The mechanisms of circFAT1 on the biological process of GBM cells

doi:10.11958/20230224

Abstract

Abstract:

Objective To investigate the regulation and mechanism of circFAT1 on proliferation, invasion and epithelial-mesenchymal transition (EMT) of glioblastoma (GBM). Methods Fifty GBM samples and 5 non-tumor brain tissue (NBT) samples were collected, and 3 GBM and 3 NBT samples were randomly selected for tissue RNA sequencing. The expression levels of circFAT1 in U87, U251, T98G, LN229 GBM cell lines and NHA astrocyte lines were detected by qRT-PCR. The cyclic properties of circFAT1 were determined by selective degradation of linear transcripts by RNase R. GBM cells were divided into the sh-control group and the sh-circFAT1 group. Cell proliferation, invasion and EMT were detected by colony formation assay, transwell assay and Western blot assay after lentivirus transfection. Fluorescence in situ hybridization (FISH) was used to detect the localization of circFAT1 in GBM cells, and the interaction between circFAT1 and miR-1182 was verified by dual-luciferase reporter and RNA immunocoprecipitation (RIP) assay. Finally, Western blot assay was performed to verify the regulatory effect of circFAT1 knockdown or inhibition of miR-1182 expression on TGF-β/Smad signaling pathway. Results The expression of circFAT1 was increased in GBM tissue and cells (P<0.05). RNase R reduced linear FAT1 expression without affecting circFAT1 (P<0.05). Knockout of circFAT1 inhibited the proliferation, invasion and EMT of GBM cells (P<0.05). Dual-luciferase reporter and RIP assay confirmed that circFAT1 targeting on miR-1182. The protein levels of TGFB2, p-SMAD2 and p-SMAD3 were decreased after circFAT1 knockdown, while protein expressions of TGFB2, p-SMAD2 and p-SMAD3 increased after inhibiting the expression of miR-1182 (all P<0.05). Conclusion Knockout of circFAT1 can inhibit proliferation, invasion and EMT of GBM cells, and its mechanism may be related to the regulation of TGF-β/Smad signaling pathway by sponge miR-1182.

Key words: glioblastoma, cell proliferation, transforming growth factor beta, microRNAs, circFAT1, epithelial-mesenchymal transition

CLC Number:

R739.41

Figures/Tables 8

References 25

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基因名称	引物序列（5′→3′）	产物大小（bp）
circFAT1	上游：GATGAGGACGCCAGAAGAGA 下游：CAAATGTCTCCCCATTGCTT	121
linerFAT1	上游：GGGCCAAAGACAAGGGAAAG 下游：TGAAAACACCAACGCCAGAG	243
β-actin	上游：CCTCTATGCCAACACAGTGC 下游：CCTGCTTGCTGATCCACATC	243
β-actin	上游：CCTCTATGCCAACACAGTGC 下游：CCTGCTTGCTGATCCACATC	206

基因名称	引物序列（5′→3′）	产物大小（bp）
circFAT1	上游：GATGAGGACGCCAGAAGAGA 下游：CAAATGTCTCCCCATTGCTT	121
linerFAT1	上游：GGGCCAAAGACAAGGGAAAG 下游：TGAAAACACCAACGCCAGAG	243
β-actin	上游：CCTCTATGCCAACACAGTGC 下游：CCTGCTTGCTGATCCACATC	243
β-actin	上游：CCTCTATGCCAACACAGTGC 下游：CCTGCTTGCTGATCCACATC	206

组别		细胞集落数/（个/视野）		侵袭率/%
sh-对照组		499±46		1.01±0.02
sh-circFAT1组		182±65		0.33±0.06
t		6.894^＊＊		19.513^＊＊
组别	E-cadherin		N-cadherin		Vimentin
sh-对照组	0.59±0.11		1.47±0.13		1.53±0.09
sh-circFAT1组	1.04±0.03		0.35±0.07		0.27±0.08
t	9.233^＊＊		6.884^＊＊		10.582^＊＊

组别		细胞集落数/（个/视野）		侵袭率/%
sh-对照组		499±46		1.01±0.02
sh-circFAT1组		182±65		0.33±0.06
t		6.894^＊＊		19.513^＊＊
组别	E-cadherin		N-cadherin		Vimentin
sh-对照组	0.59±0.11		1.47±0.13		1.53±0.09
sh-circFAT1组	1.04±0.03		0.35±0.07		0.27±0.08
t	9.233^＊＊		6.884^＊＊		10.582^＊＊

组别	TGFB2	p-SMAD2	p-SMAD3
A组	0.98±0.04	0.69±0.05	0.92±0.04
B组	0.75±0.05^a	0.36±0.01^a	0.56±0.05^a
C组	1.24±0.05^a	1.16±0.03^a	1.13±0.09^a
D组	0.92±0.02^bc	0.81±0.05^bc	0.94±0.04^bc
F	76.842^＊＊	147.204^＊＊	43.651^＊＊