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Shuai Yin,1,* Zhen-Hua Zhang,1,* Yi-Niu Chang,2,* Jin Huang,2 Ming-Li Wu,1 Qi Li,1 Jin-Qi Qiu,1 Xiao-Dong Feng,1,2 Nan Wu2 1Rehabilitation Center, The First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou, Henan, 450000, People’s Republic of China; 2School of Rehabilitation Medicine, Henan University of Chinese Medicine, Zhengzhou, Henan, 450046, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiao-Dong Feng, Rehabilitation Center, The First Affiliated Hospital of Henan University of Chinese Medicine, No. 19, Renmin Road, Zhengzhou, 450000, People’s Republic of China, Tel +8615303828605, Email fxd0502@163.com Nan Wu, School of Rehabilitation Medicine, Henan University of Chinese Medicine, No. 156, Jinshui East Road, Zhengzhou, 450046, People’s Republic of China, Tel +8613540484550, Email wunanwy@126.comPurpose: Abnormal central nervous system function is the key central pathological factor leading to chronic pain in patients with knee osteoarthritis (KOA). Acupuncture can effectively relieve the pain of KOA patients. However, the central nervous mechanism of acupuncture treating KOA is not fully understood. This trial will use functional magnetic resonance imaging (fMRI) analysis techniques to investigate the potential central nervous mechanism of acupuncture treatment of KOA.Materials and Methods: A total of 108 patients will be randomized (in a 1:1:1 ratio) into three groups, this trial will include 4-week treatment, patients in groups A and B will receive 20 acupuncture and sham acupuncture sessions, respectively, patients in group C will not receive any intervention, and all patients will receive fMRI scans before and after the intervention. The Western Ontario and McMaster Universities Osteoarthritis Index score (WOMAC) will be the primary clinical outcome. Then, we will explore the functional changes of the cognitive control network (CCN) in the brains of KOA patients through whole brain functional connectivity (FC) analysis and seed-based functional connectivity (sFC) analysis. Pearson correlation coefficient will be used to analyze the relationship between the improved value of the clinical correlation scale and the change of fMRI data.Discussion: This trial will analyze the efficacy of verum acupuncture, sham acupuncture and the waiting-list for KOA and explore the activity of the CCN in three groups of patients by fMRI, so as to reveal the central nervous mechanisms of acupuncture in the treatment of KOA.Study Registration: This study is approved by the Ethics Committee of the First Affiliated Hospital of Henan University of Traditional Chinese Medicine (No: 2019HL-133-01) and registered in the Chinese Clinical Trial Registry, ChiCTR2000038554.Keywords: knee osteoarthritis, acupuncture, cognitive control network, randomized controlled trial

PDF - 1052KB, Supplementary Figure S2. Inhibition of miR-141 upregulates MAP4K4 and promotes cell growth and invasion. (A) MiR-141 expression was downregulated after treated with miR-141 inhibitor. (B) Inhibition of miR-141 promoted the proliferation of PANC-1 cells by MTT assay. (C) Downregulation of miR-141 improved clonogenicity ability of PANC-1. (D) Invasion ability of PANC-1 cells was enhanced after decreasing of miR-141 expression. (E) MMP-2 and MMP-9 were upregulated after inhibition of miR-141 in PANC-1 by western blot analysis. *P

miRNAs are associated with various types of cancer due to their ability to affect expression of genes that modulate tumorigenesis. In this study, we explored the role of miR-141 in pancreatic cancer. The analysis of clinical characteristics showed that miR-141 was significantly downregulated in tissues and cell lines of pancreatic cancer. Moreover, the decreased miR-141 level was significantly associated with tumor size and TNM stage, as well as lymph node and distant metastasis. Meanwhile, both Kaplan–Meier and multivariate survival analysis showed decreased miR-141 were associated with overall survival. Overexpression of miR-141 in pancreatic cancer cells inhibited cell proliferation, clonogenicity, and invasion; induced G1 arrest and apoptosis; and enhanced chemosensitivity. To understand how miR-141 mediates the phenotype of pancreatic cancer cells, a bioinformatics tool was used to identify MAP4K4 as a potential target of miR-141. The Dual-Luciferase reporter gene assay showed that miR-141 binds directly to the 3′-untranslated region (3′UTR) of MAP4K4 to inhibit MAP4K4 expression. Western blot and quantitative real-time PCR (qRT-PCR) analyses revealed that MAP4K4 expression was inversely correlated with miR-141 expression both in pancreatic cancer samples and cell lines. Knockdown of MAP4K4 inhibited cell proliferation, clonogenicity, and invasion, induced G1 arrest and apoptosis, and enhanced chemosensitivity. In a nude mouse xenograft model, both overexpression of miR-141 and knockdown of MAP4K4 significantly repressed pancreatic cancer cell growth. Therefore, we conclude that miR-141 targets MAP4K4, acts as a tumor suppressor in pancreatic cancer cells, and may serve as a novel therapeutic agent for miRNA-based pancreatic cancer therapy. Mol Cancer Ther; 12(11); 2569–80. ©2013 AACR.

PDF file - 2958K, Figure S6. Overexpression of AMPKα1 rescues miR-148b-induced apoptosis and cell cycle arrest and invasion inhibition of pancreatic cancer cells.

PDF file - 3185K, Figure S3. Overexpression of AMPKα1 in pancreatic cancer tissues and cell lines and inversely correlate with miR-148b levels.

PDF - 86KB, Legends for Supplementary Figures S1-S3.

PDF - 60KB, Immunohistochemical staining analysis shows different expressions of MAP4K4 in pancreatic cancer tissues and their adjacent noncancerous tissues.

PDF file - 1881K, Figure S4. LV-shAMPKα1 suppresses tumourigenicity in nude mouse xenograft model.

PDF file - 111K

miRNAs are small noncoding RNAs that participate in a variety of biologic processes, and dysregulation of miRNA is always associated with cancer development and progression. Aberrant expression of miR-148b has been found in some types of cancer, but its expression and potential biologic role in pancreatic cancer are still largely unknown. In this study, our data showed that miR-148b was significantly downregulated in 48 pairs of human pancreatic cancer tissues and five cell lines. Furthermore, the deregulated miR-148b was correlated with increased tumor size, late tumor–node–metastasis stage, lymphatic invasion, distant metastasis, and worse prognosis in pancreatic cancer. Functional studies indicated overexpression of miR-148b dramatically suppressed the growth of cancer cells, attributable to induction of apoptosis and cell-cycle arrest at S-phase. Meanwhile, miR-148b remarkably inhibited invasion and enhanced chemosensitivity of pancreatic cancer cells. Moreover, ectopic expression of miR-148b was able to inhibit tumorigenicity in nude mice. Further studies revealed that AMPKα1 might be the direct target gene of miR-148b, and overexpressed AMPKα1 inversely correlated with miR-148b in pancreatic cancer. Silencing of AMPKα1 with RNA interference inhibited the growth of pancreatic cancer cells in vitro and in vivo and also induced apoptosis, cell-cycle arrest, and inhibited invasion of cancer cells, which is consistent with the effects of miR-148b overexpression. In conclusion, miR-148b can inhibit cell proliferation, invasion, and enhance chemosensitivity of pancreatic cancer by targeting AMPKα1. Our present results implicate the potential effects of miR-148b on prognosis and treatment of pancreatic cancer. Mol Cancer Ther; 12(1); 83–93. ©2012 AACR.

PDF - 1662KB, Supplementary Figure S1. Overexpression of MiR-141 promotes G1 phase arrest, apoptosis and chemosensitivity of MiaPaCa-2 cells. (A) Flow cytometry analysis showed that the cell cycle of MiaPaCa-2 was arrested at the G1 phase after upregulation of miR-141. The histogram indicates the percentage of cells in the G1 phase. (B) MiR-141 induced apoptosis of MiaPaCa-2 cells. (C) The bar graphs for cell cycle and apoptosis counting. (C) Overexpression of miR-141 sensitized MiaPaCa-2 cells to 5-Fu and Gemcitabine. Different concentrations were applied to the cells for 48 h and cell viability was measured by MTT assay. (D) Western blot analysis indicated that upregulation of miR-141 resulted in significant downregulation of the invasion associated proteins MMP-2 and MMP-9, the cell cycle related protein cyclinD1 and the anti-apoptotic protein Bcl-2, as well as overexpression of the pro-apoptotic protein Bax.* P

PDF - 5KB, The primer sequences of genes.

PDF - 3658KB, Supplementary Figure S3. Downregulation of MAP4K4 reverses the malignant behavior of pancreatic cancer cell lines. (A) The MAP4K4 expression was downregulated after treated with its siRNA determined by SYBR Green qRT-PCR (left) and western blot (right). (B) Knockdown of MAP4K4 suppressed the proliferation of PANC-1 and MiaPaCa-2 cells. (C) Downregulation of MAP4K4 weakened the ability of cell clonogenicity. (D) The cell invasion ability was measured at 48 h after cells seeded in chambers. Silencing of MAP4K4 decreased the ability of cell invasion. (E) Cell cycle was examined by flow cytometry. Compared to NC groups, cell cycle in siRNA-MAP4K4 groups was blocked at G1 phase. (F) The percentage of apoptotic cells increased through downregulation of MAP4K4 in pancreatic cancer cell lines. (G) Silencing of MAP4K4 increased chemosensitivity to 5-Fu and Gemcitabine based on IC50 data. (H) Western blot assay illustrated that after treated with MAP4K4 siRNA, MMP-2, cyclinD1 and Bcl-2 were downregulated while Bax was upregulated. But the changes of MMP-9 were not so significant. *P

PDF file - 3019K, Figure S2. MiR-148b suppresses tumourigenicity in nude mouse xenograft model.

PDF file - 57K, Supplemental Table S1. Sequences of qRT-PCR primers. Supplemental Table S2. Sequences of RNA oligonucleotides.

PDF - 61KB, Western Blot primary antibody.

PDF file - 2958K, Figure S5. Downregulation of AMPKα1 increases apoptosis and cell cycle arrest and inhibits invasion of pancreatic cancer cells.

PDF file - 2016K, Figure S1. MiR-148b enhances chemosensitivity of pancreatic cancer cells.

Gemcitabine (GEM) resistance is a major challenge for chemotherapy of pancreatic cancer. Previous studies have reported on the role of long noncoding RNA (lncRNA) in tumorigenesis of pancreatic cancer, however, the involvement of lncRNA in the development of GEM resistance of pancreatic cancer remains unclear. In the present study, we demonstrated that the antisense RNA1 of HIF1α (HIF1A-AS1) was significantly elevated in the GEM-resistant pancreatic cancer cells. Gain- and lost-of-function experiments validated that HIF1A-AS1 promoted GEM resistance of pancreatic cancer cells both in vitro and vivo. We further revealed that HIF1A-AS1 upregulated HIF1α expression and thus promoted glycolysis to enhance GEM resistance of pancreatic cancer cells. Mechanistically, HIF1A-AS1 facilitated the interaction between serine/threonine kinase AKT and Y-box–binding protein 1 (YB1), which promoted phosphorylation of YB1 (pYB1). Meanwhile, HIF1A-AS1 recruited pYB1 to HIF1α mRNA that consequently promoted translation of HIF1α. Furthermore, HIF1α promoted HIF1A-AS1 transcription by directly binding to the HIF1α response element in the promoter area of HIF1A-AS1 to form a positive feedback. Consistently, both HIF1A-AS1 and HIF1α were upregulated in pancreatic cancer tissues and associated with poor overall survival. Together, our results underline a reciprocal loop of HIF1A-AS1 and HIF1α that contributes to GEM resistance of pancreatic cancer and indicate that HIF1A-AS1 might serve as a novel therapeutic target for GEM resistance of pancreatic cancer.Significance:These findings show that a reciprocal feedback of HIF1A-AS1 and HIF1α promotes gemcitabine resistance of pancreatic cancer, which provides an applicable therapeutic target.

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In this paper, we provide a comprehensive description of redox-induced thermocells, including the mechanism, progress, and applications.