Ultrasound-guided Radiofrequency Ablation Circular RNA Expression Profile In Nude Mice Hepatoma Xenograft Model

Background and purpose:Liver cancer is the second leading cause of cancer-related death worldwide,and it is one of the most common and most malignant tumors in the world.There are nearly 850,000 new cases of liver cancer each year in the world,and the number of new cases and deaths in China accounts for more than 50%of the world's total population.Hepatocellular carcinoma?HCC?,as the most subtype of primary liver cancer,accounts for about 90%of primary liver cancer,and the incidence of HCC in the world is increasing year by year.Radiofrequency ablation?RFA?is currently the most widely used thermal ablation therapy for liver cancer,and it is also a representative interventional treatment for minimally invasive treatment of liver cancer.With the improvement of radiofrequency ablation apparatus and the continuous improvement of imaging diagnosis and treatment techniques,radiofrequency ablation has become increasingly prominent in the clinical treatment of HCC patients.Circular RNAs?Circular RNAs,CircRNAs?are a special type of endogenous non-coding RNAs.They are a class of non-5'-end caps and 3'-end poly?A?tails RNAs that covalently form a ring structure.Encoded RNA molecules have been shown to participate in the development of a variety of diseases.However,the expression profile of circRNA in radiofrequency ablation of liver cancer remains unknown.The purpose of this study was to use circRNA as an entry point to conduct gene chip technology to screen differentially expressed circRNA from a radiofrequency ablated nude mice hepatoma xenograft model,and to explore the possible role of circRNA in radiofrequency ablation HepG2 nude mice hepatoma xenograft models.Materials and Methods:1.Establishment of a nude mouse liver cancer modelA Balb/c nude mouse subcutaneous xenograft model was modeled by the inject of 100?l?2x10⁶ cells/ml?of HepG2 cells laterally into 8 male immunodeficient mice?Balb/c nude mice??20±2 g?.Ultrasonic?US?,color Doppler flow?CDFI?were used to evaluate the model of HepG2 subcutaneous xenograft tumor model in nude mice.2.Ultrasound-guided radiofrequency ablation in nude miceRandomly divide experimental group?N=4?and control group?N=4?.Perform incomplete ultrasound ablation of the Cool-tip cold-circulation system under RFA in the supine position in 4 Balb/c nude mice in the experimental group?RF power 30W.Ablation 10s?.After the RFA,the nude mice of the experimental group and the control group were sacrificed.Fresh tumor tissues were immediately?<10 min?taken and divided into 2?>100 mg?sections.One pathological examination was performed to observe tumor cells;the other tumor tissue was treated with phosphoric acid.The buffered saline solution was washed and the sample was immediately placed in a cryotube containing 5volumes of RNAlater protection solution.The sample was immediately placed in-80°C liquid nitrogen and frozen,and sent for sequencing.3.CircRNA sequencing analysisThe total RNA from eight samples was extracted and quantified for purity and concentration.RNA integrity was assessed by denaturing agarose gel electrophoresis.The tested samples were scanned using an Agilent Scanner G2505C for Arraystar Human CircRNA Array v2 assay analysis.R-software limma package was used to perform quantile normalization of raw data and subsequent circRNA differential expression data processing.4.CircRNA Real-time PCR?qPCR?verificationQuantitative real-time PCR?qPCR?was used to detect expression of the circRNAs with up-and down-regulations to verify the reliability of the internal chip in this study.5.Clinical expression of CircRNA in HCCThrough the big data mining technology,the Gene Expression Omnibus?GEO?database were searched,and a case-control study of clinical tissue sample expression of circRNA in HCC was analyzed retrospectively.An independent sample t-test was used to compare and analyze the expression levels of differentially expressed circRNAs between HCC and adjacent para-carcinoma tissues,and Stata12.0 was used for systematic evaluation and heterogeneity analysis to calculate standardised mean differences?SMD?.The SROC and 95%CI were combined with statistics on the GEO database chip to evaluate the expression level and clinical value of circRNA in HCC.And then,we draw the funnel plot and the Egger regression equation to assess the publication bias.Results:1.Establishment of a nude mouse liver cancer modelHep G2 nude mice underwent subcutaneous xenograft model evaluation.Two-dimensional ultrasound?US?suggested that a hypoechoic bolus was explored subcutaneously in the dorsal spine of nude mice.The size of the lesion was approximately 1.4x0.1 cm,the boundary was still clear,the shape was irregular,and the internal echo was not uniform.CDFI color Doppler imaging prompted a rich blood flow within the lesion.2.Ultrasound-guided radiofrequency ablation of nude miceAnatomy of four nude mice without radiofrequency ablation control group was performed.After the tumor tissue was incised,the tumor tissue consisted of multiple white nodular masses.The tumor does not adhere to the skin and subcutaneous tissue,there is no significant infiltration growth,easy to peel.The pathological section of HE staining showed that the tumor cells were arranged in cords or sheets.The size of the cells was different.Multinuclear tumor cells were seen.The nucleus was heavily stained and the mitotic figures were increased.Some cells were rich in cytoplasm and red stained,and the nucleus was round or oval.Radiofrequency ablation incomplete treatment of experimental group nude mice liver cancer tumor tissue incomplete damage,the tumor tissue incision,the naked eye surface of the tumor showed a white burning.Under the light microscope,the tumor cells were arranged in trabeculae.Tumor cells are large in size and are polygonal,round and columnar.The ratio of nuclear to cytoplasm of tumor cells is elevated,and the nuclei are deeply stained.Visible pathological mitoses are seen and necrosis is also been seen.3.CircRNA chip analysisThe specimens of tumor tissue in 8 male Balb/c nude mice all met the requirements of CircRNA chip quality testing.Normalized and low-expressed information was filtered on the raw data detected by the circRNA microarray.Using the RNA-seq technique,scatter plot and volcano plot analysis showed that76 transcripts that were significantly differently expressed in the unablated full and non-ablated control groups were screened?21 differentially upregulated circRNAs and 55 differentially downregulated circRNAs??|FC|>1.5,P<0.05?.4.CircRNA Real-time PCR?qPCR?verificationThe qPCR detection of circRNA hsa_circRNA₁03595 and down-regulation of circRNA hsa_circRNA₀01264 was selected.In the experimental group,hsa_circRNA₁03595 was up-regulated 1.926 times?P=0.037?,and hsa_circRNA₁03595 was down 1.554 times?P=0.531?.The result of hsa_circRNA₁03595 was up-regulated by 1.528-fold?p=0.001?and that of hsa_circRNA₁03595 was 1.591-fold?p=0.005?.5.Clinical expression of CircRNA in HCCBased on the GEO database tissue chip,21 differentially up-regulated circRNAs and 55 differentially down-regulated circRNAs were used to verify the expression of HCC clinical tissue samples.Three chips,GSE78520,GSE94508,and GSE97332,were included in this study.In the GSE94508 chip,theover-expressedcircRNAswere hsa_circRNA₀01264,hsa_circRNA₁01225,hsa_circRNA₁00139,and the low-expressing circRNAs were hsa_circRNA₁00168 and hsa_circRNA₁03392,respectively.In the GSE97332 chip,the highly expressed circRNAs were hsa_circRNA₁03666,hsa_circRNA₁03595,hsa_circRNA₁00139,hsa_circRNA₁00505,hsa_circRNA₁00168,andthelowexpressingcircRNAswere hsa_circRNA₀01264,hsa_circRNA₁04733,hsa_circRNA₁01225,hsa_circRNA₁00308,hsa_circRNA₄00101 and hsa_circRNA₁00065,respectively.The result of the normalized mean difference?SMD?obtained by combining the effect amounts of the circRNA expressing data on all three chips showed that hsa_circRNA₁00168 was lowly expressed in HCC?-2.089,95%CI:-2.089-?-3.447?,P=0.003?,SMD of other circRNAs were not statistically significant?P>0.05?.Conclusions:1.Establishment of a nude mouse liver cancer modelThe nude mouse hepatoma subcutaneous xenograft tumor model produced by HepG2 tumor cell in vivo cultivation method has a short cycle and the success rate reaches 100%.Its growth behavior is similar to that of human liver cancer and it is suitable for swollen experimental study of tumors.2.Ultrasound-guided radiofrequency ablation in nude miceCompared with the control group,residual tumor cells were seen under the microscope of the subcutaneously transplanted tumors of nude mice with hepatoma in the incomplete ablation experimental group,and the peritoneal area was necrotic,suggesting that the ablation was incomplete.Subsequent ultrasound-guided radiofrequency ablation of nude mice can be used to study circRNA expression profile.3.CircRNA chip analysisIn this experiment,incomplete ablation of nude mice liver cancer subcutaneous transplanted tumor samples,circRNA expression has undergone a differential change.4.CircRNA Real-time PCR?qPCR?verificationThe PCR results suggested that the internal chip circRNA expression data was reliable.5.Clinical expression of CircRNA in HCCThe differential expression of circRNA in the internal chip of this study may be involved in the occurrence and development of HCC and play a role.In radiofrequency ablation HepG2 nude mice hepatoma xenograft samples,this study confirmed the changes in circRNA expression in radiofrequency ablation HepG2 nude mice hepatoma xenograft models,which may play an important role in the physiological and pathological process of HCC patients after RFA.These findings not only provide a new direction for studying the molecular mechanism of RFA HCC,but also provide new possibilities for the treatment of HCC through the regulation of circRNA.