CircANXA4 (hsa_circ_0055087) regulates the miR-1256/PRM1 axis to promote tumor progression in colorectal cancer.

Colorectal cancer (CRC) incidence ranks third among malignant cancers with a high propensity for distant metastasis. Despite continuous efforts to improve treatment, the prognosis especially in patients with advanced distant metastasis is low. The mechanism of development and progression of CRC is not fully understood. Non-coding RNAs (ncRNAs) have emerged as essential regulators in cancer progression. Here, we aim to dissect the role of one critical ncRNA, circANXA4, in CRC progression. CircANXA4 expression was analyzed by the GEO database. Differentially expressed circRNAs were identified by the Limma package R software. Expression of circANXA4 and miR-1256 was detected by qRT-PCR. The regulation of circANXA4 on cell proliferation and progression was confirmed with the cell viability assay using cell counting kit-8 (CCK-8) and transwell migration assay. RNA pull-down assay, RNA immunoprecipitation (RIP), and western blot were used to determine the interaction between circANXA4, miR-1256, and protamine1 (PRM1). CircANXA4 was upregulated in both CRC tissues and cell lines. Knockdown of circANXA4 effectively reduced cell proliferation, progression, and migration. Additionally, silencing circANXA4 remarkably increased miR-1256 expression, while reducing PRM1 expression, thereby demonstrating that circANXA4 downregulates miR-1256 expression through a complementary binding site. Rescue experiments revealed the interactions between circANXA4, miR-1256, and PRM1. Pearson correlation analysis revealed that circANXA4 expression positively correlated with PRM1 expression and miR-1256 expression inversely correlated with PRM1 expression. In sum, we demonstrated that circANXA4 promotes cancer cell proliferation and progression by sponging miR-1256 and upregulating PRM1 in CRC.

Circ_0007422 Knockdown Inhibits Tumor Property and Immune Escape of Colorectal Cancer by Decreasing PDL1 Expression in a miR-1256-Dependent Manner.

Circular RNAs (circRNAs) are a group of important molecules involved in the progression of various cancers, including colorectal cancer (CRC). Here, we aim to investigate the role and molecular mechanism of circ_0007422 in regulating CRC malignant progression. The expression levels of circ_0007422, miR-1256, and PDL1 were detected by qRT-PCR. Cell viability, proliferation, apoptosis, invasion, and self-replication ability were analyzed by CCK-8, EdU, flow cytometry, transwell, and spheroid formation experiments, respectively. Protein levels were determined by western blotting assay. CRC cells were co-cultured with CD8 + T cells, phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs), or cytokine-induced killer (CIK) cells in vitro, and CD8 + T-cell apoptosis, IFN-γ and TNF-α levels, and survival rate of CRC cells were analyzed to reveal the role of circ_0007422 in antitumor immunity. The relationship between miR-1256 and circ_0007422 or PDL1 was identified by a dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. A xenograft tumor model was established to verify the function of circ_0007422 in tumor growth in vivo. Immunohistochemistry (IHC) assay was used to detect positive expression rates of Ki67, E-cadherin, N-cadherin, and PDL1 expression in primary tumors from CRC cells. Circ_0007422 was upregulated in CRC tissues and cells and its knockdown inhibited proliferation, invasion, self-replication ability, and immune escape and promoted apoptosis of CRC cells. Additionally, circ_0007422 bound to miR-1256, which was identified to target PDL1. MiR-1256 inhibition reversed the effects of circ_0007422 knockdown on the tumor properties and immune escape of CRC cells. Moreover, miR-1256 introduction interacted with PDL1 to suppress proliferation, invasion, self-replication ability, and immune escape and promote apoptosis of CRC cells. Further, circ_0007422 knockdown hampered tumorigenesis of CRC cells in vivo. Circ_0007422 knockdown inhibited tumor property and immune escape of colorectal cancer through the miR-1256/PDL1 pathway, providing a potential novel therapeutic target for CRC.

CircKRT14 upregulates E2F3 by interacting with miR-1256 to act as an oncogenic factor in esophageal cancer.

BACKGROUND: A growing number of studies have focused on the regulatory role of circular RNAs (circRNAs) in a variety of cancers. The purpose of this study was to investigate the effect of circRNA Keratin 14 (circKRT14) on the progression of esophageal cancer (EC). METHODS: The levels of circKRT14, miR-1256 and E2F transcription factor 3 (E2F3) were analyzed by real-time quantitative polymerase chain reaction (qRT-PCR) and western blot. The circular structure of circKRT14 was confirmed by RNase R digestion assay. Cell apoptosis, migration and invasion were detected by flow cytometry and transwell assay. The protein levels of related factors were determined by western blot. The relationship between miR-1256 and circKRT14 or E2F3 was verified by dual-luciferase reporter assay. The in vivo function of circKRT14 was studied by xenograft tumor assay. RESULTS: CircKRT14 was significantly increased in EC tissues and cells. CircKRT14 silencing inhibited EC cell proliferation, migration, and invasion, but promoted EC cell apoptosis in vitro. CircKRT1 acted as a sponge for miR-1256 in EC, and in-miR-1256 abolished the inhibitory effect of circKRT14 suppression on EC cell progression. E2F3 was a target of miR-1256 and functioned as an oncogene in EC cells. MiR-1256 curbed EC progression by downregulating E2F3. CircKRT14 could affect E2F3 expression by targeting miR-1256. CircKRT14 regulated EC progression in vivo through miR-1256/E2F3 axis. CONCLUSIONS: These results uncovered that circKRT14 up-regulated the expression of E2F3 and promoted the malignant development of EC through sponging miR-1256.

Circ_0008035 promotes the progression of gastric cancer via the regulation of miR-1256/CEACAM6 axis.

Gastric cancer (GC) is one of the most common malignant tumors. Circular RNA (circRNA) has been shown to be involved in the progression of GC. However, the function of circ_0008035 in GC has not been studied. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of circ_0008035, microRNA-1256 (miR-1256) and carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry, and transwell assay were used to detect cell function. Western blot examined the protein levels of Ki67, Bax, MMP-2, and CEACAM6. The relationship between miR-1256 and circ_0008035 or CEACAM6 was verified by dual-luciferase reporter assays and RNA pull down. The xenotransplantation model was established in BALB/c nude mice to study the role of circ_0008035 in vivo. Circ_0008035 and CEACAM6 were significantly high-expressed in GC tissues and cells. Silencing of circ_0008035 reduced GC cell proliferation, migration, and invasion while enhancing apoptosis. MiR-1256 was a target of circ_0008035. The inhibition effect of circ_0008035 knockdown on the malignant behavior of GC cells could be reversed by miR-1256 inhibitor. In addition, CEACAM6 was a target of miR-1256. Overexpression of CEACAM6 partially restored the inhibitory effect of miR-1256 on cell progression. Animal experiments confirmed the anti-tumor effect of circ_0008035 knockdown in vivo. Collectively, circ_0008035 regulated the expression of CEACAM6 by sponging miR-1256, thereby promoting the development of GC. Our data provided a novel targeted therapy for GC.

The lncRNA LINC00691Functions as a ceRNA for miRNA-1256 to Suppress Osteosarcoma by Regulating the Expression of ST5.

INTRODUCTION: Osteosarcoma is the most common primary malignant tumor in children and young patients. Although neoadjuvant chemotherapy and surgery could improve the prognosis of these patients, treatment outcomes are poor because of its low early diagnosis rate and high degree of malignancy as well as its tendency for early metastasis. In the field of osteosarcoma, lncRNAs have become a hot spot for studying the molecular mechanisms driving malignant biological characteristics and exploring effective treatment methods. An lncRNA is a long noncoding RNA lacking protein-encoding ability, and in its RNA form, it regulates various gene expression processes, such as epigenetic regulation, transcriptional regulation, and posttranscriptional regulation. LncRNAs play an important role in tumorigenesis and metastasis. METHODS: We used bioinformatics software to analyze the data in geo database. CCK-8 and Transwell were used to detect the effect of lncRNA LINC00691 on the proliferation and migration of osteosarcoma cells. The target gene of LINC00691 was detected by bioinformatics analysis and RNA pull down. RESULTS: In this study, we identified the lncRNA LINC00691 and confirmed its expression in osteosarcoma cells through GEO database analysis. Expression analysis showed that the levels of lncRNA LINC00691 in osteosarcoma cells were decreased compared to those of control cells. Overexpression of LINC00691 could inhibit the proliferation, migration, invasion, and induction of G1 cell cycle arrest in osteosarcoma cells, which was shown through in vitro and in vivo studies. Using bioinformatics analysis, RNA pull down experiments and luciferase reporter gene detection assays, we found that LINC00691 regulated ST5 expression by binding miR-1256. LINC00691 overexpression inhibited EMT by promoting the expression of E-cadherin and increasing the expression of ZEB1, Snail, and Fibronectin. CONCLUSION: These results suggested that overexpressed LINC00691 promoted the expression of ST5 by regulating the function of miR-1256 through a ceRNA mechanism. The LINC00691/miR-1256/ST5 pathway plays an important role in the progression and metastasis of osteosarcoma and represents a good therapeutic target.

MiR-1256 inhibits cell proliferation and cell cycle progression in papillary thyroid cancer by targeting 5-hydroxy tryptamine receptor 3A.

Aberrant expression of miR-1256 has been reported to be closely associated with the development and progression of tumors, including colon cancer and lung cancer. However, study of its expression pattern and functional role in papillary thyroid cancer (PTC) is rare. Using quantitative real time PCR analysis, we found miR-1256 was significantly down-regulated in PTC tissues and cell lines. The correlation of miR-1256 expression with clinicopathological features was statistically analyzed. The results showed miR-1256 expression was significantly correlated with tumor size (p = 0.0124) and TNM stage (p = 0.0032). Restoring miR-1256 expression significantly inhibited proliferation and cell cycle progression of PTC cells demonstrated by CCK-8 and flow cytometry assays. Luciferase reporter assay and biotin-avidin pull-down assay showed miR-1256 can directly target 5-hydroxytryptamine receptor 3A (HTR3A) in PTC cells. The expression of miR-1256 was inversely correlated with HTR3A expression in PTC tissues. Knockdown of HTR3A imitated the suppressive effects of miR-1256 in PTC cells. Ectopic expression of HTR3A can antagonize the effects of miR-1256 on PTC cells. Furthermore, the suppressive effects of miR-1256 on the expression of PCNA, CDK4, Cyclin D1, and p21 were partially reversed by HTR3A overexpression in PTC cells. In summary, our data suggested that miR-1256 could suppress PTC cellular function by targeting HTR3A, which might be a potential therapeutic target for patients with PTC.

Enhanced expression of circular RNA circ-DCAF6 predicts adverse prognosis and promotes cell progression via sponging miR-1231 and miR-1256 in gastric cancer.

Circular RNAs (circRNAs) have been reported as essential regulators in various malignancies, including gastric cancer (GC). Previously, circ-DCAF6 was screened as an elevated circRNA in GC patients' tissue samples compared with the normal tissues. To our knowledge, the role of circ-DCAF6 in human cancers is still needed to reveal. The present project is to evaluate the functions and mechanisms of circ-DCAF6 in GC progression. Upregulation of circ-DCAF6 was determined in GC tissue samples and cells by qRT-PCR. Enhanced level of circ-DCAF6 was linked to deeper tumor invasion, positive lymph node metastasis, and higher TNM stages in GC patients. Multivariate analysis further indicated circ-DCAF6 as an independent prognostic indicator. Functionally, CCK-8, clone forming, flow cytometry and transwell assays verified that circ-DCAF6 played as an oncogene in GC cells. Mechanistically, we found the negative correlation between circ-DCAF6 and miR-1231/-1256 in GC specimens. Moreover, luciferase reporter gene assay illustrated that miR-1231 and miR-1256 could be bound to circ-DCAF6. Rescue experiments revealed that circ-DCAF6 facilitated cell growth and invasion via suppressing the levels of miR-1231 and miR-1256. To sum up, circ-DCAF6 acts as an important role in GC tumor progression, and high circ-DCAF6 level may be a useful biomarker for GC.

Circular RNA circ_0026134 regulates non-small cell lung cancer cell proliferation and invasion via sponging miR-1256 and miR-1287.

Non-small cell lung cancer (NSCLC) is the major type of lung malignancy with increasing incidence worldwide. Despite progression in diagnosis and management of NSCLC, the patients' survival rate is still unfavorable. Accumulating data have shown that circular RNAs (circRNAs) act as key roles in oncogenesis. In this project, circ_0026134 expression in NSCLC tissue specimens and cells was measured by RT-qPCR. The functional roles (cell growth, apoptosis, migration, and invasion) of circ_0026134 were investigated by gain and loss-of-function assays. In addition, luciferase reporter assay was used to uncover the mechanisms of circ_0026134. Circ_0026134 was frequently upregulated in NSCLC samples and cell lines. Furthermore, we observed that downregulation of circ_0026134 attenuated NSCLC cell proliferation and metastatic properties and induced cell apoptosis. The overexpression of circ_0026134 in NSCLC cells caused the opposite effect. For the part of mechanism exploration, miR-1256 and miR-1287 were proved to be sponged by circ_0026134. Rescue experiments further indicated that the oncogenic role of circ_0026134 was dependent on its regulation of miR-1256 and miR-1287. Taken together, this study may provide a new insight and treatment target for NSCLC.

MiR-1256 suppresses proliferation and migration of non-small cell lung cancer via regulating TCTN1.

Mounting evidence has shown that miRNA expression is abnormal in various human cancers. Here, we mainly explored the biological function and the potential mechanisms of miR-1256 in non-small cell lung cancer (NSCLC). The miR-1256 mRNA expression was detected by quantitative real-time PCR and tectonic family member 1 (TCTN1) mRNA expression was detected by immunoblotting. The TCTN1 was identified to be the direct and specific target gene of miR-1256 by luciferase reporter assay. Cell proliferation was examined by methyl thiazolyl tetrazolium assay and migration was detected by transwell assay. MiR-1256 expression was downregulated in NSCLC tissues, whereas the expression of TCTN1 was upregulated, compared with normal tissues. We also found that overexpression of miR-1256 in these NSCLC cell lines inhibited cell proliferation and migration. Furthermore, TCTN1 was identified as a direct target of miR-1256 by luciferase reporter assays. Collectively, these data stated that the inhibitory effect of miR-1256 in NSCLC was realized by upregulating TCTN1, suggesting that miR-1256/TCTN1 axis may play a critical role as NSCLC therapeutic target.