Hemoglobin nanoclusters-mediated regulation of KPNA4 in hypoxic tumor microenvironment enhances photodynamic therapy in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly malignant tumor known for its hypoxic environment, which contributes to resistance against the anticancer drug Sorafenib (SF). Addressing SF resistance in HCC requires innovative strategies to improve tumor oxygenation and effectively deliver therapeutics. RESULTS: In our study, we explored the role of KPNA4 in mediating hypoxia-induced SF resistance in HCC. We developed hemoglobin nanoclusters (Hb-NCs) capable of carrying oxygen, loaded with indocyanine green (ICG) and SF, named HPRG@SF. In vitro, HPRG@SF targeted HCC cells, alleviated hypoxia, suppressed KPNA4 expression, and enhanced the cytotoxicity of PDT against hypoxic, SF-resistant HCC cells. In vivo experiments supported these findings, showing that HPRG@SF effectively improved the oxygenation within the tumor microenvironment and countered SF resistance through combined photodynamic therapy (PDT). CONCLUSION: The combination of Hb-NCs with ICG and SF, forming HPRG@SF, presents a potent strategy to overcome drug resistance in hepatocellular carcinoma by improving hypoxia and employing PDT. This approach not only targets the hypoxic conditions that underlie resistance but also provides a synergistic anticancer effect, highlighting its potential for clinical applications in treating resistant HCC.

Circular RNA circ_0000376 promotes paclitaxel resistance and tumorigenesis of non-small cell lung cancer via positively modulating KPNA4 by sponging miR-1298-5p.

BACKGROUND: Circ_0000376 could promote non-small cell lung cancer (NSCLC) progression; however, the role of circ_0000376 in paclitaxel (PTX) resistance of NSCLC is still unknown. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were applied for measuring circ_0000376, microRNA-1298-5p (miR-1298-5p), and karyopherin subunit alpha 4 (KPNA4) expression. The half inhibitory concentration (IC50) of PTX was assessed by cell counting kit-8 assay. 5-ethynyl-2'-deoxyuridine assay, wound healing assay, transwell assay, and flow cytometry were performed to measure the proliferation, migration, invasion, and apoptosis of cells. The regulatory mechanisms of circ_0000376, miR-1298-5p, and KPNA4 were validated by dual-luciferase reporter assay, RNA immunoprecipitation assay, and rescue experiments. Xenograft assay was used for the functional analysis of circ_0000376 in vivo. RESULTS: Circ_0000376 and KPNA4 expressions were upregulated, while miR-1298-5p expression was downregulated in PTX-resistant tissues and cells. Circ_0000376 depletion promoted PTX sensitivity and apoptosis, while suppressing the proliferation, migration, and invasion of PTX-resistant NSCLC cells. Furthermore, circ_0000376 could modulate KPNA4 expression by sponging miR-1298-5p. Rescue experiments showed that miR-1298-5p inhibition reversed the circ_0000376 depletion-mediated anticancer effects and PTX sensitivity. Moreover, miR-1298-5p inhibited PTX resistance and tumorigenesis of PTX-resistant cells, which were abolished by KPNA4 overexpression. In addition, circ_0000376 knockdown suppressed tumor growth and enhanced PTX sensitivity in vivo. CONCLUSION: Circ_0000376 facilitated PTX resistance and tumorigenesis of NSCLC by miR-1298-5p/KPNA4 axis, suggesting an underlying therapeutic strategy for NSCLC.

Circ_0001402 knockdown suppresses the chemoresistance and development of DDP-resistant cutaneous squamous cell carcinoma cells by functioning as a ceRNA for miR-625-5p.

Cutaneous squamous cell carcinoma (CSCC) is the most common metastatic skin cancer. Circular RNAs (circRNAs) are differentially expressed in CSCC and can sequester and sponge microRNAs. GSE74758 shows that hsa_circ_0001402 (circ_0001402) is the most overexpressed circRNA in CSCC. Expression of circ_0001402, microRNA(miR)-625-5p and karyopherin subunit alpha 4 (KPNA4) was detected by quantitative real-time polymerase chain reaction and/or Western blot. Colon formation, flow cytometry, Transwell assays and xenograft tumour model confirmed the development of CSCC cells. The competing endogenous RNA (ceRNA) interaction was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Circ_0001402 was significantly upregulated in CSCC tissues and cells, and higher expression of circ_0001402 was found in DDP-resistant samples. Functionally, circ_0001402 knockdown induced apoptosis and inhibited half maximal inhibitory concentration of DDP, colony formation, migration and invasion of DDP-resistant CSCC cells, accompanied with the depressed multi-drug resistance-1 (MDR1) and MDR-related protein-1, while miR-625-5p inhibitor could counteract these effects. Mechanically, circ_0001402 mediated the expression regulation of KPNA4 via functioning as a ceRNA for miR-625-5p. KPNA4 re-expression could abate the functions of miR-625-5p. Furthermore, circ_0001402 knockdown could hinder tumour growth of DDP-resistant CSCC. Circ_0001402 knockdown can suppress the development and chemoresistance of DDP-resistant CSCC cells at least partly through targeting miR-625-5p/KPNA4 axis.

Circular RNA hsa_circ_0008003 promotes the progression of non-small-cell lung cancer by sponging miR-548I and regulating KPNA4 expression.

OBJECTIVE: The study aimed to explore the effect of circ_0008003 on the progression of non-small-cell lung cancer (NSCLC) and its underlying regulation mechanism. METHODS: Expression of hsa_circ_0008003, miRNA (miR)-548I and karyopherin subunit α 4 (KPNA4) was examined by quantitative real-time polymerase chain reaction. Cell viability and proliferation ability were detected by cell counting kit-8 assay and 5-ethynyl-2'-deoxyuridine assay, respectively. Flow cytometry was performed to monitor cell apoptosis. Western blot assay was used to evaluate the protein levels of KPNA4, Bax, and Bcl-2. Cell migration and invasion were assessed by transwell assays. The targeted relationship between miR-548I and hsa_circ_0008003 or KPNA4 was confirmed by dual-luciferase reporter and RNA immunoprecipitation assays. Furthermore, the role of hsa_circ_0008003 in vivo was investigated by xenograft assay. RESULTS: Circ_0008003 expression was increased in NSCLC tissues and cell lines. Circ_0008003 knockdown reduced cell viability, migration, invasion, angiogenesis, and caused apoptosis in NSCLC cells. Moreover, miR-548I was targeted by circ_0008003, and miR-548I knockdown reversed the influence of circ_0008003 silence on NSCLC progression. KPNA4 was targeted by miR-548I, and miR-548I overexpression suppressed cell viability, migration, invasion, angiogenesis, and promoted cell apoptosis via decreasing KPNA4. In addition, circ_0008003 regulated KPNA4 expression via miR-548I. Circ_0008003 knockdown decreased NSCLC cell growth in the xenograft model. CONCLUSION: Circular RNA hsa_circ_0008003 promoted progression in NSCLC by sponging miR-548I and regulating KPNA4 expression, hinting that circ_0008003 participates in NSCLC pathogenesis.

Long non-coding RNA UCA1 regulates MPP+-induced neuronal damage through the miR-671-5p/KPNA4 pathway in SK-N-SH cells.

Parkinson's disease (PD) is an age-related neurodegenerative disease. Long non-coding RNA urothelial carcinoma-associated 1 (UCA1) is involved in the pathogenesis of PD. However, the pathogenesis of PD regulated by UCA1 has not been fully explained. We used 1-Methyl-4-phenylpyridinium (MPP+)-induced SK-N-SH cells for functional analysis. Expression levels of UCA1, microRNA (miR)-671-5p, and KPNA4 (karyopherin subunit alpha 4) mRNA were detected using quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability and apoptosis were analyzed using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) or flow cytometry assays. Some protein levels were measured by western blotting. The levels of pro-inflammatory cytokines were tested by ELISA (enzyme-linked immunosorbent assay). The levels of LDH (lactate dehydrogenase), MDA (malondialdehyde), and SOD (superoxide dismutase) were measured using corresponding kits. The relationship between UCA1 or KPNA4 and miR-671-5p was verified by dual-luciferase reporter assay and/or RNA immunoprecipitation (RIP) assay. MPP+ induced UCA1 expression in SK-N-SH cells in a concentration-dependent manner or time-dependent manner. UCA1 knockdown reduced MPP+-induced apoptosis, inflammation, and oxidative stress in SK-N-SH cells. MiR-671-5p was downregulated while KPNA4 was upregulated in MPP+-treated SK-N-SH cells. UCA1 sponged miR-671-5p to regulate KPNA4 expression. MiR-671-5p inhibition counteracted UCA1 knockdown-mediated influence on apoptosis, inflammation, and oxidative stress of MPP+-induced SK-N-SH cells. KPNA4 overexpression offset the inhibitory influence of miR-671-5p mimic on apoptosis, inflammation, and oxidative stress of MPP+-treated SK-N-SH cells. UCA1 inhibition reduced MPP+-induced neuronal damage through the miR-671-5p/KPNA4 pathway in SK-N-SH cells, providing a novel mechanism to understand the pathogenesis of PD.

Long noncoding RNA SNHG14 knockdown exerts a neuroprotective role in MPP+-induced Parkinson's disease cell model through mediating miR-135b-5p/KPNA4 axis.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disease resulted from the loss of dopaminergic neurons. Here, we analyzed the role of long noncoding RNA (lncRNA) small nucleolar RNA host gene 14 (SNHG14) in PD using 1-methyl-4-phenyl pyridine (MPP+)-induced PD cell model. METHODS: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were performed to determine RNA and protein expression, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry (FCM) analysis were conducted to analyze cell viability and apoptosis. Enzyme-Linked Immunosorbent Assay (ELISA) was conducted to analyze the release of inflammatory cytokines. Cytotoxicity was assessed using reactive oxygen species (ROS) assay kit, superoxide dismutase (SOD) activity assay kit and lactate dehydrogenase (LDH) activity assay kit. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to confirm the interaction between microRNA-135b-5p (miR-135b-5p) and SNHG14 or karyopherin subunit alpha 4 (KPNA4). RESULTS: MPP+ treatment elevated the expression of SNHG14 in SK-N-SH cells in a dose and time-dependent manner. SNHG14 knockdown alleviated MPP+-induced apoptosis, inflammation, and cytotoxicity in SK-N-SH cells. SNHG14 interacted with miR-135b-5p, and SNHG14 silencing-mediated effects were partly overturned by miR-135b-5p knockdown in PD cell model. Besides, miR-135b-5p interacted with the 3' untranslated region (3'UTR) of KPNA4, and KPNA4 overexpression partly reversed miR-135b-5p overexpression-induced effects in PD cell model. SNHG14 knockdown reduced the protein level of KPNA4 partly by up-regulating miR-135b-5p in SK-N-SH cells. CONCLUSION: SNHG14 promoted MPP+-induced neuro injury in PD cell model through mediating miR-135b-5p/KPNA4 axis.

Ginsenoside Rg3 inhibits osteosarcoma progression by reducing circ_0003074 expression in a miR-516b-5p/KPNA4-dependent manner.

BACKGROUND: Previous data have suggested that ginsenoside Rg3 (Rg3), isolated from the roots of Panax ginseng, plays a repressing role in multiple cancers, including osteosarcoma (OS). However, there is no any literature available about the role of circular RNA (circRNA) in Rg3-mediated OS development. The study aimed to explore the function of circ_0003074 in the anti-cancer effects of Rg3 on OS. METHODS: RNA expression of circ_0003074, miR-516b-5p and karyopherin subunit alpha 4 (KPNA4) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression was evaluated by Western blotting or immunohistochemistry assay. Cell viability, proliferation, apoptosis, migration and invasion were investigated by cell counting kit-8, 5-ethynyl-29-deoxyuridine (EdU), flow cytometry analysis, wound-healing and transwell invasion assays, respectively. Dual-luciferase reporter and/or RNA immunoprecipitation assay was performed to confirm the interplay between miR-516b-5p and circ_0003074 or KPNA4. Xenograft mouse model assay was conducted to reveal the effect of Rg3 treatment on tumor formation. RESULTS: Circ_0003074 and KPNA4 expression was significantly upregulated, while miR-516b-5p was downregulated in OS tissues and cells compared with controls. Rg3 treatment dramatically decreased circ_0003074 expression in OS cells. Rg3 treatment led to decreased cell proliferation, migration and invasion but increased cell apoptosis, which was attenuated after circ_0003074 overexpression. Besides, miR-516b-5p was a target miRNA of circ_0003074 and partially restored circ_0003074-mediated action under Rg3 treatment. Decreasing miR-516b-5p expression also promoted Rg3-treated OS cell malignancy through KPNA4, which was identified as a target mRNA of miR-516b-5p. Besides, circ_0003074 induced KPNA4 production owing to the decrease of miR-516b-5p expression. Furthermore, Rg3 treatment inhibited tumor formation by regulating circ_0003074 in vivo. CONCLUSION: Rg3 inhibited OS progression through circ_0003074/miR-516b-5p/KPNA4 axis, showing the potential of Rg3 as a therapeutic agent for OS.

Berberine Attenuates MPP+-Induced Neuronal Injury by Regulating LINC00943/miR-142-5p/KPNA4/NF-κB Pathway in SK-N-SH Cells.

Berberine plays a neuro-protective role in neurodegenerative diseases, including Parkinson's disease (PD). Long non-coding RNAs (lncRNAs) play critical roles in PD pathogenesis. The purpose of this study was to investigate whether LINC00943 was involved in the role of berberine in PD. 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) or 1-methyl-4-phenyl pyridine (MPP+) were used to construct PD mouse and cell models, respectively. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine (Edu) assays. Inflammation and cell apoptosis were assessed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry, respectively. Quantitative real-time PCR (qRT-PCR) was employed to test the expression of LINC00943, microRNA (miR)-142-5p, and karyopherin subunit alpha 4 (KPNA4) mRNA. The protein levels of NF-κB pathway-related markers and KPNA4 were measured by western blot. Oxidative stress level was assessed by corresponding kits. The interaction between miR-142-5p and LINC00943 or KPNA4 was determined via dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Berberine inhibited MPP+-induced injury in SK-N-SH cells by promoting cell proliferation and suppressing inflammation, apoptosis, and oxidative injury. LINC00943 and KPNA4 were upregulated and miR-142-5p was downregulated in PD mouse and cell models. LINC00943 (or KPNA4) overexpression or miR-142-5p inhibition abated the neuro-protective role of berberine in PD cell model. Moreover, miR-142-5p was a target of LINC00943, and KPNA4 could specially bind to miR-142-5p. Additionally, berberine inhibited NF-κB pathway by regulating LINC00943/miR-142-5p/KPNA4 axis. Berberine protected SK-N-SH cell from MPP+-induced neuronal damage via regulating LINC00943/miR-142-5p/KPNA4/NF-κB pathway, highlighting novel evidence for the neuro-protective role of berberine in PD.

KPNA4 is involved in cataract formation via the nuclear import of p53.

KPNA4 (also called importin-α3) belongs to the importin α adaptor proteins family, which orchestrates classical nuclear transport processes, importin-α/importin-ß1 pathway, and involves in cellular homeostasis. Disruption of balanced transport pathways may result in ectopic nuclear proteins and eventually cause diseases, mainly under the situation of cellular stress, such as oxidative stress. Little evidence is available on its cellular functions for high specific expression in lens. We firstly studied the role of KPNA4 in cataract formation. Lens defects were observed at an early age in kpna4 gene knockout zebrafish, generated by the CRISPR/Cas9 system. Those phenotype, including cloudy center part of the lens, via bright field microscopy, and the thinning of the LE layer, wider space between the adjacent LE and LF cells, irregular cells morphology and the increased number of holes inside the LE cells, which were detected by transmission electron microscopy, recapitulate the clinical features of cataract patients. As the p53-specific adaptor of the nuclear import, KPNA4 upregulated with the same pattern of p53 in hydrogen peroxide-induced apoptosis in human lens epithelia cells. Furthermore, the loss of Kpna4 resulted in the accumulation of p53 in the center of lens. Taken together, we showed that KPNA4 was involved in the formation of cataract, likely by mediating p53 nuclear transport.

Value of KPNA4 as a diagnostic and prognostic biomarker for hepatocellular carcinoma.

It is important to identify novel biomarkers to improve hepatocellular carcinoma (HCC) diagnosis and treatment. Herein, we reported the role of karyopherin α4 (KPNA4) in HCC patients through public data mining and examined the results using clinical samples in our center. Our results revealed that KPNA4 expression level was positively correlated with the infiltration of CD8+ T cells, B cells, dendritic cells, CD4+ T cells, neutrophils and macrophages. In addition, KPNA4 expression was significantly associated with T cell exhaustion. KPNA4 mRNA and protein expression levels were significantly higher in cancerous tissue than in normal tissue. Besides, the increased expression of KPNA4 indicated poor overall survival. Univariate and multivariate Cox regression analyses showed KPNA4 could be viewed as an independent risk factor for HCC patients. Moreover, our experimental results were consistent with those obtained from bioinformatic results. These findings revealed KPNA4 may serve as a novel prognostic biomarker and a potential therapeutic target for HCC.

Circ_ZFR contributes to the paclitaxel resistance and progression of non-small cell lung cancer by upregulating KPNA4 through sponging miR-195-5p.

BACKGROUND: A growing body of evidence has demonstrated the vital roles of circular RNAs (circRNAs) in cancer progression and drug resistance. We intended to explore the roles and mechanisms of circ_ZFR in the paclitaxel (PTX) resistance and progression of non-small cell lung cancer (NSCLC). METHODS: Two NSCLC cell lines A549 and H460 were used in this study. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was conducted to measure the levels of circ_ZFR, ZFR, miR-195-5p and karyopherin subunit alpha 4 (KPNA4) mRNA. RNase R assay was used to analyze the characteristic of circ_ZFR. MTT assay was carried out to assess PTX resistance and cell proliferation. Flow cytometry analysis was utilized to analyze cell cycle and apoptosis. Transwell assay was used to examine cell migration and invasion. Western blot assay was conducted to measure the protein levels of Ki67, Twist1, E-cadherin and KPNA4. Dual-luciferase reporter assay was adopted to verify the combination between miR-195-5p and circ_ZFR or KPNA4. Murine xenograft model assay was used to investigate the effect of circ_ZFR on PTX resistance of NSCLC in vivo. RESULTS: Circ_ZFR level was enhanced in PTX-resistant NSCLC tissues and cells. Knockdown of circ_ZFR suppressed PTX resistance, cell cycle process, proliferation, migration and invasion and induced apoptosis in PTX-resistant NSCLC cells. For mechanism analysis, circ_ZFR knockdown markedly downregulated the expression of KPNA4 by sponging miR-195-5p, thereby promoting PTX sensitivity and suppressing cell progression in PTX-resistant NSCLC cells. In addition, circ_ZFR silencing enhanced PTX sensitivity of NSCLC in vivo. CONCLUSION: Circ_ZFR knockdown played a positive role in overcoming PTX resistance of NSCLC via regulating miR-195-5p/KPNA4 axis, which might provide a possible circRNA-targeted therapy for NSCLC.

KPNA4 regulated by miR-548b-3p promotes the malignant phenotypes of papillary thyroid cancer.

AIM: Karyopherin α4 (KPNA4, importin α3) has been verified to be an oncogene in many cancers. However, its role in papillary thyroid cancer (PTC), the most frequent endocrine malignancy, is still unclear. MATERIALS AND METHODS: KPNA4 expression was analyzed in PTC tissues and cells. The effects of KPNA4 on the proliferation, invasion, and apoptosis of PTC cells were evaluated after overexpression or downregulation of KPNA4. The influence of KPNA4 on NF-κB activation was evaluated by nuclear NF-κB p65 expression and NF-κB-luciferase reporter assays. Moreover, we also explored whether KPNA4 was regulated by miR-548b-3p. Additionally, the roles of miR-548b-3p and KPNA4 were explored in a xenograft mouse model. KEY FINDINGS: KPNA4 expression was increased in PTC tissues and cells, and its expression was significantly related to patients' clinicopathologic features and overall survival. Overexpression of KPNA4 significantly promoted PTC cell proliferation and invasion, enhanced nuclear p65 expression and augmented NF-κB luciferase activity. However, KPNA4 silencing showed opposite effects on the above indexes, and induced apoptosis of PTC cells. KPNA4 was a target of miR-548b-3p, which was downregulated in PTC and inhibited proliferation and invasion, but promoted apoptosis of PTC cells. KPNA4 overexpression abrogated the suppression of miR-548b-3p on the malignant phenotypes of PTC cells. Both miR-548b-3p overexpression and KPNA4 downregulation inhibited tumor growth and Ki-67 expression, elevated numbers of Tunel-positive cells, and deceased nuclear p65 expression in mouse tumor tissues. SIGNIFICANCE: KPNA4 was negatively regulated by miR-548b-3p and promoted the development of PTC via activating the NF-κB pathway.

LncRNA ST7-AS1, by regulating miR-181b-5p/KPNA4 axis, promotes the malignancy of lung adenocarcinoma.

BACKGROUND: Growing evidence suggests that suppressor of tumorigenicity 7 antisense RNA 1 (ST7-AS1) is an oncogenic long noncoding RNA (lncRNA). However, little is known on its clinical significance, biological functions, or molecular mechanisms in lung adenocarcinoma (LUAD). METHODS: The expression of ST7-AS1 and miR-181b-5p were examined by qRT-PCR. The correlations between ST7-AS1 level and different clinicopathological features were analysed. In vitro, LUAD cells were examined for cell viability, migration and invasion by MTT, wound healing and Transwell assay, respectively. Epithelial-mesenchymal transition (EMT) biomarkers were detected by Western blot. The regulations between ST7-AS1, miR-181b-5p, and KPNA4 were examined by luciferase assay, RNA immunoprecipitation, RNA pulldown. Both gain- and loss-of-function strategies were used to assess the importance of different signalling molecules in malignant phenotypes of LUAD cells. The in vivo effect was analysed using the xenograft and the experimental metastasis mouse models. RESULTS: ST7-AS1 was upregulated in LUAD tissues or cell lines, correlated with tumours of positive lymph node metastasis or higher TNM stages, and associated with shorter overall survival of LUAD patients. ST7-AS1 essentially maintained the viability, migration, invasion, and EMT of LUAD cells. The oncogenic activities of ST7-AS1 were accomplished by sponging miR-181b-5p and releasing the suppression of the latter on KPNA4. In LUAD tissues, ST7-AS1 level positively correlated with that of KPNA4 and negatively with miR-181b-5p level. In vivo, targeting ST7-AS1 significantly inhibited xenograft growth and metastasis. CONCLUSIONS: ST7-AS1, by regulating miR-181b-5p/KPNA4 axis, promotes the malignancy of LUAD cells. Targeting ST7-AS1 and KPNA4 or up-regulating miR-181b-5p, therefore, may benefit the treatment of LUAD.

MiR-596 activated by EP300 controls the tumorigenesis in epithelial ovarian cancer by declining BRD4 and KPNA4.

BACKGROUND: Epithelial ovarian cancer (EOC), a subclass of ovarian cancer (OC), is usually diagnosed at advanced stages due to the lack of effective screening means. Mounting reports have disclosed the vitally important roles of microRNAs (miRNAs) in carcinogenesis. Here, we aimed to find out possible miRNAs participating in EOC development. METHODS: qRT-PCR ad western blot respectively examined the mRNA and protein levels of studied genes. CCK-8, colony formation, flow cytometry, TUNEL and spheroid formation assays were appropriately employed for examining cell proliferation, cell cycle, apoptosis and stemness. The interaction between molecules was affirmed by luciferase reporter, RNA pull down and ChIP assays. RESULTS: In consistent with the observation of a past study, miR-596 expression was relatively low in EOC cells. Up-regulating miR-596 suppressed EOC cell proliferation and stemness. EP300 transcriptionally activated miR-596 to serve as a tumor-repressor in EOC. Then BRD4 and KPNA4, whose knockdown led to restraining effects on cell growth and stemness, were both revealed to be targeted by miR-596 in EOC. Lastly, rescue assays affirmed the tumor-restraining role of miR-596-BRD4/KPNA4 axis in EOC. CONCLUSION: EP300-activated miR-596 hampered cell growth and stemness via targeting BRD4 and KPNA4 in EOC, proofing miR-596 as a promising therapeutic target in treating EOC patients.

STAT3-induced upregulation of circCCDC66 facilitates the progression of non-small cell lung cancer by targeting miR-33a-5p/KPNA4 axis.

Emerging evidence has manifested the critical effect of abnormally expressed circular RNAs (circRNAs) on the initiation and progression of non-small cell lung cancer (NSCLC). Although circRNA circCCDC66 has been revealed to elicit facilitating impact on cell growth and metastasis in colon cancer, the potential biological function and regulatory mechanism of it in NSCLC still require to be explored. In this study, circCCDC66 in NSCLC cells was highly expressed. Downregulation of circCCDC66 impaired cell proliferation, migration and invasion whereas boosted cell apoptosis in NSCLC. Data from molecular mechanism assays testified that circCCDC66 bound with miR-33a-5p in NSCLC cells. And miR-33a-5p inhibition could rescue the suppressive effect of circCCDC66 knockdown on NSCLC progression. In addition, karyopherin subunit alpha 4 (KPNA4) in NSCLC cells was proofed to be directly targeted by miR-33a-5p. Moreover, through rescued-function assays, we observed that upregulating KPNA4 expression could countervail the restraining function of silenced circCCDC66 on NSCLC progression. Furthermore, signal transducer and activator of transcription 3 (STAT3) was validated to activate CCDC66 transcription and thereby promote circCCDC66 expression in NSCLC cells. Briefly, STAT3-induced circCCDC66 upregulation accelerates NSCLC progression via miR-33a-5p/KPNA4 axis, suggesting circCCDC66 as a promising biomarker in NSCLC treatment.

YY1-mediated overexpression of long noncoding RNA MCM3AP-AS1 accelerates angiogenesis and progression in lung cancer by targeting miR-340-5p/KPNA4 axis.

Lung cancer is famous as an aggressive malignant tumor and is the main cause of cancer-associated mortality globally. Tumor angiogenesis is a vital part in cancer, which influences cell proliferation and metastasis. Increasing studies have claimed that long noncoding RNAs (lncRNAs) were involved in the progression of several cancers. Based on previous studies, this study focused on the role and mechanism of lncRNA MCM3AP antisense RNA 1 (MCM3AP-AS1) in lung cancer. At first, MCM3AP-AS1 expression was found to be elevated in lung cancer cells. Depletion of MCM3AP-AS1 repressed cell proliferation, migration, and angiogenesis in lung cancer cells. YY1 was confirmed to mediate MCM3AP-AS1 transcription in lung cancer cells. Moreover, the molecular mechanism investigation revealed that MCM3AP-AS1 could sponge miR-340-5p and elevate KPNA4 expression. On the basis of rescue assays, we identified that the overexpression of KPNA4 partly counteracted the suppressed effect of MCM3AP-AS1 knockdown on angiogenesis and progression in lung cancer cells. Conclusively, the YY1-mediated overexpression of MCM3AP-AS1 accelerated angiogenesis and progression in lung cancer by targeting miR-340-5p/KPNA4 axis, which highlighted the possibility of MCM3AP-AS1 as a promising therapeutic target for lung cancer.

MiR-3619-5p hampers proliferation and cisplatin resistance in cutaneous squamous-cell carcinoma via KPNA4.

Cutaneous squamous cell carcinoma (CSCC) remains the second most prevailing cancer worldwide and presents high mortality rates. Given that chemoresistance becomes an enormous obstacle to the therapy for CSCC patients, there is a pressing need to discover novel strategies for enhancing the response of CSCC cells to cisplatin. Emerging evidence has unfolded that miRNAs are participated in regulation of drug resistance in multiple cancers. MiR-3619-5p has been proofed to exert tumor inhibitive activities in human malignancies, but the biological function of miR-3619-5p in the progression of CSCC is still unclear. In this study, we observed that miR-3619-5p expression was pronouncedly dropped in cisplatin-resistant CSCC cells. Subsequently, miR-3619-5p was validated to act as a tumor suppressor in CSCC through retarding cell proliferation and cisplatin resistance. Besides, our findings certified that KPNA4 was highly expressed in cisplatin-resistant CSCC cells. Further, KPNA4 was negatively regulated by miR-3619-5p. Rescue experiments unveiled that KPNA4 counteracted the miR-3619-5p-mediated regulation of CSCC tumorigenesis. On the whole, miR-3619-5p inhibited cell proliferation and cisplatin resistance of CSCC by regulating KPNA4 expression, suggesting that miR-3619-5p/KPNA4 pathway may represent a potential promising strategy for the treatment of patients with CSCC.

Vitamin D Deficiency Accelerates Coronary Artery Disease Progression in Swine.

OBJECTIVE: The role of vitamin D deficiency in coronary artery disease (CAD) progression is uncertain. Chronic inflammation in epicardial adipose tissue (EAT) has been implicated in the pathogenesis of CAD. However, the molecular mechanism underlying vitamin D deficiency-enhanced inflammation in the EAT of diseased coronary arteries remains unknown. We examined a mechanistic link between 1,25-dihydroxyvitamin D-mediated suppression of nuclear factor-κB (NF-κB) transporter, karyopherin α4 (KPNA4) expression and NF-κB activation in preadipocytes. Furthermore, we determined whether vitamin D deficiency accelerates CAD progression by increasing KPNA4 and nuclear NF-κB levels in EAT. APPROACH AND RESULTS: Nuclear protein levels were detected by immunofluorescence and Western blot. Exogenous KPNA4 was transported into cells by a transfection approach and constituted lentiviral vector. Swine were administered vitamin D-deficient or vitamin D-sufficient hypercholesterolemic diet. After 1 year, the histopathology of coronary arteries and nuclear protein expression of EAT were assessed. 1,25-dihydroxyvitamin D inhibited NF-κB activation and reduced KPNA4 levels through increased vitamin D receptor expression. Exogenous KPNA4 rescued 1,25-dihydroxyvitamin D-dependent suppression of NF-κB nuclear translocation and activation. Vitamin D deficiency caused extensive CAD progression and advanced atherosclerotic plaques, which are linked to increased KPNA4 and nuclear NF-κB levels in the EAT. CONCLUSIONS: 1,25-dihydroxyvitamin D attenuates NF-κB activation by targeting KPNA4. Vitamin D deficiency accelerates CAD progression at least, in part, through enhanced chronic inflammation of EAT by upregulation of KPNA4, which enhances NF-κB activation. These novel findings provide mechanistic evidence that vitamin D supplementation could be beneficial for the prevention and treatment of CAD.