The Effects of <i>Herpud1</i> on Metanephric Mesenchymal Cells and Its Mechanism

doi:10.13523/j.cb.20180302

China Biotechnology

2018, Vol. 38

Issue (3): 9-15 DOI: 10.13523/j.cb.20180302

Orginal Article

The Effects of Herpud1 on Metanephric Mesenchymal Cells and Its Mechanism

Yi-man LI,Qin ZHOU(

)

The School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China

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Abstract

To explore biological function of Herpud1 in kidney development, overexpression and knockdown vector of Herpud1 gene were transfected into MK3 cells. And then RT-PCR and Western blot were used to detect expression of epithelial mesenchymal transition (EMT) marker genes E-cadherin (epithelial cells marker), Vimentin and Snail (mesenchymal cell marker) and endoplasmic reticulum (ER) stress marker genes (GRP78 and eIF2α). And cell proliferation and migration were detected by EDU cell proliferation experiment and wound healing assay. Our results showed in experimental group with Herpud1 overexpression, E-cadherin was decreased, and expression of Vimentin and Snail was increased at mRNA and protein levels compared with control group. And ER stress markers GRP78 and eIF2α were enhanced at protein levels in MK3 cells with Herpud1 overexpression. In addition, Herpud1 promoted cell migration and inhibited cell proliferation. In Herpud1 knockdown cells, expression of E-cadherin was enhanced at mRNA and protein levels, and expression of Vimentin, Snail, GRP78 and eIF2α is reduced. At the same time, Herpud1 knockdown inhibited cell migration and enhanced ability of cell proliferation. These results demonstrate Herpud1 can promote EMT of MK3 cells, cell migration and inhibit cell proliferation. The mechanism may be associate with ER stress.

Key words： Herpud1 EMT Cell proliferation ER stress Cell migration

Received: 30 October 2017 Published: 04 April 2018

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Yi-man LI,Qin ZHOU. The Effects of Herpud1 on Metanephric Mesenchymal Cells and Its Mechanism. China Biotechnology, 2018, 38(3): 9-15.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180302 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I3/9

Fig.1 Efficiency of vector of Herpud1 overexpression and knockdown in MK3 cells
(a),(b) Efficiency of Herpud1 overexpression at mRNA and protein level (c),(d) Efficiency of Herpud1 knockdown at mRNA protein level ^** P< 0.01, ^*** P< 0.001

Fig.2 Herpud1 promotes MK3 cells EMT process
(a),(b) RT-PCR and Western blot were utilized to detected expression changes of EMT marker genes E-cadherin, Vimentin and Snail at mRNA and protein levels after Herpud1 vector and control vector was transfected for 48h (c),(d) shHerpud1 knockdown vector and control vector were transfected into cells for 72h. Expression of EMT marker genes were detected at mRNA and protein level ^** P< 0.01, ^*** P< 0.001

Fig.3 Herpud1 inhibited cell proliferation of MK3 cells
After pCDH-Herpud1 vectors and shHerpud1 vectors were transfected into MK3 cells for 48h and 72h respectively, we detected MK3 cells proliferation by ERU cell proliferation Kit ^** P< 0.01, ^*** P< 0.001

Fig.4 Herpud1 promotes MK3 cells migration
(a) Effect of Herpud1 overexpression on wound healing in MK3 cells by inverted microscope (b) Quantitative analysis of cell migration rate according to Figure 4a (c) Effect of Herpud1 knockdown on wound healing in MK3 cells (d) Quantitative analysis of cell migration rate according to Figure 4c ^** P< 0.01

Fig.5 Herpud1 promotes expression of ER stress-related to protein
(a) Expression of ER stress-related protein was measured by western blot after Herpud1 overexpression vectors was transfected for 48h (b) Protein brand gray was scanned by Image J software according to Figure 5a ^*** P<0.001


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