Mechanism of downregulated PDCD10 expression promoting temozolomide resistance in human glioblastoma cell lines
10.3760/cma.j.cn115354-20240507-00279
- VernacularTitle:PDCD10表达下调导致人胶质母细胞瘤细胞系耐替莫唑胺治疗的机制研究
- Author:
Rongde ZHONG
1
;
Heng WANG
;
Yue XIAO
;
Fanfan CHEN
;
Guodong HUANG
;
Yunsheng LIU
Author Information
1. 深圳大学医学部生物医学工程学院，深圳 518060
- Keywords:
Programmed cell death 10;
Glioblastoma multiforme;
Temozolomide;
Mismatch repair;
Cell cycle
- From:
Chinese Journal of Neuromedicine
2024;23(6):541-551
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the mechanism of downregulated programmed cell death 10 ( PDCD10) expression mediating glioblastoma multiforme (GBM) resistance to temozolomide (TMZ). Methods:U87, LN229 and T98g cell lines were transfected with PDCD10 small interfering RNA or negative small interfering RNA. TMZ-resistant cell lines were constructed using 300 μmol/L TMZ (transfected T98g cell line) and 150 μmol/L TMZ (transfected U87 and LN229 cell lines), respectively: TMZ-resistant U87 cell line transfected with PDCD10 small interfering RNA (shPDCD10-U87-RG cells), TMZ-resistant U87 cell line transfected with negative small interfering RNA (EV-U87-RG cells), shPDCD10-T98g-RG cells, EV-T98g-RG cells, shPDCD10-LN229-RG cells and EV-LN229-RG cells. Flow cytometry and real-time quantitative polymerase chain reaction (qRT-PCR) were used to detect the transfection efficiency of TMZ-resistant cell lines and PDCD10 expressions; MTT assay and colony formation assay were used to verify the drug-resistant ability of TMZ-resistant cell lines. Bioinformatics analysis was performed to detect the correlations of PDCD10 with key genes ( MSH6 and PMS2) in mismatch repair (MMR) system, and drug resistant mechanism was explored by detecting the cell cycle and neurosphere formation ability of drug-resistant cells. Results:(1) qRT-PCR showed that compared with that in EV-U87-RG cells, the PDCD10 expression in shPDCD10-U87-RG cells was statistically down-regulated by (32.85±1.14)% ( t=2.925, P=0.049); compared with that in EV-T98g-RG cells, the PDCD10 expression in shPDCD10-T98g-RG cells was significantly down-regulated by (57.17±1.81)% ( t=3.179, P=0.043); compared with that in EV-LN229-RG cells, the PDCD10 expression in shPDCD10-LN229-RG cells was significantly down-regulated by (33.68±1.34)% ( t=3.085, P=0.045). (2) MTT assay showed that compared with the EV-U87-RG cells, the shPDCD10-U87-RG cells had significantly increased viability ( P<0.05); compared with the EV-T98g-RG cells, the shPDCD10-T98g-RG cells had significantly increased viability ( P<0.05). Among the same kind of cells, the viability 3 d after wash-out was significantly increased compared with that at 72 h after TMZ treatment ( P<0.05). Colony formation assay showed that cell lines with down-regulated PDCD10 expression had higher tumorigenic ability. (3) Compared with EV-U87-RG cells and EV-T98g-RG cells, cells with down-regulated PDCD10 expression (shPDCD10-U87-RG cells and shPDCD10-T98g-RG cells) escaped from TMZ-induced G2/M arrest, resulting in TMZ resistance. (4) Bioinformatics analysis revealed that the PDCD10 expression was positively correlated with MSH6 and PMS2 expressions ( r=0.262, P<0.001; r=0.327, P<0.001); qRT-PCR indicated that downregulated PDCD10 expression caused decreased MSH6 and PMS2 expressions, which disrupted the MMR system. (5) Compared with that by EV-U87 cells, number of neurospheres formed by shPDCD10-U87 cells was significantly increased ( P<0.05); compared with that by EV-U87-RG cells, number of neurospheres formed by shPDCD10-U87-RG cells was significantly increased ( P<0.05). Conclusion:PDCD10 affects the therapeutic sensitivity of GBM to TMZ by arresting cell cycle, disrupting MMR system, and increasing cell stemness.
