lncRNA TINCR对滋养层HTR-8/SVneo细胞生物学行为的影响及其机制

doi:10.3877/cma.j.issn.2095-1221.2024.03.006

目的

探讨lncRNA组织分化诱导非蛋白编码RNA （TINCR）对滋养层HTR-8/SVneo细胞增殖、迁移、侵袭、上皮间质转化和凋亡的影响及其机制。

方法

将HTR-8/SVneo细胞进行体外培养，以正常培养细胞为对照，转染NC-siRNA为阴性对照，转染TINCR-siRNA干扰TNICR的表达，采用实时荧光定量PCR （qPCR）检测细胞中TINCR表达，CCK-8法检测细胞增殖，划伤愈合实验检测HTR-8/SVneo细胞迁移，Transwell小室检测细胞迁移和侵袭，Western blot检测细胞中上皮间质转化相关蛋白E-cadherin、N-cadherin、Vimentin和Wnt/β-catenin信号通路关键蛋白Wnt5a、β-catenin表达。两组间比较采用独立样本t检验，多组间比较采用单因素方差分析，组间两两比较采用SNK-q检验。

结果

与对照及阴性对照比较，转染TINCR-siRNA细胞后TINCR表达水平（0.25 ± 0.03比1.00 ± 0.08；0.25 ± 0.03比0.98 ± 0.06）降低；与对照及阴性对照比较，转染TINCR-siRNA细胞后HTR-8/SVneo细胞中Vimentin （0.92 ± 0.08比0.33 ± 0.05，0.92 ± 0.08比0.31 ± 0.04）、N-cadherin蛋白表达水平（0.87 ± 0.07比0.29 ± 0.04，0.87 ± 0.07比0.32 ± 0.05）均升高，而E-cadherin蛋白表达水平降低；与对照及阴性对照比较，转染TINCR-siRNA细胞后Wnt5a （0.97 ± 0.06比0.48 ± 0.04，0.97 ± 0.06比0.46 ± 0.03）、β-catenin蛋白（0.89 ± 0.05比0.37 ± 0.03，0.89 ± 0.05比0.39 ± 0.03）均升高（P均< 0.05）。

结论

下调TINCR表达可能通过激活Wnt/β-catenin信号通路，促进HTR-8/SVneo细胞增殖、迁移、侵袭和上皮间质转化并抑制其凋亡。

关键词: 子痫前期, lncRNA TINCR, 细胞增殖, 迁移, Wnt/β-catenin信号通路

Objective

To investigate the effect of lncRNA tissue differentiation-inducing differentiation inducing non-protein coding RNA (TINCR) on proliferation, migration, invasion, epithelial-stromal transformation and apoptosis of trophoblast HTR-8/SVneo cells.

Methods

HTR-8/SVneo cells were cultured in vitro and then divided into three groups, which included the control group (standard culture) , negative group (transfected NC-siRNA) and interference group (transfected TINCR-siRNA) respectively. The expression level of TINCR in the cells was detected by real-time fluorescence quantitative PCR, cell proliferation was detected by CCK-8 method, the migration of HTR-8/SVneo cells was detected by scratch healing assay, and cell migration and cell invasion were tested by Transwell chamber. The expression of epithelial-mesenchymal transformation (EMT) related proteins E-cadherin, N-cadherin, Vimentin and Wnt/β-catenin key proteins Wnt5a and β-catenin in cells were checked by Western blotting.

Results

Compared with the control group and the negative group, the expression level of TINCR (0.25 ± 0.03 vs 1.00 ± 0.08; 0.25 ± 0.03 vs 0.98 ± 0.06) was decreased in the interference group; compared with the control group and negative group, the expression levels of Vimentin (0.92 ± 0.08 vs 0.33 ± 0.05, 0.92 ± 0.08 vs 0.31 ± 0.04) and N-cadherin (0.87 ± 0.07 vs 0.29 ± 0.04) . 0.87 ± 0.07 vs 0.32 ± 0.05) were increased in HTR-8/SVneo cells in interference group, while the expression level of E-cadherin protein were decreased; compared with the control group and negative group, the expression level of Wnt5a (0.97 ± 0.06 vs 0.48 ± 0.04, 0.97 ± 0.06 vs 0.46 ± 0.03) and β-catenin proteins (0.89 ± 0.05 vs 0.37 ± 0.03, 0.89 ± 0.05 vs 0.39 ± 0.03) were increased in interference group (P < 0.05) .

Conclusion

Down-regulating TINCR expression may promote the proliferation, migration, invasion and epithelial-mesenchymal transformation of HTR-8/SVneo cells and inhibit their apoptosis by activating the Wnt/β-catenin signaling pathway.

Key words: Preeclampsia, lncRNA TINCR, Cell proliferation, Migration, Wnt/β- catenin signaling pathway

表1 引物序列信息

基因	引物序列（5'→3'）	预期扩增产物长度（bp）
TINCR	上游TGTGGCCCAAACTCAGGGATACAT	216
	下游AGATGACAGTGGCTGGAGTTGTCA
GAPDH	上游CATGTTCGTCATGGGTGTGAACCA	132
	下游AGTGATGGCATGGACTGTGGTCAT

表1 引物序列信息

基因	引物序列（5'→3'）	预期扩增产物长度（bp）
TINCR	上游TGTGGCCCAAACTCAGGGATACAT	216
	下游AGATGACAGTGGCTGGAGTTGTCA
GAPDH	上游CATGTTCGTCATGGGTGTGAACCA	132
	下游AGTGATGGCATGGACTGTGGTCAT

表2 下调TINCR表达对HTR-8/SVneo细胞增殖活力的影响（

± s）

分组	实验次数	24 h	48 h	72 h
对照	3	0.36 ± 0.03	0.54 ± 0.04	0.83 ± 0.05
阴性对照	3	0.35 ± 0.03	0.52 ± 0.03	0.85 ± 0.06
TINCR-siRNA	3	0.37 ± 0.03	0.76 ± 0.06^ab	1.23 ± 0.09^ab
F值		1.000	78.492	96.592
P值		0.383	< 0.001	< 0.001

表2 下调TINCR表达对HTR-8/SVneo细胞增殖活力的影响（

± s）

分组	实验次数	24 h	48 h	72 h
对照	3	0.36 ± 0.03	0.54 ± 0.04	0.83 ± 0.05
阴性对照	3	0.35 ± 0.03	0.52 ± 0.03	0.85 ± 0.06
TINCR-siRNA	3	0.37 ± 0.03	0.76 ± 0.06^ab	1.23 ± 0.09^ab
F值		1.000	78.492	96.592
P值		0.383	< 0.001	< 0.001

图1 倒置显微镜下观察下调TINCR表达对HTR-8/SVneo细胞迁移能力的影响（×40）

图2 倒置显微镜下观察下调TINCR表达对HTR-8/SVneo细胞侵袭能力的影响（结晶紫染色，×200）

表3 下调TINCR表达对HTR-8/SVneo细胞划痕愈合率、迁移细胞数和侵袭细胞数的影响（

± s）

分组	实验次数	划痕愈合率（%）	迁移细胞数（个）	侵袭细胞数（个）
对照	3	46.35 ± 5.02	85.56 ± 6.75	67.38 ± 5.23
阴性对照	3	44.81 ± 4.76	90.25 ± 7.42	62.52 ± 5.17
TINCR-siRNA	3	65.40 ± 5.91^ab	138.85 ± 11.35^ab	105.46 ± 8.85^ab
F值		42.900	102.453	112.753
P值		< 0.001	< 0.001	< 0.001

表3 下调TINCR表达对HTR-8/SVneo细胞划痕愈合率、迁移细胞数和侵袭细胞数的影响（

± s）

分组	实验次数	划痕愈合率（%）	迁移细胞数（个）	侵袭细胞数（个）
对照	3	46.35 ± 5.02	85.56 ± 6.75	67.38 ± 5.23
阴性对照	3	44.81 ± 4.76	90.25 ± 7.42	62.52 ± 5.17
TINCR-siRNA	3	65.40 ± 5.91^ab	138.85 ± 11.35^ab	105.46 ± 8.85^ab
F值		42.900	102.453	112.753
P值		< 0.001	< 0.001	< 0.001

图3 Western blot检测Vimentin、N-cadherin和E-cadherin蛋白表达水平

表4 下调TINCR表达对细胞中Vimentin、N-cadherin和E-cadherin蛋白表达的影响（

± s）

分组	实验次数	Vimentin	N-cadherin	E-cadherin
对照	3	0.33 ± 0.05	0.29 ± 0.04	0.67 ± 0.04
阴性对照	3	0.31 ± 0.04	0.32 ± 0.05	0.70 ± 0.05
TINCR-siRNA	3	0.92 ± 0.08^ab	0.87 ± 0.07^ab	0.26 ± 0.02^ab
F值		308.829	319.900	362.600
P值		< 0.001	< 0.001	< 0.001

表4 下调TINCR表达对细胞中Vimentin、N-cadherin和E-cadherin蛋白表达的影响（

± s）

分组	实验次数	Vimentin	N-cadherin	E-cadherin
对照	3	0.33 ± 0.05	0.29 ± 0.04	0.67 ± 0.04
阴性对照	3	0.31 ± 0.04	0.32 ± 0.05	0.70 ± 0.05
TINCR-siRNA	3	0.92 ± 0.08^ab	0.87 ± 0.07^ab	0.26 ± 0.02^ab
F值		308.829	319.900	362.600
P值		< 0.001	< 0.001	< 0.001

图4 Western blot检测细胞中Wnt5a和β-catenin蛋白表达

表5 下调TINCR表达对细胞中Wnt5a和β-catenin蛋白表达水平的影响（

± s）

分组	实验次数	Wnt5a	β-catenin
对照	3	0.48 ± 0.04	0.37 ± 0.03
阴性对照	3	0.46 ± 0.03	0.39 ± 0.03
TINCR-siRNA	3	0.97 ± 0.06^ab	0.89 ± 0.05^ab
F值		369.295	545.023
P值		< 0.001	< 0.001

表5 下调TINCR表达对细胞中Wnt5a和β-catenin蛋白表达水平的影响（

± s）

分组	实验次数	Wnt5a	β-catenin
对照	3	0.48 ± 0.04	0.37 ± 0.03
阴性对照	3	0.46 ± 0.03	0.39 ± 0.03
TINCR-siRNA	3	0.97 ± 0.06^ab	0.89 ± 0.05^ab
F值		369.295	545.023
P值		< 0.001	< 0.001

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Effect of lncRNA TINCR on the biological behavior of trophoblast HTR-8/SVneo cells and its mechanism

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Effect of lncRNA TINCR on the biological behavior of trophoblast HTR-8/SVneo cells and its mechanism