RESUMO
Objective:To investigate the effect of methyltransferase like 14 (METTL14) on the proliferation and invasion of breast cancer (BC) cells by regulating cyclin L2 (Cyclin L2, CCNL2) through m6A modification.Methods:Cancer tissues and paracancerous tissues of BC patients in Yantaishan hospital were collected from Aug. 2018 to Feb. 2020. The expression levels of m6A, METTL14 and CCNL2 in tissues were detected by high performance liquid chromatography/mass spectrometry (HPLC/MS) and qRT-PCR. Dual-luciferase reporter assay, qRT-PCR, and western blot were used to verify the regulatory relationship between METTL14 and CCNL2. RIP experiments verified the regulatory relationship between YTH domain-containing family protein (YTHDF2) and CCNL2. Cell viability was detected by MTT method, and cell invasion ability was detected by Transwell.Results:Compared with normal cells (0.24±0.02) and tissues (0.18±0.02) , BC cells MCF-10A (0.47±0.03, t=11.05, P<0.001) and HS-578T (0.41±0.03, t=8.17, P=0.001) and BC tissues (0.39±0.02, t=12.86, P<0.001) m6A level increased. Compared with normal tissues (1.00±0.26) (0.84±0.07) , METTL14 mRNA (1.57±0.28, t=13.50, P<0.001) and protein levels (1.66±0.11, t=10.89, P<0.001) in BC tissues were significantly increased high. Compared with the control group (100.00±10.11) (1.00±0.12) , the BC cell invasion ability (54.15±6.21, t=6.69, P=0.003) and activity (0.64±0.06, t=4.65, P=0.010) were weakened. Compared with the control group (100±11.05) (1±0.13) , the BC cell invasion ability (175.31±13.45, t=7.49, P=0.002) and activity (2.16±0.16, t=9.75, P=0.002) in the METTL14 overexpression group were enhanced, and the effects of METTL14 on cell invasion (137.41±12.64, t=3.56, P=0.024) and activity (1.64±0.15, t=5.59, P=0.005) were partially reversed after m6A inhibitor treatment change. Compared with normal tissues, CCNL2 expression was down-regulated in BC tissues, and the interaction between CCNL2 and METTL14 was confirmed. Compared with the control group (1.00±0.1) (0.64±0.05) , knockdown of METTL14 could make CCNL2 mRNA (1.67±0.05) . 0.13, t=7.08, P=0.002) and protein (1.09±0.09, t=7.57, P=0.002) were up-regulated. METTL14 knockout enhanced the stability of CCNL2 mRNA through a YTHDF2-dependent pathway, compared with sh-METTL14 group (50.47±5.16) (0.52±0.05) , BC cell invasion ability of sh-METTL14+sh-CCNL2 group (71.69±6.41, t=4.47, P=0.011) and activity (0.64±0.05, t=2.94, P=0.042) were improved. Conclusion:METTL14 inhibits the expression of CCNL2 through m6A modification to enhance the invasion and activity of BC cells.
RESUMO
Objective:To investigate the effects of circ_000543 derived from hypoxic exosomes on proliferation and invasion of breast cancer (BC) cells.Methods:Bioinformatic website was used to predict the abnormal expression of circ_000543 in BC tissue and the transcription factor that might regulate circ_000543. Double luciferase report experiment and ChIP assay were used to confirm the regulation relationship between YY1 and circ_000543. Exosomes were separated from normal BC cells and BC cells under hypoxic condition, and qRT-PCR was adopted to detect the expression of circ_000543 in exosomes. The expression of circ_000543 and YY1 in exosomes was intervened and the exosomes were cocultured with BC cells under normoxia. CCK8 and Transwell assay was used to detect the proliferation and invasive ability individually.Results:qRT-PCR experiment found that, compared with MCF-10A cells (1±0.11) and exosomes isolated from normoxic cells (1±0.10), circ_000543 expression was up-regulated in BC cells (1.59±0.13) and exosomes derived from cells under hypoxic condition (1.63±0.12) ( t=6.001, P=0.004; t=6.986, P=0.002). Exosomes derived from cells under hypoxic condition promoted proliferation and invasion of BC cells under normoxia. Inhibition of circ_000543 partially offset the effects of exosomes. YY1 induced the expression of circ_000543 in BC cells as a transcription factor. The expression of circ_000543 was inhibited when YY1 expression was down-regulated; at the same time, down-regulation of YY1 inhibited the effects of exosomes on proliferation and invasion of BC cells. Conclusion:The transcription factor YY1 promoted proliferation and invasion of BC cells by inducing hypoxic breast cancer-derived exosomal circ_000543.
