Construction of human SMARCAL11 gene over-expressed lentiviral vector and its effect on proliferation of embryonic kidney cells

doi:10.12307/2021.300

Abstract

Abstract: BACKGROUND: Studies have found that SMARCAL1 is selectively expressed in a variety of cells in human developing and mature kidneys, indicating that it plays an important role in kidney development.
OBJECTIVE: To investigate the effect of SMARCAL1 overexpression on the proliferation of embryonic kidney cells.
METHODS: 293T and HEK293 cells at passage 3 were selected. pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO vector was used to construct lentiviral vector carrying human SMARCAL1 gene. The human SMARCAL1 gene was over-expressed using lentiviral vector and transfected into 293T cells. There were three groups: blank group untreated HEK293 cells; negative control group, in which HEK293 cells were transfected with empty virus; SMARCAL1 overexpression group, in which HEK293 cells were transfected with SMARCAL1 overexpressing virus. Stably transfected cell lines were screened using puromycin. Virus titer was determined based on the transfection rate. The stable transfection of SMARCAL1 into embryonic kidney cells was verified by quantitative real-time PCR and western blot. The effect of SMARCAL1 overexpression on the proliferation of HEK293 cells was assessed by cell counting kit-8 and EdU assays. Fluorogenic quantitative PCR was further used to evaluate the effect of SMARCAL1 overexpression on Wnt pathway.
RESULTS AND CONCLUSION: The overexpressed SMARCAL1 plasmid gene sequence was consistent with the target gene. The titer of the SMARCAL1 overexpressed lentivirus was 1×108 TU/mL and 3×108 TU/mL for the control lentivirus. The mRNA and protein levels of SMARCAL1 in HEK293 cells were significantly increased after transfection with SMARCAL1 overexpressed lentivirus (P < 0.05). Up-regulation of SMARCAL1 expression gradually inhibited the proliferation of HEK293 cells (P < 0.05). The mRNA expression levels of CTNNB1 and WNT4 genes in the Wnt pathway were also decreased (P < 0.05). The SMARCAL1 overexpressed lentiviral vector is successfully constructed and expressed in HEK293 cells. The SMARCAL1 gene may be involved in regulating the proliferation of embryonic kidney cells through the Wnt signaling pathway and may affect the development of kidney.

Key words: lentivirus vector, SMARCAL1, HEK293 cells, Wnt signaling pathway, cell proliferation

CLC Number:

Shi Shujuan, Li Cheng, Qiao Lingyan, Yang Binyi, Li Tang. Construction of human SMARCAL11 gene over-expressed lentiviral vector and its effect on proliferation of embryonic kidney cells[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(35): 5682-5687.

Figures/Tables 8

References

[1] SARIN S, JAVIDAN A, BOIVIN F, et al. Insights into the renal pathogenesis in Schimke immuno-osseous dysplasia: A renal histological characterization and expression analysis. J Histochem Cytochem. 2015;63(1):32-44.
[2] SHAN J, JOKELA T, SKOVORODKIN I, et al. Mapping of the fate of cell lineages generated from cells that express the Wnt4 gene by time-lapse during kidney development. Differentiation. 2010;79(1):57-64.
[3] IGLESIAS DM, HUEBER PA, CHU L, et al. Canonical WNT signaling during kidney development. Am J Physiol Renal Physiol. 2007;293(2):F494-500.
[4] KATO H, SUSZTAK K. Repair problems in podocytes: Wnt, Notch, and glomerulosclerosis. Semin Nephrol. 2012,32(4):350-356.
[5] DAI C, STOLZ DB, KISS LP, et al. Wnt/beta-catenin signaling promotes podocyte dysfunction and albuminuria. J Am Soc Nephrol. 2009;20(9):1997-2008.
[6] HE W, TAN RJ, LI Y, et al. Matrix metalloproteinase-7 as a surrogate marker predicts renal Wnt/β-catenin activity in CKD. J Am Soc Nephrol. 2012;23(2):294-304.
[7] MAEZAWA Y, ONAY T, SCOTT RP, et al. Loss of the podocyte-expressed transcription factor Tcf21/Pod1 results in podocyte differentiation defects and FSGS. J Am Soc Nephrol. 2014;25(11):2459-2470.
[8] Yang X, Wang X, Nie F, et al. miR-135 family members mediate podocyte injury through the activation of Wnt/β-catenin signaling. Int J Mol Med. 2015;36(3):669-677.
[9] NAVES MA, REQUIÃO-MOURA LR, SOARES MF, et al. Podocyte Wnt/ß-catenin pathway is activated by integrin-linked kinase in clinical and experimental focal segmental glomerulosclerosis. J Nephrol. 2012;25(3):401-409.
[10] WAREJKO JK, TAN W, DAGA A, et al. Whole Exome Sequencing of Patients with Steroid-Resistant Nephrotic Syndrome. Clin J Am Soc Nephrol. 2018;13(1):53-62.
[11] MORIMOTO M, MYUNG C, BEIRNES K, et al. Increased Wnt and Notch signaling: a clue to the renal disease in Schimke immuno-osseous dysplasia? Orphanet J Rare Dis. 2016;11(1):149.
[12] 邱晓,韦荣飞,张令强,等.肾脏发育中信号通路的调控作用[J].遗传,2015, 37(1):1-7.
[13] DEKEL B, METSUYANIM S, GOLDSTEIN N, et al. Schimke immuno-osseous dysplasia: expression of SMARCAL1 in blood and kidney provides novel insight into disease phenotype. Pediatr Res. 2008;63(4):398-403.
[14] 文雅蕾,吕柯孬,徐小康, 等.退火解旋酶SMARCAL1在维持基因组稳定中的作用与机制[J].遗传,2019,41(12):1084-1098.
[15] PUGLIESE GM, SALARIS F, PALERMO V, et al. Inducible SMARCAL1 knockdown in iPSC reveals a link between replication stress and altered expression of master differentiation genes. Dis Model Mech. 2019;12(10):dmm039487.
[16] BANSAL R, HUSSAIN S, CHANANA UB, et al. SMARCAL1, the annealing helicase and the transcriptional co-regulator. IUBMB Life. 2020;72(10):2080-2096.
[17] HAOKIP DT, GOEL I, ARYA V, et al. Transcriptional Regulation of Atp-Dependent Chromatin Remodeling Factors: Smarcal1 and Brg1 Mutually Co-Regulate Each Other. Sci Rep. 2016;6:20532.
[18] SARIN S, JAVIDAN A, BOIVIN F, et al. Insights into the renal pathogenesis in Schimke immuno-osseous dysplasia: A renal histological characterization and expression analysis. J Histochem Cytochem. 2015;63(1):32-44.
[19] SHARMA T, BANSAL R, HAOKIP DT, et al. SMARCAL1 Negatively Regulates C-Myc Transcription By Altering The Conformation Of The Promoter Region. Sci Rep. 2015;5:17910.
[20] BANDINO A, COMPAGNONE A, BRAVOCO V, et al. Beta-catenin triggers nuclear factor kappaB-dependent up-regulation of hepatocyte inducible nitric oxide synthase. Int J Biochem Cell Biol. 2008;40(9):1861-1871.
[21] PEIFER M, POLAKIS P. Wnt signaling in oncogenesis and embryogenesis--a look outside the nucleus. Science. 2000;287(5458):1606-1609.
[22] KÜHL M, SHELDAHL LC, PARK M, et al. The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape. Trends Genet. 2000;16(7):279-283.
[23] KÜHL M, SHELDAHL LC, MALBON CC, et al. Ca(2+)/calmodulin-dependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xenopus. J Biol Chem. 2000;275(17):12701-12711.
[24] ADLI M, PARLAK M, LI Y, et al. Epigenetic States of nephron progenitors and epithelial differentiation. J Cell Biochem. 2015;116(6):893-902.
[25] PARK JS, VALERIUS MT, MCMAHON AP. Wnt/beta-catenin signaling regulates nephron induction during mouse kidney development. Development. 2007;134(13):2533-2539.
[26] KARNER CM, CHIRUMAMILLA R, AOKI S, et al. Wnt9b signaling regulates planar cell polarity and kidney tubule morphogenesis. Nat Genet. 2009;41(7):793-799.
[27] HALT K, VAINIO S. Coordination of kidney organogenesis by Wnt signaling. Pediatr Nephrol. 2014;29(4):737-744.
[28] MENG P, ZHU M, LING X, et al. Wnt signaling in kidney: the initiator or terminator? J Mol Med (Berl). 2020;98(11):1511-1523.
[29] SCHMIDT-OTT KM, BARASCH J. WNT/beta-catenin signaling in nephron progenitors and their epithelial progeny. Kidney Int. 2008;74(8):1004-1008.
[30] MORIMOTO M, MYUNG C, BEIRNES K, et al. Increased Wnt and Notch signaling: a clue to the renal disease in Schimke immuno-osseous dysplasia? Orphanet J Rare Dis. 2016;11(1):149.