| Gastric cancer（GC）has been one of the most common malignant tumors worldwide.There were 1,033,701 new cases of gastric cancer and782,685 deaths worldwide in 2018.The morbidity and mortality are high and increasing as time goes on.Due to special eating habits（prefer pickled foods,share bowls and disehes,etc.）,Chinese ingested more nitrites,and the proportion of patients infected with H.pylori is large,resulting in the morbidity and mortality are higher than that of many other countries.Among the patients with gastric cancer,the middle and advanced cases account for the vast majority,which brings great challenges to the treatment of gastric cancer.For the advanced gastric cancer,the current treatment is mainly based on surgical resection and radiochemotherapy,supplemented by immunotherapy and molecular targeted therapy.Especially for patients whose tumor cannot be resected at advanced stage or accompanied by peritoneal implantation metastasis or distant metastases such as liver metastases and lung metastases.Although immunotherapy and molecular targeted therapy have gradually become the main treatment tools,which can only extend the survival time of patients to a certain extent.Therefore,searching for markers of early metastasis of gastric cancer and effective molecular therapeutic targets has crucial clinical guiding meaning for the treatment of advanced gastric cancer.CircRNAs（circular RNAs）are a special class of non-coding RNA molecules which are made into circles by reverse splicing.It has been more than 40 years since its discovery in the 1990 s and it was first considered a "by-product" of transcripts from the beginning and gradually found to have many important biological functions.Its main functions are as follows: 1.Regulation of downstream gene expression by sponging mi RNA（micro RNA,micro RNA）;2.Regulation of downstream gene expression by adsorbing RNA-binding proteins;3.Regulating functional proteins by binding with them;4.Part of the exon circular RNA has the function of translation into protein and so on.Previously,we found a circ RNA that was downregulated in gastric cancer by analyzing the circ RNA expression chip:hascirc0004873（named circ-OXCT1 after its parent gene OXCT1）.After analyzing the clinical data,we found that circ-OXCT1 expression level was closely related to the tumor T stages,TNM stages and lymph node metastasis in gastric cancer,especially in patients with advanced lymph node metastasis,the lower the expression levels of circ-OXCT1,the later stages they were in.Therefore,how does circ-OXCT1 affect the stages of gastric cancer and lymph node metastasis? This is worth for our investigation.Then considering the important role of epithelial-mesenchymal transition（EMT）in assisting tumor cell atypia and invasion and metastasis in the middle and advanced stages of malignant tumors,we tried to find out whether circ-OXCT1 can inhibit the staging and invasion and metastasis of gastric cancer through the EMT function of gastric cancer cells.We hope to find a new therapeutic marker for the invasion and metastasis of advanced gastric cancer,so as to provide a new therapeutic target for the control and treatment of invasion and metastasis of advanced gastric cancer.The purpose of this study was to explore the relationship between circ-OXCT1 and gastric cancer cell EMT,so as to provide new treatment options for patients with advanced gastric cancer especially accompanied with invasion and metastasis.This study mainly consists of the following three parts:Part Ⅰ Expression profile and clinical significance of circ-OXCT1 in gastric cancerOBJECTIVE:To explore the expression of circ-OXCT1 in clinical specimens of gastric cancer and the relationship between the expression level of circ-OXCT1 and the clinicopathological features and prognosis of patients with gastric cancer.METHODS:（1）74 pairs of gastric and adjacent tissue specimens were collected from our hospital.These specimens were used to extract total RNA,and to detect the expression level of circ-OXCT1 in cancer and adjacent tissues by real-time fluorescence quantitative PCR（q RT-PCR）;（2）Analyze the relationship between the expression level of circ-OXCT1 and the clinicopathological characteristics of GC patients;（3）Explore the correlation between circ-OXCT1 and patient prognosis by Overall Survival（OS）analysis.RESULTS:（1）The expression of circ-OXCT1 in gastric cancer tissues was significantly lower than that in adjacent tissues（p <0.00）,and the expression level in adjacent cancer tissues was approximately 3.9 times that of cancer tissues;（2）Clinicopathological analysis showed that circ-OXCT1 expression is related to the T stages（p=0.021）,TNM stages（p = 0.017）and N stages（perigastric lymph node metastasis）（p = 0.024）: The lower the level of circ-OXCT1,the severer the T stages,TNM stages and N stages,but there was no certain correlation between age,gender,the degree of cell differentiation and Borrmann classification;（3）Survival analysis showed that patients with lower expression of circ-OXCT1 had a shorter 5-year overall survival rate.CONCLUSIONS:（1）Circ-OXCT1 is lower expressed in gastric cancer tissues than that of paracancerous tissues;（2）Lower expressed circ-OXCT1 indicates that patients are more likely to progress to advanced gastric cancer and more lymph nodes metastasis;（3）Lower expressed circ-OXCT1 indicates a shorter survival time and a worse prognosis,which can be used as an index for the recurrence,lymph node metastasis,and distant metastasis of gastric cancer.Part Ⅱ Circ-OXCT1 regulates SMAD4 expression in gastric cancer cells through sponging mi R-136OBJECTIVE:To investigate the effects of circ-OXCT1 on the proliferation and invasion of gastric cancer cells,and to study the effects of circ-OXCT1 overexpressing gastric cancer cells on tumor formation and lung metastasis in nude mice and to explore its potential related mechanisms.METHODS:（1）QRT-PCR was used to detect the expression of circ-OXCT1 in gastric cancer cell lines（AGS,MKN28,MKN45,MGC803,BGC823）,and the two cells with the lowest expression and the highest expression were selected;（2）Construction of lentiviral plasmid to overexpress circ-OXCT1 in GC cell lines;construction of si NRAs to knockdown circ-OXCT1;（3）Use cell function experiments（clone formation,CCK-8,scratches and Transwell assyas）to verify the effects of overexpression or knockdown of circ-OXCT1 on the malignant biological behaviors of gastric cancer cell proliferation and invasion;（4）Use nude mice experiment to verify the effect of circ-OXCT1 overexpression on subcutaneous tumor formation and lung metastasis nodules;（5）Circ Interactome and Star Base v3.0 network prediction tools are used to predict mi RNAs which can interact with circ-OXCT1 and have higher binding efficiencices.The predicted mi RNAs are screened by double luciferase experiment and FISH experiment;（6）Mi Randa and Star Base v3.0 are used to predict the downstream binding genes of the selected mi RNAs and find possible genes that may be related to epithelial-mesenchymal transition;RESULTS:（1）Circ-OXCT1 was expressed lowest AGS and MGC803 and highest in MKN28 and MKN45 through q RT-PCR experiments;（2）Circ-OXCT1 increased to about 8 times in AGS and 4 times in MGC803 after overexpressing by lentvirus;while decresed to 1/30 in MKN28 and 1/20 in MKN45 after knockdown by si-circ-OXCT1-1.（3）Plate cloning experiment,CCK-8 and subcutaneous tumor formation experiments in nude mice found that the overexpression or knockdown of circ-OXCT1 had no statistical effect on the proliferation of gastric cancer cells,however,wound healing assay,Transwell assay and nude mice lung metastasis assay confirmed that overexpression of circ-OXCT1 can significantly inhibit the migration and invasion of gastric cancer cells and the number of lung metastases in nude mice.Knockdown of circ-OXCT1 can significantly promote the migration and invasion of gastric cancer cells;（4）According to Circ Interactome and Star Base v3.0,mi RNAs predicted to bind to circ-OXCT1 and have higher binding efficiencies are:mi R-136,145 and 665.The dual-luciferase experiment and FISH experiment confirmed that only mi R-136 binds well to circ-OXCT1,and the binding site is mainly in the cytoplasmic region;（5）Mi Randa and Star Base v3.0 predict that mi R-136 can bind to the the coding sequence（CDS）of SMAD4.Knockdown or overexpression mi R-136 can downregulate or upregulate SMAD4 protein expression level.CONCLUSIONS:（1）circ-OXCT1 expressed lowest in AGS and MGC803,highest in MKN28 and MKN45;（2）Overexpression or knockdown of circ-OXCT1 didn’t change the proliferation ability in GC cells,however,circ-OXCT1 overexpression significantly inhibited GC cell migration,invasion and the nude mice lung metastasis,and circ-OXCT1 knockdown significantly promoted GC migration and invasion.Part Ⅲ Circ-OXCT1 inhibits the EMT function of gastric cancer cells by blockading the TGF-β pathway through the mi R-136-SMAD4 axisOBJECTIVE:To explore the way through which circ-OXCT1 acts on epithelial-mesenchymal transition of gastric cancer and how it affects the migration and invasion of gastric cancer cells.METHODS:（1）Treat circ-OXCT1 overexpressing gastric cancer cell lines with mi R-136 mimics and observe the changes in the expression levels of EMT-related proteins;（2）Treatment of circ-OXCT1 knockdown gastric cancer cell line with mi R-136 inhibitor,and observe the change of expression level of EMT-related proteins;（3）Treat gastric cancer cell lines with TGF-β receptor inhibitor LY2109761,and then add circ-OXCT1 overexpression plasmid to treat these cells,and observe whether circ-OXCT1 can reverse the effect of LY2109761 on the expression level of EMT-related proteins;（4）Establish the circ-OXCT1-mi RNAs-EMT connection and present it using AI diagrams.RESULTS:（1）After overexpressing circ-OXCT1 by lentivirus in gastric cancer cell line AGS,E-cadherin was significantly reduced,while SMAD4,N-cadherin and vimentin were significantly increased.After treating these cells with mi R-136 mimics,it was found that the changes of EMT-related proteins caused by overexpression of circ-OXCT1 were reversed;（2）E-cadherin in gastric cancer cell line KMN28 was significantly increased after Si-circ-OXCT1-1 treatment,while N-cadherin and vimentin were significantly reduced.After treating these cells with mi R-136 inhibitor,it was found that the changes of EMT-related proteins caused by circ-OXCT1 knockdown were reversed;（3）Circ-OXCT1 reversed the effects of E-cadherin upregulation,N-cadherin and vimentin downregulation caused by TGF-β receptor inhibitor LY2109761.CONCLUSION:（1）Mi R-136 mimics can restore the effect of circ-OXCT1 overexpression on the expression of EMT-related proteins in gastric cancer cells;（2）Mi R-136 inhibitor can restore the effect of circ-OXCT1 knockdown on the expression of EMT-related proteins in gastric cancer cells;（3）TGF-β receptor inhibitor LY2109761 can significantly inhibit the migration and invasion of gastric cancer cells;（4）Circ-OXCT1 can restore the effect of TGF-β receptor inhibitor LY2109761 on the expression of EMT-related proteins in gastric cancer cells.Through the above experiments,we confirmed the existence of the circ-OXCT1-mi R-136-SMAD4-EMT axis,which provides new therapeutic targets and ideas for patients with advanced gastric cancer,especially those with lymph nodes or distant metastases. |
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