Analysis of the Effect of microRNA 29a on Gene Expression in Small Intestinal Epithelial Cells of Bamei Piglets (Sus scrofa) Based on RNA-seq Technology
1 College of Agriculture and Animal Husbandry, Qinghai University, Xining 810016, China; 2 Animal Husbandry and Veterinary Station, Penglai District, Yantai 265699, China; 3 School of Life Sciences, Qilu Normal University, Ji'nan 250200, China
Abstract：Small intestinal epithelial cells (IECs) are important basis for the small intestine to play an important role in digestion and absorption of nutrients and intestinal barrier. microRNA 29a (miR-29a) plays an important role in cell apoptosis, differentiation and proliferation during animal growth and development. In order to clarify the effect of miR-29a on the gene expression of small intestinal epithelial cells of piglets (Sus scrofa), miR-29a mimics group, miR-29a inhibitor group, and negative control group were constructed. The IECs were collected after transfection 48 h, extracted RNA to construct libraries and performed Illumina sequencing; The differential expressed genes (DEGs) were screened and subjected to GO functional annotation and KEGG pathway analysis; Venn analysis was used to screen target differential genes and construct target gene co-expression network, 4 genes were selected randomly to verify the accuracy of transcriptome data by qPCR. A total of 9 transcriptome libraries were obtained in this study and total clean base was 55.42 Gb, and the comparison rate with the reference genome reached more than 95%. The DESeq2 software was used to analyze the expressed genes obtained by sequencing, and the results showed that 7 102 DEGs were expressed between the miR-29a inhibitor group (VPA) and the negative control group (VPC), of which 2 558 up-regulated genes and 3 544 down-regulated genes were found in VPA; there were 2 829 DEGs between the two groups, of which 1 563 genes were up-regulated genes in miR-29a mimics group (VPT), and 1 266 genes were down-regulated in the VPT; there were 503 DEGs in the VPA and VPT. There were 197 up-regulated and 306 down-regulated genes in VPA. The GO functional annotation and KEGG pathway enrichment analysis of the differential genes showed that the IECs after miR-29a treatment were in a very active stage of cell proliferation and differentiation and the obtained DEGs were mainly enriched in DNA replication and cell cycle pathways. qPCR results were consistent with the sequencing data. The above results suggested that miR-29a might be regulated cell apoptosis by affecting genes such as CDKN2C, E2F1, MCM family and POLE in IECs, thereby affecting intestinal absorption and intestinal barrier function. This study provides basic for studying the effect of miR-29a on the intestinal of Bamei piglets.
[1] 邓路, 何志伟, 朱昌毫, 等. 2021. CDCA7通过与MCM3相互作用介导胰腺癌细胞增殖、侵袭及迁移[J]. 肿瘤, 41(01): 24-35. (Deng L, He Z W, Zhu C H, et al.2021. CDCA7 mediates proliferation, invasion and migration of pancreatic cancer cells by interacting with MCM3[J]. Tumor, 41(01): 24-35.) [2] 郭伟伟, 张晓鹏, 李霞, 等. 2020. MiRNA-138抑制JNK/p38 MAPK通路保护新生大鼠心肌细胞凋亡[J]. 中山大学学报(医学版), 41(01): 86-94. (Guo W W, Zhang X P, Li X, et al.2020. MiRNA-138 protecting cardiomyocyte apoptosis of neonatal rats by inhibiting JNK/p38 MAPK pathway[J]. Journal of Sun Yat-sen University (Medical Sciences), 41(01): 86-94.) [3] 靳继鹏. 2021. 母体日粮粗蛋白水平对互助八眉哺乳仔猪空肠发育的作用及其机制[D]. 博士学位论文, 甘肃农业大学, 导师: 张利平, 贾建磊, pp. 1-2. (Jin J P.2021. Effect and mechanism of maternal dietary crude protein level on jejunum development in Huzhu Bamei suckling piglets[D]. Thesis for Ph. D., Gansu Agricultural University, Supervisor: Zhang L P, Jia J L, pp. 1-2.) [4] 李娜苗. 2020. POLE2基因沉默通过调控PLK1表达抑制肺腺癌细胞的增殖[D]. 硕士学位论文, 延安大学, 导师: 李建英, pp. 4-5. (Li N M.2020. POLE2 gene silencing inhibits the proliferation of lung adenocarcinoma cells by regulating PLK1 expression[D]. Thesis for M.S., Yan'an University, Supervisor: Li J Y, pp. 4-5.) [5] 苏嘉明. 2019. ESPL1在前列腺癌中的表达及其预后价值[D].硕士学位论文,遵义医科大学, 导师: 孙兆林, 朱建国, pp. 22-23. (Su J M.2019. Prognostic value of ESPL1 expressions in human prostate cancer[D]. Thesis for M.S., Zunyi Medical University, Supervisor: Sun Z L, Zhu J G, pp. 22-23.) [6] 陶新, 徐子伟. 2013. miRNAs对肠道健康的调控作用及机理[J]. 动物营养学报, 25(09): 1911-1915. (Tao X, Xu Z W.2013. miRNAs: Regulation on intestinal health and mechanism[J]. Acta Zoonutrimenta Sinica, 25(09): 1911-1915.) [7] 王慧玲, 林兴滔, 区应亮, 等. 2017. miR-29a调控HDAC4促肝癌细胞增殖和转移的机制研究[J]. 胃肠病学和肝病学杂志, 26(04):381-385. (Wang H L, Lin X T, Ou Y L, et al.2017. The mechanism of miR-29a regulating the proliferation and metastasis of hepatocellular carcinoma cells through HDAC4[J]. Chinese Journal of Gastroenterology and Hepatology, 26(04): 381-385.) [8] 王可葭. 2014. 下调FEN1基因表达对胃癌SGC-7901细胞增殖和凋亡的影响[D]. 硕士学位论文, 重庆医科大学, 导师: 陈道荣, pp. 30-31. (Wang K J.2014. Effects of FEN1 gene silencing on proliferation and apoptosis of gastric cancer cell line SGC-7901[D]. Thesis for M.S., ChongQing Medical University, Supervisor: Chen H R, pp. 30-31.) [9] 王明月. 2019. IGFBP3在肺腺癌中的差异表达及其对肺腺癌细胞的增殖、凋亡和侵袭等的影响[D]. 硕士学位论文, 锦州医科大学, 导师: 黄波, pp. 17-18. (Wang Y M.2019. Differential expression of IGFBP3 in lung adenocarcinoma and its effects on proliferation, apoptosis and invasion of lung adenocarcinoma cells[D]. Thesis for M.S., Jinzhou Medical University, Supervisor: Huang B, pp. 17-18.) [10] 吴国芳, 周继平, 王磊, 等. 2019. 日粮能量水平对八眉三元杂交猪生长性能、屠宰性能及肉质性状的影响[J]. 家畜生态学报, 40(04): 27-31. (Wu G F, Zhou J P, Wang L, et al.2019. Effects of dietary energy level on growth performance, slaughter performance and meat quality of Bamei Ternary Hybrid Pig[J]. Journal of Domestic Animal Ecology, 40(04): 27-31.) [11] 吴慧荣. 2020. 亮氨酸调控猪肠道紧密连接的分子机制[D].硕士学位论文, 湖南师范大学, 导师: 何善平, pp. 2-3. (Wu H R.2020. Molecular mechanism of tight junction regulated by leucine in porcine intestinal[D]. Thesis for M.S., Hunan Normal University, Supervisor: He S P, pp. 2-3.) [12] 张力, 徐勃兴, 李杰, 等. 2016. MAPK抑制剂对骨碎补总黄酮促进转基因成肌细胞成骨分化的影响[J]. 中国组织工程研究, 20(51): 6.(Zhang L, Xu B X, Li J, et al. 2016. Influence of mitogen-activated protein kinase inhibitor in the process of assemble flavone of rhizome drynaria promoting the osteogentic differentiation of myoblasts[J]. Journal of Clinical Rehabilitative Tissue Engineering Research, 20(51): 6.) [13] 朱玉华. 2017. 谷氨酰胺通过抑制microRNA-29a和细胞自噬调控宫内生长受限仔猪肠道发育的研究[D]. 博士学位论文, 中国农业大学, 导师: 王军军, pp. 48-49. (Zhu Y H.2017. Regulation of glutamine on the small intestinal development in IUGR pigs via suppressing intestinal microRNA-29a and autophagy activity[D]. Thesis for Ph.D., China Agricultural University, Supervisor: Wang J J, pp. 48-49.) [14] Bartel D. P.2004. microRNAs: Genomics, biogenesis, mechanism, and function[J]. Cell, 116(2), 281-297. [15] Buckley A, Turner J R.2017. Cell biology of tight junction barrier regulation and mucosal disease[J]. Cold Spring Harbor Perspectives in Biology, 10(1): a029314. [16] Chassin C, Kocur M, Pott J, et al.2010. miR-146a mediates protective innate immune tolerance in the neonate intestine[J]. Cell Host and Microbe, 8(4): 358-368. [17] Dang Y M, Huang G, Chen Y R, et al.2014. Sulforaphane inhibits the proliferation of the BIU87 bladder cancer cell line via IGFBP-3 elevation[J]. Asian Pacific Journal of Cancer Prevention, 15(4): 1517-1520. [18] Doench J G.2004. Specificity of microRNA target selection in translational repression[J]. Genes & Development, 18(5): 504-511. [19] Goto Y, Kiyono H.2011. Epithelial cell microRNAs in gut immunity[J]. Nature Immunology, 12(3): 195-197. [20] Kuhn K A, Pedraza I, Demoruelle M K.2014. Mucosal immune responses to microbiota in the development of autoimmune disease[J]. Rheumatic Diseases Clinics of North America, 40(4): 711-725 [21] Kuo K K, Chen Y L, Chen L R, et al.2011. Involvement ofphorbol-12-myristate-13-acetate-induced protein 1 in goniothalamin-induced TP53-dependent and-independent apoptosis in hepatocellular carcinoma-derived cells[J]. Toxicology & Applied Pharmacology, 256(1): 8-23. [22] Liu Y W.2004. MicroRNAs: Genomics, biogenesis, mechanism, and function[J]. Cell, 116(2): 281-297. [23] Macdonald T T, Monteleone G.2005. Immunity, inflammation, and allergy in the gut[J]. Science, 307(5717): 1920-1925. [24] Morimoto Y, Mizushima T, Wu X, et al., et al.2020. miR-4711-5p regulates cancer stemness and cell cycle progression via KLF5, MDM2 and TFDP1 in colon cancer cells[J]. British Journal of Cancer, 122(7): 1037-1049. [25] Nguyen H, Dalmasso G, Yan Y T, et al.2010. Microrna-7 modulates CD98 expression during intestinal epithelial cell differentiation[J]. Journal of Biological Chemistry, 285(2): 1479-1489 [26] Peng J M, Tseng R H, Shih T C, et al.2021. CAMK2N1 suppresses hepatoma growth through inhibiting E2F1-mediated cell-cycle signaling[J]. Cancer Letters, 497: 66-76. [27] Sansom S E, Nuovo G J, Martin M M, et al.2010. miR-802 regulates human angiotensin Ⅱ type 1 receptor expression in intestinal epithelial C2BBe1 cells[J]. AJP Gastrointestinal and Liver Physiology, 299(3): G632-42. [28] Stelekati E, Cai Z, Manne S, et al.2022. MicroRNA-29a attenuates CD8 T cell exhaustion and induces memory-like CD8 T cells during chronic infection[J].Proceeding of the National Academy of Sciences of the USA, 119(17): e2106083119. [29] Wu G, Bazer F W, Wallace J M, et al.2006. Board-invited review: Intrauterine growth retardation: Implications for the animal sciences[J]. Journal of Animal Science, 84(9): 2316-2337. [30] Yang Z H, Bo Liao, Xiang X Y, et al.2020. miR‐21‐5p promotes cell proliferation and G1/S transition in melanoma by targeting CDKN2C[J]. FEBS Open Bio, 10(5): pp. 752-760. [31] Zhou Q Q, Costinean S, Croce C M, et al.2015. MicroRNA 29 targets nuclear factor-κB-repressing factor and claudin 1 to increase intestinal permeability[J]. Gastroenterology, 148(1): 158-169. e8. [32] Zhu H L, Wang H B, Wang S H, et al.2018. Flaxseed oil attenuates intestinal damage and inflammation by regulating necroptosis and TLR4/NOD signaling pathways following lipopolysaccharide challenge in a piglet model[J]. Molecular Nutrition & Food Research, 62(9): e1700814.
