| Object:Gastric cancer is one of the common malignant tumors.Worldwide,gastric cancer incidence accounts malignant tumors for fourth and its death rate ranks No.3.The incidence of gastric cancer is higher in China,especially in southeast coastal areas.Gastric cancer is a major disease with serious impact on China’s national health.Due to lack of specific symptoms,it is difficult to detect in earlier times and stage Ⅲ,Ⅳpatients accounted for the majority clinically.At present,treatments of gastric cancer are limited.The proportion of patients who can undergo radical surgery is small and efficacy of chemotherapy is poor.Positive clinical effect of targeted therapy only achieved by trastuzumab and apatinib whose crowd is limited and there is also a problem of drug resistance.Tumor treatment has entered the era of precision medical.We need to find new therapeutic targets and develop new therapeutic drugs.The ROS1 gene is a monomeric receptor tyrosine kinase which widely expresses in human kidney,cerebellum,gastrointestinal and other organizations.The current study found that it was activated by gene rearrangement in lung cancer,ovarian cancer,sarcoma,cholangiocellular cancer and inflammatory myofibroblastic cancer.And in the clinical study of non-small cell lung cancer,the drug for ROS1 gene rearrangement kozotinib was found making this gene an important target for accurate treatment of malignant tumors.ROS1 gene rearrangement has been detected by FISH in clinical specimens of gastric cancer in China and abroad and ROS1 gene rearrangement was associated with poor clinical phenotypes such as poorly differentiated,lymph node metastases.Although ROS1 gene expression in gastric cancer is not high,there are certain effective drugs.It is still necessary to further study its role in gastric cancer.ROS1 gene appeared by rearrangement of the fusion gene.Previous studies focused on the structure and function of ROS1 fusion gene,and how to detect the fusion gene.But each fusion gene function was not exactly the same.In this study,only the ROS1 gene was studied and its fusion partner was ignored.Gastric cancer cell was for the first timeto be studied with the ROS1 gene on the biological behavior and mechanism of action.To further study the gene as the targetfor the treatment of basic research data.Methods:First,the expression of ROS1 protein in gastric cancer cells BGC-823,MGC-803,SGC-7901 and HGC-27 was detected by Western blot and ROS1 high expression cell lines were found.Two cell lines with the highest expression were selected as the study objects.Then we constructed the ROS1 gene interfering RNA plasmid and irrelevant sequence plasmid.Its gene fragments were respectively ROS1 shRNA:5’-GAGGAGACCTTCTTACTTAT-3’,NC shRNA:5’-GTTCTCCGAACGTGTCACGT-3’.The plasmid was identified by sequencing.The two plasmids were successfully transfected into two cell lines.After 24 hours of culture,G418-containing medium was used for selection and culture.After two weeks,the gastric cancer cell lines stably transfected with the two plasmids were obtained.Real-time PCR and Western blot were used to identify the mRNA and protein expression.If the rate of knockdown was 70% or more,experiments were followed-up.The growth curve of gastric cancer cells was determined by MTT assay.The ability of cell cloning was determined by clone formation assay.The cell cycle was detected by flow cytometry.After ROS1 gene knockdown,the growth and proliferation of gastric cancer cells were determined.Apoptotic cells were detected by flow cytometry to investigate the effect of ROS1 gene on the apoptosis of gastric cancer cells.Cell migration ability was determined by scratch healing experiment.Invasive ability of gastric cancer cells was determined by transwell chamber assay to study the invasion and metastasis of gastric cancer cells after ROS1 gene knockout.Western blot was used to detect the expressions of cleaved-caspase-3,cleaved PARP,Bax and bcl-2 proteins to investigate the ROS1 gene-induced apoptosis pathway.Western blot was used to detect the expressions of E-cadherin,vimentin and N-cadherin to study whether the ROS1 gene was involved in gastric cancer EMT.Western blot was used to detect the expressions of p-PI3 K,PI3K,p-AKT and AKT to study whether the ROS1 gene played a role in the activation of PI3 K / AKT pathway.Real-time PCR was used to detect the expressions of Snail,Slug,Twist and MMP9 in epithelial-mesenchymal transition to study whether ROS1 gene can induce invasion and metastasis of gastriccancer cells by inducing EMT in tumor cells.At last,inorder to study the relationship between the expression of ROS1 protein and clinicopathological features,qRT-PCR assay was used to detect ROS1 mRNA in37GC and normal gastric mucosa.TCGA database was applied to analyze the expression pattern,the relationship with clinicopathological factors and prognosis in gastric cancer.Then,immunohistochemical method was used to detect the expression of ROS1 in126 cases of gastric carcinoma.Results:1.Western blot results showed that the relative expression of ROS1 protein in BGC-823 cells and SGC-7901 cells was higher than others and they were selected as further experimental objects.After transfection of the ROS1-interfering RNA plasmid and the irrelevant sequence plasmid,stable transfections of the two plasmids of gastric cancer cell lines were observed.Western blot and real-time PCR were used to identify the protein and gene expression.ROS1 gene knockout rate was confirmed 75% or more and the experiment was continued to complete.Appellate experiments were conducted simultaneously for both cell lines.The groups were as follows: BGC-823,BGC-823-NC,BGC-823-shROS1;SGC-7901,SGC-7901-NC,SGC-7901-shROS1.2.MTT assay showed that after ROS1 knockdown BGC-823-shROS1 cells and SGC-7901-shROS1 cells had lower cell growth curves than the control cells.Growth rates decreased by 37% and 30% than BGC-823 cells and SGC-7901 cells(P < 0.01).Clone formation assay showed that the number of BGC-823-shROS1 cells and SGC-7901-shROS1 cells cloning formation was significantly reduced compared with the two control groups,respectively.The clonogenic rates were 19.5%±1.8%,37.33%±2.25%,36.17%±1.76% and 16.00%±2.00%,33.50%±1.50%,31.17%±2.52%(P<0.01).Cell cycle assays by flow cytometry showed that G0/G1 phase of BGC-823-shROS1 cell and SGC-7901-shROS1 cell rose and S phase(55.77%±2.70%vs 61.08%±0.87%;55.33%±0.88% vs 67.43%±0.65%),G2 phase declined significantly compared with BGC-823 cells and SGC-7901 cells(23.25%±0.61%;24.55%±3.15% vs13.98±0.59)and there were significant differences(P<0.01).Apoptosis was detected by flow cytometry showed that early cell apoptosis(2.99%±0.11% vs 15.94%±0.85%;5.78%±0.42% vs 16.19%±1.01%)and late cell apoptosis(0.42%±0.03% vs0.42%±0.03%;1.18%±0.09% vs 9.13±0.62%)rates of BGC-823-shROS1 cell and SGC-7901-shROS1 cell significantly increased compared with BGC-823 cells and SGC-7901 cells(P<0.01).Transwell assay showed that the number of cells through the microporous membrane from the upper chamber into the lower chamber of BGC-823-shROS1 and SGC-7901-shROS1 significantly increased compared with BGC-823 cells and SGC-7901 cells(21.4±2.07 vs 43.2±4.15;20.60±2.30 vs 61.4±5.18).3.Western blot was used to detect apoptosis-related proteins.The result showed that the expressions of cleaved-caspase-3 and cleaved PARP of BGC-823-shROS1 and SGC-7901-shROS1 significantly increased compared with BGC-823 and SGC-7901.The relative expression of them are 3.71±0.27,1.58±0.04,1.49±0.03,1.91±0.08.The expressions of Bax(1.43±0.13,1.52±0.07)significantly increased and bcl-2(0.50±0.02,0.34±0.02)significantly decreased compared with BGC-823 and SGC-7901.The results of cytoskeleton-related protein detection showed that the expression of E-cadherin of BGC-823-shROS1 and SGC-7901-shROS1 significantly increased(2.14±0.13,2.52±0.04).The expression of vimentin(0.52±0.04,0.49±0.07)and N-cadherin(0.48±0.04,0.38±0.02)significantly decreased.In signal transduction pathways,the expressions of PI3 K and AKT of BGC-823-shROS1 and SGC-7901-shROS1 had no significant difference(P>0.05).The expression of p-PI3K(0.33±0.04,0.42±0.02)and p-AKT(0.51±0.01,0.54±0.01)significantly decreased(P < 0.01)(The above results were calculated with the corresponding protein expression of BGC-823 and SGC-7901 cells as "1").4.Real-time PCR was used to detect EMT-related genes in gastric cancer cells.The result showed that the expressions of Twist,Snail,Slug in BGC-823-shROS1 and SGC-7901-shROS1 significantly decreased compared with BGC-823 and SGC-7901.But different degrees of cell decline were not the same.The relative expression of three genes in BGC-823-shROS1 cells were Twist(1.59vs2.36),Snail(0.43 vs 0.76),Slug(1.40vs2.06).The relative expression of three genes in SGC-7901-shROS1 cells were Twist(0.84vs1.00),Snail(0.84vs1.00),Slug(0.57vs1.00).The above results were statistically significant(P<0.01).5.Real-time PCR method was used to detect MMP9 gene expression in gastric cancer cells.The result showed that MMP9 expression in BGC-823-shROS1 andSGC-7901-shROS1 significantly decreased.The value of BGC-823-shROS1 and SGC-7901-shROS1 and their control cell were 0.97 vs 1.29 and 0.43vs1.00.The results were statistically significant(P<0.01).6.TCGA database demonstrated that the expression of ROS1 in gastric cancer was significantly higher than that in adjacent normal tissues(P < 0.0001).The high expression of ROS1 was correlated with higher histological grade(P=0.014),higher T stage,N stage,M stage and TNM stage(P<0.0001).GC patients with high ROS1 expression had bad prognosis compared with low-expression of ROS1.Only 2 in 393 gastric cancer patients had been found ROS1 gene amplification.7.qRT-PCR assay showed that ROS1 mRNA was up-regulated in 37 GC samples than that in normal gastric tissues.8.Immunochemistry showed the five ROS1 high-expressed samples in 126 gastric cancer patients.The common characters were age>60,owning lymphatic matastasis and II stage of gastric cancer.Conclusion:1.ROS1 gene was high-expressed in gastric cancer BGC-823 and SGC-7901 cells.Knocking-down ROS1 gene can suppress gastric cancer cell proliferation,invasion and metastasis and induce gastric cancer cell apoptosis.2.After knockdown of ROS1 gene,bcl-2/ bax ratio imbalanced this led to activation of caspase-PARP pathway and increasing apoptosis.3.ROS1 gene directly or indirectly down-regulated E-cadherin and promoted gastric cancer cells EMT which increased their invasion and migration capacity through activation of intracellular Twist,Snail and Slug gene.ROS1 gene was found activating Twist,Snail,and Slug gene with different degrees in different cells at the first time.4.ROS1 gene activated PI3 K / AKT signal transduction pathway in gastric cancer to initiate tumor proliferation signal inhibit apoptosis andpromote EMT in gastric cancer cell.5.TCGA database showed the expression of ROS1 in gastric cancer tissues was significantly higher than that in adjacent normal tissues.The expression of ROS1 in gastric cancer tissues was correlated with histological grade,T stage,N stage,M stage and TNM stage.High ROS1 expression in gastric cancer is associated with poorprognosis.The rare rate of ROS1 gene amplification has been found in gastric cancer.Compared with normal gastric mucosa,ROS1 mRNA was high-expressed in gastric cancer.6.Immunochemistry showed the high expression of ROS1 protein in gastric cancer tissue was 3.97%. |
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