Lnczc3h7a靶向CTHRC6对肠癌细胞的增殖和迁移的影响

doi:10.3877/cma.j.issn.1674-0785.2023.03.015

目的

探讨结直肠癌治疗的潜在靶点，研究Lnczc3h7a靶向CTHRC6对结直肠癌细胞的表达以及增殖和迁移能力的影响。

方法

选择结直肠癌细胞株（HT-29、SW-60、HCT-116、Lovo、COLO320DM和DLD-1）及正常结直肠上皮细胞FHC，采用荧光定量PCR法检测Lnczc3h7a在结直肠癌细胞株及正常结直肠上皮细胞株中的表达。根据结直肠癌细胞HCT-116转染方式分为Lnczc3h7a mimics组、Lnczc3h7a inhibitor组和使用空白blank plasmid进行转染的空白对照组。采用CCK₈法和划痕实验分别检测并比较三组细胞的增殖能力和迁移能力，通过CRISP-Cas9技术敲除Lnczc3h7a后采用Western blot法检测Lnczc3h7a对CTHRC6蛋白表达的影响。

结果

HT-29、SW-60、HCT-116、Lovo、COLO320DM和DLD-1中的Lnczc3h7a表达水平分别为：0.64±0.02、0.67±0.03、0.59±0.04、0.68±0.03、0.72±0.04、0.62±0.05，均显著低于正常结直肠上皮细胞的FHC（1.00±0.00），转染后，Lnczc3h7a mimics组中的Lnczc3h7a表达水平为（1.36±0.05）明显高于Lnczc3h7a inhibitor组（0.57±0.04）和空白对照组（1.02±0.03），差异有统计学意义（P＜0.05）。转染1~3d后，3组细胞增殖能力差异不显著；转染第4、5d，Lnczc3h7a inhibitor组（0.84±0.06、1.08±0.06）结直肠癌细胞增殖能力明显高于空白对照组（0.73±0.06、0.96±0.06）和Lnczc3h7a mimics组（0.67±0.05、0.91±0.04），同时，空白对照组结直肠癌细胞增殖能力高于Lnczc3h7a mimics组（P值均＜0.05），差异均有统计学意义。细胞培养24 h、48 h时，Lnczc3h7a inhibitor组（447.96 ±46.19、467.23±45.36）的结直肠癌细胞迁移能力高于对照组（416.57±59.48、425.17±58.34）和Lnczc3h7a mimics组（416.57±59.48、425.17±58.34），同时，对照组结直肠癌细胞迁移能力高于Lnczc3h7a mimics组（P值均＜0.05），差异均有统计学意义。对敲除的基因进行RT-PCR检测，结果显示在mRNA水平上，Lnczc3h7a的表达已大幅度下降（P＜0.01），同时Westerm botting结果显示已经完全没有Lnczc3h7a蛋白的表达。Westerm botting结果显示，Lnczc3h7a inhibitor组CTHRC6蛋白的表达水平显著高于空白对照组（P＜0.05），与对照组相比，CTHRC6在Lnczc3h7a mimics组中的蛋白表达水平显著下降（P＜0.05），同时Lnczc3h7a敲除组的CTHRC6的表达水平明显高于其他几组（P＜0.05）。

结论

Lnczc3h7a在结直肠癌细胞株中的表达降低，同时Lnczc3h7a的过度表达可以抑制结直肠癌的增殖和迁移，并且Lnczc3h7a可能通过对CTHRC6蛋白的靶向抑制，从而抑制结直肠癌的生长和转移。

关键词: Lnczc3h7a, CTHRC6, 结直肠癌, 生物学行为

Objective

To investigate the effect of Lnczc3h7a on colorectal cancer cell proliferation and migration to provide a potential target for colorectal cancer treatment.

Methods

Colorectal cancer cell lines (HT-29, SW-60, HCT-116, Lovo, COLO320DM, and DLD-1) and normal colorectal epithelial cell line (FHC) were used in this study, and the expression of Lnczc3h7a in these cell lines was detected by fluorescence quantitative PCR. HCT-116 cells were divided into three groups and transfected with Lnczc3h7a mimic, Lnczc3h7a inhibitor, and empty plasmid, respectively. The proliferation and migration of the three groups of cells were detected and compared by the CCK₈ method and scratch assay, respectively. The effect of Lnczc3h7a on CTHRC6 protein expression was detected by Western blot after knockdown of Lnczc3h7a using the CRISP-Cas9 technique.

Results

The expression levels of Lnczc3h7a in HT-29, SW-60, HCT-116, Lovo, COLO320DM, and DLD-1 cells were 0.64±0.02, 0.67±0.03, 0.59±0.04, 0.68±0.03z, 0.72±0.04, and 0.62±0.05, respectively, which were significantly lower than that in normal colorectal epithelial cells (FHC; 1.00±0). After transfection, the expression level of Lnczc3h7a in the Lnczc3h7a mimic group was (1.36±0.05), significantly higher than that in the Lnczc3h7a inhibitor group (0.57±0.04) and the blank control group (1.02±0.03) (P＜0.05). After transfection for 1~3 days, the difference in cell proliferation was not significant among the three groups; on the 4th and 5th days of transfection, the proliferation of colorectal cancer cells in the Lnczc3h7a inhibitor group (0.84±0.06 and 1.08±0.06) was significantly higher than that in the blank control group (0.73±0.06 and 0.96±0.06) and the Lnczc3h7a mimic group (0.67±0.05 and 0.91±0.04), and the proliferation of colorectal cancer cells in the blank control group was higher than that in the Lnczc3h7a mimic group (P＜0.05). At 24 h and 48 h of cell culture, the migration of colorectal cancer cells in the Lnczc3h7a inhibitor group (447.96±46.19 and 467.23±45.36) was higher than that in the control group (416.57±59.48 and 425.17±58.34) and the Lnczc3h7a mimics group (416.57±59.48 and 425.17±58.34), and the migration ability of colorectal cancer cells in the control group was higher than that in the Lnczc3h7a mimic group (all P＜0.05). After knockdown of Lnczc3h7a using the CRISP-Cas9 technique, RT-PCR showed that the expression of Lnczc3h7a was substantially decreased at the mRNA level (P＜0.01), while Western blot analysis showed that there was no expression of Lnczc3h7a protein. Western blot analysis showed that the Lnczc3h7a inhibitor group had significantly higher CTHRC6 protein expression levels than the blank control group (P＜0.05), and CTHRC6 protein expression levels were significantly decreased in the Lnczc3h7a mimic group compared to the control group (P＜0.05), while the Lnczc3h7a knockout group had significantly higher CTHRC6 expression than other groups (P＜0.05).

Conclusion

The expression of Lnczc3h7a is reduced in colorectal cancer cell lines. Overexpression of Lnczc3h7a could inhibit the proliferation and migration of colorectal cancer cells. Lnczc3h7a inhibits the growth and metastasis of colorectal cancer possibly by targeting CTHRC6 expression.

Key words: Lnczc3h7a, CTHRC6, Colorectal cancer, Biological behavior

图1 Lnczc3h7a在结直肠癌细胞株中的表达水平（与FHC细胞相比，*P＜0.05。）

图2 转染后，Lnczc3h7a的表达水平（与空白对照组相比，*P＜0.05。）

表1 3组不同时间点结直肠癌细胞增殖能力对比（

x ¯

±SD）

组别	第1天	第2天	第3天	第4天	第5天
空白对照组	0.11±0.01	0.16±0.01	0.20±0.04	0.73±0.06	0.96±0.06
Lnczc3h7a inhibitor组	0.12±0.01	0.15±0.02	0.22±0.03	0.84±0.06^∆	1.08±0.06^∆
Lnczc3h7a mimics组	0.11±0.02	0.16±0.02	0.19±0.03	0.67±0.05^∆▲	0.91±0.04^∆▲
F值	1.000	0.667	1.235	13.794	15.614
P值	0.391	0.528	0.319	＜0.001	＜0.001

表1 3组不同时间点结直肠癌细胞增殖能力对比（

x ¯

±SD）

组别	第1天	第2天	第3天	第4天	第5天
空白对照组	0.11±0.01	0.16±0.01	0.20±0.04	0.73±0.06	0.96±0.06
Lnczc3h7a inhibitor组	0.12±0.01	0.15±0.02	0.22±0.03	0.84±0.06^∆	1.08±0.06^∆
Lnczc3h7a mimics组	0.11±0.02	0.16±0.02	0.19±0.03	0.67±0.05^∆▲	0.91±0.04^∆▲
F值	1.000	0.667	1.235	13.794	15.614
P值	0.391	0.528	0.319	＜0.001	＜0.001

图3 不同时间点中Lnczc3h7a对三组结直肠癌细胞增殖能力的影响

表2 3组不同时间点结直肠癌细胞迁移能力对比（

x ¯

±SD）

组别	0 h	24 h	48 h
空白对照组	341.26±42.17	416.57±59.48	425.17±58.34
Lnczc3h7a inhibitor组	342.09±41.95	447.96±46.19^∆	467.23±45.36^∆
Lnczc3h7a mimics组	341.84±42.37	196.26±42.68^∆▲	179.48±34.25^∆▲
F值	0.001	45.792	65.539
P值	0.999	＜0.001	＜0.001

表2 3组不同时间点结直肠癌细胞迁移能力对比（

x ¯

±SD）

组别	0 h	24 h	48 h
空白对照组	341.26±42.17	416.57±59.48	425.17±58.34
Lnczc3h7a inhibitor组	342.09±41.95	447.96±46.19^∆	467.23±45.36^∆
Lnczc3h7a mimics组	341.84±42.37	196.26±42.68^∆▲	179.48±34.25^∆▲
F值	0.001	45.792	65.539
P值	0.999	＜0.001	＜0.001

图4 不同时间点中Lnczc3h7a对3组结直肠癌细胞迁移能力的影响

图5 Lnczc3h7a水平检测（图a为RT-PCR检测Lnczc3h7a mRNA水平，图b为Westerm botting检测Lnczc3h7a蛋白表达。与空白对照组相比，^##P＜0.01。）

图6 Lnczc3h7a对结直肠癌细胞中CTHRC6蛋白表达的影响Westerm botting图和CTHRC6蛋白表达量化图（A.空白对照组；B.Lnczc3h7a敲除组；C.Lnczc3h7a inhibitor组；D.Lnczc3h7a mimics组。与Lnczc3h7a mimics组相比，#P＜0.05；与空白对照组相比，^*P＜0.05。）

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Lnczc3h7a inhibits proliferation and migration of intestinal cancer cells by targeting CTHRC6

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Lnczc3h7a inhibits proliferation and migration of intestinal cancer cells by targeting CTHRC6