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Journal of Peking University (Health Sciences) ›› 2022, Vol. 54 ›› Issue (5): 846-852. doi: 10.19723/j.issn.1671-167X.2022.05.012

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Kindlin-2 regulates endometrium development via mTOR and Hippo signaling pathways in mice

Jing ZHANG1,Jia-gui SONG1,2,Zhen-bin WANG1,Yu-qing GONG1,Tian-zhuo WANG1,Jin-yu ZHOU1,Jun ZHAN1,*(),Hong-quan ZHANG1,*()   

  1. 1. Department of Human Anatomy, Histology and Embryology, Peking University School of Basic Medical Sciences, Beijing 100191, China
    2. Center of Basic Medical Research, Institute of Medical Innovation and Research, Cancer Center, Peking University Third Hospital, Beijing 100191, China
  • Received:2022-06-16 Online:2022-10-18 Published:2022-10-14
  • Contact: Jun ZHAN,Hong-quan ZHANG E-mail:Zhanjun@bjmu.edu.cn;Hongquan.zhang@bjmu.edu.cn
  • Supported by:
    the National Natural Science Foundation of China(81730071);the National Natural Science Foundation of China(82172972);the National Natural Science Foundation of China(31170711);the National Natural Science Foundation of China(81670626);the National Natural Science Foundation of China(81902840);Peking University Double First-Class Construction Funds(PKU2021LCXQ023);Peking University Double First-Class Construction Funds(BMU2022XKQ004)

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Abstract:

Objective: To investigate the effects and mechanisms of Kindlin-2 on uterus development and reproductive capacity in female mice. Methods: Cdh16-Cre tool mice and Kindlin-2flox/flox mice were used to construct the mouse model of uterus specific knockout of Kindlin-2, and the effects of Kindlin-2 deletion on uterine development and reproduction capacity of female mice were observed. High expression and knockdown of Kindlin-2 in endometrial cancer cell lines HEC-1 and Ish were used to detect the regulation of mammalian target of rapamycin (mTOR) signaling pathway. In addition, uterine proteins of the female mice with specific knockout of Kindlin-2 and female mice in the control group were extracted to detect the protein levels of key molecules of mTOR signaling pathway and Hippo signaling pathway. Results: The mouse model of uterine specific knockout of Kindlin-2 was successfully constructed. The knockout efficiency of Kindlin-2 in mouse uterus was identified and verified by mouse tail polymerase chain reaction (PCR), Western blot protein identification, immunohistochemical staining (IHC) and other methods. Compared with the control group, the female mice with uterus specific deletion of Kindlin-2 lost weight, seriously impaired reproductive ability, and the number of newborn mice decreased, but the proportion of the female mice and male mice in the newborn mice did not change. Hematoxylin eosin staining (HE) experiment showed that the endometrium of Kindlin-2 knockout group was incomplete and the thickness of uterine wall became thinner. In terms of mechanism, the deletion of Kindlin-2 in endo-metrial cancer cell lines HEC-1 and Ish could downregulate the protein levels of mTOR, phosphorylated mTOR, adenosine monophosphate-activated protein kinase (AMPK), phosphorylated AMPK and phosphorylated ribosomal protein S6 (S6), and the mTOR signal pathway was inhibited. It was found that the specific deletion of Kindlin-2 could upregulate the protein levels of Mps one binding 1 (MOB1) and phosphorylated Yes-associated protein (YAP) in the uterus of the female mice, and the Hippo signal pathway was activated. Conclusion: Kindlin-2 inhibits the development of uterus by inhibiting mTOR signal pathway and activating Hippo signal pathway, thereby inhibiting the fertility of female mice.

Key words: Kindlin-2, Mouse model, specific knockout, Endometrium, mTOR signaling pathway, Hippo signaling pathway

CLC Number: 

  • R34

Figure 1

Identification of endometrial specific knockout Kindlin-2 mouse model A, mouse tail DNA identification, red boxes show the experimental group; B, the protein level of Kindlin-2 was detected by Western bot; C, immunohistochemistry staining was used to detect the expression and localization of Kindlin-2 in endometrium and glandular epithelium of female mice.Red box, mice in the experimental group; Red arrows, the expression of Kindlin-2 in the endometrium (upper) and glands (lower) in the control group; Black arrows, expression of Kindlin-2 in the endometrium (upper) and glands (lower) in the KO group. Ctrl, control; KO, knockout; WT, wild type."

Figure 2

Endometrial specific knockout of Kindlin-2 caused fertility decline in female mice A, the number of newborn mice was counted; B, observation of uterine morphology and uterine wall thickness; C, statistics of uterine morphology and uterine wall thickness. # P < 0.01. Ctrl, control; KO, knockout."

Table 1

Number of male and female newborn mice in the Kindlin-2 KO group and the control group"

Items Female of newborn mice, n Male of newborn mice, n Total, n
Ctrl 56 58 114
Kindlin-2 KO 28 25 53
Total 84 83 167

Figure 3

Endometrium specific knockout of Kindlin-2 leads to abnormal uterine development in female mice A, HE staining of uterus of female mice in control group and Kindlin-2 KO group at proestrus stage, estrus stage, metestrus stage and diestrus stage; B, statistics of endometrial thickness. *P < 0.05. Ctrl, control; KO, knockout."

Figure 4

Endometrial specific knockdown of Kindlin-2 inhibits mTOR signaling pathway and activates Hippo signaling pathway A, B, proliferation effects of overexpression of Kindlin-2 and knockdown of Kindlin-2 in endometrial cancer cell line HEC-1A, *P < 0.05, #P < 0.01, △P < 0.001, vs.FLAG-Kindlin-2; C, D, detection of mTOR signaling pathway related molecules after overexpression and knockdown of Kindlin-2 in endometrial cancer cell lines HEC-1A and Ish; E, F, the protein levels of mTOR signaling pathway and Hippo signaling pathway related molecules in Kindlin-2 KO and control mice were detected by Western blot.mTOR, mammalian target of rapamycin; AMPK, adenosine monophosphate-activated protein kinase; S6, ribosomal protein; MOB1, Mps one binding 1; YAP, Yes-associated protein; Ctrl, control; KO, knockout."

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