Dual-luciferase Reporter Gene Experiment Research Articles

Design, synthesis, and biological evaluation of benzo[b]thiophene 1,1-dioxide derivatives as potent STAT3 inhibitors.

As a member of the signal transducer and activator of transcription (STAT) family, STAT3 plays a critical role in several biological pathways such as cell proliferation, migration, survival, and differentiation. Due to abnormal continuous activation in tumors, inhibition of STAT3 has emerged as an attractive approach for the treatment of various cancer cells. Herein, we report a series of novel STAT3 inhibitors based on benzo[b]thiophene 1,1-dioxide scaffold and evaluated their anticancer potency. Among them, compound 8b exhibited the best activity against cancer cells. Compound 8b induced apoptosis and blocked the cell cycle. Meanwhile, 8b reduced intracellular ROS content and caused the loss of mitochondrial membrane potential. Further research revealed that 8b significantly blocked STAT3 phosphorylation and STAT3-dependent dual-luciferase reporter gene experiments showed that compound 8b has a marked inhibition of STAT3-mediated Firefly luciferase activity. Molecular modeling studies revealed compound 8b occupied the pocket well with the SH2 domain in a favorable conformation.

MicroRNA-148 is involved in NF-κB signaling pathway regulation after LPS stimulation by targeting IL-1β in miiuy croaker

The inflammatory response is a protective process to clear detrimental stimuli, constitutes the defense against infectious pathogens. Clearing pathogen infection requires appropriate immune and inflammatory response, but excessive inflammatory response can lead to uncontrolled inflammation, autoimmune disease, or pathogen transmission. Accumulating evidences show that miRNAs are important and multifunctional regulators of innate immunity and inflammation. However, in the inflammatory response of lower vertebrates, the miRNAs regulatory networks are largely unknown. In this study, a combination of bioinformatics and experimental techniques were used to investigate the functions of miR-148. IL-1β is a hypothetical target gene of miR-148 predicted by bioinformatics. In addition, dual-luciferase reporter gene experiment was used to verify the targeting effect of miR-148 on IL-1β-3'UTR. miR-148 inhibits IL-1β expression in a dose-dependent manner at protein and mRNA levels. It is important that miR-148 participates in regulation of LPS-induced the NF-κB signaling pathway by inhibiting IL-1β. These results will improve our understanding of the regulation of miRNAs in fish on the immune response.

Propofol Downregulates lncRNA MALAT1 to Alleviate Cerebral Ischemia-Reperfusion Injury.

Propofol (PPF) is reported to play a protective role in ischemia/reperfusion (I/R) injury, including cerebral ischemia-reperfusion injury (CIRI). This study aims to investigate the mechanism by which PPF ameliorates CIRI. Kunming mice were used to establish the middle cerebral artery occlusion (MCAO)/reperfusion mouse model in vivo. PPF pre-treatment was performed before CIRI. Lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) levels were detected to evaluate the tissue injury. PC12 cells were exposed to hypoxia/reoxygenation (H/R) to construct the in vitro CIRI model, and PC12 cells were pre-treated with PPF before H/R. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to detect the expression of lncRNA MALAT1 and miR-182-5p. Flow cytometry was used to detect the apoptosis of PC12 cells. Bioinformatics analysis, qRT-PCR, dual-luciferase reporter gene experiments, and RNA immunoprecipitation (RIP) experiments were performed to predict and validate the targeting relationship between MALAT1 and miR-182-5p. Western blot was used to detect Toll-like receptor 4 (TLR4) expression at protein level. PPF pre-treatment remarkably inhibited LDH and CPK levels in the serum of the mice with CIRI, and reduced the apoptosis of PC12 cells exposed to H/R. Besides, PPF pre-treatment markedly suppressed MALAT1 expression in both in vivo and in vitro models and upregulated miR-182-5p expression. MiR-182-5p was validated to be a downstream target gene of MALAT1, and MALAT1 could increase the expression of TLR4 by suppressing miR-182-5p. The effects of PPF on the injury of the mice brain and PC12 cells were partly counteracted by the restoration of MALAT1. PPF protects the brain against I/R-induced injury by regulating MALAT1/miR-182-5p/TLR4 axis.

F-Box and Leucine-Rich Repeat Protein 20 (FBXL20), Negatively Regulated by microRNA (miR)-195-5p, Accelerates the Malignant Progression of Ovarian Cancer.

Ovarian cancer (OC) is one of the most common cancers among women, characterized by various histological subtypes. Here, we aimed to investigate the biological function of F-box and leucine-rich repeat protein 20 (FBXL20) in the malignant phenotype of OC cells and its related mechanism. The expression of FBXL20 in OC tissue and normal tissue samples was analyzed through the GEPIA database. Quantitative real-time PCR (qRT-PCR), immunohistochemistry (IHC) and Western blot were employed to detect the expression of miR-195-5p and FBXL20 in OC tissues and cell lines. Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) experiment and flow cytometry were applied to detect cell proliferation, cell cycle and apoptosis. Bioinformatics analysis and dual-luciferase reporter gene experiments were adopted to predict and validate the targeting relationship between miR-195-5p and FBXL20 mRNA 3'-untranslated region (3'UTR). Correlation between the expressions of miR-195-5p and FBXL20 mRNA was analyzed by Pearson correlation analysis. FBXL20 expression was upregulated in OC, and its high expression level was significantly associated with higher International Federation of Gynecology and Obstetrics (FIGO) stage and poor tumor differentiation. Functionally, overexpression of FBXL20 promoted proliferation, inhibited apoptosis and accelerated the cell cycle in OC cells in comparison to control group, and knockdown of FBXL20 exerted the opposite effects. Mechanistically, miR-195-5p directly targeted FBXL20 and negatively regulated its expression. Pearson correlation analysis indicated that miR-195-5p was negatively correlated with FBXL20 mRNA expression. In addition, overexpression of miR-195-5p reversed the above biological functions of FBXL20 in OC cells. FBXL20, negatively regulated by miR-195-5p, accelerates the proliferation and cell cycle progression of OC cells, and inhibits cell apoptosis, which might act as a prospective prognostic biomarker and a promising therapeutic target for OC.

Circ_0000317/microRNA-520g/HOXD10 axis affects the biological characteristics of colorectal cancer.

Circular RNAs (circRNAs) are a class of noncoding RNAs that are widely expressed in cancer tissues and play a pro- or anticancer role in modulating cancer progression. This work is aimed to probe the biological role of circ_0000317 in colorectal cancer (CRC) and its underlying mechanism. Circ_0000317 was selected from the circRNA microarray datasets (GSE121895). Quantitative real-time polymerase chain reaction was utilized to examine circ_0000317, microRNA (miR)-520g, and homeobox D10 (HOXD10) mRNA expression in CRC. Cell Counting Kit-8 and Transwell experiments were conducted to examine the effects of circ_0000317 on proliferation, migration, and invasion of CRC cells. Bioinformatic analysis and dual-luciferase reporter gene experiments were implemented to predict and validate the targeting relationship between circ_0000317 and miR-520g, miR-520g, and HOXD10. Western blot was employed to examine HOXD10 expression at protein level in CRC cells. Circ_0000317 and HOXD10 mRNA expression were unveiled to be down-modulated and miR-520g expression was up-modulated in CRC. Functionally, circ_0000317 overexpression repressed CRC cell proliferation, migration, and invasion. Mechanistically, miR-520g was a direct target of circ_0000317 and miR-520g specifically modulated HOXD10 expression. Furthermore, miR-520g mimics partially counteracted the suppressing effect of circ_0000317 on malignant phenotype of CRC cells. Circ_0000317 represses CRC progression by targeting miR-520g and modulating HOXD10 expression. Hence, circ_0000317 may be a promising diagnostic biomarker and a therapeutic target for CRC.

Hsa_circ_0069244 acts as the sponge of miR-346 to inhibit non-small cell lung cancer progression by regulating XPC expression

Circular RNAs (circRNAs) play a significant role in the progression of diverse malignancies. Here, we aimed to probe the function and mechanism of circ_0069244 in non-small cell lung cancer (NSCLC). In the present study, circ_0069244 was selected from the circRNA microarray datasets (GSE112214). Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to examine circ_0069244, miR-346 and XPC complex subunit, DNA damage recognition and repair factor (XPC) expression levels. Kaplan-Meier curve was employed to analyze the association between circ_0069244 expression and overall survival of NSCLC patients. Cell counting kit-8 (CCK-8) and 5-Bromo-2'-deoxyuridine (BrdU) experiments were utilized to examine the proliferation of NSCLC cells. Scratch healing and Transwell experiments were executed to examine the migration of NSCLC cells. Western blot was conducted to detect XPC expression at protein level in NSCLC cells. Bioinformatics analysis, dual-luciferase reporter gene and RNA immunoprecipitation (RIP) experiments predicted and validated the targeting relationships of circ_0069244 and miR-346, as well as miR-346 and 3'untranslated region (UTR) of XPC mRNA, respectively. We reported that circ_0069244 was remarkably down modulated in NSCLC and was linked to shorter survival and poor tumor histological grade in NSCLC patients. Functionally, circ_0069244 repressed NSCLC cell proliferation and migration. Furthermore, miR-346-5p was unveiled to be a downstream target of circ_0069244, and miR-346-5p specifically modulated XPC expression. Rescue experiments indicated that the inhibitory effect of circ_0069244 was abolished by co-expression of miR-346-5p mimics. Taken together, circ_0069244 restrained NSCLC progression by modulating the miR-346-5p/XPC axis.

MicroRNA-449 targets histone deacetylase 1 to regulate the proliferation, invasion, and apoptosis of synovial fibroblasts in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic joint disease. The study aimed to explore the effects of microRNA (miR)-449 and histone deacetylase 1 (HDAC1) on the proliferation, invasion, and apoptosis of synovial fibroblasts in rheumatoid arthritis. Synovial tissue was collected from 20 patients with RA and 20 patients with osteoarthritis (OA) who underwent joint replacement surgery. RA synovial fibroblasts (RASFs) and OA synovial fibroblasts (OASFs) were isolated and cultured. Real-time quantitative PCR was used to detect the expression levels of miR-449 and HDAC1 in synovial tissues and cells. Western blot was performed to detect the cellular expression levels of HDAC1 protein, and apoptosis and invasion-related proteins. The proliferation, invasion, and apoptosis of RASFs were detected by MTT assay, Transwell assay, and flow cytometry. The dual-luciferase reporter gene was used to test the targeting relationship between inflammatory miR-449 and HDAC1. Compared with normal synovial tissue and OASFs, the levels of HDAC1 messenger RNA in RA synovial tissue and RASF cells were significantly increased (P<0.01), while the expression levels of miR-449 were significantly decreased (P<0.01). The dual-luciferase reporter gene experiment confirmed that miR-449 could specifically bind to the 3' untranslated region of HDAC1 to inhibit its luciferase activity (P<0.05). HDAC1 inhibition or miR-449 overexpression significantly inhibited the proliferation and invasion of RASFs (P<0.001), while inducing their apoptosis (P<0.001). HDAC1 overexpression reversed the biological effects of miR-449 on RASFs (P<0.001). miR-449 inhibits the proliferation and invasion of RASFs and induces their apoptosis by targeting HDAC1, thereby exerting a protective effect against RA.

MiR-16-5p Regulates PTPN4 and Affects Cardiomyocyte Apoptosis and Autophagy Induced by Hypoxia/Reoxygenation.

Objectives To explore the effects of miR-16-5p and PTPN4 on the apoptosis and autophagy of AC16 cardiomyocytes after hypoxia/reoxygenation treatment. Methods AC16 cells were divided into the control group (NC), hypoxia/reoxygenation group (H/R), knockdown miR-16-5p negative control group (NC inhibitor), knockdown miR-16-5p group (miR-16-5p inhibitor), overexpression miR-16-5p negative control group (NC mimics), overexpression miR-16-5p group (miR-16-5p mimics), silent PTPN4 negative control group (sh-NC), silent PTPN4 group (sh-PTPN4), and silent PTPN4 + knockdown miR-16-5p group (sh-PTPN4 + miR-16-5p inhibitor). Real-time fluorescent quantitative PCR (RT-qPCR) and western blotting (WB) were used to measure the expression level of miR-16-3p, miR-16-5p, protein tyrosine phosphatase nonreceptor type 4 (PTPN4), and autophagy-related proteins (beclin-1, LC3 II/I, and P26) in AC16 cells. The apoptosis level of AC16 cells in each group was measured by flow cytometry and TUNEL. The dual-luciferase reporter gene experiment was also used to verify the targeting relationship between miR-16-5p and PTPN4. Results After H/R treatment, the levels of myocardial injury markers including LDH and CK-MB in AC16 cells were increased significantly (P < 0.05), and the levels of cell apoptosis and autophagy also increased significantly (P < 0.05). The level of miR-16-3p in AC16 cells did not change significantly after H/R treatment, whereas the level of miR-16-5p was increased significantly (P < 0.05). After miR-16-5p was knocked down, the levels of LDH and CK-MB in AC16 cells treated with H/R were significantly reduced (P < 0.05), and the rates of cell apoptosis and autophagy were also significantly reduced (P < 0.05). miR-16-5p negatively regulated the expression level of PTPN4 protein in AC16 cells (P < 0.05), and the dual-luciferase reporter gene experiment confirmed that PTPN4 was the downstream target of miR-16-5p. Silencing of PTPN4 significantly increased the damage of AC16 cells induced by H/R treatment (P < 0.05), but simultaneously inhibiting the expression of PTPN4 and miR-16-5p reversed the protective effect of miR-16-5p knockdown on AC16 cells (P < 0.05). Conclusions The expression of miR-16-5p is upregulated in AC16 cells after H/R treatment and the knockdown which can protect AC16 cells from H/R-induced cell damage that may be due to its regulation on the expression of PTPN4.

Serine/Arginine Repetitive Matrix 2 Antisense RNA 1 Negatively Regulates miR-370-3p and Promotes Hyperplasia, Migration, and Aggression of the Colon Cancer Cell Line

The purpose of this study is to explore the effect and possible machine-processing of the long non-coding RNA (lncRNA) SRRM2-AS1 in the development and pathogenesis of colorectal cancer. LncRNA plays an important role in tumorigenesis and development. LncRNA can regulate gene transcription and translation, cell proliferation, differentiation and apoptosis by affecting gene expression pathways of various coding proteins. SRRM2-AS1 is a kind of lncRNA. Studies have confirmed that the expression of SRRM2-AS1 is increased in colon adenocarcinoma tissues of colon cancer patients and is closely related to the prognosis of patients. However, the influence and molecular mechanism of SRRM2-AS1 on the malignant biological behavior of colon cancer cells are no yet clear. SRRM2-AS1 may interact with miR-370-3p. Studies have confirmed that overexpression of miR-370-3p can inhibit the proliferation and epithelial-mesenchymal transition of colon cancer cells in vitro. However, it is not yet clear whether SRRM2-AS1 can target miR-370-3p to affect the occurrence and development of tumors. In this study, RT-qPCR was employed to detect levels of SRRM2-AS1 and miRNA-370-3p in carcinoma tissues and corresponding paracarcinoma tissues from 41 patients with colon cancer. SW1116 colon cancer cells were cultured in vitro and separated into 4 groups: (1) si-NC group, (2) si-SRRM2-AS1 group, (3) si-SRRM2-AS1+anti-miRNA-NC group, and (4) si-SRRM2-AS1+anti-miRNA-370-3p group. The CCK-8 assay and colony formation experiment was employed to gauge cell proliferation. The scratch test was used to detect cell migration while the transwell assay was used to detect cell invasion. Finally, Western blot analysis was employed to detect levels of Ki67, E-cadherin, and N-cadherin proteins in colorectal cancer cells. The dual-luciferase reporter gene experiment verified that SRRM2-AS1 regulates miRNA-370-3p. The study found that compared to paracarcinoma tissue, levels of SRRM2-AS1 in colon cancer tissues was increased (P &lt; 0.05). Compared to the si-NC group, the SW1116 cell OD value, number of colonies formed, scratch healing rate, number of invasive cells, and expression levels of Ki67 and N-cadherin protein in the si-SRRM2-AS1 group were all decreased (P &lt; 0.05). However, E-cadherin protein levels were elevated (P &lt; 0.05). SRRM2-AS1 negatively regulates levels of miRNA-370-3p in SW1116 cells. Compared to the si-SRRM2-AS1+anti-miRNA-NC group, SW1116 cell OD value, number of colonies formed, scratch healing rate, number of invasive cells, and Ki67 and N-cadherin protein levels were increased (P &lt; 0.05) in the si-SRRM2-AS1+anti-miRNA-370-3p group. Conversely, E-cadherin protein levels were decreased (P &lt; 0.05). These findings indicate that SRRM2-AS1 is predominately expressed in cancerous colon tissues. Attenuating expression of SRRM2-AS1 may curb the hyperplasia of colon carcinoma cell line SW1116 and promote cell apoptosis by regulating miRNA-370-3p expression.

LncRNA GAS5 modulates the progression of non-small cell lung cancer through repressing miR-221-3p and up-regulating IRF2

BackgroundLong non-coding RNA growth arrest specific 5 (GAS5) is a regulator in non-small cell lung cancer (NSCLC) progression. Nonetheless, the mechanism by which GAS5 exerts its biological function in NSCLC cells remains unclear.MethodsGAS5, miR-221-3p relative expression levels in NSCLC tissues and cells were examined by qPCR. After gain-of-function and loss-of-function models were established, the viability of H1299 and A549 cells were examined by CCK-8 and EdU assays. Cell migration and invasion were examined by the Transwell experiment. The binding sequence of GAS5 for miR-221-3p was confirmed by the dual-luciferase reporter gene experiment. The regulatory function of GAS5 and miR-221-3p on IRF2 was investigated by Western blot.ResultsGAS5 expression was down-modulated in NSCLC tissues and cell lines. GAS5 overexpression restrained the proliferation, migration and invasion of NSCLC cells, while miR-221-3p, which was targeted and negatively modulated by GAS5, worked oppositely. Restoration of miR-221-3p markedly reversed the effects of GAS5 on NSCLC cells. Additionally, GAS5 increased IRF2 expression in NSCLC cells by repressing miR-221-3p.ConclusionsGAS5 blocks the progression of NSCLC partly via increasing IRF2 expression level via repressing miR-221-3p.

The GWAS Analysis of Body Size and Population Verification of Related SNPs in Hu Sheep.

Body size is an important indicator of growth and health in sheep. In the present study, we performed Genome-Wide Association Studies (GWAS) to detect significant single-nucleotide polymorphisms (SNPs) associated with Hu sheep’s body size. After genotyping parental (G1) and offspring (G2) generation of the nucleus herd for meat production of Hu sheep and conducting GWAS on the body height, chest circumference, body length, tail length, and tail width of the two groups, 5 SNPs associated with body height and 4 SNPs correlated with chest circumference were identified at the chromosomal significance level. No SNPs were significantly correlated to body length, tail length, and width. Four out of the 9 SNPs were found to be located within the 4 genes. KITLG and CADM2 are considered as candidate functional genes related to body height; MCTP1 and COL4A6 are candidate functional genes related to chest circumference. The 9 SNPs found in GWAS were verified using the G3 generation of the nucleus herd for meat production. Nine products were amplified around the 9 sites, and 29 SNPs were found; 3 mutation sites, G > C mutation at 134 bp downstream of s554331, T > G mutation at 19 bp upstream of s26859.1, and A > G mutation at 81 bp downstream of s26859.1, were significantly correlated to the body height. Dual-luciferase reporter gene experiments showed that the 3 SNPs could significantly impact dual-luciferase and gene transcription activity.

LINC00958 promotes proliferation, migration, invasion, and epithelial-mesenchymal transition of oesophageal squamous cell carcinoma cells.

Oesophageal cancer is one of the deadliest cancers in the world. Oesophageal squamous cell carcinoma (ESCC) is the most prevalent histological type of oesophageal cancer. Oesophageal cancer has a poor prognosis because of its invasiveness. Thus, it is especially important to seek effective treatment methods. Research indicates that long non-coding RNAs (lncRNAs) play a significant role in the occurrence and development of oesophageal cancer. The aim of this study was to describe the role of LINC00958 in ESCC. Bioinformatics and real-time quantitative polymerase chain reaction (RT-qPCR) methods were utilized to predict and verify the expression of LINC00958 in ESCC. Related functional experiments, including cell proliferation, migration and invasion, were performed. In addition, a western blot and a dual luciferase reporter gene experiment were used to study the detailed carcinogenic mechanism of LINC00958. The results indicated there was a high expression of LINC00958 in ESCC, which promoted proliferation, migration, invasion and Epithelial-Mesenchymal Transition (EMT) of ESCC cells, and this effect may be via regulating miR-510-5p.

LINC00958 promotes endometrial cancer cell proliferation and metastasis by regulating the miR‐145‐3p/TCF4 axis

Long noncoding RNAs (lncRNAs) exert an essential regulatory role in cancer progression. This work focuses on the role of LINC00958 in endometrial cancer (EC). LINC00958 expression in EC tissues was examined by GEPIA database and TCGA-UCEC dataset. LINC00958, miR-145-3p, and TCF4 mRNA expression levels in EC tissues and cells were examined by qRT-PCR. Western blot was employed to determine TCF4, E-cadherin, and N-cadherin protein expression levels. After LINC00958 was overexpressed or silenced, cell proliferation was determined using Cell Counting Kit 8 (CCK-8) and bromodeoxyuridine (BrdU) incorporation experiments. Cell migration and invasion were examined by Transwell experiment. Dual-luciferase reporter gene or RNA immunoprecipitation (RIP) experiments were executed to validate the targeting relationships among LINC00958 and miR-145-3p and TCF4. The effects of LINC00958 on EC cell proliferation and metastasis were investigated in vivo using a nude mouse subcutaneous graft model and a caudal vein injection model. LINC00958 was remarkably upmodulated in EC. Moreover, its overexpression was strongly linked to unfavorable overall survival of the patients. Functional experiments confirmed that in vitro knockdown of LINC00958 suppressed EC cell proliferation and metastasis. LINC00958 was validated to decoy miR-145-3p and repressed its expression, and TCF4 was uncovered to be a target gene of miR-145-3p and negatively modulated by miR-145-3p. Furthermore, the function of LINC00958 was dependent on its regulation of miR-145-3p and TCF4. LINC00958 acts as an oncogenic lncRNA to regulate EC progression by modulating the miR-145-3p/TCF4 axis. Knockdown of LINC00958 impedes tumor growth and metastasis in vitro and in vivo, opening a new avenue for therapeutic intervention.

MicroRNA-125b and its downstream Smurf1/KLF2/ATF2 axis as important promoters on neurological function recovery in rats with spinal cord injury.

The purpose of this study is to investigate the role of microRNA‐125b (miR‐125b) and its mechanism in spinal cord injury (SCI) by targeting Smurf1. After loss‐ and gain‐function approaches were conducted in SCI rat models and neural stem cells (NSCs) isolated from foetal rats, the Basso‐Beattie‐Bresnahan (BBB) score was calculated, and related protein expression was determined by Western blot analysis and cell apoptosis by TUNEL staining. NSC viability was detected by CCK‐8, migration abilities by Transwell assay and apoptosis by flow cytometry. The relationship between miR‐125b, Smurf1 and KLF2 was evaluated by dual‐luciferase reporter gene experiments, Co‐IP and in vivo ubiquitin modification assays. Inhibition of miR‐125b and KLF2 and the up‐regulation of Smurf1 and ATF2 were observed in SCI rats. BBB scores were elevated, the expression of Nestin, NeuN, GFAP, NF‐200 and Bcl‐2 protein was enhanced but that of Bax protein was reduced, and cell apoptosis was inhibited in SCI rats after up‐regulating miR‐125b or silencing ATF2. Smurf1 was a target gene of miR‐125b, which promoted KLF2 degradation through its E3 ubiquitin ligase function, and KLF2 repressed the expression of ATF2 in NSCs. The results in vivo were replicated in vitro. miR‐125b overexpression promotes neurological function recovery after SCI.

Effects of silencing circRNA ABCB10 expression on biological properties of colorectal cancer cells

Objective: To investigate the expression of circular ribonucleic acid ABCB10 (circABCB10) in colorectal cancer tissues and cells and its effects on cell biological behavior, radiosensitivity and growth of subcutaneous xenografts. Methods: The tumor tissue and adjacent tissue from colorectal cancer patients treated in Henan People's Hospital were collected from January 2018 to December 2018. Quantitative polymerase chain reaction (qPCR) was used to detect the expressions of circABCB10 and miR-217, cell viability was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT), cell apoptosis rate was detected by flow cytometry, cell migration and invasion were detected by Transwell method, cell radiosensitivity was detected by colony formation assay. The downstream miRNAs of circABCB10 were predicted by Circular RNA Interactome and verified by the dual luciferase reporter gene experiment. The effect of circABCB10 on the growth of transplanted tumor was examined in nude mice. Results: The expression level of circABCB10 mRNA in colorectal cancer tissues was (3.97±2.12), higher than (1.13±0.64) in adjacent tissues (P<0.05). The expression level of circABCB10 mRNA in FHC cells was (1.00±0.09), lower than that (4.53±0.44) in SW480, (3.12±0.32) in HCT116 and (3.51±0.36) in HT29 cells, respectively (all P<0.05). The MTT results showed that the absorbance values of SW480 cells in si-circABCB10-1 group at 48 and 72 hours after transfection were (0.36±0.04) and (0.43±0.04), lower than (0.48±0.05) and (0.82±0.08) in circ-negative control (NC) group, respectively (all P<0.05). The number of migrating cells and invasive cells in si-circABCB10-1 group were (45±8) and (34±7), lower than (106±21) and (84±15) in circ-NC group, respectively (all P<0.01). The radiosensitization ratio was 1.632. The results of subcutaneous transplantation assay showed that the tumor volume and tumor weight of the si-circABCB10-1 group were significantly lower than circ-NC group after 8 days of inoculation ( all P<0.05). MiR-217 is a target gene of circABCB10. Inhibition of miR-217 reversed the inhibitory effect of circABCB10 silencing on cell proliferation, migration, invasion and subcutaneous xenograft growth in nude mice and the radiosensitization activity. Conclusion: Silence of circABCB10 can up-regulate the expression of miR-217 to inhibit the proliferation, migration, invasion and growth of subcutaneous xenografts and increase the radiosensitivity of SW480 cells, which reveals the underlying molecular mechanism of colorectal cancer progression and provides a new sensitizing target for clinical radiotherapy of colorectal cancer.

MicroRNA-181a promotes the proliferation of hypertrophic scar fibroblasts and inhibits their apoptosis via targeting phosphatase and tensin homolog.

This study aimed to compare the expression of microRNA (miR)-181a and phosphatase and tensin homolog (PTEN) in hypertrophic scar tissue and cells and to explore the effects of miR-181a and PTEN on the proliferation and apoptosis of the human scar fibroblast cell line HSFb. HSFb cells were transfected with miR-negative control (miR-NC), miR-181a mimics, miR-181a inhibitor, pcDNA3.1-PTEN (pc-PTEN), or small interfence-PTEN (si-PTEN) plasmid using a Lipofectamine 2000 transfection kit. The effects of miR-181a and PTEN on the proliferation and apoptosis of HSFb were determined using a Cell Counting Kit (CCK)-8 experiment and flow cytometry, respectively. The effects of miR-181a and PTEN on the expression of apoptosis-related proteins in HSFb, including type I collagen (Col-1) and type III collagen (Col-3), were measured by western blot. Finally, the relationship between miR-181a and PTEN was explored by the dual-luciferase reporter gene experiment. The miR-181a in hypertrophic scar tissues and HSFb were significantly up-regulated compared to embryo skin fibroblast (ESF-1) cells and normal tissues (P<0.05), whereas the opposite results were seen for PTEN expression (P<0.05). Inhibiting miR-181a or upregulating the expression of PTEN significantly suppressed the proliferation of HSFb (P<0.05) and induced their apoptosis (P<0.05). Western blot revealed that inhibiting and upregulating miR-181a and PTEN, respectively, decreased the expression of the B-cell lymphoma-2 (Bcl-2), Col-1, and Col-3 proteins in HSFb, but significantly up-regulated the expression of Bcl-2-associated X protein (Bax), cleaved caspase-3 (c-caspase-3), and cleaved caspase-9 (c-caspase-9) (P<0.05). The dual-luciferase reporter gene experiment results confirmed PTEN to be the downstream target gene of miR-181a. Simultaneous upregulation of miR-181a and PTEN expression had no significant effect on the proliferation and apoptosis of HSFb. miR-181a promotes the up-regulation of Col-1 and Col-3, and regulates the proliferation and apoptosis of HSFb by targeting PTEN, thereby enhancing the formation of hypertrophic scarring (HS). Therefore, miR-181a and PTEN may be potential therapeutic targets for the treatment of HS.

Circ_0001806 Promotes the Proliferation, Migration and Invasion of NSCLC Cells Through miR-1182/NOVA2 Axis.

BackgroundCircular RNAs (circRNAs) are non-coding RNAs with a covalently closed loop. circRNAs affect the progression of diverse cancers. Nonetheless, circ_0001806 expression and function in non-small cell lung cancer (NSCLC) are undefined.MethodsqRT-PCR was executed to examine circ_0001806, miR-1182 and NOVA2 mRNA expression levels in NSCLC tissues and cells. CCK-8, EdU, cell scratch test and Transwell assay were conducted to examine the viability, multiplication, migration and invasion of NSCLC cell lines H1650 and HCC827. The binding sites between circ_0001806 and miR-1182, miR-1182 and NOVA2 mRNA were predicted by the circular RNA Interactome and TargetScan databases, and the dual-luciferase reporter gene experiment was employed for verification. Western blot was implemented to examine NOVA2 expression.ResultsCirc_0001806 expression in NSCLC tissues and cell lines was substantially augmented, while miR-1182 expression was markedly decreased. Circ_0001806 facilitated the multiplication, migration and invasion of H1650 and HCC827 cells, while miR-1182 exerted the opposite effect. Circ_0001806 indirectly enhanced NOVA2 expression by specifically down-modulating miR-1182.ConclusionCirc_0001806 augments NOVA2 expression by targeting miR-1182 to enhance the multiplication, migration and invasion of NSCLC cells.

Effect of Long Non-Coding RNA Small Nucleolar RNA Host Gene 14 Targeting miR-93-5p on Oxidized Low-Density Lipoprotein-Induced Endothelial Cell Injury and Its Mechanism

To explore the influence and mechanism of lncRNA SNHG14 on oxidized low-density lipoprotein (oxLDL)-induced vascular endothelial cell injury, cellular levels of SNHG14 and miRNA-93-5p were detected in oxLDL-induced vascular endothelial cells by RT-qPCR, and the levels of MDA, SOD and GSH-Px were detected by ELISA. Flow cytometry was used to detect cellular apoptosis, and western blot analysis was used to determine the abundance of Bcl-2 and Bax proteins. SNHG14 small interfering RNA or miRNA-93-5p mimics were transfected into vascular endothelial cells to interfere with SNHG14 expression or overexpress miRNA-93-5p. To study the effects of interfering with SNHG14 expression or overexpression of miRNA-93-5p, the levels of MDA, SOD and GSH-Px, apoptosis and the levels of Bcl-2 and Bax protein were studied in oxLDL-induced endothelial cells with either altered SNHG14 expression or overexpressed miRNA-93-5p. A dual-luciferase reporter gene experiment verified the regulatory connection between SNHG14 and miRNA-93-5p. After oxLDL acted on vascular endothelial cells, the expression levels of SNHG14 were significantly increased, while the expression levels of miRNA-93-5p were significantly reduced, MDA levels were increased, and SOD and GSH-Px activities were reduced. Both the apoptosis rate and Bax protein levels were significantly increased, and Bcl-2 expression was reduced. Interference with SNHG14 expression or overexpression of miRNA-93-5p can reduce the MDA content in oxLDL-induced vascular endothelial cells, increase the activity of SOD and GSH-Px, and reduce the apoptosis rate and Bax protein levels, and promote Bcl-2 expression. SNHG14 targeted to negatively regulate miRNA-93-5p expression, inhibited miRNA-93-5p expression and reversed the interference of SNHG14 expression with oxLDL-induced variation in MDA, SOD and GSH-Px levels, apoptosis rate, and Bax and Bcl-2 protein levels. Interference of SNHG14 expression may reduce oxidative stress and apoptosis of vascular endothelial cells induced by oxLDL by negatively regulating the expression of miRNA-93-5p.

MiR-378a-5p inhibits the proliferation of colorectal cancer cells by downregulating CDK1

BackgroundMicroRNAs (miRNAs) play an important role in tumor occurrence. The role of miR-378a-5p and CDK1 in colorectal cancer (CRC) was investigated in this study.MethodsInvestigation of TCGA database and the detection of miR-378a-5p expression in colorectal cancer pathological tissues and colorectal cancer cell lines were undertaken by using qRT-PCR. We performed cell function experiments (CCK-8 assay, EdU assay, colony formation assay, wound healing assay, transwell assay, cell apoptosis assessment, and cell cycle assessment) and nude mouse tumor formation experiments to evaluate the effects of miR-378a-5p on proliferation, metastasis, and invasion to explore the role of miR-378a-5p in vivo and in vitro. Next, through TCGA database, immunohistochemical staining of pathological tissues, and cell function experiments, the role of the target gene CDK1 of miR-378a-5p was verified by database prediction, and dual luciferase reporter gene experiments in colorectal cancer cells were performed. Finally, whether upregulation of CDK1 restores the inhibitory effect of overexpression of miR-378a-5p on the proliferation of CRC cells was studied by overexpression of CDK1.ResultsBioinformatic analysis showed significant downregulation of miR-378a-5p levels in colorectal cancer (CRC). Cell function experiments and tumor xenograft mouse models confirmed the low expression of miR-378a-5p within CRC tissues, which indicated the tumor suppressive role of miR-378a-5p in CRC. To better explore the regulation of miR-378a-5p in CRC, we predicted and validated cell cycle-dependent protein kinase 1 (CDK1) as the miR-378a-5p target gene and observed that miR-378a-5p suppressed CRC cell proliferation by targeting CDK1.ConclusionThe results of this study help to elucidate the mechanism by which miR-378a-5p can be used as a tumor marker to inhibit the growth of colorectal cancer and CDK1, which is related to the prognosis of colorectal cancer patients. MiR-378a-5p inhibits CRC cell proliferation by suppressing CDK1 expression, which may become a possible therapeutic target for treatment of CRC.

LINC00649 promotes bladder cancer malignant progression by regulating the miR‑15a‑5p/HMGA1 axis.

The aim of the present study was to explore the effects of LINC00649 on the proliferation, migration and invasion of bladder cancer (BC) and identify possible mechanisms. Through TCGA database analysis of LINC00649 expression in bladder cancer and the association of LINC00649 with the BC patient prognosis, RT-qPCR was employed for detecting LINC00649 expression in 60 clinical tissue specimens and cell lines of bladder cancer. The lentivirus stable transfection or small interfering RNA was used to increase or decrease the LINC00649 expression level in T24 and UM-UC-3 cells. CCK8 and clone formation assay were utilized to observe the effects of LINC00649 on the proliferation and colony formation of BC cells. Transwell experiment was performed to detect the effects of LINC00649 on the migration and invasion of bladder cancer. Bioinformatics database was used to identify the possible downstream targets of LINC00649 while RT-qPCR, western blot analysis and dual luciferase reporter gene experiments were carried out to verify the possible molecular mechanism. The TCGA database analysis revealed a significantly high expression of LINC00649 in bladder cancer and an association of LINC00649 expression with overall survival rate of BC patients. As shown by RT-qPCR detection, LINC00649 expression was notably upregulated in BC tissues and BC cell lines. In addition, statistical analyses unveiled that highly expressed LINC00649 was clearly associated with poor overall survival of bladder cancer. Based on the in vitro cell experiment, upregulated LINC00649 considerately enhanced the proliferation, migration and invasion of BC cells, as opposed to those in T24 and UM-UC-3 cells by suppressing LINC00649. Mechanically, LINC00649 may promote the malignant progression of bladder cancer by regulating miR-15a-5p to promote the HMGA1 expression axis. Overall, LINC00649 upregulates HMGA1 expression by binding to miR-15a-5p to enhance the proliferation, migration and invasion of BC cells. Thus, LINC00649 is a potential biomarker and therapeutic target for bladder cancer.