The Study Of Expression Levels Of The Long Non-coding RNA NEAT1,CASC9 In Gastric Cancer And The Influence And Mechanism On The Biological Behaviors Of Gastric Cancer

Objective:Although the incidence of gastric cancer is declining benefited from the tremendous advances in therapeutic methods in recent years,it is still one of the most common and lethal malignant tumors worldwide.Early diagnosis and treatment are the most important means to prolong the postoperative survival time of gastric cancer patients.However,as the onset of gastric cancer is relatively insidious,the disease often has entered the advanced stage or even the latest stage when diagnosed.Although after radical resection and enough regional lymph nodes dissection,combined with standard postoperative adjuvant treatments,the prognosis of gastric cancer patients is still not as ideal as expected.Therefore,in order to improve the postoperative survival rate of gastric cancer patients,we need to find the specific molecular mechanisms involved in the occurrence,development and progress of gastric cancer,and find new molecular markers with high sensitivity and specificity for early diagnosis and prognosis evaluation.Genetic factor plays an important role in the development of gastric cancer.The Human Genome Project revealed that there are no more than 20,000 genes which can actually encode proteins,accounting for only about 2%of the total.While more than90%of transcripts do not encode proteins,called non-coding RNA（ncRNA）.Among them,the long non-coding RNA（lncRNA）has been paid more and more attention by cancer researchers in recent years.Most of the lnc RNAs are transcribed by RNA polymerase II.The lncRNA neither has an open reading frame nor encodes proteins,but is widely distributed in various kinds of eukaryotes.In the field of malignant tumors,lncRNA has been proved to be involved in a variety of activities of tumor occurrence and development process,and plays a key role in them.Lnc RNA NEAT1 has been proved to be overexpressed in esophageal cancer,non-small cell lung cancer and bladder cancer.Prior to the beginning of this study,the study of NEAT1 in the field of gastric cancer was still largely blank.In addition,we also found another lncRNA CASC9 which is dysregulated in gastric cancer by microarray assays.The study of CASC9 in the field of gastric cancer was also largely blank prior to the beginning of this study.Therefore,we selected NEAT1 and CASC9 as the research objects of our study,and we focused on the detection of the expression levels of these two lncRNAs in gastric cancer and the relationship between the expression levels of them and the diagnosis and clinicopathological data of gastric cancer.Besides,we also tried to explore the effects of NEAT1 and CASC9 on the biological behaviors of gastric cancer（including proliferation,invasion,apoptosis,chemoresistance,etc.）,and find the specific molecular mechanisms that may be involved in the influence of NEAT1 and CASC9 on the biological behaviors of gastric cancer,so as to provide the basis for further elucidating the molecular biological mechanisms of the occurrence and development of gastric cancer and for the corresponding targeted treatments of gastric cancer in the future.Methods:In this study,76 gastric cancer tissues and matched paracancerous tissues from patients who underwent radical resection of gastric cancer at the Department of Surgical Oncology,the First Affiliated Hospital of China Medical University from February 2016to October 2016 were selected,and the expression levels of NEAT1 and CASC9 in 76gastric cancer tissues and matched paracancerous tissues were detected by Real-time Polymerase Chain Reaction（Real-time PCR）.Wilcoxon test was used to analyze the differences of expression levels of these two kinds of lncRNAs between gastric cancer tissues and matched paracancerous tissues.Meanwhile,we used the non-parametric test（Manner-Whitney U test was used for the comparison between two groups,and Kruskal-Wallis H test was used for the comparison between multiple groups）to analyze the relationship between the expression levels of NEAT1 and CASC9 and the clinicopathological data of 76 gastric cancer patients.Using Real-time PCR to detect the expression levels of NEAT1 and CASC9 in serum exosomes of 77 gastric cancer patients and 24 healthy volunteers,Wilcoxon test was used to analyze the differences.Under the condition that the expression levels had significant statistically differences,the receiver operating characteristic curve（ROC curve）was drawn to explore whether the serum exosomal NEAT1 and CASC9 can be used as molecular markers for early diagnosis of gastric cancer.Using Real-time PCR to detect the expression levels of NEAT1 in human normal gastric mucosal epithelial cell line GES-1,gastric cancer cell line SGC7901 and adriamycin-resistant gastric cancer cell line SGC7901/ADR,then comparing the differences in expression levels.Using Real-time PCR to detect the expression levels of CASC9 in human normal gastric mucosal epithelial cell line GES-1,gastric cancer cell lines SGC7901 and BGC823,drug-resistant gastric cancer cell lines SGC7901/DR and BGC823/DR,then comparing the differences in expression levels.We constructed a gastric cancer cell line SGC7901 and an adriamycin-resistant gastric cancer cell line SGC7901/ADR with silenced NEAT1 expression using Lipofectamine 3000 reagent and a constructed RNA（siRNA）vector containing specific interference sequences.The same method was used to construct the drug-resistant gastric cancer cell lines BGC823/DR and SGC7901/DR with silenced CASC9 expression.The negative control group（NC,cell line transfected of blank vector without any interference sequence）and the control group（control,cell line without any transfection reagent and vector）were set in all transfection experiments.The silencing expression effects were verified by Real-time PCR.The differences in proliferation ability among the silenced NEAT1 and CASC9 gastric cancer cells,the corresponding NC group and control group were examined by MTT assay,and the effects of NEAT1 and CASC9 on the proliferation of gastric cancer cells were determined.The differences in invasion ability among the silenced NEAT1 and CASC9 gastric cancer cells,the corresponding NC group and control group were examined by Transwell assay,and the effects of NEAT1and CASC9 on the invasion of gastric cancer cells were determined.The differences in apoptosis ability among the silenced NEAT1 and CASC9 gastric cancer cells,the corresponding NC group and control group were examined by flow cytometry and Annexin V-FITC/PI staining,and the effects of NEAT1 and CASC9 on the apoptosis of gastric cancer cells were determined.The MTT assay was performed after adding different gradients of chemotherapeutic drugs to different wells of the cell culture plate to measure the absorbance（OD,Optical Density）between the treated and non-medicated negative control wells（NC）to calculate the cell suppression rates under different concentrations of chemotherapeutic drugs(cell suppression rate=(1-OD_treated/OD_NC)×100%).The Probit regression model was used to calculate the half maximal inhibitory concentrations（IC50）of chemotherapeutic drugs on the silenced NEAT1 and CASC9 drug-resistant gastric cancer cell lines,the corresponding NC group and control group,and the differences were compared to determine the effects of NEAT1and CASC9 on chemoresistance of drug-resistant gastric cancer cells.After adding chemotherapy drugs,flow cytometry and Annexin V-FITC/PI staining were used to detect the differences of apoptosis ability among the silenced NEAT1adriamycin-resistant gastric cancer cell line SGC7901/ADR,the NC group and control group,to determine whether the effect of NEAT1 on chemoresistance of adriamycin-resistant gastric cancer cells could be achieved by influencing the apoptosis induced by chemotherapeutic drugs.Western blot was used to detect the expression levels of multidrug resistance protein 1（MDR1）among the silenced CASC9drug-resistant gastric cancer cell lines BGC823/DR and SGC7901/DR,the corresponding NC group and control group,to determine whether CASC9 affects the chemoresistance of drug-resistant gastric cancer cells to various chemotherapeutic drugs by regulating the MDR1 protein.Results:The expression level of NEAT1 in 76 gastric cancer tissues was significantly higher than that in matched paracancerous tissues（P<0.01）.The 2^-?CT?CT value of NEAT1in gastric cancer tissues was 0.97±1.57,and the 2^-?CT?CT value of NEAT1 in matched paracancerous tissues was 0.16±0.17.The relative expression level of NEAT1 was positively associated with TNM stage（P=0.003）and serosal invasion（P=0.020）in gastric cancer patients.The serum exosomal NEAT1 was significantly higher in 77patients with confirmed gastric cancer compared with 24 healthy volunteers（P=0.012）.The 2^-?CT?CT value of serum exosomal NEAT1 in gastric cancer patients was 0.16±0.36,and the 2^-?CT?CT value of serum exosomal NEAT1 in healthy volunteers was 0.13±0.11.Based on the results of ROC curve,the AUC was calculated to be 0.424（P=0.262）.Compared with the human normal gastric mucosal epithelial cell line GES-1,NEAT1were highly expressed in the gastric cancer cell line SGC7901 and the adriamycin-resistant gastric cancer cell line SGC7901/ADR（P<0.05）.After transfection of RNA containing specific interference sequences,the expression levels of NEAT1 in gastric cancer cell line SGC7901 and adriamycin-resistant gastric cancer cell line SGC7901/ADR were all significantly downregulated compared with the corresponding NC group and control group（P<0.05）.Through MTT assay,it was proved that the proliferation ability of silenced NEAT1 gastric cancer cells was significantly lower than the NC group and control group（P<0.05）.Through Transwell assay,it was proved that the invasion ability of silenced NEAT1 gastric cancer cells was significantly lower than the NC group and control group（P<0.05）.Through flow cytometry and Annexin V-FITC/PI staining,it was proved that the apoptosis ability of silenced NEAT1 gastric cancer cells was significantly higher than the NC group and control group（P<0.05）.By adding different gradients of chemotherapeutic drugs to different wells of cell culture plates,and then performing MTT assay,it was proved that the IC50 of adriamycin on silenced NEAT1 adriamycin-resistant gastric cancer cells was significantly lower than the NC group and control group（P<0.05）.After adding chemotherapy drugs,flow cytometry and Annexin V-FITC/PI staining were used to detect apoptosis,it was proved that under the induction of chemotherapy drugs,the apoptosis ability of silenced NEAT1adriamycin-resistant gastric cancer cells was significantly higher than the NC group and control group（P<0.05）,which indicted that NEAT1 can increase the chemoresistance of adriamycin-resistant gastric cancer cells by reducing the apoptosis induced by chemotherapy drugs.The expression level of CASC9 in 76 gastric cancer tissues was significantly higher than that in matched paracancerous tissues（P<0.01）.The 2^-?CT?CT value of CASC9in gastric cancer tissues was 0.47±0.38,and the 2^-?CT?CT value of CASC9 in matched paracancerous tissues was 0.21±0.24.The relative expression level of CASC9 was positively associated with TNM stage（P=0.018）and lymph node metastasis（P=0.049）in gastric cancer patients.There was no significant statistical difference in serum exosomal CASC9 between 77 patients with confirmed gastric cancer and 24 healthy volunteers（P=0.136）.The 2^-?CT?CT value of serum exosomal CASC9 in gastric cancer patients was1.72±1.16,and the 2^-?CT?CT value of serum exosomal CASC9 in healthy volunteers was1.49±1.50.Compared with the human normal gastric mucosal epithelial cell line GES-1,CASC9 were highly expressed in gastric cancer cell lines SGC7901,BGC823,the drug-resistant gastric cancer cell lines SGC7901/DR and BGC823/DR（P<0.05）.After transfection of RNA containing specific interference sequences,the expression levels of CASC9 in drug-resistant gastric cancer cell lines BGC823/DR and SGC7901/DR were all significantly downregulated compared with the corresponding NC group and control group（P<0.05）.Through MTT assay,it was proved that the proliferation ability of silenced CASC9 gastric cancer cells was significantly lower than the corresponding NC group and control group（P<0.05）.Through Transwell assay,it was proved that the invasion ability of silenced CASC9 gastric cancer cells was significantly lower than the corresponding NC group and control group（P<0.05）.Through flow cytometry and Annexin V-FITC/PI staining,it was proved that the apoptosis ability of silenced CASC9gastric cancer cells was significantly higher than the corresponding NC group and control group（P<0.05）.By adding different gradients of chemotherapeutic drugs to different wells of cell culture plates,and then performing MTT assay,it was proved that the IC50of adriamycin and paclitaxel on silenced CASC9 drug-resistant gastric cancer cells were all significantly lower than the corresponding NC group and control group（P<0.05）.Western blot showed that the MDR1 in the silenced CASC9 drug-resistant gastric cancer cell lines BGC823/DR and SGC7901/DR were all significantly downregulated compared with the corresponding NC group and control group（P<0.05）.CASC9 can increase the chemoresistance of drug-resistant gastric cancer cells to various chemotherapy drugs by upregulating the expression level of MDR1 protein.Conclusion:The expression level of NEAT1 in gastric cancer tissues is significantly higher than that in matched paracancerous tissues,the relative expression level of NEAT1 is positively associated with TNM stage and serosal invasion.Serum exosomal NEAT1 has no obvious value in the early diagnosis of gastric cancer.The expression levels of NEAT1 in gastric cancer cell lines are significantly higher than that in human normal gastric mucosal epithelial cell line.NEAT1 can significantly enhance the proliferation and invasion of gastric cancer cells,and significantly inhibit the apoptosis of gastric cancer cells.NEAT1 can significantly enhance the chemoresistance of adriamycin-resistant gastric cancer cells to adriamycin.NEAT1 can reduce the apoptosis induced by chemotherapy drugs,thereby increasing the chemoresistance of adriamycin-resistant gastric cancer cells to adriamycin.The expression level of CASC9 in gastric cancer tissues is significantly higher than that in matched paracancerous tissues,the relative expression level of CASC9 is positively associated with TNM stage and lymph node metastasis.Serum exosomal CASC9 has no obvious value in the early diagnosis of gastric cancer.The expression levels of CASC9 in gastric cancer cell lines are significantly higher than that in human normal gastric mucosal epithelial cell line.CASC9 can significantly enhance the proliferation and invasion of gastric cancer cells,and significantly inhibit the apoptosis of gastric cancer cells.CASC9 can significantly enhance the chemoresistance of drug-resistant gastric cancer cells to adriamycin and paclitaxel.Maybe CASC9 can increase the chemoresistance of drug-resistant gastric cancer cells to various chemotherapeutic drugs by upregulating the expression level of MDR1 protein.