Effects of Sp5 silencing on Wnt signaling pathway related factors and proliferative ability in mEPMCs

doi:10.11958/20230521

Abstract

Abstract:

Objective To investigate the effect of transcription factor specific protein5 (Sp5) silencing on Wnt signaling pathway correlated factors and cell proliferation ability in mouse embryo palatal mesenchymal cells (mEPMCs). Methods mEPMCs of 14.5 d pregnant C57BL/6J mice were isolated and cultured in vitro. Cell source was identified by immunofluorescence staining. Lentivirus transfection technique was used to silence the expression of Sp5 gene in mEPMCs, and the transfection efficiency was verified by Western blot assay. Follow-up experiments were set up with the blank control group, the no-load virus group and the slience group (the Sp5-shRNA group). The protein and mRNA expression levels of β-catenin, GSK-3β, Wnt3a and CyclinD1 were detected by Western blot assay and RT-qPCR after transfection for 72 h in each group. Cell proliferation capacity was detected by CCK-8. The proliferation rate of 5-Ethynyl-2′-deoxyuridine (EdU) positive cells was detected by immunofluorescence assay. Cell cycle was detected by flow cytometry. Results mEPMCs were successfully isolated, and Sp5 expression was silenced. Western blot and RT-qPCR results showed that the protein and mRNA expressions of β-catenin, GSK-3β, Wnt3a and CyclinD1 were significantly higher in the Sp5-shRNA group than those in the blank control group and the no-loaded virus group (P < 0.05). The proliferative ability and the proliferative rate of EdU positive cells were higher in the Sp5-shRNA group than those in the blank control group and the no-loaded virus group (P < 0.05). The proportion of mEPMCs in S phase was higher in the Sp5-shRNA group than that in the blank control group and the no-loaded virus group (P < 0.05). Conclusion Sp5 in silenced mEPMCs can participate in palate development and promote the proliferation of mEPMCs by regulating Wnt signaling pathway.

Key words: Sp transcription factors, Wnt signaling pathway, cell proliferation, embryopalatal mesenchymal cells, Sp5 gene

CLC Number:

R782.22

Figures/Tables 13

References 24

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序列名称	碱基序列	病毒滴度/ （TU/mL）
shRNA4	CCCGTCGGACTTTGCACAGTA	1.5×10⁸
shRNA5	AGTGCGGCAAACGCTTCATGC	1×10⁸
shRNA6	TGGGTTCACCCTCCAGACTTT	1×10⁸
空载病毒对照	TTCTCCGAACGTGTCACGTAA	2×10⁸

序列名称	碱基序列	病毒滴度/ （TU/mL）
shRNA4	CCCGTCGGACTTTGCACAGTA	1.5×10⁸
shRNA5	AGTGCGGCAAACGCTTCATGC	1×10⁸
shRNA6	TGGGTTCACCCTCCAGACTTT	1×10⁸
空载病毒对照	TTCTCCGAACGTGTCACGTAA	2×10⁸

组别	MOI值=10 （病毒原液＋完全培养基）	MOI值=30 （病毒原液＋完全培养基）	MOI值=50 （病毒原液＋完全培养基）
空载病毒组	0.40＋49.55	1.20＋48.75	2.00＋47.95
shRNA4组	0.33＋49.62	1.60＋48.35	2.60＋47.35
shRNA5组	0.80＋49.15	2.40＋47.55	4.00＋45.95
shRNA6组	0.80＋49.15	2.40＋47.55	4.00＋45.95

组别	MOI值=10 （病毒原液＋完全培养基）	MOI值=30 （病毒原液＋完全培养基）	MOI值=50 （病毒原液＋完全培养基）
空载病毒组	0.40＋49.55	1.20＋48.75	2.00＋47.95
shRNA4组	0.33＋49.62	1.60＋48.35	2.60＋47.35
shRNA5组	0.80＋49.15	2.40＋47.55	4.00＋45.95
shRNA6组	0.80＋49.15	2.40＋47.55	4.00＋45.95

基因名称	引物序列（5′→3′）	产物大小/bp
β-catenin	上游：AGCTGGCCTGGTTTGATACT	125
β-catenin	下游：CCATTCCCACCCTACCAAGT	125
GSK-3β	上游：ACCAGGAGCAGGACATTTCAC	197
GSK-3β	下游：CAGGTGTGTCTCGCCCATT	197
Wnt3a	上游：CCTGGTCTACTACGAGGCCT	201
Wnt3a	下游：ACGTAGCAGCACCAATGGAA	201
CyclinD1	上游：TGCGTGCAGAAGGAGATTGT	241
	下游：AGATGCACAACTTCTCGGCA	241
GAPDH	上游：TCACCATCTTCCAGGAGCGAGAC	303
	下游：TGAGCCCTTCCACAATGCCAAAG	303