Role of human pleiotrophin in the metastasis of malignant peripheral nerve sheath tumor

doi:10.3969/j.issn.1674-8115.2022.09.009

Abstract

Abstract:

Objective ·To investigate the role of pleiotrophin (PTN) in the proliferation, migration and invasion of malignant peripheral nerve sheath tumor (MPNST). Methods ·The expression of PTN was verified by immunochemistry and bioinformatics analysis. By lentivirus transfection, the PTN-overexpressing and the PTN-knocked-down MPNST cell lines were established. To validate the role of PTN in MPNST migration and invasion, cell wound healing assay and Transwell migration/invasion assays were performed in boththe PTN-overexpressing MPNST cells and the PTN-knocked-down cells. By CCK8 assay, EdU assay and colony formation assay, the effects of PTN on the proliferation and the colony formation capabilities were evaluated. The pathway expression pattern regulated by PTN was validated by Western blotting and high-throughput transcriptome sequencing. To identify the biological function of PTN in vivo, an MPNST lung metastatic model in Balb/c nude mice was established. By mouse tail intravenous injection technique, PTN-knocked-down MPNST cells (experimental group) and negative control cells (control group) were injected into Balb/c nude mice, respectively. Results ·According to the immunochemistry staining results of the MPNST tissue samples from 49 patients and the normal nerve tissue samples from 3 non-MPNST people, PTN was significantly down-regulated in the MPNST tissues. In cell wound healing assay and Transwell migration/invasion assay, knocking down PTN significantly enhanced the migration and invasion capabilities of MPNST cells, while PTN overexpression inhibited them. The results of CCK8 assay, EdU assay and colony formation assay indicated that PTN overexpression hindered the proliferation of MPNST cells while colony formation was barely impacted; no significant differences in these assays were detected in the PTN-knocked-down cells. According to Western blotting assay, PTN overexpression activated mitogen activated protein kinase (MAPK) pathway while knocking down PTN showed mild influence. Transcriptome high-throughput sequencing showed that the expression levels of the genes related with programmed cell death pathway changed in the PTN-overexpressing cells. In the in vivo experiments, there was no significant difference in the tumor formation rate; however, the quantity and the volume of lung metastasis lesions were both larger in the experimental group. The most mice in the experimental group were diagnosed with severe pulmonary interstitial disease, and their growth rate of body mass also decreased. Conclusion ·PTN is down-regulated in the MPNST tissue and overexpressing PTN inhibites proliferation, migration and invasion of the MPNST cells in vitro. The ability of PTN-knocked-down MPNST cells to metastasize to the lungs in mice is enhanced.

Key words: malignant peripheral nerve sheath tumor (MPNST), pleiotrophin (PTN), tumor metastasis, neurofibromatosis type 1, plexiform neurofibroma

CLC Number:

R739.43

CUI Xiwei, CHUNG Manhon, AIMAIER Rehanguli, WANG Zhichao, LI Qingfeng. Role of human pleiotrophin in the metastasis of malignant peripheral nerve sheath tumor[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(9): 1225-1238.

Figures/Tables 9

TrendMD

References 46

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Antibody	Lot.	Manufacturer
Anti-PTN primary antibody （IHC）	Sc-74443	Santa Cruz
Anti-PTN primary antibody	Ab79411	Abcam
Anti-ACTB primary antibody	D190826	Sangon
p44/42 MAPK primary antibody	4695	CST
Phospho-p44/42 MAPK primary antibody	4370	CST
MEK1/2 primary antibody	4694	CST
Phospho-MEK1/2 primary antibody	2338	CST
AKT （pan） primary antibody	4691	CST
Phospho-AKT primary antibody	4060	CST
Anti N-cadherin primary antibody	Ab76011	Abcam
Anti E-cadherin primary antibody	Ab40722	Abcam
Anti-Slug primary antibody	Ab51772	Abcam
Anti-Snail primary antibody	Ab53519	Abcam
Anti-vimentin primary antibody	Ab92547	Abcam
Anti-α-SMA primary antibody	Ab7817	Abcam
Anti-FAK primary antibody	Ab40794	Abcam
Anti-pFAK （Y397） primary antibody	Ab81298	Abcam
Anti-SRC primary antibody	Ab109381	Abcam
Anti-pSRC （Y419） primary antibody	Ab185617	Abcam
DYKDDDDK （flag） primary antibody	14793	CST
Donkey to goat secondary antibody	Ab97110	Abcam
Goat to rabbit secondary antibody	Ab97080	Abcam
Rabbit to mouse secondary antibody	Ab6728	Abcam

Antibody	Lot.	Manufacturer
Anti-PTN primary antibody （IHC）	Sc-74443	Santa Cruz
Anti-PTN primary antibody	Ab79411	Abcam
Anti-ACTB primary antibody	D190826	Sangon
p44/42 MAPK primary antibody	4695	CST
Phospho-p44/42 MAPK primary antibody	4370	CST
MEK1/2 primary antibody	4694	CST
Phospho-MEK1/2 primary antibody	2338	CST
AKT （pan） primary antibody	4691	CST
Phospho-AKT primary antibody	4060	CST
Anti N-cadherin primary antibody	Ab76011	Abcam
Anti E-cadherin primary antibody	Ab40722	Abcam
Anti-Slug primary antibody	Ab51772	Abcam
Anti-Snail primary antibody	Ab53519	Abcam
Anti-vimentin primary antibody	Ab92547	Abcam
Anti-α-SMA primary antibody	Ab7817	Abcam
Anti-FAK primary antibody	Ab40794	Abcam
Anti-pFAK （Y397） primary antibody	Ab81298	Abcam
Anti-SRC primary antibody	Ab109381	Abcam
Anti-pSRC （Y419） primary antibody	Ab185617	Abcam
DYKDDDDK （flag） primary antibody	14793	CST
Donkey to goat secondary antibody	Ab97110	Abcam
Goat to rabbit secondary antibody	Ab97080	Abcam
Rabbit to mouse secondary antibody	Ab6728	Abcam

Primer	Sequence
PPIA forward	5'-CCCACCGTGTTCTTCGACATT-3'
PPIA reverse	5'-GGACCCGTATGCTTTAGGATGA-3'
PTN forward	5'-GAAGTCTGAAGCGAGCCCTG-3'
PTN reverse	5'-GCTTGAGGTTTGGGCTTGGT-3'

Primer	Sequence
PPIA forward	5'-CCCACCGTGTTCTTCGACATT-3'
PPIA reverse	5'-GGACCCGTATGCTTTAGGATGA-3'
PTN forward	5'-GAAGTCTGAAGCGAGCCCTG-3'
PTN reverse	5'-GCTTGAGGTTTGGGCTTGGT-3'

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