NR2F2基因调控猪PK15细胞增殖和凋亡的研究

doi:10.11843/j.issn.0366-6964.2023.08.011

Abstract

Abstract: This study aimed to explore the effect of NR2F2 gene on the proliferation and apoptosis of porcine PK15 cells. In this study, the effect of overexpression of NR2F2 gene on cell proliferation was detected by qRT-PCR, Western blot, CCK-8, EdU, flow cytometry, scratch assay and other methods in porcine PK15 cells. CO-IP technology was used to verify the binding ability of NR2F2 to Smad4 protein. The effect of NR2F2 gene on Smad4 gene expression and the expression of downstream gene Cyclin B, CDK1, CDK4, Cyclin D1 were detected by qRT-PCR. The expression of downstream genes and proliferating genes Ki67, PCNA were detected by rescue experiment overexpressed NR2F2 gene and interfered with Smad4 gene. The results showed that overexpression of NR2F2 gene significantly upregulated the expression of Ki67(P<0.01), and significantly upregulated the expression of PCNA(P<0.05), promoted cell cycle progression, inhibited cell apoptosis, and promoted cell proliferation. The CO-IP results proved that NR2F2 and Smad4 proteins were physically bound. After overexpression of NR2F2 gene, the expression of Smad4 gene increased significantly (P<0.01), and the expression of downstream regulated genes by Smad4 gene Cyclin B, CDK1, CDK4 and Cyclin D1 significantly increased (P<0.01). After interfering with NR2F2 gene, the expression of Smad4 gene significantly decreased (P<0.01), and the expression of downstream genes regulated by Smad4 gene was significantly decreased (P<0.01). The rescue experiments showed that after overexpression of NR2F2 gene, interfering with the expression of Smad4 gene could significantly reduce the expression of Cyclin D1, Cyclin B, CDK1, Ki67 and PCNA (P<0.01,P<0.05). In summary, overexpression of NR2F2 gene promoted cell proliferation and inhibited apoptosis, and NR2F2 protein could bind to Smad4 protein and affect the expression of Smad4, which in turn affected the expression of downstream genes.

Key words: NR2F2 gene, Smad4 gene, cell proliferation, PK15 cell

CLC Number:

S828.2

ZHANG Wanfeng, ZHAO Tianzhi, LI Jiao, YOU Ziwei, YANG Yang, CAI Chunbo, GAO Pengfei, CAO Guoqing, GUO Xiaohong, LI Bugao. Study on NR2F2 Gene Regulating Proliferation and Apoptosis of Porcine PK15 Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3242-3251.

References 41

[1]	TAKEUCHI T, NAKAMURA H.Cell proliferation and development[J].Dev Growth Differ, 2014, 56(5):323.
[2]	JIANG M Z, XU S, BAI M, et al.The emerging role of MEIS1 in cell proliferation and differentiation[J].Am J Physiol Cell Physiol, 2021, 320(3):C264-C269.
[3]	SUN Y, LIU Y, MA X L, et al.The influence of cell cycle regulation on chemotherapy[J].Int J Mol Sci, 2021, 22(13):6923.
[4]	GURSKA L M, AMES K, GRITSMAN K.Signaling pathways in leukemic stem cells[J].Adv Exp Med Biol, 2019, 1143:1-39.
[5]	NUSSE R, CLEVERS H. Wnt/β-Catenin Signaling, Disease, and Emerging Therapeutic Modalities[J].Cell, 2017, 169(6):985-999.
[6]	ZHANG Y, WANG X.Targeting the Wnt/β-catenin signaling pathway in cancer[J].J Hematol Oncol, 2020, 13(1):165.
[7]	LIU J Q, XIAO Q, XIAO J N, et al.Wnt/β-catenin signalling:function, biological mechanisms, and therapeutic opportunities[J]. Signal Transduct Target Ther, 2022, 7(1):3.
[8]	SPRINZAK D, BLACKLOW S C.Biophysics of Notch signaling[J].Annu Rev Biophys, 2021, 50:157-189.
[9]	WANG H F, ZANG C Z, LIU X S, et al.The role of Notch receptors in transcriptional regulation[J].J Cell Physiol, 2015, 230(5):982-988.
[10]	TZAVLAKI K, MOUSTAKAS A.TGF-β signaling[J].Biomolecules, 2020, 10(3):487.
[11]	PENG D D, FU M Y, WANG M N, et al.Targeting TGF-β signal transduction for fibrosis and cancer therapy[J].Mol Cancer, 2022, 21(1):104.
[12]	COONEY A J, TSAI S Y, O'MALLEY B W, et al.Chicken ovalbumin upstream promoter transcription factor (COUP-TF) dimers bind to different GGTCA response elements, allowing COUP-TF to repress hormonal induction of the vitamin D3, thyroid hormone, and retinoic acid receptors[J].Mol Cell Biol, 1992, 12(9):4153-4163.
[13]	KLEPSCH V, HERMANN-KLEITER N, BAIER G.Beyond CTLA-4 and PD-1:orphan nuclear receptor NR2F6 as T cell signaling switch and emerging target in cancer immunotherapy[J].Immunol Lett, 2016, 178:31-36.
[14]	POLVANI S, PEPE S, MILANI S, et al.COUP-TFII in health and disease[J].Cells, 2020, 9(1):101.
[15]	MAURI F, SCHEPKENS C, LAPOUGE G, et al.NR2F2 controls malignant squamous cell carcinoma state by promoting stemness and invasion and repressing differentiation[J].Nat Cancer, 2021, 2(11):1152-1169.
[16]	XIA B L, HOU L J, KANG H, et al.NR2F2 plays a major role in insulin-induced epithelial-mesenchymal transition in breast cancer cells[J].BMC Cancer, 2020, 20(1):626.
[17]	LI L P, XIE X, QIN J, et al.The nuclear orphan receptor COUP-TFII plays an essential role in adipogenesis, glucose homeostasis, and energy metabolism[J].Cell Metab, 2009, 9(1):77-87.
[18]	XU Z, YU S T, HSU C H, et al.The orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II is a critical regulator of adipogenesis[J].Proc Natl Acad Sci U S A, 2008, 105(7):2421-2426.
[19]	CHEN G X.Roles of vitamin A metabolism in the development of hepatic insulin resistance[J].ISRN Hepatol, 2013, 2013:534972.
[20]	OKAMURA M, KUDO H, WAKABAYASHI K I, et al.COUP-TFII acts downstream of Wnt/β-catenin signal to silence PPARγ gene expression and repress adipogenesis[J].Proc Natl Acad Sci U S A, 2009, 106(14):5819-5824.
[21]	LENG X, COONEY A J, TSAI S Y, et al.Molecular mechanisms of COUP-TF-mediated transcriptional repression:evidence for transrepression and active repression[J].Mol Cell Biol, 1996, 16(5):2332-2340.
[22]	MA L, HUANG M, LIAO X H, et al.NR2F2 regulates cell proliferation and immunomodulation in Whartons' jelly stem cells[J].Genes (Basel), 2022, 13(8):1458.
[23]	QIN J, WU S P, CREIGHTON C J, et al.COUP-TFII inhibits TGF-β-induced growth barrier to promote prostate tumorigenesis[J]. Nature, 2013, 493(7431):236-240.
[24]	WANG C Q, ZHOU Y, RUAN R Y, et al.High expression of COUP-TF II cooperated with negative Smad4 expression predicts poor prognosis in patients with colorectal cancer[J].Int J Clin Exp Pathol, 2015, 8(6):7112-7121.
[25]	WANG H, NIE L, WU L, et al.NR2F2 inhibits Smad7 expression and promotes TGF-β-dependent epithelial-mesenchymal transition of CRC via transactivation of miR-21[J].Biochen Biophys Res Commun, 2017, 485(1):181-188.
[26]	XU Q J, JIANG S J.miR-194-5p serves a suppressive role in human keloid fibroblasts via targeting NR2F2[J].Mol Med Rep, 2021, 23(1):57.
[27]	SAJINOVIC T, BAIER G.New insights into the diverse functions of the NR2F nuclear orphan receptor family[J].Front Biosci (Landmark Ed), 2023, 28(1):13.
[28]	QIN J, TSAI S Y, TSAI M J.The critical roles of COUP-TFII in tumor progression and metastasis[J].Cell Biosci, 2014, 4(1):58.
[29]	ZHENG J, QIN W J, JIAO D, et al.Knockdown of COUP-TFII inhibits cell proliferation and induces apoptosis through upregulating BRCA1 in renal cell carcinoma cells[J].Int J Cancer, 2016, 139(7):1574-1585.
[30]	KIM B J, TAKAMOTO N, YAN J, et al.Chicken Ovalbumin Upstream Promoter-Transcription Factor Ⅱ (COUP-TFII) regulates growth and patterning of the postnatal mouse cerebellum[J].Dev Biol, 2009, 326(2):378-391.
[31]	BOUDOT A, LE DILY F, PAKDEL F.Involvement of COUP-TFs in cancer progression[J].Cancers (Basel), 2011, 3(1):700-715.
[32]	YU C T, TANG K, SUH J M, et al.COUP-TFII is essential for metanephric mesenchyme formation and kidney precursor cell survival[J].Development, 2012, 139(13):2330-2339.
[33]	POLVANI S, TAROCCHI M, TEMPESTI S, et al.Nuclear receptors and pathogenesis of pancreatic cancer[J].World J Gastroenterol, 2014, 20(34):12062-12081.
[34]	SYED V.TGF-β signaling in cancer[J].J Cell Biochem, 2016, 117(6):1279-1287.
[35]	HERNANDA P Y, CHEN K, DAS A M, et al.SMAD4 exerts a tumor-promoting role in hepatocellular carcinoma[J].Oncogene, 2015, 34(39):5055-5068.
[36]	SHENG S H, XIE L, WU Y Y, et al.MiR-144 inhibits growth and metastasis in colon cancer by down-regulating SMAD4[J].Biosci Rep, 2019, 39(3):BSR20181895.
[37]	HAEGER S M, THOMPSON J J, KALRA S, et al.Smad4 loss promotes lung cancer formation but increases sensitivity to DNA topoisomerase inhibitors[J].Oncogene, 2016, 35(5):577-586.
[38]	ZHAO H D, WEI J, SUN J.Roles of TGF-β signaling pathway in tumor microenvirionment and cancer therapy[J].Int Immunopharmacol, 2020, 89:107101.
[39]	JAHN S C, LAW M E, CORSINO P E, et al.TGF-beta antiproliferative effects in tumor suppression[J].Front Biosci (Schol Ed), 2012, 4(2):749-766.
[40]	LIU J Q, YUAN B, CAO J, et al.AMBRA1 promotes TGFβ signaling via nonproteolytic polyubiquitylation of Smad4[J].Cancer Res, 2021, 81(19):5007-5020.
[41]	LIU L, LI Q Q, YANG L, et al.Smad4 feedback activates the canonical TGF-β family signaling pathways[J].Int J Mol Sci, 2021, 22(18):10024.

Study on NR2F2 Gene Regulating Proliferation and Apoptosis of Porcine PK15 Cells

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