The Role And Mechanism Of LncRNA LIFR-AS1 In Gastric Cancer

Objective: This study aims to analyze the expression of lncRNA LIFR-AS1 in gastric cancer and the relationship between it and the prognosis of patients,and to explore the influence of lncRNA LIFR-AS1 on gastric cancer and the molecular mechanism involved,so as to provide a reliable theoretical basis and a new direction for clinical diagnosis,treatment and prognosis evaluation of gastric cancer.Methods:1.In the first part,RNA-Seq sequencing analysis showed that there was down-regulation of lncRNA LIFR-AS1 expression in gastric cancer;Through sample expression analysis,the expression changes of lncRNA LIFR-AS1 in gastric cancer samples,gastric cancer tissues and cell lines in the database were further demonstrated.2.In the second part,the function of lncRNA LIFR-AS1 in gastric cancer was analyzed,and the influence of lncRNA LIFR-AS1 expression on the proliferation,apoptosis,invasion,metastasis and tumor formation of gastric cancer cells was mainly analyzed through overexpression of LncRNA LIFR-AS1.3.In the third part,the specific mechanism of the action of lncRNA LIFR-AS1 on gastric cancer was explored by means of molecular biology and cell biology.Results:1.In the first part of the study,in order to identify gene differential expression profiles in gastric cancer,tissue microarray analysis was performed on 4 groups of gastric cancer tissues and their corresponding paracancerous tissues.Compared with paracancerous tissues,we found that lncRNA LIFR-AS1 was low expressed in gastric cancer.In addition,we also found that miR-4698,Cdc25 B,CD133,SOX2,NANOG,ER and other genes were highly expressed in gastric cancer.In order to further demonstrate our sequencing the reliability of the results,we by qPCR further examined the cancer of the stomach in the corresponding tissue adjacent to carcinoma tissues and the expression of these genes in RNA level changes,the results showed that with the sequencing results are consistent,lncRNA LIFR-AS1 show lower expression in gastric cancer,miR-4698 and Cdc25 B,CD133,SOX2,NANOG,ER gene is highly expressed in gastric cancer.These results indicate that the transformation of gastric tissue from normal state to aggressive malignant gastric cancer not only has the change of coding protein genes,but also has the change of a large number of non-coding RNA(lncRNA,miRNA),among which the change of lncRNA LIFR-AS1 is particularly obvious.Next,we will focus on the study of lncRNA LIFR? AS1 to explore the significance of lncRNA in the occurrence and development of gastric cancer and its specific molecular mechanism.In order to study the biological role of lncRNA LIFR-AS1 in gastric cancer,gene expression data of normal and tumor tissues were obtained from TCGA.Through analysis,we found that lncRNA LIFR-AS1 was down-regulated in gastric cancer tissues compared with normal tissues,and the difference was statistically significant(P<0.05).The expression of lncRNA LIFR-AS1 in 41 GC tissues and adjacent tissues was detected by RT-qPCR,and the results showed that lncRNA LIFR-AS1 was indeed low expressed in GC tissues,and the difference was statistically significant(P<0.05).We also further analyzed the relationship between the expression of lncRNA LIFR-AS1 and the clinical characteristics of gastric cancer,and the results showed that lncRNA LIFR-AS1 was down-regulated more significantly in gastric cancer tissues with lower differentiation,higher malignant degree and more obvious lymphatic metastasis,with statistical significance(P<0.05).Subsequently,we used the TCGA database to analyze the relationship between the expression of lncRNA LIFR-AS1 and the overall survival time of gastric cancer patients.The results showed that the overall survival time of patients with high expression of lncRNA LIFR-AS1 was significantly higher than that of gastric cancer patients with low expression of lncRNA LIFR-AS1,and the difference was statistically significant(P<0.05).In addition,we also analyzed the expression of lncRNA LIFR-AS1 in normal gastric mucosal cells GES-1 and gastric cancer cell lines(MKN45 and AGS),and the results showed that the expression of lncRNA LIFR-AS1 in MKN45 and AGS cells was lower than that in GES-1 cells,with statistical significance(P<0.05).These results suggested that the expression of lncRNA LIFR-AS1 was negatively correlated with the occurrence and progression of gastric cancer,and lncRNA LIFR-AS1 could inhibit the progression of gastric cancer.2.In the second part of the study,we transfected LncRNA LIFR-AS1 into gastric cancer cells MKN45 and AGS by Lipofectamine?3000transfection method,and detected the expression of LncRNA LIFR-AS1 by qPCR.The results showed that,compared with the control group,the expression of lncRNA LIFR-AS1 in MKN45 and AGS was significantly up-regulated after transfection,and the difference was statistically significant(P<0.05).Next,we will use these two gastric cancer cell lines overexpressing lncRNA LIFR-AS1 to detect cell biological functions such as proliferation,apoptosis,invasion and metastasis,and tumor formation.We used CCK-8 and Ed U assay to detect the effect of overexpression of LncRNA LIFR-AS1 on proliferation of MKN45 and AGS in gastric cancer cells.The results showed that compared with the control group,the overexpression of lncRNA LIFR-AS1 could significantly inhibit the proliferation of gastric cancer cells,and the difference was statistically significant(P<0.05).This suggested that the overexpression of lncRNA LIFR-AS1 could inhibit the proliferation of gastric cancer cells.The effect of overexpression of lncRNA LIFR-AS1 on the clonal formation of MKN45 and AGS in gastric cancer cells was detected by clonal formation assay.The results showed that,compared with the control group,the overexpression of lncRNA LIFR-AS1 significantly inhibited the formation of gastric cancer cell clones,and the difference was statistically significant(P<0.05).This suggested that the overexpression of lncRNA LIFR-AS1 could inhibit the clonal formation of gastric cancer cells.We used flow cytometry to detect the effect of overexpression of lncRNA LIFR-AS1 on apoptosis of MKN45 and AGS in gastric cancer cells.The results showed that compared with the control group,the overexpression of lncRNA LIFR-AS1 significantly promoted the apoptosis of gastric cancer cells,and the difference was statistically significant(P<0.05).This suggests that the overexpression of lncRNA LIFR-AS1 can promote the apoptosis of gastric cancer cells.Transwell assay was used to detect the effect of overexpression of lncRNA LIFR-AS1 on invasion of MKN45 and AGS in gastric cancer cells.The results showed that compared with the control group,the overexpression of lncRNA LIFR-AS1 could significantly inhibit the invasion of gastric cancer cells,and the difference was statistically significant(P<0.05).This suggested that the overexpression of lncRNA LIFR-AS1 could inhibit the invasion of gastric cancer cells.The effect of overexpression of lncRNA LIFR-AS1 on migration of MKN45 and AGS in gastric cancer cells was detected by cell scratch test.The results showed that compared with the control group,the overexpression of lncRNA LIFR-AS1 could significantly inhibit the migration of gastric cancer cells,and the difference was statistically significant(P<0.05).This suggested that the overexpression of lncRNA LIFR-AS1 could inhibit the migration of gastric cancer cells.The effect of lncRNA LIFR-AS1 expression on tumor formation of MKN45 cells was detected in a subcutaneous tumor formation model in nude mice,apoptosis of tumor cells was detected by TUNEL staining,and proliferation of tumor cells was detected by Ki67 staining.The results showed that,compared with the control group,the overexpression of lncRNA LIFR-AS1 significantly inhibited the formation of tumors in MKN45 cells,and the Ki67 signal of tumor cells was significantly decreased and the TUNEL signal was significantly increased after the overexpression of lncRNA LIFR-AS1,with statistical significance(P<0.05).These results indicate that lncRNA LIFR-AS1 can inhibit the formation of gastric cancer cells,inhibit the proliferation of gastric cancer cells,and promote the apoptosis of gastric cancer cells.3.In the third part of the study,we predicted the predicted binding sites of miR-4698 in lncRNA LIFR-AS1.In order to verify the sequence binding sites predicted by lncRNA LIFR-AS1 and miR-4698,luciferase reporter gene assay was performed to further confirm their association.The results showed that the luciferase activity of MKN45 and AGS cells was decreased after transfection of LIFR-AS1-WT and miR-4698 simulants,and the difference was statistically significant(P<0.05).Notably,overexpression of lncRNA LIFR-AS1 decreased the expression of miR-4698 in MKN45 and AGS cells,with statistical significance(P<0.05).In addition,compared with normal tissues and cells,the expression of miR-4698 was up-regulated in gastric cancer tissues and gastric cancer cell lines,with statistical significance(P<0.05).Pearson correlation test showed that lncRNA LIFR-AS1 was negatively correlated with miR-4698.These results indicate that lncRNA LIFR-AS1 is negatively correlated with miR-4698,and lncRNA LIFR-AS1 may inhibit the occurrence of gastric cancer by down-regulating miR-4698.Target Scan reveals that the 3’-UTR of MTUS1 contains the complementary region of miR-4698.The changes in luciferase activity were evaluated after co-transfection with miR-4698 mimics and MTUS1-WT or MTUS1-MUT fragments.The results showed that luciferase activity was inhibited in cells co-transfected with miR-4698 mimics and MTUS1-WT,and the difference was statistically significant(P<0.05).The correlation between miR-4698 and MTUS1 was monitored after transfection of gastric cancer cells with miR-4698 overexpression and inhibitory vectors(miR-4698 mimics and miR-4698 inhibitors).The expression of miR-4698 was significantly enhanced after the transfection of miR-4698 mimics,while the expression of miR-4698 was inhibited by the transfection of miR-4698 inhibitors.Western-blot results showed that miR-4698 overexpression could inhibit the expression of MTUS1,while miR-4698 inhibition could increase the expression of MTUS1,and the difference was statistically significant(P<0.05).In addition,qPCR detection showed that compared with normal tissues and cells,the expression of MTUS1 was down-regulated in gastric cancer tissues and gastric cancer cell lines(MKN45 and AGS),and the difference was statistically significant(P<0.05).Pearson correlation test further confirmed that there was a negative correlation between MTUS1 and miR-4698,and a positive correlation between MTUS1 and lncRNA LIFR-AS1.These results indicate that MTUS1 is a new potential target gene of miR-4698 and is also positively regulated by lncRNA LIFR-AS1.Cells were transfected with pc DNA-LIFR-AS1 and si-MTUS1 expression vectors to explore the changes in downstream signaling pathways.Notably,compared with the control group,overexpression of lncRNA LIFR-AS1 significantly inhibited the phosphorylation of MEK and ERK in gastric cancer cell lines(MKN45 and AGS),with statistical significance(P<0.05).Next,we transfected gastric cancer cells with si-MTUS1 vector to inhibit the expression of MTUS1 and explore the relationship between lncRNA LIFR-AS1 and MTUS1.The results showed that,compared with the control group,the overexpression of lncRNA LIFR-AS1 could significantly up-regulate the expression of MTUS1 in gastric cancer cell line MKN45 and AGS cells,with statistical significance(P<0.05).In contrast,inhibition of MTUS1 expression significantly increased the phosphorylation levels of MEK and ERK,and the difference was statistically significant(P<0.05).In addition,si-MTUS1 reversed the phosphorylation of MEK and ERK in gastric cancer cell lines(MKN45 and AGS)after co-transfection with pc DNA-LIFR-AS1 and si-MTUS1,and the difference was statistically significant(P<0.05).These results suggest that the overexpressed lncRNA LIFR-AS1 in gastric cancer cells can inhibit the activation of the MEK/ERK signaling pathway by down-regulating MTUS1.We also found that overexpression of lncRNA LIFR-AS1 inhibited CDC25 B expression in gastric cancer cell lines(MKN45and AGS)compared to control cells.Compared with the control group,the overexpression of lncRNA LIFR-AS1 promoted the phosphorylation level of CDK1 in gastric cancer cell lines(MKN45 and AGS),and the difference was statistically significant(P<0.05).In addition,si-MTUS1 reversed CDC25 B expression inhibition and CDK1 phosphorylation induction in gastric cancer cell lines(MKN45 and AGS)after co-transfection with pc DNA-LIFR-AS1 and si-MTUS1.These results suggest that lncRNA LIFR-AS1 may promote CDK1 phosphorylation by inhibiting CDC25 B activity.We analyzed the effect of lncRNA LIFR-AS1 expression on the activity of MEK/ERK signaling pathway in tumor tissues formed by MKN45 by Western-blot and immunohistochemistry.The results showed that,compared with the control group,overexpression of lncRNA LIFR-AS1 not only promoted the expression of MTUS1,but also inhibited the phosphorylation of MEK and ERK,further indicating that lncRNA LIFR-AS1 inhibited the activation of MEK/ERK signaling pathway by upregulating MTUS1.Conclusions: LncRNA LIFR-AS1 is abnormally low expressed in gastric cancer and is associated with the prognosis of patients.In gastric cancer,lncRNA LIFR-AS1 inhibited the activation of the MEK/ERK signaling pathway by down-regulating miR-4698 and upregulating MTUS1,promoted cell apoptosis,and inhibited cell proliferation,invasion and metastasis,as well as tumor formation.Therefore,lncRNA LIFR-AS1 could be a potential target for the treatment of gastric cancer.