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Background In this study, we determine the potential roles and uncover the regulatory mechanisms of circCCDC66 in regulating cell growth and cell metastasis of glioma. Methods qRT-PCR was used to detect the expressions of circCCDC66 in gliomas and tissues. The biological function of circCCDC66 in glioma cell lines was elucidated by functional experiments. Cell counting kit-8 and transwell were used to detect the effect of circCCDC66 on the proliferation, migration and invasion of glioma cells. Bioinformatics analysis was applied to reveal the targets of circCCDC66. Results The results showed circCCDC66 was overexpressed in glioma and acted as an oncogene. CircCCDC66 knockdown suppressed the proliferation, migration, and invasion of glioma cells. We constructed a circCCDC66 regulating miRNA network and revealed miR-320a was a potential target of circCCDC66, which was down-regulated in high-grade gliomas compared to low-grade gliomas. Bioinformatics analysis showed circCCDC66-miR-320a/b axis was involved in regulating multiple cancer-related pathways. Furthermore, we identified FOXM1 as a key target of circCCDC66, which was involved in regulating DNA damage response pathways. In mechanism study, circCCDC66 could sponge miR-320a, thereby increasing the expression of FOXM1. Conclusions CircCCDC66 could facilitate glioma cells proliferation, invasion and migration by down-regulating miR-320a and up-regulating FOXM1.

Cutaneous squamous cell carcinoma (cSCC) is a highly prevalent skin malignancy, and the effective therapy still remains a challenge. Crocin can be used for cSCC therapy. This study explored the effects of cSCC cells treatment with crocin in vitro and in vivo. The study used A431 and SCL-1 cells lines, cSCC human samples and BALB/C nude mice for investigations. Apoptosis was determined by MTT assays, while miR-320a and ATG2B expressions were validated through RT-qPCR. Interaction of miR-320a with ATG2B was examined via dual luciferase reporter assay. The autophagy and apoptosis proteins expressions were further confirmed through western blot and immunofluorescence staining assays. The results indicated a significantly upregulated miR-320a, but a down-regulated ATG2B expression in the cSCC clinical samples. Crocin significantly repressed cSCC cells growth, and induced apoptosis through autophagy. Furthermore, miR-320a expression was inhibited and ATG2B expression was increased. Dual luciferase reporter assay revealed that miR-320a regulated ATG2B expression directly. Additionally, the upregulation of ATG2B expression in cSCC cells inhibited cell proliferation and led to cell apoptosis. Crocin also reduced tumor growth and stimulated the apoptosis in vivo. In conclusion, miR-320a is upregulated and ATG2B is down-regulated in cSCC, Crocin suppresses the proliferation and induces apoptosis of cSCC cells. Further, Crocin increases autophagy while miR-320a hinders autophagy and the apoptotic effects of crocin on cSCC cells. MiR-320a binds ATG2B directly, and ATG2B expression is upregulated by crocin. Finally, Crocin triggers cSCC cells apoptosis in vivo. Crocin can target ATG2B/miR-320a and may be an effective alternative for cSCC treatment., GRAPHICAL ABSTRACT

Osteosarcoma is a primary malignant bone tumor, which affects children, adolescents, and young adults commonly. MicroRNAs (miRNAs) have been proved to be dysregulated in different cancers, including osteosarcoma. Although miR‐320a has been implicated in many types of malignancies, little is known about the role of miR‐320a in osteosarcoma. In this study, we show that the overexpression of miR‐320a or knockdown of cytoplasmic polyadenylation element‐binding protein 1 (CPEB1) inhibited osteosarcoma cell migration and invasion. miR‐320a downregulates CPEB1 expression by directly targeting the CPEB1 3′‐UTR. Furthermore, CPEB1 reintroduction reversed the antiproliferation, antimigration, and antiinvasion roles of miR‐320a, indicating that miR‐320a might function as a tumor suppressor in osteosarcoma through CPEB1. In conclusion, our study demonstrates that miR‐320a plays a critical role in osteosarcoma progression and may provide a potential therapeutic target for osteosarcoma., Our study demonstrates that miR‐320a plays a critical role in osteosarcoma progression and may provide a potential therapeutic target for osteosarcoma.

Background Curcumin has a potential therapeutic role in ovarian cancer. However, whether curcumin plays anti-cancer role in ovarian cancer by mediating the circular RNA (circRNA)/microRNA (miRNA)/mRNA network is still unclear. Methods The expression of circ-PLEKHM3, miR-320a, and suppressor of morphogenesis in genitalia 1 (SMG1) was detected via qRT-PCR. Cell viability, colony-formation ability and apoptosis were analyzed via cell counting kit-8 assay, colony formation analysis, and flow cytometry. Protein expression was measured using western blot. The in vivo experiments were performed using a xenograft model. Target association was evaluated via dual-luciferase reporter analysis and RIP assay. Results Curcumin suppressed ovarian cancer cell proliferation and promoted apoptosis. Circ-PLEKHM3 was downregulated in ovarian cancer, and its expression could be promoted by curcumin treatment. Circ-PLEKHM3 overexpression exacerbated the effect of curcumin on ovarian cancer cell proliferation and apoptosis, as well as anti-tumor effect. MiR-320a was targeted by circ-PLEKHM3. The inhibition effect of circ-PLEKHM3 overexpression on cell proliferation and the enhancing effect on cell apoptosis could be reversed by miR-320a mimic. SMG1 was targeted by miR-320a, and its knockdown also reversed the regulation of miR-320a inhibitor on the proliferation and apoptosis of ovarian cancer cells. In addition, circ-PLEKHM3 could upregulate SMG1 expression via sponging miR-320a. Conclusion Curcumin restrained proliferation and facilitated apoptosis in ovarian cancer by regulating the circ-PLEKHM3/miR-320a/SMG1 axis.

AFAP1‐AS1 is a long non‐coding RNA that is associated with tumorigenesis and poor prognosis in a variety of cancers. We have been suggested that AFAP1‐AS1 increases tumorigenesis in laryngeal carcinoma specifically by enhancing stemness and chemoresistance. We assessed AFAP1‐AS1 expression in human laryngeal specimens, paired adjacent normal tissues and human HEp‐2 cells. Indeed, we found not only that AFAP1‐AS1 was up‐regulated in laryngeal carcinoma specimens and cells, but also that stemness‐associated genes were overexpressed. Silencing of AFAP1‐AS1 promoted HEp‐2 cell chemoresistance under cisplatin treatment. Expression of AFAP1‐AS1 was increased in drug‐resistant Hep‐2 cells. We then probed the mechanism of AFAP1‐AS1 activity and determined that miR‐320a was a potential molecular target of AFAP1‐AS1. Luciferase reporter and qRT‐PCR assays of AFAP1‐AS1 and miR‐320a levels in human specimens and cell cultures indicated that AFAP1‐AS1 negatively regulates miR‐320a. To discover the molecular mechanism of miR‐320a, we again used the DIANA Tools algorithm to predict its genetic target, RBPJ. After cloning the 3′‐untranslated regions (3′‐UTR) of RBPJ into a luciferase reporter, we determined that miR‐320a did in fact reduce RBPJ mRNA and protein levels. Ultimately, we determined that AFAP1‐AS1 increases RBPJ expression by negatively regulating miR‐320a and RBPJ overexpression rescues stemness and chemoresistance inhibited by AFAP1‐AS1 silencing. Taken together, these results suggest that AFAP1‐AS1 can serve as a prognostic biomarker in laryngeal carcinoma and that miR‐320a has the potential to improve standard therapeutic approaches to the disease, especially for cases in which cancer cell stemness and drug resistance present significant barriers to effective treatment.

Background Vascular homeostasis abnormalities may involve in doxorubicin induced cardiotoxicity. Methods Enhanced cardiac miR-320a expression, reduced cardiac microvessel density and impaired cardiac function were observed in mice treated by anthracycline doxorubicin. To further explore the role of miR-320a in doxorubicin induced cardiotoxicity, microRNA mimics/inhibitor in vitro and rAAV administration in vivo were employed in mice. Results Knockdown of miR-320a not only resulted in enhanced proliferation and inhibited apoptosis in cultured endothelial cells, but also attenuated cardiac abnormalities induced by doxorubicin. On the contrary, overexpression of miR-320a enhanced apoptosis in vitro, and aggravated vessel abnormalities in heart and subsequent cardiac dysfunction in mice. Furthermore, Western blot assays showed that VEGF-A was a potential target of miR-320a, which was verified by anti-Ago2 co-immunoprecipitation. Moreover, as same as miR-320a, siRNA against VEGF-A reinforced doxorubicin induced endothelial cells injury. Finally, the negative effects of miR-320a on vascular homeostasis and cardiac function were alleviated by VEGF-A re-expression in doxorubicin treated mice. Conclusion Our observations demonstrate that miR-320a play important roles in doxorubicin induced cardiotoxicity via vessel homeostasis in heart and thus, inhibition of miR-320a may be applied to the treatment of cardiac dysfunction induced by anthracycline.

Background Lung cancer is among the most frequently diagnosed types of cancer worldwide, with high morbidity and mortality. Metastasis accounts for the deadliest and most poorly understood feature of lung cancer. Herein, we demonstrate that SND1 (also known as p100) acts as a candidate metastasis activator and is targeted by microRNA‐320a (miR‐320a) in lung cancer cells. Methods p100 expression in lung cancer cell lines and tissues was determined by quantitative real time‐PCR and Western blot. RNA interference was applied to investigate the functions of p100 in lung cancer cell migration, reflected by wound healing and transwell assays. Luciferase reporter assay, quantitative real time‐PCR, and Western blot were finally used to examine miR‐320a targeting of p100 in lung cancer cells. Results p100 expression was significantly higher in lung cancer cell lines and tissues compared to normal human bronchial epithelial cells and matched normal lung tissues. Downregulation of p100 by RNA interference obviously inhibited lung cancer cell migration in vitro. Moreover, we validated p100 as a direct target of miR‐320a, a tumor suppressing microRNA repressing lung cancer cell migration. Finally, we showed an inversely expressed correlation between p100 and miR‐320a in tested lung cancer tissues and cell lines, both of which acted as important prognostic factors in lung cancer. Conclusion Our findings identify that p100, targeted by tumor suppressing miR‐320a, is a key metastasis activator in lung cancer, and both p100 and miR‐320a could be considered as biomarkers for prognosis of lung cancer patients.

// Huining Li 1, 2, 3, * , Lin Yu 4, * , Jing Liu 1, 2, 3, * , Xiuwu Bian 5 , Cuijuan Shi 1, 2, 3 , Cuiyun Sun 1, 2, 3 , Xuexia Zhou 1, 2, 3 , Yanjun Wen 1, 2, 3 , Dan Hua 1, 2, 3 , Shujun Zhao 1, 2, 6 , Linlin Ren 1, 2, 3 , Tongling An 1, 2, 3 , Wenjun Luo 1, 2, 3 , Qian Wang 1, 2, 3 , Shizhu Yu 1, 2, 3 1 Department of Neuropathology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China 2 Tianjin Key Laboratory of Injuries, Variations and Regeneration of the Nervous System, Tianjin, China 3 Key Laboratory of Post-trauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin, China 4 Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences of Tianjin Medical University, Tianjin, China 5 Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing, China 6 Laboratory of Hormone and Development, Ministry of Health, Institute of Endocrinology, Tianjin Medical University, Tianjin, China * These authors have contributed equally to this work Correspondence to: Shizhu Yu, email: tjyushizhu@yahoo.com Qian Wang, email: wangqiantni@163.com Keywords: gliomas, miR-320a, SND1, β-catenin, prognosis Received: April 04, 2016 Accepted: January 03, 2017 Published: February 01, 2017 ABSTRACT miR-320a downexpression contributes to tumorigenesis in several human cancers. However, the relevance of miR-320a to prognosis, proliferation and invasion in gliomas remains unclear. In this study, we demonstrated that miR-320a expression was decreased in human glioma tissues and cell lines. Moreover, miR-320a expression was inversely correlated with glioma grades and Ki-67 index, but positively correlated with patients’ survival. Contrarily, SND1 and β-catenin expressions were positively correlated with glioma grades and Ki-67 index, but inversely correlated with miR-320a expression and patients’ survival. Furthermore, two subgroups with distinct prognoses in our glioma patients of different grade, IDH status, age and KPS were identified according to expression of miR-320a, SND1 or β-catenin. Cox regression showed that miR-320a and SND1 were independent predictors and β-catenin was an auxiliary predictor for patients’ survival. miR-320a overexpression suppressed the G1/S phase transition, proliferation, migration and invasion of glioblastoma cells. Mechanistically, we validated SND1 and β-catenin as direct targets of miR-320a, and found that miR-320a overexpression increased SND1-inhibited tumor suppressor p21 WAF1 and decreased Smad2, Smad4, MMP2, MMP7 and cyclinD1, the pivotal downstream effectors of SND1 or β-catenin. Our findings demonstrate the potential values of miR-320a, SND1 and β-catenin as prognostic biomarkers and therapeutic candidates for malignant gliomas.

// Nan Xie 1, 3, * , Cheng Wang 2, 3, * , Zehang Zhuang 2, 3 , Jinson Hou 2, 3 , Xiqiang Liu 2, 3 , Yue Wu 2, 3 , Haichao Liu 2, 3 , Hongzhang Huang 2, 3 1 Department of Oral Pathology, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong 510055, China 2 Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong 510055, China 3 Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong 510055, China * These authors have contributed equally to this work Correspondence to: Hongzhang Huang, email: drhuang52@163.com Keywords: tongue squamous cell carcinoma, tumor budding, miR-320a, Suz12, invasion and metastasis Received: June 06, 2016 Accepted: August 15, 2016 Published: August 25, 2016 ABSTRACT We aimed to determine the specific miRNA profile of tumor budding cells and investigate the potential role of miR-320a in invasion and metastasis of tongue squamous cell carcinoma (TSCC). We collected tumor budding cells and paired central tumor samples from five TSCC specimens with laser capture microdissection and examined the specimens using a miRNA microarray. The specific miRNA signature of tumor budding cells was identified. We found that miR-320a was dramatically decreased in tumor budding cells. Knockdown of miR-320a significantly enhanced migration and invasion of TSCC cell lines. Suz12 was shown to be a direct target of miR-320a. Similar results were also observed in nude mouse models. Multivariate analysis indicated that miR-320a was an independent prognostic factor. Kaplan–Meier analysis demonstrated that decreased miR-320a and high intensity of tumor budding were correlated with poor survival rate, especially in the subgroup with high-intensity tumor budding and low expression of miR-320a. We concluded that decreased expression of miR-320a could promote invasion and metastasis of tumor budding cells by targeting Suz12 in TSCC. A combination of tumor budding and miR-320a may serve as an index to identify an aggressive sub-population of TSCC cells with high metastatic potential.

// Ying Zhu 1, * , Yan Zhang 1, * , Zhenhua Sui 1 , Yi Zhang 1 , Min Liu 1 and Hua Tang 1 1 Tianjin Life Science Research Center, Department of Pathogen Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, 300070, China * These authors contributed equally to this work Correspondence to: Hua Tang, email: htang2002@yahoo.com , tangh@tijmu.edu.cn Keywords: vimentin, USP14, miR-320a, gastric cancer Received: January 27, 2016 Accepted: June 01, 2016 Published: July 19, 2016 ABSTRACT Vimentin plays important roles in the epithelial-to-mesenchymal transition (EMT). In this study, we found that vimentin was highly expressed in human gastric cancer (GC) tissues and cell lines and significantly promoted cell growth, migration and invasion. Ubiquitin-specific protease 14 (USP14) interacted with the vimentin protein, which led to its de-ubiquitination. miR-320a was found to bind to the 3′UTR of both vimentin and USP14 transcripts and downregulate the expression of both proteins. The downregulation of miR-320a upregulates vimentin expression by directly binding to the 3′UTR of vimentin to derepress expression and indirectly by augmenting USP14 to increase vimentin stability in GC cells. Taken together, these results provide new insight into malignancy in gastric cancers.

Advanced prostate cancer (PCa) metastasizes to bone and lymph nodes, and currently available treatments cannot prevent the progression and metastasis of the disease. Therefore, an improved understanding of the molecular mechanisms of the progression and metastasis of advanced PCa using current genomic approaches is needed. Our miRNA expression signature in castration-resistant prostate cancer (CRPC) revealed that microRNA-320a (miR‑320a) was significantly reduced in cancer tissues, suggesting that miR‑320a may be a promising anticancer miRNA. The aim of this study was to investigate the functional roles of miR‑320a in naive PCa and CRPC cells and to identify miR‑320a-regulated genes involved in PCa metastasis. The expression levels of miR‑320a were significantly reduced in naive PCa, CRPC specimens, and PCa cell lines. Restoration of mature miR‑320a in PCa cell lines showed that miR‑320a significantly inhibited cancer cell migration and invasion. Moreover, we found that lysosomal-associated membrane protein 1 (LAMP1) was a direct target of miR‑320a in PCa cells. Silencing of LAMP1 using siRNA significantly inhibited cell proliferation, migration, and invasion in PCa cells. Overexpression of LAMP1 was observed in PCa and CRPC clinical specimens. Moreover, downstream pathways were identified using si-LAMP1-transfected cells. The discovery of tumor-suppressive miR‑320a-mediated pathways may provide important insights into the potential mechanisms of PCa metastasis.

// Juan Yu 1, * , Ji-Gang Wang 1, 2, * , Lei Zhang 1 , Hai-Ping Yang 3 , Lei Wang 1 , Di Ding 1 , Qi Chen 1 , Wen-Lin Yang 1 , Ke-Han Ren 1 , Dan-Mei Zhou 1 , Qiang Zou 4 , Yi-Ting Jin 4 , Xiu-Ping Liu 1, 5 1 Department of Pathology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China 2 Department of Pathology, The Affiliated Hospital of Qingdao University, Qingdao 266003, China 3 Department of Pathology, People’s Hospital, Linzi District, Zibo 255400, China 4 Department of Breast Surgery, Huashan Hospital, Fudan University, Shanghai 200040, China 5 Department of Pathology, The Fifth People’s Hospital, Fudan University, Shanghai 200240, China * These authors contributed equally to this work and are co-first authors Correspondence to: Xiu-Ping Liu, email: xpliu1228@fudan.edu.cn Yi-Ting Jin, email: clara_raky@aliyun.com Keywords: miR-320a, MTDH, metastasis, breast cancer Received: September 05, 2015 Accepted: May 01, 2016 Published: May 24, 2016 ABSTRACT Dysregulated microRNAs play important pathological roles in carcinogenesis that are yet to be fully elucidated. This study was performed to investigate the biological functions of microRNA-320a (miR-320a) in breast cancer and the underlying mechanisms. Function analyses for cell proliferation, cell cycle, and cell invasion/migration, were conducted after miR-320a silencing and overexpression. The specific target genes of miR-320a were predicted by TargetScan algorithm and then determined by dual luciferase reporter assay and rescue experiment. The relationship between miR-320a and its target genes was explored in human breast cancer tissues. We found that miR-320a overexpression could inhibit breast cancer invasion and migration abilities in vitro , while miR-320a silencing could enhance that. In addition, miR-320a could suppress activity of 3′-untranslated region luciferase of metadherin ( MTDH ), a potent oncogene. The rescue experiment revealed that MTDH was a functional target of miR-320a. Moreover, we found that MTDH was negatively correlated with miR-320a expression, and it was related to clinical outcomes of breast cancer. Further xenograft experiment also showed that miR-320a could inhibit breast cancer metastasis in vivo . Our findings clearly demonstrate that miR-320a suppresses breast cancer metastasis by directly inhibiting MTDH expression. The present study provides a new insight into anti-oncogenic roles of miR-320a and suggests that miR-320a/MTDH pathway is a putative therapeutic target in breast cancer.

In the present study, we investigated the roles and molecular mechanisms of miR-320a in human nasopharyngeal carcinoma (NPC). miR-320a expression was strongly reduced in NPC tissues and cell lines. Overexpression of miR-320a significantly suppressed NPC cell growth, migration, invasion and tumor growth in a xenograft mouse model. A luciferase reporter assay revealed that miR-320a could directly bind to the 3′ UTR of BMI-1. Overexpression of BMI-1 rescued miR-320a-mediated biological function. BMI-1 expression was found to be up-regulated and inversely correlated with miR-320a expression in NPC. Collectively, our data indicate that miR-320a plays a tumor suppressor role in the development and progression of NPC and may be a novel therapeutic target against NPC.