The Role And Mechanism Of Long Non-coding RNA In The Occurrence And Development Of Gastric Cancer

IntroductionGastric cancer is a malignant cancer of gastroenterology system with high incidence rate and high mortality rate.There are about 1 million cases of gastric cancer diagnosed worldwide every year and gastric cancer has been a huge threat to human health and life quality.The mechanism of gastric cancer is very complex and it is regulated by many factors.Although the clinical treatment and basic study have achived some progress,the specific mechanism of gastric cancer has not been illustrated and the effect of clinical treatment is not good.So the fundermental molecular regulating gastric cancer is the key point of finding new way to diagnosis and treat gastric cancer.Long non-coding RNA(lncRNA)is a RNA longer than 200bp and it is usually transcrited by RNA polymerase ?(RNAP ?).It can not translated protein,but it can regulate transcription,translation,and epigenetics.Now,lncRNA is found to play the important role in many diseases.LncRNA has been proved to regulate many cancers,including lung cancer,gastric cancer,liver cancer,breast cancer,and prostate cancer,et al.With the progress of microarray,more and more scientists get the whole lncRNA profile.H19 is a very important IncRNA in cancer study.It is as long as 2.3kb,and it is at llp15.5 of human chrosome.The role of H19 in cancer is still controversial.In the beginning,H19 was proved to incrase cancer proliferation rate and accelerate cancer invation.Hoever,some recent study found that H19 promoted cancer.So it is necessary to study how H19 regulates cancer.Objective1.To screen the abberrant IncRNA expression profile in gastric cancer.2.To find a new target of H19/miR-675.3.To validate the role of H19/miR-675 in proliferation and apoptosis of gastric cancer.4.To study how H19/miR-675 regulate target gene and the downstreat pathway.Methods1.RNA was extracted from 6 gastric cancer tissues and its non-cancerous tissues and we conduct quality measurement.2.We screen significantly different expressed lncRNA in samples with high quality.3.We conducted GO analysis,KEGG analysis,and transctritional facter analysis to find the molecular mechanism of these screened lncRNA.4.Bioinformation analysis website was used to predict potential target gene of miR-675.5.MiR-675 mimic and inhibitor were constructed and used to transfect cells.RT-PCR and WB were used to detect expression level of the target gene.6.H19 plasmid and siRNA were constructed and used to transfect cells.We then used RT-PCR to measure expression level of the target gene.7.We construceted wild type and mutant type luciferase plasmid and co-transfected them with H19 plasmid,H19 inhibitor,miR-675 mimic,miR-675 inhibitor,and their controls.Then we measure the level of fluorescence.8.RT-PCR was conducted to measure H19,miR-675,and target gene expression in gastric cancer and paracancer tissures.We also measure H19,miR-675,and target gene expression in normal gastric epithelial cell,gastric cancer cell,and cisplatin resistant gastric cancer cell line.9.We transfect H19 plasmid and siRNA to check the role of H19 in proliferation and apoptosis of gastric cancer.In addition,miR-675 inhibitor and mimic were co-transfected to see if H19 regulates gastric cancer partially via producing miR-675.10.We transfect miR-675 inhibitor and mimic to check the role of miR-675 in proliferation and apoptosis of gastric cancer.In addition,plasmid and siRNA of target gene were co-transfected to see if miR-675 regulates gastric cancer partially via regulating the target gene.11.Tumor formation experiment in nude mice was used to observe the influence of H19 and miR-675 on gastric cancer cell proliferation and apoptosis in vivo.Gastric cancer cells infected with LV-nc,LV-shH 19,and LV-shH19+LV-miR675 were injected subcutaneous and we measure the tumor growth and the weight after 28 days.12.WB was used to validate the target gene of H19/miR-675 and downstream pathway.Results1.The six pairs of gastric cancer tissue samples were valided of high qulity.2.LncRNA microarray revealed that 1581 lncRNAs were significantly differently expressed,including 422 up-regulated IncRNAs and 1159 down-regulated IncRNAs.3.We did GO analysis,KEGG analysis,and transctrition factor analysis,and found differently expressed genes were rich in cell cycle.In addition,many associated important transcritption factors were found,such as JUND,E2F1,SP1,MYC,IRF3.4.We predicted FADD as the target of miR-675 with the help of TargetScan.5.We found both RNA and protein of FADD were inversely asscociated with miR-675.6.Both RNA and protein of FADD were found inversely asscociated with H19.7.We construceted FADD wild type luciferase plasmid and co-transfected them with H19 plasmid,H19 inhibitor,miR-675 mimic,miR-675 inhibitor,and their controls.Then we measure the level of fluorescence and found both H19 and miR-675 could decrease the level of fluorescence.However,the phenomenon disappeared for the mutant type of luciferase plasmid.8.RT-PCR was conducted to measure H19,miR-675,and target gene expression in gastric cancer and paracancer tissures.We found both H19 and miR-675 were up-regulated in gastric cancer,but FADD was decreased.We also measure H19,miR-675,and target gene expression in normal gastric epithelial cell,gastric cancer cell,and cisplatin resistant gastric cancer cell line.We found both H19 and miR-675 were up-regulated in gastric cancer cell lines,but FADD was decreased.9.We found H19 could increase proliferation and inhibit apoptosis of gastric cancer.In addition,H19 regulates gastric cancer partially via producing miR-675.10.We found miR-675 could increase proliferation and inhibit apoptosis of gastric cancer.In addition,miR-675 regulates gastric cancer partially via targeting FADD.11.Low-expression of H19 inhibit proliferation of gastric cancer cells in vivo,and co-infection of miR-675 could promote gastric cancer cell proliferation.In addition,low-expression of H19 decreased tumor weight and co-infection of miR-675 could increase tumor weight.12.We found H19/miR-675 could regulate FADD and its downstream caspase pathway to regulate proliferation and apoptosis of gastric cancer.Conclusions1.LncRNA microarray revealed that 1581 lncRNAs were significantly differently expressed,and differently expressed genes were associated with cell cycle and many important transcritption factors.2.FADD was validated to be the target of H19/miR-675.3.H19/miR-675 promoted gastric cancer proliferation and inhibited apoptosis both in vivo and in vitro.4.H19/miR-675 could regulate FADD and its downstream caspase pathway to regulate proliferation and apoptosis of gastric cancer.