Notch2 regulates proliferation, invasion and migration of tongue squamous cell carcinoma stem cells

doi:10.3969/j.issn.2095-4344.2016.45.010

Abstract

Abstract:

BACKGROUND: Previous studies have found that the expression of Notch2 is up-regulated in tongue squamous cell carcinoma (TSCC) stem cells, but the effect of Notch2 on TSCC remains unclear.
OBJECTIVE: To explore the effect of Notch2 on the proliferation, invasion and migration of TSCC stem cells (ALDH^br).
METHODS: ALDHbr cells were enriched and separated by fluorescence-activated cell sorting technique from Tca8113 cells cultured in using serum-free medium, and Notch2 expression in ordinary TSCC cells (ALDH^low) and ALDH^br cells was detected by western blot. The ALDH^brcells transfected with Notch2 shRNA (sh-Notch2) by liposome served as experimental group, and ALDH^br cells transfected with sh-control were as control group. The effects of Notch2 on the proliferation, invasion and migration of ALDH^br cells were detected by cell counting kit-8 kit and Transwell assay, respectively. In addition, the expression levels of Ki67, matrix metalloproteinases 2 and 9 protein in Notch2-silenced ALDH^br cells and control cells were detected by western blot.
RESULTS AND CONCLUSION: The expression level of Notch2 in ALDH^br cells was significantly higher than that in ALDH^low cells (P < 0.05). The ALDHbr cell proliferation ability and Ki67 protein expression in the experimental group were obviously lower than those in the control group (P < 0.05). The ALDH^br cell invasion and migration abilities in the experimental group were significantly lower than those in the control group (P < 0.05). The expression levels of matrix metalloproteinases 2 and 9 protein in the experimental group were obviously lower than those in the control group (P < 0.05). All these results show that Notch2 can promote the proliferation, invasion and migration of ALDH^br cells, which suggests that Notch2 plays an important role in the occurrence and development of TSCC, and it may be a potential molecular target for the treatment of TSCC.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Neoplastic Stem Cells, Cell Biology, Tissue Engineering

CLC Number:

R394.2

Ge Zhen-qiong, Yan En-li, Sun Xue-rong, Ren Xue-mei, Xiao Hai. Notch2 regulates proliferation, invasion and migration of tongue squamous cell carcinoma stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(45): 6753-6759.

Figures/Tables 1

2.1 培养不同时间ALDHbr细胞所占的比例通过无血清培养基培养舌鳞癌细胞株Tca8113来富集ALDHbr细胞，流式细胞仪检测发现，ALDHbr细胞比例随着无血清培养基培养时间的延长而逐渐增加，刚开始增加较为缓慢，培养15-20 d的短时间内，ALDHbr细胞所占比例迅速增加；20 d时，ALDHbr细胞所占比例最高，之后又逐渐降低。在无血清培养第1，5 ，10，15，20，25，30天的细胞中，ALDHbr细胞所占的比例分别为1.1%、2.5%、4.5%、10.1%、31.2%、26.7%和23.2%，见图1。这一发现表明无血清培养20 d是分选ALDHbr细胞的最佳时机。后续实验所用到的ALDHbr和ALDHlow细胞均为无血清培养20 d时所分选得到的。 2.2 Notch2蛋白在ALDHbr中表达上调通过免疫磁珠分选无血清培养20 d的舌鳞癌细胞株Tca8113得到了ALDHbr和ALDHlow细胞，为了证实Notch2在ALDHbr细胞中表达上调，执行了蛋白免疫印迹实验，结果显示Notch2蛋白在ALDHbr细胞的表达水平显著高于其在ALDHlow细胞中的表达水平，证实Notch2蛋白在ALDHbr细胞中的确表达上调，见图2。 2.3 Notch2促进ALDHbr细胞增殖为了探究Notch2对ALDHbr细胞增殖的影响，首先通过脂质体转染构建Notch2沉默的ALDHbr细胞和对照细胞，将这2种细胞置于含体积分数10%胎牛血清的RPMI 1640培养基中培养48 h，然后加入含有CCK-8的混合液，继续培养2 h，之后检测这两种细胞在450，650 nm的吸光度，参照公式计算这两种细胞的相对增殖能力，发现实验组细胞的相对增殖能力明显低于对照组，见图3A。此外，还采用免疫印迹检测这两组细胞中Ki67蛋白的表达水平，结果显示实验组细胞中Ki67蛋白的表达水平明显低于对照组，见图3B。这些结果表明Notch2促进ALDHbr细胞的增殖。 2.4 Notch2促进ALDHbr细胞侵袭和迁移为了探究Notch2对ALDHbr细胞侵袭和迁移的影响，采用Transwell小室实验检测了Notch2沉默ALDHbr细胞和对照细胞的侵袭和迁移能力。结果显示，实验组细胞的侵袭和迁移能力均显著低于对照组，见图4A，B。此外，还执行了蛋白免疫印迹实验检测侵袭标志物蛋白基质金属蛋白酶2和基质金属蛋白酶9在这2种细胞中的表达水平，结果显示，基质金属蛋白酶2和基质金属蛋白酶9在实验组中的表达水平显著低于其在对照组中的表达水平，见图4C。这些结果表明，Notch2促进ALDHbr细胞的侵袭和迁移。"

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