Effect Of LncRNA AFAP1-AS1 On Proliferation And Invasion Of Gastric Cancer Cells And Its Correlation With Clinical Prognosis

Background:Gastric cancer is one of the most common malignant tumors.In the world,the incidence and mortality of gastric cancer are the fourth and second respectively.The diagnosis and treatment of gastric cancer is still facing great challenges.It is urgent to further understand the molecular mechanism of gastric cancer and find new biomarkers and therapeutic targets.Noncoding RNA plays an important biological role in cell development,metabolism,tumour and it also plays an important biological role in the occurrence and development of many diseases such as tumor.LncRNAs are non-coding RNAs with a length of more than 200 nucleotides,lacking an open reading framework,with fewer exons and lower expression levels than protein coding genes.Antisense LncRNAsN are a class of LncRNAss transcribed from the opposite strands of protein or non-protein coding genes,which also play an important role in the carcinogenesis and development of tumors.Actin filament-associated protein 1-antisense RNA1 lncRNA AFAP1-AS1 is a kind of antisense LncRNAs,which was first found in Barrett's esophagus and esophageal adenocarcinoma.The high expression of AFAP1-AS1 is related to tumor size,vascular invasion and poor prognosis,and may affect the ability of tumor cell proliferation and promote tumor metastasis.However,there is no evidence that AFAP1-AS1 can affect the development of gastric cancer.Object:To detect the expression of actin filament-associated protein 1-antisense RNA 1(AFAP1-AS1)in tumor tissues and adjacent normal tissues of patients with gastric cancer,and to analyze the correlation between the expression of AFAP1-AS1 and clinical prognosis.To explore the effect of AFAP1-AS1 on the proliferation and invasion of gastric cancer cells,offer a new diagnostic markers and therapeutic targets for gastric cancer.Methods:1.Cancer tissues and adjacent normal tissues(above 5cm)in 91 patients with gastric cancer and their detailed clinical data were collected.The expression of AFAP1-AS1in tumor tissues and adjacent tissues of gastric cancer patients was detected by QRT-PCR method.The correlation between AFAP1-AS1 expression and sex,age,tumor size,lymph node metastasis and TNM staging was analyzed statistically.The correlation between the expression of AFAP1-AS1 and clinicopathological features was analyzed by?~2test.The effect of AFAP1-AS1 expression on survival time was analyzed by K-M survival curve.2.The expression of AFAP1-AS1 in four kinds of human gastric cancer cell lines(AGS,BGC-823,MGC-803,SGC-7901)and human gastric epithelial immortalized cell line(GES-1)was analyzed by QRT-PCR method.3.After transfection of SGC-7901 and BGC-823 cells with plasmid mediated si-AFAP1-AS1-1 and si-AFAP1-AS1-2,the expression of AFAP1-AS1 in BGC-823and SGC-7901 of gastric cancer cells was was detected.To investigate the effect of AFAP1-AS1 on cell proliferation,the OD value of 450nm absorbance was measured by CCK-8 experiment and the growth curve was plotted at3,4,5 days after siRNA interference.4.After transfection of SGC-7901 and BGC-823 cells with plasmid mediated si-AFAP1-AS1-1 and si-AFAP1-AS1-2,the effect of AFAP1-AS1 knockdown on SGC-7901 and BGC-823 cell cycle was observed by flow cytometry.5.After transfecting SGC-7901 and BGC-823 cells with plasmid mediated si-afap1-as1-1 and si-afap1-as1-2,Transwell migration test was used to detect the effect of afap1-as1 knockout on the oriented invasion ability of the cells.6.The expression of EMT related gene E-cadherin and vimentin in SGC-7901 and BGC-823 cells transfected with si-AFAP1-AS1-1 and si-AFAP1-AS1-2 was detected by western blot.Results:1.The mean relative expression of AFAP1-AS1 was 2.15±0.23 in gastric cancer and0.97±0.13 in adjacent normal tissues.The expression level of AFAP1-AS1 in gastric cancer was significantly higher than that in normal paracancerous tissues.There was a positive correlation between the expression of AFAP1-AS1 and lymph node metastasis and the stage of gastric cancer.The expression of AFAP1-AS1 in stage?~?was significantly higher than that in patients with stage?~?(P 0.006).However,there was no significant correlation with other factors,such as age(p=0.190),sex(p=0.876),histological differentiation(p=0.916).The median survival time of patients with low expression of AFAP1-AS1 was 33±2.887m.The median survival time of patients with high expression of AFAP1-AS1 was 19±1.635m.K-M survival curve analysis shows,Compared with the low expression of AFAP1-AS1,the prognosis of patients with high expression of AFAP1-AS1 was worse,and the survival time was shorter.The chi-square value was 14.01,p<0.05,figure 5).2.Compared with immortalized human gastric epithelial cell line GES-1,the expression of Lnc AFAP1-AS1 in four human gastric cancer cell lines(AGS,BGC-823MGC-803 and SGC-7901)increased significantly(P<0.01).3.After transfection of si-AFAP1-AS1-1 and si-AFAP1-AS1-2 into SGC-7901 and BGC-823 cells,the AFAP1-AS1 expression level of SGC-7901 or BGC-823 in gastric cancer cells was significantly inhibited.The results of CCK8 proliferation test showed,compared with The Si-NC group,The difference of 450nm absorbance of SGC-7901and BGC-823 cell groups is obvious at the 3,4,5 days.And the results were statistically significant(P<0.05).These results approved AFAP1-AS1 can promote the proliferation of gastric cancer cells,and knockout AFAP1-AS1 can significantly inhibit the proliferation of gastric cancer cells SGC-7901 or BGC-823.4.After transfection of si-AFAP1-AS1-1 and si-AFAP1-AS1-2 into SGC-7901 and BGC-823 cells,the number of G0/G1 phase increased,meanwhile,S phase and G 2/M phase cells decreased.It was confirmed that AFAP1-AS1 knockout induced G0/G1cell cycle arrest and inhibited S and G 2/M cell cycle progression,suggesting that AFAP1-AS1 could enhance cell proliferation by regulating cell cycle distribution.5.After transfection of si-AFAP1-AS1-1 and si-AFAP1-AS1-2 into SGC-7901 and BGC-823 cells,the transwell assay was used to detect the directional invasion ability of the cells.The results showed that the number of migratory cells decreased significantly after AFAP1-AS1 knockout.Compared with the si-NC group,the difference was statistically significant(P<0.05).6.After transfection of si-AFAP1-AS1-1 and si-AFAP1-AS1-2 into SGC-7901 and BGC-823 cells,the expression of EMT related gene E-cadherin increased and vimentin decreased by Western blot assay.Conclusion:1.LncRNA AFAP1-AS1 is highly expressed in gastric cancer tissues and cell lines,and may play a role of oncogene in the carcinogenesis and development of gastric cancer.2.The expression of LncRNA AFAP1-AS1 was positively correlated with lymph node metastasis and staging in patients with gastric cancer.3.LncRNA AFAP1-AS1 could enhance the proliferation and invasion ability of gastric cancer cells in vitro,and affect the cell cycle distribution of gastric cancer cells.The proliferation of gastric cancer cells after knocking down AFAP1-AS1 was obviously inhibited.4.LncRNA AFAP1-AS1 may enhance the invasiveness of gastric cancer cells by influencing the EMT process,that is,down-regulating E-cadherin and up-regulating the expression of vimentin.5.LncRNA AFAP1-AS1 can be used as a potential biomarker and therapeutic target for gastric cancer.