KRT17调节Wnt/β-catenin信号通路对膀胱癌细胞增殖、凋亡及上皮间质转化的影响

doi:10.11958/20241498

摘要/Abstract

摘要：

目的探讨敲低角蛋白17（KRT17）通过调节Wnt/β-连环蛋白（β-catenin）信号通路对膀胱癌细胞增殖、凋亡及上皮间质转化（EMT）的影响。方法 qRT-PCR及Western blot检测膀胱癌组织、癌旁组织及膀胱癌细胞系（5637、T24、UM-UC-3）及人永生化尿路上皮细胞系SV-HUC-1细胞中KRT17 mRNA及蛋白表达。免疫组化染色检测组织中KRT17表达。通过转染NC siRNA、KRT17 siRNA至细胞，标记为NC siRNA组、KRT17 siRNA组；以20 mmol/L LiCl处理T24细胞，标记为LiCl组；以20 mmol/L LiCl处理转染KRT17 siRNA的T24细胞，标记为KRT17 siRNA+LiCl组，不转染的细胞为空白组。CCK-8、克隆形成实验、流式细胞术检测细胞增殖及凋亡；qRT-PCR检测KRT17 mRNA表达；Western blot检测KRT17、β-catenin、细胞周期蛋白D1（Cyclin D1）、EMT相关蛋白Vimentin、E-cadherin、Snail蛋白表达。结果 KRT17 mRNA及蛋白表达在膀胱癌组织及细胞中明显升高（P<0.05）；KRT17 siRNA组KRT17 mRNA及蛋白表达、细胞增殖率及菌落数、细胞侵袭数、β-catenin、Cyclin D1、Vimentin、Snail表达较NC siRNA组和空白组降低，凋亡率、E-cadherin表达增加（P<0.05）；LiCl逆转了敲低KRT17对膀胱癌恶性行为的抑制。结论敲低KRT17通过抑制Wnt/β-catenin信号通路抑制膀胱癌细胞增殖及EMT，促进其凋亡。

关键词: 膀胱肿瘤, 上皮-间质转化, 细胞增殖, 凋亡, KRT17, Wnt/β-catenin信号通路

Abstract:

Objective To investigate the impacts of knocking-down Keratin 17 (KRT17) on proliferation, apoptosis and epithelial mesenchymal transition (EMT) of bladder cancer cells by regulating Wnt/β-catenin signaling pathway. Methods The expression of KRT17 mRNA and protein in bladder cancer tissue, adjacent tissue, bladder cancer cell lines (5637, T24 and UM-UC-3) and human immortalized urothelial cell line SV-HUC-1 were detected by qRT-PCR and Western blot assay. Immunohistochemical staining was used to detect the expression of KRT17 in the tissues.Cells transfected with NC siRNA and KRT17 siRNA were labeled as the NC siRNA group and the KRT17 siRNA group, respectively. T24 cells treated with 20 mmol/L LiCl were labeled as the LiCl group. T24 cells transfected with KRT17 siRNA and treated with 20 mmol/L LiCl were labeled as the KRT17 siRNA+LiCl group. The non transfected cells were used as the blank group. CCK-8, cloning formation experiment and flow cytometry were applied to detect cell proliferation and apoptosis. QRT-PCR was applied to detect KRT17 mRNA expression. Western blot assay was applied to detect the expression levels of KRT17, β-catenin, Cyclin D1, EMT related proteins Vimentin, E-cadherin and Snail1 proteins. Results The expression of KRT17 mRNA and protein was greatly increased in bladder cancer tissue and cells (P<0.05). The cell proliferation, colony count, KRT17 mRNA and protein expression, β-catenin, Cyclin D1, Vimentin, and Snail expression were lower in the KRT17 siRNA group than those in the NC siRNA group and the blank group, while apoptosis and E-cadherin expression were higher (P<0.05). LiCl reversed the inhibition of KRT17 knockdown on the malignant behavior of bladder cancer. Conclusion Knocking-down KRT17 inhibits the proliferation and EMT of bladder cancer cells and promotes their apoptosis by inhibiting Wnt/β-catenin signaling pathway.

Key words: urinary bladder neoplasms, epithelial-mesenchymal transition, cell proliferation, apoptosis, KRT17, Wnt/β-catenin signaling pathway

中图分类号:

R737.14

图/表 14

图1 Western blot检测膀胱癌组织和癌旁组织中KRT17蛋白表达

Fig.1 Western blot analysis of KRT17 protein expression in bladder cancer tissue and adjacent tissue

表1 膀胱癌组织和癌旁组织中KRT17 mRNA及蛋白表达水平比较（n=80,$\bar{x}±s$）

Tab.1 Comparison of KRT17 mRNA and protein expression levels between bladder cancer tissue and adjacent tissue

组别	KRT17 mRNA	KRT17/β-actin
癌旁组织	0.94±0.10	0.42±0.05
膀胱癌组织	1.64±0.17	0.87±0.09
t	31.744^＊＊	39.094^＊＊

图2 膀胱癌和癌旁组织中KRT17表达（免疫组化染色，×200）

Fig.2 Expression of KRT17 in bladder cancer tissue and paracancer tissue (immunohistochemical staining, ×200)

图3 Western blot检测膀胱癌细胞中KRT17蛋白表达 A：SV-HUC-1细胞；B：5637细胞；C：UM-UC-3细胞；D：T24细胞。

Fig. 3 Western blot analysis of KRT17 protein expression in bladder cancer cells

表2 膀胱癌细胞中KRT17 mRNA及蛋白表达水平比较（n=6,$\bar{x}±s$）

Tab.2 Comparison of KRT17 mRNA and protein expression levels between bladder cancer cells

细胞	KRT17 mRNA	KRT17/β-actin
SV-HUC-1	1.02±0.11	0.37±0.04
5637	1.57±0.16^a	0.54±0.06^a
UM-UC-3	1.61±0.17^a	0.51±0.07^a
T24	2.02±0.21^abc	0.84±0.09^abc
F	36.545^＊＊	51.560^＊＊

图4 Western blot检测各组T24细胞中KRT17蛋白表达 A：空白组；B：NC siRNA组；C：KRT17 siRNA组；D：LiCl组； E：KRT17 siRNA+LiCl组。

Fig. 4 Western blot analysis of KRT17 protein expression in T24 cells in each group

表3 各组T24细胞中KRT17 mRNA及蛋白表达水平比较（n=6,$\bar{x}±s$）

Tab.3 Comparison of KRT17 mRNA and protein expression between five groups of T24 cells

组别	KRT17 mRNA	KRT17/β-actin
空白组	1.06±0.12	0.78±0.08
NC siRNA组	1.07±0.11	0.72±0.08
KRT17 siRNA组	0.47±0.05^ab	0.37±0.04^ab
LiCl组	1.98±0.21	1.45±0.15
KRT17 siRNA+LiCl组	1.12±0.12^cd	0.69±0.07^cd
F	99.960^＊＊	112.371^＊＊

表4 各组T24细胞中增殖及菌落数比较（n=6,$\bar{x}±s$）

Tab.4 Comparison of proliferation and colony number of T24 cells between the five groups

组别	增殖率/%	菌落数/个
空白组	88.64±8.91	201.42±20.27
NC siRNA组	89.04±8.97	211.50±21.29
KRT17 siRNA组	48.62±4.93^ab	124.62±12.66^ab
LiCl组	128.64±12.94	308.51±31.02
KRT17 siRNA+LiCl组	87.24±8.81^cd	198.54±19.97^cd
F	55.977^＊＊	54.063^＊＊

图5 各组T24细胞中菌落形成情况（结晶紫染色）

Fig. 5 Colony formation in T24 cells of each group (crystal violet staining)

表5 各组T24细胞凋亡率和细胞侵袭数比较（n=6,$\bar{x}±s$）

Tab.5 Comparison of apoptosis rate and cell invasion number of T24 cells between five groups

组别	凋亡率/%	侵袭数/（个/视野）
空白组	9.73±0.10	134.57±13.61
NC siRNA组	10.04±1.02	133.94±13.52
KRT17 siRNA组	28.43±2.91^ab	82.02±8.33^ab
LiCl组	5.24±0.55	216.24±21.72
KRT17 siRNA+LiCl组	11.18±1.21^cd	134.05±13.55^cd
F	213.361^＊＊	63.661^＊＊

图6 各组T24细胞流式凋亡图

Fig. 6 Flow cytometry of T24 cells in each group

图7 结晶紫染色观察各组T24细胞侵袭能力（×200）

Fig.7 The invasion ability of T24 cells in each group observed by crystal violet staining （×200）

图8 Western blot检测各组T24细胞中β-catenin、Cyclin D1、Vimentin、E-cadherin、Snail蛋白表达 A：空白组；B：NC siRNA组；C：KRT17 siRNA组；D：LiCl组； E：KRT17 siRNA+LiCl组。

Fig.8 The protein expressions of β-catenin, Cyclin D1, Vimentin, E-cadherin and Snail in T24 cells of each group detected by Western blot assay

表6 各组T24细胞中β-catenin、Cyclin D1、Vimentin、E-cadherin、Snail表达比较（n=6,$\bar{x}±s$）

Tab.6 The expression of β-catenin, Cyclin D1, Vimentin, E-cadherin and Snail in T24 cells of each group

组别	β-catenin/β-actin		Cyclin D1/β-actin
空白组	0.67±0.07		0.59±0.06
NC siRNA组	0.63±0.07		0.55±0.06
KRT17 siRNA组	0.28±0.03^ab		0.24±0.03^ab
LiCl组	0.94±0.10		0.84±0.09
KRT17 siRNA+LiCl组	0.71±0.08^cd		0.52±0.06^cd
F	62.357^＊＊		69.197^＊＊
组别	Vimentin/ β-actin	E-cadherin/ β-actin	Snail/ β-actin
空白组	0.71±0.08	0.57±0.06	0.89±0.09
NC siRNA组	0.79±0.08	0.52±0.06	0.85±0.09
KRT17 siRNA组	0.37±0.04^ab	0.78±0.08^ab	0.58±0.06^ab
LiCl组	1.44±0.15	0.24±0.03	1.47±0.15
KRT17 siRNA+LiCl组	0.75±0.08^cd	0.49±0.05^cd	0.78±0.08^cd
F	104.702^＊＊	65.912^＊＊	68.273^＊＊

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