RESUMO
Objective:To investigate the effects of regulation of GATA6 expression by long non-coding RNA(lncRNA)GATA6 antisense RNA 1(GATA6-AS1)on the proliferation,apoptosis,and metastasis of 5-fluorouracil(5-FU)resistant gastric cancer(GC)cells SGC7901/5-FU,and to explore the underlying mechanisms. Methods:Parental SGC7901 cells and SGC7901/5-FU cells were used as controls(treated with blank culture medium).Empty vector pcDNA3.1(control for GATA6-AS1 overexpression),recombinant vector pcDNA3.1-GATA6-AS1(for GATA6-AS1 overexpression),shNC(negative control for GATA6-AS1 silencing)or shGATA6-AS1(for GATA6-AS1 silencing)were transfected into SGC7901/5-FU cells by Lipofectamine 2000 to overexpress or silent GATA-AS1.The mRNA expression of GATA6-AS1 and GATA6 in cells of different treatment groups were examined by real-time fluorescence quantitative PCR.After 5-FU treatment,the proliferation,migration,invasion and apoptosis were analyzed by CCK-8 assay,wound-healing assay,Transwell assay,and flow cytometry(FCM)assay,respectively.The protein expression levels of Bax,Bcl-2,Caspase 3 and GATA6 in different treatment groups were examined by Western blotting.The stability of GATA6 mRNA was evaluated after α-amanitin treatment.The relationship between GATA6-AS1 and GATA6 was studied by RNA pull-down experiment.Furthermore,the SGC7901 cell transplantation tumor model was established using Balb/c nude mice,and the grouping was the same as in vitro experiments with 12 mice in each group.The tumor growth was recored and the tumor inhibition rate was calculated after 5-FU treatment.The histopathological changes of tumor tissue in each group was assessed by HE staining.The protein expression of proliferating cell nuclear antigen(PCNA)in tumor tissues of nude mice in each group was detected by Western blotting. Results:Compared with parental SGC7901 cells(control group),the expression of GATA6-AS1 and GATA6 mRNA was significantly downregulated in SGC7901/5-FU cells(P＜0.05);the proliferation,migration and invasion of SGC7901/5-FU cells was significantly decreased while the apoptosis of SGC7901/5-FU cells was significantly decreased(P＜0.05);the protein expression levels of GATA6,Bax and Caspase 3 were significantly decreased while that of Bcl-2 was significantly increased(P＜0.05).Compared with SGC7901/5-FU-pcDNA group,the above changes showed opposite trends in SGC7901/5-FU cells overexpressing GATA6-AS1.Compared with SGC7901/5-FU-shNC group,the expression of GATA6-AS1 and GATA6 mRNA was significantly downregulated in GATA6-AS1-silencing SGC7901/5-FU cells(P＜0.05);the proliferation,migration and invasion of GATA6-AS1-silencing SGC7901/5-FU cells was significantly decreased while apoptosis of GATA6-AS1-silencing SGC7901/5-FU cells was significantly decreased(P＜0.05);the protein expression levels of GATA6,Bax and Caspase 3 were significantly decreased while that of Bcl-2 was significantly increased(P＜0.05).GATA6-AS1 can increase the stability of GATA6 mRNA and positively regulate the expression of GATA6.In vivo characterization showed that,compared with mice transplanted with parental SGC7901 cells,the tumor volume and PCNA protein expression in tumor tissues were significantly increased in mice transplanted with drug resistant SGC7901/5-FU cells after 5-FU treatment(P＜0.05).Mice in SGC7901/5-FU-GATA6-AS1 group showed significantly reduced tumor volume and PCNA protein expression compared with the SGC7901/5-FU-pcDNA group(P＜0.05),and pathological analysis of the tumor tissues revealed milder development of GC in SGC7901/5-FU-GATA6-AS1 group.Whereas,mice in SGC7901/5-FU-shGATA6-AS1 group showed significantly increased tumor volume and PCNA protein expression compared with the SGC7901/5-FU-shNC group(P＜0.05),and histopathological analysis of the tumor tissues revealed severer development of GC in SGC7901/5-FU-shGATA6-ASl group. Conclusion:Overexpression of GATA6-AS1 can promote the expression of GATA6,inhibit the growth of GC cells,and thus reverse the resistance of SGC7901/5-FU cells to 5-FU.