The Effect Of Hsa＿circ＿0005230/miR-1299/RHOT1 Axis On The Pathobiological Behavior Of Gastric Cancer And Relevant Mechanism

Objective: Gastric cancer(GC),as one of the leading malignant tumors of the digestive system in the world in terms of incidence and mortality.Since the pathogenesis of gastric cancer and biological behavioral mechanisms such as invasion and metastasis are not very clear,the prognosis evaluation indexes such as overall survival time and the patients’ life quality have not improved significantly despite continuous improvement through radical surgery,radiotherapy,and molecular targeted therapy.The recurrence and metastasis of GC are the main causes of patient death.Circular RNAs(circRNAs)are a group of non-coding RNAs that are closed into a special loop-like configuration.They have various biological functions,as one of the ce RNAs,it can sponge miRNAs to affect the transcription of mRNA;it can bind RBPs(RNA binding proteins)to take part in transcriptional expression or translations.miRNAs are a group of small non-coding RNAs,approximately 22 nucleotides in length,which are broadly involved in the post-transcriptional regulation of genes.miRNAs do not possess coding functions and perform regulation mainly through complete or incomplete pair binding to target genes.mRNAs play multiple roles in tumor development as messenger RNAs.RBPs can take part in the formation of circRNAs and enrich the functions of circRNAs,which play an important role in RNA transcription and translations.RBPs can bind or interact with circRNAs and play a role in circRNAs biogenesis,localization,special splicing and folding,and promoting expression stabilization.EMT(Epithelial-Mesenchymal Transition)is thought to be the source of tumor cells proliferation by invasion and metastasis.In EMT phenotypic transformation,non-coding RNAs can influence tumor cell proliferation and metastasis by epigenetically controlling the expression of tumor-associated genes.This study aimed to discover and explore the tumor-related genes and their effects on the pathobiological behavior of GC.It could provide a theoretical basis for the early diagnosis,treatments,and prognosis assessments of GC,and provide theoretical supports for personalized and precise therapeutic targets.Methods:1.The human immortalized normal gastric mucosal epithelial cell line(GES-1)and human GC cell lines SGC-7901,BGC-823,AGS,HGC-27,and MKN-45 were routinely cultured,130 cases of surgically resected fresh GC tissues and paired distal cancer normal gastric mucosal tissues were collected from the Department of Gastrointestinal Oncology of The First Affiliated Hospital of China Medical University.The total RNA was extracted and reverse transcribed into cDNA,and the expression of hsa＿circ＿0005230 at the cell and tissue level of gastric cancer was detected by qRT-PCR.The relationship between the different hsa＿circ＿0005230 expression in GC tissues and various clinicopathological factors was analyzed.2.Synthesize siRNA and silencing hsa＿circ＿0005230 in AGS and HGC-27 cell lines constructed functional cell lines.The effects were assessed silencing hsa＿circ＿0005230on the proliferation ability of GC cells by CCK-8 and clone formation assays;the effects of silencing hsa＿circ＿0005230 on the cell cycle changes of GC cells were assessed by flow cytometry assay;the effects of silencing hsa＿circ＿0005230 on the migration of GC cells were assessed by scratch healing assay and transwell migration assay;the effect of silencing hsa＿circ＿0005230 on the invasion ability of GC cells was assessed by transwell invasion assay.Western Blotting(WB)was applied to detect the expression trend of EMT-related proteins after silencing hsa＿circ＿0005230 in GC cells.3.Hsa＿circ＿0005230 was predicted to sponge miR-1299 by the Circinteractome website.The expression of miR-1299 in GES-1 and five GC cell lines and 33 cases of GC tissues and its paired normal gastric mucosal tissues were detected by qRT-PCR.The expression correlation between hsa＿circ＿0005230 and miR-1299 in GC tissues was analyzed.Survival analysis of miR-1299 was done by the Kaplan-Meier Plotter website.The miRWalk and Target Scan websites were used to predict the downstream mRNAs to bind miR-1299.GES-1 and five GC cell lines and 51 cases of GC tissues and its paired normal gastric mucosa tissues of RHOT1 mRNA expression were detected by qRT-PCR,and correlation analysis was done between RHOT1 mRNA expression level of GC tissue and clinicopathological factors.The correlation of expression between hsa＿circ＿0005230,miR-1299,and RHOT1 mRNA in GC tissues was analyzed;the changes of miR-1299 and RHOT1 mRNA expression after silencing hsa＿circ＿0005230 were verified in siRNA functional cell lines.The dual-luciferase reporter gene assay was performed to confirm that there were binding sites between miR-1299 and RHOT1 that could be targeted for binding.To clarify whether RHOT1 protein as an effector protein influence the biological behavior of GC,the expression of RHOT1 protein in 49 cases of fresh GC and its paired normal gastric mucosa tissues were detected by Western Blotting(WB),and immunohistochemical staining was applied to detect the expression of RHOT1 protein in155 GC and normal gastric mucosa tissues and the relationship was analyzed between the two groups of expression and the clinicopathological factors.4.RBP FUS with potential binding sites to hsa＿circ＿0005230 was predicted by the Starbase website.qRT-PCR was applied to detect the expression of FUS mRNA in GES-1 and five GC cell lines and 32 human GC tissues and their paired normal gastric mucosa.The correlation was analyzed between FUS mRNA and hsa＿circ＿0005230 and RHOT1 mRNA.To analyze the relationship between FUS mRNA expression in GC tissues and various clinicopathological factors.5.The data were processed by statistical analysis using SPSS 22.0 software and Graph Pad Prism 8.0.2.The measurement data were expressed by mean±SD,and paired t-test was used for comparison between two groups;the count data were statistically analyzed by chi-square test,survival analysis by Kaplan-Meier Log-rank test,and correlation analysis by Spearman test,and P< 0.05 was statistically significant.Results: 1.The qRT-PCR showed that the expression of hsa＿circ＿0005230 was significantly up-regulated in five human GC cell lines compared with GES-1 and hsa＿circ＿0005230 expression was up-regulated in 130 GC tissues compared with paired normal gastric mucosal tissues(P=0.0477).The high expression of hsa＿circ＿0005230 in GC tissues was closely correlated with the Histological grade(X~2=4.186,P= 0.014),lymph node metastasis(X~2=7.754,P=0.021)and pTNM stage(X~2=7.486,P=0.006)of GC clinicopathological factors;among the lymph node metastasis factors,hsa＿circ＿0005230 expression was significantly upregulated when the group with numbers of lymph node metastasis(N2-3)compared with the group without lymph node metastasis(N0)(X~2=4.873,P=0.027).Other clinicopathological factors such as gender,age,tumor size,Gross types,WHO’s histological types,Lauren’s types,Depth of invasion,distant metastasis,and Location were not associated with the relative expression level of hsa＿circ＿0005230.2.Synthetic siRNA could effectively be silencing hsa＿circ＿0005230.Silencing hsa＿circ＿0005230 assessed the suppression of proliferation ability of GC cell lines by CCK-8 assay.Silencing hsa＿circ＿0005230 assessed the diminished clonogenic ability of GC cells by clone formation assay.Flow cytometry after silencing hsa＿circ＿0005230assessed GC cell cycle arrest at G0/G1 phase.Silencing hsa＿circ＿0005230 assessed diminished cell migration ability and reduced cell scratch healing rate by Scratch healing assay.The diminished cell migration ability of GC cell lines was assessed by transwell migration assay after silencing hsa＿circ＿0005230.Transwell invasion assay after silencing hsa＿circ＿0005230 assessed that the cell invasion ability of GC cell lines was significantly reduced.Silencing hsa＿circ＿0005230,the WB results showed that the expression of epithelial phenotype protein E-cadherin was increased and mesenchymal phenotype proteins such as N-cadherin,Vimentin,and Snail were all down-regulated.3.Compared with GES-1,the expression of miR-1299 was significantly down-regulated in GC cell lines and the expression of RHOT1 mRNA was significantly up-regulated in GC cell lines.Compared with paired normal gastric mucosal tissues,miR-1299 expression was down-regulated in 33 GC tissues(P=0.0179),and RHOT1 mRNA expression was up-regulated in 51 GC tissues(P=0.045).The expression level of miR-1299 was increased and RHOT1 mRNA expression level was down-regulated in GC cells after silencing hsa＿circ＿0005230.There was a negative correlation between hsa＿circ＿0005230 and miR-1299 expression levels in GC tissues(P=0.0228,r=-0.4078);miR-1299 and RHOT1 expression levels were negatively correlated(P=0.0237,r=-0.4053);hsa＿circ＿0005230 and RHOT1 mRNA expression levels were positively correlated(P=0.0044,r=0.4971);hsa＿circ＿0005230/miR1299/RHOT1 axial molecules held by expression correlations.The specific binding site between RHOT1 and miR-1299 was confirmed by the dual-luciferase reporter gene experiment.4.RHOT1 mRNA expression levels in GC tissues correlated with lymph node metastasis(X~2=6.708,P=0.035)and pTNM stage(X~2=8.209,P=0.042),where RHOT1 high expression was significantly increased in the group with numbers of lymph node metastasis(N3)compared with the group without lymph node metastasis(N0)(X~2=4.297,P=0.038).The RHOT1 expression level of GC was not associated with other pathological factors such as gender,age,Gross types,Histological grade,WHO’s histological types,Lauren’ types,depth of invasion,distant metastasis,and location.5.RHOT1 protein expression was up-regulated in GC tissues.Immunohistochemical staining showed that RHOT1 protein was localized in the cytoplasm,and the positive expression rate of RHOT1 protein was significantly higher in GC tissues compared with normal gastric mucosa(P<0.01).Statistical analysis of different RHOT1 protein expression and clinicopathological data of 155 tissues revealed that the protein expression of RHOT1 in GC was correlated with clinicopathological factors such as histological grade(X~2=7.045,P=0.03),Lauren’s types(X~2=8.861,P=0.012),depth of invasion(X~2=6.534,P=0.038),lymph node metastasis(X~2=5.929,P=0.015),and pTNM stage(X~2=14.74,P<0.01).In Lauren’ types,compared with intestinal type,the high expression of RHOT1 protein in diffuse GC tissues was significant(X~2=5.397,P=0.02),the high expression of RHOT1 protein in mixed type GC tissues was also significantly increased(X~2=8.607,P=0.003);among the depth of invasion factors(T),RHOT1 protein expression significantly differed between different invasion depths(X~2=4.958,P=0.026).While other pathological factors such as gender,age,tumor size,Gross types,WHO’s histological types,distant metastasis,and location were not associated with RHOT1 protein expression in GC.6.Kaplan-Meier log-rank had analyzed the survival of hsa＿miR-1299 GC patients.The result was showed that patients were divided into miR-1299 low and high expression groups according to the best cutoff value,and showed that the overall survival time of patients with miR-1299 high expression GC at low mutational load(38.43 months)was prolonged(P=0.014)compared with the low expression group(17.77 months),and patients had a better prognosis;there was no significant difference between the two groups at high mutational load.Kaplan-Meier plotter website survival analysis of the GEO database GSE29272 dataset,which included 592 valid patients data was divided into RHOT1 mRNA high and low expression groups based on median expression,the results showed that the overall survival time was significantly longer in the RHOT1 mRNA low expression group(28.7 months)compared to the high expression group(20.6months),patients had a better prognosis(P=0.0035).7.FUS was predicted as an RBP may affect the hsa＿circ＿0005230/miR-1299/RHOT1 axis through the EMT pathway to affect the pathobiological behavior of GC.Conclusion: 1.Hsa＿circ＿0005230 is upregulated in GC cell lines and tissues and suggesting that hsa＿circ＿0005230 is expected to be a potential tumor biomarker,providing a theoretical basis for GC diagnosis,accurately assessing prognosis,and finding a personalized therapeutic target.The high expression of hsa＿circ＿0005230 in GC correlates with histological grade,lymph node metastasis,and pTNM staging,and it is speculated that hsa＿circ＿0005230 is differentially expressed circRNA may affect the biological behavior of GC.2.Hsa＿circ＿0005230 could be effectively silenced by synthesized siRNA in GC cells.Silencing hsa＿circ＿0005230 could significantly inhibit the proliferation,invasion,and migration ability of GC cells,reversion the expression of main proteins of EMT phenotype,suggesting that hsa＿circ＿0005230 could affect the malignant biological behavior of GC cells.3.The expression of RHOT1 mRNA and protein levels in GC tissues and cells are both up-regulated and the high expression of RHOT1 mRNA correlates with lymph node metastasis and TNM stage.Upregulated hsa＿circ＿0005230 could regulate the miR-1299/RHOT1 axis to influence the invasive metastatic behavior of GC by the EMT phenotypic pathway in GC cells.These suggest that RHOT1,as well as miR-1299,were expected to be new targets for the precise treatment of GC patients.4.FUS mRNA expression is up-regulated in GC tissues and GC cells and correlate with lymph node metastasis and pTNM stage clinicopathological factors with poor patient prognosis.FUS has the potential to influence hsa＿circ＿0005230 expression and regulated the miR-1299/RHOT1 axis to influence the pathobiological behavior of GC by the EMT pathway.