuin-1 small interfering RNA (SIRT1 siRNA), then stimulated with Palmitic acid (PA) followed by the treatment of Sal B. Then, inflammatory response, apoptosis, and autophagy of ATDC5 cells in different groups were detected.ResultsSal B reduced the body weight, decreased the levels of inflammatory markers, and improved cartilage damage in OA mice fed with HFD. KCNQ1OT1 was downregulated in OA mice fed with HFD, and PA-stimulated ATDC5 cells. Sal B protected ATDC5 cells against PA-mediated inflammation, apoptosis, and the inhibition of autophagy, while knockdown of KCNQ1OT1 reversed these results. KCNQ1OT1 was found to be functioned as a ceRNA to bind and downregulate the expression of miR-128-3p that was upregulated in PA-induced cells. Furthermore, SIRT1 was verified as a target of miR-128-3p. MiR-128-3p overexpression reversed the effects of Sal B on inflammatory response, apoptosis, and autophagy in PA-stimulated cells, and knockdown of SIRT1 displayed the similar results.ConclusionSal B exerted a chondroprotective effect by upregulating KCNQ1OT1, which indicates Sal B can used for a therapeutic agent in obesity-related OA.

Dexamethasone attenuates dry eye-induced pyroptosis by regulating the KCNQ1OT1/miR-214 cascade

Dry eye disease (DED) is an inflammatory disorder of the ocular surface seriously affecting the quality of life of patients. Topical dexamethasone (Dex) administration protects the cornea from the hyperosmotic stress (HS) induced by tears. Pyroptosis participates in the activation of epithelial inflammation during DED. However, it remains unclear whether Dex attenuates the progression of DED through pyroptosis. In this study, we aimed to investigate the effect of Dex on DED using both cell and animal models and its underlying mechanism. The inflammatory factors contained in tears were detected using a cytokine assay. The pyroptosis in DED mice and human corneal epithelial cells (HCECs) treated with hyperosmotic medium under various treatments was evaluated by immunohistochemical assays (IHC) or western blotting (WB). RNA expression was manipulated with siRNA or agomir microRNAs and measured using a polymerase chain reaction. The scratch assay was used to assess the migration rate of HCECs. Remaining corneal defects were evaluated using fluorescein staining and photographed using a digital camera. Dex could suppress the release of inflammatory factors and notably attenuate pyroptosis, KCNQ1OT1 expression, and NF-κB activation induced by HS injury in vivo and in vitro. KCNQ1OT1 upregulation could activate pyroptosis by sponging miR-214. Furthermore, KCNQ1OT1 knockdown and miR-214 overexpression reversed the effect of HS, promoted the migration of HCECs, and accelerated corneal wound healing. Dex effectively suppressed HS-induced pyroptosis through the KCNQ1OT1/miR-214/caspase-1 signaling axis by inhibiting the NF-κB activation. Our results provide a novel understanding of the mechanism of Dex as an anti-inflammatory drug in DED.

Long noncoding RNA KCNQ1OT1 inhibits osteoclast differentiation by regulating the miR-128-3p/NFAT5 axis.

Noncoding RNAs play an important role in regulating osteoclast differentiation. We investigated whether and how potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1), a long noncoding RNA, regulates osteoclast differentiation. We found that the expression of KCNQ1OT1 was downregulated in osteoporotic bone tissue. Then transfection of KCNQ1OT1 overexpression vectors or small interfering RNAs showed that the proliferation, migration, and osteoclast differentiation of RAW 264.7 cells were inhibited by KCNQ1OT1 upregulation, while they were promoted by KCNQ1OT1 knockdown. Interestingly, we found and confirmed that miR-128-3p was a target of KCNQ1OT1 using online databases, dual luciferase reporter assays and quantitative real-time polymerase chain reaction, and that it inhibited the expression of miR-128-3p. Moreover, we confirmed that miR-128-3p directly targeted nuclear factor of activated T cell 5 (NFAT5), a protein that combines with osteoprotegerin and thus regulates osteoclastogenesis with the presence of the receptor activator of nuclear factor κB ligand. Furthermore, we demonstrated that both the knockdown of KCNQ1OT1 and the overexpression of miR-128-3p attenuate the expression of NFAT5, while upregulating the osteoclastogenesis markers c-Fos, NFATc1, and Ctsk. The results from overexpression of KCNQ1OT1 and the inhibition of miR-128-3p were contrary to the above. Finally, we found that the inhibition of osteoclast differentiation by KCNQ1OT1 overexpression could be rescued using a miR-128-3p mimic, while the enhancement of migration and osteoclast differentiation by si-NFAT5 could be reversed with a miR-128-3p inhibitor. These results suggested that KCNQ1OT1 regulates the osteoclast differentiation via the miR-128-3p/NFAT5 axis.

Silencing of LncRNA KCNQ1OT1 confers an inhibitory effect on renal fibrosis through repressing miR-124-3p activity

ABSTRACT LncRNA have been increasingly shown that plays pivotal roles in the development of various diseases, including renal fibrosis. Nevertheless, the pathological function of Long non-coding RNA KCNQ1OT1 (KCNQ1OT1) in the renal fibrosis remains obscure. Unilateral ureteral obstruction (UUO) was used to induce renal fibrosis. We detected the expression levels of KCNQ1OT1 in the TGF-β1-induced HK-2 cells via RT-qPCR analysis. The functions of KCNQ1OT1 on the progression of renal fibrosis were examined by CCK-8, EdU, dual-luciferase reporter, and immunofluorescence analyses. In the present study, we found that sh-KCNQ1OT1 obviously attenuated UUO-induced renal fibrosis. Moreover, production of extracellular matrix (ECM), including α-SMA and Fibronectin levels, was significantly increased in kidney and HK-2 cells after UUO or TGF-β stimulation. Knockdown of KCNQ1OT1 inhibited cell proliferation and inhibits the α-SMA and Fibronectin expression of TGF-β1-induced HK-2 cells. In addition, bioinformatics analysis and dual-luciferase reporter assay indicated that miR-124-3p was a target gene of KCNQ1OT1. Mechanistically, silencing miR-124-3p abolished the repressive effects of KCNQ1OT1 on TGF-β1-induced HK-2 cells. In conclusion, KCNQ1OT1 knockdown plays an anti-fibrotic effect through promotion of miR-124-3p expression in renal fibrosis, which provides a promising therapeutic target for the treatment of renal fibrosis.

Downregulation of Kcnq1ot1 attenuates β-cell proliferation and insulin secretion via the miR-15b-5p/Ccnd1 and Ccnd2 axis.

To examine the effect of lncRNA Kcnq1ot1 on pancreatic β cells in the development of diabetes. The expression levels of Kcnq1ot1 were detected in the islets of diabetes mouse models and the serum of patients with type 2 diabetes by qRT-PCR. CCK8, Ki67 staining, immunohistochemical analyses, glucose-stimulated insulin secretion and intraperitoneal glucose tolerance test were performed to detect the effect of Kcnq1ot1 on β-cell proliferation and insulin secretion in vitro and in vivo. The relationship between Kcnq1ot1 and miR-15b-5p was predicted by bioinformatics prediction, which was confirmed by luciferase reporter assay. Kcnq1ot1 was more abundant in the pancreas. The expression of Kcnq1ot1 was decreased in the islets of db/db mice and diet-induced obese mice and in the serum of patients with type 2 diabetes. Silencing Kcnq1ot1 inhibited the β-cell proliferation concomitant with a reduction in the levels of Ccnd1 and Ccnd2. Insulin synthesis and secretion were impaired, along with the decreased expression of Ins1, Ins2, and insulin-related transcription factors. Moreover, Kcnq1ot1 knockdown in vivo reduced glucose tolerance and decreased insulin secretion, consistent with the reduction in the relative islet area and Ki67-positive β-cells detected by immunochemistry and immunofluorescence staining, respectively. Mechanistically, Kcnq1ot1 directly targeted miR-15b-5p which regulated β-cell proliferation and insulin secretion through Ccnd1 and Ccnd2. Notably, the suppression of miR-15b-5p attenuated the inhibition of Min6 proliferation and insulin production induced by Kcnq1ot1 knockdown. Kcnq1ot1 regulated β-cell proliferation and insulin secretion via the miR-15b-5p/Ccnd1 and Ccnd2 axis, which is worthy of further investigation considering its potential in diabetes treatment.

LncRNA KCNQ1OT1-mediated cervical cancer progression by sponging miR-1270 as a ceRNA of LOXL2 through PI3k/Akt pathway.

Dysregulated noncoding RNAs participated in progressions of cervical cancer. To verify impacts of KCNQ1OT1 on modulating progressions of cervical cancer cells. Expressions of KCNQ1OT1, miR-1270, and LOXL2 were analyzed through RT-qPCR and protein expressions of LOXL2, p-AKT, and AKT were validated using western blot. Bindings of miR-1270 with KCNQ1OT1 or LOXL2 were verified using luciferase reporter assay. CCK-8 and flow cytometry evaluated cell viability and apoptosis, respectively. The PI3K/AKT signaling pathway suppressor, LY294002, was applied to treat the cells and the changes of KCNQ1OT1 expression and LOXL2, p-AKT, and AKT protein expressions were examined. KCNQ1OT1 expression was the highest in HeLa cells but lowest in SiHa cells whose upregulation improved the viability but inhibited the apoptosis in SiHa cells while knockdown of KCNQ1OT1 caused opposite results in HeLa cells. MiR-1270 was sponged and negatively modulated by KCNQ1OT1. MiR-1270 mimics caused low viability and high apoptosis of SiHa cells but miR-1270 inhibitor reverse its roles in HeLa cells. LOXL2, the target of miR-1270, positively interplayed with KCNQ1OT1 but had negative interaction with miR-1270. LOXL2 overexpression promoted viability and decreased apoptosis of SiHa cells but knockdown of LOXL2 restored its effects in HeLa cells. Moreover, LOXL2 and phosphorylated AKT (p-AKT) protein expressions were downregulated by suppressed KCNQ1OT1 and LOXL2 and miR-1270 mimics but promoted by overexpressed KCNQ1OT1 and LOXL2 and miR-1270 inhibitor. Additionally, LY294002 treatment caused low KCNQ1OT1 RNA expression and decreased LOXL2 and p-AKT protein expressions. KCNQ1OT1/miR-1270/LOXL2 axis modulated viability and apoptosis of cervical cancer cells.

KCNQ1OT1 polymorphism rs35622507 and methylation status of KCNQ1OT1 promoter influence the drug resistance to L-OHP.

Background: LncRNA potassium voltage-gated channel subfamily Q member 1 opposite strand/antisense transcript 1 (KCNQ1OT1) has been reported to promote resistance to chemotherapy in colon cancer by inhibiting the expression of miR-34a. And the methylation of KCNQ1OT1 was also reported in the pathogenesis of various diseases. In this study, we aimed to study the combined effect of allele variation of KCNQ1OT1 polymorphism rs35622507 and methylation status of KCNQ1OT1 promoter in the treatment of colon cancer.Methods: The expression levels of KCNQ1OT1, miR-34a, and ATG4B mRNA were assessed by qRT-PCR. ATG4B protein expression was analyzed by Western blot analysis. TUNEL and MTT assay were performed to examine the cell apoptosis and viability. Luciferase assays revealed the relationship between KCNQ1OT1, miR-34a and ATG4B.Results: Carrier of allele 10 and methylated promoter in KCNQ1OT1 was associated with decreased KCNQ1OT1/ATG4B expression, increased miR-34a expression and enhanced apoptosis in colon cancer tissue samples. And subsequent luciferase assay showed that miR-34a could bind to KCNQ1OT1 and ATG4B at specific binding sites. The knockdown of KCNQ1OT1 significantly suppressed the KCNQ1OT1/ATG4B expression, improved the miR-34a expression and reduced the viability of HCT116 and SW480 cells. The over-expression of ATG4B notably restored the cell viability loss and apoptosis increase induced by the knockdown of KCNQ1OT1. Moreover, oxaliplatin (L-OHP) treatment elevated the apoptosis of HCT116 and SW480 cells.Conclusions: The drug resistance in the treatment of colon cancer is most reduced in patients carrying allele 10 and methylated in KCNQ1OT1 promoter. This function is accomplished by the signaling pathway of KCNQ1OT1/miR-34a/ATG4B.

Long Non-Coding RNA KCNQ1OT1 Regulates Protein Kinase CK2 Via miR-760 in Senescence and Calorie Restriction.

Long non-coding RNAs (lncRNAs) play important biological roles. Here, the roles of the lncRNA KCNQ1OT1 in cellular senescence and calorie restriction were determined. KCNQ1OT1 knockdown mediated various senescence markers (increased senescence-associated β-galactosidase staining, the p53-p21Cip1/WAF1 pathway, H3K9 trimethylation, and expression of the senescence-associated secretory phenotype) and reactive oxygen species generation via CK2α downregulation in human cancer HCT116 and MCF-7 cells. Additionally, KCNQ1OT1 was downregulated during replicative senescence, and its silencing induced senescence in human lung fibroblast IMR-90 cells. Additionally, an miR-760 mimic suppressed KCNQ1OT1-mediated CK2α upregulation, indicating that KCNQ1OT1 upregulated CK2α by sponging miR-760. Finally, the KCNQ1OT1–miR-760 axis was involved in both lipopolysaccharide-mediated CK2α reduction and calorie restriction (CR)-mediated CK2α induction in these cells. Therefore, for the first time, this study demonstrates that the KCNQ1OT1–miR-760–CK2α pathway plays essential roles in senescence and CR, thereby suggesting that KCNQ1OT1 is a novel therapeutic target for an alternative treatment that mimics the effects of anti-aging and CR.

LncRNA Kcnq1ot1 promotes bone formation by inhibiting miR-98-5p/Tbx5 axis in MC3T3-E1 cells.

Long non-coding (lnc)RNA KCNQ1 opposite strand/antisense transcript 1 (Kcnq1ot1) has been shown to regulate multiple biological processes. However, the functional role of Kcnq1ot1 in osteoporosis and the underlying mechanism are still unclear. The present study aimed to investigate the function of lncRNA Kcnq1ot1 in osteogenic differentiation. Alkaline phosphatase (ALP) activity was measured using an ALP assay kit. Western blotting was performed to assess the expression levels of osteogenic differentiation-associated proteins. Reverse transcription-quantitative PCR was performed to detect Kcnq1ot1, microRNA (miR)-98-5p and T-box transcription factor 5 (Tbx5) expression levels. The binding of Kcnq1ot1 with miR-98-5p and that of miR-98-5p with Tbx5 were predicted by starBase and TargetScan databases, respectively, and verified using dual luciferase reporter assays. The mineralization of MC3T3-E1 cells was observed using an Alizarin red S staining assay. The results revealed that expression of Kcnq1ot1 was increased and that of miR-98-5p was decreased during osteogenic differentiation. Additionally, Kcnq1ot1 was shown to target miR-98-5p and inhibit its expression. Inhibiting miR-98-5p reversed the inhibitory effect of Kcnq1ot1 knockdown on osteogenic differentiation and mineralization of MC3T3-E1 cells. Furthermore, Kcnq1ot1 regulated Tbx5 expression via miR-98-5p. Overexpressing miR-98-5p or downregulating Tbx5 expression reversed the promotive effect of Kcnq1ot1 overexpression on osteogenic differentiation and mineralization of MC3T3-E1 cells. In conclusion, these findings suggested that Kcnq1ot1 may promote bone formation by inhibiting miR-98-5p and upregulating Tbx5. Kcnq1ot1, miR-98-5p and Tbx5 may therefore serve as promising targets for the treatment of osteoporosis.

LncRNA KCNQ1OT1 regulated high glucose-induced proliferation, oxidative stress, extracellular matrix accumulation, and inflammation by miR-147a/SOX6 in diabetic nephropathy (DN).

Long non-coding RNAs (lncRNAs) have been proved to play critical roles in diabetic nephropathy (DN). This study aimed to investigate the functions and underlying mechanism of potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) in DN. Blood samples were obtained from 33 DN patients and 30 healthy volunteers. Kidney biopsies tissues of DN patients (n = 10) and patients with normal kidney morphology (n = 10) were collected. We found that KCNQ1OT1 was markedly overexpressed in the blood and kidney biopsies tissues of DN patients, as well as in high glucose (HG)-cultured human glomerular mesangial (HGMC) cells. Knockdown of KCNQ1OT1 suppressed proliferation, extracellular matrix (ECM) accumulation, inflammation, and oxidative stress in HG-treated HGMC cells in vitro. KCNQ1OT1 functioned as a sponge for microRNA-147a (miR-147a), and SRY-Box Transcription Factor 6 (SOX6) was directly targeted by miR-147a. Downregulation of miR-147a or upregulation of SOX6 partly overturned the prohibitive effects of KCNQ1OT1 knockdown or miR-147a overexpression on proliferation, ECM accumulation, inflammation, and oxidative stress in HG-treated HGMC cells. Altogether, KCNQ1OT1 mediated the proliferation, ECM accumulation, inflammation, and oxidative stress in HG-treated HGMC cells via miR-147a/SOX6 axis, which might be a novel target for DN therapy.

LncRNA KCNQ1OT1 (potassium voltage-gated channel subfamily Q member 1 opposite strand/antisense transcript 1) aggravates acute kidney injury by activating p38/NF-κB pathway via miR-212-3p/MAPK1 (mitogen-activated protein kinase 1) axis in sepsis

ABSTRACT Acute kidney injury (AKI), a common complication of sepsis, is characterized by a rapid loss of renal excretory function. A variety of etiologies and pathophysiological processes may contribute to AKI. Previously, mitogen-activated protein kinase 1 (MAPK1) was reported to regulate cellular processes in various sepsis-associated diseases. The current study aimed to further explore the biological function and regulatory mechanism of MAPK1 in sepsis-induced AKI. In our study, MAPK1 exhibited high expression in the serum of AKI patients. Functionally, knockdown of MAPK1 suppressed inflammatory response, cell apoptosis in response of lipopolysaccharide (LPS) induction in HK-2 cells. Moreover, MAPK1 deficiency alleviated renal inflammation, renal dysfunction, and renal injury in vivo. Mechanistically, MAPK1 could activate the downstream p38/NF-κB pathway. Moreover, long noncoding RNA potassium voltage-gated channel subfamily Q member 1 opposite strand/antisense transcript 1 (KCNQ1OT1) was identified to serve as a competing endogenous RNA for miR-212-3p to regulate MAPK1. Finally, rescue assays indicated that the inhibitory effect of KCNQ1OT1 knockdown on inflammatory response, cell apoptosis, and p38/NF-κB pathway was reversed by MAPK1 overexpression in HK-2 cells. In conclusion, KCNQ1OT1 aggravates acute kidney injury by activating p38/NF-κB pathway via miR-212-3p/MAPK1 axis in sepsis. Therefore, KCNQ1OT may serve as a potential biomarker for the prognosis and diagnosis of AKI patients.