m6A甲基转移酶KIAA1429转染对结肠癌细胞增殖的影响及机制探讨

Effect and mechanism of m6A methyltransferase KIAA1429 transfection on the proliferation of colon cancer cells

目的 观察m6A甲基转移酶KIAA1429转染对结肠癌细胞增殖的影响,并探讨其机制。方法 通过慢病毒转染的方法在人结肠癌细胞RKO中构建稳定敲低KIAA1429的对照组和实验组细胞系,将其分别命名为sh-NC组、shKIAA1429组,在人结肠癌细胞LS180中构建稳定过表达KIAA1429的对照组和实验组细胞系,将其分别命名为OE-NC组、OE-KIAA1429组。采用Western blotting法检测KIAA1429蛋白来验证转染效率,采用CCK-8实验和平板克隆实验检测KIAA1429转染对结肠癌细胞增殖的影响,通过差异基因的富集分析预测KIAA1429转染对结肠癌细胞增殖的影响机制,采用流式细胞术检测KIAA1429转染对细胞周期的影响,采用Western blotting法观察OE-NC组和OE-KIAA1429组细胞内E2F1、CyclinB1、p21蛋白变化。结果 sh-NC组、sh-KIAA1429组KIAA1429蛋白相对表达量分别为0.979±0.028、0.299±0.138,两组比较P=0.002;OE-NC组、OE-KIAA1429组KIAA1429蛋白相对表达量分别为0.446±0.104、0.899±0.153,两组比较P=0.026。CCK-8实验和平板克隆实验结果显示,KIAA1429显著促进了结肠癌细胞的增殖(P均<0.05)。KEGG富集分析提示差异基因主要于细胞周期相关的信号通路得到富集,GO富集分析提示差异基因的功能与遗传物质的复制及细胞周期相关。流式细胞术结果显示,RKO细胞中sh-NC组和sh-KIAA1429组的G0/G1期所占百分比分别为52.07%±1.00%、58.57%±0.98%,两组相比P=0.003;LS180细胞中OE-NC组和OE-KIAA1429组的G0/G1期所占百分比分别为68.37%±0.82%、62.40%±1.34%,两组相比P=0.006。OE-NC组CyclinB1、E2F1、p21蛋白相对表达量分别为0.491±0.116、0.661±0.132、1.007±0.058,OE-KIAA1429组分别为1.254±0.169、1.125±0.135、0.725±0.110,两组相比P均<0.05。结论 m6A甲基转移酶KIAA1429通过调控细胞周期信号通路进而促进结肠癌细胞增殖。

Objective To observe the effect of m6A methyltransferase KIAA1429 transfection on the proliferation of colon cancer cells and to investigate its mechanism.Methods The control group and experimental group cell lines stably knockdown of KIAA1429 were constructed in human colon cancer cell RKO by lentivirus transfection,which were named sh-NC group and shKIAA1429 group respectively.The control and experimental group cell lines with stable overexpression of KIAA1429 were constructed in human colon cancer cell LS180,which were named OE-NC group and OE-KIAA1429 group respectively.Transfection efficiency was detected by Western blotting.The effect of KIAA1429 transfection on cell proliferation activity was detected by CCK-8 assay and plate cloning experiment.The potential mechanism of the effect of KIAA1429 transfection on the proliferation of colon cancer cells was predicted by enrichment analysis of differential genes.The effect of KIAA1429 transfection on cell cycle was detected by flow cytometry.Changes of the expression of E2F1,Cy⁃clinB1 and p21 in OE-NC group and OE-KIAA1429 group were detected by Western blotting.Results The relative ex⁃pression of KIAA1429 protein in sh-NC group and sh-KIAA1429 group were 0.979±0.028 and 0.299±0.138,respec⁃tively,P=0.002.The relative expression of KIAA1429 protein in OE-NC group and OE-KIAA1429 group were 0.446±0.104 and 0.899±0.153,respectively,P=0.026.The results of CCK-8 assay and plate cloning experiment showed that KIAA1429 significantly promoted the proliferation of colon cancer cells(all P<0.05).KEGG enrichment analysis showed that the differential genes were mainly enriched in the signal pathway related to cell cycle,and GO enrichment analysis showed that the function of differential genes was mainly related to the replication of genetic material and cell cycle.The re⁃sults of flow cytometry showed that the percentages of G0/G1 phase in sh-NC group and sh-KIAA1429 group were 52.07%±1.00%and 58.57%±0.98%,respectively,P=0.003.The percentages of G0/G1 phase in OE-NC group and OE-KI⁃AA1429 group were 68.37%±0.82%and 62.40%±1.34%,respectively,P=0.006.The relative expression levels of cyclinB1,E2F1 and p21 protein in OE-NC group were 0.491±0.116,0.661±0.132 and 1.007±0.058,the proteins in OE-KIAA1429 group were 1.254±0.169,1.125±0.135 and 0.725±0.110 respectively,there were statistically sig⁃nificant between the two groups(all P<0.05).Conclusion The m6A methyltransferase KIAA1429 can promote the pro⁃liferation of colon cancer cells by regulating cell cycle signal pathway.