LINC-p21 Regulates Pancreatic ß-Cell Function in Type 2 Diabetes Mellitus.

This study aimed to investigate the underlying mechanism and assess the biological role of long intergenic non-coding RNA (LINCRNA)-p21 in type 2 diabetes mellitus (T2DM). LINC-p21 and miR-335-3p expression levels were evaluated in blood from T2DM patients, healthy individuals, and mouse islet ß-cell line MIN6 cells grown in a high glucose environment. Apoptosis-related proteins, iNOS, and IGF-1 were detected in vitro and in vivo. Bioinformatics was used to predict that miR-335-3p had complementary binding sites to IGF-1, and a dual-luciferase reporter confirmed the targeting link between LINC-p21 and miR-335-3p. LINC-p21 was highly expressed in the T2DM serum and cells, and LINC-p21 was significantly associated with T2DM prognosis. In vitro and in vivo dysfunction of ß-cells was reduced by LINC-p21 knockdown. MiR-335-3p and IGF-1 may be potential targets of LINC-p21 and miR-335-3p, respectively, after the prediction of the target of LINC-p21 was verified by dual-luciferase assay. Anti-miR-335-3p made LINC-p21 knockdown function again; however, interference of IGF-1 mRNA restored the function of LINC-p21. The miR-335-3p/IGF-1 axis may have a role in the functional protection of pancreatic ß-cells by LINC-p21 silencing, boosting insulin production, and slowing the course of diabetes.

miR-335-3p improves type II diabetes mellitus by IGF-1 regulating macrophage polarization.

Previous studies have found that miR-335 is highly expressed in type II diabetes mellitus (T2DM) models and is related to insulin secretion, but there are few studies on the regulatory effects of miR-335-3p on insulin resistance and macrophage polarization in T2DM patients. This study aims to explore the effects of miR-335-3p on insulin resistance and macrophage polarization in T2DM patients. Blood glucose (insulin tolerance tests, glucose tolerance tests) and body weight of the T2DM model were measured; macrophages from adipose tissue were isolated and cultured, and the number of macrophages was detected by F4/80 immunofluorescence assay; the Real-time quantitative polymerase chain reaction (qPCR) assay and Western blot assay were used to detect the miR-335-3p expression levels, insulin-like growth factor 1 (IGF-1), M1-polarizing genes (inducible nitric oxide synthase [iNOS] and TNF-α) as well as M2-polarizing genes (IL-10 and ARG-1). The targeting link between miR-335-3p and IGF-1 was confirmed using bioinformatics and dual luciferase assay. The results showed that miR-335-3p expression level in adipose tissue of the T2DM model was significantly decreased, and the mice's body weight and blood glucose levels dropped considerably, miR-335-3p inhibited the number of macrophages, inhibiting the iNOS and TNF-α relative mRNA expression levels, and up-regulated the IL-10 and ARG-1 relative mRNA expression levels, miR-335-3p negatively regulated target gene IGF-1, IGF-1 significantly increased the iNOS and TNF-α mRNA and protein expression levels, decreasing the IL-10 and ARG-1 mRNA and protein expression levels, indicating that miR-335-3p could affect the T2DM process by regulating macrophage polarization via IGF-1.

METTL3 Mediates Microglial Activation and Blood-Brain Barrier Permeability in Cerebral Ischemic Stroke by Regulating NLRP3 Inflammasomes Through m6A Methylation Modification.

Cerebral ischemic stroke (CIS) is the main cause of disability. METTL3 is implicated in CIS, and we explored its specific mechanism. Middle cerebral artery occlusion (MCAO) rat model and oxygen-glucose deprivation/reperfusion (OGD/R) HAPI cell model were established and treated with LV-METTL3 or DAA, oe-METTL3, miR-335-3p mimics, or DAA, to assess their effects on MCAO rat neurological and motor function, cerebral infarction area, brain water content, microglial activation, blood-brain barrier (BBB) permeability, and NLRP3 inflammasome activation. METTL3, pri-miR-335-3p, mature miR-335-3p, and miR-335-3p mRNA levels were assessed by RT-qPCR; M1/M2 microglial phenotype proportion and M1/M2 microglia ratio, inflammatory factor levels, and m6A modification were assessed. MCAO rats manifested cerebral ischemia injury. METTL3 was under-expressed in CIS. METTL3 overexpression inhibited microglial activation and M1 polarization and BBB permeability in MCAO rats and inhibited OGD/R-induced microglial activation and reduced M1 polarization. METTL3 regulated miR-335-3p expression and inhibited NLRP3 inflammasome activation. m6A methylation inhibition averted METTL3's effects on NLRP3 activation, thus promoting microglial activation in OGD/R-induced cells and METTL3's effects on BBB permeability in MCAO rats. Briefly, METTL3 regulated miR-335-3p expression through RNA m6A methylation and inhibited NLRP3 inflammasome activation, thus repressing microglial activation, BBB permeability, and protecting against CIS.

CircRNA ARHGAP10 promotes osteogenic differentiation through the miR-335-3p/RUNX2 pathway in aortic valve calcification.

Background: Calcific aortic valve disease (CAVD) is a common cardiovascular disease with high morbidity and mortality, and no effective prevention or treatment is available. In recent years, increasing evidence has shown that noncoding RNAs (ncRNAs) play an important role in the pathogenesis and prognosis of CAVD. Several associated circular RNAs (circRNAs) have been reported to be involved in CAVD, such as circRIC3 and TGFBR2. However, the limited number of circRNAs identified in CAVD warrants further in-depth investigation, and the comprehensive elucidation of their role in the key mechanisms of this disease is needed. Methods: The expression of circRNAs and microRNAs (miRNAs) were analyzed by RNA sequencing. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to analyze the expression of circRNA ARHGAP10 (circARHGAP10), miR-335-3p, and RUNX2. Luciferase reporter assay, pull-down assay, and RNA binding protein immunoprecipitation (RIP) assay were performed to evaluate the binding of miR-335-3p to circARHGAP10 or RUNX2. Alizarin red S staining showed the formation of calcified nodules in valve interstitial cells (VICs). The expression of circARHGAP10 and miR-335-3p was altered through lentivirus infection. Alkaline phosphatase (ALP) activity was used to verify the correlation between circARHGAP10 and miR-335-3p. The expression of proteins was assessed via Western blot. RNA fluorescence in situ hybridization (FISH) was used to confirm the localization of circARHGAP10 in the cytoplasm of VICs. Immunofluorescence was used to detect the expression level of RUNX2. ApoE-/- mice were used to construct a CAVD model, circARHGAP10 short hairpin RNA (shRNA) and miR-335-3p inhibitor lentivirus were intraperitoneally injected, and scramble and inhibitor normal control (NC) lentivirus were injected as controls, followed by hematoxylin and eosin (HE) staining. Results: Through RNA sequencing, we found that circARHGAP10 (hsa_circ_0008975) was highly expressed in calcific aortic valves. CircARHGAP10 knockdown effectively inhibited the extent of osteogenic differentiation of VICs. We then found that circARHGAP10 was a competing endogenous RNA (ceRNA) of miR-355-3p and that miR-355-3p targeted RUNX2. In vitro experiments confirmed that circARHGAP10 regulated the osteogenic differentiation of VICs through the miR-355-3p/RUNX2 pathway, and this was validated in vivo using an ApoE-/- mouse model. Conclusions: These findings provide a foundation for circRNA-directed diagnostics and therapeutics for CAVD.

MiR-335-3p/miR-155-5p Involved in IGFBP7-AS1-Enhanced Odontogenic Differentiation.

BACKGROUND: The differentiation of stem cells from exfoliated deciduous teeth (SHEDs) into odontoblasts determines the regeneration of dentin-pulp complex. Non-coding RNAs (ncRNAs), including microRNA (miRNA) and long non-coding RNA (lncRNA), participate in many multiple biological processes, but the specific miRNAs involved in odontogenesis are incompletely defined. It was confirmed that lncRNA IGFBP7-AS1 could positively regulate odontogenetic differentiation in SHEDs. To investigate the downstream mechanisms of this process, miR-335-3p and miR-155-5p were found to be closely related with SHED odontogenic differentiation through whole-genome sequencing. The aim of the current study was to determine the role of miR-335-3p/miR-155-5p in IGFBP7-AS1-enhanced SHED differentiation and explore the potential mechanism of IGFBP7-AS1-mediated odontogenesis. METHODS: Putative miR-335-3p/miR-155-5p binding sites within IGFBP7-AS1 were identified by bioinformatics analysis, and the binding of miR-335-3p/miR-155-5p to these sites was confirmed by dual-luciferase reporter gene assays. The effects of miR-335-3p/miR-155-5p in odontogenesis were detected by tissue nonspecific alkaline phosphatase staining, Alizarin red staining, quantitative real-time polymerase chain reaction (qRT-PCR) analyses, and western blot testing. The molecular mechanisms of miR-335-3p/miR-155-5p involved in IGFBP7-AS1-mediated odontogenesis were analysed by qRT-PCR and western blot testing. RESULTS: Dual-luciferase reporter gene assays showed that miR-335-3p/miR-155-5p could directly bind to IGFBP7-AS1. MiR-335-3p and miR-155-5p both could down-regulate dentin sialophosphoprotein expression, and both miRNAs could inhibit IGFBP7-AS1-mediated SHED odontogenetic differentiation via suppression of the extracellular signal-regulated kinase (ERK) pathway. CONCLUSIONS: Both miR-335-3p and miR-155-5p were negative regulators to IGFBP7-AS1-enhanced odontogenic differentiation of SHED through suppression of the ERK pathway.

Yin Yang 1 (YY1)-induced long intergenic non-protein coding RNA 472 (LINC00472) aggravates sepsis-associated cardiac dysfunction via the micro-RNA-335-3p (miR-335-3p)/Monoamine oxidase A (MAOA) cascade.

As a leading complication of sepsis, sepsis-induced cardiac dysfunction (SICD) contributed to the high mortality of patients with sepsis. Long non-coding RNA (LncRNA) LINC00472 has been reported to be in sepsis-induced disease. Nonetheless, its biological function and underlying molecular in SICD remain largely unknown. In this study, in vivo and in vitro SICD models were established via LPS treatment. H&E staining was employed for the evaluation of myocardial injury. ELISA assay was performed to detect cardiac Troponin I (cTnI), creatine kinase-MB (CK-MB), interleukin (IL)-1ß, and tumor necrosis factor-α (TNF-α) levels. Cardiomyocyte viability and apoptosis were assessed via CCK-8 and flow cytometry assays. The transcriptional regulation of YY1 on LINC00472 was demonstrated via ChIP assay. Besides, the interaction between YY1 and LINC00472, as well as the association between miR-335-3p and LINC00472 or MAOA were verified via luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Herein, highly expressed LINC00472 was observed in both in vivo and in vitro SICD models. LINC00472 knockdown substantially attenuated LPS-induced inhibition on cardiomyocyte viability and reversed cardiomyocyte apoptosis and inflammatory response mediated by LPS treatment. YY1 induced LINC00472 upregulation, thereby promoting cardiomyocyte dysfunction induced by LPS. In addition, MAOA upregulation or miR-335-3p inhibition could partly reverse the suppressive effect on LPS-induced cardiomyocyte dysfunction mediated by LINC00472 knockdown. Based on our results, it seemed that YY1-activated LINC00472 might contribute to SICD progression via the miR-335-3p/MAOA pathway.

LncRNA-HAGLR motivates triple negative breast cancer progression by regulation of WNT2 via sponging miR-335-3p.

BACKGROUND: Triple negative breast cancer (TNBC) is a group of highly heterogeneous mixed breast cancer at the level of gene expression profile. Therefore, it is of great clinical significance to explore the molecular mechanism of TNBC and find a targeted therapeutic approach from the molecular level. METHODS: Long non-coding RNA (lncRNA) HAGLR expression level was measured by and qRT-PCR in TNBC tissues and cell lines. EdU, MTT, wound healing and Transwell assays were performed to explore the role of HAGLR on the malignancy of TNBC cells. Luciferase assay was used to clarify the binding between miR-335-3p with HAGLR and WNT2. The tumor formation experiment in nude mice was used to explore the function of HAGLR in vivo. RESULTS: HAGLR was increased in TNBC tissues and cell lines. Silencing of HAGLR inhibited viability, proliferation, migration, and invasion of BT549 cells. Furthermore, HAGLR acted as a sponge of miR-335-3p and inhibited its expression. And miR-335-3p directly targeted WNT2. Functionally, forced expression of miR-335-3p or knockdown of WNT2 removed the promoted effects of lncRNA HAGLR on TNBC development. In vivo tumorigenesis experiments indicated HAGLR accelerated tumor growth via miR-335-3p/WNT2 axis. CONCLUSION: Our study revealed that HAGLR promoted the growth of TNBC, which was mediated by miR-335-3p/WNT2 axis.

MiR-335-3p inhibits cell proliferation and induces cell cycle arrest and apoptosis in acute myeloid leukemia by targeting EIF3E.

Here, we aimed to investigate the biological roles and the regulatory mechanisms of miR-335-3p in acute myeloid leukemia (AML). We first found miR-335-3p was significantly downregulated in blood samples from leukemia patients and cell lines using reverse transcription quantitative polymerase chain reaction. Through CCK-8 assay and flow cytometry, we observed that miR-335-3p overexpression significantly inhibited cell proliferation and induced cell cycle G0/G1 arrest and apoptosis in AML cell lines (THP-1 and U937). Moreover, miR-335-3p directly targets EIF3E and negatively regulated its expression. More importantly, EIF3E overexpression reversed the effects of miR-335-3p on cell proliferation, G1/S transition, and apoptosis. Furthermore, miR-335-3p overexpression obviously downregulated the expression of CDK4, Cyclin D1, and Bcl-2, while upregulated the expression of p21 and Bad, which were significantly rescued by the cotransfection of pcDNA3.1-EIF3E. Collectively, our study proposes that miR-335-3p/EIF3E axis could be a promising therapeutic target to mitigate the progression of AML.

LncRNA LOC100129620 promotes osteosarcoma progression through regulating CDK6 expression, tumor angiogenesis, and macrophage polarization.

Osteosarcoma is a malignant tumor with high mortality in children and adolescents. The mechanism of osteosarcoma metastasis is currently unclear. Abnormal expression of long non-coding RNA (lncRNA) plays an important role in tumor metastasis. We used bioinformatics to analyze the differences in gene expression between osteosarcoma in situ and osteosarcoma lung metastases. CCK-8 was used to detect the effect of lncRNA LOC100129620 on the proliferation of osteosarcoma cells. The effect of LOC100129620 on the invasion of osteosarcoma cells was assessed by Transwell assay. The regulatory effect of LOC100129620 on miR-335-3p was examined using RNA pull-down and luciferase reporter gene assays. The effect of LOC100129620 on the polarization of macrophages was detected by quantitative real-time fluorescent PCR. The results show that LOC100129620 can promote the proliferation and migration of osteosarcoma cells. LOC100129620 can promote the proliferation of osteosarcoma in vivo. LOC100129620 can bind to miR-335-3p and regulate its function. MiR-335-3p mediates the regulatory effects of LOC100129620 on CDK6. LOC100129620 promotes the formation of blood vessels and the polarization of macrophages. The LOC100129620/miR-335-3p/CDK6 signaling pathway promotes the metastasis of osteosarcoma by regulating the proliferation of osteosarcoma cells, angiogenesis, and macrophage polarization.

MicroRNAs Possibly Involved in the Development of Bone Metastasis in Clear-Cell Renal Cell Carcinoma.

Bone metastasis in clear-cell renal cell carcinoma (ccRCC) leads to substantial morbidity through skeletal related adverse events and implicates worse clinical outcomes. MicroRNAs (miRNA) are small non-protein coding RNA molecules with important regulatory functions in cancer development and metastasis. In this retrospective analysis we present dysregulated miRNA in ccRCC, which are associated with bone metastasis. In particular, miR-23a-3p, miR-27a-3p, miR-20a-5p, and miR-335-3p specifically correlated with the earlier appearance of bone metastasis, compared to metastasis in other organs. In contrast, miR-30b-3p and miR-139-3p were correlated with less occurrence of bone metastasis. These miRNAs are potential biomarkers and attractive targets for miRNA inhibitors or mimics, which could lead to novel therapeutic possibilities for bone targeted treatment in metastatic ccRCC.

Long noncoding RNA CRNDE functions as a diagnostic and prognostic biomarker in osteosarcoma, as well as promotes its progression via inhibition of miR-335-3p.

BACKGROUND: This study was performed to evaluate the diagnostic and prognostic value, as well as the role of long-chain noncoding RNA (lncRNA) colorectal neoplasia differentially expressed (CRNDE) in osteosarcoma (OS). MATERIALS AND METHODS: A quantitative real-time polymerase chain reaction assay was to determine lncRNA CRNDE and microRNA-335-3p (miR-335-3p) expressions. The Kaplan-Meier analysis was to analyze the relationship between lncRNA CRNDE expression and survival in patients with OS. Receiver operating characteristic curves were to evaluate the diagnostic value of lncRNA CRNDE in OS. Bioinformatics analysis and luciferase reporter assays were used to predict and confirm the relationship between lncRNA CRNDE and miR-335-3p. Cell counting Kit-8 and transwell migration assays assessed the role of lncRNA CRNDE and miR-335-3p in OS cells. RESULTS: lncRNA CRNDE expression was upregulated and miR-355-3p expression was downregulated in OS. In patients with OS, low lncRNA CRNDE expression demonstrated higher overall survival, whereas high lncRNA CRNDE expression was an independent poor prognostic factor. Furthermore, increased lncRNA CRNDE expression was associated with distant metastasis and the tumor-node-metastasis stage in patients with OS, which can be considered as an independent diagnostic biomarker in OS. We revealed that miR-335-3p was the target of lncRNA CRNDE. It also demonstrated that knockdown of lncRNA CRNDE inhibited OS cell proliferation, migration, and invasion, and inhibition of miR-355-3p promoted this effect. Finally, miR-335-3p partially mediated the stimulatory effects of lncRNA CRNDE in OS. CONCLUSION: We demonstrated that lncRNA CRNDE is a potential diagnostic and prognostic biomarker for OS, and the lncRNA CRNDE/miR-335-3p axis participates in OS progression.

miR-708 and miR-335-3p Inhibit the Apoptosis of Retinal Ganglion Cells Through Suppressing Autophagy.

This study aimed to clarify the regulation role of miR-708 and miR-335-3p in retinal ganglion cell (RGC) autophagy and apoptosis in glaucoma. Chronic glaucoma mice were established by laser photocoagulation. RGCs were isolated and transfected with a series of plasmids and the cultured in 60 mmHg pressure. miR-335-3p, miR-708, and ATG3 mRNA expressions were detected by qRT-PCR. Protein levels of ATG3, autophagy-related protein LC3, and p62 were detected by Western blot. The apoptosis of RGCs was detected by flow cytometry. The regulation role of miR-335-3p/miR-708 in ATG3 was confirmed by the dual-luciferase reporter gene. The expressions of several miRNAs were measured in retinal tissues from chronic glaucoma mice and RGCs under pressure conditions, and results showed that both miR-335-3p and miR-708 were down-regulated. Besides, the inhibition of miR-708 and miR-335-3p induced the apoptosis of RGCs through promoting autophagy. Also, miR-708 and miR-335-3p could bind to ATG3 and targeted regulated ATG3. Furthermore, the interference with miR-708/miR-335-3p induced RGC apoptosis by up-regulating ATG3 to promote autophagy. In general, the down-regulation of miR-708 and miR-335-3p contributed to the apoptosis of RGCs through promoting autophagy in glaucoma.

Upregulated lncRNA CASC9 Contributes to Progression of Non-Small Cell Lung Cancer Through Inhibition of miR-335-3p and Activation S100A14 Expression.

INTRODUCTION: Non-small cell lung cancer (NSCLC) is a deadly cancer type worldwide and the main sub-type of lung cancer. Cancer susceptibility candidate-9 (CASC9) was reported to be a key player in cancer progression. However, its function and underlying mechanism in NSCLC remain unclear. MATERIALS AND METHODS: Expression level of CASC9 in NSCLC tissues and cells was measured with RT-qPCR. Biological roles of CASC9 in NSCLC were analyzed with a series of in vitro experiments. Potential mechanisms of CASC9 in NSCLC were analyzed by predicting and validating the possible targets of CASC9 in NSCLC. RESULTS: In this study, we found CASC9 expression was upregulated in NSCLC tissues and cell lines. High CASC9 expression was identified as a predictor for poorer overall survival of NSCLC patients. Furthermore, functional assays showed CASC9 knockdown suppressed NSCLC cell proliferation, migration, and invasion, while CASC9 overexpression caused opposite effects. We also found microRNA-335-3p (miR-335-3p) could act as a target of CASC9 in NSCLC and the inhibition effect of CASC9 knockdown on NSCLC progression required the activity of miR-335-3p. In addition, we identified S100 calcium-binding protein A14 (S100A14) acts as a target of miR-335-3p. DISCUSSION: Taken together, our study suggested CASC9 could promote NSCLC progression via miR-335-3p/S100A14 axis. The CASC9/miR-335-3p/S100A14 regulatory triplets identified in this work might provide new therapeutic strategies for NSCLC treatment.

Upregulation of miR-335-3p by NF-κB Transcriptional Regulation Contributes to the Induction of Pulmonary Arterial Hypertension via APJ during Hypoxia.

Pulmonary arterial hypertension (PAH) is a cardiopulmonary disease that can lead to heart failure and eventually death. MicroRNAs (miRs) play essential roles during PAH progression; however, their exact mechanism of action remains unclear. Apelin is a small bioactive peptide with a key protective function in the pathogenesis of PAH mediated by binding to the APJ gene. The aim of the present study was to investigate the role of miR-335-3p in chronic normobaric hypoxia (CNH)-induced PAH in mice and the potential underlying regulatory mechanism. Adult male C57BL/6 mice were exposed to normoxia (~21% O2) or CNH (~10% O2, 23 h/d) for 5 weeks. MiR-335-3p was significantly increased in lung tissue of CNH-induced PAH mice. Blocking miR-335-3p attenuated CNH-induced PAH and alleviated pulmonary vascular remodeling. Bioinformatics analysis and luciferase reporter assay indicated that nuclear factor-kappa beta (NF-κB) acted as a transcriptional regulator upstream of miR-335-3p. Pyrrolidine dithiocarbamate treatment reversed the CNH-induced increase in miR-335-3p expression and diminished CNH-induced PAH. Moreover, p50-/- mice were resistant to CNH-induced PAH. Finally, APJ was identified as a direct targeting gene downstream of miR-335-3p, and pharmacological activation of APJ by its ligand apelin-13 reduced CNH-induced PAH and improved pulmonary vascular remodeling. Our results indicate that NF-κB-mediated transcriptional upregulation of miR-335-3p contributes to the inhibition of APJ and induction of PAH during hypoxia; hence, miR-335-3p could be a potential therapeutic target for hypoxic PAH.

Dysregulation of miR-335-3p, targeted by NEAT1 and MALAT1 long non-coding RNAs, is associated with poor prognosis in childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most prevalent cancer among children, and multidrug efflux mediated by overexpression of ABC transporters is the major impediment to successful chemotherapy in this malignancy. The goal of this study is to identify the non-coding RNAs (ncRNAs) which may affect the expression levels of ABCA3; the previously identified prognostic biomarker for multidrug resistance (MDR) in childhood ALL (cALL). Bone marrow samples from 64 cALLs, including 46 de novo and 18 relapsed patients, in addition to 30 non-cancer controls were collected, and ncRNAs were nominated using in silico studies. Quantitative RT-PCR showed low expression profiles of miR-335-3p in cALLs compared with the control group (P = 0.018). Inverse correlation was determined between the miR-335-3p and ABCA3 mRNA expression profiles in cALL patients (r = 0.5019, P = 0.002). Moreover, it was shown that the expression levels of miR-335-3p was downregulated in the drug-resistant samples (MDR group) compared with the drug-sensitive patients (mrd- group), (P = 0.0005, AUC = 0.801). On the other hand, negative correlations were identified between the expression levels of miR-335-3p and the selected LncRNAs, NEAT1 and MALAT1, in the MDR group compared with the mrd- patients (P = 0.009), suggesting a sponge effect for these LncRNAs. The current study showed a potential regulatory role for miR-335-3p in ABCA3 expression targeted by NEAT1 and MALAT1 long non-coding RNAs. This negative impact may possibly contribute to the development of chemoresistance in childhood ALL, and provide an exceptional insight to new therapeutic approaches.

LINC00969 promotes the degeneration of intervertebral disk by sponging miR-335-3p and regulating NLRP3 inflammasome activation.

Long noncoding RNAs (LncRNAs) may serve as miRNA sponges to regulate the expressions of miRNA target genes. LncRNA LINC00969 has been indicated to be upregulated in intervertebral disk degeneration. However, the regulatory mechanism of LINC00969 in intervertebral disk degeneration progression remains unclear. Differently expressed LINC00969, miR-335-3p, and thioredoxin-interacting protein (TXNIP) were determined in nucleus pulposus (NP) tissues and cells isolated from patients with intervertebral disk degeneration. The interaction between LINC00969, miR-335-3p, and TXNIP was also assessed. In this study, we demonstrated that LINC00969 was highly expressed, whereas miR-335-3p was aberrantly downregulated in NP tissues and cells of intervertebral disk degeneration patients. In addition, our results suggested that LINC00969 enhanced NP cell apoptosis. More importantly, LINC00969 was identified to function as a competitive endogenous RNA (ceRNA) for miR-335-3p to positively regulate TXNIP expression in vitro. Our study provided evidence for the cross-talk between LINC00969, miR-335-3p, and TXNIP, shedding light on the therapy for intervertebral disk degeneration. © 2018 IUBMB Life, 71(5):611-618, 2019.