AVIL过表达载体构建及对GES-1细胞株增殖及迁移影响

Construction of AVILoverexpression vector and primarily investigation on its proliferation and migration of GES-1 cell lines

目的生物学分析显示AVIL基因表达与胃癌患者预后密切相关,本研究旨在探讨胃正常粘膜细胞AVIL基因过表达对其增殖迁移的影响,为进一步研究AVIL在胃癌中的作用机制提供参考。方法正常人胃黏膜上皮细胞GES-1购自中国科学院细胞库。采用PCR技术扩增目的基因,经酶切将目的基因转入GV358载体中,得到重组慢病毒载体,用PCR和测序对其进行鉴定后与慢病毒包装系统质粒共同转染293T细胞。用重组病毒颗粒感染GES-1细胞株,筛选过表达AVIL的细胞株。qRT-PCR检测稳转细胞AVIL mRNA的表达水平,CCK-8、划痕和Transwell迁移实验检测AVIL过表达后的细胞增殖迁移能力,统计数据以x-±s表示,多组间均数比较采用单因素方差分析,组内两两比较采用t检验。结果测序结果显示,AVIL过表达慢病毒载体构建成功,GV358-AVIL重组病毒和GV358-NC病毒液浓度滴度是1×10^8 TU/mL,转染GES-1后荧光显微镜下可观察到绿色荧光蛋白的表达。PCR结果显示,转染AVIL过表达组AVIL表达水平均明显增高,均P<0.001。GES-1-AVIL组24、48和72h增殖能力分别为(197.18±1.87)%、(297.91±3.62)%和(574.84±20.35)%,GES-1组分别为(150.48±11.28)%、(238.72±12.44)%和(349.45±16.70)%,GES-1-NC组分别为(160.59±3.92)%、(260.23±6.81)%和(386.88±9.96)%,GES-1-AVIL组和GES-1组、GES-1-NC组差异均有统计学意义,均P<0.05。GES-1-AVIL组细胞愈合率为(59.0±5.6)%,高于GES-1组的(20.3±1.5)%和GES-1-NC组的(17.6±4.1)%,P<0.001。GES-1-AVIL穿过上室的细胞数为81.0±4.0,高于GES-1组的39.7±7.0和GES-1-NC组的39.3±1.5,P<0.01。结论成功克隆、扩增AVIL基因,构建过表达AVIL慢病毒载体,该载体能稳定转染GES-1细胞株并对其增殖迁移有促进作用,为后续AVIL在胃癌细胞中的相关研究奠定基础。

OBJECTIVE Biological analysis showed that AVILexpression was closely related to the prognosis of gastric cancer patients.This paper aimed to explore the effect of AVILgene overexpression on the proliferation and migration of gastric normal mucosal cells,so as to lay a foundation for further study on the mechanism of AVILin gastric cancer.METHODS Normal gastric mucosa epithelial cells GES-1 were purchased from the cell bank of Chinese Academy of Sciences.Polymerase chain reaction(PCR)was used to amplify the code region of AVIL.The amplified AVILfragment and GV358 vector were digested with restriction enzymes.The recombinant lentivirus vector was obtained,identified by PCR and DNA sequencing.GES-1 cell lines were transfected with recombinant lentivirus vector and cell line with stable AVILover-expressing was established.Then detect the expression of AVILby Real Time qPCR and access the cell proliferation and migration by CCK-8 assay and wound-healing assay.RESULTS Sequencing results showed that the over-expressed AVILlentivirus vector was successfully constructed.GV358 and GV358-NC-AVILrecombinant viruses poison concentration drops was 1×10^8 TU/ml.Transfection GES-1 could be observed under fluorescence microscope after the expression of green fluorescent protein.The PCR results showed transfection AVILexpression level in expressed group were significantly higher(P<0.001).The proliferative ability of ges-1-AVILgroup was enhanced at 24,48 and 72 hcompared with the GES-1 and GES-1-NC group,and the difference was statistically significant(both P<0.05).In addition,the cell healing rate of GES-1-AVILgroup was higher than that of GES-1 group and GES-NC group〔(59.0±5.6)%vs(20.3±1.5)%,(59.0±5.6)%vs(17.6±4.1)%,P<0.001〕.The number of GES-1-AVILgroup cells passing through the upper compartment was higher than that in GES-1 group and GES-NC group(81.0±4.0 vs 39.7±7.0,81.0±4.0 vs39.3±1.5,P<0.01).CONCLUSION The AVILgene was successfully cloned and amplified,and an over-expressed AVILlentivirus was constructed,which could stably transfected the GES-1 cell line and promote its proliferation and migration,laying a foundation for the subsequent studies on AVILin gastric cancer cells.