| Gastric cancer(GC)is the fourth most common malignancy in the world and second leading cause of cancer death worldwide,especially in East Asia.Diffuse-type gastric carcinoma(DGC)is one of major histological types(diffuse and intestinal)according to the classification scheme originally described by Lauren in 1965.Despite the recent advances in the surgical techniques and medical treatment of DGC,the prognosis of patients with DGC remains relatively poor,in the absence of reliable early diagnostic markers and therapeutic targets for suppression of the tumor metastasis.The five-year survival rate remains at a low level.DGC has a poorer prognosis and occurs more frequently in younger individuals in GC.Previous studies have reported many oncogenes and tumor suppressor genes closely associated with DGC,but the highly complex molecular mechanisms underlying its invasion and metastasis are still obscure.Therefore,the identification of reliable biomarkers that are associated with DGC early diagnosis,effective therapy,and prognosis evaluation would be of great clinical relevance.Different from the conventional coding genes,long noncoding RNAs(lncRNAs)are a class of RNA transcripts longer than 200 nucleotides with no or limited protein-coding potential.Recently,Emerging evidence demonstrates that lncRNAs were closely involved in many biological processes including stem cell pluripotency,cell differentiation,cell growth,cell apoptosis and cancer metastasis.The dysregulation of lncRNAs has been reported in the different kind of cancers including DGC.This also makes the abnormal expression of lncRNA in the diagnosis and prognosis of the tumor,drug research has become the focus of the study.However,the mechanism of action of lncRNA in tumors remains unclear and is still in the early stages of research.LncRNA RP11-357H14.17 located in the 17q21.32.The role in the tumor of RP11-357H14.17 at home and abroad has not yet been reported.So its function and mechanism in the DGC know little about it.In order to investigate the biological mechanism of LncRNA RP11-357H14.17 in DGC and the initial mechanism of invasion and metastasis,this study was divided into three parts.First,the LncRNA RP11-357H14.17 was screened and verified by high-throughput sequencing.And the clinical significance of LncRNA RP11-357H14.17 was discussed.The effect of LncRNA-RP11-357H14.17 on the biological function of DGC was discussed by in vitro and in vivo functional experiments.Finally,it was transfer mechanism for preliminary study.Part One: Screening and Verification of LncRNA in the diffuse-type gastric carcinoma【Methods】1、Clinical specimens were selected to collect the clinical diagnosis of poorly differentiated gastric cancer in 6 patients with cancer tissues and adjacent normal gastric mucosal tissues.2、The expression of lnc RNA in cancer tissues and adjacent normal gastric mucosa tissues of 6 patients with diffuse-type gastric cancer were analyzed by high-throughput sequencing.3、The expression of lnc RNAs in the sequencing were analyzed,and the lncRNAs were screened by stratified cluster analysis.4、42 patients with poorly differentiated gastric cancer were selected for qRT-PCR and the expression level of lncRNA RP11-357H14.17 were further verified.【Result】1、High-throughput sequencing results showed that 112 differentially expressed lncRNAs were found in the tumor group compared with the normal group by Fold Change≥2 and p-value <0.05 as the screening criteria.Among them,73 lncRNAs were up-regulated,39 LncRNA expression downregulated.Including 11 lincRNAs(18 down-regulated,53 up-regulated),30 anti-sense lncRNA(16 down-regulated,14 up-regulated),11 intronic lncRNA(5 down-regulated,6 up-regulated).2、The lncRNAs were identified by stratified cluster analysis in high-throughput sequencing,and found 10 differentially expressed lncRNAs.We further identified Fold Change ≥ 8 and p-value <0.05,and the expression was elevated as the screening criteria for 10 lncRNAs.XIST,PVT1 and RP11-357H14.17 were found to meet the criteria in which XIST and PVT1 have been reported High expression in gastric cancer,which also demonstrates our sequencing results and the reliability of screening criteria.Then we will put RP11-357H14.17 as the research object.3、qRT-PCR results showed that in 42 cases of diffuse-type gastric carcinoma patients,lncRNA RP11-357H14.17 relative expression increased by 32,the relative expression of 10 reduction.The expression level of lncRNA RP11-357H14.17 was significantly increased in cancer tissues compared to adjacent normal gastric mucosal tissues(P <0.05).QRT-PCR results and sequencing results have the same trend,for the next step RP11-357H14.17 in invasive gastric cancer function to lay the foundation.Part Two: The clinical value of LncRNA RP11-357H14.17 and the biological function of diffuse-type gastric carcinoma【Methods】1、Combined with clinical pathological data on lncRNA RP11-357H14.17,analysis of diffuse-type gastric carcinoma patients with age,sex,tumor size,invasion depth,lymph node metastasis and TNM staging.2、The expression of lncRNA RP11-357H14.17 in 5 gastric cancer cell lines(well differentiated AGS,intermediate differentiated SGC-7901,low differentiated MKN-45 and MGC-803,normal gastric mucosa GES-1)was detected by qRT-PCR.3、si-RP11-357H14.17 virus was prepared and packaged by si-RNA technology,and the low differentiation gastric cancer cell lines MGC-803 and MKN-45 were transfected with si-RP11-357H14.17 virus.The lncRNA RP11-357H14.17 was down-expression in cell lines.4、The establishment of experimental group:the normal target cell group(CON),normal plus negative control virus infection of the target cell group(NC),normal lncRNA target gene virus infection of the target cell group(KD).5、MTT and clone formation assay were used to detect the proliferation and growth of three experimental cells.6、The cell cycle and apoptosis rate of three experimental cells were detected by flow cytometry.7、Scratch test,Transwell chamber test the invasion and migration ability of 3 groups experimental cells.8 、 The effect of lncRNA RP11-357H14.17 on gastric cancer was detected by subcutaneous tumor growth in nude mice.【Result】1 、 There was no significant correlation between the expression of lncRNA RP11-357H14.17 and the age and sex of patients in diffuse-type gastric carcinoma(P> 0.05).The expression of lncRNA RP11-357H14.17 was associated with gross tumor volume,invasion depth,lymphatic spread and TNM staging(P <0.05).2、qRT-PCR results show that the expression of RP11-357H14.17 in gastric cancer cell lines MGC-803,MKN-45,SGC-7901,AGS was significantly higher than that in normal gastric mucosa cell line GES-1(P<0.05).Differences were statistically significant.The expression of MGC-803 in gastric cancer cell lines MGC-803,MKN-45,SGC-7901 and AGS was the highest relative to normal gastric mucosa cell line GES-1.MKN-45 expression followed.MGC-803 and MKN-45 is diffuse-type gastric carcinoma cell lines,which is consistent with RP11-357H14.17 expression in diffuse-type gastric carcinoma.This also confirms the feasibility of our study and the next step in the study of cell lines.3 、 After successfully prepared and packaged si-RP11-357H14.17 virus,si-RP11-357H14.17 virus transfected low differentiation gastric cancer cell lines MGC-803 and MKN-45.The knockout efficiency of MGC-803 and MKN-45 cells was detected by qRT-PCR.The knockout efficiency of lncRNA gene was 42.8% in si-RP11-357H14.17 group,and the difference was statistically significant(P <0.05).4、The MTT results showed that the absorption rate of light at the wavelength of 490 nm was markedly lower than wavelength of the NC group and the normal target cell group CON on the fourth day after the addition of MTT.And with time,the difference is more obvious.The difference was statistically significant(P <0.05).The results of colony formation assay showed that there was no significant change in the clone formation count of si-RP11-357H14.17 group(P> 0.05)compared with control CON and NC group after 12 days of siRNA lentivirus infection in MGC-803 and MKN-45 cells.5、The results of flow cytometry showed that in the cell cycle experiment,the proportion of RP11-357H14.17 in the si-RP11-357H14.1 group was significantly higher in the S and G2/M phases than in the control group NC;the percentage of cells in G1 was observably decreased.The difference was statistically significant(P <0.05).In the apoptotic experiment,the number of cell transfer in si-RP11-357H14.17 group was observably reduced than the number of control group(P <0.05),and the difference was statistically significant(P <0.05).6、The results of scratches showed that the number of cells in the si-RP11-357H14.17 group was markedly reduced than the number of the CON group at 24 hours,which was statistically significant(P <0.05).The number of cell transfer in si-RP11-357H14.17 group was markedly reduced than the number of control group(P <0.05),and the number of cell metastasis was markedly reduced than the number of control group(P <0.05).7、Nude mice subcutaneous tumor test results show that: compared to the blank control group,si-RP11-357H14.17 group significantly reduced tumor and the difference was statistically significant(P <0.05).Part Three: Preliminary mechanism of lncRNA RP11-357H14.17 promoting invasion and metastasis of diffuse-type gastric carcinoma【Methods】1、The expression of lncRNA RP11-357H14.17 in MGC-803 and MKN-45 cells was down-regulated by si RNA transfection and reduced the expression of lncRNA RP11-357H14.17 in cell line MGC-803 and MKN-45.2、The establishment of experimental group: the normal target cell group(NC)and normal lncRNA target gene virus infection of the target cell group(KD)3、The expression of E-cadherin,N-cadherin and Vimentin in EMT markers were detected by qRT-PCR.4、The expression of E-cadherin,N-cadherin and Vimentin in EMT markers were detected by immunoblotting(WB).【Result】1 、 qRT-PCR test results show that: The expression levels of E-cadherin and N-cadherin in MGC-803 and MKN45 cell lines were markedly higher than those in NC group and the low expression of RP11-357H14.17 in si-lncRNA group,and the expression level of Vimentin was markedly decreased.The difference was statistically significant(P<0.01).2、Western blotting(WB)test results show that: The expression levels of E-cadherin and N-cadherin in MGC-803 and MKN45 cell lines were markedly higher than those in NC group and the low expression of RP11-357H14.17 in si-lncRNA group,and the expression level of Vimentin was markedly decreased.The difference was statistically significant(P<0.01).【Conclusion】1、LncRNA RP11-357H14.17 was screened out by sequencing and stratified cluster analysis in diffuse-type gastric carcinoma.The expression level of LncRNA RP11-357H14.17 in diffuse-type gastric carcinoma was significantly higher than that in adjacent normal gastric mucosa tissues.2、The expression of LncRNA RP11-357H14.17 in gastric cancer cell line in vitro showed that RP11-357H14.17 could promote the proliferation of tumor cells in diffuse-type gastric carcinoma,affect cell cycle of gastric carcinoma,inhibit cell apoptosis and promote cell invasion and migration.In vivo experiments showed that RP11-357H14.17 could promote the growth of nude mice.3、LncRNA RP11-357H14.17 may affect the invasion and metastasis by influencing EMT.4 、 LncRNA RP11-357H14.17 play an carcinogenic role in diffuse-type gastric carcinoma,which is expected to be a marker for the diagnosis and prognosis of diffuse-type gastric carcinoma,and to provide effective targets for treatment. |
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