Luciferase Reporter Gene Experiment Research Articles

MiR-137 promotes TLR4/NF-κB pathway activity through targeting KDM4A, inhibits osteogenic differentiation of human bone marrow mesenchymal stem cells and aggravates osteoporosis

PurposeAs the global population ages rapidly, osteoporotic fractures have become an important public health problem. Previous studies have suggested that miR-137 is involved in the regulation of bone formation, but its specific regulatory mechanism remains unclear. In this study, we aimed to explore the expression, role, and regulatory mechanism of miR-137 in the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).MethodshBMSCs were induced into osteoblasts at first, and the expression level of miR-137 at different time points was detected. After knockdown and overexpression of miR-137, the effect of miR-137 on the osteogenic differentiation of hBMSCs was examined through alkaline phosphatase (ALP) staining and Alizarin Red staining. Western blotting was performed to detect the expression of runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) pathway. Bioinformatics websites were used to predict the target binding sites for miR-137 and KDM4A, and the results were validated using luciferase reporter gene experiments. Moreover, the ALP activity, calcium nodule formation, and activation of Runx2, OCN, and TLR4/NF-κB pathways were observed after knockdown of KDM4A.ResultsThe expression of miR-137 decreased during osteogenic differentiation. Knockdown of miR-137 expression increased the osteogenic ability of hBMSCs, while overexpression of it weakened the ability. Through the activation of the TLR4/NF-κB pathway, miR-137 inhibited osteogenic differentiation. KDM4A was identified as a predicted target gene of miR-137. After knocking down KDM4A expression, the osteogenic ability of hBMSCs was diminished, and the TLR4/NF-κB pathway was activated. Furthermore, the osteogenic ability of hBMSCs was partially restored and the activation level of TLR4/NF-κB was reduced after miR-137 knockdown.ConclusionMiR-137 enhances the activity of the TLR4/NF-κB pathway by targeting KDM4A, thereby inhibiting the osteogenic differentiation of hBMSCs and exacerbating osteoporosis.

RETRACTED ARTICLE: MiR-28-5p Promotes Osteosarcoma Development by Suppressing URGCP Expression.

The purpose is to investigate the role of miR-28-5p in osteosarcoma (OS) development. The expressions of miR-28-5p and URGCP in OS tissues (n = 30) and cells (MG-63 and U2OS) were discovered by q-PCR assay. MiR-28-5p mimic, sh-URGCP, pcDNA3.1-URGCP, and their controls were transfected by lipofectamine ™2000. CCK8 and tunel experiments were processed for proliferation and apoptosis testing. Migration and invasion were monitored by transwell assay. Western blot was undertaken to show the levels of Bax and Bcl-2. The target between miR-28-5p and URGCP was confirmed via a luciferase reporter gene experiment. Finally, the rescue assay further verified the function of miR-28-5p and URGCP in OS cells. MiR-28-5p expressed lower (P < 0.001) in OS tissue and cells. MiR-28-5p mimics suppressed (P < 0.05) proliferation and migration ability, and it accelerated apoptosis of osteosarcoma cells. MiR-28-5p targeted and negatively regulated URGCP expression. Sh-URGCP suppressed (P < 0.01) proliferation and migration ability, while it improved apoptosis of OS cells. Overexpression of miR-28-5p obviously accelerated (P < 0.05) Bax expression, while it reduced (P < 0.05) the Bcl-2 level. Interestingly, pcDNA3.1-URGCP rescued the process. Up-regulated URGCP rescued the effects of miR-28-5p mimic in vitro. MiR-28-5p accelerates the proliferation and migration of osteosarcoma cells, and inhibits tumor cell apoptosis by suppressing URGCP expression, which could be regarded as a potential target for OS treatment.

BORIS variant SF2(C2/A4) promotes the malignant development of liver cancer by activating epithelial-mesenchymal transition and hepatic stellate cells.

The underlying mechanisms of metastasis and recurrence of liver cancer remain largely unknown. Here, we found that Brother of the Regulator of Imprinted Sites (BORIS) variant SF2(C2/A4) was highly expressed in high metastatic potential hepatocellular carcinoma (HCC) cells and clinical tumor samples, related to the formation of satellite nodules. Its over expression promoted self-renewal, the expression of tumor stem cell markers, chemoresistance, wound healing rate, invasion and metastasis of HepG2 and Hep3B cells; reinforced epithelial-mesenchymal transition (EMT), decreased the expression of E-cadherin and increased N-cadherin and Vimentin. Subcellular localization experiment showed that BORIS SF2(C2/A4) was localized in nucleus and cytoplasm. Further double luciferase reporter gene experiment confirmed that it bound to TWIST1 gene promoter and significantly increased latter expression. BORIS SF2(C2/A4) knock down induced apoptosis of HCCLM3 and PLC/PRF/5 cells, and increased the protein content of cleaved caspase 3. Additionally, BORIS SF2(C2/A4) over expression increased the expression of fibroblast growth factor 2 (FGF2) in HepG2 and Hep3B cells. FGF2 expressed higher in HCC tumor tissues than in paired peri-tumor tissues, and its expression was positively correlated with BORIS SF2(C2/A4). Interestingly, high expression of FGF2 is also associated with the formation of satellite nodules. Moreover, using the medium from BORIS SF2(C2/A4) overexpressed cell lines to coculture hepatic stellate cell (HSCs) line LX-2, the latter could be activated and increased the expression of CD90 and PIGF, which is consistent with the effect of adding bFGF alone. These results indicate that BORIS SF2(C2/A4) plays a role in deterioration of liver cancer by regulating TWIST1 to induce EMT, and by FGF2 to activate HSCs.

The effect of miR-155-5p on M1 polarization of Kupffer cells and immune response during liver transplantation through regulating the expression of KDM5D.

To explore the effect and its specific mechanism of miR-155-5p on M1 polarization of Kupffer cells (KCs) and immune response in liver transplantation (LT) through KDM5D. Primary KCs were isolated from Wistar rats and identified by cell culture, ink-swallowing test and flow cytometry. The cells identified as KCs were induced into LT acute rejection (AR) model cells by LPS/IFN-γ, flow cytometry was used for cell sorting and apoptosis detection. Enzyme-linked immunosorbent assay (ELISA) kit was used to detect the levels of inflammatory factors, macrophages and liver function markers. RT-qPCR detected the expression of miR-155-5p and KDM5D mRNA. The protein expression of KDM5D was detected by Western blot. Dual luciferase reporter gene experiment verified the targeting relationship between miR-155-5p and KDM5D. The separated KCs adhered after being cultured for 24h, had pseudopodia and phagocytosis, and the proportion of F4/80 positive cells was more than 90%. The expression of miR-155-5p was increased in LPS/IFN-γ-induced KCs. And knockdown of miR-155-5p inhibited H3K4me3 and H3K27me3 of TNF-α promoter, M1 polarization of KCs and the immune response of AR model cells by upregulating KDM5D. In animal experiments, knockdown of miR-155-5p was found to inhibit liver damage and immune response in rats with allogeneic orthotopic LT. These results confirmed that miR-155-5p inhibited M1 polarization of KCs induced by LPS/IFN-γ, thereby alleviating AR and liver function impairment after LT by upregulating KDM5D.

MicroRNA-22-3p Regulates the Expression of Kruppel-like Factor 6 to Affect the Cardiomyocyte-like Differentiation of Bone Marrow Mesenchymal Stem Cell

Objective To explore the effect of microRNA-22-3p (miR-22-3p) regulating the expression of Kruppel-like factor 6 (KLF6) on the cardiomyocyte-like differentiation of bone marrow mesenchymal stem cell (BMSC). Methods Rat BMSC was isolated and cultured,and the third-generation BMSC was divided into a control group,a 5-azacytidine(5-AZA)group,a mimics-NC group,a miR-22-3p mimics group,a miR-22-3p mimics+pcDNA group,and a miR-22-3p mimics+pcDNA-KLF6 group.Real-time fluorescent quantitative PCR (qRT-PCR) was carried out to determine the expression of miR-22-3p and KLF6 in cells.Immunofluorescence staining was employed to detect the expression of Desmin,cardiac troponin T (cTnT),and connexin 43 (Cx43).Western blotting was employed to determine the protein levels of cTnT,Cx43,Desmin,and KLF6,and flow cytometry to detect the apoptosis of BMSC.The targeting relationship between miR-22-3p and KLF6 was analyzed by dual luciferase reporter gene assay. Results Compared with the control group,5-AZA up-regulated the expression of miR-22-3p (q=7.971,P<0.001),Desmin (q=7.876,P<0.001),cTnT (q=10.272,P<0.001),and Cx43 (q=6.256,P<0.001),increased the apoptosis rate of BMSC (q=12.708,P<0.001),and down-regulated the mRNA (q=20.850,P<0.001) and protein (q=11.080,P<0.001) levels of KLF6.Compared with the 5-AZA group and the mimics-NC group,miR-22-3p mimics up-regulated the expression of miR-22-3p (q=3.591,P<0.001;q=11.650,P<0.001),Desmin (q=5.975,P<0.001;q=13.579,P<0.001),cTnT (q=7.133,P<0.001;q=17.548,P<0.001),and Cx43 (q=4.571,P=0.037;q=11.068,P<0.001),and down-regulated the mRNA (q=7.384,P<0.001;q=28.234,P<0.001) and protein (q=4.594,P=0.036;q=15.945,P<0.001) levels of KLF6.The apoptosis rate of miR-22-3p mimics group was lower than that of 5-AZA group (q=8.216,P<0.001).Compared with the miR-22-3p mimics+pcDNA group,miR-22-3p mimics+pcDNA-KLF6 up-regulated the mRNA(q=23.891,P<0.001) and protein(q=13.378,P<0.001)levels of KLF6,down-regulated the expression of Desmin (q=9.505,P<0.001),cTnT (q=10.985,P<0.001),and Cx43 (q=8.301,P<0.001),and increased the apoptosis rate (q=4.713,P=0.029).The dual luciferase reporter gene experiment demonstrated that KLF6 was a potential target gene of miR-22-3p. Conclusion MiR-22-3p promotes cardiomyocyte-like differentiation of BMSC by inhibiting the expression of KLF6.

LncRNA FAM138B inhibits the progression of non-small cell lung cancer through miR-105-5p

ABSTRACT As a type of lung cancer, non-small cell lung cancer (NSCLC) has the characteristics of high mortality and high recurrence rate, which poses a great threat to human life and health. Due to the high risk of surgical treatment and the slow recovery of wounds, non-coding RNAs, especially lncRNAs are used as new potential clinical prognostic markers to prevent and treat cancer in advance. This study aims to explore the role of FAM138B in NSCLC and its possibility as a prognostic biomarker. Real-timequantitative polymerase chain reaction (RT-qPCR) was used to detect the expression and overexpression level of lncRNA FAM138B (FAM138B) in cells and tissues. The CCK-8, Transwell migration and invasion methods were performed to observe the cell transfection.The interaction between FAM138B and miR-105-5p was predicted by the bioinformatics tool starBase v2.0, and verified by the luciferase reporter gene experiment. Kaplan-Meier and Cox regression analyses were used to determine the prognostic significance of FAM138B in NSCLC. The expression of FAM138B is down-regulated in NSCLC cells and tissues. Overexpression of FAM138B can inhibit the expression level of miR-105-5p in NSCLC cells, and the ability of NSCLC cells to proliferate, migrate and invade is downregulated. FAM138B targets miR-105-5p, and there is a negative correlation between FAM138B and miR-105-5p. It is confirmed that FAM138B inhibits the progression of NSCLC by targeting miR-105-5p and can be a potential prognostic biomarker for NSCLC.

Machine learning and experimental validation to construct a metastasis-related gene signature and ceRNA network for predicting osteosarcoma prognosis

ObjectiveOsteosarcoma (OS) is more common in adolescents and significantly harmful, and the survival rate is considerably low, especially in patients with metastatic OS. The identification of effective biomarkers and associated regulatory mechanisms, which predict OS occurrence and development as well as improve prognostic accuracy, will help develop more refined protocols for OS treatment.MethodsIn this study, genes showing differential expression in metastatic and non-metastatic types of OS were identified, and the ones affecting OS prognosis were screened from among these. Following this, the functions and pathways associated with the genes were explored via enrichment analysis, and an effective predictive signature was constructed using Cox regression based on the machine learning algorithm, least absolute shrinkage and selection operator (LASSO). Next, a correlative competing endogenous RNA (ceRNA) regulatory axis was constructed after verification by bioinformatics analysis and luciferase reporter gene experiments conducted based on the prognostic signature.ResultsOverall, 251 differentially expressed genes were identified and screened using bioinformatics and double luciferase reporter gene experiments. An effective prognostic signature was constructed based on 15 genes associated with OS metastasis, and upstream non-coding RNAs were identified to construct the “NBR2/miR-129-5p/FKBP11” regulatory axis based on the ceRNA networks, which helped identify candidate biomarkers for the OS clinical diagnosis and treatment, drug research, and prognostic prediction, among other applications. The findings of this study provide a novel strategy for determining the mechanism underlying OS occurrence and development and the appropriate treatment.

Bta-miR-199a-3p Inhibits LPS-Induced Inflammation in Bovine Mammary Epithelial Cells via the PI3K/AKT/NF-κB Signaling Pathway.

Mastitis is characterized by inflammatory damage to mammary gland tissue, which could decline milk production and quality and significantly affect the economic benefits of ranching. MicroRNAs (miRNAs), such as miR-199a-3p, are novel therapeutic targets in inflammation, and their regulation is an effective strategy for inflammation control. Despite its importance in humans and animals, the molecular mechanism of bovine miR-199a-3p (bta-miR-199a-3p) in dairy cow mastitis and bovine mammary epithelial cell (bMEC) inflammation is unclear. In our study, a bovine mammary epithelial cell line (MAC-T) induced by lipopolysaccharide (LPS) was used as an inflammatory cell model to investigate the molecular mechanism of bta-miR-199a-3p in the MAC-T inflammatory response. bta-miR-199a-3p was up-regulated in the LPS-induced MAC-T cells, while CD2-associated protein (CD2AP) was revealed as its target gene in a double luciferase reporter gene experiment. In addition, the overexpression of bta-miR-199a-3p negatively regulated the expression of CD2AP and the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/nuclear factor kappa-B (NF-κB) signaling pathway. These subsequently inhibited the secretion of related inflammatory factors (TNF-α, IL-1β, and IL-6) and the expression of apoptotic genes (CASP3 and CASP9), thereby alleviating the LPS-challenged inflammatory response in the MAC-T cells. Silencing of bta-miR-199a-3p, however, reversed the above effects. Thus, bta-miR-199a-3p inhibits LPS-induced inflammation in bMECs by directly targeting CD2AP and regulating the PI3K/AKT/NF-κB signaling pathway. This study reveals the potential regulatory mechanism of bta-miR-199a-3p in bMEC inflammatory immune response and may serve as a useful target for the treatment of mastitis.

Lncrna FEZf1-as1 negatively regulates ETNK1 to promote malignant progression of renal cell carcinoma.

To explore the role of LncFEZF1-AS1 in renal cell carcinoma (RCC) tissues and cells, and the possible molecular mechanism. Expressions of LncFEZF1-AS1 in RCC tissues and adjacent ones were detected. The association of LncFEZF1-AS1 level with clinical data of RCC patients was also analyzed. Besides, the differential expressions of LncFEZF1-AS1 in a variety of RCC cell lines were also determined. Then the LncFEZF1-AS1 knockdown model was constructed in RCC cell line to further determine the influences of LncFEZF1-AS1 on the proliferative ability and migration of RCC cells through CCK8 and Transwell experiments. Furthermore, luciferase reporter gene experiment were used to validate the combination of LncFEZF1-AS1 to ETNK1. Results suggested that expression of LncFEZF1-AS1 was noticeably higher in RCC tumor tissues and the RCC cells. Clinical pathological data analysis also suggested that high LncFEZF1-AS1 expression was in correlation with the pathological stage and the incidence of distant metastasis in RCC patients, and the poor overall survival rate. In vitro experiments demonstrated that knocking down of LncFEZF1-AS1 markedly repressed the proliferation and migration of RCC cell lines. Bioinformatics suggested that LncFEZF1-AS1 can interact with the downstream target gene ETNK1, which was confirmed by the luciferase reporter gene experiments. Western Blot results revealed that knocking down of LncFEZF1-AS1 markedly enhanced ETNK1. qRT-PCR analysis indicated that ETNK1 level was under-expressed in RCC tissues and in negative correlation with LncFEZF1-AS1. Further experiments suggested that knockdown of ETNK1 partially reversed the inhibitory effects of LncFEZF1-AS1 silencing on the proliferative and migrative abilities of RCC cells. LncFEZF1-AS1 could facilitation the proliferative and migration of RCC cells by regulating the expression of ETNK1. Therefore, FEZF1-AS1 might function as a cancer-promoting factor and possible new therapeutic target for RCC.

Transcription factor NFE2L3 promotes the proliferation of esophageal squamous cell carcinoma cells and causes radiotherapy resistance by regulating IL-6

ObjectiveTo scrutinize the impact of overexpression and interference of NFE2L3 on radiosensitivity of esophageal squamous cell carcinoma cells (ESCC) and its downstream mechanism and to assess whether NFE2L3 expression alters in vivo radiosensitivity of ESCC by developing a subcutaneous tumor model in mice. MethodsThrough RNA-Seq, we compared the differentially expressed genes between the ECA-109R cell line and its parent ECA-109 cell line. The differentially expressed genes were selected and verified by qRT-PCR. Transfection of ESCC cell lines with NFE2L3 inhibitor or mimic lentivirus constructs was done to study the activity of NFE2L3. To assess the effect of NFE2L3 on cellular growth and proliferation, clonogenic survival assay, EdU incorporation assay, and CCK-8 assay were done after irradiation. To probe how many irradiated DNA double-strand breaks were produced, the corresponding intensity of γ-H2AX foci were detected by immunofluorescence. Apoptotic cells were assayed by flow cytometry assay after irradiation; To investigate the downstream genes of NFE2L3, we knocked NFE2L3, and RNA-Seq was used to find out the downstream genes. qRT-PCR and western blot ensued to score associated protein profiles. The in vivo ESCC cell radiosensitivity was scrutinized by nude mouse xenograft models. ResultsThe differential genes between ECA-109R cells and its parent ECA-109 cells were compared by qRT-PCR to unveil a significant increase in NFE2L3 expression. Functional analysis indicated that NFE2L3 increased radioresistance in ESCC cells. Then, through high-throughput sequencing and bioinformatics analysis, IL-6 was found to be a hub gene that played a role downstream of NFE2L3 and was verified by qRT-PCR, western blot, and double luciferase reporter gene experiment. NFE2L3 could regulate ESCC cell radiosensitivity via the IL-6-STAT3 signaling pathway, and downregulation of IL-6 expression could reverse the effects of highly expressed NFE2L3. In vivo tumor xenograft experiments confirmed that NFE2L3 affects the sensitivity to radiation therapy. ConclusionNFE2L3 can affect the radiosensitivity of ESCC cells through IL-6 transcription and IL-6/STAT3 signaling pathway. This makes NFE2L3 a putative target to regulate ESCC cell radiosensitivity.

Circular RNA circRara promote the innate immune responses in miiuy croaker, Miichthys miiuy

With the in-depth study of circRNA, more and more biological studies have shown that circRNAs play an important role in mammals, such as cell proliferation, apoptosis, invasion, development and disease state. However, the regulatory mechanism of circRNA in lower vertebrates remains unclear. Here, we found a new circular RNA and named it circRara. We carried out the experimental study on its antiviral and antibacterial response, cell proliferation and activity. The results showed that circRara had a positive regulatory effect on the antiviral and antibacterial response, cell proliferation and activity in miiuy croaker. First, we found that the expression of circRara could be up-regulated under the stimulation of LPS and poly (I: C), but not the expression of linear Rara. In addition, the increase of circRara can increase the production of inflammatory factors and antiviral genes, which was confirmed by double luciferase reporter gene experiment and qPCR. These results will help to further understand the immunomodulatory mechanism of circRNA in teleost fish.

GATA3 Exerts Distinct Transcriptional Functions to Regulate Radiation Resistance in A549 and H1299 Cells

Background Radiation resistance of lung cancer cells is a vital factor affecting the curative effect of lung cancer. Transcription factor GATA3 is involved in cell proliferation, invasion, and migration and is significantly expressed in a variety of malignancies. However, the molecular mechanism governing GATA3 regulation in lung cancer cells' radiation resistance is unknown. Methods Radiation-resistant cell models (A549-RR and H1299-RR) were made using fractionated high-dose irradiation. Use clone formation, CCK-8, F-actin staining, cell cycle detection, and other experiments to verify whether the model is successfully constructed. Cells were transiently transfected with knockdown or overexpression plasmid. To explore the relationship between GATA3/H3K4me3 and target genes, we used ChIP-qPCR, ChIP-seq, and dual luciferase reporter gene experiments. Xenograft tumor models were used to evaluate the effect of GATA3 depletion on the tumorigenic behavior of lung cancer cells. Results We report that transcription factors GATA3 and H3K4me3 coactivate NRP1 gene transcription when A549 cells develop radiation resistance. However, the mechanism of radiation resistance in H1299 cells is that GATA3 acts as a transcription inhibitor. The decrease of GATA3 will promote the increase of NRP1 transcription, in which H3K4me3 does not play a leading role. Conclusions GATA3, an upstream transcriptional regulator of NRP1 gene, regulates the radioresistance of A549 and H1299 cells by opposite mechanisms, which provides a new target for radiotherapy of lung cancer.

Effects of miR-124-3p Silencing on Neuronal Damage in the Hippocampus of Depression Rats by Regulating STAT3 Gene.

Objective A large amount of evidence shows that the abnormal expression of miRNA plays an important role in the development of depression. Therefore, we investigated the effect of miR-124-3p on neuronal damage in the hippocampus of depression rats. Methods The target genes of miR-124-3p were predicted by the database; the depression model was prepared by subcutaneous injection of corticosterone (CORT), and LV-miR-124-3p asponge lentiviral suspension was given to determine the weight of rats and open-field test, sugar preference experiment, Serum CORT, 5-HT, DA, and NE were measured, observe and record the behavior of rats, including behavior, diet, and hair. The expression of miR-124-3p, STAT3, Bcl-2, and Bax in rat hippocampus was measured. The rat hippocampal neuron cells were extracted and transfected with miR-124-3p inhibitor; the cells were cultured with CORT, and the cell survival rate was evaluated by MTT experiment, and the expressions of miR-124-3p, STAT3, Bcl-2, and Bax in the cells were detected. Luciferase reporter gene verifies the targeted regulation of miR-124-3p on STAT3. Results Compared with depression rats, silencing miR-124-3p increased the weight of the rats, increased the number of open-field activities, and significantly improved the general state and pathological state of the rats. The sugar water preference rate was significantly increased, the CORT content in the serum of rats decreased significantly, and the levels of 5-HT, DA, and NE increased significantly. After the treatment of silencing miR-124-3p, the expression level of miR-124-3p was decreased, while the STAT3 mRNA and protein expression levels were increased. And the protein and mRNA expression levels of Bcl-2 were increased, and the Bax protein and mRNA expression were decreased. Cell experiments verified that silencing miR-124-3p increased cell survival, the expression level of miR-124-3p decreased remarkably, while the expression levels of STAT3 mRNA and protein increased significantly. Silencing miR-124-3p reversed the effects of CORT treatment on miR-124-3p and STAT3 in neuronal cells. The luciferase reporter gene experiment confirmed that miR-124-3p targets and regulates STAT3 expression. Conclusion Silencing miR-124-3p may protect hippocampal neurons from damage in depression rats by upregulating STAT3 gene.

1468-P: Effect of Kcnq1ot1 on Influencing the Function of Pancreatic ß-Cells Targeting miR-92a-1-5p

Objective: To study the role and mechanism of Kcnq1ot1 by targeting to regulate miR-92a-1-5p to effect insulin secretion in pancreatic β cells. Methods: The qRT-PCR was used to detect the expression of Kcnq1ot1 in different tissues of C57BL/6J mice, Min6 with PA intervention, islets of T2D model mice and the serum of T2D patients. The biological information website was used to predict that Kcnq1ot1 had complementary binding sites with miR-92a-1-5p, which was verified by the luciferase reporting gene system. The si-Kcnq1ot1, miR-92a-1-5p mimics, miR-92a-1-5p inhibitors and their respective controls were respectively transfected into Min6, and the effect of Ins1, Ins2, Insr, Nkx6.1, Irs1 and Ngn3 were detected by qRT-PCR, the Western blot method was used to detect the expression of insulin and Insr, and the GSIS experiment was used to detect its effect on insulin secretion. Results: The expression of Kcnq1ot1 in pancreas was significantly higher than that in other tissues. Compared with the control group, the expression of Kcnq1ot1 in T2D patients' serum, Min6 after PA treatment and the islets of T2D model mice were lower. And the expression of Kcnq1ot1 in serum is negatively correlated with FBG or HbA1c. Low expression of Kcnq1ot1 led to remarkable reduction of insulin secretion in Min6 cells. Down-regulation of Kcnq1ot1 or overexpression of miR-92a-1-5p reduced the mRNA expression levels of Ins1, Ins2, Insr, Nkx6.1, Irs1 and Ngn3, and the protein levels of insulin and Insr. We screened miR-92a-1-5p as targets of Kcnq1ot1, and their interaction was validated with dual luciferase reporter gene experiment. After knocking down Kcnq1ot1, the expression of miR-92a-1-5p was found to increase. Further rescue experiments showed that down-regulation of miR-92a-1-5p could reverse the effect of low Kcnq1ot1 expression on the function of pancreatic β-cells. Conclusion: Kcnq1ot1 may regulate the function of pancreatic β cells by targeting to negatively regulate miR-92a-1-5p. Disclosure Y.Chen: None. Y.Li: None. W.Xu: None. B.Lin: None. X.Mai: None. Y.Peng: None. D.Chen: None. Y.Su: None. W.Li: n/a. Funding Science &amp; Technology Project of Guangzhou（202102010175）

SQLE facilitates the pancreatic cancer progression via the lncRNA-TTN-AS1/miR-133b/SQLE axis.

Studies have shown that SQLE is highly expressed in a variety of tumours and promotes tumour progression. However, the role of SQLE in pancreatic cancer (PC) has not been reported. Here, we aim to study the role and molecular mechanism of SQLE in PC. Immunohistochemistry and functional experiments showed that SQLE was highly expressed in PC tissues and promoted the proliferation and invasion of PC cells. Terbinafine, an inhibitor of SQLE, inhibited this effect. In order to further study the upstream mechanism that regulates SQLE, we used bioinformatics technology to lock miR‐133b and lncRNA‐TTN‐AS. In situ hybridization was used to detect the expression of miR‐133b and lncRNA‐TTN‐AS1 in PC tissues. The luciferase reporter gene experiment was used to confirm the binding of miR‐133b and lncRNA‐TTN‐AS1. The results showed that miR‐133b was down‐regulated in PC tissues and negatively correlated with the expression of SQLE. LncRNA‐TTN‐AS1 was upregulated in pancreatic cancer tissues and positively correlated with the expression of SQLE. Luciferase gene reporter gene analysis confirmed lncRNA‐TTN‐AS1 directly binded to miR‐133b. Therefore, we propose that targeting the lncRNA‐TTN‐AS1/miR‐133b/SQLE axis is expected to provide new ideas for the clinical treatment of PC patients.

MiR-145-5p targets MMP2 to protect brain injury in hypertensive intracerebral hemorrhage via inactivation of the Wnt/β-catenin signaling pathway.

BackgroundDifferences in microRNA (miRNA) expression after hypertensive intracerebral hemorrhage (HICH) have been reported in human and animal models. miRNA-145 plays an important role in vascular endothelial cells. The purpose of this work was to determine the role of miR-145-5p in HICH and the molecular mechanisms by which it acts.MethodsIn this study, we constructed a model of hemoglobin-induced HICH in rats and used thrombin-treated human brain microvascular endothelial cells (hBMECs) to create a HICH cell model. To determine brain damage, we tested the rats’ neurological performance and measured the cerebral water level of their brain tissue. Cell counting kit 8 (CCK8) was used to determine the viability of cells. Apoptosis was detected using the terminal TdT-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry (FCM). Starbase and TargetScan were used to predict and confirm target genes. Luciferase reporter gene experiments were used to confirm the predictions. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to identify the associated genes and proteins.ResultsWe observed a reduction in miRNA-145-5p expression in both the HICH cell model and the rat model. miRNA-145-5p overexpression increased cell survival and preserved newly created blood vessels and vascular permeability in hBMECs. MiRNA-145-5p has been predicted to target matrix metalloproteinase 2 (MMP2). Additionally, MMP2 was identified as a miR-145-5p target gene and shown to be substantially expressed in the thrombin-treated hBMECs. MMP2 overexpression suppressed miR-145-5p-mediated effects and increased hBMECs’ malfunction. In comparison with controls, the HICH + AAV-miR-145-5p group performed better on behavioral tests and had less brain water. Additionally, miR-145-5p injection increased ZO-1 and occludin expressions, as determined by immunohistochemical staining in the HICH rat model.ConclusionsmiRNA-145-5p protects against brain injury following HICH by targeting MMP2, suggesting a possible therapeutic mechanism for HICH.

Circular RNA circLRCH3 Inhibits Proliferation, Migration, and Invasion of Colorectal Cancer Cells Through miRNA-223/LPP Axis.

PurposeColorectal cancer (CRC) is one of the most common carcinomas worldwide with a high mortality rate. Numerous studies suggest that circular RNA (circRNA) plays a crucial role in the progression of various carcinomas, including CRC. The present work focused on exploring the role and underlying molecular mechanism of action of the circRNA circLRCH3 in CRC.MethodsReal-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was conducted to detect expression levels of circLRCH3, miR-233, and lipoma preferred partner (LPP). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure the proliferation of CRC cells and the transwell assay was used to evaluate cell migration and invasion capacity. A flow cytometry assay was used to analyze the effect of circLRCH3 on the distribution of the cell cycle and apoptosis of CRC cells. The expression of LPP was analyzed using Western blotting or an RT-qPCR assay. The relationship between miR-223 and circLRCH3, and that between miR-223 and LPP, was predicted and examined using bioinformatics analysis and luciferase reporter gene experiments. A xenograft tumor formation assay was also performed.ResultsWe found that the expression level of circLRCH3 was downregulated in CRC cells and negatively correlated with miR-223. The overexpression of circLRCH3 or silencing of miR-223 inhibited the growth, invasion, and migration of CRC cells, but promoted their apoptosis. In contrast, overexpression of miR-223 and depletion of LPP severally abrogated the tumor suppressive roles of circLRCH3 and miR-223 knockdown in CRC cells in vitro. The xenograft experiments in nude mice also proved the antitumor effect of circLRCH3.ConclusionThese results suggested that the circLRCH3/miR-223/LPP axis likely plays a critical role in CRC.

Effect of Carvacrol on the Biological Behavior of Leukemia Cells and Its Mechanism

To explore the effect of carvacrol on the biological behavior of leukemia cells and its regulation to circ-0008717/miR-217 molecular axis. Human acute lymphoblastic leukemia cells Molt-4 were cultured in vitro, and different concentrations of carvacrol were added to the cells. si-NC and si-circ-0008717 were transfected into Molt-4 cells (si-NC group, si-circ-0008717 group). pcDNA, pcDNA-circ-0008717, anti-miR-NC, anti-miR-217 were transfected into Molt-4 cells and then added to carvacrol-treated cells (carvacrol+pcDNA group, carvacrol+pcDNA-circ-0008717 group, carvacrol+anti-miR-NC group, carvacrol+anti-miR-217 group). MTT, plate clone formation experiment, and flow cytometry were used to detect the viability of the cell, colony formation number, and apoptosis rate of cells, respectively. The RT-qPCR method was used to detect the expression levels of circ-0008717 and miR-217. The dual luciferase reporter gene experiment was used to detect the targeting relationship between circ-0008717 and miR-217. After carvacrol treatment, the cell viability decreased significantly (r=-0.9405), expression level of circ-0008717 decreased (r=-0.9117), colonies formed number decreased (r=-0.9256), while the cell apoptosis rate increased (r= 0.8464), and the expression level of miR-217 increased (r=0.9468). Compared with the si-NC group, the expression level of miR-217 in si-circ-0008717 group increased (P＜0.001), the cell apoptosis rate increased (P＜0.001), while cell viability decreased (P＜0001), the number of colonies formed decreased (P＜0.001). Compared with the carvacrol+pcDNA group, the cell viability of the carvacrol+pcDNA-circ-0008717 group increased (P＜0.001), the number of colonies formed increased (P＜0.001), while the cell apoptosis rate decreased (P＜0.001). circ-0008717 could target miR-217. The cell viability of the carvacrol+anti-miR-217 group increased (P＜0.001), and the number of colonies formed increased (P＜0.001), while the cell apoptosis rate decreased (P＜0001) as compared with the carvacrol+anti-miR-NC group. Carvacrol can promote the expression of miR-217 by down-regulating the expression of circ-0008717, thereby reducing the proliferation and cloning ability of leukemia cells and promoting cell apoptosis.

MiR-16 Inhibits Extracellular Signal-Regulated Kinases (ERK) Mitogen-Activated Protein Kinases (MAPK) Signaling to Affect Epithelial-Mesenchymal Transition (EMT) and Invasion of Glioma Cells

Abnormal MEK1 expression is associated with tumor cell EMT, invasion and metastasis. Decreased miR-16 level is associated with glioma. Bioinformatics analysis showed a relationship between miR-16 and MEK1. This study assessed whether miR-16 regulates MEK1 expression and affects glioma cell EMT and invasion. The tumor tissues and adjacent glioma tissues were collected to measure miR-16 and MEK1 mRNA. The dual luciferase assay validated the relation of miR-16 with MEK1. U251 cells were cultured and assigned into NC group and mimic group, followed by analysis of cell biological behaviors, and MEK1, p-ERK1/2, E-cadherin, N-Cadherin expression. Compared with adjacent tissues, miR-16 expression was significantly decreased and MEK1 was elevated in glioma tissues. Compared with HEB, miR-16 in glioma U251 and SHG44 cells was decreased and MEK1 was increased. Dual luciferase reporter gene experiments confirmed the relation of miR-16 with MEK1. Transfection of miR-16 mimic significantly down-regulated MEK1, p-ERK1/2 and N-cadherin in U251 cells, upregulated E-cadherin, inhibited cell proliferation, promoted apoptosis, and attenuated EMT and invasion of glioma cells. In conclusion, decreased miR-16 expression and increased MEK1 expression is related to glioma pathogenesis. Overexpression of miR-16 can inhibit MEK1 expression, ERK/MAPK signaling, glioma cell proliferation, promote apoptosis, and attenuate EMT and invasion.

MiR-200a-3p promoted cell proliferation and metastasis by downregulating SOX17 in non-small cell lung cancer cells.

Lung cancer has high mortality and incidence rates in which non-small cell lung cancer (NSCLC) is the primary type of lung cancer that accounts for about 80%-85% of total patients. It has been demonstrated that microRNAs (miRNAs) are critical in the incidence and progression of tumors, while the role and inner mechanism of miR-200a-3p, one type of essential miRNAs, in NSCLC have yet to be revealed. Herein, we investigated the in vitro and vivo pro-/antiproliferative influence of miR-200a-3p on NSCLC cells and utilized bioinformatic programs to further predict the SOX17 gene as miR-200a-3p's potential target. A double luciferase reporter gene experiment was performed to confirm that miR-200a-3p interacts with theSOX17 3'-UTR region specifically. On the basis of the results of Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR), miR-200a-3p impacted the posttranscriptional levels of SOX17 rather than influencing its mRNA expression. In the end, we found that overexpressed SOX17 can reverse miR-200a-3p's impact on NSCLC cell proliferation and metastasis. Therefore, this study demonstrated that miR-200a-3p influences NSCLC cell proliferation and metastasis by modulating the levels of SOX17.