RNA Binding Protein Immunoprecipitation Assay Research Articles

Hes1 is associated with long non-coding RNAs in colorectal cancer.

Long noncoding RNAs (lncRNAs) play important roles in the development and pathophysiology of colorectal cancer (CRC). Our previous study showed that Hes1 was involved in the self-renewal and tumorigenicity of stem-like cancer cells in CRC. ArrayStar Human LncRNA/mRNA Expression Microarray Version 3.0 was used to detect lncRNA expression in CRC tissues compared with their matched non-tumoral tissues. RNA-binding protein immunoprecipitation and sequencing (RIP-seq) assay was used to detect lncRNAs binding to Hes1. Real-time qPCR was used to detect expression of specific lncRNAs in CRC tissues. We found significantly up-regulated as well as down-regulated lncRNAs in CRC tissues compared with their matched non-tumoral tissues. We also screened a number of lncRNAs interacting with Hes1 in CRC cells. Interestingly, we found several lncRNAs binding to Hes1 (such as, GNAS-AS1, RP11-89K10.1, and RP11-465L10.10) were up-regulated in CRC tissues showed by the tissue microarray. Next, we confirmed that Hes1 directly interacted with these lncRNAs using RIP-qPCR and RNA pulldown assay. Finally, we verified the expression of these lncRNAs in 32 CRC samples as well as the adjacent non-tumoral tissues using real-time qPCR. Based on these, we speculate that Hes1 interacts with one or more lncRNAs which contribute to the development and progression of CRC.

<p>Long noncoding RNA ZFAS1 promotes progression of papillary thyroid carcinoma by sponging miR-590-3p and upregulating HMGA2 expression</p>

BackgroundThyroid cancer is the most common endocrine malignancy, papillary thyroid carcinoma (PTC) is the main form of thyroid cancer. The long non-coding RNA (lncRNA) zinc finger antisense 1 (ZFAS1) is highly expressed in various cancer tissues and it has been shown to function as a tumor promoter in various cellular processes. However, the role of ZFAS1 in PTC is not well understood currently. Thus, this study aimed to explore the potential roles of ZFAS1 in the development and progression of PTC.Material and methodsPTC tissues (n=80) and noncancerous tissues were collected. Gain- and loss-of-function assays were performed to determine the effect of ZFAS1 on proliferation in K-1 and TPC-1 cells. The ZFAS1/mir-590-3P/HMGA2 aixs were analysed in PTC cell lines.ResultsWe found that the expression of ZFAS1 was increased in PTC tissues and four PTC cell lines (B-CPAP, IHH-4, TPC-1, and K-1). The gain- and loss-of-function assays showed that overexpressing ZFAS1 promoted cell proliferation and inhibited cell apoptosis in PTC cells in vitro. We demonstrated that knockdown of ZFAS1 inhibits tumor growth and upregulation of ZFAS1 promotes tumor growth in vivo. Bioinformatics analysis revealed that miR-590-3p targeted the 3ʹ-UTR of ZFAS1. The double luciferase reporter and RNA-binding protein immunoprecipitation assay demonstrated that miR-590-3p is a target of ZFAS1. Rescue experiments confirmed that miR-590-3p could reverse the effect of ZFAS1 on PTC cells. Moreover, we identified high mobility group AT-hook 2 (HMGA2) to be a downstream target of miR-590-3p and ZFAS1 which activates HMGA2 expression by sponging to miR-590-3p.ConclusionHigh ZFAS1 expression level was associated with the progression of PTC, and ZFAS1 contributed to PTC progression via miR-590-3p/HMGA2 regulatory aixs. Therefore, ZFAS1 might be a potential therapeutic target for PTC intervention.

MiR-204 suppresses cell proliferation and promotes apoptosis in ovarian granulosa cells via targeting TPT1 in polycystic ovary syndrome.

MicroRNA (miR)-204 is known to be associated with several different diseases. Polycystic ovary syndrome (PCOS) has the highest incidence rate among the endocrine disorders in females between the ages of 18 and 44. We aimed to illustrate the miR-204 function in PCOS. MiR-204 expression levels in tissue and cell were examined through RT-qPCR. Colony formation assay and MTT assay were applied to detect the cell viability. Flow cytometry was employed to examine the apoptosis and cell cycle in cells. RNA binding protein immunoprecipitation assay and luciferase reporter assay were provided to demonstrate the direct interaction between translationally controlled tumor protein (TPT1) and miR-204. The expression of miR-204 was declined in KGN cells and ovarian cortex tissues of PCOS patients. MiR-204 enhanced the colony formation capacity and cell proliferation in KGN cells. Cell cycle and apoptosis were also influenced by miR-204. Since miR-204 has direct interaction with TPT1, TPT1 overexpression suppressed the miR-204-induced apoptosis and cell cycle alteration in KGN cells. MiR-204 inhibits the cell viability and induces apoptosis and cell cycle arrest by directly interacting with TPT1, indicating a role of miR-204 to be a potential target in the PCOS patients.

LncRNA GAS5 regulates myocardial infarction by targeting the miR‐525‐5p/CALM2 axis

Long noncoding RNAs (lncRNAs) play critical roles in the pathogenesis of cardiovascular diseases, especially in myocardial infarction (MI). However, the underlying molecular mechanism of how lncRNA involves and affect MI still remains unclear. This study aimed to investigate the expression of lncRNA growth arrest-specific transcript 5 (GAS5) and its effects on myocardial cells' proliferation, cell cycle, and apoptosis. The possible mechanisms involved in GAS5, calmodulin 2 (CALM2), and microRNA (miR)-525-5p were also explored. The messenger RNA (mRNA) level of CALM2, GAS5, and miR-525-5p in postmyocardial infarction (MI) and normal cells were examined by quantitative real-time polymerase chain reaction (RT-qPCR). Western blot analysis assay was conducted to detect the protein levels of CALM2. The changes of cell cycle/apoptosis and cell viability of post-MI myocardial cells (PMMC) were determined by flow cytometry analysis and MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay after knockdown of GAS5 or CALM2, respectively. Dual luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) assay were performed to verify the targeting relationship between miR-525-5p and GAS5, CALM2 in myocardial. Hypoxic preconditioning was performed in normal cells, which constructed a simulated MI environment, and the effect of GAS5 on cardiomyocyte apoptosis was detected. Our data showed that the expression of GAS5 and CALM2 in PMMC was significantly upregulated, while the expression of miR-525-5p was downregulated. Overexpression of GAS5 and CALM2 profoundly promoted the apoptosis of myocardial cell. However, the proliferation of myocardial cell was inhibited by the upregulation of GAS5 and CALM2. Moreover, GAS5 was proved to be the target of miR-525-5p and GAS5 downregulated the expression of miR-525-5p and CALM2. In addition, lncRNA GAS5 promotes MI, while CALM2 induced MI can be reversed by miR-525-5p. These data suggested that lncRNA GAS5 promoted the development and progression of MI via targeting of the miR-525-5p/CALM2 axis and it has the potential to be explored as a therapeutic target for the treatment of MI in the future.

Exercise Reduces Insulin Resistance in Type 2 Diabetes Mellitus via Mediating the lncRNA MALAT1/MicroRNA-382-3p/Resistin Axis

Insulin resistance (IR) is the primary pathological mechanism underlying type 2 diabetes mellitus (T2DM). Here, the study aimed to ascertain whether and how exercise mediates IR in T2DM. An in vivo mouse model of high-fat diet-induced IR and an in vitro high-glucose-induced IR model were constructed. High long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) expression was detected in T2MD and was positively correlated with HOMA-IR and resistin levels. Then, short hairpin RNA targeting MALAT1 (sh-MALAT1) or pcDNA-MALAT1 was delivered into human umbilical vein endothelial cells (HUVECs) to knock down or upregulate its expression, respectively. Silencing of MALAT1 resulted in reduced levels of resistin, Ang II, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), endothelin-1 (ET-1), and p-insulin receptor substrate-1 (p-IRS)/ISR-1, and decreased cell migration, as well as enhanced glucose uptake and levels of nitric oxide (NO) and p-Akt/Akt. In the IR mouse model, exercise was observed to downregulate MALAT1 to reduce resistin, whereby exercise reduced homeostatic model assessment-insulin resistance (HOMA-IR). Besides, exercise also elevated microRNA-382-3p (miR-382-3p) expression in the serum of IR mice. Dual-luciferase reporter and RNA binding protein immunoprecipitation (RIP) assays identified that MALAT1 could bind to miR-382-3p to upregulate resistin. Collectively, the key observations of the study provide evidence that inhibition of MALAT1 elevates miR-382-3p to repress resistin, which consequently underlies the mechanism of exercise protecting against IR, highlighting a direction for T2DM therapy development.

LINC01116 promotes proliferation, invasion and migration of osteosarcoma cells by silencing p53 and EZH2.

The aim of this study was to elucidate the expression pattern and potential function of LINC01116 in regulating the progression of osteosarcoma. Expression levels of LINC01116 in osteosarcoma tissues (n=52) and adjacent normal tissues (n=52) were detected by quantitative Real-time polymerase chain reaction (qRT-PCR). Survival analysis and univariate analysis were performed in osteosarcoma patients based on the relative expression levels of LINC01116 and clinical data. Overexpression or silence of LINC01116 in osteosarcoma cells was achieved by transfection of plasmid complementary deoxyribonucleic acid (pcDNA)-LINC01116 or si-LINC01116, respectively. Subsequently, the regulatory effects of LINC01116 on cellular behaviors of osteosarcoma cells were examined by cell counting kit-8 (CCK-8), transwell and flow cytometry. Meanwhile, the potential mechanism of LINC01116 in regulating the progression of osteosarcoma was explored by RNA binding protein immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP) and Western blot. Potential target genes in osteosarcoma were searched, and their functions were clarified through a series of rescue experiments. LINC01116 expression in osteosarcoma tissues was significantly higher than adjacent normal tissues. The expression of LINC01116 was negatively correlated with overall survival, whereas positively correlated with tumor size and clinical grade of osteosarcoma patients. Transfection of pcDNA-LINC01116 significantly enhanced proliferative, migratory and invasive abilities of U2OS cells, shortened G0/G1 phase period, and inhibited cell apoptosis. However, transfection of si-LINC01116 in MG63 cells obtained the opposite trends in the above-mentioned cellular behaviors. Furthermore, RIP assay confirmed the binding of enhancer of zeste homolog 2 (EZH2) to LINC01116. Knockdown of LINC01116 significantly up-regulated the expressions of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and p53. Moreover, EZH2 knockdown could reverse the inhibitory effect of LINC01116 on carcinogenesis of osteosarcoma. LINC01116 is highly expressed in osteosarcoma. Up-regulated LINC01116 can promote cell proliferation, invasion and cell cycle progression, while inhibiting the apoptosis of osteosarcoma cells. Furthermore, LINC01116 is involved in the development of osteosarcoma by binding to EZH2 to regulate expressions of PTEN and p53.

LncRNA DLEU2 aggravates the progression of hepatocellular carcinoma through binding to EZH2

To explore whether lncRNA deleted in lymphocytic leukemia 2 (DLEU2) could accelerate the migratory, invasive and proliferative abilities, thus influencing the progression of HCC. DLEU2 level in HCC tissues and adjacent normal tissues was firstly determined. Its level in HCC tissues with different tumor sizes (≤ 5 cm or > 5 cm), different tumor stages (stage I-II or III-IV) and either with vascular invasion or not was determined. Potential influences of DLEU2 on proliferative, migratory and invasive abilities of SMMC7721 and HCLM3 cells were assessed. The interaction between DLUE2 and enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) was evaluated by RNA Binding Protein Immunoprecipitation (RIP) assay. Finally, the effect of DLEU2/EZH2 regulatory loop on proliferative ability of HCC cells was detected. DLEU2 was upregulated in HCC tissues, especially in those larger than 5 cm in tumor size, accompanied with vascular invasion and in worse tumor stage. Knockdown of DLEU2 attenuated proliferative, migratory and invasive abilities of SMMC7721 and HCLM3 cells. RIP assay proved that DLEU2 could interact with EZH2. Knockdown of EZH2 attenuated the inhibited proliferation in HCC cells with DLEU2 knockdown. DLEU2 accelerates the proliferative, migratory and invasive abilities of HCC cells via binding to EZH2, thus aggravating the progression of HCC.

LncRNA HOTAIR targets miR-126-5p to promote the progression of Parkinson's disease through RAB3IP.

Parkinson's disease (PD) is a common neurological disorder characterized by dopaminergic (DA) neuron degeneration and death in the midbrain, and the long noncoding RNA HOTAIR has been shown to affect disease progression in PD. In this study, we aimed to further illustrate the molecular mechanism of HOTAIR in PD. Bioinformatics analysis was utilized to determine the potential downstream targets of HOTAIR in PD. Luciferase assay and the RNA Binding Protein Immunoprecipitation (RIP) assay were used to validate the existence of binding sites between competing endogenous RNAs (ceRNAs). Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting indicated that HOTAIR and RAB3IP increased while miR-126-5p decreased in PD cells and PD mice. Additionally, the CCK-8 assay and flow cytometric analysis indicated that the knockdown of HOTAIR and RAB3IP and the overexpression of miR-126-5p significantly increased cell proliferation and reduced apoptosis in PD cells. Furthermore, the results of in vivo experiments suggested that knockdown of HOTAIR expression increased the number of TH-positive cells and the number of α-synuclein-positive cells decreased while reducing the apoptosis rate among DA neurons. Our study confirmed that HOTAIR promotes PD progression by regulating miR-126-5p and RAB3IP in a ceRNA-dependent manner and further clarified how HOTAIR works in PD.

Downregulated Long Noncoding RNA GAS5 Fails to Function as Decoy of CEBPB, Resulting in Increased GDF15 Expression and Rapid Ovarian Cancer Cell Proliferation.

Introduction: Growth differentiation factor 15 (GDF15), a newly identified member of transforming growth factor (GDF) superfamily, is upregulated in ovarian (OV) cancer. Upregulated GDF15 positively correlates with poor prognosis of OV cancer. Thus, elucidation of the mechanism underlying GDF15 overexpression is important. Method and Results: PROMO and JASPAR prediction software were used to find transcription factors for GDF15 expression. Data from TCGA database were analyzed to find long noncoding RNAs (lncRNAs) that were also abnormally expressed in OV cancer and had associations with GDF15 expression. Transcription factor CEBPB was predicted as an important regulator of GDF15, confirmed by luciferase reporter assay. However, CEBPB expression was not significantly changed in OV cancer. Data from TCGA database showed that lncRNA GAS5 is downregulated in OV cancer and its expression is negatively correlated with GDF15 expression. RPISeq showed high affinity of GAS5 to CEBPB and this was confirmed by RNA-binding protein immunoprecipitation assay. GAS5 overexpression increased its binding to CEBPB and consequently downregulated GDF15. GAS5 overexpression and GDF15 knockdown decreased viability and increased apoptosis of OV cancer cells, but CEBPB overexpression had opposite effects. However, simultaneous GAS5 and CEBPB overexpression or CEBPB overexpression together with GDF15 knockdown had no effect on cell viability and apoptosis. Conclusion: GAS5 functions as decoy of CEBPB, blocking transcription-promoting effect of CEBPB on GDF15.

Circ_0001730 promotes proliferation and invasion via the miR-326/Wnt7B axis in glioma cells

Aim: To study the role of circRNA (circ_0001730) in glioblastoma. Materials & methods: The interaction between circ_0001730 and miR-326 was confirmed by FISH, RNA pull down, RNA-binding protein immunoprecipitation and luciferase reporter assays. Cell proliferation and growth were determined by MTT, EdU and colony formation assays. Cell migration was assessed by the Boyden assay. Results: The levels of circ_0001730 were elevated in glioblastoma cell lines and tissues. circ_0001730 downregulation suppressed migration and proliferation in glioblastoma cells. SP1 bounds to the promoter of circ_0001730 host gene EPHB4 thereby increasing the expression of circ_0001730. circ_0001730 activated the Wnt/β-catenin pathway via the miR-326/Wnt7B axis. Conclusion: circ_000173 promoted growth and invasion in glioblastoma cells via the miR-326/Wnt7B axis.

Long non-coding RNA MEG3 inhibits cervical cancer cell growth by promoting degradation of P-STAT3 protein via ubiquitination

BackgroundMaternally expressed 3 (MEG3) plays an important role in cervical cancer development, but its exact role remains unclear. Here, we explored the specific regulatory mechanism of MEG3 and its downstream proteins in cervical cancer cells.MethodsThe effect of MEG3 on tumor formation ability of cervical cancer cells was determined in nude mice. The direct binding of MEG3 to phosphorylated signal transducer and activator of transcription 3 (P-STAT3) was detected by RNA pull-down and RNA-binding protein immunoprecipitation (RIP) assays. Cycloheximide (CHX)-chase and ubiquitination assays were performed to determine the regulatory effect of MEG3 on P-STAT3 ubiquitination. Clone formation assay and flow cytometry were used to evaluate the effect of the MEG3-STAT3 regulatory axis on cell proliferation and apoptosis.ResultsIn vivo tumor formation experiments showed that MEG3 inhibited the tumor formation ability of cervical cancer cells. RNA pull-down and RIP assays demonstrated that MEG3 bound directly to P-STAT3 protein. CHX-chase and ubiquitination assay results showed that MEG3 promoted P-STAT3 degradation via ubiquitination. Clone formation assay and flow cytometry analysis results revealed that the inhibitory effect of MEG3 on P-STAT3 promoted apoptosis and inhibited proliferation of cervical cancer cells.ConclusionMEG3 binds to P-STAT3 in cervical cancer cells, resulting in P-STAT3 ubiquitination and degradation and apoptosis and inhibition of proliferation of tumor cells. The in-depth elaboration of the MEG3-STAT3 regulatory axis in cervical cancer may clarify the mechanism of action of MEG3 and provide new ideas for cervical cancer treatment.

LncRNA SNHG1 overexpression regulates the proliferation of acute myeloid leukemia cells through miR-488-5p/NUP205 axis.

To elucidate the function of long non-coding RNA (lncRNA) SNHG1 in acute myeloid leukemia (AML) and to explore its biological mechanism. Expression levels of lncRNA SNHG1 and miR-488-5p in AML cell lines pAML and THP-1 were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Proliferative potential and cell cycle progression of pAML and THP-1 cells were detected after SNHG1 knockdown. Potential binding sites of SNHG1 and miR-488-5p were predicted by bioinformatics. Through RNA Binding Protein Immunoprecipitation (RIP) assay and luciferase reporter gene assay, we evaluated the binding condition between SNHG1 and miR-488-5p. MiR-488-5p expression in pAML and THP-1 cells with SNHG1 knockdown was detected by qRT-PCR to further verify their interaction. Subsequently, binding sites of NUP205 and miR-488-5p were predicted. Both mRNA and protein levels of NUP205 in pAML and THP-1 cells were examined. The regulatory effects of overexpressed NUP205 on proliferative potential and cell cycle progression of pAML and THP-1 cells transfected with si-SNHG1 were explored by gain-of-function experiments. LncRNA SNHG1 was highly expressed in pAML and THP-1 cells, while miR-488-5p was lowly expressed. SNHG1 knockdown in pAML and THP-1 cells inhibited proliferative ability and arrested cell cycle in G0/G1 phase. Knockdown of SNHG1 markedly upregulated the miR-488-5p expression in pAML and THP-1 cells. Furthermore, RIP and luciferase reporter gene assay confirmed the binding between SNHG1 and miR-488-5p. NUP205 was highly expressed in pAML and THP-1 cells at both mRNA and protein levels. Moreover, luciferase reporter gene assay indicated that NUP205 could bind to miR-488-5p. More importantly, the overexpression of NUP205 in pAML and THP-1 cells reversed the inhibitory effect of SNHG1 knockdown on proliferative ability and cell cycle progression. LncRNA SNHG1 promotes the development of AML through miR-488-5p/NUP205 axis.

IGF2-AS affects the prognosis and metastasis of gastric adenocarcinoma via acting as a ceRNA of miR-503 to regulate SHOX2.

Disorder of long non-coding RNAs (LncRNAs) is found in various types of cancers and demonstrated to be associated with tumor occurrence and development. Our study found that lncRNA insulin growth factor 2 antisense (IGF2-AS) is up-regulated in gastric adenocarcinoma (GAC) tissues and correlated with poor prognosis in patients with GAC. Cell counting kit-8 (CCK8), colony formation, wound healing and transwell assays revealed that knockdown of IGF2-AS in BGC823 and SGC7901 cells significantly suppressed cell proliferation, migration and invasion. While, overexpression of IGF2-AS in AGS and MGC803 cells exhibited the opposite effects. RNA-FISH and subcellular fractionation assay found that most IGF2-AS was distributed in the cytoplasm, suggesting that IGF2-AS functioned as a potential ceRNA. RNA binding protein immunoprecipitation (RIP) assays further confirmed this assumption. By informatics prediction and luciferase reporter assay, we found that IGF2-AS functioned as an efficient miR-503 sponge and the level of miR-503 showed an inverse correlation with IGF2-AS. Short stature homeobox 2 (SHOX2) is predicted and verified as a target of miR-503. Moreover, IGF2-AS expression exhibited a negative correlation with miR-503 and a positive correlation with IGF2-AS. Subsequent rescue assay revealed that down-regulation of miR-503 or restoration of SHOX2 canceled IGF2-AS depletion-induced depression in proliferation and motility of BGC823 and SGC7901 cells. Meanwhile, up-regulation of miR-503 or down-regulation of SHOX2 decreased IGF2-AS overexpression induced promotion in proliferation and motility of AGS and MGC803 cells. In vivo tumorigenicity assay showed that knockdown of IGF2-AS significantly reduced tumor volume. Taken together, our results demonstrated that IGF2-AS takes important regulatory parts in GAC development by functioning as a ceRNA to regulate SHOX2 via sponging miR-503.

Novel mechanisms for osteogenic differentiation of human aortic valve interstitial cells

ObjectiveAortic valve calcification is common in aging populations without effective pharmacologic interventions. Our previous in vitro data revealed a critical role for long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 as a positive regulator of osteogenic differentiation in aortic valve calcification pathogenesis. The current study sought to determine the mechanism by which metastasis-associated lung adenocarcinoma transcript 1 is regulated in aortic valve calcification. MethodsThe stability assay was used to examine the effect of human antigen R on metastasis-associated lung adenocarcinoma transcript 1 expression. Aortic valves from patients with aortic stenosis and normal controls were subjected to determination of RNA-binding protein human antigen R expression. Mineralized bone matrix formation was assessed by Alizarin Red staining. The interaction between metastasis-associated lung adenocarcinoma transcript 1 and miR-191-3p was confirmed via RNA pull-down, luciferase reporter, and RNA-binding protein immunoprecipitation assays. ResultsIn cultured human aortic valvular interstitial cells, we found human antigen R enhanced metastasis-associated lung adenocarcinoma transcript 1 stability and thus increased its concentration. Moreover, human antigen R was significantly upregulated in human calcific aortic valves and valvular interstitial cells after osteogenic induction. Human antigen R partly relied on metastasis-associated lung adenocarcinoma transcript 1 to positively regulate osteogenic differentiation of valvular interstitial cells. Luciferase reporter assays validated human antigen R as the direct target of miR-191-3p. Metastasis-associated lung adenocarcinoma transcript 1 positively regulated the expression of human antigen R through sponging miR-191-3p. ConclusionsThis study demonstrates the existence of a regulatory loop between metastasis-associated lung adenocarcinoma transcript 1 and human antigen R during osteogenic differentiation of valvular interstitial cells. Our findings provide novel mechanistic insights into a critical role of human antigen R in the aortic valve calcification progression and shed new light on RNA-binding protein-directed diagnostics and therapeutics in aortic valve calcification.

Circular RNA hsa_circ_0001649 inhibits hepatocellular carcinoma progression via multiple miRNAs sponge.

Circular RNA (circRNA) exerts an essential role in tumor development. Hsa_circ_0001649 (circ-0001649) was produced at the SHPRH gene locus containing exon 26-29. This study analyzed the specific mechanism of circ-0001649 in influencing the development of hepatocellular carcinoma (HCC). Relative levels of circ-0001649 in HCC cell lines and tissues were examined by qRT-PCR. The direct binding between circ-0001649 and miR-127-5p/miR-612/miR-4688 were verified through Dual-luciferase reporter gene assay, RNA Binding Protein Immunoprecipitation (RIP) assay and western blot detection. In vitro and in vivo regulatory roles of circ-0001649 in proliferative and migratory abilities of HCC were evaluated by EdU, Transwell and tumourigenicity assay, respectively. Results showed that circ-0001649 was markedly decreased in hepatocellular carcinoma cell lines and tumor tissues. Overexpression of circ-0001649 greatly inhibited proliferation and migration of HCC in vitro and in vivo. More importantly, we confirmed that circ-0001649 regulated cellular behaviors of HCC cells by targeting SHPRH. Furthermore, we determined that circ-0001649 served as a ceRNA to sponge miR-127-5p, miR-612 and miR-4688, thus activating SHPRH. In summary, our study showed that circ-0001649 was lowly expressed in HCC and inhibited HCC progression via multiple miRNAs sponge.

LncRNA H19 Knockdown in Human Amniotic Mesenchymal Stem Cells Suppresses Angiogenesis by Associating with EZH2 and Activating Vasohibin-1.

Human amniotic mesenchymal stem cells (HAMSCs) are promising seed cells with great advantages in promoting angiogenesis. However, the mechanisms underlying angiogenesis facilitated by HAMSCs are still unclear. Long noncoding RNA H19 is involved in many biological processes, such as enhancing angiogenesis and proliferation, invasion, and migration of cancer cells. In this study, we constructed HAMSCs of stable low-expression H19 (HAMSC-shH19) and the scramble control (HAMSC-shNC) using lentiviral vectors, and in a three-dimensional coculture with human umbilical vein endothelial cells (HUVECs) to investigate the effect of H19 knockdown in HAMSCs on angiogenesis. Our results demonstrated that H19 knockdown significantly inhibited the angiogenic function of HAMSCs at an early stage in vitro and in vivo. The results of CCK8 and transwell assays demonstrated that the conditioned medium secreted by HAMSCs reduced proliferation and migration of HUVECs after downregulating H19. The angiogenesis factors expressed and secreted by HAMSC-shH19 were decreased compared with those secreted by the control, while angiogenesis inhibitors were elevated. Furthermore, we conducted chromatin immunoprecipitation and RNA-binding protein immunoprecipitation assays and found that H19 could interact with the histone methyltransferase Enhancer of Zeste homolog 2 (EZH2) and that H19 knockdown inhibited the ability of EZH2 to recruit methyl groups to the promoter region of the angiogenesis inhibitor gene vasohibin-1 (VASH1), thus increasing VASH1 expression and secretion of HAMSCs, suppressing angiogenesis. In summary, our study identified H19 as an important regulator in HAMSCs for promoting angiogenesis, which would help to construct ideal gene-modified seed cells to enhance angiogenesis in regenerative medicine.

LncRNA CCAT1 enhances radiosensitivity of human pancreatic cancer cells PANC-1 by targeting miR-130b-3p

Objective To investigate the effect of lncRNA CCAT1 and miR-130b-3p on the radiosensitivity of human pancreatic cancer cells PANC-1. Methods Real-time PCR was used to detect the relative expression levels of CCAT1 and miR-130b-3p in pancreatic cancer tissues and cell lines including PANC-1 cells irradiated with 2 Gy X-rays. After silencing CCAT1 and/or inhibiting miR-130b-3p expression, cell apoptosis rate, Caspase 3 activity and cell survival were detected by flow cytometry, Caspase 3 activity detection kit and colony formation assay, respectively. Cell survival curve was stimulated by the multi-target single-hit model. Based on the starBase v2.0 online analysis, the luciferase reporter gene assay, RNA-binding protein immunoprecipitation assay (RIP) and Real-time PCR assay were applied to verify the relationship between CCAT1 and miR-130b-3p. Results CCAT1 expression was up-regulated (t=6.322-8.555, P<0.05), but miR-130b-3p expression was down-regulated (t=3.950-18.795, P<0.05) in the radiation-resistant pancreatic cancer tissues, pancreatic cancer cell lines and 2 Gy-irradiated PANC-1 cells. When the CCAT1 silenced PANC-1 cells were irradiated with 2 Gy, cell survival fraction decreased (t=2.929, 5.047, 5.234, 5.125, P<0.05), apoptosis rate and Caspase 3 activity increased (t=6.953, 6.836, P<0.05). CCAT1 could selectively regulate miR-130b-3p expression. Inhibition of miR-130b-3p expression could enhance PANC-1 cell survival (t=4.564, 6.736, 8.656, P<0.05), but reduced apoptosis rate (t=5.234, P<0.05) and Caspase 3 activity (t=10.440, P<0.05). Conclusions Silencing CCAT1 promotes the expression of miR-130b-3p and enhances radiosensitivity of PANC-1 cells. Key words: lncRNA CCAT1; miR-130b-3p; Pancreatic cancer; Radiosensitivity

Long Noncoding RNA MALAT1 Acts as a Competing Endogenous RNA to Regulate TGF-β2 Induced Epithelial-Mesenchymal Transition of Lens Epithelial Cells by a MicroRNA-26a-Dependent Mechanism.

The aim of the present study was to characterize whether the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/miR-26a/Smad4 axis is involved in epithelial–mesenchymal transition (EMT) of lens epithelial cells (LECs). Primary human LECs were separated and cultured. Microarray analysis showed that a total of 568 lncRNAs are differentially expressed in primary HLECs in the presence of TGF-β2 and MALAT1 is mostly significantly dysregulated lncRNAs, which is increased by nearly 17-fold. In addition, upregulation of MALAT1 and downregulation of miR-26a were detected in human posterior capsule opacification (PCO) attached LECs and the LECs obtained from patients with anterior polar cataracts by quantitative RT-PCR (qRT-PCR). Next, our results showed that TGF-β2 induces overexpression of EMT markers in primary HLECs via a MALAT1-dependent mechanism. The mechanism is that MALAT1 negatively regulates miR-26a and miR-26a directly targets Smad4 by luciferase reporter assays and RNA-binding protein immunoprecipitation assay. In summary, TGF-β2 induces MALAT1 overexpression, which in turn MALAT1 acts as a ceRNA targeting Smad4 by binding miR-26a and promotes the progression of EMT of LECs.

ZFP36L1 regulates osteoarthritis by modulating HSPA1A

Purpose: Osteoarthritis (OA) is a whole-joint disease characterized by cartilage destruction and other whole-joint pathological changes. There is currently no effective disease-modifying therapy. Here, we investigated the post-transcriptional mRNA regulation of OA-modulating proteins in chondrocytes by focusing on RNA-binding proteins. Methods: Human OA cartilage was sourced from individuals undergoing arthroplasty. C57BL/6J mice were used for the experimental OA studies. ZFP36l1+/- mice (8-bp insertion/42-bp deletion in exon 2) were generated by ToolGen, Inc. For mRNA decay assay, primary-culture chondrocytes were infected with 800 MOI of Ad-HSPA1A to overexpress HSPA1A. The cells were co-infected with 800 MOI of Ad-C or Ad-ZFP36L1 for 12 hours, and exposed to actinomycin D (1 μg ml-1) for the indicated time periods. The RNA-binding protein immunoprecipitation assay was performed using a Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. Results: Microarray analysis revealed that the ZFP36 family member, ZFP36L1, is specifically upregulated in OA chondrocytes and OA cartilage of humans and mice. Adenovirus-mediated overexpression of ZFP36L1 alone in mouse knee-joint tissue did not modulate OA pathogenesis. However, genetic ablation or silencing of Zfp36l1 significantly abrogated experimental OA in mice. Knockdown of Zfp36l1 increased the mRNA expression of two heat shock protein 70 (HSP70) family members, which act as its direct targets. Furthermore, overexpression of HSPA1A in joint tissues protected mice against experimental OA by inhibiting chondrocyte apoptosis. Conclusions: Our results collectively indicate that the RNA-binding protein, ZFP36L1, regulates HSP70 family members that appear to protect against OA pathogenesis by inhibiting chondrocyte apoptosis.

Highly expressed microRNA-940 promotes early abortion by regulating placenta implantation by targeting ZNF672.

Our study aims to explore whether microRNA-940 could participate in early abortion by inhibiting placenta implantation and its underlying mechanism. Expressions of microRNA-940 and ZNF672 in the placental villi of 6 early abortion pregnancies and 6 normal pregnancies were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Expressions of microRNA-940 and ZNF672 in trophoblast cells (BeWo, JEG3, Wish and HTR-8) were also detected. Cell counting kit-8 (CCK-8) assay was performed to detect the effect of microRNA-940 on the proliferation of trophoblast cells after being transfected with microRNA-940 inhibitor or mimics, respectively. Dual-Luciferase reporter gene assay and RNA binding protein immunoprecipitation (RIP) assay were conducted to demonstrate the binding condition of microRNA-940 to ZNF672. MicroRNA-940 was highly expressed in placental villi of early abortion pregnancies, whereas ZNF672 was lowly expressed. HTR-8 cells expressed the highest level and BeWo cells expressed the lowest level of microRNA-940. After transfection of microRNA-940 inhibitor in HTR-8 cells, the proliferative capacity was remarkably increased. On the contrary, the transfection of microRNA-940 mimic downregulated the proliferation of BeWo cells. Dual-Luciferase reporter gene assay demonstrated that microRNA-940 targets ZNF672. RIP results further indicated that microRNA-940 binds to ZNF672. MicroRNA-940 is highly expressed in the placental villi of early abortion pregnancies and promotes the occurrence of early abortion by inhibiting the proliferation of trophoblast cells by targeting ZNF672.