Study On The Molecular Mechanism Of Circ-FIG4/miR-99a/ABHD4 Axis Affecting The Progression Of Human Esophageal Cancer

1 BackgroundEsophageal carcinoma(ESCA)is a most prevalent malignant tumor and the eighth incidence and the sixth among cancer-related mortality.China is one of the places with high incidence of esophageal cancer.Because the early symptoms are not obvious,most patients with esophageal cancer are diagnosed with advanced stage,often accompanied by lymph node metastasis or distant metastasis.Due to the lack of effective treatment and specific targeted drugs,the 5-year survival rate of patients with esophageal cancer is only 30%.Therefore,through the study of the mechanism of the occurrence and development of esophageal cancer,in order to find effective diagnostic markers and specific treatment targets,it is of great significance to reduce the mortality and improve the quality of life of patients with esophageal cancer.Circular RNA is a special type of endogenous non-coding RNA.Different from the traditional linear RNA,the 3' end and 5'end of circRNA connect to form a closed ring structure,which makes it more conservative and stable,so as to avoid being degraded by RNA cleavage enzyme.At the same time,this property also gives circRNA many special biological functions.In recent years,circRNA has attracted much attention in the study of malignant tumors.More and more circRNA are abnormally expressed in malignant tumors and participate in the occurrence and development of tumors.At present,there is no in-depth study on the function and mechanism of circRNA in esophageal cancer,so screening and identifying circRNA,which plays an important role in the progression of esophageal cancer and exploring its regulatory mechanism are of great significance for the development of molecular markers and potential therapeutic targets for the diagnosis or prognosis of esophageal cancer.The effect of circRNA on tumor progression is often realized through ceRNA network.CeRNA networks include circRNA,lncRNA,pseudogenes,coding protein RNA,etc.,these molecules,as miRNA response elements(MicroRNA Response Element,MRE),can compete to bind to the same miRNA to regulate their expression levels.As an important molecule in the ceRNA network,circRNA can absorb a large amount of its corresponding miRNA through the action of miRNA sponge,and then regulate the expression of corresponding genes at the post-transcriptional level,which controlling the occurrence and development of tumors and affecting the biological behavior of malignant tumors.Some recent studies have shown that circRNA can regulate the expression of downstream miRNA through molecular "sponge action" to inhibit or promote the malignant progression of esophageal cancer.Such as:circ-0006168 is highly expressed in esophageal cancer,and it can target to regulate mir-100/mTOR and mir-339-5p/CDC25A pathway,thus promoting the progress of esophageal cancer.Circ-0004771 fraction highly expressed in plasma of esophageal cancer patients and esophageal cancer cell lines EC9706 and TE1,can regulate the malignant progress of esophageal cancer through mir-339-5p/CDC25A pathway.In esophageal cancer tissue the highly expressed sire-0000654 can sponge absorb mir-149-5p to promote the expression of STAT3 protein,and then promote the proliferation,migration and invasion of esophageal cancer cells,and inhibit their apoptosis.The expression of circ-0004370 is significantly increased in esophageal cancer tissues,and it can promote the malignant progress of esophageal cancer through the mir-1294/laspl axis;the highly expressed sire-0006948 in esophageal cancer tissues and cell lines can directly Combined with mir-490-3p,3'UTR targeting HMGA2 can induce EMT,which also plays an important role in the development of esophageal cancer.Therefore,in-depth study of the influence of circRNA/microRNA signal axis in the occurrence and development of esophageal cancer is of high scientific value to promote the development of early diagnosis and treatment of esophageal cancer.In the first part,this study confirmed that circ-0077607(circ-FIG4)was significantly overexpressed in esophageal cancer.The expression level of circ-FIG4 and the analysis of clinical characteristics of esophageal cancer patients showed that high expression of circ-FIG4 was significantly associated with TNM stage,tumor size,lymph node metastasis and distant metastasis of esophageal cancer,and survival analysis suggested that esophageal cancer patients with high expression of circ-FIG4 had shorter survival time,suggesting that circ-FIG4 may be a potential oncogene in the progression of esophageal cancer.To explore the effect of circ-FIG4 on the development of esophageal cancer,this study conducted a series of functional tests in vivo and in vitro.In vitro experiments,this study confirmed that the proliferation,DNA synthesis,clonal formation,migration and invasion of EC9706 and KYSE30 cells were significantly lower than those of the negative control group after silencing circ-FIG4.In the same time,the apoptosis of circ-FIG4 silencing esophageal cancer cells was increased.In vivo,the growth rate of tumor and Ki-67 level were significantly decreased after circ-FIG4 silenced.The above results confirm that circ-FIG4 has the biological functions of promoting the proliferation,invasion and apoptosis of esophageal cancer and plays the role of oncogene.circRNA,as a non-coding RNA,plays a biological role mainly in influencing the activity and expression of encoded proteins,one of which is the regulation network mechanism of ceRNA.To clarify the involvement of circ-FIG4 in the progression of esophageal cancer through ceRNA network,this study screened and validated the circ-FIG4/miR-99a/ABHD4 regulatory network based on bioinformatics and molecular biology experiments in the second part of this study,as well as its impact on the biological behavior of esophageal cancer cells.This study first screened and predicted that circ-FIG4 could upregulate the expression of ABHD4 by competitively binding to miR-99a,and then RT-qPCR,Western blotting and dual luciferase reporter assay further confirmed the direct binding and regulatory role of circ-FIG4/miR-99a/ABHD4 regulatory axis to each other.In the third part,thish study analyzed the effect of circ-FIG4/miR-99a/ABHD4 regulatory network on the proliferation and invasion of esophageal cancer using functional replenishment test.Functional replenishment test revealed that circ-FIG4 could indirectly promote the expression of ABHD4 through miR-99a to promote malignant phenotypes such as proliferation and invasion of esophageal cancer cells.Silencing miR-99a or overexpressing ABHD4 could partially reverse the inhibition of proliferation and invasion caused by circ-FIG4 silencing.The mechanism exploration found that circ-FIG4/miR-99a/ABHD4 regulated the malignant phenotype of esophageal cancer by regulating the epithelial-mesenchymal transition(EMT)signaling pathway.In the last part,this study firstly analyzed the expression and clinical significance ABHD4 though TCGA database and tissue microarray.Then,the function of ABHD4 on proliferation and invasion of esophageal cancer cells were analyzed by in vitro experiment.The results showed that ABHD4 was significantly overexpressed in esophageal cancer.Esophageal cancer Patients with high expression of ABHD4 had shorter survival time.Functional experiments confirmed that the proliferation,DNA synthesis,clone formation,migration and invasion abilities of EC9706 and KYSE30 cells were significantly down-regulated after ABHD4 silencing compared with the negative control group.It is suggested that ABHD4 plays an oncogenic role in esophageal cancer.In summary,this study is the first identify the biological function of circ-FIG4/miR-99a/ABHD4 ceRNA regulatory network in the progression of esophageal cancer.it reveals a new mechanism of esophageal cancer pathogenesis.This study has a great clinical significance and social value in searching for new drug treatment targets for esophageal cancer.2 Methods2.1 Part 12.1.1 RT-qPCR was used to detect the expression of circ-0077607(circ-FIG4)in 120 esophageal cancer and paracancerous tissues;the expression of circ-0077607(circ-FIG4)in 4 esophageal cancer cell lines(TE-13,KYSE140,EC9706,KYSE30)and one esophageal epithelial cell line(Het-1A)was detected.2.1.2 The relative expression level of circ-FIG4 in esophageal cancer tissues was determined by RT-qPCR assay,and the correlation between circ-FIG4 expression level and clinical characteristics(TNM stage,tumor size,lymph node metastasis and distant metastasis)and survival prognosis of esophageal cancer patients was analyzed in combination with clinical information.2.1.3 Design circ-FIG4 reverse and forward primers to identify the circular RNA characteristics of circ-FIG4 by PCR experiments;confirm the localization of circ-FIG4 in esophageal cancer cells by nucleic acid in situ hybridization experiments.2.1.4 A circular RNA circ-FIG4 silenced esophageal cancer cell line was constructed by lentiviral transfection,and the effect of gene interference was identified by RT qPCR.2.1.5 To observe the changes of proliferation,migration,invasion and apoptosis phenotype of esophageal cancer cells after inhibiting circ-FIG4 expression by silencing lentivirus in esophageal cancer cell lines with high circ-FIG4 expression.2.1.6 To construct a subcutaneous transplanted tumor model in nude mice using circ-FIG4 stably silenced esophageal cancer cell line and observe the changes of tumor proliferation rate and volume in nude mice.2.2 Part 22.2.1 Screening and validation of miR-99a regulated by circ-FIG4 using bioinformatics analysis and RT-qPCR.2.2.2 RT-qPCR was used to analyze the expression level of miR-99a after overexpression or silencing of circ-FIG4.Dual luciferase reporter gene experiment was used to confirm that circ-FIG4 can directly bind to miR-99a.2.2.3 To analyze the expression of circ-FIG4 and its correlation with the expression of miR-99a in esophageal cancer tissues.2.2.4 Bioinformatics analysis,RT-qPCR,Western blotting and dual luciferase reporter gene experiments were used to predict and confirm that miR-99a can bind to and inhibit the expression of ABHD4.2.2.5 Western blotting analysis that circ-FIG4 promotes ABHD4 expression by adsorbing miR-99a.2.3 Part 32.3.1 The esophageal cancer cells were divided into six groups:transfection negative control group(NC),circ-FIG4 silencing group(circ-FIG4),circ-FIG4 silencing&miR-99a inhibitor group(sh-circ-FIG4&miR-99a inhibitor),ABHD4 silencing group(sh-ABHD4),ABHD4 silencing Group&miR-99a inhibitor group(sh-ABHD4&miR-99a inhibitor)and circ-FIG4 silencing&ABHD4 overexpression group(sh-circ-FIG4&FIG4 inhibitor).&ABHD4),to observe the effects of circ-FIG4/miR-99a/ABHD4 axis on the proliferation,migration,invasion and apoptosis of esophageal cancer cells.2.3.2 Subcutaneous transplanted tumors were constructed using the above groups of cells,and the effects of circ-FIG4/miR-99a/ABHD4 axis on the proliferation growth curve and tumor weight of subcutaneous transplanted tumors in nude mice were observed.2.3.3 Using bioinformatics to predict the downstream potential signaling pathways of circ-FIG4/miR-99a/ABHD4 axis involved in the progression of esophageal cancer.the Epithelial-mesenchymal transition(EMT)signaling pathway was validated by Western blotting experiments.2.4 Part 42.4.1 Relative expression levels of ABHD4 in esophageal cancer tissues were determined by RT-qPCR,Western blotting and immunohistochemistry expreiments,and the relationship between ABHD4 expression levels and prognosis of esophageal cancer patients was analyzed in combination with clinical follow-up information.2.4.2 The stably silenced esophageal cancer cell line ABHD4 was constructed by lentiviral transfection and the silencing efffect was detected.2.4.3 Functional experiments to analyze the changes of proliferation and invasion phenotype of esophageal cancer cells after ABHD4 silencing.3 Results3.1 Part 13.1.1 The expression of circ-FIG4 is significantly increased in esophageal carcinoma.It is closely related to the clinical malignant phenotypes such as tumor size,distant metastasis and TNM grade.The survival time of patients with high expression of circ-FIG4 was significantly shorter than that of patients with low expression of circ-FIG4.3.1.2 After circ-FIG4 knockdown,in vitro experiments showed that the ability of proliferation,migration and invasion were significantly decreased.3.1.3 After circ-FIG4 knockdown,the proliferative ability,tumor weight and Ki-67 expression of subcutaneous xenografts of esophageal carcinoma in nude mice were significantly decreased.3.2 Part 23.2.1 circ-FIG4 can bind and adsorb miR-99a,which is significantly low expressed in esophageal cancer and negatively correlated with the expression of circ-FIG4.3.2.2 miR-99a can bind and inhibit the expression of ABHD4,and the expression of miR-99a and ABHD4 is significantly negatively correlated.3.2.3 circ-FIG4 can promote the expression of ABHD4 by adsorbing miR-99a,and the expression of circ-FIG4 is significantly positively correlated with ABHD4.3.3 Part 33.3.1 Functional replenishment experiments confirmed that silencing circ-FIG4 could significantly inhibit the proliferation,DNA synthesis,migration and invasion ability of esophageal cancer cells EC9706 and KYSE30,this function could be partially reversed after inhibiting or overexpressing ABHD4 by miR-99a.3.3.2 Functional replenishment experiments confirmed that silencing circ-FIG4 could significantly inhibit the proliferation rate of nude mice subcutaneous xenograft tumor model in vivo,this function could be partially reversed after inhibiting or overexpressing ABHD4 by miR-99a.3.3.3 circ-FIG4/miR-99a/ABHD4 regulates the malignant phenotype of esophageal cancer through EMT signaling pathway.Mechanistic replenishment experiments confirmed that silencing circ-FIG4 could significantly inhibit the EMT phenotype of esophageal cancer cells,while it could be partially reversed after inhibiting or overexpressing ABHD4 by miR-99a.3.4 Part 43.4.1 TCGA data analysis confirmed that the expression of ABHD4 was significantly increased in esophageal carcinoma and was closely related to the clinical prognosis of patients.3.4.2 The tissue microarray analysis of esophageal cancer further confirmed that the expression of ABHD4 protein was significantly increased in esophageal carcinoma and was closely related to the clinical prognosis of patients.3.4.3 The proliferation,clonogenic and invasive abilities of esophageal carcinoma cells with ABHD4 silencing significantly decreased.4 Conclusion4.1 Circ-FIG4 is highly expressed in esophageal carcinoma,which is closely related to clinical malignant phenotype and survival prognosis,and is a potential oncogene.4.2 Circ-FIG4 can promote the expression of ABHD4 by adsorbing miR-99a to form a ceRNA regulatory network,and the circ-FIG4/miR-99a/ABHD4 axis regulates the biological process of esophageal cancer by affecting the EMT pathway.4.3 ABHD4 is highly expressed in esophageal carcinoma,which is closely related to clinical malignant phenotype and survival prognosis,and is a potential oncogene.In conclusion,this study confirms that circ-FIG4/miR-99a/ABHD4 regulatory axis may regulate the occurrence and development of esophageal cancer,and maybe a new target for early diagnosis and treatment of esophageal cancer.