结直肠癌细胞中lnczc3h7a的表达及对肿瘤细胞增殖和迁移的作用及机制

目的研究结直肠癌细胞中Lnczc3h7a的表达及对肿瘤细胞增殖和迁移的作用机制。方法选择6种结直肠癌细胞株人结肠癌细胞(HT-29),人结直肠癌细胞(HCT-116),人结肠癌细胞(SW-60),人结直肠腺癌细胞(COLO320DM),人结肠癌细胞(Lovo)和人结直肠腺癌上皮细胞(DLD-1)与正常结直肠上皮细胞系。以Lnczc3h7a模拟物(mimics)、Lnczc3h7a抑制剂(inhibitor)及空白对照分别转染结直肠癌细胞SW60,采用细胞计数试剂盒(CCK-8)法和细胞划痕实验分别检测转染Lnczc3h7a mimics和Lnczc3h7a inhibitor后结直肠癌细胞的增殖和迁移能力,免疫印迹试验(Western blot)法检测结直肠癌细胞SW60中胶原三股螺旋重复蛋白6(CTHRC6)的表达。分别使用过表达体系和siRNA分别改变细胞系中Lnczc3h7a和CTHRC6)的表达,检测结直肠癌细胞的增殖和迁移能力。再采用CRISP-Cas9技术敲除Lnczc3h7a和CTHRC6),检测结直肠癌细胞的增殖和迁移能力。结果HT-29、HCT-116、SW-60、COLO320DM、Lovo和DLD-1中的Lnczc3h7a表达水平分别为0.65±0.02、0.67±0.02、0.84±0.04、0.58±0.02、0.70±0.02、0.52±0.30,均显著低于正常结直肠上皮细胞系(1.00±0.00,F=5.298、5.462、8.172、4.698、7.012、4.463,P值均<0.05),差异均有统计学意义。细胞转染后第3、4、5天,模拟物组(0.33±0.02、0.58±0.01、0.65±0.02)及对照组(0.41±0.05、0.71±0.02、0.90±0.04)结直肠癌细胞增殖能力均低于抑制剂组(0.51±0.03、0.88±0.04、1.07±0.08),且模拟物组结直肠癌细胞增殖能力低于对照组(F=5.104、6.873、8.284,P<0.05),差异均有统计学意义。细胞培养24、48 h时,模拟物组(184.92±34.82、157.71±27.47)及对照组(411.86±62.35、412.94±63.19)的结直肠癌细胞迁移能力低于抑制剂组(466.92±47.37、470.83±47.82),且模拟物组结直肠癌细胞迁移能力低于对照组(F=1.482、5.104,P值均<0.05),差异均有统计学意义。细胞转染24 h时模拟物组及对照组的CTHRC6蛋白表达分别为0.31±0.04、0.62±0.13,均低于抑制剂组(0.86±0.17),且模拟物组细胞CTHRC6蛋白表达低于对照组(t=41.772,P值均<0.05),差异均有统计学意义。结论结直肠癌细胞中Lnczc3h7a存在明显低表达,其可能具有抑制结直肠癌细胞增殖、迁移的作用,且作用机制可能和抑制CTHRC6蛋白表达密切相关。

Expression of lnczc3h7a in colorectal cancer cells and its mechanism on proliferation and migration of tumor cells

Objective To study the expression of lnczc3h7a in colorectal cancer(CRC)cells and its mechanism on the proliferation and migration of tumor cells.Methods Atotal of 6 CRC cell lines(ht-29,hct-116,sw-60,COLO320DM,Lovo and dld-1)were selected to be paired with normal colorectal epithelial cell lines.Colorectal cancer cells SW60 were transfected with lnczc3h7a mimics,lnczc3h7a inhibitor and blank control,respectively.The proliferation and migration ability of CRC cells transfected with lnczc3h7a mimics and lnczc3h7a inhibitor were detected by cell counting kit-8(CCK-8)and cell scratch test,respectively.The expression of collagen-triple helix repeat protein 6(CTHRC6)in CRC cells SW60 was detected by Western blotting.The expression of lnczc3h7a and CTHRC6 in cell lines was altered using the overexpressed system and small interfering RNA(siRNA),respectively,and then the proliferation and migration ability of CRC cells were measured.lnczc3h7a and CTHRC6 were knocked out by crisp-cas9 technique,and the proliferation and migration ability of colorectal cancer cells were measured.Results The expression levels of lnczc3h7a in ht-29,hct-116,sw-60,COLO320DM,Lovo and dld-1 cells were 0.65±0.02,0.67±0.02,0.84±0.04,0.58±0.02,0.70±0.02 and 0.52±0.30,respectively,which were significantly lower than those in normal colorectal epithelial cell lines(1.00±0.00,F=5.298,5.462,8.172,4.698,7.012,4.463,all P<0.05).At 3rd,4th and 5th day after transfection,the proliferation capacity of CRC cells in the mimic group(0.33±0.02,0.58±0.01,0.65±0.02)and the control group(0.41±0.05,0.71±0.02,0.90±0.04)was lower than that in the inhibitor group(0.51±0.03,0.88±0.04,1.07±0.08),and the proliferation capacity of CRC cells in the mimic group was lower than that in the control group(F=5.104,6.873,8.284,all P<0.05).At 24th and 48th h of cell culture,the migration ability of CRC cells in the mimic group(184.92±34.82,157.71±27.47)and control group(411.86±62.35,412.94±63.19)was lower than that in the inhibitor group(466.92±47.37,470.83±47.82),and the migration ability of CRC cells in the mimic group was lower than that in the control group(F=1.482,5.104,all P<0.05).At 24th h after cell transfection,CTHRC6 protein expression in the mimic group and control group was(0.31±0.04)and(0.62±0.13)respectively,lower than that in the inhibitor group(0.86±0.17),and CTHRC6 protein expression in the mimic group was lower than that in the control group(t=41.722,all P<0.05).Conclusion Lnczc3h7a expression in CRC cells is significantly low,which may inhibit the proliferation and migration of CRC cells,and the action mechanism may be closely related to the inhibition of CTHRC6 protein expression.