Microrna-128 Mediates CB1 Expression and Regulates NF-KB/P

Gou et al. J Transl Med (2020) 18:284 https://doi.org/10.1186/s12967-020-02406-9 Journal of Translational Medicine RESEARCH Open Access microRNA-128 mediates CB1 expression and regulates NF-KB/p-JNK axis to infuence the occurrence of diabetic bladder disease Xin Gou1†, Jing Wu2†, Mingqing Huang3, Yuting Weng1, Tongxin Yang1, Tao Chen1, Guiqing Li1 and Kewei Fang1* Abstract Background: Diabetic bladder disease is common complications of diabetes, its symptoms are diverse, can be due to diferent stages. In this study we investigate the mechanism of miR-128 targeting CB1 expression to mediate the occurrence of diabetic bladder disease. Methods: Bioinformatics analysis predicts related regulatory factors of miR-128 in diabetic bladder disease. Models of diabetic bladder lesions were constructed in male SD rats by intraperitoneal injection of streptozotocin at 65 mg/kg body weight. The expression of miR-128 and CB1 mRNA in bladder tissues of each group was detected by RT-qPCR, and CB1, NF-KB, p-JNK and Bcl2 protein expression was detected by Western Blotting. We tested the function of the bladder by urodynamics, detected the pathological characteristics of the ARTICLEbladder tissue by HE staining, and verifed the targeting relationship between miR-128 and CB1 through the prediction of the biological website, dual luciferase reporter gene assay and RIP. Results: miR-128 was highly expressed in the bladder tissue of diabetic rats. Inhibition of miR-128 could improve the occurrence of diabetic bladder lesions in rats. miR-128 could target the inhibition of CB1 expression, and high expression of CB1 could antagonize miR-128 against diabetic bladder. In the diabetic bladder, miR-128 can regulate the expression of NF-KB and p-JNK through CB1 and afect the level of apoptosis. miR-128 regulates NF-KB/p-JNK through CB1, thus afecting the occurrence of diabetic bladder disease. Conclusion: The high expression of miR-128 can down-regulate the expression of CB1, promote the activation of NF-KB and p-JNK, increase the level of apoptosis and promote the occurrence of diabetic bladder disease. Keywords: microRNA-128, CB1, Diabetes, Bladder disease, NF-KB, P-JNK, Apoptosis Background of bladder flling and weakened contractility changes in Diabetic bladder disease is common complications of urination characteristics, and may be complicated by uri- diabetes, its symptoms are diverse, can be due to difer- nary tract infection and bladder urination Tube refux, ent stages, but mainly manifested by impaired feeling hydronephrosis, kidney stones, and eventually uremia [1]. Te number of people with diabetes has continued to increase in recent years [2]. Diabetic cystitis (DCP) is a *Correspondence: [email protected] systemic disease associated with diabetes in the urinary †Xin Gou, and Jing Wu are co-frst authors system of humans and animals, accounting for more than 1 Department of Urology, The Second Afliated Hospital of Kunming Medical University, No. 374, Dianmian Dadao, Kunming, Yunnan 650101, 80% of people with diabetes [3]. Te pathogenesis of DCP People’sRETRACTED Republic of China is still unclear, it is a complex and has many incentives, Full list of author information is available at the end of the article and the course of the disease is closely related to time. © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons.org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons.org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gou et al. J Transl Med (2020) 18:284 Page 2 of 14 Myogenic, currently considered neurogenic and ure- regulatory genes. Te co-expression relationship of thral epithelial changes are the main causes of DCP [4, downstream genes was obtained through Chipbase v2.0 5]. Many studies have tried to elucidate the mechanism website (http://rna.sysu.edu.cn/chipb ase/). of DCP, but it is still unclear. Terefore, the treatment of DCP is greatly restricted [6]. Establishment of Diabetic Mellitus (DM) rat model miRNAs are a group of highly conserved small RNA A total of 100 SPF male SD rats (purchased from the molecules that can regulate gene expression functions Experimental Animal Center of China Medical Univer- More and more studies show that miRNAs play an sity), weighing 180–250 g, were randomly divided into 3 important role in many physiological processes [7, 8]. groups after fasting for 12 h: normal control group (NC Mutations or disorders of miRNAs are related to a variety group, n = 16); Polyuria control group (PU group, n = 16), of human tumors. Over control protein coding to play a hypertonic polyuria induced with 5% sucrose water as role in promoting or suppressing cancer [9]. Studies have drinking water; diabetes group (DM group, n = 68), sin- shown that miR-128 expression in tumors of the nervous gle intraperitoneal injection of streptozotocin (STZ; system, breast and prostate cancer, tumor expression is dissolved at pH = 4.2 in 0.1 mol/L framic acid bufer; down-regulated [10], and through its target genes such as Sigma, St. Louis, MO) at 65 mg/kg body weight. Te the oncogene Bmi-1 [11], EGFR [12], p70S6K1 [13], E2F3 rats in the NC group and the PU group were intraperi- [14], miR-128 play a role in suppressing cancer. However, toneally injected with the same dose of normal saline. At level of miR-128a and its downstream regulated signals in 4 weeks after STZ injection, the blood glucose level was diabetes have not been reported. confrmed to be above 300 mg/dl, and the rats with urine Recent years more reports were focused on the role glucose test strip ++++ were diabetic rats. All rats were of miR-144, also like miR-128 carcinogenesis because free to eat and drink. At 6 and 12 weeks, 8 rats were ran- it is dysregulated and involved in the tumorigenesis of domly selected from each group, placed in a metabolic various cancer, such as lung cancer [15], osteosarcoma cage, and urine of rats was collected and weighed for [16], hepatocellular carcinoma [17], thyroid cancer [18], 24 h, and thenARTICLE performed with urodynamic experiments. bladder cancer [19] and colorectal carcinoma [20]. How- Tis experimental procedure and animal use protocol ever, the biological functions and underlying molecular have been approved by the Animal Ethics Committee mechanism of miR-144 in DCP are not yet described. of the Second Afliated Hospital of Kunming Medical Terefore, we investigate the biological role and poten- University. tial mechanism of miR-144 in DCP by several experiments in vitro and tumor growth of xenograft in vivo and Grouping and processing of experimental animals in vitro. Te rats in the NC group, the PU group, and the suc- cessfully modeled DM group were anaesthetized with Methods 200 g/L urethane (injection dose 1.5 g/kg) after 6 h of Bioinformatics analysis fasting. Ureter is inserted through the urethra to empty Trough bioinformatics website microT (http:// the urine after anesthesia. Te rats in the NC group and diana .imis.athen a-innov ation .gr/Diana Tools /index the PU group were perfused with 0.2 ml of PBS solution .php?r=micro T_CDS/), TargetScan (http://www.targe through the ureter. Tere were 56 of DM group success- tscan .org/vert_72/), miRWalk (http://mirwa lk.umm.uni- fully modeled rats, and the modeling success rate was heide lberg .de/) and RNAInter (http://www.rna-socie 93.33%. Te modeled rats were divided into the following ty.org/rnain ter/), the downstream target genes of miR- groups, 8 in each group: mimic NC group (urinary cath- 128 in rats were jointly predicted using diferent bind- eter perfusion with 0.2 mL mimic NC), miR-128 mimic ing site matching algorithms. We used the jvenn tool group (urinary catheter perfusion with 0.2 mL miR-128 (http://jvenn .toulo use.inra.fr/app/examp le.html) to take mimic), inhibitor NC group (urinary catheter perfusion the intersection of four predicted results for target gene with 0.2 mL inhibitor NC), miR-128 inhibitor group (uri- screening. Te interaction between genes was analyzed nary catheter perfusion with0.2 mL miR-128 inhibitor), through the STRING website (https ://strin g-db.org/), mimic NC + oe-NC group (urinary catheter perfusion and theRETRACTED results of the interaction analysis were visual- with mimic NC and oe-NC lentivirus 0.2 mL), miR-128 ized using Cytoscape 3.5.1. In order to further predict the mimic + oe-NC group (urinary catheter perfusion with downstream regulatory factors of genes, we set the key- miR-128 mimic and oe-NC lentivirus 0.2 mL), and miR- word “diabetic bladder disease” through the GeneCards 128 mimic + oe-CB1 group (urinary catheter perfusion database (https ://www.genec ards.org/) to fnd related with miR-128 mimic and oe-CB1 0.2 mL each).

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