Effects And Regulatory Mechanism Of Long Non-coding RNA AFDN-DT In Proliferation And Metastasis Of Gastric Carcinoma

Background: Gastric cancer is one of the most threatening human tumors,and the mechanism of its development has not been fully elucidated.Current researches show that lnc RNA plays an important role in the development of diseases,especially tumors.Studying the relationship between lnc RNA and gastric cancer will help reveal the regulation of gastric cancer,develop new pathological mechanisms,and provide new targets for clinical diagnosis and treatment.Part one: Screening of gastric carcinoma differential expressed lnc RNAObjective: This study aimed to screen if lnc RNA expressed differentially in gastric cancer tissues.Methods: In this study,the expressions of lnc RNA in normal tissues and gastric cancer tissues were determined by Agilent Human lnc RNA 4*180K,and the differentially expressed lnc RNA was screened and verified by RT-q PCR.Results: The lnc RNA was differentially expressed in the gastric cancer group and the control group.The results were verified in enlarged samples by RT-q PCR.The expressions of lnc RNA AFDN-DT in gastric cancer were significantly lower than those in normal tissues.Conclusion: Lnc RNA is dysregulated in gastric cancer,and lnc RNA AFDN-DT is down-regulated in gastric cancer,which may become new target for clinical diagnosis and treatment.Part two: Relationship between expression of long non-coding RNA AFDN-DT and metastasis and prognosis of gastric carcinomaObjective: This study aimed to investigate the relationship between the expression of lnc RNA AFDN-DT and clinical,pathological features and prognosis of patients with gastric cancerMethods: RT-q PCR was used to detect the expression level of lnc RNA AFDN-DT in cancer tissues of 98 gastric cancer patients and lymph nodes with gastric cancer metastasis samples.The correlation between the expression level of AFDN-DT and clinical,pathological characteristics and prognosis of gastric cancer patients was analyzed.Results: The expression level of AFDN-DT decreased in gastric cancer tissues;and the expression level of AFDN-DT in lymph nodes with gastric cancer metastasis was significantly lower than that in non-metastatic lymph nodes;Advanced TNM staging was associated with lymph node metastasis;gastric cancer patients with low AFDN-DT expression had lower overall survival rates than gastric cancer patients with higher AFDN-DT expression level.Cox proportional hazard regression model analysis indicated that low AFDN-DT expression level is an independent prognostic factor for gastric cancer patients.Conclusion: AFDN-DT is lowly expressed in gastric cancer tissues,and its expression is related to gastric cancer TNM stage and lymph node metastasis.AFDN-DT may be a predictor of poor prognosis of gastric cancer.Part three: Influence of long non-coding RNA AFDN-DT to biological effects of gastric carcinoma cells and mechanism studyObjective: This study aimed to investigate the biological effects of lnc RNA AFDN-DT on gastric cancer cells in vitro and in vivo,and the possible mechanism of AFDN-DT affecting the growth and metastasis of gastric cancer.Methods: The effect of AFDN-DT on the growth of gastric cancer cells was detected with CCK8 kit;the effect of AFDN-DT on the cell cycle was detected using flow cytometry;the scratching test and Transwell test were used to detect the impact of AFDN-DT on the migration and invasion ability of gastric cancer cells.The effect of AFDN-DT on the growth of gastric cancer in vivo was measured in a nude mouse tumor formation model.RNA pull-down experiments and LC-MS/MS analysis were used to analyze the proteins that interacted with AFDN-DT in gastric cancer cells;Ch IRP-seq experiments was performed to explore AFDN-DT binding sites;RNA sequencing was performed to identify target genes regulated by AFDN-DT.Results: We demonstrated that AFDN-DT overexpression inhibited gastric cancer proliferation and metastasis using in vitro and in vivo models.We found that AFDNDT was located in the nucleus and interacted with histones such as HIST1H4 A,HIST1H1C,and HIST1H2 BK.Ch IRP-seq and RNA-seq analysis showed that AFDN-DT directly binded to the promoter region and regulated the expression of genes related to malignant transformation of gastric cancer such as EGR1,JUN and FOS.Conclusion: Tumor suppressor long noncoding RNA AFDN-DT inhibits the proliferation and metastasis of gastric cancer through transcriptional regulation.