Potential of miR-181a-5p and miR-630 as clinical biomarkers in NSCLC.

BACKGROUND: The development of drug resistance and high mortality rates are the major problems observed in non-small cell lung cancer (NSCLC). Biomarkers indicating and predicting disease development towards these unfavorable directions are therefore on high demand. Many studies have demonstrated that changes in miRNAs expression may be associated with a response to treatment and disease prognosis, thus suggesting its potential biomarker value for a broad spectrum of clinical applications. The aim of the present study was to investigate the expression level of miR-181a-5p, miR-630, and its targets in NSCLC tumor tissue and plasma samples; and to analyze its association with NSCLC patient's response to treatment and disease prognosis. METHODS: The study was performed in 89 paired tissue specimens and plasma samples obtained from NSCLC patients who underwent surgical treatment at the Department of Thoracic Surgery and Oncology of the National Cancer Institute. Analysis of miR-181a-5p and miR-630 expression was performed by qRT-PCR using TaqMan miRNA specific primers. Whereas BCL2, LMO3, PTEN, SNAI2, WIF1 expression levels were identified with KAPA SYBR FAST qPCR Kit. Each sample was examined in triplicate and calculated following the 2-ΔΔCt method. When the p-value was less than 0.05, the differences were considered statistically significant. RESULTS: It was found that miR-181a-5p and miR-630 expression levels in NSCLC tissue and plasma samples were significantly decreased compared with control samples. Moreover, patients with low miR-181a-5p expression in tumor tissue and plasma had longer PFS rates than those with high miRNA expression. Decreased miR-630 expression in tumor was statistically significantly associated with better NSCLC patients' OS. In addition, the expression of miR-181a-5p, as well as miR-630 in tumor tissue, are the statistically significant variables for NSCLC patients' OS. Moreover, in NSCLC patient plasma samples circulating miR-181a-5p can be evaluated as significant independent prognostic factors for OS and PFS. CONCLUSIONS: Our findings indicate the miR-181a-5p and miR-630 expression levels have the potential to prognose and predict and therefore improve the treatment individualization and the outcome of NSCLC patients. Circulating miR-181a-5p has the potential clinical value as a non-invasive biomarker for NSCLC.

Acovenoside A as a novel therapeutic approach to boost taxol and carboplatin apoptotic and antiproliferative activities in NSCLC: Interplay of miR-630/miR-181a and apoptosis genes.

The aim of the present study is to explore the potential anticancer effect of the cardenolide; acovenoside A against non-small cell lung cancer (NSCLC), understand its molecular mechanism in inducing apoptosis and show the effect of its combination with carboplatin and taxol. MTT assay showed that the combination of acovenoside A with taxol and carboplatin caused 78.9% cytotoxicity reflecting the synergistic effect. The triple combination showed the best growth inhibition efficiency where the number of cells at the G2/M phase was decreased and boosted up apoptotic and necrotic activity. The combination also showed the most remarkable increase in gene expression of Bax and p53 and the least level of Bcl2. The gene expression of miRNA181a and miRNA630 was significantly upregulated in cell lines treated with the combination. The present study has proven that the underlying mechanism of acovenoside A is partially attributed to the upregulation of miR-630 and miR-181a gene expressions which in turn targets the intrinsic apoptosis genes as p53, Bax and Bcl2 as well as caspase 3. The present study is the first to address the valuable effect of using acovenoside A together with carboplatin and taxol in the treatment of NSCLC via exerting apoptotic, antiproliferative, and cytotoxic effects..

Silencing of circARHGAP12 inhibits the progression of atherosclerosis via miR-630/EZH2/TIMP2 signal axis.

Atherosclerosis (AS) is a common disease that seriously threatens human health. So far, the pathogenesis of AS has not been fully understood. This project investigates the effects of circARHGAP12 on AS and its regulatory mechanism. ApoE-/- knockout mice (ApoE) were adopted and reared with a high-fat diet to construct an AS model. Lentivirus was established to knock down the expression of circARHGAP12 in mice. After 12 weeks, the aorta was removed and the expression of circARHGAP12 was detected. Vascular oil red O staining was used to detect the degree of AS. The expression of inflammatory factors was detected by ELISA. Aortic smooth muscle cells (MASMCs) were cultured to evaluate the effects of circARHGAP12 on the phenotype of MASMCs. RNA pull-down and luciferase assay were used to verify the downstream target genes of circARHGAP12. In addition, the effects of circARHGAP12 on MASMCs proliferation and migration were detected by MTT and transwell assay. Compared with the normal group, the expression of circARHGAP12 in the MASMCs under ox-LDL treatment was elevated, and circARHGAP12 silencing could inhibit AS in vitro and in vivo. The results of the mechanism study showed that circARHGAP12 can directly bind with miR-630. In addition, miR-630 can also target EZH2 to modulate the transcription of TIMP2 and to influence the migration of MASMCs. circARHGAP12 is upregulated in AS. CircARHGAP12 knockdown can inhibit the progression of AS. This study expands on the role of circRNA in AS and provides potential targets for the treatment of AS.

The importance of miRNA-630 in human diseases with an especial focus on cancers.

miR-630 is encoded by MIR630 gene (NC_000015.10) on 15q24.1. This miRNA is mostly associated with cytokine signaling in immune system. Several neoplastic as well as non-neoplastic conditions have been linked with dysregulation of miR-630. It is an oncogenic miRNA in renal cell carcinoma, multiple myeloma, colorectal cancer, acute lymphoblastic leukemia, ovarian cancer and prostate cancer. On the other hand, it is a putative tumor suppressor miRNA in lung, cervical, breast, thyroid and esophageal tissues. In a number of other tissues, data regarding the role of miR-630 in the carcinogenesis is conflicting. Expression levels of miR-630 can be used as markers for prediction of cancer course. Moreover, miR-630 can influence response to chemoradiotherapy. This miRNA is also involved in the pathoetiology of IgA nephropathy, obstructive sleep apnea, age-related nuclear cataract and vitiligo. In the present review, we discuss the role of miR-630 in these conditions.

MiR-630 suppresses non-small cell lung cancer by targeting vimentin.

OBJECTIVE: This study aimed to clarify the function of miR-630 on non-small cell lung cancer (NSCLC) cells. METHODS: Quantitative real-time PCR was utilized to detect the mRNA expression of miR-630 and vimentin (VIM) in NSCLC tissues and cells. The protein expression of VIM, P53, Caspase-3, Bcl-2, Bax and JAK2/STAT3 was evaluated via Western blot. Dual-luciferase reporter assay was applied to evaluate whether VIM is the target gene of miR-630. The migration, invasion, proliferation and apoptosis of NSCLC cells were examined by wound-healing assay, transwell assay, CCK-8 assay, and flow cytometry, respectively. RESULTS: MiR-630 was lowly expressed in NSCLC tissues and cells, while VIM was highly expressed in NSCLC cells. Dual-luciferase reporter assay data validated that miR-630 directly targeted VIM. MiR-630 overexpression inhibited VIM expression, but the inhibition of miR-630 upregulated VIM expression. Besides, miR-630 mimics restrained cell migration, invasion, and proliferation, and promoted NSCLC cell apoptosis. Whereas, VIM overexpression partly attenuated the inhibitory effect of miR-630 on NSCLC cells. Moreover, miR-630 mimics impeded p-JAK2 and p-STAT3 protein expression; and miR-630 inhibitor upregulated p-STAT3 and VIM protein expression, which was reversed after the addition of STAT3 inhibitor C188-9. CONCLUSION: MiR-630 constrained the progression of NSCLC by inhibiting JAK2/STAT3 pathway and downregulating VIM expression.

CircCERS6 Suppresses the Development of Epithelial Ovarian Cancer Through Mediating miR-630/RASSF8.

Accumulating evidence have demonstrated that circular RNAs (circRNAs) exert important roles in tumor initiation and progression. Nevertheless, the role and mechanism of circRNA ceramide synthase 6 (circCERS6) in epithelial ovarian cancer (EOC) remain to be clarified. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay, we measured RNA and protein expression. We analyzed the effects of circCERS6/microRNA-630 (miR-630)/Ras-association domain family member 8 (RASSF8) axis in cell proliferation, migration, and invasion by 5-Ethynyl-2'-deoxyuridine (EdU) assay, colony formation assay, scratch test, and transwell assay. Using RNA-pull down assay and dual-luciferase reporter assay, the interaction between miR-630 and circCERS6 or RASSF8 was verified. The in vivo role of circCERS6 in tumor growth was analyzed using xenograft mice model. CircCERS6 expression was markedly reduced in EOC tissues and cell lines. CircCERS6 overexpression hampered the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of EOC cells. CircCERS6 directly interacted with miR-630. miR-630 expression was up-regulated in EOC tissues and cell lines. CircCERS6 overexpression-induced anti-tumor effects in EOC were largely reversed by the overexpression of miR-630. RASSF8 was a direct target of miR-630. RASSF8 level was decreased in EOC tissues and cell lines. CircCERS6 up-regulated RASSF8 expression by acting as a molecular sponge for miR-630 in EOC cells. CircCERS6 overexpression-mediated suppressive effects in EOC cells were largely overturned by the silence of RASSF8. CircCERS6 overexpression blocked tumor growth in vivo. CircCERS6 overexpression hampered the proliferation, migration, invasion, and EMT of EOC cells by up-regulating RASSF8 via sponging miR-630.

Effect of miR-630 expression on esophageal cancer cell invasion and migration.

BACKGROUND: Esophageal cancer (EC) is a common malignancy of the digestive tract, with high incidence. The objective of this study was to investigate the effect of miR-630 expression on esophageal cancer (EC) cell invasion and migration. METHODS: The study group comprised 58 EC patients admitted to our hospital from April 2014 to 2016, and the control group comprised 60 healthy people visiting the hospital during the same period. miR-630 levels in the peripheral blood of the two groups were compared, and the diagnostic value of miR-630 for EC was analyzed. EC cell lines were used to evaluate the influence of miR-630 expression on EC cell invasion and migration. RESULTS: miR-630 expression was low in EC (p < 0.050). A receiver operating characteristic curve analysis showed that miR-630 expression had a good diagnostic value for EC (p < 0.050) and was associated with disease course, pathological stage, differentiation degree, tumor metastasis, and patient prognosis and survival (p < 0.05). The ROC curve analysis showed that when cutoff value was 5.38, the diagnostic sensitivity and specificity of miR-630 for EC were 73.33% and 76.67%, respectively; area under the ROC curve was 0.778 (95%CI 0.695-0.861). Transfection of miR-630 into EC cells indicated that miR-630 overexpression can reduce EC cell invasion and migration (p < 0.05). miR-630 expression is low in EC and has good diagnostic value for EC. CONCLUSION: miR-630 overexpression can reduce EC cell invasion and migration, showing a possible key role of miR-630 in EC diagnosis and treatment in the future.

circMTDH.4/miR-630/AEG-1 axis participates in the regulation of proliferation, migration, invasion, chemoresistance, and radioresistance of NSCLC.

Astrocyte elevated gene-1 (AEG-1) plays a critical role in the development, progression, and metastasis of a variety of cancers, including non-small-cell lung cancer (NSCLC). The objective of the current study is to unravel the upstream signaling of AEG-1. A cohort of 28 NSCLC tissues and 30 normal tissues were collected. Quantitative reverse transcription-polymerase chain reaction and Western blotting were used to examine AEG-1, migration, and invasion related markers in NSCLC cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay coupled with colony formation assay were conducted to monitor cell growth. Transwell assay was performed to determine cell migration and invasion. Apoptotic cells were detected by costaining with Annexin-V-fluorescein isothiocyanate and propidium iodide. Immunofluorescent staining was used to observe the levels of migration and invasion related markers. Xenograft models were used to investigate tumor formation in vivo. Dual-luciferase reporter assay and RNA immunoprecipitation were carried out to determine the interaction between circMTDH.4 and miR-630, as well as the associated between miR-630 and AEG-1. AEG-1 was highly expressed in NSCLC tissues and cell lines. Silencing of AEG-1 inhibited cell proliferation, migration, invasion, and chemoresistance/radioresistance in NCI-H1650 and A549 cells. circMTDH.4 regulated AEG-1 expression via sponging miR-630. Knockdown of circMTDH.4 and/or overexpression of miR-630 inhibited chemoresistance and radioresistance in NSCLC cells, whereas overexpression of AEG-1 or knockdown of miR-630 exerted rescue effects. circMTDH.4/miR-630/AEG-1 axis is responsible for chemoresistance and radioresistance in NSCLC cells.

High-throughput microRNA profile in adult and pediatric primary glioblastomas: the role of miR-10b-5p and miR-630 in the tumor aggressiveness.

Glioblastoma (GBM) is the most common primary malignant neoplasm of the central nervous system and, despite the standard therapy; the patients' prognoses remain dismal. The miRNA expression profiles have been associated with patient prognosis, suggesting that they may be helpful for tumor diagnosis and classification as well as predictive of tumor response to treatment. We described the microRNA expression profile of 29 primary GBM samples (9 pediatric GBMs) and 11 non-neoplastic white matter samples as controls (WM) by microarray analysis and we performed functional in vitro assays on these 2 most differentially expressed miRNAs. Hierarchical clustering analysis showed 3 distinct miRNA profiles, two of them in the GBM samples and a group consisting only of cerebral white matter. When adult and pediatric GBMs were compared to WM, 37 human miRNAs were found to be differentially expressed, with miR-10b-5p being the most overexpressed and miR-630 the most underexpressed. The overexpression of miR-630 was associated with reduced cell proliferation and invasion in the U87 GBM cell line, whereas the inhibition of miR-10b-5p reduced cell proliferation and colony formation in the U251 GBM cell line, suggesting that these miRNAs may act as tumor-suppressive and oncogenic miRNAs, respectively. The present study highlights the distinct epigenetic profiling of adult and pediatric GBMs and underscores the biological importance of mir-10b-5p and miR-630 for the pathobiology of these lethal tumors.

LncRNA NOC2L-4.1 functions as a tumor oncogene in cervical cancer progression by regulating the miR-630/YAP1 pathway.

Long noncoding RNA (lncRNA) is a new class of noncoding RNA playing an indispensable role in different diseases by regulating miRNA. Our previous studies have suggested that miR-630 was decreased in patients with cervical cancer. Recently, studies have shown that lncRNA NOC2L-4.1 was abnormally expressed in patients with cervical cancer and can target miR-630. Therefore, we wanted to identify the integrated relationship between lncRNA NOC2L-4.1 and miR-630 in the pathological processes regarding cervical cancer either in vitro or in vivo. Quantitative reverse transcription-polymerase chain reaction detection shows that compared with human normal cervical epithelial cell, the expression of lncRNA NOC2L-4.1 was significantly increased and the expression of miR-630 was decreased in cell lines of cervical cancer. Moreover, luciferase reporter assay showed that miR-630 was a target of lncRNA NOC2L-4.1. The in vitro study found that downregulation of lncRNA NOC2L-4.1 suppressed cervical cancer cell migration (transwell assays) and proliferation (cell counting kit-8 and cloning formation assays). miR-630 specific inhibitor treatment reversed the inhibitory effect of lncRNA NOC2L-4.1 on cell proliferation and migration. Further studies also found that yes-associated protein 1 (YAP1) was the target of miR-630. Overexpression YAP1 suppressed miR-630 overexpression induced cell proliferation and inhibition of migration. Tumors induced by implantation of lncRNA NOC2L-4.1-knockdown Hela cells in nude mice showed that lncRNA NOC2L-4.1 silencing decreased the growth of tumors in both volume and weight by regulation of miR-630/YAP1. Taken together, our study reveals the important role of lncRNA NOC2L-4.1/miR-630/YAP1 regulatory network in cervical cancer, which provides new insights concerning the pathogenesis of cervical cancer.

MicroRNA-630 inhibits breast cancer progression by directly targeting BMI1.

MicroRNAs (miRNAs) play critical roles in breast cancer cell biological processes, including proliferation and apoptosis by inhibiting the expression of their target genes. Herein, we reported that miR-630 overexpression initiates apoptosis, blocks cell cycle progression and suppresses cell proliferation in breast cancer cells. Furthermore, BMI1, a member of polycomb group family, was identified as a direct target of miR-630, and there was a negative correlation between the expression levels of BMI1 and miR-630 in human breast cancer samples. With a series of biology approaches, subsequently, we proved that BMI1 was a functional downstream target of miR-630 and mediated the property of miR-630-dependent inhibition of breast cancer progression. Taken together, these findings provide further evidence on the tumor-suppression function of miR-630 in breast cancer, and clarify BMI1 as a novel functional target gene of miR-630.

MicroRNA-630 inhibitor sensitizes chemoresistant ovarian cancer to chemotherapy by enhancing apoptosis.

MicroRNA-630 (miR-630) has been implicated in the development and progression of multiple cancers. The current study aimed to investigate the role of miR-630 in chemoresistant epithelial ovarian cancer. MiR-630 expression levels were detected in ovarian cancer cell line SKOV3 and paclitaxel-resistant SKOV3 (SKOV3-TR) via microarray and qRT-PCR. MiR-630 inhibitors and negative controls were transfected into SKOV3 and SKOV3-TR cells. Wound healing, invasion, chemosensitivity, and cell apoptosis assays were performed to determine proliferation and migration rates. Chemoresistant patient-derived xenograft (PDX) models were established and utilized to verify the effect of miR-630 on chemoresistant ovarian cancer. Inhibition of miR-630 decreased cell proliferation and enhanced the sensitivity of SKOV3-TR and SKOV3 cells to paclitaxel. In the chemosensitivity assay, we observed that the miR-630 inhibitor exhibited a synergistic effect with paclitaxel on SKOV3-TR cells. Inhibition was correlated with enhanced expression of apoptosis-related proteins. APAF-1 was predicted to be a potential target of miR-630. An in vivo PDX study showed that the miR-630 inhibitor sensitized chemoresistant ovarian cancer to paclitaxel. Thus, miR-630 inhibitor sensitizes chemoresistant epithelial ovarian cancer to chemotherapy by enhancing apoptosis. Our findings suggest that miR-630 might be a potential therapeutic target for chemotherapy-resistant ovarian cancer.

MiR-630 inhibits invasion and metastasis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is among the most aggressive malignancies and has a high incidence in China. MicroRNAs (miRNAs) are small endogenous RNAs that regulate multiple tumorigenic processes, including proliferation, invasion, metastasis and prognosis. Using miRNA expression profiling analysis, we found that miR-630 was markedly down-regulated in three ESCC tissue samples compared with that in paired normal esophageal tissues. Differential miR-630 expression was subsequently confirmed using quantitative real-time PCR. To determine whether miR-630 down-regulation could be considered as a diagnostic indicator and adverse prognostic factor, we investigated the association between miR-630 and clinicopathological characteristics in patients with ESCC. It was found that decreased miR-630 expression was associated with poor overall survival in these patients. In addition, we also explored the biological function of miR-630 by targeting Slug and investigated the correlation between miR-630 expression and epithelial-mesenchymal transition (EMT) progression in vivo and in vitro Ectopic miR-630 expression could inhibit proliferation, invasion and metastasis, whereas miR-630 knockdown induced proliferation, invasion, metastasis and EMT traits. Overall, our study supports a role for miR-630 as a critical novel modulator in ESCC.

E2F1-regulated DROSHA promotes miR-630 biosynthesis in cisplatin-exposed cancer cells.

DNA damage may regulate microRNA (miRNA) biosynthesis at the levels of miRNA transcription, processing and maturation. Although involvement of E2F1 in the regulation of miRNA gene activation in response to DNA damage has been documented, little is known about the role of E2F1 in miRNA processing. In this study we demonstrate that E2F1 enhances miR-630 biosynthesis under cisplatin (CIS) exposure through promoting DROSHA-mediated pri-miR-630 processing. Northern blot and RT-qPCR revealed that CIS exposure caused not only an increase in pri-miR-630 but also much more increase in pre-miR-630 and mature miR-630. The increases in pri-miR-630 and pre-miR-630 expression in unmatched proportion indicated that primary transcript processing was involved in CIS-stimulated miR-630 biosynthesis. Furthermore, combination of reporter enzyme assay with mutation and over-expression of E2F1 showed that induction of DROSHA promoted miR-630 expression, in which CIS-induced E2F1 activated DROSHA gene expression by recognizing and binding two E2F1 sites at the positions -214/-207 and -167/-160 of the DROSHA promoter. The increased binding of E2F1 to the DROSHA promoter in CIS-exposed cells was further evidenced by chromatin immunoprecipitation assay. Together, E2F1-regulated DROSHA promotes pri-miR-630 processing, thereby, contributes to CIS-stimulated miR-630 expression. The involvement of E2F1-dependent DROSHA activation in pri-miRNA processing under DNA damage stress will provide further insight into the regulation of miRNA biosynthesis. These data also give us a deeper understanding of E2F1 role in response to DNA damage.

CircMAP3K4 Suppresses H2O2-Induced Human Lens Epithelial Cell Injury by miR-630/ERCC6 Axis in Age-Related Cataract.

BACKGROUND: Dysregulated circular RNAs (circRNAs) is involved in the pathogenesis of age-related cataract (ARC). Here, this study aimed to explore the function and mechanism of circMAP3K4 in ARC. METHODS: Human lens epithelial cells were exposed to hydrogen peroxide (H2O2) for functional experiments. qRT-PCR and western blotting analyses were used for the expression detection of genes and proteins. Cell proliferation was tested using cell counting kit-8 and EdU. Flow cytometry was applied to analyze cell apoptosis and cell cycle. The oxidative stress was evaluated by detecting the production of malondialdehyde (MDA), reactive oxygen species (ROS), and superoxide dismutase (SOD). The target relationship between miR-630 and circMAP3K4 or Excision repair cross-complementing group 6 (ERCC6) was analyzed by dual-luciferase reporter assay and RIP assay. RESULTS: CircMAP3K4 was lowly expressed in ARC patients and H2O2-induced HLECs. Functionally, forced expression of circMAP3K4 protected HLECs against H2O2-evoked proliferation inhibition, cell cycle arrest and the promotion of cell apoptosis and oxidative stress. Mechanistically, circMAP3K4 acted as a sponge for miR-630 to regulate the expression of its target ERCC6. MiR-630 was highly expressed while ERCC6 was lowly expressed in ARC patients and H2O2-induced HLECs. Up-regulation of miR-630 could reverse the protective effects of circMAP3K4 on HLECs under H2O2 treatment. In addition, inhibition of miR-630 suppressed H2O2-induced HLEC injury, which was abolished by ERCC6 silencing. CONCLUSION: Forced expression of circMAP3K4 protected HLECs against H2O2-evoked apoptotic and oxidative injury via miR-630/ERCC6 axis, suggesting that circMAP3K4 may function as a potential therapeutic target for ARC.

CircFAM114A2 inhibits the progression of hepatocellular carcinoma via miR-630/HHIP axis.

BACKGROUND: Many studies have shown that circular RNAs (circRNAs) are abnormally expressed in various tumor tissues and served as a key regulator in the occurrence and development of cancer. However, in hepatocellular carcinoma (HCC), the molecular mechanism of circRNAs in body fluids remains to be further explored. METHODS: The expression levels of genes and proteins were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. Cell counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), wound healing assay, Transwell assays, flow cytometry, and tumor formation models in nude mice were conducted to investigate the effects of circFAM114A2 on HCC cells both in vitro and in vivo. RNA antisense purification (RAP), dual luciferase reporter assays and rescue assays were carried out to verify the interaction between circFAM114A2, miR-630 and HHIP. RESULTS: CircFAM114A2 was significantly downregulated in HCC tissues and was associated with microvascular invasion and lymph node metastasis of HCC patients. We also observed that circFAM114A2 was lowly expressed in HCC plasma, which may serve as an effective biomarker to screen HCC patients from healthy controls (area under curve (AUC)=0.922). In vitro, circFAM114A2 overexpression significantly blunted HCC cell proliferation, migration, invasion, and promoted apoptosis, whereas circFAM114A2 silencing posed opposite effects. In vivo, circFAM114A2 overexpression inhibited the growth of HCC cells. Mechanistically, circFAM114A2 could increase the expression of the tumor suppressor HHIP via acting as a sponge for miR-630. CONCLUSIONS: CircFAM114A2 exerts a tumor suppressor role in HCC through miR-630/HHIP axis, and may be served as a potential diagnostic and therapeutic biomarker for HCC patients.

MiR-630 Promotes Radioresistance by Induction of Anti-Apoptotic Effect via Nrf2-GPX2 Molecular Axis in Head-Neck Cancer.

Head and neck cancer (HNC) ranks among the top ten prevalent cancers worldwide. Radiotherapy stands as a pivotal treatment component for HNC; however, radioresistance in cancerous cells often leads to local recurrence, becoming a substantial factor in treatment failure. MicroRNAs (miRNAs) are compact, non-coding RNAs that regulate gene expression by targeting mRNAs to inhibit protein translation. Although several studies have indicated that the dysregulation of miRNAs is intricately linked with malignant transformation, understanding this molecular family's role in radioresistance remains limited. This study determined the role of miR-630 in regulating radiosensitivity in HNC. We discovered that miR-630 functions as an oncomiR, marked by its overexpression in HNC patients, correlating with a poorer prognosis. We further delineated the malignant function of miR-630 in HNC cells. While it had a minimal impact on cell growth, the miR-630 contributed to radioresistance in HNC cells. This result was supported by decreased cellular apoptosis and caspase enzyme activities. Moreover, miR-630 overexpression mitigated irradiation-induced DNA damage, evidenced by the reduced levels of the γ-H2AX histone protein, a marker for double-strand DNA breaks. Mechanistically, the overexpression of miR-630 decreased the cellular ROS levels and initiated Nrf2 transcriptional activity, resulting in the upregulation of the antioxidant enzyme GPX2. Thus, this study elucidates that miR-630 augments radioresistance by inducing an anti-apoptotic effect via the Nrf2-GPX2 molecular axis in HNC. The modulation of miR-630 may serve as a novel radiosensitizing target for HNC.

CircPAG1 Inhibits the High Glucose-Induced Lens Epithelial Cell Injury by Sponging miR-630 and Upregulating EPHA2.

Background: Circular RNAs (circRNAs) have been considered as vital regulators in the progression of human ocular diseases, including diabetic cataract (DC). This report was designed to research the biological role of circRNA phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (circPAG1) in high glucose (HG)-induced lens epithelial damages.Methods: Lens epithelial damage in DC was investigated by the effects of 25 mM glucose (HG) on human lens epithelial cells (HLE-B3). CircPAG1, microRNA-630 (miR-630), and ephrin type-A receptor 2 (EPHA2) levels were examined by the quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation analysis was performed by 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) assay and colony formation assay. Cell apoptosis was measured through flow cytometry. Protein levels were detected using western blot. Oxidative stress was determined by malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) levels via the corresponding kits. Dual-luciferase reporter and RNA immunoprecipitation (RIP) and RNA pull-down assays were used for target binding analysis.Results: CircPAG1 expression was downregulated in lens samples of DC patients and HG-treated lens epithelial cells. HG inhibited cell growth but promoted apoptosis and oxidative stress in HLE-B3 cells, while circPAG1 overexpression relieved these damages. Moreover, circPAG1 was identified as a molecular sponge for miR-630. HG-induced cell injury was also attenuated by the inhibition of miR-630, and the function of circPAG1 was related to its sponge effect on miR-630. In addition, miR-630 directly targeted EPHA2 and circPAG1 could regulate the EPHA2 expression via sponging miR-630. Furthermore, we found that the protective role of circPAG1 against the HG-induced cell injury was ascribed to the upregulation of EPHA2.Conclusion: Our evidence suggested that circPAG1 alleviated cell damages in HG-treated human lens epithelial cells by regulating the miR-630/EPHA2 axis.

4-Aminobiphenyl inhibits the DNA homologous recombination repair in human liver cells: The role of miR-630 in downregulating RAD18 and MCM8.

4-Aminobiphenyl (4-ABP), a well-known human carcinogen, has been shown to cause oxidative DNA damage and induce miR-630 expression in HepG2 cells treated with 18.75 µM-300 µM for 24 h. However, the underlying mechanism regarding the epigenetic regulation of miR-630 on DNA damage repair in liver cells is still not understood and needs to be investigated. In present study, our results showed that miR-630 was upregulated, resulting in mediating a decrease of DNA homologous recombination (HR) repair in L-02, HepG2 or Hep3B cells. Results from a luciferase reporting experiment showed that RAD18 and MCM8 were the potential targets of miR-630 during DNA damage induction. The downregulation of RAD18 or MCM8 by miR-630 was accompanied by inhibition of HR repair. Conversely, inhibiting miR-630 enhanced the expression of RAD18 and MCM8, and rescued HR repair. Additionally, we proved that the transcription factor CREB was related to miR-630 biogenesis in liver cells. Moreover, the levels of CREB, miR-630 expression, and double-strand breaks (DSBs) were attenuated by 5 mM N-acetyl-L-cysteine (NAC) pretreatment, indicating that reactive oxygen species (ROS)-dependent CREB-miR-630 was involved in DSB repair. These findings indicated that the ROS/CREB/-miR-630 axis plays a relevant role in the regulation of RAD18 and MCM8 in HR repair, which may facilitate our understanding of molecular mechanisms regarding the role of miR-630 downregulating DNA damage repair in liver cells.

A low microRNA-630 expression confers resistance to tyrosine kinase inhibitors in EGFR-mutated lung adenocarcinomas via miR-630/YAP1/ERK feedback loop.

Purpose: MicroRNA-630 plays dual roles in apoptosis and drug resistance in human cancers. However, the role of miR-630 in resistance to tyrosine kinase inhibitors (TKIs) in lung adenocarcinoma remains to be elucidated. Methods: Manipulation of miR-630 and its targeted gene YAP1 and/or combination of inhibitor treatments was performed to explore whether low miR-630 could confer TKI resistance due to de-targeting YAP1, and this could decrease proapoptotic protein Bad expression through the miR-630/YAP1/ERK feedback loop. A retrospective study was conducted to examine whether the expression of miR-630 and YAP1 could be associated with TKI therapeutic response in patients with lung adenocarcinoma. Results: Low miR-630 expression may confer TKI resistance via increased SP1 binding to the miR-630 promoter due to ERK activation by YAP1 de-targeting. Persistent activation of ERK signaling via the miR-630/YAP1/ERK feedback loop may be responsible for TKI resistance in EGFR-mutated cells. Moreover, a decrease in Bad expression by its phosphorylation at Serine 75 through ERK activation conferred low miR-630-mediated TKI resistance by modulating the apoptotic pathway. Xenographic tumors induced by miR-630-knockdown PC9 and PC9GR cells in nude mice were nearly suppressed by the combination of gefitinib with the YAP1 inhibitor verteporfin or an MEK/ERK inhibitor AZD6244. Patients with low miR-630 and high YAP1 expressing tumors had a higher prevalence of unfavorable responses to TKI therapy and poorer outcomes when compared with their counterparts. Conclusion: MiR-630 may be a potential biomarker for the prediction of TKI therapeutic response and outcome in patients with lung adenocarcinoma.