| BackgroundOesophageal carcinoma(EC)is the sixth leading cause of cancer-related mortality and the eighth most common cancer worldwide.It accounts for the fourth highest incidence of malignancy in China and is one of the best malignant tumors.LncRNA has become a key regulator of cancer.LincRNA-p21 is a transcript downstream of the p53 gene.Studies have shown that the abnormal expression of LincRNA-p21 plays an important role in the development of a variety of tumors,but its mechanism in esophageal cancer is still unknown.This study intends to explore the relationship between LincRNA-p21 and the pathogenesis of EC.Objectives1.To compare the expression of LincRNA-p21 in EC cells EC109,EC9706 with esophageal immortalized epithelial cells and the expression of LincRNA-p21 in EC tissues with adjacent tissues,and to explore the relationship between LincRNA-p21 and EC risk factors.2.To analysis affect of LincRNA-p21 on the biological function of EC cell line EC109.3.To explore the mechanism of LincRNA-p21 regulating target p21 protein on the development of EC so that provide more experimental basis for basic research of EC Methods1.The expression of LincRNA-p21 in EC and its clinical significance64 patients with pathologically diagnosed EC patients were recruited from the First People's Hospital of Huai'an City during 2011-2012.The expression of LincRNA-p21 inEC cells EC109,EC9706 and EC immortalized epithelial cells Het-1A were detected by RT-qPCR,as well as the levelof LincRNA-p21 in EC tissues and the cerresponding adjacent non-tumortissues.Then correlation between the expression of LincRNA-p21 and the clinical pathological parameters of EC was analyzed conditional logistic regression and chi-square analysis.2.Bioinformatics prediction of LincRNA-p212.1 Predict LincRNA-p21 target gene using on-line lncRNA database such as ChIPBase,NONCODE,starBase,LncRNADisease,DIANA-LncBase,Genecards and so on.2.2 Implicating bioinformatics analysis software: catRAPID(http://service.tartaglialab.com/page/catRAPID).The binding of p21 to LincRNA-p21 was predicted online,and the binding ability and binding reliability were analyzed.3.Effect of LincRNA-p21 on the biological function of EC EC109 cells3.1 EC109 ells were transfected with defective lentivirus targeting LincRNA-p21 or control lentivirus to determine the effect of LincRNA-p21 expression on tumorigenesis.Promycin was used to screen and RT-qPCR to verify the transfection efficiency.To evaluate the effects of LincRNA-p21 on the biology behaviors of esophageal cancer cells(cell proliferation,apoptosis,cell cycle,cell migration and invasion)were detected by CCK-8,flow cytometry,Transwell migration and invasion.3.2 The distribution of LincRNA-p21 in the nucleus and cytoplasm were analyzed by FISH and nuclear isolation kit.4.The mechanism of LincRNA-p21 affecting the proliferation of esophageal cancer cells4.1 The expression of p21 in EC tissues and cells were detected by RT-qPCR and Western Blot after up-regulating LincRNA-p21 in EC109 cell.4.2 Downstream cell cycle-ralated protein were detected by Western Blot.4.3 The proliferation,apoptosis and cycle changes of EC109 cell were detected using CCK-8,EDU and flow cytometry after p21 inhibited by the inhibitor UC2288.Results:1.The expression of LincRNA-p21 in EC and its clinical significance1.1 The expression of LincRNA-p21 in EC was significantly lower than in matching adjacent non-tumor tissues(P<0.05).The low expression of LincRNA-p21 increased the risk of esophageal cancer(hazard rate,HR=3.76,95% CI 2.20-9.18).Aberrant expression of LincRNA-p21 differs in the distribution of lymph node metastasis and tumor location(P<0.05).However,LincRNA-p21 was not associated with smoking and drinking.1.2 The results of RT-qPCR showed that the levels of LincRNA-p21 were 18.3% in EC109 and32.5% in EC9706 of Het-1A(P<0.05),respectively.The mRNA levels of p21 were 48.0% in EC109 and 42.3% in EC9706 of Het-1A(P<0.05),respectively.LincRNA-p21 increased the level mRNA of p21 to 2.83 times(P<0.05).2.LincRNA-p21 bioinformatics prediction results2.1 LincRNA-p21 was 2882 bp in chr6: 36664421-36667296.The analysis of UCSC gene showed that p21/Cdkn1 a was located downstream of LincRNA-p21.A total of 10 targets of LincRNA-p21 were predicted in the database including 9 proteins and 1 miRNA.There are 12 targets reported in the literature,including 5 protein targets and 7 miRNA targets.2.2 RNA-protein binding prediction software: catRAPID(http://service.tartaglialab.com/page/catrapid_group)online prediction found that LincRNA-p21 at its fragment 1724-2750 nt and p21 protein 20-80 amino acid have the strongest binding ability.3.Effect of LincRNA-p21 on the biological function of EC EC109 cells3.1 The results of CCK-8 and EDU staining showed that the cell proliferation rates of LincRNA-p21 and its negative control group were(64.69±3.78)% and(44.40±1.78)%,respectively.3.2 The results of flow cytometry(PI staining)showed that the cell cycle cell rates of LincRNA-p21 and its negative control group were: G1 phase(59.63±0.22)% and(51.76±1.30)%,S phase 24.80±0.28)% and(30.66±0.64)% respectively;and G2(15.57±0.67)% and(17.57±0.76)%.Statistical analysis showed that the differences were statistically significant(P<0.05).Cells increased in G1 phase but decreased in S phase so that synthesizing cell DNA was inhibited which contribute to inhibition of proliferation of esophageal cancer cell.LincRNA-p21 arrested EC109 cell cycle in the G1/S phase.3.3 Flow cytometry(Annexin V-7AAD/VPC marked)results showed that the apoptotic rates of LincRNA-p21 and its negative control group were(10.43±0.25)% and(4.87±0.78)%respectively;LincRNA-p21 overexpression could inhibit EC cell apoptosis(P<0.05).3.4 Transwell migration assay showed that the number of transmembrane cells in LincRNA-p21 and negative control groups were(20.90±1.57)and(32.70±2.26),respectively.LincRNA-p21 inhibited cell migration.3.5 Transwell chamber assay showed that the number of MMPs in LincRNA-p21 and negative control groups was(11.70±2.35)and(22.20±2.72),respectively.LincRNA-p21 inhibited cell invasion.3.6 Western Blot results showed that LincRNA-p21 promoted the expression of cdc-25 c and p-cdc-2 proteins and inhibited the expression of cyclin D protein.3.7 FISH and nucleoplasm separation RT-qPCR results showed that LincRNA-p21 mainly localized in cytoplasm.4.LincRNA-p21 induced G1/ S arrest in EC109 cells by upregulating p214.1 LincRNA-p21 up-regulated p21 at both mRNA and protein levels.4.2 Western Blot results showed that LincRNA-p21 promoted the expression of cdc-25 c and p-cdc-2 proteins and inhibited the expression of cyclin D protein.4.3 Proliferation and G1/S phase block of EC109 cells overexpressing LincRNA-p21 were reversed after the inhibition of p21 protein.Conclusions:1.The expression of LincRNA-p21 was downregulated in EC tissues and plays a role as tumor suppressor in EC cells.LincRNA-p21 changed the cell cycle distribution to induce G1/S arrest and inhibited the proliferation,migration and invasion ability of EC109 cells.2.Mechanistic studies found that LincRNA-p21 promotes p21 expression at mRNA and protein levels,besides inhibits cyclin D protein expression and promotes cdc-2phosphorylation to induce G1/S arrest of EC cells and thereby inhibits cell proliferation.3.The subcellular localization of LincRNA-p21 by FISH combined with nuclear isolation kit found that it located in the EC109 cytoplasm.In summary,LincRNA-p21 may be a new auxiliary marker for the diagnosis and treatment of esophageal cancer. |
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