猪miR-331-3p过表达载体的构建及其对细胞增殖的影响
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摘要
本研究旨在阐明猪miR-331-3p对细胞增殖的影响,探讨其对细胞增殖的作用机制首先构建了miR-331-3p的过表达载体pcDNA 3.1 (+)-miR-331-3p,并将将PK15细胞分为4组,分别为实验组、实验组对照组、抑制剂组和抑制剂对照组。实验组和对照组分别转染pcDNA 3.1(+)-miR-331-3p和pcDNA 3.1(+)。抑制剂组和抑制剂对照组分别转染miR-331-3p Inhibitor和miR-331-3p阴性对照(miR-331-3p NC)。通过在各组添加CCK-8试剂绘制细胞增殖曲线,并使用PI染色检测细胞所处周期比例。同时,利用实时荧光定量PCR(Quantitative real-time PCR,qPCR)检测生长抑制蛋白家族成员5 (Inhibitor of growth family member 5,ING5)、细胞周期蛋白依赖性激酶2 (Cyclin dependent kinase 2,CDK2)、细胞周期蛋白依赖性激酶3 (Cyclin dependent kinase 3,CDK3)、细胞周期蛋白依赖性激酶4 (Cyclin dependent kinase 4,CDK4)、细胞周期蛋白B (Cyclin B)和细胞周期蛋白依赖性激酶抑制剂1A(Cyclindependentkinaseinhibitor1A,CDKN1A)的表达变化。结果表明,实验组miR-331-3p表达量显著升高,细胞增殖曲线表明48 h和72 h时细胞数目均呈现出实验组>实验对照组和抑制剂对照组>抑制剂组的趋势(P<0.05)。与实验对照组相比,实验组处于G0/G1期的细胞比例下调,S期和G2/M细胞的比例上调,抑制剂对照组趋势与之相反;同时,实验组中与促进增殖的基因CDK2、CDK3、CDK4和CyclinB的mRNA表达水平均显著升高,而抑制增殖的基因ING5和CDKN1A均表现出显著下降的趋势。本研究成功构建了miR-331-3p过表达载体,且发现miR-331-3p具有促进猪肾上皮细胞增殖的能力,研究结果为深入研究miR-331-3p在猪生长发育中的作用机制奠定了基础。
To investigate the effect of miR-331-3 p on the proliferation of porcine renal epithelial cells(PK15) and its mechanism, the pcDNA 3.1(+) overexpression vector of miRNA-331-3 p(pcDNA 3.1(+)-miR-331-3 p) was constructed. PK15 cells were divided into four groups, including experimental group, experimental control group, inhibitor group and inhibitor control group. Experimental group and experimental control group were transfected with pcDNA 3.1(+)-miR-331-3 p and pcDNA 3.1(+), respectively. Inhibitor group and inhibitor control group were transfected with miR-331-3 p inhibitor and miR-331-3 p negative control(miR-331-3 p NC), respectively. Above all, CCK-8 reagent was used to plot the cell proliferation curve and Propidium(PI) staining was used to detect the proportion of cell stages. Secondly, its expression change were detected by quantitative real-time PCR that included the growth inhibitory protein family member 5(ING5), cyclin-dependent kinase 2(CDK2), cyclin-dependent kinase 3(CDK3), cyclin-dependent kinase 4(CDK4), Cyclin B and cyclin-dependent kinase inhibitor 1 A(CDKN1 A). The results showed that the expression of miRNA-331-3 p was significantly increased in the experimental group. The cell proliferation curve showed that the number of cells in experimental group was significantly higher than that in experimental control group or inhibitor control group at 48 h and 72 h(P<0.05). Simultaneously, Inhibitor group was significantly lower than experimental control group or inhibitor control group in the number of cells at 48 h and 72 h(P<0.05), but there was no significant difference between the experimental group and the control group. Compared with the experimental control group, the proportion of cells of experimental group in G0/G1 phase decreased, the proportion of S phase and G2/M phase increased, and the inhibitor control group showed the opposite trend. Simultaneously, the expression levels of CDK2, CDK3, CDK4 and Cyclin B genes in the experimental group were significantly increased, while ING5 and CDKN1 A genes inhibiting proliferation showed a significant downward trend. These results demonstrate that the miR-331-3 p overexpression vector was successfully constructed, and miR-331-3 p has the ability to promote the proliferation of PK15 cells. The study lays a solid foundation for further research for its role in pig growth and development.
To investigate the effect of miR-331-3 p on the proliferation of porcine renal epithelial cells(PK15) and its mechanism, the pcDNA 3.1(+) overexpression vector of miRNA-331-3 p(pcDNA 3.1(+)-miR-331-3 p) was constructed. PK15 cells were divided into four groups, including experimental group, experimental control group, inhibitor group and inhibitor control group. Experimental group and experimental control group were transfected with pcDNA 3.1(+)-miR-331-3 p and pcDNA 3.1(+), respectively. Inhibitor group and inhibitor control group were transfected with miR-331-3 p inhibitor and miR-331-3 p negative control(miR-331-3 p NC), respectively. Above all, CCK-8 reagent was used to plot the cell proliferation curve and Propidium(PI) staining was used to detect the proportion of cell stages. Secondly, its expression change were detected by quantitative real-time PCR that included the growth inhibitory protein family member 5(ING5), cyclin-dependent kinase 2(CDK2), cyclin-dependent kinase 3(CDK3), cyclin-dependent kinase 4(CDK4), Cyclin B and cyclin-dependent kinase inhibitor 1 A(CDKN1 A). The results showed that the expression of miRNA-331-3 p was significantly increased in the experimental group. The cell proliferation curve showed that the number of cells in experimental group was significantly higher than that in experimental control group or inhibitor control group at 48 h and 72 h(P<0.05). Simultaneously, Inhibitor group was significantly lower than experimental control group or inhibitor control group in the number of cells at 48 h and 72 h(P<0.05), but there was no significant difference between the experimental group and the control group. Compared with the experimental control group, the proportion of cells of experimental group in G0/G1 phase decreased, the proportion of S phase and G2/M phase increased, and the inhibitor control group showed the opposite trend. Simultaneously, the expression levels of CDK2, CDK3, CDK4 and Cyclin B genes in the experimental group were significantly increased, while ING5 and CDKN1 A genes inhibiting proliferation showed a significant downward trend. These results demonstrate that the miR-331-3 p overexpression vector was successfully constructed, and miR-331-3 p has the ability to promote the proliferation of PK15 cells. The study lays a solid foundation for further research for its role in pig growth and development.
引文
[1]Lund E,Güttinger S,Calado A,et al.Nuclear export of MicroRNA precursors.Science,2004,303(5654):95-98.
[2]Zhuang M,Qiu XB,Cheng D,et al.MicroRNA-524promotes cell proliferation by down-regulating PTENexpression in osteosarcoma.Cancer Cell Int,2018,18:114.
[3]Pogue AI,Cui JG,Li YY,et al.Micro RNA-125b(miRNA-125b)function in astrogliosis and glial cell proliferation.Neurosci Lett,2015,476(1):18-22.
[4]Chang RM,Yang H,Fang F,et al.MicroRNA-331-3p promotes proliferation and metastasis of hepatocellular carcinoma by targeting PH domain and leucine-rich repeat protein phosphatase.Hepatology,2015,60(4):1251-1263.
[5]Guo XB,Guo L,Ji J,et al.miR-331-3p directly targets E2F1 and induces growth arrest in human gastric cancer.Biochem Bioph Res Commun,2010,398(1):1-6.
[6]Giles KM,Barker A,Zhang PM,et al.MicroRNAregulation of growth factor receptor signaling in human cancer cells//Wu W,ed.Micro RNA and Cancer.Totowa,NJ:Humana Press,2011.
[7]Cao YY,Chen J,Wang D,et al.Upregulated in Hepatitis B virus-associated hepatocellular carcinoma cells,miR-331-3p promotes proliferation of hepatocellular carcinoma cells by targeting ING5.Oncotarget,2015,6(35):38093-38106.
[8]Ge YJ,Chen J.MicroRNAs in skeletal myogenesis.Cell Cycle,2011,10(3):441-448.
[9]Walia R,Dardari R,Chaiyakul M,et al.Porcine circovirus-2 capsid protein induces cell death in PK15cells.Virology,2014,468-470:126-132.
[10]Li CH,Chen W,Hu JQ,et al.miRNA-29a targets COL3A1 to regulate the level of type III collagen in pig.Gene,2016,592(1):140-147.
[11]Shi T,Yan XR,Qiao LY,et al.MiR-330-5p negatively regulates ovine preadipocyte differentiation by targeting branched-chain aminotransferase 2.Anim Sci J,2018,89(6):858-867.
[12]Mi L,Chen YS,Zheng XL,et al.MicroRNA-139-5p suppresses 3T3-L1 preadipocyte differentiation through notch and IRS1/PI3K/Akt insulin signaling pathways.J Cell Biochem,2015,116(7):1195-1204.
[13]Xu LN,Qi YL,Lv LW,et al.In vitro anti-proliferative effects of Zuojinwan on eight kinds of human cancer cell lines.Cytotechnology,2014,66(1):37-50.
[14]Fujii T,Shimada K,Asano A,et al.MicroRNA-331-3p suppresses cervical cancer cell proliferation and E6/E7 expression by targeting NRP2.Int J Mol Sci,2016,17(8):1351-1364.
[15]Lai YJ,He S,Ma LM,et al.HOTAIR functions as a competing endogenous RNA to regulate PTENexpression by inhibiting miR-19 in cardiac hypertrophy.Mol Cell Biochem,2017,432(1/2):179-187.
[16]Li J,Zhang YY.Research progress of miR-331-3p in malignant tumors.Prac Oncol J,2018,32(2):125-129(in Chinese).李静,张云艳.miR-331-3p在恶性肿瘤中的研究进展.实用肿瘤学杂志,2018,32(2):125-129.
[17]Ye P,Ke XP,Zang XH,et al.Up-regulated MiR-27-3p promotes the G1-S phase transition by targeting inhibitor of growth family member 5 in osteosarcoma.Biomed Pharmacother,2018,101:219-227.
[18]Tsai LH,Lees E,Faha B,et al.The cdk2 kinase is required for the G1-to-S transition in mammalian cells.Oncogene,1993,8(6):1593-1602.
[19]Sherr CJ,Roberts JM.Inhibitors of mammalian G1cyclin-dependent kinases.Genes Dev,1995,9(10):1149-1163.
[20]Ito M.Factors controlling cyclin B expression.Plant Mol Biol,2000,43(5/6):677-690.
[21]Zhang YY,Xu ZN.miR-93 enhances cell proliferation by directly targeting CDKN1A in nasopharyngeal carcinoma.Oncol Lett,2018,15(2):1723-1727.
[2]Zhuang M,Qiu XB,Cheng D,et al.MicroRNA-524promotes cell proliferation by down-regulating PTENexpression in osteosarcoma.Cancer Cell Int,2018,18:114.
[3]Pogue AI,Cui JG,Li YY,et al.Micro RNA-125b(miRNA-125b)function in astrogliosis and glial cell proliferation.Neurosci Lett,2015,476(1):18-22.
[4]Chang RM,Yang H,Fang F,et al.MicroRNA-331-3p promotes proliferation and metastasis of hepatocellular carcinoma by targeting PH domain and leucine-rich repeat protein phosphatase.Hepatology,2015,60(4):1251-1263.
[5]Guo XB,Guo L,Ji J,et al.miR-331-3p directly targets E2F1 and induces growth arrest in human gastric cancer.Biochem Bioph Res Commun,2010,398(1):1-6.
[6]Giles KM,Barker A,Zhang PM,et al.MicroRNAregulation of growth factor receptor signaling in human cancer cells//Wu W,ed.Micro RNA and Cancer.Totowa,NJ:Humana Press,2011.
[7]Cao YY,Chen J,Wang D,et al.Upregulated in Hepatitis B virus-associated hepatocellular carcinoma cells,miR-331-3p promotes proliferation of hepatocellular carcinoma cells by targeting ING5.Oncotarget,2015,6(35):38093-38106.
[8]Ge YJ,Chen J.MicroRNAs in skeletal myogenesis.Cell Cycle,2011,10(3):441-448.
[9]Walia R,Dardari R,Chaiyakul M,et al.Porcine circovirus-2 capsid protein induces cell death in PK15cells.Virology,2014,468-470:126-132.
[10]Li CH,Chen W,Hu JQ,et al.miRNA-29a targets COL3A1 to regulate the level of type III collagen in pig.Gene,2016,592(1):140-147.
[11]Shi T,Yan XR,Qiao LY,et al.MiR-330-5p negatively regulates ovine preadipocyte differentiation by targeting branched-chain aminotransferase 2.Anim Sci J,2018,89(6):858-867.
[12]Mi L,Chen YS,Zheng XL,et al.MicroRNA-139-5p suppresses 3T3-L1 preadipocyte differentiation through notch and IRS1/PI3K/Akt insulin signaling pathways.J Cell Biochem,2015,116(7):1195-1204.
[13]Xu LN,Qi YL,Lv LW,et al.In vitro anti-proliferative effects of Zuojinwan on eight kinds of human cancer cell lines.Cytotechnology,2014,66(1):37-50.
[14]Fujii T,Shimada K,Asano A,et al.MicroRNA-331-3p suppresses cervical cancer cell proliferation and E6/E7 expression by targeting NRP2.Int J Mol Sci,2016,17(8):1351-1364.
[15]Lai YJ,He S,Ma LM,et al.HOTAIR functions as a competing endogenous RNA to regulate PTENexpression by inhibiting miR-19 in cardiac hypertrophy.Mol Cell Biochem,2017,432(1/2):179-187.
[16]Li J,Zhang YY.Research progress of miR-331-3p in malignant tumors.Prac Oncol J,2018,32(2):125-129(in Chinese).李静,张云艳.miR-331-3p在恶性肿瘤中的研究进展.实用肿瘤学杂志,2018,32(2):125-129.
[17]Ye P,Ke XP,Zang XH,et al.Up-regulated MiR-27-3p promotes the G1-S phase transition by targeting inhibitor of growth family member 5 in osteosarcoma.Biomed Pharmacother,2018,101:219-227.
[18]Tsai LH,Lees E,Faha B,et al.The cdk2 kinase is required for the G1-to-S transition in mammalian cells.Oncogene,1993,8(6):1593-1602.
[19]Sherr CJ,Roberts JM.Inhibitors of mammalian G1cyclin-dependent kinases.Genes Dev,1995,9(10):1149-1163.
[20]Ito M.Factors controlling cyclin B expression.Plant Mol Biol,2000,43(5/6):677-690.
[21]Zhang YY,Xu ZN.miR-93 enhances cell proliferation by directly targeting CDKN1A in nasopharyngeal carcinoma.Oncol Lett,2018,15(2):1723-1727.