| Gastric cancer is one of the most common malignant tumors in the world,which seriously endangers human health.Its incidence ranks fifth in the world and its mor-tality ranks third in the world.There are about one million new cases of gastric cancer every year in the world.About 42%of the patients in the developing countries are 2/3 in China.Early diagnosis and treatment are of great significance to the condition and prognosis of patients with gastric cancer.The occurrence and development of gastric cancer involves abnormal regulation of multiple genes and multi-step participation,which eventually leads to abnormal cell biological behavior.It has been found that the genesis and development of gastric cancer are closely related to genes.The genetic correlation between some genes in gastric cancer patients and normal people has changed.These mutated genes may be the key to the occurrence of gastric cancer.When abnormal cell behavior is based on changes in gene level,it can cause irre-versible changes in cell structure and function,and ultimately lead to cancer.In addi-tion,distant metastasis and recurrence are the main causes of death in patients with gastric cancer,which have an important impact on clinical efficacy and prognosis.Therefore,the in-depth study of the pathogenesis of gastric cancer and the specific molecular mechanism of gastric cancer metastasis can provide theoretical basis for the early diagnosis and prognosis evaluation of gastric cancer,as well as for the explora-tion of new diagnostic methods and discovery of new therapeutic targets for gastric cancer.Previous studies on gastric cancer mainly focus on traditional protein coding genes,mainly involving epigenetics,genetics and related signaling pathways.With the development of high-throughput sequencing technology,researchers found that the proportion of non coding RNA(Non-coding RNA)(ncRNA)in the human ge-nome accounts for over 95%.The complex interaction between protein encoded RNA and functional non coding RNA plays an important role in the occurrence and devel-opment of gastric cancer.Long non-coding RNA(IncRNA),as an important compo-nent of ncRNA,is attracting more and more attention.Long non-coding RNA is usually located in the nucleus or cytoplasm.The tran-script length is usually greater than 200 nucleotides,and is rarely involved in protein coding.It mainly regulates gene expression at epigenetic,transcriptional and post transcriptional levels in RNA form.LncRNA plays an important role in genomic im-printing,chromatin modification,chromosome silencing,transcriptional activation and interference of RNA,post-transcriptional regulation,proto-oncogene activation and nuclear transport.It not only activates the expression of certain protein coding genes,but also inhibits the expression of protein coding genes.In recent years,long non-coding RNA has attracted more and more attention in the study of cancer mecha-nism.Up to now,many IncRNAs are closely related to the proliferation,invasion,metastasis and recurrence of cancer cells.In previous studies of gastric cancer,it has been reported that there are significant differences in the expression of IncRNA in gastric cancer tissues and adjacent normal tissues.Some IncRNAs,such as HULC and H19,can affect the proliferation and invasiveness of gastric cancer cells by regulating downstream target genes,leading to the occurrence and development of gastric cancer.At present,the research of IncRNA in gastric cancer is still in its infancy.Exploring and discovering new IncRNA and its specific regulatory mechanism can provide a new theoretical basis for the study of the pathogenesis of gastric cancer.In this study,we used bioinformatics analysis,high-throughput sequencing and classical molecular biology techniques to explore the effects of long non-coding RNA Inc-Z38 on biological effects of gastric cancer at molecular level,cell level and clini-cal samples.Part Ⅰ Expression of lncRNAZ38 in Gastric Cancer Tissues and Cell Lines and Its Clinical SignificanceBackgroundGastric cancer is one of the most common malignant tumors in the world.In China,the incidence of male gastric cancer ranks the second in all malignant tumors.The in-cidence of female is the fifth,which seriously endangers the national health of our country.The occurrence and development of gastric cancer need to go through a mul-ti-stage and multi-factor process.Recent studies show that long non-coding RNA is closely related to the occurrence and development of tumors.It can regulate the ex-pression of tumor related genes at transcriptional,post transcriptional and translational levels.At present,it has been found that there are many abnormal expression of IncRNA in gastric cancer tissue,and these abnormal expression of IncRNA plays an important regulatory role in the occurrence and development of gastric cancer.How-ever,the specific mechanisn of the role of lncRNA in gastric cancer has not been fully elucidated,and needs to be further explored.Methods and resultsWe performed high throughput sequencing of IncRNA expression profiles in 5 pairs of gastric cancer tissues and adjacent tissues.Bioinformatics analysis showed that lncRNAZ38 was highly expressed in gastric cancer tissues;qPCR was used to verify the high expression of lncRNAZ38 in 100 cases of gastric cancer and adjacent tissues;the correlation between the expression level of lncRNAZ38 and clinicopatho-logical parameters in 100 cases of gastric cancer was analyzed.We found that the ex-pression level of lncRNAZ38 was significantly correlated with tumor size,lymph node metastasis,TNM stage and distant metastasis,and was not related to sex and age.At the same time,we detected the expression of lncRNAZ38 in normal gastric epithe-lial cell line(GES-1)and gastric cancer cell line(KATO Ⅲ,SGC 7901,AGS,MKN45,MKN74)by qPCR.It was found that the expression of lncRNAZ38 in five cancer cells of gastric cancer cell line was higher than that in normal gastric epithelial cell line.SummaryThis study found that the expression of lncRNAZ38 in gastric cancer tissues was significantly higher than that in adjacent tissues(P<0.05),and the expression level was significantly correlated with tumor size,lymph node metastasis,TNM stage and distant metastasis.Compared with normal gastric mucosal epithelial cells,the expres-sion of InRNAZ38 was higher in gastric cancer cells.This study provides an im-portant reference for evaluating the development,prognosis and potential tumor markers of gastric cancer.It also provides a basis for further study of the role of lncRNAZ38 in gastric cancer and its regulatory mechanism.Part Ⅱ Effect of IncRNAZ38 on biological behavior of gastric cancer cellsBackgroundThe occurrence and development of tumors are influenced by many factors and are closely related to genes.Tumor metastasis is the main cause of death in patients with malignant tumors,accounting for more than 90%of the cancer-related mortality.Distant tissue or organ metastasis is an important marker of poor prognosis in cancer patients.The occurrence,development and metastasis of tumors are very complex processes,including transformation,growth,angiogenesis,invasion,transmission and survival of cancer cells,as well as subsequent adhesion and colonization,involv-ing numerous different genes and pathways.Long non-coding RNA has long been regarded as a non-functional sequence without protein coding ability,and has not been paid attention to for a long time.However,in recent years,more and more stud-ies have found that IncRNA can regulate gene expression at the level of transcription,post-transcription and translation,thus exerting its biological functions,affecting the biological behavior of tumor cells such as proliferation,apoptosis,invasion and me-tastasis.In the previous experiments,we found that the expression of lncRNAZ38 was significantly higher in gastric cancer tissues and was significantly correlated with tu-mor size,lymph node metastasis,TNM stage and distant metastasis.At the same time,previous studies have found that the expression of lncRNAZ38 in breast cancer is in-creased,which is an indicator of poor prognosis of breast cancer.Then we studied the effects of lncRNAZ38 on the proliferation,invasion and apoptosis of gastric cancer cells.Methods and results1.The gastric cancer cell lines AGS and MKN74 stably interfering with lncRNAZ38 were successfully constructed.2.CCK-8 and plate cloning assay showed that the proliferation and cloning abil-ity of gastric cancer cell lines AGS and MKN74,which interfered with the expression of lncRNAZ38,were significantly reduced.3.Trans-well test and scratch test showed that the migration and invasion ability of gastric cancer cell lines AGS and MKN74,which interfered with the expression of lncRNAZ38,decreased significantly.4.Detection of apoptosis and relative activity of cysteine asparagine showed that interfering with the expression of lncRNAZ38 could promote apoptosis by increasing the activity of Caspase-3 and caspase-9.SummaryIn this study,we found that lncRNAZ38 promotes the proliferation,invasion and metastasis of gastric cancer cells and inhibits the apoptosis of gastric cancer cells.Part Ⅲ Molecular Mechanisms of lncRNAZ38 Involving in the Development of Gastric CancerBackgroundPrevious studies on the mechanism of gastric cancer development focused on traditional protein-coding genes,mainly involving genetics,epigenetics and the regu-lation of related signaling pathways.In the past decades,high-throughput sequencing technology has been greatly developed,non-coding RNA,especially long non-coding RNA,has attracted more and more researchers’ attention in the study of tumorigenesis mechanism.Up to now,many IncRNAs have been reported to be highly correlated with the proliferation,invasion and metastasis of tumor cells and recurrence of tumors.Similarly,in the study of gastric cancer,it has been reported that there are distinct differentially expressed IncRNA profiles in gastric cancer tissues and adjacent tissues,and some IncRNAs,such as H19 and HULC,can regulate the proliferation or inva-siveness of gastric cancer cells through different mechanisms,thus participating in the occurrence and development of gastric cancer.However,the study of IncRNA in gas-tric cancer is still in its infancy.Exploring and discovering new lncRNA and its spe-cific regulatory mechanism can provide a new theoretical basis for the study of the pathogenesis of gastric cancer.In previous experiments,we found that lncRNAZ38 can promote the invasion and metastasis of gastric cancer.Next,we studied the regu-lation mechanism of lncRNAZ38 on the occurrence and development of gastric can-cer.Methods and results1.The stable overexpression of lncRNAZ38 in gastric cancer cell lines AGS and MKN74 was successfully constructed.2.High-throughput sequencing was performed on gastric cancer cell line AGS with stable over-expression of lncRNAZ38 and its parallel control cells.The results suggested that LncRNAZ38 could regulate gastric cancer through TGF-beta signaling pathway.3.We selected up-regulated genes ID3,BMP6 and down-regulated genes GDF-5 in TGF-beta signaling pathway to verify.Western Blot assay showed that the results were consistent with those of high throughput sequencing,suggesting that LncRNAZ38 could regulate gastric cancer through the TGF-beta signaling pathway.SummaryIn this study,we found that lncRNAZ38 can regulate the occurrence and devel-opment of gastric cancer through TGF-beta signaling pathway. |
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