Levels Of miR-99a Research Articles

MiRNA99a as a Potential target in P13K/Akt1/mTOR signaling pathway in progression of OSCC

BackgroundOral cancer presents a significant global health challenge, driving ongoing research to enhance diagnostics and treatments. MicroRNAs, particularly miRNA99a, have emerged as key players in oral cancer's initiation, progression, and advanced development. However, their precise molecular mechanisms remain unclear. We analyze miRNA99a in modulating the PI3K/Akt1/mTOR signaling pathway within oral squamous cell carcinoma through in silico data analysis. Additionally, we examined miRNA99a levels in both oral submucous fibrosis (OSMF) and oral squamous cell carcinoma (OSCC). Materials and MethodsA comprehensive approach utilizing various insilico tools identified potential target genes regulated by miRNA99a and examined their interactions within the PI3K/Akt1/mTOR signaling pathway. The study involved meticulous screening, functional enrichment analyses, and network analyses to understand the regulatory networks influenced by miRNA99a. Additionally, RT-PCR was used to measure the CT levels of miR-99a in OSMF, OSCC and NM samples. ResultsThe analysis revealed a cohort of putative target genes regulated by miRNA99a, demonstrating their involvement in crucial cellular processes linked to OSCC progression. Functional enrichment analyses highlighted the significant association of these target genes with the PI3K/Akt1/mTOR pathway, indicating their potential impact on pivotal oncogenic signaling pathways. Network analyses revealed complex regulatory networks orchestrated by miRNA99a, its action within the PI3K/Akt1/mTOR signalling pathway and influencing OSCC development. RT-PCR analysis revealed a significant downregulation of miR-99a in OSCC and OSMF samples compared to NM, with mean CT values of 39.0940 and 38.3986 respectively, versus 33.7540 in NM (p = 0.000). ConclusionmiRNA99a's potential as a crucial regulator of the PI3K/Akt1/mTOR pathway in OSCC. The identified target genes and their interactions offer a foundation for further experimental validations, presenting opportunities for discovering novel therapeutic avenues or prognostic markers in managing OSCC. Integrating multi-omics data reinforces the significance of miRNA99a-mediated regulatory mechanisms in the intricate landscape of oral cancer biology.

Clinical importance of serum miRNA levels in breast cancer patients

There is limited data on the relationship of miRNAs with parameters that may affect surgical management or reflect tumour prognosis. It was aimed to evaluate serum miRNA levels in breast carcinoma cases and reveal the relationship between these levels and prognosis-related factors such as the histological type of the tumour, estrogen receptor, progesterone receptor, Ki-67 index, HER-2neu, E-cadherin, tumour size, CK5/6, CA15.3 levels, number of tumour foci, number of metastatic lymph nodes, and status of receiving neoadjuvant therapy. Thirty-five patients with a histopathologically confirmed breast carcinoma diagnosis in the case group and 35 healthy individuals in the control group were examined. miR-206, miR-17-5p, miR-125a, miR-125b, miR-200a, Let-7a, miR-34a, miR-31, miR-21, miR-155, miR-10b, miR-373, miR-520c, miR-210, miR-145, miR-139-5p, miR-195, miR-99a, miR-497 and miR-205 expression levels in the serum of participants were determined using the Polymerase Chain Reaction method. While serum miR-125b and Let-7a expression levels were significantly higher in breast cancer patients, miR-17-5p, miR-125a, miR-200a, miR-34a, miR-21, miR-99a and miR-497 levels were significantly lower in them. The Let-7a expression level had a statistically significant relationship with breast cancer histological type and HER-2neu parameters, miR-17-5p, miR-125b, Let-7a, miR-34a, miR-21 and miR-99a levels with E-cadherin, miR-34a, miR-99a and miR-497 with CA15.3, miR-125b, miR-200a and miR-34a with the number of metastatic lymph nodes, miR-125a with the number of tumour foci and miR-200a with the status of having the neoadjuvant therapy. Serum miR-17-5p, miR-125a, miR-125b, miR-200a, Let-7a, miR-34a, miR-21, miR-99a and miR-497 expression levels were determined to have predictive and prognostic importance in breast cancer.

MiRNAs as predictive biomarkers of response to treatment in pediatric patients with acute lymphoblastic leukemia.

MicroRNAs (miRNAs/miRs) are promising prognostic biomarkers in pediatric acute lymphoblastic leukemia (ALL). The present study aimed to identify miRNAs that could serve as prognostic biomarkers or as novel therapeutic targets in ALL. The expression levels of 84 miRNAs were assessed in the bone marrow aspirates of 10 pediatric patients with newly diagnosed ALL at diagnosis and on day 33 of induction of the ALL Intercontinental Berlin-Frankfurt-Münster 2009 protocol, and associations with established prognostic factors were evaluated. The levels at diagnosis of 25 miRNAs were associated with ≥2 prognostic factors. Higher expression levels of let-7c-5p, miR-106b-5p, miR-26a-5p, miR-155-5p, miR-191-5p, miR-30b-5p and miR-31-5p were significantly associated with a good prednisone response. The expression levels of miR-125b-5p, miR-150-5p and miR-99a-5p were significantly higher in standard- or intermediate-risk patients compared with those in high-risk patients (P=0.017, P=0.033 and P=0.017, respectively), as well as in those with a complete response at the end of induction (P=0.044 for all three miRNAs). The change in expression levels between diagnosis and the end of induction differed significantly between risk groups for three miRNAs: miR-206, miR-210 and miR-99a (P=0.033, P=0.047 and P=0.008, respectively), with the post induction levels of miR-206 increased in high-risk patients, whilst miR-210 and miR-99a levels were increased in intermediate/standard risk patients. Therefore, miRNAs that could be integrated into the risk stratification of pediatric ALL after further evaluation in larger patient cohorts were identified.

Preoperative Plasma miRNA Levels Predict Prognosis in Early-stage Malignant Melanoma.

Non-invasive circulating tumor biomarkers in liquid biopsy, such as microRNAs (miRNA), provide for better personalization of treatment strategies. The aim of our study was to assess the prognosis of patients with melanoma undergoing tumor resection with curative intent based on analysis of selected circulating miRNAs. A total of 22 patients with stage I to III melanoma were enrolled into this prospective study. Plasma samples were obtained pre-surgery and early post-surgery from peripheral blood draws. A panel of 23 candidate miRNAs was designed and expression of miRNAs were analyzed by reverse transcription-quantitative polymerase chain reaction with exogenous reference control cel-miR-39-3p. Higher preoperative expression levels of miR-99a (p=0.008), miR-320 (p=0.009), miR-1908 (p=0.001), miR-494 (p=0.018) and miR-4487 (p=0.048) were associated with a shorter disease-free interval. Similarly, higher preoperative plasma levels of miR-99a (p=0.017), miR-221 (p=0.026), miR-320 (p=0.016), miR-494 (p=0.009), miR-1260 (p=0.026) and miR-1908 (p=0.024) were associated with worse overall survival. No significant differences between pre- and postoperative plasma miRNA levels were observed. Liquid biopsy is a minimally-invasive approach which can lead to a better understanding of cancer behavior and offers the possibility of precise patient prognosis, allowing selection of the most appropriate treatment. Our study showed that preoperative plasma levels of miR-99a, miR-221, miR-320, miR-494, miR-1908 and miR-4487 were associated with disease-free interval and overall survival of patients with early-stage melanoma. This approach may help in decision-making about the appropriateness of modern adjuvant treatment administration in patients with resectable melanoma.

MiR-99a alleviates apoptosis and extracellular matrix degradation in experimentally induced spine osteoarthritis by targeting FZD8

BackgroundOur previous study identified miR-99a as a negative regulator of early chondrogenic differentiation. However, the functional role of miR-99a in the pathogenesis of osteoarthritis (OA) remains unclear.MethodsWe examined the levels of miR-99a and Frizzled 8 (FZD8) expression in tissue specimens. Human SW1353 chondrosarcoma cells were stimulated with IL-6 and TNF-α to construct an in vitro OA environment. A luciferase reporter assay was performed to analyze the relationship between miR-99a and FZD8. CCK-8 assays, flow cytometry, and ELISA assays were used to assess cell viability, apoptosis, and inflammatory molecule expression, respectively. Percutaneous intra-spinal injections of papain mixed solution were performed to create an OA Sprague–Dawley rat model. Alcian Blue staining, Safranin O Fast Green staining, and Toluidine Blue O staining were performed to detect the degrees of cartilage injury.ResultsMiR-99a expression was downregulated in the severe spine OA patients when compared with the mild spine OA patients, and was also decreased in the experimentally induced in vitro OA environment when compared with the control environment. Functionally, overexpression of miR-99a significantly suppressed cell apoptosis and extracellular matrix degradation stimulated by IL-6 and TNF-α. FZD8 was identified as a target gene of miR-99a. Furthermore, the suppressive effects of miR-99a on cell injury induced by IL-6 and TNF-α were reversed by FZD8 overexpression. Moreover, the levels of miR-99a expression were also reduced in the induced OA model rats, and miR-99a agomir injection relieved the cartilage damage. At the molecular level, miR-99a overexpression downregulated the levels of MMP13, β-catenin, Bax, and caspase-3 protein expression and upregulated the levels of COL2A1 and Bcl-2 protein expression in the in vitro OA-like chondrocyte model and also in the experimental OA model rats.ConclusionsOur data showed that miR-99a alleviated apoptosis and extracellular matrix degradation by targeting FZD8, and thereby suppressed the development and progression of experimentally induced spine osteoarthritis.

Down-regulated expression of miR-99a is associated with lymph node metastasis and predicts poor outcome in stage IB cervical squamous cell carcinoma: a case-control study.

Background Lymph node metastasis (LNM) accounts for the most important route of metastasis for cervical cancer. Yet, the status of LNM is different in patients with similar clinico-pathological variables. It has been revealed that microRNAs are widely involved in the occurrence and development of various malignancies, and the tumor-suppressive or promoting effects of microRNA-99 (miR-99) family have been previously reported. This study sought to investigate the predictive value of miR-99a for lymphogenous spread and its effect on the survival of patients with early-stage cervical squamous cell cancer (CSCC).MethodsPatients with stage IB squamous cervical cancer who were treated surgically between October 2015 and November 2018 were enrolled. A total of 21 formalin-fixed paraffin-embedded tissues of pathologically confirmed positive lymph nodes were retrieved, and an additional 21 tissues of negative lymph nodes from patients well-matched on baseline characteristics were collected as the control group. TaqMan real-time quantitative polymerase chain reaction was used to examine the expression levels of miR-99a in the samples. Differential expression levels of miR-99a were compared between the 2 groups using independent sample t-test. Furthermore, the associations between miR-99a expression level and clinico-pathological parameters of these 42 patients was evaluated by Chi-square test or Fisher’s exact-probability method, and their effects on survival were assessed using Kaplan-Meier product-limit method.ResultsThere were no significant differences in baseline clinico-pathological parameters between the 2 groups (P>0.05). The expression levels of miR-99a in the node-positive group and control group were 1.61±3.09 and 16.77±30.40, respectively (P=0.029). Downregulated expression of miR-99a was closely related to depth of invasion (DOI) and lymph-vascular space invasion (P<0.05). Univariate analysis revealed that downregulated miR-99a and deeper DOI were associated with worse 5-year disease-free survival, while multivariate analysis showed that only the expression level of miR-99a was an independent factor for disease-free survival (HR =0.120; 95% CI: 0.015–0.979; P=0.048). Patients with downregulated miR-99a tended to have more unfavorable overall survival, but the difference did not reach statistical significance.Conclusions MiR-99a plays an inhibitory role in the pathogenesis of lymph node metastasis and may serve as a novel prognostic biomarker for patients with CSCC.

MiRNA-99a alleviates inflammation and oxidative stress in lipopolysaccharide-stimulated PC-12 cells and rats post spinal cord injury

ABSTRACT Spinal cord injury (SCI) is caused by spinal fracture after the displacement of the spine or broken bone fragments protruding into the spinal canal, resulting in different degrees of injury to the spinal cord or spinal nerves. Expression levels of miR-99a and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) in cerebrospinal fluid of SCI patients were analyzed. Rat adrenal gland pheochromocytoma cell line PC-12 were stimulated with lipopolysaccharide (LPS) to mimic the in vitro environment of SCI. A rat mode of SCI was established by laminectomy. Reactive oxygen species (ROS) levels were measured by 2’,7’-Dichlorodihydrofluorescein diacetate staining assay. Western blot was conducted to evaluate the expression levels of apoptotic indexes and proinflammatory cytokines. The interaction between miR-99a and NOX4 was verified by dual-luciferase reporter assay. The expression level of miR-99a was reduced while NOX4 expression was upregulated in cerebrospinal fluid of SCI patients and LPS-treated PC-12 cells. LPS impeded cell viability and promoted inflammation, apoptosis and ROS levels of PC-12 cells. Overexpression of miR-99a significantly promoted cell viability and reduced inflammation, apoptosis and oxidative stress in LPS-stimulated PC-12 cells. Dual-luciferase reporter assays verified that NOX4 was a target of miR-99a. Moreover, the expression of NOX4 was reduced in PC-12 cells after transfection with miR-99a mimic. Overexpression of NOX4 partly abolished the protective effect of miR-99a in LPS-treated PC-12 cells. To sum up, miR-99a suppresses NOX4 expression to relieve the LPS-induced inflammation, apoptosis and the progression of oxidative stress in SCI.

MicroRNA-99a Restricts Replication of Hepatitis C Virus by Targeting mTOR and de novo Lipogenesis.

In this study, we investigated the role of microRNA-99a (miR-99a) in hepatitis C virus (HCV) replication and lipogenesis in hepatocytes. Cell-culture-derived HCV (HCVcc) infection caused down-regulation of miR-99a in Huh-7 cells, and the relative levels of miR-99a were significantly lower in the sera of the HCV-infected patients than in those of healthy controls. Transfection of miR-99a-5p mimics resulted in a decrease in the intracellular and secreted HCV RNA levels. It also caused a decreased mammalian target of rapamycin (mTOR) protein level and phosphorylation of its downstream targets in HCV-replicating cells. Sterol regulatory element binding protein (SREBP)-1c expression and intracellular lipid accumulation decreased when either miR-99a-5p mimics or si-mTOR was transfected in oleic acid-treated Huh-7 cells. Overexpression of mTOR rescued HCV RNA replication and lipid droplet accumulation in miR-99a-5p mimics-transfected HCV replicon cells. Our data demonstrated that miR-99a ameliorates intracellular lipid accumulation by regulating mTOR/SREBP-1c and causes inefficient replication and packaging of intracellular HCV.

Transcriptional factor Yin Yang 1 facilitates the stemness of ovarian cancer via suppressing miR-99a activity through enhancing its deacetylation level

The promoting effects of transcriptional factor Yin Yang 1 (YY1) have been confirmed in various tumors, however, its roles in ovarian cancer (OC) progression are still unclear. Here, Kaplan-Meier Plotter analysis was used to determine the correlation between YY1 expression and the survival of OC patients. It was found that YY1 expression was negatively correlated with the overall survival, progression-free survival and post-progression survival of OC patients. Functional experiments indicated that overexpression of YY1 facilitated the stemness of OC cells, while YY1 knockdown reduced it. MiRNAs-based RNA-sequencing analysis showed that miR-99a was the mostly upregulated miRNA in RNA extracted from OC cells with YY1 knockdown. Mechanistic studies revealed that YY1 recruited (Histone deacetylase) HDAC5 to the promoter of miR-99a, and subsequently enhanced miR-99a deacetylation level and decreased miR-99a level. Additionally, overexpression of miR-99a or knockdown of HDAC5 attenuated the promoting effects of YY1 on the stemness of OC cells. This work firstly indicated a novel YY1/miR-99a axis, which promotes the stemness of OC cells.

Plasma level of miR-99b may serve as potential diagnostic and short-term prognostic markers in patients with acute cerebral infarction.

ObjectiveThe aim of the present study is to explore the potential diagnostic and prognostic value of plasma levels of miR‐99 family for patients with acute cerebral infarction (ACI).MethodsA total of 112 patients who have been diagnosed with ACI were enrolled in this study, and 112 healthy volunteers were served as the controls. The plasma of the patients and controls were collected, and total RNAs were isolated, and the expression levels of miR‐99a, miR‐99b, and miR‐100 in the plasma of patients and controls were compared determined by RT‐qPCR methods; moreover, the receiver operating characteristic (ROC) curve has been drawn to determine whether the plasma levels of miR‐99b can distinguish patients with ACI from the controls; furthermore, the short‐term prognosis of the patients was evaluated by glasgow outcome scale (GOS), and the correlation between the plasma levels of miR‐99b and the GOS of the patients was evaluated. Finally, the correlation between the plasma level of miR‐99 and VEGF of ACI patients was analyzed.ResultsIt was observed that miR‐99b was significantly decreased in the plasma of ACI patients compared with the healthy controls (P < .01), while the plasma levels of miR‐99a and miR‐100 showed no significant differences between the patients with ACI and the healthy controls; moreover, the area under the curve (AUC) of miR‐99b for the diagnosis of ACI was 0.8882 (95% confidence interval (CI), 0.8451‐0.9313), suggesting that plasma level of miR‐99b is a sensitive marker to distinguish patients with ACI from the healthy volunteers; furthermore, the serum level of miR‐99b was negatively correlated with GOS score of the patients (r = −.56, P < .001); finally, the plasma level of miR‐99b was negatively correlated with the levels of VEGF (r = −.3013, P = .0012).ConclusionmiR‐99b was down‐regulated in plasma of patients with ACI, and plasma level of miR‐99b may be a potential diagnostic and prognostic marker for the diagnosis and treatment of ACI.

Study of serum microRNA-99a relative expression as a diagnostic and prognostic noninvasive biomarker of breast cancer in Egyptian females

BackgroundBreast cancer (BC) is the most significant cause of malignancy deaths among females in Egypt as well as in other countries. Despite the evident miR-99a deregulation in many malignancies, including breast cancer, its serum level and clinical significance in breast cancer are still vague. This study assessed the role of dysregulation of serum levels of miR-99a in the diagnosis and prognosis of breast cancer in Egyptian females. MethodsBlood samples were obtained from 60 breast cancer females diagnosed as invasive ductal carcinoma and 40 healthy females of matched age. Samples were evaluated for miR-99a relative expression using SYBR green-based real-time RT-PCR technology. Also, the association between serum miR-99a expression and CA 15–3, the known tumor marker of breast cancer, was investigated. ResultsThe serum expression level of MiR-99a was lower in the breast cancer group than that of the control group (P < 0.001). MiR-99a was 76.7% sensitive and 95% specific in the diagnosis of breast cancer and its level correlated with many clinical parameters of breast cancer, including primary tumor extent (P = 0.021), TNM stage (P < 0.001), and histological grade of cancer (P = 0.003). Regression analysis revealed that lower miRNA 99a expression was considered as an independent predictor for the higher BC tumor stage. ConclusionExpression of serum miR-99a was downregulated in breast cancer offering early diagnosis and indicating poor prognosis of cases.

Influence and mechanism of miR-99a suppressing development of colorectal cancer (CRC) with diabetes mellitus (DM).

ObjectiveThis study aimed to identify the changes of miRNAs in colorectal cancer (CRC) complicated with diabetes mellitus (DM) (CRC + DM) tissues and their potential effects.MethodsThe changes of miRNAs in CRC + DM tissues were determined by miRNA microarray. The expression levels of miR-99a in 40 clinical specimens and 6 CRC cell lines were determined by qRT-PCR. The capacity for miR-99a to induce cell proliferation and invasion was examined with miR-99a-overexpressing HCT-116 cells. The relative mTOR mRNA and protein levels were determined by qRT-PCR and Western blotting, respectively, in HCT-116 cells transfected with miR-99a. The dual luciferase assay was performed to confirm the direct regulation of miR-99a on mTOR 3′-UTR. The HCT-116 cells were treated with 100 mg/L advanced glycation end products (AGEs); then, the mTOR expression levels were determined by qRT-PCR, Western blotting, and immunohistochemistry.ResultsSeventeen miRNAs were found to be differentially expressed among normal tissue, CRC tissue, and CRC with DM tissue, including 15 upregulated and 2 downregulated with fold changs of more than 2 times. qRT-PCR confirmed that miR-99a was downregulated in CRC and CRC + DM tissues. In addition, miR-99a overexpression remarkably impaired CRC cell proliferation and metastasis, and negatively regulated mTOR signaling through direct binding to the 3′-UTR of mTOR. AGEs could suppress miR-99a and stimulate mTOR signaling in CRC cells. Increased mTOR was also identified in CRC with DM tissues.ConclusionOur findings indicate that miR-99a is a potential marker and therapeutic target of CRC complicated with DM, and that AGEs impair miR-99a-overactivated mTOR signaling in CRC with DM patients, which promotes CRC development.

MiR-99a suppresses cell migration and invasion by regulating IGF1R in gastric cancer.

Gastric cancer is a common kind of gastrointestinal malignancies. Increasing evidence indicates dysregulation of microRNA-99a (miR-99a) in gastric cancer, and has been extensively investigated in terms of cancer formation, progression, diagnosis, therapy, and prognosis. The purpose of this study is to explore how miR-99a worked in gastric cancer on migration and invasion. The mRNA and protein levels of miR-99a and insulin-like growth factor 1 receptor (IGF1R) in gastric cancer were measured by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot. Transwell assay was employed to analyze the migratory and invasive capacities. The Dual-Luciferase reporter assay was performed to confirm miR-99a mediated the expression of IGF1R by directly targeting its mRNA 3'-untranslated regions (3'-UTR) in gastric cancer cells. MiR-99a was discovered to be significantly downregulated while IGF1R was upregulated in gastric cancer tissues and cell lines. The expression of miR-99a had a negative correlation with the IGF1R expression in gastric cancer tissues. Moreover, miR-99a was low expressed in gastric cancer cells HGC-27 and MGC-803 compared to the normal cell line. MiR-99a suppressed the migration and invasion through directly binding to the 3'-UTR of IGF1R mRNA in HGC-27 cells. In addition, IGF1R could reverse partial roles of miR-99a on migration and invasion in gastric cancer. MiR-99a inhibited the migratory and invasive abilities by regulating the expression of IGF1R. MiR-99a was downregulated while IGF1R was upregulated in gastric cancer cell lines. The newly identified miR-99a/IGF1R axis provides novel insight into the pathogenesis of gastric cancer.

MiR-99a suppressed cell proliferation and invasion by directly targeting HOXA1 through regulation of the AKT/mTOR signaling pathway and EMT in ovarian cancer.

Ovarian cancer (OC) is the third frequently tumor worldwide. MicroRNA-99a (miR-99a), acting as a tumor suppressor, has been reported to be downregulated in multiple tumors. We aimed at exploring the significant roles of miR-99a in ovarian cancer. Quantitative Real-time polymerase chain reaction (qRT-PCR) and Western blotting were applied to calculate the mRNA and protein levels of miR-99a and its target genes. Kaplan-Meier method was conducted to evaluate the overall survival of ovarian cancer patients. CCK8 and transwell assays were performed to measure the proliferative and invasive abilities. miR-99a, acting as a prognosis predictor, was downregulated in ovarian cancer tissues and cell lines. miR-99a mediated the expression of homeobox A1 (HOXA1) through directly targeting to the 3'-untranslated region (3'-UTR) of its mRNA in ovarian cancer cell lines. miR-99a inhibited the proliferation of ovarian cancer by AKT/mTOR pathway in vitro and in vivo, and it suppressed the invasion-mediated epithelial-mesenchymal transition (EMT) through direct targeting to the 3'-UTR of HOXA1 mRNA. miR-99a suppressed the proliferation through AKT/mTOR signaling pathway and the invasion-mediated EMT in ovarian cancer. The newly identified miR-99a/HOXA1/AKT/mTOR axis provides novel insight into the pathogenesis of ovarian cancer.

Diagnostic and prognostic value of serum miR-99a expression in oral squamous cell carcinoma.

Despite progress in the treatment of oral squamous cell carcinoma (OSCC) over past years, the prognosis for OSCC patients remains dismal. MicroRNA-99a (miR-99a) has been found to involve in the development of many cancer types, but its clinical role in OSCC is unclear. The aim of this study was to explore the clinical implications of serum miR-99a in OSCC. This study detected serum miR-99a levels in 121OSCC cases and 55healthy controls by using quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. The data showed that serum miR-99a expression was significantly decreased in OSCC patients compared with normal controls. OSCC patients with low miR-99a expression experienced more frequent poor differentiation and advanced clinical stage. Furthermore, in screening OSCC cases from normal controls, miR-99a could yield a receiver-operating characteristic (ROC) area under the curve (AUC) of 0.911 with 83.6% specificity and 80.2% sensitivity. Notably, patients with high miR-99a expression had longer overall survival and recurrence free survival. Finally, serum miR-99a was identified to be an independent prognostic indicator for OSCC. These results suggested that miR-99a might be a valuable marker for the prediction of early detection and prognosis in OSCC.

Abstract 4655: Elevated level of serum miR-99a is correlated with serous epithelial ovarian cancer and can be a potential biomarker

Abstract Objective: There is a critical need for improved diagnostic markers to detect ovarian high grade serous cancer (HGSC). microRNAs (miRNAs) stably exist in circulating blood, reflecting tissue or organ conditions and present in circulating microvesicles such as exosomes. We aimed to identify which miRNAs are highly produced from HGSCs and analyze whether serum miRNA can discriminate patients with HGSC from healthy controls. Methods: Secreted exosomes from ovarian cancer cell lines were collected and exosomal miRNAs extracted. miRNA microarray was performed and several elevated miRNAs specific to ovarian cancer cells were picked up. Among these, we focused on miR-99a. Sera were collected from 15 patients with HGSC (stage III and IV) and 10 healthy controls. Expression level of miR-99a was determined by miRNA RT-qPCR. Results: miRNA microarray revealed several miRNAs were highly expressed in exosomes in patients with HGSC. In patients, serum miR-99a levels were significantly increased (4.8 fold) compared with those of healthy controls. ROC analysis showed that at the cut-off of 1.4, the sensitivity and specificity were 73 % and 80 % respectively for detecting HGSC (AUC = 0.85). Conclusion: Exosomal miRNAs can be detected in sera of patients with HGSC and have the potential to predict ovarian cancer. Citation Format: Akihiko Yoshimura, Kenjiro Sawada, Koji Nakamura, Yasuto Kinose, Erika Nakatsuka, Seiji Mabuchi, Tadashi Kimura. Elevated level of serum miR-99a is correlated with serous epithelial ovarian cancer and can be a potential biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4655. doi:10.1158/1538-7445.AM2017-4655

Upregulation of Mir-99a Is Associated with Poor Prognosis of Acute Myeloid Leukemia and Promotes Myeloid Leukemia Cell Expansion

Acute myeloid leukemia (AML) is a hereditarily and clinically heterogeneous disease driven by a subpopulation of leukemic stem cells (LSCs) that generate the bulk of leukemic cells. LSCs can resist available treatments and result in disease progression and relapse. High number of LSCs or expression of a LSC related gene signature is independently associated with poor prognosis in AML, supporting the notion for LSCs as important targets for therapeutic intervention. It is now evident that miRNAs contribute significantly to the leukemogenic phenotype. To dissect the miRNA expression profile of LSCs, we performed miRNA array analysis in enriched LSCs cells and non-LSCs (leukemic blast cells) from bone marrow (BM) cells of the same AML patient at different disease stages. We identified that miR-99a was significantly upregulated in the LSCs obtained at relapse compared to the LSCs collected at the time of initial diagnosis. We also found that miR-99a were upregulated in LSCs compared to non-LSCs in a larger cohort of AML patients. The cohort were divided into two groups based on the median expression level of miR-99a in LSCs reference to non-LSCs, and correlation analysis indicated that higher expression levels of miR-99a in LSCs were significantly correlated with worse overall survival and event-free survival in these AML patients. Compared with cord blood CD34+ HSPCs, miR-99a was significantly highly expressed in LSCs from AML patients. The expression level of miR-99a was also higher in K562/A02 (multidrug resistant counterpart of K562 cell line) and K562/G01 (Gleevec resistant counterpart of K562 cell line) than the parental K562 cells. These results point to the importance of a novel role of miR-99a in LSC activity and disease progression.By gain-of-function studies via stable lentiviral transduction, we demonstrated that miR-99a functionally regulated the growth and survival of leukemic cells in vitro and in vivo. Enforced miR-99a expression significantly increased the clonogenic capacity in primary AML LSCs. Further studies in myeloid leukemia cell lines revealed that ectopic miR-99a led to accelerated cell-cycle progression and cell proliferation. MiR-99a overexpression (OE) also promotes leukemic cell survival after exposure to chemotherapeutic agents in AML cell lines. In vivo study in a subcutaneous xenograft model showed that a significant lower tumor inhibition rate was observed overtime in the miR-99a/OE cell injected mice compared to the Ctrl cell injected mice when treated with Ara-C. All these results demonstrated that miR-99a, the miRNA enriched from LSCs at relapse stage, plays an important role in myeloid leukemic cell survival and expansion.To explore the mechanism underlying the enhanced proliferation and chemoresistance meditated by miR-99a, we identified differential gene expression profiles by microarray analysis. The results provide a potential regulatory pathway that links the downregulated targets of miR-99a (i.e., PPP3CA) to the dysregulated key genes associated with leukemic cell expansion. Overexpression of miR-99a led to upregulation of CCNE1, and downregulation of CDKN2A which mediate G1 cell cycle arrest by stabilizing the structure of p53 or inducing p53 activation. All these results suggest an important role of miR-99a in cell cycle entry and progression, leading to the accelerated proliferation and overcoming of chemotherapeutic-agents mediated cell cycle arrest.Collectively, our study provides strong evidence that miR-99a plays an important role in altering biological properties, which may lead to the pathogenesis of myeloid leukemia and chemoresistance induction. The levels of miR-99a may be used as a biomarker for the prognosis and progression of AML. DisclosuresNo relevant conflicts of interest to declare.

Upregulation of miR-99a is associated with poor prognosis of acute myeloid leukemia and promotes myeloid leukemia cell expansion.

Leukemia stem cells (LSCs) can resist available treatments that results in disease progression and/or relapse. To dissect the microRNA (miRNA) expression signature of relapse in acute myeloid leukemia (AML), miRNA array analysis was performed using enriched LSCs from paired bone marrow samples of an AML patient at different disease stages. We identified that miR-99a was significantly upregulated in the LSCs obtained at relapse compared to the LSCs collected at the time of initial diagnosis. We also found that miR-99a was upregulated in LSCs compared to non-LSCs in a larger cohort of AML patients, and higher expression levels of miR-99a were significantly correlated with worse overall survival and event-free survival in these AML patients. Ectopic expression of miR-99a led to increased colony forming ability and expansion in myeloid leukemia cells after exposure to chemotherapeutic drugs in vitro and in vivo, partially due to overcoming of chemotherapeutic agent-mediated cell cycle arrest. Gene profiling and bioinformatic analyses indicated that ectopic expression of miR-99a significantly upregulated genes that are critical for LSC maintenance, cell cycle, and downstream targets of E2F and MYC. This study suggests that miR-99a has a novel role and potential use as a biomarker in myeloid leukemia progression.

Abstract 1086: Elevated level of circulating microRNA-99a correlates with serous epithelial ovarian cancer and can be used as a potential biomarker

Abstract [Objective] There is a critical need for improved diagnostic markers for ovarian high grade serous cancer (HGSC). Emerging evidence have shown that microRNAs (miRNAs) stably exist in circulating blood, reflecting tissue or organ conditions and these are present in circulating microvesicles such as exosomes. The purpose of this study was to identify which miRNAs are highly produced from HGSCs and to analyze whether circulating miRNA can discriminate patients with HGSC from healthy volunteers. [Methods] Secreted exosomes from serous ovarian cancer cell lines were collected and exosomal miRNAs were extracted. miRNA microarray was performed and several elevated miRNAs specific to ovarian cancer cells were picked-up. Among these, we focused on miR-99a in this study. Sera were collected from 9 consecutive patients with HGSC and 5 healthy volunteers. Expression level of miR-99a was determined by miRNA RT-qPCR. [Results] miRNA microarray revealed several miRNAs were highly expressed in exosomes in patients with HGSC. In patients, serum miR-99a levels were significantly increased (7.2 fold) compared with healthy controls. Receiver operating curve (ROC) analysis for miR-99a showed that at the cut-off of 2.1 times compared with control, the sensitivity and specificity of this marker were 77.8% and 100%, respectively for detecting HGSC (AUC = 0.84). Serum miR-99a levels decreased after surgery by 0.2 fold, indicating that serum miR-99a was originated from ovarian cancer. [Conclusion] Elevated serum miR-99a were detected in sera of patients with HGSC and reflected the disease condition. This miRNA profiling in serum can be used as a diagnostic biomarker, extending its utility to screening asymptomatic populations. Citation Format: Akihiko Yoshimura. Elevated level of circulating microRNA-99a correlates with serous epithelial ovarian cancer and can be used as a potential biomarker. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1086.

Inhibition of the glucocorticoid receptor results in an enhanced miR-99a/100-mediated radiation response in stem-like cells from human prostate cancers.

Radiation therapy is a major primary treatment option for both localized early stage prostate cancer, and for advanced, regionally un-resectable, cancer. However, around 30% of patients still experience biochemical recurrence after radiation therapy within 10 years. Thus, identification of better biomarkers and new targets are urgently required to improve current therapeutic strategies. The miR-99 family has been shown to play an important role in the regulation of the DNA damage response, via targeting of the SWI/SNF chromatin remodeling factors, SMARCA5 and SMARCD1 in cell line models. In the present study, we have demonstrated that low expression of miR-99a and miR-100 is present in cell populations which are relatively radiation insensitive, for example in prostate cancer stem cells and in castration-resistant prostate cancer. Additionally, treatment of cells with the synthetic glucocorticoid, Dexamethasone resulted in decreased miR-99a and 100 expression, suggesting a new mechanism of miR-99a and 100 regulation in androgen-independent prostate cells. Strikingly, treatment of prostate cells with the glucocorticoid receptor inhibitor, Mifepristone was found to sensitize prostate cells to radiation by increasing the levels of miR-99a and miR-100. These results qualify the miR99 family as markers of radiation sensitivity and as potential therapeutic targets to improve efficiency of radiotherapy.