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Knockout of the Placenta Specific 8 Gene Affects the Proliferation and Migration of Human Embryonic Kidney 293T Cell

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Abstract

Candidate oncogene placenta specific 8 (PLAC8) has been identified to participate in different cellular process and human diseases. However, the effects of PLAC8 on cell proliferation and migration in human kidney cancer (KC) remained unclear. In current study, physiological effects of PLAC8 in immortalized human embryonic kidney cell line (HEK293T) were investigated in vitro. Two PLAC8 knockout (KO) cell lines were established via CRISPR/Cas9-mediated methods combined with fluorescence activated single cell sorting. To classify the characteristic of PLAC8 during cell proliferation and migration in HEK293T, cellular proliferative activity was analyzed by cell counting and colony formation assay. Cell cycle distribution was analyzed by flow cytometry. Cellular motile activity was analyzed by wound-healing and migration assay. Further underlying molecular mechanism was explored via western blot. With the KO cell lines, it was found that PLAC8 KO could decrease cell proliferation. Moreover, the inhibitory effects of PLAC8 KO on cell proliferation were associated with a G2/M arrest in cell cycle progression concomitant with a remarkable inhibition of Cyclin B1 and elevation of Cyclin A. The alteration of cell cycle proteins and E-cadherin might further associate with the enhancement of cell motility. Our study revealed a novel role for PLAC8 in cell proliferation and migration of HEK293T cells, which might shed light on further study of PLAC8 on human KC.

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Data Availability

All data generated and analyzed in this study are available upon reasonable request from the corresponding author.

Abbreviations

ccRCC:

clear cell renal cell carcinoma

CDC:

cell division cycle

CDK:

cyclin-dependent kinase

EMT:

epithelial-mesenchymal transition

FACS:

fluorescence activated cell sorting

FITC:

fluorescein isothiocyanate

gRNA:

guide RNA

HEK293T:

embryonic kidney cell line

KC:

kidney cancer

KO:

knockout

NC:

nitrocellulose

PBS:

phosphate-buffered saline

PI:

propidium iodide

PLAC8:

Placenta specific 8

WT:

wildtype

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Acknowledgements

The authors thank all the colleagues in Institute for Medical Biology for their technical support.

Funding

This project was supported by Fund for Key Laboratory Construction of Hubei Province (Grant No. 2018BFC360), the Natural Science Foundation of Hubei Province, China (Grant No. 2018CFB594 to LX), “the Fundamental Research Funds for the Central Universities”, South-Central University for Nationalities (Grant No. CZD19003 to LX) and the China Scholarship Council (Grant No. 201808420069 to LX). The funding body had no role in the design of the study and collection, analysis, and interpretation of data or in writing the manuscript.

Authors’ Contributions

J.S., Q.H.L. and L.X. conceived and designed the experiments. X.H.Q., H.X.W. and L.M. performed the experiments. X.H.Q. and L.X. analyzed the data and generated the figures. L.X. wrote the manuscript. All authors gave final approval for the submitted version.

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Authors and Affiliations

  1. Institute for Medical Biology and Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China, College of Life Sciences, South-Central University for Nationalities, 182 Minyuan Road, Hongshan District, Wuhan, 430074, China

    Xu-Hui Qin, Hai-Xia Wang, Liqun Ma, Jinhua Shen, Qing-Hua Liu & Lu Xue

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  1. Xu-Hui Qin
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Correspondence to Lu Xue.

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These authors contributed equally: Xu-Hui Qin, Hai-Xia Wang and Liqun Ma

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Qin, XH., Wang, HX., Ma, L. et al. Knockout of the Placenta Specific 8 Gene Affects the Proliferation and Migration of Human Embryonic Kidney 293T Cell. Cell Biochem Biophys 78, 55–64 (2020). https://doi.org/10.1007/s12013-019-00893-2

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