| Gastric cancer is one of the most common malignant tumors all over the world,with surgical resection remaining the most effective curative method.Due to the insidious symptoms,early diagnosis of gastric cancer is very difficult.Most patients are already in the middle and late stage when they are diagnosed with gastric cancer for the first time,and they have lost the chance of curative treatment.For patients with gastric cancer in the advanced stage,there is currently no effective targeted therapy.It is of great significance to investigate the molecular mechanism of gastric carcinogenesis and to find key genes that mediate the progression of gastric cancer,and to find molecular biomarkers and potential therapeutic targets for early diagnosis and treatment of gastric cancer.In recent years,the role of miRNAs in the development of gastric cancer has gradually arose great attentions.miRNAs play an important role in the development of gastric cancer by inhibiting the expression of downstream target genes at the post-transcriptional level,activating or inhibiting the signaling pathways which mediate biological functions of gastric cancer cells such as growth,metastasis,drug resistance and tumor stem cell formation.Finding out miRNAs with abnormal expression in gastric cancer tissues,clarifying their biological functions in the development and progression of gastric cancer,and further exploring the downstream molecular signaling mechanism,are of great significance for early diagnosis,prognosis evaluation and threatment of gastric cancer.Among numerous miR NAs,miR-216 a has been found to be abnormally expressed in various tumors,and has important regulatory effects on biological behaviors such as tumor growth and metastasis.Objective: This study collected clinical tissue specimens and clinical pathological data,and molecular experiments including qRT-PCR,western blot and luciferase reporter gene experiment,functional assays including scratch healing assay and Transwell assay,animal experiments including tail vein injection liver metastasis model and and other molecular biological techniques,to systematically explore the expression level,clinical significance,biological function and molecular signaling mechanism of miR-216 a in gastric cancer.Method:(1)By collecting 90 pairs of gastric cancer tissues and adjacent tissues,and extracting miRNAs from tissue samples,this stdudy detected the expression level of miR-216 a in tissues by qRT-PCR;(2)The clinical and pathological data and survival data of 90 patients were collected,and the enrolled patients were grouped based on the expression level of miR-216 a in gastric cancer tissues.The correlation between miR-216 a expression level and clinicopathological features and prognosis of gastric cancer patients was analyzed.(3)After transfecting miR-216 a mimic or inhibitor into gastric cancer cells,the changes of the ability of the migration and invasion of gastric cancer cells were assessed by scratch healing assay and Transwell assay after overexpression or knockdown of miR-216 a.(4)The effect of miR-216 a overexpression on the metastatic ability of MGC-803 cells in nude mice was evaluated using the tail vein injection liver metastasis model;(5)Using western blot and immunofluorescence staining to detect changes in the expression of epithelial-mesenchymal transition markers after altering the expression of miR-216 a in cells;(6)Through luciferase reporter gene assay,qRT-PCR,western blot and immunohistochemical staining to determine the regulatory effect of miR-216 a on JAK2 protein expression and activation of JAK2/STAT3 signaling pathway;(7)JAK2 overexpression plasmid and JAK2 inhibitor SAR317461 were used to treat gastric cancer cells,to confirm that miR-216 a regulated the metastatic abilities of gastric cancer cells including cell migration,invasion and epithelial-mesenchymal transition by modulating JAK2/STAT3 pathway;Result:(1)qRT-PCR data revealed that the level of miR-216 a in the clinical specimens of gastric cancer tissues was decreased compared with that in adjacent tissues(P<0.05);correlation analysis showed that no significant correlation was found betwen miR-216 a expression level in gastric cancer tissues and age(p=0.280),gender(p=0.512),tumor size(p=0.634)tumor differentiation(p=0.527).Low expression ofmiR-216 a in gastric cancer tissues was correlated the lymph node metastasis of gastric cancer(p=0.003),venous infiltration(p=0.019),depth of invasion(p=0.004),and TNM stage(p=0.035).Comparision of the survival of gastric patients using Kaplanmeier analysis showed that the overall survival of gastric cancer patients with relatively low level of miR-216 a was significantly decreased(P=0.016).COX regression analysis demonstrated that occurrence of lymph node metastasis of gastric cancer(p=0.035),gastric cancer infiltration depth of gastric cancer(p=0.007),TNM stage of gastric cancer(p=0.006),and miR-216 a expression level(p=0.018)were five independent factors affecting the overall survival of gastric cancer patients.(2)qRT-PCR for gastric cancer cell lines and normal gastric epithelial cells showed that the expression level of miR-216 a in four gastric cancer cell lines including MGC-803 cells,BGC-823 cells,MKN-28 cells and SGC-7901 cells was significantly decreased in comparision with that of normal gastric epithelial GES-1 cells.(P<0.05);among five gastric cancer cell lines,the expression level of miR-216 a was the lowest in MGC-803,but the highest in SGC-7901.(3)Transfection of pre-miR-216 a overexpression plasmid significantly increased the expression of miR-216 a in MGC-803 cells(P<0.05);cell scratch healing assay and Transwell assay showed that compared with control cells,the migration and invasion ability of MGC-803 cells were significantly decreased after overexpression of miR-216a(P<0.05).The transfection of miR-216 a knockdown plasmid significantly decreased the expression of miR-216 a in SGC-7901 cells(P<0.05).The migratory and invasive ability of SGC-7901 cells were increased significantly after miR-216 a knockdown(P<0.05).(4)The results of tail vein injection experiment showed that the number of liver metastases formed by MGC-803 cells overexpressing miR-216 a in nude mice was significantly lower than that of the control cells(P<0.05).(5)Western blot was performed in gastric cancer cell lines including MGC-803 cells and SGC-7901 cells and showed that compared with MGC-803 cells in control group,the relative expression level of E-cadherin was elevated in MGC-803 cells after miR-216 a overexpression and the relative expression levels of N-cadherin and Vimentin were reduced after forced expression of miR-216a(P<0.05).The relative level of E-cadherin in gastric cancer cell line SGC-7901 cell was significantly decreased after miR-216 a knockdown and the relative expression levels of N-cadherinand Vimentin were significantly increased in SGC-7901 cells after knocking-down of miR-216 a,compared with cells in negative control group.The results of immunofluorescence showed that the expression level of E-cadherin in MGC-803 cells was increased and the level of vimentin was decreased after miR-216 a overexpression.The expression level of E-cadherin in gastric cancer cells line SGC-7901 cell was decreased after miR-216 a knockdown and Vimentin expression was elevated after knocking-down of miR-216 a in SGC-7901 cells.(6)TargetScan database showed that complementary sequences were found between miR-216 a and JAK2 3’-UTR;overexpression of miR-216 a significantly reduced the level of JAK2 mR NA and protein in MGC-803 and BGC-823 cells(P<0.05);luciferase reporter assay in gatric cancer cells lines revealed that overexpression of miR-216 a in gastric cance cells significantly decreased the luciferase activity of wild-type JAK2 3’-UTR(P<0.05),while had no effect on that of mutant JAK2 3’-UTR.(7)qRT-PCR was performed in clinical specimens of gastric cancer cells and found that the level of miR-216 a in the clincal tissues of gastric cancer tissues was negatively correlated with JAK2 mR NA level in gastria cancer tissus(P<0.05).The Immunohistochemicol staining for JAK2 in gatric cancer tissues showed that compared with that in tisseus with high miR-216 a level,the expression level of JAK2 in gastric cancer tissues with low expression of miR-216 a was significantly increased(P<0.05).(8)Western blot results showed that after overexpression of miR-216 a,the protein levels of JAK-and p-STAT3 were significantly decreased in MGC-803 cells(P<0.05),and the expression of EMT-related transcription factors including Slug,Snail and Twist was significant decreased(P<0.05);after knocking down miR-216 a,the expression levels of JAK2 and p-STAT3 protein in SGC-7901 cells were increased(P<0.05),and the expression of EMT-related transcription factors including Slug,Twist and Snail proteins was significant increased(P<0.05).(9)After overexpression of JAK2,the decrease of p-STAT3,the mesenchymal marker including N-cadherin and Vimentin,and the up-regulation of the epithelial marker E-cadherin,as well as the decrease of migration and invasion of gastric cancer cells,induced by miR-216 a overexpression,was blocked.The JAK2 inhibitor SAR317461 abrogared the promoting effects of miR-216 a knockdown on the cell migration,invasion,and epithelial-mesenchymal transition of SGC-7901 cells.Conclusion: The results of this study indicate that miR-216 a is significantly down-regulated in gastric cancer,and the low expression of miR-216 a is associated with poor clinical pathological features and prognosis of gastric cancer patients.Functionally,miR-216 a can decrease the invasion and metastasis ability of gastric cancer cells and inhibit the occurrence of epithelial-mesenchymal transition.In terms of molecular mechanism,miR-216 a can inhibit the expression of JAK2 protein in gastric cancer cells and prevent the activation of JAK2/STAT3 signaling pathway,thereby inhibiting the epithelial-mesenchymal transition process of gastric cancer cells and reducing the migration and invasion ability of gastric cancer cells.miR-216 a is expected to become a novel biomarker for the disease evaluation and prognosis prediction for gastric cancer patients,and it is expected to become a attractive target for treatment of gastric cancer. |
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