The Role And Mechanism Of CircTRPS1-2 On The Proliferation And Migration Of Esophageal Squamous Cell Carcinoma By Influencing Ribosome

Esophageal cancer is a common digestive tract cancer with high incidence and mortality.The latest reports show that the prevalence of esophageal cancer ranked seventh worldwide and its mortality is in the sixth position in 2020,what’s more,China is the disease area with high incidence of esophageal cancer.The pathological types were esophageal squamous cell carcinoma（ESCC）and esophageal adenocarcinoma and the ESCC is the leading pathological type of esophageal cancer in China.The intermediate or advanced stage ESCC is common in the clinical practice due to the low early diagnosis rate caused by fewer early consultations.The survival time of the patients with ESCC was greatly reduced by the usually concomitant lymph node metastases in the mid-stage and advanced stage.The prognosis of the ESCC is still not satisfactory because the relapse and metastasis rate after surgery remain high in spite of the rapid development of multiple therapies including surgery,radiochemotherapy and immunotherapy for the intermediate or advanced stage ESCC.Consequently,it is very important for the improvement of the diagnosis and treatment to deepen the study concerning the mechanism of development and metastasis of ESCC,find out novel targets and biomarkers,and develop the new drugs.Currently,circular RNAs（circRNAs）are one kind of non-coding RNAs which have definite biological significance and are particularly worthy of studying.CircRNAs involve a plenty of diseases including diabetes mellitus,nervous system diseases,cardiovascular disease,chronic inflammatory diseases,and cancers and in particular,circRNAs play an increasingly important role in the development of the neoplastic diseases.There was the specific expression of circRNAs in the development and progression of the ESCC,which is related to the clinical and pathological features.It means that circRNAs possess the potential for serving as a biomarker of disease diagnosis and prognosis prediction and so on.However,there are a large amount of unexplored circRNAs in the ESCC currently.Our research focused on the expression profile of circRNAs and the mechanism of action of differentially expressed circTRPS1-2 in the ESCC.The results achieved are as follows.Part Ⅰ The detection and screening of circRNAs in the ESCC tissues1.The establishment of expression profile of ESCC-related circRNAs:7566 circRNAs in total were found through high-throughput sequencing of three pairs of ESCC and paracancer tissue samples including 2685 up-regulated genes and 4881 down-regulated genes.Using fold change≥2 and P<0.05 as the screening criterion,46 circRNAs with significantly statistic difference were identified through initial screening,among which 5 genes were up-regulated and 41 genes were down-regulated.Based on tumor correlation and statistical differences in expression,5 up-regulated circRNAs and 10 down-regulated circRNAs were selected to conduct a tissue validation.2.Screening of the differentially expressed circRNAs:Firstly,we used 4 pairs of new tissue samples to filter out 9 circRNAs with poor primers specificity and no significant differences.6 remaining circRNAs were further validated by 26 new pairs of tissue samples and the results showed that there were a significantly high expression of hsa_circ₀006156 and significantly low expressions of hsa circ₀026782,hsa_circ₀085362,hsa_circ₀078299,hsa_circ₀000099 and hsa_circ₀008967 in the ESCC tissues;the hsa_circ₀085362 was selected as the subject investigated for the sequent assays through comprehensive analysis of results of high-throughput sequencing and qRT-PCR based on the tissues.3.The expression of circTRPS1-2 in the ESCC and paracancerous normal tissues and their ROC curve analysis:BaseScope assay found that compared with the normal esophageal epithelial tissues.According to the median relative expression level of circTRPS1-2 in ESCC,it can be divided into low expression group and high expression group.The overall survival time analysis results show that the overall survival rate of low expression group is significantly lower than that of high expression group,suggesting that the low expression of circTRPS1-2 indicates bad prognosis and has clinical value in predicting the prognosis of ESCC.4.Selection of experimental cell strain and loop domain identification of circTRPS1-2:We finally selected esophageal squamous cell lines K150 and E109 as the main cell lines for this study by using qRT-PCR,because the expression level of circTRPS1-2 in these cell lines was significantly lower than that of human normal esophageal epithelial cells.In order to verify the loop structure of circTRPS1-2,we used Sanger sequencing to confirm that the sequence of the product of qRT-PCR was consistent with the reverse splicing sequence of circTRPS1-2.agarose gel electrophoresis results showed that reverse splicing gene segment of circTRPS1-2 only was amplified in the cDNA,while the above gene segment was not observed in the gDNA.RNase R digestion assay showed that circTRPS1-2 can significantly resist the digestion of RNase R.5.Subcellular localization of circTRPS1-2:RNA FISH showed that circTRPS1-2 distributed in the cytoplasm and cell nucleus,but the fluorescence intensity in the cytoplasm was stronger than that in the cell nucleus;nucleocytoplasmic RNA isolation assay showed that circTRPS1-2 can be detected in the cytoplasm and cell nucleus,but the content in the cytoplasm was much higher than that in the cell nucleus.In summary,circTRPS1-2 is mainly distributed in cytoplasm and a small amount in nucleus.Part Ⅱ The inhibition of circTRPS1-2 on the proliferation and migration of ESCC cells1.Overexpression and knock-down of the circTRPS1-2:qRT-PCR results showed that overexpression plasmid and siRNAs of circTRPS1-2 was constructed successfully,and both cannot influence the expression of the parental gene TRPS1 significantly.2.The inhibition of the proliferation by circTRPS1-2 in ESCC cells:EdU testified that the proportion of mitotic cell in the overexpression group decreased significantly compared with that in the control group,while the knockdown group showed the opposite effect of inhibiting the proliferation of esophageal squamous cells.The growth curve drawn through CCK-8 assay showed that the overexpression of the circTRPS1-2 can significantly slowed down the growth rate of the cells,while the knock-down of the circTRPS 1-2 can significantly increase the growth rate of the two ESCC cell lines.Colony formation assay found that the circTRPS1-2 can inhibit the colony formation,which was showed by that the number of the colony formation decreased significantly in the overexpression group,while the number increased prominently in the knock-down group.3.The inhibition of the migration by circTRPS1-2 in ESCC cells:wound scratch healing assay showed that wound scratch healing rate of the overexpression group was significantly slower than that of the control group,while the rate of the knock-down group was significantly faster than that of the control group.Transwell invasion assay indicated that during the same time period,the number of cells invading through the membrane in overexpression group was significantly lower than that in the control group,while the number in the knock-down group was significantly higher than that in the control group.4.Animal model validation concerning inhibition of the growth and metastasis of circTRPS1-2 in ESCC:subcutaneous xenograft model results showed that compared with the control group,tumor volume was significantly smaller and the growth rate was slower in the circTRPS1-2 overexpression group.whereas the volume in the knock-down group was bigger and the rate was faster than that in the control group.Animal models of metastatic tumor showed that the number of the metastatic tumor in the circTRPS1-2 overexpression group was significantly fewer than that in the control group,while just the reverse in the knock-down group.Part Ⅲ The role and mechanism of circTRPS1-2 regulating ribosome in ESCC1.Results concerning total protein spectrum of stable transformation cell strain with the overexpression of the circTRPS1-2:circTRPS1-2 can lead to the changes of multiple downstream proteins and affect the multiple pathways.2.Identification of circTRPS1-2 binding protein:15 specific binding proteins were identified through the mass spectrometry analysis of the binding proteins obtained by RNA pulldown assay.The above proteins were closely associated with the generation of the ribosome,which was indicated by the bioinformatics analysis results.3.Binding of circTRPS1-2 to ribosomal proteins and subcellular localization of ribosomal proteins:The results showed that circTRPS1-2 can specifically bind to ribosomal proteins S4X,S8,S24,L7 and L11 through forward validation with western blot after RNA pulldown and reverse validation with qRT-PCR after RIP assay.The results of immunofluorescence showed that the subcellular localization of ribosomal proteins was consistent with the spatial distribution of circTRPS1-2.4.The effect of the circTRPS1-2 on the number of the ribosome and prediction of functional fragments:electron microscopy and absorbance detection suggested that overexpression of the circTRPS1-2 can cause the decrease in the number of the ribosomes and reduced nucleolar diameter,while the opposite results occurred in the knock-down group.The sequence containing cyclization site can inhibit the proliferation and migration of the ESCC cells as well as induce the reduction of the number of the ribosome,which was proved by the CCK-8 assay,colony formation assay and transwell invasion assay and we speculated that this sequence may contain the functional site of the circTRPS1-2.The following conclusions were drawn according to the above results:1.The expression profile of ESCC-related circRNAs was established successfully,and the expression of circTRPS1-2 was significantly low in ESCC.The expression of circTRPS1-2 was positively correlated with the prognosis of ESCC.2.The circTRPS1-2 was a circRNA formed by end-to-end reverse shear of the 2-5 exon sequence of TRPS1 gene and mainly located in the cytoplasm and a few in the cell nucleus.3.Overexpression of circTRPS1-2 inhibited the proliferation and migration of ESCC cells,and the effect was opposite after knockdown.4.CircTRPS1-2 could bind with ribosome protein S4X,S8,S24,L7 and L11,reduce the number of ribosomes,and inhibit the proliferation and migration of ESCC.