Expression Of miR-663b Research Articles

Evaluation of the biomarker potential of miR-650 and miR-663b in tumor tissues and plasma specimens of colon cancer patients living in northwest of Iran

BackgroundColorectal cancer (CRC) is considered as one of the most common malignancy and the fourth leading cause of cancer-related deaths, worldwide. Here, we aimed to investigate the expression of miR–663b and miR–650 in CRC tissue and plasma specimens.MethodsIn this case–control study, tumor specimens, non-tumoral adjacent tissues, and matched-plasma samples were obtained from forty patients with CRC living in the northwestern of Iran. Plasma of healthy patients was also collected as control. Total RNA was extracted from all specimens and studied by real-time PCR. Furthermore, the correlation between the expression of microRNAs and clinico-pathological features were also studied.ResultsOur data illustrated that miR-650 and miR-663b are down-regulated and up-regulated in tumor samples compared to non-tumoral margins, respectively (p < 0.001). However, the results did not show any significant difference in patient’s plasmas compared to controls. Further analysis disclosed that the expression of miR-663b is significantly associated with tumor size, lymph node metastasis, and tumor stage, while miR-650 is remarkably related to TNM stage, lymph node metastasis, distant metastasis, tumor size, and age. (p < 0.05) Furthermore, receiver operating characteristic (ROC) analyses revealed that miR-650 and miR-663b are potential biomarkers in differentiating CRC patients from healthy controls.ConclusionIn conclusion, our data illustrated the potential of miR-650 and miR-663b as biomarkers in colorectal cancer. However, further studies are needed to confirm the employment of these microRNAs in the diagnosis and/or prognosis of colorectal malignancies.

Multi-Drug Resistance against Second-Line Medication and MicroRNA Plasma Level in Metastatic Breast Cancer Patients.

Circulating microRNAs (miRNAs) can help to predict the chemotherapy response in breast cancer with promising results. The aim of the present study was to investigate the relationships between the miR-199a, miR-663a, and miR-663b expression and chemotherapy response in metastatic breast cancer patients. This study is a case-control study performed at Yasuj University of Medical Sciences (2018-2021). The expression levels of miR-663a, miR-663b, and miR-199a in the serum of 25 patients with metastatic breast cancer versus 15 healthy individuals were determined by the real-time polymerase chain reaction method. The response to treatment was followed up in a 24-month period. All patients were treated with second-line medications. Two or more combinations of these drugs were used: gemcitabine, Navelbine®, Diphereline®, Xeloda®, letrozole, Aromasin®, and Zolena®. Statistical analyses were performed in SPSS 21.0 and GraphPad Prism 6 software. The expression levels were presented as mean±SD and analyzed by Student's t test. The results and clinicopathological features of patients were analyzed by t test. The statistical analysis showed that miR-663a expression was related to human epidermal growth factor receptor 2 (HER2) status and was significantly lower in the HER2+ than HER2- group (P=0.027). Moreover, the expression of miR-199a and miR-663b was significantly correlated with the response to treatment, in which the expression of miR-199a was higher in the poor-response group (P=0.049), while the higher expression of miR-663b was seen in the good-response group (P=0.009). These findings state that the high plasma level of miR-199a and the low plasma level of miR-663b may be related to chemoresistance in patients with metastatic breast cancer.

The effect of microRNA-663b in the inhibition of interleukin-1-induced nucleus pulposus cell apoptosis and inflammatory response.

The aim of this study was to explore the role and pathological mechanism of microRNA-663b in interleukin-1beta (IL-1β)-induced inflammation and apoptosis of nucleus pulposus cells. First, the best concentration and time to construct the nucleus pulposus cell inflammation model was screen out. Overexpression or inhibition of miR-663b expression was performed by adding microRNA-663b mimic or microRNA-663b inhibitor. 293T cells were transfected according to experimental requirements. The luciferase activity of each group was detected to analyze the targeted regulation of microRNA-663b on interleukin-1 receptor (IL1R1). Compared with the mimic negative control (NC) group, the expression of inflammatory factors in the microRNA-663b overexpression group was inhibited (P<0.05), and the expression of type 2 collagen and polysaccharide protein increased (P<0.05), and the apoptosis of nucleus pulposus cells was inhibited (P<0.01), and the number of TUNEL-positive cells decreased significantly (P<0.01), and the microRNA and protein expression of IL1R1, the ratio of P-P65/P65 and phospho-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (P-IκBα)/nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) protein expression were significantly decreased (P<0.05). The expression of inflammatory factors in the miR-663b inhibitor group was significantly higher than that in the inhibitor NC group (P<0.01), and the expression of type 2 collagen and polysaccharide protein was significantly decreased (P<0.01), and the number of apoptosis cells and TUNEL staining positive cells increased (p<0.01). The expression of IL1R1 gene and protein was significantly increased (P<0.01). The ratio of P-P65/P65 and P-IκBα/IκBα protein expression increased (P<0.05). IL1R1 is a downstream target gene of microRNA-663b. MicroRNA-663b may down-regulate the expression of IL1R1 at the transcriptional level by targeting IL1R1, inhibit the inflammatory response of nucleus pulposus cells, and slow down the degeneration of nucleus pulposus cells.

The effect of microRNA-663b in the inhibition of interleukin-1-induced nucleus pulposus cell apoptosis and inflammatory response.

The aim of this study was to explore the role and pathological mechanism of microRNA-663b in interleukin-1beta (IL-1β)-induced inflammation and apoptosis of nucleus pulposus cells. First, the best concentration and time to construct the nucleus pulposus cell inflammation model was screen out. Overexpression or inhibition of miR-663b expression was performed by adding microRNA-663b mimic or microRNA-663b inhibitor. 293T cells were transfected according to experimental requirements. The luciferase activity of each group was detected to analyze the targeted regulation of microRNA-663b on interleukin-1 receptor (IL1R1). Compared with the mimic negative control (NC) group, the expression of inflammatory factors in the microRNA-663b overexpression group was inhibited (P<0.05), and the expression of type 2 collagen and polysaccharide protein increased (P<0.05), and the apoptosis of nucleus pulposus cells was inhibited (P<0.01), and the number of TUNEL-positive cells decreased significantly (P<0.01), and the microRNA and protein expression of IL1R1, the ratio of P-P65/P65 and phospho-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (P-IκBα)/nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) protein expression were significantly decreased (P<0.05). The expression of inflammatory factors in the miR-663b inhibitor group was significantly higher than that in the inhibitor NC group (P<0.01), and the expression of type 2 collagen and polysaccharide protein was significantly decreased (P<0.01), and the number of apoptosis cells and TUNEL staining positive cells increased (p<0.01). The expression of IL1R1 gene and protein was significantly increased (P<0.01). The ratio of P-P65/P65 and P-IκBα/IκBα protein expression increased (P<0.05). IL1R1 is a downstream target gene of microRNA-663b. MicroRNA-663b may down-regulate the expression of IL1R1 at the transcriptional level by targeting IL1R1, inhibit the inflammatory response of nucleus pulposus cells, and slow down the degeneration of nucleus pulposus cells.

Second-line therapy and MicroRNA plasma level in metastatic lung cancer patients: case-control study

The first cause of cancer death was lung cancer among men and women. MicroRNAs are small noncoding regulatory RNAs with a length of 18–24 nucleotides. MicroRNAs could target more than 100 mRNAs, and they can affect many cellular processes. The objective of this study is investigating the role of miR-663a and miR-663b, in metastatic lung cancer patients compare with healthy people. The relationship between these microRNAs and chemoresistance was investigated through Real time polymerase chain reaction methodology used to determine the levels expression of miR-663a and miR-663b in the serum of 30 patients with metastatic lung cancer were treated with second line therapy versus 25 healthy individuals who follow up for one year. The resulted data showed that miR-663a expression was related to type of lung cancer and was significantly higher in metastatic squamous lung cancer group (P=0.012). While the expression of miR-663b was significantly correlated with the response to treatment, in which the expression of miR-663b was higher in the poor-response group (P=0.016). Inconclusion, this study showed that the high plasma level of miR-663a miR-663b may be related to chemoresistance in patients with metastatic lung cancer.

Effect of microRNA-663b on migration, invasion and epithelial‑mesenchymal transition of oral squamous cell carcinoma cells.

To explore the effect of microRNA-663b (miR-6636) on migration, invasion and epithelial-mesenchymal transition (EMT) of oral squamous cell carcinoma cells (OSCC). Use R Studio of gene expression omnibus (GEO) database to analyze expressions of miR-663b in the OSCC and adjacent normal tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-663b in tissues and cells. The transfection efficiency of HN30 cells with miR-663b knockout plasmid was detected. Transwell assay was used to detect the effect of the migration and invasion ability. Bioinformatics method was used to predict the targeted mRNA that may bind to miR-663b and double luciferase assay was used to verify the binding. Western blot assay was used to detect the expression of EMT-related markers. The expression of miR-663b was up-regulated in OSCC tissues and higher in HN30, CAL27 and SCC-9 cells than in HOEC cells (P<0.05). Knockout of miR-663b could inhibit migration and invasion of HN30 cells (P<0.05) and inhibit the occurrence of EMT. Bioinformatics prediction software predicts that SH3BP2 was the target gene of miR-663b, and patients with low SH3BP2 expression had a poor prognosis (P<0.05). MiR-663b could bind to SHBP2 (P<0.05). The expression of SH3BP2 was increased and the occurrence of EMT was inhibited in HN30 cells with miR-663b knocked out. Knockout of miR-663b can inhibit the migration, invasion and EMT of OSCC by targeting SH3BP2.

Exosomal miR‑663b exposed to TGF‑β1 promotes cervical cancer metastasis and epithelial‑mesenchymal transition by targeting MGAT3.

Transforming growth factor (TGF)-β1 is a key cytokine affecting the pathogenesis and progression of cervical cancer. Tumor-derived exosomes contain microRNAs (miRNAs/miRs) that interact with cancer and stromal cells, thereby contributing to tissue remodeling in the tumor microenvironment (TME). The present study was designed to clarify how TGF-β1 affects tumor biological functions through exosomes released by cervical cancer cells. Deep RNA sequencing found that TGF-β1 stimulated cervical cancer cells to secrete more miR-663b-containing exosomes, which could be transferred into new target cells to promote metastasis. Further studies have shown that miR-663b directly targets the 3′-untranslated regions (3′-UTR) of mannoside acetylglucosaminyltransferase 3 (MGAT3) and is involved in the epithelial-mesenchymal transition (EMT) process. Remarkably, the overexpression of MGAT3 suppressed cervical cancer cell metastasis promoted by exosomal miR-663b, causing increased expression of epithelial differentiation marker E-cadherin and decreased expression of mesenchymal markers N-cadherin and β-catenin. Throughout our study, online bioinformation tools and dual luciferase reporter assay were applied to identify MGAT3 as a novel direct target of miR-663b. Exosome PKH67-labeling experiment verified that exosomal miR-663b could be endocytosed by cervical cancer cells and subsequently influence its migration and invasion functions which were measured by wound healing and Transwell assays. The expression of miR-663b and MGAT3 and the regulation of the EMT pathway caused by MGAT3 were detected by quantitative real-time transcription-polymerase chain reaction (qPCR) and western blot analysis. These results, thus, provide evidence that cancer cell-derived exosomal miR-663b is endocytosed by cervical cancer cells adjacent or distant after TGF-β1 exposure and inhibits the expression of MGAT3, thereby accelerating the EMT process and ultimately promoting local and distant metastasis.

Melatonin levels and microRNA (miRNA) relative expression profile in the follicular ambient microenvironment in patients undergoing in vitro fertilization process.

Intrafollicular fluid (IFF) melatonin plays a decisive role in maintaining granulosa cells' DNA integrity and protects them against apoptosis. It reduces oxidative stress and improves the oocyte quality with a higher fertilization rate. This prospective study investigated the antioxidant property of IFF melatonin and its impact on IVF outcome parameters. We also explored the relative expression of five microRNAs (miR-663b, miR-320a, miR-766-3p, miR-132-3p, miR-16-5p) and levels of cell-free DNA (cfDNA) by real-time PCR in unexplained infertile patients. We collected 425 follicular fluid (FF) samples containing mature oocytes from 295 patients undergoing IVF. Patients were subgrouped based on IFF melatonin concentration (group A ≤ 30pg/mL, group B > 70 to ≤ 110pg/mL, group C > 111 to ≤ 385pg/mL). Our results showed that patients with ≤ 30pg/mL IFF melatonin levels have significantly higher oxidative stress markers, cfDNA levels, and lower relative expression of miR-663b, miR-320a, miR-766-3p, miR-132-3p, and miR-16-5p compared to other subgroups (p < 0.001). Similarly, they have a low fertilization rate and a reduced number of high-quality day 3 embryos. Findings suggest that the therapeutic use of melatonin produces a considerable rise in the number of mature oocytes retrieved, fertilization rate, and good-quality embryo selection. Furthermore, miRNA signature enhances the quality of embryo selection, thus, may allow us to classify them as non-invasive biomarkers to identify good-quality embryos.

MiR-663b promotes colorectal cancer progression by activating Ras/Raf signaling through downregulation of TNK1.

MiR-663b has been demonstrated to be abnormally expressed in several cancer types and was involved in the progression of cancer. Although overexpression of miR-663b in colorectal cancer was observed, the role of miR-663b in colorectal cancer cells has not been identified yet. In this study, we analyzed expression of miR-663b in colorectal tumors and explored the molecular mechanism of miR-663b in colorectal cancer cells. MiR-663b was significantly overexpressed in colorectal tumors and cell lines. Downregulation of miR-663b inhibited cell proliferation and sphere forming ability in colorectal cancer cells. In addition, miR-663b downregulation inactivated Ras/Raf signaling activity and subsequently decreased YAP1 and CD44 expression in colorectal cancer cells. Using TargetScan software, TNK1, a negative regulator of Ras/Raf signaling, was predicted to be a target gene of miR-663b. Western blotting and RT-qPCR showed that TNK1 expression was negatively regulated by miR-663b. In addition, the direct binding of miR-663b to TNK1 mRNA was proved by dual luciferase reporter assay. Furthermore, downregulation of miR-663b inhibited colorectal cancer cell proliferation and stemness, which was reversed after siRNA-mediated silencing of TNK1. In conclusion, the current study revealed a pivotal role of miR-663b in the progression of colorectal cancer.

MicroRNA-663b promotes cell proliferation and epithelial mesenchymal transition by directly targeting SMAD7 in nasopharyngeal carcinoma.

MicroRNAs (miRs) serve important roles in the development of various types of human cancer, including nasopharyngeal carcinoma (NPC). In the present study, the expression levels of miR-663b in NPC were investigated and its role and underlying mechanisms were examined. Reverse transcription-quantitative polymerase chain reaction was performed to assess miR-663b expression levels in NPC tissues and C666-1 cells. TargetScan was applied to predict the putative targets of miR-663b and the dual luciferase reporter assay was used to confirm the predictions. To investigate the role of miR-663b in NPC, the NPC C666-1 cell line was transfected with miR-663b mimics, miR-663b inhibitors or negative control. The Cell Counting kit-8 assay was performed for cell proliferation detection and western blot analysis was applied to determine the expression levels of epithelial mesenchymal transition (EMT)-associated proteins. Results indicated that when compared with the adjacent normal tissues and the normal nasopharyngeal epithelial cells, miR-663b expression levels were significantly upregulated in the NPC tissues and the NPC cells (P<0.01). Notably, SMAD7 is a target gene of miR-663b and may be inhibited by miR-663b. Results indicated that NPC cell proliferation was significantly promoted by miR-663b mimics and significantly inhibited by miR-663b inhibitors (P<0.05 and P<0.01). In addition, the results indicated that, when compared with the negative control group the expression levels of E-cadherin were significantly decreased, whereas the expression levels of N-cadherin, Vimentin and matrix metalloproteinase-9 were significantly increased in the cells of the miR-663b mimics group (P<0.05 and P<0.01). However, cells in the miR-663b inhibitors group exhibited the opposite effects. In conclusion, the results of the present study indicated that miR-663b functions as a tumor promoter in NPC via promoting NPC cell proliferation and EMT by directly targeting SMAD7.

Screening of miRNAs in human follicular fluid reveals an inverse relationship between microRNA-663b expression and blastocyst formation.

Are miRNAs found in follicular fluid related to blastocyst formation from the corresponding oocytes? In this study, 91 individual follicular fluid samples from single follicles containing mature oocytes from 91 women were collected and classified into group 1 (n = 38) with viable blastocysts, and group 2 (n = 53) with no blastocyst. TaqMan human miRNA cards and quantitative reverse transcription polymerase chain reaction were used to identify differently expressed follicular fluid miRNAs between the two groups. We found MIR-663B to be significantly differentially expressed in follicular fluid of oocytes that yielded viable blastocysts versus those that did not develop into blastocysts (14.16 ± 7.00 versus 23.68 ± 17.02; P = 0.019), as well as for those which develop into blastocysts with good morphology versus those with poor morphology (11.69 ± 3.49 versus 20.16 ± 9.33; P = 0.003). MIR-663B expression levels in human follicular fluid samples were significantly negatively related to viable blastocyst formation and may become an objective evaluation criterion for embryo development potential after IVF.

Blockade of miR-663b inhibits cell proliferation and induces apoptosis in osteosarcoma via regulating TP73 expression.

This study aimed to investigate the exact role of miR-663b in osteosarcoma (OS) progression and further explore the underlying molecular mechanisms. The miR-663b expression in human OS cell lines was determined by qRT-PCR, and the results suggested that miR-663b was highly expressed in human OS cells. TargetScan was used to predict the potential targets of miR-663b, and the prediction was confirmed by dual-luciferase reporter assay. To investigate the role of miR-663b in OS, miR-663b was down-regulated in U2OS cells using miR-663b inhibitor. CCK8 and flow cytometry were preformed to investigate the proliferation and apoptosis of U2OS cells. Moreover, qRT-PCR and western blot analysis were performed to measure the mRNA and protein expression. We found that miR-663b directly targets TP73 and negatively regulates TP73 expression. MiR-663b inhibitor significantly decreased the proliferation ability of U2OS cells, while the percentage of apoptotic cells was markedly increased. The level of Bcl-2 was notably inhibited by miR-663b inhibitor, while Bax expression was significantly enhanced. Moreover, miR-663b down-regulation promoted p53 and p21 expression in U2OS cells. MiR-663b down-regulation represses proliferation and induces apoptosis in OS by targeting TP73. Therefore, we provide a potential therapeutic target for OS treatment (Fig. 6, Ref. 27).

MiR-663b promotes tumor cell proliferation, migration and invasion in nasopharyngeal carcinoma through targeting TUSC2.

The authors' previous study revealed that the serum levels of microRNA (miR)-663b are significantly increased in patients with nasopharyngeal carcinoma (NPC), and are associated with NPC progression and poor prognosis. However, the molecular mechanism of underlying NPC growth and metastasis remains unclear. In the present study, quantitative polymerase chain reaction and western blot analyses were performed to examine changes to mRNA and protein expression, respectively. MTT, wound healing and Transwell assays were used to examine cell proliferation, migration and invasion, respectively. Luciferase reporter gene assays were performed to identify target genes of miR-663b. It was demonstrated that miR-663b was significantly upregulated in NPC tissue compared with non-tumor nasopharyngeal epithelial tissue samples. Furthermore, miR-663b expression gradually increased with advancing stages of NPC, with the highest expression being observed in the latest stage IV. The increased expression of miR-663b was associated with advanced clinical stage and lymph node metastasis. In addition, miR-663b expression was increased in NPC cell lines compared with normal nasopharyngeal epithelial NP69 cells. Knockdown of miR-663b resulted in a significant reduction in the proliferation, migration and invasion of NPC CNE1 cells. Tumor suppressor candidate 2 (TUSC2) was identified as a novel target gene of miR-663b. It was further demonstrated that TUSC2 was significantly downregulated in NPC tissue samples and cell lines. miR-663b negatively regulated the expression of TUSC2 at the post-transcriptional level in CNE1 cells. Additionally, inhibition of TUSC2 expression attenuated the suppressive effects of miR-663b downregulation on the proliferation, migration and invasion of CNE1 cells. To the best of our knowledge, this is the first study to demonstrate that miR-663b, which is upregulated in NPC, promotes the proliferation, migration and invasion of NPC cells, partially through the inhibition of TUSC2 expression. Therefore, it is suggested that miR-663b is a promising therapeutic target for the treatment of patients with NPC.

Pterostilbene suppresses human endometrial cancer cells in vitro by down-regulating miR-663b.

Resveratrol has long been known as an antioxidant and a chemopreventive agent. Similar to resveratrol, pterostilbene (PT) is also a phenolic compound extracted from the Vitis species. However, there are few studies on the antitumor effect of PT. Thus, we investigated the effects of PT on the endometrial cancer (EC) cells in vitro and the related molecular mechanisms. Treatment of EC cell lines HTB-111 and Ishikawa with PT (25-100 μmol/L) dose-dependently suppressed the cell viability and induced apoptosis. Using miR microarrays, we examined the miR expression profile in Ishikawa cells with or without PT, and revealed that miR-663b was the most decreased in PT-treated Ishikawa cells. Furthermore, we predicted and verified that the pro-apoptosis factor BCL2L14 is the direct target of miR-663b. Over-expression of miR-663b and knock-down of BCL2L14 counteracted the suppressing effects of PT on HTB-111 and Ishikawa cells. In addition, we evaluated the miR-663b levels in EC tissues of 51 patients using an in situ hybridization technique. With the median of the score of miR-663b as a cut-off value, these EC patients were divided into two groups, and the patients with high miR-663b expression had significantly poor prognosis.

Epigenetic inhibition of miR-663b by long non-coding RNA HOTAIR promotes pancreatic cancer cell proliferation via up-regulation of insulin-like growth factor 2.

Pancreatic cancer is one of the most deadly cancers with a poor prognosis. Although microRNAs are involving in the carcinogenesis and development of pancreatic cancer, little information is known regarding the role of miR-663b in pancreatic cancer. In this study, the expression of miR-663b in pancreatic cancer cells was down-regulated by hypermethylation in its putative promoter region, and overexpression of miR-663b repressed cell proliferation, invasion and migration, and induced apoptosis in pancreatic cancer cells. Bioinformatics analysis, luciferase report assay and rescue experiments showed that insulin-like growth factor 2 (IGF2) was a direct target of miR-663b. Results from clinical samples showed that the expression level of miR-663b correlated with the pathological grading, and the expression of miR-663b was down-regulated and was inversely correlated with IGF2 expression level in pancreatic cancer tissues. More importantly, the long non-coding RNA, HOX transcript antisense RNA (HOTAIR), was up-regulated in both pancreatic cancer cells and tissues, and HOTAIR suppressed the expression of miR-663b in pancreatic cancer by histone modification on H3K4me3 and H3K27me3 on miR-663b promoter. Further in vivo studies demonstrated that the stable overexpression of miR-663b or knock-down of HOTAIR inhibited tumor growth and was associated with IGF2 expression. In summary, our studies demonstrated that miR-663b is epigenetically repressed by HOTAIR and exerts its tumor-suppressive function via targeting IGF2 in pancreatic cancer.