Circ-CSNK1G1 promotes cell proliferation, migration, invasion and glycolysis metabolism during triple-negative breast cancer progression by modulating the miR-28-5p/LDHA pathway.

BACKGROUND: Circular RNAs (circRNAs) play a vital role in cancer progression. However, there are still numerous circRNAs that have not been functionally explored. Our study aimed to disclose the role of circ-CSNK1G1 in triple-negative breast cancer (TNBC). METHODS: The expression of circ-CSNK1G1, miR-28-5p and lactate dehydrogenase A (LDHA) mRNA was measured by quantitative real-time polymerase chain reaction (qPCR), and the expression of LDHA protein was measured by western blot. Cell proliferation was assessed using MTT assay and colony formation assay. Cell apoptosis was monitored using flow cytometry assay. Cell migration and cell invasion were investigated using transwell assay. Glycolysis progression was assessed according to glucose consumption, lactate production and ATP/ADP ratio. Tumor formation assay in nude mice was conducted to verify the role of circ-CSNK1G1 in vivo. The interplays between miR-28-5p and circ-CSNK1G1 or LDHA were confirmed by dual-luciferase reporter assay. RESULTS: Circ-CSNK1G1 was upregulated in TNBC tissues and cells. Circ-CSNK1G1 knockdown suppressed cancer cell proliferation, migration, invasion and glycolysis energy metabolism, promoted cell apoptosis in vitro, and blocked tumor growth in vivo. Mechanism analysis showed that circ-CSNK1G1 positively regulated LDHA expression by suppressing miR-28-5p. Rescue experiments presented that circ-CSNK1G1 played functions by targeting miR-28-5p, and miR-28-5p participated in TNBC progression by degrading LDHA. CONCLUSION: Circ-CSNK1G1 promotes cell proliferation, migration, invasion and glycolysis metabolism during TNBC development by regulating the miR-28-5p/LDHA pathway.

miR-28-5p improved carotid artery stenosis by regulating vascular smooth muscle cell proliferation and migration.

OBJECTIVES: Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are involved in carotid artery stenosis. The purpose of this study was to investigate the diagnostic value of serum miR-28-5p in asymptomatic carotid artery stenosis and its regulation on the proliferation and migration of VSMCs. METHODS: Serum miR-28-5p levels in 65 healthy controls and 68 asymptomatic carotid artery stenosis patients were detected by qRT-PCR. The receiver-operating characteristic curve was applied to elucidate the diagnostic value of serum miR-28-5p for carotid artery stenosis patients. The specificity of miRNA targets was detected by luciferase reporter assay. CCK-8 and Transwell assay were applied to detect proliferation and migration of cells. Pearson correlation test was used to investigate the correlation between Forkhead box subclass O 1 (FOXO1) and serum miR-28-5p. RESULTS: Serum miR-28-5p was significantly reduced in asymptomatic carotid artery stenosis patients. Moreover, miR-28-5p could distinguish asymptomatic carotid artery stenosis patients from healthy controls, with sensitivity and specificity of 86.8% and 81.5%, respectively, indicating its high diagnostic value. The overexpression of miR-28-5p inhibited the proliferation and migration of VSMCs, while inhibition of miR-28-5p resulted in the opposite effect. What is more, FOXO1, a direct target of miR-28-5p, was significantly increased in asymptomatic carotid artery stenosis patients. Inhibition of miR-28-5p in VSMCs reversed the reduction of FOXO1 levels in patients. CONCLUSIONS: miR-28-5p is a valuable diagnostic biomarker for asymptomatic carotid artery stenosis and can affect the proliferation and migration of VSMCs by regulating FOXO1.

The miR-28-5p-CAMTA2 axis regulates colon cancer progression via Wnt/ß-catenin signaling.

BACKGROUND: Colon cancer is the third most commonly diagnosed cancer with high morbidity and mortality. Calmodulin-binding transcription activator 2 (CAMTA2) belongs to the calmodulin-binding transcription activator protein family. The functional role of CAMTA2 in colon cancer development remains unclear. Our research found out that CAMTA2 was high-level expressed in colon cancer, and the upregulated CAMTA2 expression was markedly correlated with poor survival. Functional experiments showed that knockdown of CAMTA2 repressed colon cancer cell proliferation/migration in vitro and attenuated proliferation in vivo. In additional, CAMTA2 expression was controlled by miR-28-5p via posttranscriptional regulation and miR-28-5p expression was reversely correlated with CAMTA2 expression in colon cancer. Moreover, enforced miR-28-5p expression downregulated the expression of CAMTA2 significantly and the restoration of CAMTA2 expression abolished the inhibitory effect of miR-28-5p on colon cancer cell proliferation and metastasis. Mechanistically, overexpression of miR-28-5p suppressed Wnt/ß-catenin signaling and the inhibitory could be partly abolished by overexpression of CAMTA2. In summary, our findings reveal that miR-28-5p/CAMTA2 axis plays a critical role in human colon cancer, which might be a promising diagnosis and therapeutic target for colon cancer treatment.

LncRNA LOXL1-AS1/miR-28-5p/SEMA7A axis facilitates pancreatic cancer progression.

Pancreatic cancer (PC), one of the most aggressive and lethal human malignancies, is associated with a deplorable prognosis despite progressive therapeutic strategies. Emerging evidence manifests that miR-28-5p is involved in several cancers, and its descending expression is associated with poor prognosis. Nevertheless, the function of miR-28-5p in PC remains unclear. Thus, the underlying regulatory mechanism of miR-28-5p in PC is urgent to be clarified. In the present study, we first recognized miR-28-5p was downregulated in PC, and miR-28-5p overexpression inhibited cell proliferation and migration in PC. Then miR-28-5p was verified to act as a molecular sponge of LOXL1-AS1. Therefore, the function of LOXL1-AS1 was further explored in PC, presenting that LOXL1-AS1 suppression inhibited cell proliferation and migration. What is more, SEMA7A was found to be a target gene for miR-28-5p and was upregulated in PC. In addition, LOXL1-AS1 could positively regulate SEMA7A expression while miR-28-5p could negatively regulate SEMA7A expression. According to rescue experiments, SEMA7A overexpression partially neutralized LOXL1-AS1 silence-mediated inhibitory function on progression in PC. Taken together, all the data demonstrated that LOXL1-AS1/miR-28-5p/SEMA7A axis facilitated pancreatic cancer progression, which may be regarded as an innovative therapeutic target for PC treatment. SIGNIFICANCE OF THE STUDY: Our findings constitute the first report to delineate that lncRNA LOXL1-AS1/miR-28-5p/SEMA7A axis facilitates PC progression. According to our experimental results, we found the expression of miR-28-5p was downregulated in PC cells and miR-28-5p overexpression inhibited cell proliferation and migration in PC. LOXL1-AS1 could sponge miR-28-5p and then upregulate the expression of SEMA7A. Thus, LOXL1-AS1/miR-28-5p/SEMA7A axis facilitated PC progression. This initially proposed point might provide a novel molecular target for PC treatment.

miR-28-5p targets MTSS1 to regulate cell proliferation and apoptosis in esophageal cancer.

Esophageal cancer (EC) is one of the most common aggressive malignant diseases worldwide. miR-28-5p plays important regulatory roles in many cancers including human EC. However, the molecular mechanism and potential role of miR-28-5p in EC remain uncertain. In this study, qRT-PCR and western blot analysis revealed that miR-28-5p expression was up-regulated and metastasis suppressor-1 (MTSS1) was down-regulated in EC tissues relative to matched para-cancer tissues. Cell counting kit-8 (CCK-8) assay demonstrated that miR-28-5p mimics increased cell viability, and miR-28-5p inhibitor decreased it. Flow cytometry (FCM) assay indicated that miR-28-5p mimics promoted cell cycle entry, while miR-28-5p inhibitor reduced it and induced cell apoptosis. Moreover, miR-28-5p mimics up-regulated the expressions of cyclin A, cyclin dependent kinase 2 (CDK2), cyclin D1, and cyclin E but down-regulated the expressions of cleaved caspase-3 and cleaved caspase-9, which was abolished by miR-28-5p inhibitor. Furthermore, luciferase reporter assay verified that miR-28-5p directly targeted MTSS1 3'UTR and down-regulated its expression. MTSS1 overexpression in TE-1 cells inhibited cell proliferation and promoted apoptosis induced by miR-28-5p mimics, whereas silencing of MTSS1 reversed cell progression induced by miR-28-5p inhibitor. We also demonstrated that miR-28-5p could promote esophageal tumor formation in vivo. Hematoxylin-eosin staining, immunohistochemistry, and TUNEL assays confirmed that miR-28-5p antagomir inhibited cell growth and accelerated apoptosis. Our results suggest that miR-28-5p may induce cell proliferation and suppress apoptosis to promote EC tumor formation via decreasing MTSS1 expression. Thus, miR-28-5p may be a potential target for human EC therapy.

LncRNA-UCA1 modulates progression of colon cancer through regulating the miR-28-5p/HOXB3 axis.

Emerging evidence has shown that the long noncoding RNA urothelial carcinoma-associated 1 (UCA1) plays a tumor-promoting role in colorectal cancer, while miR-28-5p shows tumor-inhibitory activity in several tumor types. However, the mechanisms both of these in colon cancer progression are still unknown. In this work, the detailed roles and mechanisms of UCA1 and its target genes in colon cancer were studied. The results showed that UCA1 was upregulated in colon cancer tissues when compared with the adjacent nonhumorous tissues, as well as in the various colon cancer cell lines, but the expression of miR-28-5p showed an opposite trend. Furthermore, a high UCA1 level in colon cancer tissues is positively associated with the tumor size and advanced tumor stages. Functional assays revealed that both UCA1 knockdown and miR-28-5p overexpression could inhibit colon cancer cell growth and migration. Further mechanistic studies indicated that UCA1 knockdown played tumor suppressive roles in SW480 and HT116 cells through binding with miR-28-5p. We also, for the first time, identified HOXB3 as the target gene of miR-28-5p and that HOXB3 overexpression could mediate the functions of UCA1 in cell proliferation and migration of colon cancer cells. In conclusion, our data provided evidence for the regulatory network of UCA1/miR-28-5p/HOXB3 in colon cancer, suggesting that UCA1, miR-28-5p, and HOXB3 are the potential targets for colon cancer therapy.

Strand-specific miR-28-3p and miR-28-5p have differential effects on nasopharyngeal cancer cells proliferation, apoptosis, migration and invasion.

BACKGROUND: MicroRNAs (miRNAs) play crucial roles in varieties of cancers, particularly in tumorigenesis, progression, and migration. Dysregulation of miR-28 was reported to occur in various types of human malignancies. In humans, two different mature miRNA sequences are excised from opposite arms of the stem-loop pre-miR-28, hsa-miR-28-3p and hsamiR-28-5p. However, the expression and distinct role of miR-28-3p and miR-28-5p in nasopharyngeal carcinoma (NPC) remain undetermined. METHODS: The expressions of miR-28-3p/-5p in human NPC tissues were tested by quantitative real-time PCR. miR-28-3p/-5p were overexpressed by mimics and silenced by inhibitors. The roles of miR-28-3p/-5p in NPC development were studied using cultured HONE-1 cells. RESULTS: The mRNA expression levels of miR-28-3p and -5p were significantly decreased in NPC tissues in comparison with adjacent normal tissues. Overexpression of miR-28-5p suppressed NPC cell proliferation and induced cell cycle arrest and apoptosis, while miR-28-3p promoted NPC cell migration and invasion. The miRNAs effected on different signal pathways: miR-28-5p altered expression of cyclin D1 and influenced the PI3K/AKT signaling pathway. In contrast, miR-28-3p downregulated Nm23-H1 and accelerated the process of EMT. CONCLUSION: miR-28-3p and -5p were both downregulated in NPC tissues but had distinct biological effects in NPC cells. They may serve as potential prognostic markers and therapeutic targets for NPC.

NDRG2 mRNA levels and miR-28-5p and miR-650 activity in chronic lymphocytic leukemia.

BACKGROUND: NDRG2 is identified as a tumor suppressor gene in many tumors, and functions in cell proliferation, differentiation and apoptosis. Recent data indicate that NDRG2 expression is up-regulated by TP53. Moreover, proposed mechanisms of NDRG2 inactivation include epigenetic silencing of the NDRG2 promoter and down-regulation by microRNAs (miRNAs). However, few studies have ever been done on the role of NDRG2 and the NDRG2-regulating miRNAs interference in chronic lymphocytic leukemia (CLL). METHODS: NDRG2 and microRNAs mRNA levels in CLL subjects were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The dual-luciferase reporter assay was performed to determine NDRG2-related miRNAs. Low expression of mature exogenous miRNAs in CLL cells was established by transient transfection. NDRG2 protein levels in CLL cells were detected by western blot. In addition, flow cytometry was conducted to examine the apoptosis of CLL cells. RESULTS: Lower expression of NDRG2 was found in the B-cells from 102 CLL patients compared the 40 normal subjects (P < 0.001). Patients with advanced Binet stage (P = 0.001), high lactate dehydrogenase (LDH) level (P = 0.036), un-mutated immunoglobulin heavy chain variable region gene (IGHV) (P = 0.004) and those with p53 aberrations (P < 0.001) had a markedly lower levels of NDRG2 mRNA. This decrease was associated with briefer time-to-treatment (P = 0.001) and poorer survival (P < 0.001). High expression of miR-28-5p and miR-650 was associated with Binet B/C stage (P = 0.044) and IGHV un-mutated (P = 0.011), as well as Binet B/C stage (P = 0.013) and p53 aberrations (P = 0.037), respectively. Inhibition of miR-28-5p or miR-650 could induce more apoptosis in CLL cells with germline TP53. CONCLUSIONS: NDRG2 mRNA levels might be a useful prognostic variable for patients of CLL and up-regulating NDRG2 transcription may be a therapy approach in CLL without p53 aberrations.

miR-28-5p Involved in LXR-ABCA1 Pathway is Increased in the Plasma of Unstable Angina Patients.

BACKGROUND: Previous studies confirmed that the intronic miRNAs participated in regulating host gene-primed biological processes. The coordinated roles of miR-28 with its host gene, LIM domain lipoma-preferred partner (LPP), remain unknown in atherosclerosis. METHODS: In this study, we determined to assess circulating levels of miR-28-5p in unstable angina patients, compared with age- and sex- matched control subjects by quantitative PCR. Furthermore, we attempted to explore whether miR-28-5p could influence the expression of ATP-binding cassette transporter A1 (ABCA1) and liver X receptor (LXR), major mediators of high density lipoprotein (HDL) synthesis and transportation in hepatic cells and macrophages. RESULTS: It was found that plasma levels of miR-28-5p were significantly increased in unstable angina patients with or without type 2 diabetes mellitus. Notably, miR-28-5p upregulated ABCA1 expression at transcription and translation levels, strongly correlated with translational activation of LXRα in HepG2 and THP-1-derived macrophages. CONCLUSIONS: Our findings suggest that circulating miR-28-5p, involved in LXRα-ABCA1 pathway, may be a potential biomarker for diagnosis and prognosis of unstable angina.

Transplantation of MiR-28-5p-Modified BMSCs Promotes Functional Recovery After Spinal Cord Injury.

Traumatic spinal cord injury (TSCI) is a prevalent central nervous system condition that imposes a significant burden on both families and society, affecting more than 2 million people worldwide. Recently, there has been increasing interest in bone marrow mesenchymal stem cell (BMSC) transplantation as a promising treatment for spinal cord injury (SCI) due to their accessibility and low immunogenicity. However, the mere transplantation of BMSCs has limited capacity to directly participate in the repair of host spinal cord nerve function. MiR-28-5p, identified as a key differentially expressed miRNA in spinal cord ischemia-reperfusion injury, exhibits differential expression and regulation in various neurological diseases. Nevertheless, its involvement in this process and its specific regulatory mechanisms in SCI remain unclear. Therefore, this study aimed to investigate the potential mechanisms through which miR-28-5p promotes the neuronal differentiation of BMSCs both in vivo and in vitro. Our results indicate that miR-28-5p may directly target Notch1, thereby facilitating the neuronal differentiation of BMSCs in vitro. Furthermore, the transplantation of lentivirus-mediated miR-28-5p-overexpressed BMSCs into SCI rats effectively improved footprint tests and Basso, Beattie, and Bresnahan (BBB) scores, ameliorated histological morphology (hematoxylin-eosin [HE] and Nissl staining), promoted axonal regeneration (MAP2 and growth-associated protein 43 [GAP43]), and facilitated axonal remyelination (myelin basic protein [MBP]). These findings may suggest that miR-28-5p-modified BMSCs could serve as a therapeutic target to enhance the behavioral and neurological recovery of SCI rats.

Ginsenoside Rh2 attenuates the progression of non-small cell lung cancer by sponging miR-28-5p/STK4 axis and inactivating Wnt/ß-catenin signaling.

BACKGROUND: Ginsenoside Rh2 (G-Rh2) exerts anti-tumor activity in non-small cell lung cancer (NSCLC). microRNAs (miRNAs, miRs) play pivotal roles in NSCLC. We aimed to investigate whether G-Rh2 inhibited NSCLC progression by targeting miRNA. METHODS: Cell viability, apoptosis and cycle were determined by Cell Counting Kit-8, 6-diamidino-2-phenylindole (DAPI) staining and flow cytometry. The potential target miRNAs of G-Rh2 were screened by real-time quantitative polymerase chain reaction (RT-qPCR). The difference in miR-28-5p expression between lung adenocarcinoma (LUAD) tissues and normal tissues or lung squamous cell carcinoma (LUSC) tissues and normal tissues was retrieved from TCGA-LUAD and TCGA-LUSC, respectively. Kaplan-Meier Plotter was conducted to analyze the survival rate for different serine/threonine-protein kinase 4 (STK4) expressions with different prognostic risks. immunohistochemistry of STK4 expression in non-tumor and tumor tissues was analyzed from the HPA database. RT-qPCR and Western blot were adopted for detecting mRNA and protein expression. TargetScan V7.2, miRanda and PITA were adopted for predicting targets of miR-28-5p, overlapped genes were subjected to GO analysis. The interactions of miR-28-5p-Wnt and miR-28-5p-STK4 were detected by TOP/FOP luciferase reporter assay and dual luciferase reporter assay, respectively. RESULTS: Current study observed that G-Rh2 reduced miR-28-5p expression in NSCLC cells dose-dependently. miR-28-5p was upregulated in NSCLC tissues and cells. The target genes of miR-28-5p were enriched in negative regulation of Wnt signaling. miR-28-5p inhibitor inactivated Wnt signaling, inhibited cell viability and cell cycle, while enhanced cell apoptosis of NSCLC cells by targeting STK4. G-Rh2 exerted the similar effects with miR-28-5p inhibitor by reducing miR-28-5p. G-Rh2 and miR-28-5p inhibitor exerted a synergistic effect on inhibiting NSCLC tumor growth. CONCLUSION: In conclusion, G-Rh2 attenuates NSCLC development by affecting miR-28-5p/STK4 axis and inactivating Wnt signaling. Taken together, we project out a novel therapeutic target for NSCLC.

miR-28-5p inhibits cholangiocarcinoma progression and predicts good prognosis of patients.

Cholangiocarcinoma (CCA) is one of the most common hepatic and biliary malignancies. The overall five-year survival rate for cholangiocarcinoma is less than 15%. miR-28-5p has been reported to participate the development of various human cancer types. But whether miR-28-5p is associated with the clinical course of CCA patients has not been clarified. Herein, we observed that miR-28-5p was reduced in CCA tissues and predicts the poor prognosis of CCA patients. Treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-AZA) restored miR-28-5p expression in CCA cell lines. Furthermore, up-regulated miR-28-5p inhibited CCA cells growth and metastasis. Mechanistically, miR-28-5p suppressed CCA cells growth and metastasis via directly targeting CD44 molecular. Specific CD44 special siRNA abrogated the discrepancy of the proliferation and metastasis capacity between miR-28-5p-overexpression CCA cells and their control cells, which further confirmed that CD44 was required in miR-28-5p-inhibited CCA cell growth and metastasis.

MicroRNA-28-5p as a potential diagnostic biomarker for chronic periodontitis and its role in cell proliferation and inflammatory response.

Background/purpose: Recent studies have pointed to the crucial role of microRNAs (miRNAs) in chronic periodontitis (CP). This study investigated the regulation and potential mechanisms of miR-28-5p in CP patients and lipopolysaccharide (LPS)-induced periodontal ligament cells (PDLCs). Materials and methods: 76 CP patients and 71 periodontally healthy subjects were included. RT-qPCR was employed to examine miR-28-5p and sphingosine kinase -1 (SPHK1) in subjects' gingival sulcus fluid and PDLCs. The diagnostic performance was evaluated by measuring the area under the curve (AUC) of the receiver operating characteristic (ROC) analysis. Pearson correlation coefficient (r) was adopted to explore the statistical relation between indicators. PDLCs proliferation and inflammation factors were determined by CCK-8 and ELISA assay. The direct target gene was validated by a dual-luciferase reporter assay. Results: miR-28-5p was lowly expressed in CP patients and LPS-induced PDLCs (P < 0.05). AUC for miR-28-5p was 0.937, which had certain diagnostic value. Additionally, miR-28-5p was negatively correlated with periodontal clinical indicators and inflammatory factors. Cell proliferation of PDLCs was inhibited and inflammation was promoted under LPS induction, however, elevated miR-28-5p diminished the effect of LPS (P < 0.05). SPHK1 acts as a miR-28-5p target and the elevation of SPHK1 caused by LPS treatment was inhibited by the increased miR-28-5p. Conclusion: Present study revealed that miR-28-5p could be served as a potential diagnostic biomarker for CP. And miR-28-5p may participate in CP progression by targeting SPHK1 to regulate the proliferation and inflammation of PDLCs. This study may offer insights into CP treatment and diagnosis.

The E6 Oncoprotein of HPV16 AA-c Variant Regulates Cell Migration through the MINCR/miR-28-5p/RAP1B Axis.

The E6 oncoprotein of HPV16 variants differentially alters the transcription of the genes involved in migration and non-coding RNAs such as lncRNAs. The role of the lncRNA MINCR in cervical cancer and its relationship with variants of oncogenic HPV remain unknown. Therefore, the objective of this study was to analyze the effect of the E6 oncoprotein of the AA-c variant of HPV16 in cell migration through the MINCR/miR-28-5p/RAP1B axis. To explore the functional role of MINCR in CC, we used an in vitro model of C33-A cells with exogenous expression of the E6 oncoprotein of the AA-c variant of HPV16. Interfering RNAs performed MINCR silencing, and the expression of miR-28-5p and RAP1B mRNA was analyzed by RT-qPCR. We found that C33-A/AA-c cells expressed MINCR 8-fold higher compared to the control cells. There is an inverse correlation between the expression of miR-28-5p and RAP1B in C33-A/AA-c cells. Our results suggest that MINCR might regulate the expression of RAP1B through the inhibition of miR-28-5p in CC cells expressing the E6 oncoprotein of HPV16 AA-c. We report, for the first time, that the MINCR/miR-28-5p/RAP1B axis positively regulates cell migration in CC-derived cells that express the E6 oncoprotein of the AA-c variant of HPV16.

circAGFG1 sponges miR-28-5p to promote non-small-cell lung cancer progression through modulating HIF-1α level.

Circular RNAs (circRNAs) have gained much attention for their crucial regulatory roles in human diseases and cancers. However, the role and the mechanism of circRNA ArfGAP with FG repeats 1 (circAGFG1) in non-small-cell lung cancer (NSCLC) are still largely unknown. circAGFG1 was highly expressed in NSCLC, and high expression of circAGFG1 was closely related to the low survival rate of NSCLC patients. circAGFG1 knockdown inhibited the proliferation, migration, and invasion and promoted the apoptosis of NSCLC cells. circAGFG1 bound to miR-28-5p in NSCLC cells, and circAGFG1 promoted NSCLC progression partly through sponging miR-28-5p in vitro. HIF-1α was a target of miR-28-5p, and miR-28-5p overexpression-mediated influences in NSCLC cells were partly overturned by the addition of HIF-1α overexpression plasmid. circAGFG1/miR-28-5p/HIF-1α axis regulated cellular glycolytic metabolism in NSCLC cells. circAGFG1 silencing restrained the xenograft tumor growth in vivo. circAGFG1 promoted the proliferation, migration, and invasion and suppressed the apoptosis of NSCLC cells through accelerating the glycolysis via miR-28-5p/HIF-1α axis.

LncRNA CDKN2B­AS1 sponges miR­28­5p to regulate proliferation and inhibit apoptosis in colorectal cancer.

Long noncoding RNA (lncRNA) CDKN2B­antisense RNA 1 (AS1) functions as a tumor oncogene in numerous cancers. However, the roles and mechanism of CDKN2B­AS1 in colorectal cancer (CRC) have not been explored. The present study aimed to investigate whether and how CDKN2B­AS1 contributes to CRC progression. The data revealed that CDKN2B­AS1 expression was upregulated in CRC tissues. Loss­of­function assays demonstrated that CDKN2B­AS1 in CRC modulated cell proliferation and apoptosis, which was mediated by cyclin D1, cyclin­dependent kinase (CDK) 4, p­Rb, caspase­9 and caspase­3. Bioinformatics analysis and luciferase reporter assays indicated direct binding of microRNA (miR)­28­5p to CDKN2B­AS1. Moreover, the results herein revealed that the expression of miR­28­5p was negatively correlated with that of CDKN2B­AS1 in CRC tissue. Moreover, CDKN2B­AS1 acted as a miR­28­5p competing endogenous RNA (ceRNA) to target and regulate the expression of URGCP. These findings indicated that CDKN2B­AS1 plays roles in CRC progression, providing a potential therapeutic target or novel diagnostic biomarker for CRC.

Circ-0001068 is a novel biomarker for ovarian cancer and inducer of PD1 expression in T cells.

Ovarian cancer is a primary gynecological malignancy with a global 5-year survival rate of 44%. The majority of patients present with advanced disease at initial diagnosis because of the lack of an effective early detection screening test. Circular RNAs (circRNAs) within exosomes in the circulatory system are effective diagnostic and therapeutic biomarkers for many diseases, especially tumors. In this study, we used microarrays to identify 6 circRNAs that were upregulated and 37 circRNAs that were downregulated in exosomes from ovarian cancer patients as compared to healthy volunteers. We validated the accumulation trends for the 6 upregulated circRNAs in the training set using qRT-PCR and found that circ-0001068 was significantly higher in the serum exosomes from the ovarian cancer patients as than healthy volunteers. Circ-0001068 was next evaluated further in a larger cohort. As with the training set, results from the larger cohort revealed that levels of circ-0001068 in the exosomes were significantly higher in ovarian cancer patients than healthy volunteers. Circ-0001068 was also delivered into T cells and induced PD1 expression by acting as a competing endogenous RNA (ceRNA) for miR-28-5p through the exosomes.

miR-28-5p inhibits carcinogenesis in colon cancer cells and is necessary for erastin-induced ferroptosis.

BACKGROUND: Ferroptosis is a newly discovered type of regulated cell death, the underlying mechanisms of which need to be further illuminated. The regulatory activity of miR-28-5p in ferroptosis in colon cancer cells is currently unclear. This study set out to investigate the effect of miR-28-5p on ferroptosis in colon cancer cells and determine its underlying mechanism. METHODS: Biochemical Kits were used to measure iron concentration, malondialdehyde (MDA) concentration, glutathione (GSH) concentration and glutathione peroxidase (GPX) vitality. Cell counting kit 8 (CCK8) assays were conducted to evaluate cell viability. Flow cytometry was conducted to assess apoptosis. Transwell™ assays were used to measure the migratory and invasive abilities of HCT116 cells. Western blotting was used to measure the protein relative expression of NEDD4 binding protein 1 (N4BP1). Quantitative real-time polymerase chain reaction (RT-PCR) was used to measure the RNA relative expression of N4BP1 and miR-28-5p. RESULTS: Ferroptosis was induced in HCT116 cells by erastin in a dose- and time-dependent manner, which caused significant inhibition of proliferation, migration, and invasion in HCT116 cells; however, there was no obvious effect on apoptosis. miR-28-5p expression was decreased in colon cancer cells compared with the normal colon cells but was upregulated in erastin-treated HTC116 cells. Additionally, when overexpressed via the transfection of miR-28-5p mimics, miR-28-5p had an inhibitive effect on proliferation, migration, and invasion, while promoting apoptosis, in HCT116 cells. erastin-induced ferroptosis was also increased by miR-28-5p overexpression. Compared with normal colon cells, following erastin treatment, NEDD4 binding protein 1 (N4BP1) expression was increased in colon cancer cells and further decreased in HTC116 cells. miR-28-5p overexpression also inhibited N4BP1 mRNA and protein expression in HTC116 cells. CONCLUSIONS: miR-28-5p plays an important role in ferroptosis by targeting N4BP1 and could serve as a potential therapeutic approach for colon cancer.

Long noncoding RNA LINC00514 accelerates pancreatic cancer progression by acting as a ceRNA of miR-28-5p to upregulate Rap1b expression.

BACKGROUND: Pancreatic cancer (PC) is one of the most aggressive cancers and has an extremely poor prognosis worldwide. Long noncoding RNA (lncRNA) has been reported to be a potential prognostic biomarker in the initiation and prognosis of PC. Nevertheless, the biological functions and the detailed molecular mechanism of LINC00514 in PC remain unclear. METHODS: We measured the expression level of LINC00514 in PC tissues and cell lines by quantitative real-time PCR. Gain- and loss-of-function experiments were performed to explore the bioeffects of LINC00514 on PC development both in vitro and in vivo. Subcellular fractionation, luciferase reporter assay, RNA immunoprecipitation assay, pull-down assay and western blotting were performed to investigate the oncogenic molecular mechanisms of LINC00514. RESULTS: In this study, LINC00514 was shown to be upregulated in PC tissues and cell lines. Increased LINC00514 expression was significantly associated with the clinical progression and prognosis of PC patients. In addition, silencing LINC00514 inhibited PC cell proliferation, migration and invasion, while LINC00514 overexpression promoted these processes. Moreover, LINC00514 knockdown remarkably inhibited PC development and metastasis in vivo. Deeper investigations indicated that LINC00514 acted as a sponge for microRNA-28-5p (miR-28-5p) in PC and that Rap1b was a downstream target of miR-28-5p. Furthermore, the positive correlation of LINC00514 and Rap1b and the negative correlation between miR-28-5p and LINC00514 (or Rap1b) were revealed. Based on the rescue assays, Rap1b inhibition partially suppressed the oncogenic effect of LINC00514 overexpression on PC cell proliferation, migration and invasion. CONCLUSIONS: This study is the first to characterize the oncogenic function of the long noncoding RNA LINC00514 in pancreatic cancer progression by acting as a competing endogenous RNA (ceRNA) of miR-28-5p to upregulate Rap1b expression. Understanding this molecular mechanism might contribute to further discoveries of better diagnostic and therapeutic options for pancreatic cancer.

Long Noncoding RNA LUADT1 Is Upregulated in Melanoma and May Sponge miR-28-5p to Upregulate RAP1B.

Background: Long noncoding RNA (lncRNA) LUADT1 is a known oncogenic lncRNA in lung cancer. This study aimed to explore the roles of LUADT1 in melanoma. Materials and Methods: Sixty pairs of melanoma and nontumor tissues were obtained from 60 melanoma patients (37 men and 23 women, 38-68 years, 52.1 ± 4.9 years) at the First Affiliated Hospital of Zhejiang University School of Medicine. Gene expression was analyzed by quantitative polymerase chain reaction and western blot. Cell transfections were performed to analyze gene expression. Results: We found that LUADT1 was upregulated in melanoma and high levels of LUADT1 predicted poor survival. RNA interaction prediction showed that LUADT1 can form base pairing with miR-28-5p. In melanoma cells, LUADT1 overexpression mediated the upregulated Ras-related protein Rap-1b (RAP1B). Cell proliferation assay showed that LUADT1 and RAP1B overexpression mediated the increased proliferation rate of melanoma cells. In addition, miR-28-5p overexpression played opposite roles attenuating the effects of LUADT1 overexpression on both RAP1B expression and cancer cell proliferation. Conclusions: LUADT1 in melanoma and may sponge miR-28-5p to upregulate RAP1B, thereby promoting cancer cell proliferation.