RNA Binding Protein Immunoprecipitation Assay Research Articles

Effect of m6A Methylation Modification on IncRNA ENST00000425005 in Doxorubicin Resistance and Epithelial-Mesenchymal Transition Progression in Lung Cancer Cells

Lung cancer is the leading cause of cancer-related mortality worldwide, accounting for 18.4% of all cancer deaths. This study aims to investigate the underlying mechanism by which long non-coding RNA (lncRNA) ENST00000425005 mediates doxorubicin resistance and epithelial-mesenchymal transition (EMT) in lung cancer cells. The expression levels of ENST00000425005 in non-small cell lung cancer (NSCLC) cells were determined using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The protein levels of alkB homolog 5 (ALKBH5) and EMT markers (including Snail1, E-cadherin, N-cadherin, and Vimentin) were assessed using Western Blot analysis. RNA binding protein immunoprecipitation assays were conducted to detect the interaction between ENST00000425005 and ALKBH5. Cell viability was evaluated using cell counting kits assay, and cell invasion was determined by transwell assay. It was found that ENST00000425005 expression was downregulated, while ALKBH5 expression was upregulated in NSCLC cells. Additionally, ALKBH5 bound to ENST00000425005 and downregulated its expression. Overexpression of ALKBH5 reduced m6A modification and RNA levels of ENST00000425005. Moreover, co-overexpression of ENST00000425005 and ALKBH5 rescued loss of NSCLC cell viability, invasion, and doxorubicin resistance caused by overexpression of ENST00000425005. Furthermore, this co-overexpression rescued ENST00000425005-induced changes in expression of E-cadherin, Snail1, N-cadherin, and Vimentin. The reduction of m6A methylation modification on lncRNA ENST00000425005 caused by binding to ALKBH5 promoted doxorubicin resistance and EMT progression in NSCLC cells. In summary, targeting lncRNA ENST00000425005 holds promise as a therapeutic strategy for NSCLC.

LncRNA LINC01278 Regulates the Prognosis and Related Mechanisms of Gastric Cancer by Targeting miR-129-5p.

Gastric cancer, a prevalent malady within the digestive tract, has a complex pathological mechanism and numerous patients. The regulation of gastric cancer process by long non-coding RNA (lncRNA) presented new prospects for the study of its molecular mechanism and the treatment of patients. The abnormal expressed genes in gastric cancer were screened by GSE193109 dataset. The correlation between LINC01278 and the likelihood of survival in patients suffering from gastric cancer was investigated by Kaplan-Meier survival curve and multivariate Cox analysis. LINC01278 in gastric cancer tissue samples and cells was verified via RT-qPCR. The cell counting kit-8 (CCK-8) and transwell assay were selected to detect the growth activity of gastric cancer cells. The association between LINC01278 and miR-129-5p was validated through luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) assay. Correlation analysis of clinical features revealed an association between LINC01278 and the prognosis in gastric cancer patients. LINC01278 was actively expressed in gastric cancer, which exerts a tumor-promoting effect. Silencing LINC01278 suppressed the biological function of tumor cells through spongiform miR-129-5p. LINC01278 has the potential to serve as a novel biomarker, offering new avenues of research for the prognosis and treatment of gastric cancer.

Suppression of the lncRNA TUG1 alleviates neuropathic pain in rats with chronic contractile injury via the miR-29b-3p/HMGB1 axis.

The present research focused on the function of lncRNA taurine upregulated 1 (TUG1) in a rat neuropathic pain (NP) model constructed by chronic contractile injury (CCI). Construction of the NP rat model was performed by CCI surgery. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were applied to examine the NP behavior. RT-qPCR was established to explore the levels of TUG1, microRNA (miR)-29b-3p, and HMGB1. ELISA was carried out to evaluate the concentrations of interleukin (IL)-6, IL-1β, tumor necrosis factor α (TNF-α), IL-4, and IL-6. The underlying mechanisms of TUG1 were explored by RNA-binding protein immunoprecipitation (RIP) and dual-luciferase reporter (DLR) assay. TUG1 and HMGB1 were statistically elevated in the tissue of CCI rats, while miR-29b-3p was reduced. TUG1 competitively binds to miR-29b-3p to upregulate HMGB1 levels. Suppression of TUG1 persistently decreased PWL and PWT along with increased frequency of paw-lifting, whereas this alleviation was typically rescued by the abrogated miR-29b-3p. Analogously, knockdown of TUG1 inhibited CCI-induced overproduction of IL-6, IL-1β, and TNF-α, and reduction of IL-4 and IL-6, but this inhibition was partially abrogated by the reduction of miR-29b-3p. Suppression TUG1 can alleviate NP hypersensitivity and neuroinflammation in CCI rats by competitively binding miR-29b-3p to weaken HMGB1.

IGF2BP1 facilitates non-small cell lung cancer progression by regulating the KIF2A-mediated Wnt/β-catenin pathway.

Insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) are crucially implicated in the cancer progression. The current study intends to excavate and clarify the mechanisms of the key IGF2BPs in non-small cell lung cancer (NSCLC). The expression of IGF2BPs and kinesin family member 2A (KIF2A) was examined using immunohistochemistry, real-time quantitative polymerase chain reaction, and western blot in NSCLC tissue samples or cell lines. NSCLC cell viability was examined using a cell counting kit-8 assay. Cell apoptotic rate was assessed using flow cytometry analysis. The migration and invasion of H1299 cells were subject to scratch test and Transwell assays, respectively. Starbase 2.0 was used to detect the downstream factors of the IGF2BP1 protein. The binding of IGF2BP with KIF2A was detected using RNA binding protein immunoprecipitation assays. Ki-67 immunohistochemistry assay and TUNEL assays were applied for the evaluation of proliferation and apoptosis in vivo, respectively. IGF2BP1 was upregulated in NSCLC tissue samples and cells. Functionally, IGF2BP1 overexpression promoted the proliferative ability, migration, and invasiveness of H1299 cells, while inhibiting cell apoptosis in vitro. In vivo studies revealed that overexpression of IGF2BP1 promoted tumor growth of NSCLC. Mechanistically, IGF2BP1 was involved in KIF2A mRNA stabilization. KIF2A exerted the same functions as IGF2BP1 via the Wnt/β-catenin signaling. In conclusion, IGF2BP1 enhances NSCLC malignant progression by stabilizing KIF2A to modulate the Wnt/β-catenin pathway.

CircGPRC5A enhances colorectal cancer progress by stabilizing PPP1CA and inducing YAP dephosphorylation

BackgroundWith the advancements in bioinformatic technology, an increasing number of circular RNAs (circRNAs) have been discovered and their crucial roles in the development and progression of various malignancies have been confirmed through multiple pathways. However, the specific mechanisms involving protein-binding circRNAs in colorectal cancer (CRC) remain largely unexplored.MethodsDifferential circRNA expression was assessed using a human circRNA microarray in five CRC tissue and paired normal samples. CircGPRC5A expression was then confirmed in the CRC tissues and paired normal samples using qRT-PCR. The biological function of circGPRC5A in CRC were studied in vitro and in vivo. Western blotting, fluorescence in situ hybridization, immunofluorescence, RNA pulldown, mass spectrometry, immunoprecipitation, quantitative phosphoproteomics, and RNA-binding protein immunoprecipitation assays were used to study circGPRC5A.ResultsOur analysis revealed that circGPRC5A expression was higher in CRC tissues compared to normal tissues and was associated with tumor size, tumor stage and lymph node status. CircGPRC5A promoted CRC cell proliferation, migration, and metastasis in vitro and in vivo. CircGPRC5A could stabilize PPP1CA protein by inhibiting the binding between UBA1 and PPP1CA, and increasing YAP dephosphorylation.ConclusionsOur study revealed that circGPRC5A plays an essential function in CRC progression by stabilizing PPP1CA protein and enhancing YAP dephosphorylation. CircGPRC5A could act as a novel and potential target for CRC.

HnRNPA2B1 promotes the occurrence and progression of hepatocellular carcinoma by downregulating PCK1 mRNA via a m6A RNA methylation manner

BackgroundN6-methyladenosine (m6A) is the most prevalent RNA modification. Although hnRNPA2B1, as a reader of m6A modification, has been reported to promote tumorigenesis in a few types of tumors, its role in hepatocellular carcinoma (HCC) and the underlying molecular mechanism remains unclear.MethodsMultiple public databases were used to analyze the expression of hnRNPA2B1 in HCC and its correlation with survival prognosis. We employed a CRISPR-Cas9 sgRNA editing strategy to knockout hnRNPA2B1 expression in HCC cells. The biological function of hnRNPA2B1 in vitro in HCC cells was measured by CCK8, colony formation, migration, and invasion assay. The tumorigenic function of hnRNPA2B1 in vivo was determined by a subcutaneous tumor formation experiment and a HCC mouse model via tail injection of several plasmids into the mouse within 5s-7s. RNA binding protein immunoprecipitation (RIP) experiment using hnRNPA2B1 was performed to test the target genes of hnRNPA2B1 and methylated RNA immunoprecipitation (MeRIP) assay was performed to explore the m6A methylated mRNA of target genes.ResultshnRNPA2B1 highly expressed in HCC tissues, correlated with high grades and poor prognosis. Its knockout reduced HCC cell proliferation, migration, and invasion in vitro, while overexpression promoted these processes. hnRNPA2B1-knockout cells inhibited tumor formation in graft experiments. In HCC mice, endogenous knockout attenuated hepatocarcinogenesis. RNA-seq showed downregulated gluconeogenesis with high hnRNPA2B1 expression. hnRNPA2B1 negatively correlated with PCK1, a key enzyme. RIP assay revealed hnRNPA2B1 binding to PCK1 mRNA. hnRNPA2B1 knockout increased m6A-methylation of PCK1 mRNA. Interestingly, PCK1 knockout partially counteracted tumor inhibition by hnRNPA2B1 knockout in mice.ConclusionOur study indicated that hnRNPA2B1 is highly expressed in HCC and correlated with a poor prognosis. hnRNPA2B1 promotes the tumorigenesis and progression of HCC both in vitro and in vivo. Moreover, hnRNPA2B1 downregulates the expression of PCK1 mRNA via a m6A methylation manner. More importantly, the ability of hnRNPA2B1 to induce tumorigenesis and progression in HCC is dependent on its ability to decrease the expression of PCK1. Therefore, this study suggested that hnRNPA2B1 might be a diagnostic marker of poor prognosis of HCC and a potential therapeutic target for HCC patients.

Alkylation Repair Homolog 5 Regulates N(6)-methyladenosine (m6A) Methylation of Mitsugumin 53 to Attenuate Myocardial Infarction by Inhibiting Apoptosis and Oxidative Stress.

N(6)-methyladenosine (m6A) methylation modification is involved in the progression of myocardial infarction (MI). In this study, we investigated the effects of demethylase alkylation repair homolog 5 (ALKBH5) on cell apoptosis and oxidative stress in MI. The ischemia/reperfusion (I/R) injury mouse model and hypoxia/reoxygenation (H/R) cell model were established. The levels of ALKBH5 and mitsugumin 53 (MG53) were measured by quantitative real-time polymerase chain reaction, immunohistochemical, and immunofluorescence analysis. Apoptosis was evaluated by TUNEL assay, flow cytometry, and western blot. Oxidative stress was assessed by antioxidant index kits. Methylation was analyzed by RNA binding protein immunoprecipitation (RIP), MeRIP, and dual-luciferase reporter assay. We observed that ALKBH5 and MG53 were highly expressed in MI. Overexpression of ALKBH5 inhibited H/R-induced cardiomyocyte apoptosis and oxidative stress in vitro, and it inhibited I/R-induced collagen deposition, cardiac function, and apoptosis in vivo. ALKBH5 could bind to MG53, inhibit m6A methylation of MG53, and increase its mRNA stability. Silencing of MG53 counteracted the inhibition of apoptosis and oxidative stress induced by ALKBH5. In conclusion, ALKBH5 suppressed m6A methylation of MG53 and inhibited MG53 degradation to inhibit apoptosis and oxidative stress of cardiomyocytes, thereby attenuating MI. The results provided a theoretical basis that ALKBH5 is a potential target for MI treatment.

Downregulated lncRNA HOTAIR inhibits the proliferation of granulosa cells in endometriosis by upregulating p21.

This study was carried out to elucidate the biological function of HOTAIR in granulosa cells of endometriosis and the underlying mechanism. Granulosa cells were extracted from endometriosis patients and subjects with fallopian tube factor alone who received IVF-ET. Relative levels of HOTAIR and p21 in the extracted granulosa cells were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, HOTAIR level in endometriosis patients in stage I-II or III-IV was determined. Regulatory effects of HOTAIR on the proliferation of KGN cells were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), 5-Ethynyl-2'- deoxyuridine (EdU) and colony formation assay. Flow cytometry was conducted to evaluate the potential influence of HOTAIR on apoptosis of KGN cells. The interaction between HOTAIR and EZH2, SUV12 was detected by RNA binding protein immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP) assay. Finally, the potential role of the HOTAIR/p21 axis in mediating cellular behaviors of KGN cells was explored. HOTAIR was downregulated in granulosa cells extracted from endometriosis patients relative to those with fallopian tube factor alone who received IVF-ET. Knockdown of HOTAIR suppressed the proliferative ability and induced apoptosis of KGN cells. RIP and ChIP assay showed that silence of HOTAIR released EZH2 to suppress the DNA methylation of p21. Knockdown of p21 could reverse the regulatory effect of HOTAIR on the proliferative change of KGN cells. Downregulated HOTAIR suppresses the proliferative ability and induces apoptosis of granulosa cells in endometriosis by upregulating p21.

CircMETTL14(11)S upregulated METTL14 and induced CXCR4 to aggravate endothelial inflammation and atherosclerosis

Endothelial inflammatory response can induce a variety of cardiovascular diseases, including atherosclerosis (AS). As a member of the m6A methyltransferase family, methyltransferase like 14 (METTL14) was reported to propel endothelial inflammation and aggravate AS. In this study, qRT-PCR and western blot analyses were performed to detect the RNA and protein levels of genes. To analyze the cyclic structure and localization of circMETTL14(11)S, agarose gel electrophoresis, subcellular fractionation and FISH assays were conducted. The role of circMETTL14(11)S on endothelial inflammation was exposed by monocyte adhesion assay. Luciferase reporter, chromatin immunoprecipitation (ChIP), pull-down and RNA binding protein immunoprecipitation (RIP) assays were conducted to explore the mechanism of circMETTL14(11)S on endothelial inflammation and AS. We found that circMETTL14(11)S (hsa_circ_0125169) expressed highly in TNF-α-induced endothelial inflammation and positively regulated the expression of METTL14 in human umbilical vein endothelial cells (HUVECs). CircMETTL14(11)S facilitated endothelial inflammation of HUVECs by METTL14. Based on the nuclear location, circMETTL14(11)S was found to activate METTL14 transcription via cooperating with SRY-box transcription factor 2 (SOX2). METTL14 accelerated the m6A methylation and stabilization of C-X-C motif chemokine receptor 4 (CXCR4) mRNA. Further, the facilitation of circMETTL14(11)S/METTL14/CXCR4 on TNF-α-induced endothelial inflammation of HUVECs was verified. Collectively, circMETTL14(11)S/METTL14/CXCR4 axis aggravated endothelial inflammation and AS.

Loss of DDX3X Function Promotes CLL Progression By Facilitating NOTCH1 mRNA Translation

A variety of driver genes are present in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), and those with high mutation frequencies include SF3B1, NOTCH1, and TP53, which are involved in the development of CLL/SLL and are associated with poor prognosis. The biological significance of some driver genes, such as DDX3X, has not yet been elucidated. DDX3X is located on the X chromosome. As one of the members of the DEAD-box helicase family, it is expressed ubiquitously in human tissues and participates in many biological processes. Here, we described the mapping of the DDX3X abnormalities and demonstrated its clinical significance in CLL/SLL. We also explored the effect and mechanism of DDX3X dysregulation on tumorigenesis and development of CLL by in vitro experiments. To examine the mutational landscape of DDX3X, we performed next-generation sequencing on 402 CLL/SLL patients, and 17 patients (4.2%) had DDX3X mutations. Six patients were frameshift mutations and 2 were nonsense mutation, which caused truncation of DDX3X. Among the 7 cases of missense mutations, 4 cases involved the ATP-binding or C-terminal domain, which may affect the function of DDX3X. The remaining two cases are deletion mutation and splicing mutation. Next, we evaluated the clinical significance and prognostic role of DDX3X mutation. The patients with DDX3X mutations are mostly male (94.1% vs 66.5%, P=0.017), and had more adverse prognostic indicators: unmutated IGHV (88.2% vs 48.6%, P=0.002), 17p deletion (42.9% vs 11.4%, P=0.041), and 11q deletion (46.7% vs 15.7%, P=0.006). DDX3X mutation was an independent risk factor for disease specific survival of CLL/SLL in the competing-risk regression. Also, we found all (14/14) the mutated patients and 15.2% (5/33) of the non-mutated patients had low expression of DDX3X in bone marrow samples by immunohistochemistry. Overall, these results indicates that dysregulation of DDX3X are common in CLL/SLL and may contribute to its progression. To delineate the functional consequences of DDX3X loss in CLL, we used CRISPR/cas9 system to construct stable DDX3X knockout (DDX3X-KO) MEC-1 and JVM-3 cells. DDX3X-KO cells showed significantly increased proliferation and reduced spontaneous apoptosis compared to vector control. Additionally, DDX3X-KO cells showed higher rates of cell viability and reduced apoptosis after treatment of ibrutinib or venetoclax. We further found that DDX3X-KO cells had higher expression level of phosphorylated BTK and MCL-1, which may explain the resistance to ibrutinib and venetoclax, respectively. Moreover, DDX3X-KO cells showed increased expression level of CXCR4 and enhanced chemotaxis in response to CXCL-12. In summary, loss of DDX3X function promotes progression of CLL by mediating more invasive biological behaviors of CLL cells. To evaluate the mechanisms by which DDX3X deletion affects CLL, we detected key signaling pathways in the tumorigenesis and development of CLL in DDX3X-KO and control cells. We found increased expression of NOTCH1 and its downstream target proteins in DDX3X-KO cells (Figure 1), indicating that loss of DDX3X activated the NOTCH1 pathway. Considering that DDX3X is an RNA-binding protein and mainly regulates translation, we speculated that DDX3X may regulate the translation of NOTCH1. We performed RNA binding protein immunoprecipitation assay and found that DDX3X directly interacted with NOTCH1 mRNA (Figure 2A, B). Dual-luciferase reporter assay showed enhanced activity of NOTCH1 5‘ UTR in DDX3X-KO cells (Figure 2C), further suggesting that DDX3X may regulate translation of NOTCH1 mRNA through binding to the 5‘ UTR. Finally, polysome profiling demonstrated that DDX3X knockout resulted in increased NOTCH1 mRNAs in polysome fractions (Figure 2D), confirming that loss of DDX3X function facilitates NOTCH1 mRNA translation. In conclusion, DDX3X abnormalities are common in CLL and associated with poor prognosis. DDX3X dysregulation is involved in the progression of CLL by facilitating NOTCH1 mRNA translation through binding to the 5' UTR.

Targeting G3BP2 Sensitizes Acute Myeloid Leukemia to Venetoclax Treatment Via ELF1-MCL1 Axis

Venetoclax-based combination therapies have emerged as promising treatments for elderly acute myeloid leukemia (AML) patients. However, venetoclax resistance renders the treatment ineffective, mainly due to the upregulation of MCL1. Thus, new venetoclax-based combination therapy is under development to circumvent venetoclax resistance. The Ras-GTPase-activating protein SH3-domain binding protein 2 (G3BP2) is upgraded in several solid tumors. Targeting G3BP2 re-sensitized tumor cells to chemotherapy. To date, whether targeting G3BP2 sensitizes venetoclax treatment in AML and its role in leukemogenesis has not been defined. To this end, the primary AML cells and AML cells were collected and cultured with compound C108 (G3BP2 inhibitor) and venetoclax alone or both. The result showed that co-culture with compound C108 and venetoclax decreased the cell viability in primary AML and AML cells, indicating the potent synergy of G3BP2 inhibitor and venetoclax. The Quantitative real-time PCR (RT-qPCR) showed that G3BP2 was overexpressed in AML patients and was associated with poor prognosis. Knockdown (KD) of G3BP2 decreased proliferation and induced apoptosis in AML cells. In addition, G3BP2 inhibition impeded the tumorigenicity in the subcutaneous AML xenograft tumor model. These data demonstrated that G3BP2 sensitized AML to venetoclax treatment and is critical in AML development. The RNA-seq analysis was performed to investigate the role of G3BP2 in venetoclax resistance and leukemogenesis. The results showed that differentially expressed genes (DEG) were enriched in apoptotic processes when G3BP2 were downregulated. The immunoblotting results indicated that G3BP2 knockdown decreased the expression of MCL1 and BCL2, and vice versa. Moreover, we found that the transcription factor ELF1 was positively correlated with G3BP2 by the immunoblotting and RNA-seq analysis. The result was further validated by the Pearson correlation analysis based on the online database. The expression of ELF1 was decreased when G3BP2 were downregulated. Thus, we concluded that G3BP2 is an upstream gene of ELF1 and MCL1. To further investigate the relationship among G3BP2, ELF1, and MCL1, the RNA binding protein immunoprecipitation assay (RIP) and mRNA stability analysis was performed. The results suggested that G3BP2 interacted with the ELF1 mRNA to enhance its stability. We employed bioinformatics analysis via the JASPAR database and RPISeq tools and found ELF1 may facilitate the transcription of MCL1, which was validated in the following study. The expression of MCL1 was restored in the G3BP2 KD cells when ELF1 was overexpressed. We subsequently performed CHIP-qPCR, dual-luciferase reporter gene assays with truncated MCL1 promoter fragments. A 576bp of the binding region upstream of MCL1 promoter was defined, indicating that ELF1 regulated MCL1 expression by modulating its transcription activity. Taken together, our data suggested that targeting G3BP2 decreases MCL1 expression by disturbing ELF1 mRNA stability, thus increasing the sensitivity to venetoclax treatment. We finally established the intra-venous tumorigenesis xenograft model to verify the role of G3BP2 downregulation in venetoclax-treated leukemia mice. The NCG immunodeficient mice were transplanted with U937 cells and administrated with venetoclax daily from 7-21 days post transplantation (Figure 1A). The data showed that G3BP2 KD in combination with venetoclax reduced clinical scores and prolonged the overall survival of AML mice (combination vs. venetoclax: p<0.01; combination vs. G3BP2 KD: p<0.05, Figure 1B). The reduced leukemic burden was observed when targeting G3BP2 in combination with venetoclax. Meanwhile, we verified the decreased expression of ELF1 and MCL1 in mice targeting G3BP2 in combination with venetoclax by immunofluorescence (IF) and flow cytometry analysis. These data supported the synergic effect of G3BP2 inhibition and venetoclax treatment in AML. Our findings emphasize the critical role of G3BP2 in conferring resistance to venetoclax in AML by stabilizing ELF1 mRNA to regulate MCL1 expression. Targeting G3BP2 represents an appealing therapeutic strategy for patients with venetoclax-resistant AML.

IGF2BP2‑dependent STIM1 inhibition protects against LPS‑induced pneumonia invitro by alleviating endoplasmic reticulum stress and the inflammatory response.

Pneumonia is a disease caused by inflammation and has high morbidity and mortality rates. Stromal interaction molecule 1 (STIM1) is involved in the regulation of inflammatory processes. However, to the best of the authors' knowledge, the role of STIM1 in pneumonia has not yet been reported. In the present study, lipopolysaccharide (LPS) was administered to A549 cells to construct a cell damage model. The expression of STIM1 in the model cells was detected by western blotting and reverse transcription-quantitative PCR. Then, STIM1 expression was inhibited and cell survival was detected by Cell Counting Kit-8 and flow cytometry. The expression of inflammatory factors was detected by enzyme-linked immunosorbent assay and endoplasmic reticulum stress (ERS)-related proteins were detected by immunofluorescence and western blotting. Subsequently, the relationship between insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) and STIM1 was verified by RNA-binding protein immunoprecipitation assay and actinomycin D treatment. Finally, the regulatory mechanism of IGF2BP2 and STIM1 in LPS-induced A549 cells was further investigated. The results of the present study demonstrated that STIM1 expression was increased in LPS-induced A549 cells and that STIM1 knockdown inhibited LPS-induced A549 cell apoptosis and alleviated LPS-induced A549 cell inflammation and ERS. In addition, IGF2BP2 enhanced the stability of STIM1 mRNA and knockdown of IGF2BP2-regulated STIM1 expression alleviated LPS-induced ERS and inflammatory responses in A549 cells. In conclusion, knockdown of IGF2BP2-regulated STIM1 improved cell damage in the LPS-induced pneumonia cell model by alleviating ERS and the inflammatory response.

Circular RNA PTP4A2 regulates microglial polarization through STAT3 to promote neuroinflammation in ischemic stroke.

Microglial polarization plays a critical role in neuroinflammation and may be a potential therapeutic target for ischemic stroke. This study was to explore the role and underlying molecular mechanism of Circular RNA PTP4A2 (circPTP4A2) in microglial polarization after ischemic stroke. C57BL/6J mice underwent transient middle cerebral artery occlusion (tMCAO), while primary mouse microglia and BV2 microglial cells experienced oxygen glucose deprivation/reperfusion (OGD/R) to mimic ischemic conditions. CircPTP4A2 shRNA lentivirus and Colivelin were used to knock down circPTP4A2 and upregulate signal transducer and activator of transcription 3 (STAT3) phosphorylation, respectively. Microglial polarization was assessed using immunofluorescence staining and Western blot. RNA pull-down and RNA binding protein immunoprecipitation (RIP) were applied to detect the binding between circPTP4A2 and STAT3. The levels of circPTP4A2 were significantly increased in plasma and peri-infarct cortex in tMCAO mice. CircPTP4A2 knockdown reduced infarct volume, increased cortical cerebral blood flow (CBF), and attenuated neurological deficits. It also decreased pro-inflammatory factors levels in peri-infarct cortex and plasma, and increased anti-inflammatory factors concentrations 24 h post-stroke. In addition, circPTP4A2 knockdown suppressed M1 microglial polarization and promoted M2 microglial polarization in both tMCAO mice and OGD/R-induced BV2 microglial cells. Moreover, circPTP4A2 knockdown inhibited the phosphorylation of STAT3 induced by oxygen-glucose deprivation. In contrast, increased phosphorylation of STAT3 partly counteracted the effects of circPTP4A2 knockdown. RNA pull-down and RIP assays further certified the binding between circPTP4A2 and STAT3. These results revealed regulatory mechanisms of circPTP4A2 that stimulated neuroinflammation by driving STAT3-dependent microglial polarization in ischemic brain injury. CircPTP4A2 knockdown reduced cerebral ischemic injury and promoted microglial M2 polarization, which could be a novel therapeutic target for ischemic stroke.

Circ-Slain2 Alleviates Cartilage Degradation and Inflammation of TMJOA

Temporomandibular joint osteoarthritis (TMJOA) is a degenerative disease with the cessation of matrix anabolism and aggravation of inflammation, which results in severe pain and impaired joint function. However, the mechanisms are not well understood. Circular RNAs (circRNAs) are reported to have various biological functions and participate in the development, diagnosis, prognosis, and treatment of different diseases. This study aimed to investigate the roles and mechanisms of circ-slain2 in TMJOA. We first established TMJOA mouse models and found circ-slain2 was lowly expressed in the cartilage of TMJOA through sequencing data. We observed that circ-slain2 is predominantly localized in the cytoplasm and downregulated in mouse condylar chondrocytes (mCCs) treated with tumor necrosis factor α (TNFα) and interferon γ (IFNγ). Micro–computed tomography and histological examination showed that intra-articular injection of circ-slain2 overexpressing adeno-associated virus could alleviate cartilage catabolism and synovial inflammation to relieve TMJOA in vivo. In addition, elevated circ-slain2 also showed anticatabolic and anti-inflammatory effects on IFNγ- and TNFα-stimulated mouse condylar chondrocytes (mCCs). Functional enrichment analysis indicated that protein processing in endoplasmic reticulum (ER) was associated with TMJOA, and further functional experiments confirmed that circ-slain2 could suppress ER stress in OA mCCs. RNA binding protein immunoprecipitation assay revealed an overt interaction between activating transcription factor 6 (ATF6) and circ-slain2. Inhibition of the expression of both ATF6 and circ-slain2 resulted in dilation of the ER and enhanced the expression of ER stress markers, whose ER stress level was higher than inhibition of ATF6 but lower than knockdown of circ-slain2 expression. Collectively, our research demonstrated that circ-slain2 could regulate ATF6 to relieve ER stress, reducing temporomandibular joint cartilage degradation and synovial inflammation. These findings provide prospects for developing novel osteoarthritis therapies based on circ-slain2 by focusing on reducing the inflammation of synovium and the imbalance between matrix synthesis and degradation.

A1BG-AS1 promotes the biological functions of osteosarcoma cells via regulating the microRNA-148a-3p/USP22 axis and stabilizing the expression of SIRT1 through deubiquitinase function

ABSTRACT Background The study aims to explore the role of A1BG antisense RNA 1 (A1BG-AS1), microRNA (miR)-148a-3p and ubiquitin-specific protease 22 (USP22) on osteosarcoma (OS) cell growth. Research design & methods A1BG-AS1, miR-148a-3p, USP22, and silent information regulator 2 homolog 1 (SIRT1) levels in OS tissues and cells were determined. The effects of A1BG-AS1, miR-148a-3p, and USP22 on the biological functions of OS cells were examined by functional assays. In vivo assay was conducted to observe the effect of A1BG-AS1 on OS growth in vitro. The relationship of A1BG-AS1, miR-148a-3p, and USP22 was analyzed by bioinformatics analysis, RNA-fluorescence in situ hybridization, luciferase activity, and RNA binding protein immunoprecipitation assays. The relation between USP22 and SIRT1 was evaluated by immunoprecipitation. Results A1BG-AS1 and USP22 were highly expressed, and miR-148a-3p was lowly expressed in OS tissues and cells. Down-regulation of A1BG-AS1 and USP22 or up-regulation of miR-148a-3p impaired the malignant behaviors of OS cells. A1BG-AS1 sponged miR-148a-3p, and miR-148a-3p targeted USP22, thereby inhibiting USP22 expression. Up-regulating USP22 reversed the A1BG-AS1 suppression-induced phenotypic inhibition of OS cells. USP22 affected the biological functions of OS cells by deubiquitinating SIRT1. Conclusion A1BG-AS1 facilitates the biological functions of OS cells via mediating the miR-148a-3p/USP22 axis.

LncRNA-LINC00472 suppresses the malignant progression of non-small cell lung cancer via modulation of the miRNA-1275/Homeobox A2 axis.

Long non-coding RNAs (lncRNAs) are increasingly observed as regulatory factors for the initiation and progression of varying kinds of cancers. However, studies on lncRNAs in non-small cell lung cancer (NSCLC) progression are currently lacking. We intended to determine the role of lncRNA LINC00472 and its downstream regulatory mechanism in NSCLC, thus providing novel ideas for targeted therapies for NSCLC. The target signaling axis comprising the lncRNA/microRNA/mRNA was identified through bioinformatics analysis. Subcellular localization of LINC00472 was assessed with fluorescence in situ hybridization (FISH). Cellular function experiments were conducted to examine the proliferation, migration, invasion, and apoptosis of NSCLC cells, and dual-luciferase and RNA binding protein immunoprecipitation assays were performed to validate the binding relationship. Quantitative real-time polymerase chain reaction (qPCR) and western blot were utilized to assess the expression levels of the investigated gene and protein, respectively. The LINC00472 expression was markedly decreased in NSCLC tissues and cells. The FISH, combined with nuclear-cytoplasm separation assay, demonstrated that LINC00472 was mainly located in the cytoplasm. The overexpression of LINC00472 restrained proliferation and metastasis of NSCLC in vitro. The LINC00472 could target and repress miR-1275 level, and overexpression of LINC00472 reduced the miR-1275-dependent malignant cell phenotype in NSCLC. Further study revealed that HOXA2 was a downstream target of miR-1275 and was negatively modulated by miR-1275. Rescue assays exhibited that the overexpression of miR-1275 or inhibition of HOXA2 reversed the impact of LINC00472 overexpression on the malignant progression of NSCLC cells. The LINC00472 repressed the epithelial-mesenchymal transition (EMT) of NSCLC cells through miR-1275/HOXA2. The LINC00472 functioned as a competing endogenous RNA to modulate HOXA2 level by sponging miR-1275 in NSCLC. Simultaneously, the LINC00472/miR-1275/HOXA2 axis may be a possible therapeutic target and biomarker for NSCLC.

Circ_0021350 plays an oncogene role by regulating miR-1207-3p/PIK3R3 in glioblastoma

BackgroundGlioblastoma (GBM) is the most malignant glioma, with poor survival rates and prognosis. Several studies have reported the abnormal expression of circular RNAs (circRNAs) and their functions in the malignant biological behavior of GBM. However, such research is still in the preliminary stages, and further study is needed to confirm the therapeutic potential of circRNAs in GBM.MethodsRNA-seq was performed using four tumor tissues from patients with GBM and their adjacent non-tumor brain tissues to screen differentially expressed circRNAs. Fluorescence in situ hybridization assay was used to examine the location of circ_0021350 in glioma cells. In addition, a series of biological function assays were employed to verify the oncogenic role of circ_0021350 in GBM. Quantitative reverse transcription PCR was used to examine circular, micro- (miRNA), and messenger RNA (mRNA) levels. Furthermore, dual-luciferase reporter, RNA pull-down, and RNA binding protein immunoprecipitation assays were applied to verify the interaction between circ_0021350 and its downstream effectors.ResultsCirc_0021350 was significantly elevated in GBM tissues and glioma cells. Overexpression of circ_0021350 promoted glioma cell proliferation and metastatic ability; silencing of circ_0021350 had the opposite effect. Mechanistic analysis revealed that circ_0021350 sponged miR-1207-3p to regulate PIK3R3, whose overexpression reversed the reduction in the malignant biological behavior of glioma cells caused by silencing circ_0021350.ConclusionOur findings suggest that circ_0021350 is an oncogenic circRNA in GBM, and the circ_0021350/miR-1207-3p/PIK3R3 axis may serve as a potential therapeutic target in GBM treatment.

A novel LINC00478 serves as a tumor suppressor in endometrial carcinoma progression.

Long noncoding RNAs (lncRNAs) are involved in the pathogenesis and progression of various cancers, but their roles in endometrial cancer (EC) are largely unknown. The expressions of LINC00478 and PTBP1 in EC tissues were determined by RT-qPCR. Cell counting kit-8, flow cytometry and Transwell assays were executed for detecting the roles of LINC00478 in EC cells proliferation, migration and invasion. The mouse-xenograft models were established by subcutaneous injection in vivo. The interaction between LINC00478 and PTBP1 was confirmed by RNA pull-down assay and RNA-binding protein immunoprecipitation assay. LINC00478 was significantly down-regulated in EC tissues while compared to that in their paracancerous samples, and a higher expression level of LINC00478 was negatively correlated with clinical progress of EC patients. Functional experiments in vivo and in vitro revealed that LINC00478 overexpression could dramatically retard the proliferation of EC cells, decrease the rate of colony formation, suppress the migration and invasion abilities of EC cells in vitro and inhibit tumor growth in vivo. Mechanistically, LINC00478 regulated the expression of PTBP1, a key factor in the Warburg effect, and affected the metabolic process of EC cells. LINC00478 acts as a tumor suppressor in EC by negatively controlling PTBP1 expression and influencing the Warburg effect, providing a potential biomarker and therapeutic target for patients with EC.

Circular RNA circSP5 promotes liver metastasis of colorectal cancer via SP5-mediated BAMBI transcription.

Liver metastasis of colorectal cancer (CRC) is a major cause of cancer morbidity and mortality. Circular RNAs (circRNAs) have been widely reported to be implicated in cancer metastasis. This study aims to investigate the effect of circSP5 (has_circ_0057010) on liver metastasis of CRC. Quantitative real-time PCR (RT-qPCR) analysis was performed to detect gene expression. The level of proteins was measured by western blot. The migration and invasion of CRC cells were assessed by wound healing assay and transwell assay. In vivo assays were performed after the construction of the CRC xenograft model and CRC model with liver metastasis. Mechanism analyses were performed via RNA-binding protein immunoprecipitation (RIP), RNA pulldown, luciferase reporter, chromatin immunoprecipitation (ChIP), and DNA pulldown assays. We found that circSP5 is significantly overexpressed in CRC with liver metastasis and its depletion suppresses the progression of CRC with liver metastasis in vitro and in vivo. Moreover, circSP5 enhances the expression of Sp5 transcription factor (SP5) via competitively sponging microRNA (miR)-1249-3p and could regulate BMP and activin membrane-bound inhibitor (BAMBI) via transcriptional activation. CircSP5 promotes the migration, invasion, and epithelial-mesenchymal transition (EMT) of CRC cells via BAMBI. In sum, circSP5 promotes liver metastasis of CRC by up-regulating SP5-mediated BAMBI transcription.

Long noncoding RNA BBOX1-AS1 increased radiotherapy sensitivity in colorectal cancer by stabilizing and activating PFK1

PurposeOur study explored the effect of long noncoding RNA BBOX1-AS1 on colorectal cancer (CRC) radiosensitivity in vivo and in vitro. MethodsDifferentially expressed lncRNAs in CRC were screened using a bioinformatics database and an online prediction website. The expression of BBOX1-AS1 in tissue samples was analyzed via real-time quantitative PCR (RT-qPCR). Subcellular localization of BBOX1-AS1 in CRC cells was analyzed using fluorescence in situ hybridization (FISH). The correlation between BBOX1-AS1 and PFK1 expression levels in CRC tissues was analyzed via Pearson's correlation coefficient. The effect of BBOX1-AS1 on PFK1 stability was investigated using RNA and protein stability testing. RNA Binding Protein Immunoprecipitation (RIP) and RNA pull-down assays were used to confirm the binding of BBOX1-AS1 to PFK1. ResultsBBOX1-AS1 was highly expressed in CRC and associated with poor prognosis. Similarly, it was highly expressed in CRC tissues and CRC cell lines. In addition, BBOX1-AS1 promoted the proliferation, invasion, migration, and glycolysis of CRC cells and inhibited apoptosis. RIP and RNA pull-down experiments confirmed that BBOX1-AS1 bound to PFK1. RNA stability and protein stability experiments showed that BBOX1-AS1 affected the stability of PFK1 mRNA and protein. Furthermore, we confirmed that BBOX1-AS1 increased radiation resistance through the regulation of PFK1 expression. ConclusionsBBOX1-AS1 promoted the proliferation, invasion, migration, and glycolysis of CRC cells through stabilization of the expression of PFK1. BBOX1-AS1 also inhibited CRC cell apoptosis and increased radiotherapy resistance in CRC cells.