Animal Bioscience

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
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ssc-miR-185 targets cell division cycle 42 and promotes the proliferation of intestinal porcine epithelial cell

  • Wang, Wei (College of Animal Science and Technology, Gansu Agricultural University) ;
  • Wang, Pengfei (College of Animal Science and Technology, Gansu Agricultural University) ;
  • Xie, Kaihui (College of Animal Science and Technology, Gansu Agricultural University) ;
  • Luo, Ruirui (College of Animal Science and Technology, Gansu Agricultural University) ;
  • Gao, Xiaoli (College of Animal Science and Technology, Gansu Agricultural University) ;
  • Yan, Zunqiang (College of Animal Science and Technology, Gansu Agricultural University) ;
  • Huang, Xiaoyu (College of Animal Science and Technology, Gansu Agricultural University) ;
  • Yang, Qiaoli (College of Animal Science and Technology, Gansu Agricultural University) ;
  • Gun, Shuangbao (College of Animal Science and Technology, Gansu Agricultural University)
  • Received : 2020.05.11
  • Accepted : 2020.09.02
  • Published : 2021.05.01
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Abstract

Objective: microRNAs (miRNAs) can play a role in a variety of physiological and pathological processes, and their role is achieved by regulating the expression of target genes. Our previous high-throughput sequencing found that ssc-miR-185 plays an important regulatory role in piglet diarrhea, but its specific target genes and functions in intestinal porcine epithelial cell (IPEC-J2) are still unclear. We intended to verify the target relationship between porcine miR-185 and cell division cycle 42 (CDC42) gene in IPEC-J2 and to explore the effect of miR-185 on the proliferation of IPEC-J2 cells. Methods: The TargetScan, miRDB, and miRanda software were used to predict the target genes of porcine miR-185, and CDC42 was selected as a candidate target gene. The CDC42-3' UTR-wild type (WT) and CDC42-3'UTR-mutant type (MUT) segments were successfully cloned into pmirGLO luciferase vector, and the luciferase activity was detected after co-transfection with miR-185 mimics and pmirGLO-CDC42-3'UTR. The expression level of CDC42 was analyzed using quantitative polymerase chain reaction and Western blot. The proliferation of IPEC-J2 was detected using cell counting kit-8 (CCK-8), methylthiazolyldiphenyl-tetrazolium bromide (MTT), and 5-ethynyl-2'-deoxyuridine (EdU) assays. Results: Double enzyme digestion and sequencing confirmed that CDC42-3'UTR-WT and CDC42-3'UTR-MUT were successfully cloned into pmirGLO luciferase reporter vector, and the luciferase activity was significantly reduced after co-transfection with miR-185 mimics and CDC42-3'UTR-WT. Further we found that the mRNA and protein expression level of CDC42 were down-regulated after transfection with miR-185 mimics, while the opposite trend was observed after transfection with miR-185 inhibitor (p<0.01). In addition, the CCK-8, MTT, and EdU results demonstrated that miR-185 promotes IPEC-J2 cells proliferation by targeting CDC42. Conclusion: These findings indicate that porcine miR-185 can directly target CDC42 and promote the proliferation of IPEC-J2 cells. However, the detailed regulatory mechanism of miR-185/CDC42 axis in piglets' resistance to diarrhea is yet to be elucidated in further investigation.

Keywords

References

  1. Huang X, Yang Q, Yuan J, et al. Effect of genetic diversity in swine leukocyte antigen-DRA gene on piglet diarrhea. Genes 2016;7:36. https://doi.org/10.3390/genes7070036
  2. Dai C, Yang L, Jin J, Wang H, Wu S, Bao W. Regulation and molecular mechanism of TLR5 on resistance to Escherichia coli F18 in weaned piglets. Animals 2019;9:735. https://doi.org/10.3390/ani9100735
  3. Xia B, Yu J, He T, et al. Lactobacillus johnsonii L531 ameliorates enteritis via elimination of damaged mitochondria and suppression of SQSTM1-dependent mitophagy in a Salmonella infantis model of piglet diarrhea. FASEB J 2020;34:2821-39. https://doi.org/10.1096/fj.201901445RRR
  4. Huang XY, Sun WY, Yan ZQ, et al. Novel insights reveal anti-microbial gene regulation of piglet intestine immune in response to Clostridium perfringens infection. Sci Rep 2019;9: 1963. https://doi.org/10.1038/s41598-018-37898-5
  5. Huang X, Sun W, Yan Z, et al. Integrative analyses of long non-coding RNA and mRNA involved in piglet ileum immune response to Clostridium perfringens type C infection. Front Cell Infect Microbiol 2019;9:130. https://doi.org/10.3389/fcimb.2019.00130
  6. Yan Z, Cai L, Huang X, et al. Histological and comparative transcriptome analyses provide insights into small intestine health in diarrheal piglets after infection with Clostridium perfringens type C. Animals 2019;9:269. https://doi.org/10.3390/ani9050269
  7. Wu M, Zhang Q, Yi D, et al. Quantitative proteomic analysis reveals antiviral and anti-inflammatory effects of puerarin in piglets infected with porcine epidemic diarrhea virus. Front Immunol 2020;11:169. https://doi.org/10.3389/fimmu.2020.00169
  8. Ambros V. The functions of animal microRNAs. Nature 2004; 431:350-5. https://doi.org/10.1038/nature02871
  9. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281-97. https://doi.org/10.1016/S0092-8674(04)00045-5
  10. Chang W, Zhang L, Xian Y, Yu Z. MicroRNA-33a promotes cell proliferation and inhibits apoptosis by targeting PPARα in human hepatocellular carcinoma. Exp Ther Med 2017;13: 2507-14. https://doi.org/10.3892/etm.2017.4236
  11. Liu Y, Liang H, Jiang X. miR-1297 promotes apoptosis and inhibits the proliferation and invasion of hepatocellular carcinoma cells by targeting HMGA2. Int J Mol Med 2015;36: 1345-52. https://doi.org/10.3892/ijmm.2015.2341
  12. Zhu T, Fan D, Ye K, et al. Role of miRNA-542-5p in the tumorigenesis of osteosarcoma. FEBS Open Bio 2020;10:627-36. https://doi.org/10.1002/2211-5463.12824
  13. Chandan K, Gupta M, Sarwat M. Role of host and pathogen-derived micrornas in immune regulation during infectious and inflammatory diseases. Front Immunol 2020;10:3081. https://doi.org/10.3389/fimmu.2019.03081
  14. Wang P, Huang X, Yan Z, et al. Analyses of miRNA in the ileum of diarrheic piglets caused by Clostridium perfringens type C. Microb Pathog 2019;136:103699. https://doi.org/10.1016/j.micpath.2019.103699
  15. Wang YP, Huang Y, Hou T, Lu M. LncRNA XIST acts as a ceRNA sponging miR-185-5p to modulate pancreatic cancer cell proliferation via targeting CCND2. Transl Cancer Res 2020;9:1427-38. https://doi.org/10.21037/tcr.2020.01.26
  16. Lin G, Zhang C, Chen X, et al. Identification of circulating miRNAs as novel prognostic biomarkers for bladder cancer. Math Biosci Eng 2019;17:834-44. https://doi.org/10.3934/mbe.2020044
  17. Ge P, Cao L, Yao YJ, Jing RJ, Wang W, Li HJ. lncRNA FOXD2-AS1 confers cisplatin resistance of non-small-cell lung cancer via regulation of miR185-5p-SIX1 axis. Onco Targets Ther 2019;12:6105-17. https://doi.org/10.2147/OTT.S197454
  18. Ostadrahimi S, Valugerdi MA, Hassan M, et al. miR-1266-5p and miR-185-5p promote cell apoptosis in human prostate cancer cell lines. Asian Pac J Cancer Prev 2018;19:2305-11. https://doi.org/10.22034/APJCP.2018.19.8.2305
  19. Tan B, Li Y, Zhao Q, Fan L, Wang D. ZNF139 increases multi-drug resistance in gastric cancer cells by inhibiting miR-185. Biosci Rep 2018;38:BSR20181023. https://doi.org/10.1042/BSR20181023
  20. Liu L, Zhu Y, Liu AM, Feng Y, Chen Y. Long noncoding RNA LINC00511 involves in breast cancer recurrence and radioresistance by regulating STXBP4 expression via miR-185. Eur Rev Med Pharmacol Sci 2019;23:7457-68. https://doi.org/10.26355/eurrev_201909_18855
  21. Zhang Q, Chen Y, Liu K. miR-185 inhibits cell migration and invasion of hepatocellular carcinoma through CDC42. Oncol Lett 2018;16:3101-7. https://doi.org/10.3892/ol.2018.8971
  22. Yuan M, Zhang X, Zhang J, et al. DC-SIGN-LEF1/TCF1-miR-185 feedback loop promotes colorectal cancer invasion and metastasis. Cell Death Differ 2020;27:379-95. https://doi.org/10.1038/s41418-019-0361-2
  23. Liu Y, Chen SH, Jin X, Li YM. Analysis of differentially expressed genes and microRNAs in alcoholic liver disease. Int J Mol Med 2013;31:547-54. https://doi.org/10.3892/ijmm.2013.1243
  24. Ma D, Cao Y, Wang Z, et al. CCAT1 lncRNA promotes inflammatory bowel disease malignancy by destroying intestinal barrier via downregulating miR-185-3p. Inflamm Bowel Dis 2019;25:862-74. https://doi.org/10.1093/ibd/izy381
  25. Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature 2002;420:629-35. https://doi.org/10.1038/nature01148
  26. Sahai E, Marshall CJ. RHO-GTPases and cancer. Nat Rev Cancer 2002;2:133-42. https://doi.org/10.1038/nrc725
  27. Liu M, Lang N, Qiu M, et al. miR-137 targets Cdc42 expression, induces cell cycle G1 arrest and inhibits invasion in colorectal cancer cells. Int J Cancer 2011;128:1269-79. https://doi.org/10.1002/ijc.25452
  28. Liu M, Lang N, Chen X, et al. miR-185 targets RhoA and Cdc42 expression and inhibits the proliferation potential of human colorectal cells. Cancer Lett 2011;301:151-60. https://doi.org/10.1016/j.canlet.2010.11.009
  29. Lewis BP, Shih I, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of mammalian microRNA targets. Cell 2003;115: 787-98. https://doi.org/10.1016/S0092-8674(03)01018-3
  30. Chen Y, Wang X. miRDB: an online database for prediction of functional microRNA targets. Nucleic Acids Res 2020;48: D127-31. https://doi.org/10.1093/nar/gkz757
  31. Betel D, Wilson M, Gabow A, Marks DS, Sander C. The microRNA.org resource: targets and expression. Nucleic Acids Res 2008;36(Suppl 1):D149-53. https://doi.org/10.1093/nar/gkm995
  32. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 2001;25:402-8. https://doi.org/10.1006/meth.2001.1262
  33. Chernichenko N, Omelchenko T, Deborde S, et al. Cdc42 mediates cancer cell chemotaxis in perineural invasion. Mol Cancer Res 2020;18:913-25. https://doi.org/10.1158/15417786.MCR-19-0726
  34. Gu G, Wang L, Zhang J, Wang H, Tan T, Zhang G. MicroRNA-384 inhibits proliferation migration and invasion of glioma by targeting at CDC42. Onco Targets Ther 2018;11:4075-85. https://doi.org/10.2147/OTT.S166747
  35. Yang T, Chen T, Li Y, et al. Downregulation of miR-25 modulates non-small cell lung cancer cells by targeting CDC42. Tumor Biol 2015;36:1903-11. https://doi.org/10.1007/s13277014-2793-0
  36. Li Y, Zhu X, Xu W, Wang D, Yan J. miR-330 regulates the proliferation of colorectal cancer cells by targeting Cdc42. Biochem Biophys Res Commun 2013;431:560-5. https://doi.org/10.1016/j.bbrc.2013.01.016
  37. Niu Y, Tang G. miR-185-5p targets ROCK2 and inhibits cell migration and invasion of hepatocellular carcinoma. Oncol Lett 2019;17:5087-93. https://doi.org/10.3892/ol.2019.10144
  38. Fang M, Li Y, Wu Y, Ning Z, Wang X, Li X. miR-185 silencing promotes the progression of atherosclerosis via targeting stromal interaction molecule 1. Cell Cycle 2019;18:682-95. https://doi.org/10.1080/15384101.2019.1580493
  39. Fan L, Tan B, Li Y, et al. Upregulation of miR-185 promotes apoptosis of the human gastric cancer cell line MGC803. Mol Med Rep 2018;17:3115-22. https://doi.org/10.3892/mmr.2017.8206
  40. Zou Q, Wu H, Fu F, Yi W, Pei L, Zhou M. RKIP suppresses the proliferation and metastasis of breast cancer cell lines through up-regulation of miR-185 targeting HMGA2. Arch Biochem Biophys 2016;610:25-32. https://doi.org/10.1016/j.abb.2016.09.007
  41. Singaravelu R, O'Hara S, Jones DM, et al. MicroRNAs regulate the immunometabolic response to viral infection in the liver. Nat Chem Biol 2015;11:988-93. https://doi.org/10.1038/nchembio.1940