PCAT1 Inhibits The Expression Of Mir-329-3p Targeted VEGFA And Regulates The Development Of Hepatocellular Carcinoma

Objective: Hepatocellular carcinoma(HCC)is a kind of malignant tumor seriously harmful to health.Due to the late onset of symptoms,the best treatment opportunity has been missed,and the prognosis is poor and the treatment is limited.HCC grows rapidly and may be accompanied by multiple organ metastases in liver,lung,bone and other organs.These pathological processes are closely related to gene expression and regulation.Recent studies have been looking for biomarkers related to hepatocellular carcinoma in peripheral blood.Peripheral blood transcriptome RNA is extremely important for the study of molecular biological mechanisms of disease.Transcriptome RNAs include messenger RNA(m RNA)and non-coding RNAs,such as long non-coding RNA(lnc RNA)and circular RNA(circ RNA).Sequencing technology can be used to detect the expression of RNA related to the biological process of disease,including the expression data of m RNA,lnc RNA and other levels.This study intends to detect the differential expression of lnc RNA and m RNA in peripheral blood of HCC patients by RNA sequencing technology,analyze the related mechanism of key genes and biological behavior of HCC by GO,KEGG and other methods,and select the angiogenesis-related pathways related to the occurrence and development of HCC.lnc RNA-PCAT1 with large differences before and after TACE treatment was screened,as well as m RNA-VEGFA regulating VEGF pathway,and PCAT1-targeted m RNA-VEGFA was predicted.By detecting PCAT1 in peripheral blood and transfecting PCAT1 and its inhibitor into HUH-7 cell line model,the effects of PCAT1 on proliferation,apoptosis,migration and invasion of HCC cells were observed,and the mi R-329-3p binding sites with PCAT1 and VEGFA were predicted by software.Cell experiments verified that PCAT1 can serve as a Competitive endogenous RNA(ce RNA)and inhibit the expression level of mi R-329-3p targeted VEGFA through mi RNA sponges.Methods: First,peripheral blood of HCC patients before and after TACE was collected,and RNA sequencing was performed on 5 of them.The "Facto Mine R","Pheatmap" and "Limma" packages of R language were used for analysis and screening,and the differences of lnc RNA and m RNA before and after TACE treatment were screened.The biological process was analyzed,and GO and KEGG analysis were performed.The 15 most differentially expressed lnc RNAs were detected in the peripheral blood of 30 patients,and it was found that the differentially expressed lnc RNA-PCAT1 was the most obvious.The m RNA-VEGFA that can regulate angiogenesis was selected,both of which are involved in the regulation of angiogenesis.Lncrna-pcat1 is predicted to regulate VEGFA.Next,PCAT1 regulates the biological function of liver cancer and VEGFA expression.Serum of patients before and after TACE treatment was used to evaluate the correlation between PCAT1 and tumor stage,and HUH7 cell lines with significant differences in PCAT1 expression before and after hypoxia were screened out.HUH7 cell lines were transfected with control and si-PCAT1.Control transfection and PCAT1 transfection were carried out in hypoxic HUH7 cells.CCK-8 cell proliferation assay,Transwell assay and Annexin V-PI assay were performed to verify that PCAT1 could affect the biological function of HCC cells by promoting VEGFA expression.This process was verified in nude mice,and it was found that the weight and volume of tumor tissue in si-PCAT1 group was lower than that in the control group,and the metastasis ability was decreased.The expression of VEGFA in si-PCAT1 group was lower than that in the control group by staining.Finally,the mechanism of PCAT1 regulating HCC cell function was explored.STARBASE3 software was further used to predict that PCAT1 had specific binding sites with mi R-329-3p,and MIRDB software was used to predict that VEGFA and mi R-329-3p had specific binding sites,which were verified by immunofluorescence assay.HUH7 cells were transfected with control,mi R-329-3p and mi R-329-3p inhibitor,and transfection efficiency was detected by rt-PCR.After transfection,CCK-8 cell proliferation assay,Transwell assay and Annexin V-PI assay were performed to verify that mi R-329-3p inhibited VEGFA expression and affected the biological function of HCC cells.HUH7 cells were further transfected into groups,and the proliferation level of HUH7 cells was detected by CCK8 assay,and the expression levels of VEGFA,PCAT1 and mi R-329-3p were detected by rt-PCR.Finally,it was verified that PCAT1 regulates the expression of VEGFA through mi R-329-3p and affects the biological function of HCC cells.Results: 1.Differential expression existed in serum of patients before and after TACE,including 179 down-regulated lnc RNAs and 244 up-regulated lnc RNAs.2.GO and KEGG analysis were conducted on 50 lnc RNAs with significant differential expression,and it was found that they were involved in regulating a variety of biological processes,among which phosphorylation regulation,phosphatase activity regulation,angiogenesis and cell adhesion regulation were closely related to the treatment and progression of HCC.3.In serum of HCC patients before and after TACE treatment,15 lnc RNAs with the most significant differential expression were detected,and PCAT1 was significantly down-regulated after TACE treatment,which may regulate VEGF signaling pathway.4.There were several m RNA differentially expressed in serum of patients before and after TACE,including m RNA-VEGFA.5.Down-regulation of PCAT1 expression is correlated with AFP content and TUMOR BCLC grade.Patients with higher tumor grade and AFP content are more likely to have down-regulation of PCAT1 expression in serum after TACE treatment.6.Cell experiments confirmed that lnc RNA-PCAT1 was down-regulated after hypoxia and promoted the proliferation,migration and invasion of HCC cells.7.In vivo experiments have demonstrated that PCAT1 can regulate VEGFA expression and affect the biological function of HCC cells.Inhibition of PCAT1 reduces tumor volume,tumor size,VEGFA staining and metastasis ability.8.HUH7 cells were divided into four groups,transfected with si-NC+control,si-NC+mi R-329-3p inhibitor,si-PCAT+control and si-PCAT1+mi R-329-3p inhibitor,respectively.After transfection,CCK8 assay and rt-PCR detection showed that mi R-329-3p inhibitor significantly promoted cell proliferation,up-regulated expression of PCAT1 and VEGFA,and si-PCAT1 down-regulated cell proliferation.The expression levels of PCAT1 and VAGFA were down-regulated,and the expression levels of mi R-329-3p were up-regulated.The co-transfection of PCAT1 and VAGFA on cell proliferation and the effects on PCAT1,mi R-329-3p and VEGFA were basically offset.9.HUH7 cells were divided into four groups,which were transfected with vector+control,vector+mi R-329-3p,PCAT1+control and PCAT1+mi R-329-3p,respectively.After transfection,CCK8 test and rt-PCR detection were performed.The results showed that PCAT1 significantly promoted cell proliferation,up-regulated the expression of PCAT1 and VEGFA,and down-regulated the expression of mi R-329-3p.mi R-329-3p down-regulated cell proliferation and down-regulated the expression of PCAT1 and VEGFA.The effects on cell proliferation and PCAT1,mi R-329-3p and VEGFA were basically offset.Conclusion: 1.There were a large number of differentially expressed RNAs in serum of patients before and after TACE treatment,including 179 down-regulated ln RNAs and244 up-regulated lnc RNAs.After treatment,592 m RNAs were significantly down-regulated and 688 m RNAs were significantly up-regulated.2.Differentially expressed lnc RNAs before and after TACE treatment may be involved in regulating a variety of biological functions and signaling pathways.3.PCAT1 was significantly down-regulated after TACE treatment,which may regulate VEGF signaling pathway.4.PCAT1 can significantly promote the proliferation level of HCC cells,inhibit the apoptosis ability of HCC cells,enhance the metastasis ability of HCC cells,and promote the expression of VEGFA.5.PCAT1 can attenuate the phenomenon of proliferation inhibition,increased apoptosis and reduced migration of HCC cells caused by hypoxia.6.Mir-329-3p has binding sites with PCAT1 and VEGFA.Upregulation of Mir-329-3p can reduce the proliferation level of HCC cells,increase the apoptosis ability of HCC cells,down-regulate the migration effect of HCC cells,inhibit the expression of VAGFA,and inhibit the proliferation level of HCC cells.VAGFA expression was promoted by inhibiting the apoptosis of HCC cells,up-regulating the migration of HCC cells.7.PCAT1 can act as a competitive endogenous RNA to inhibit mi R-329-3p targeted VEGFA expression through mi RNA sponges.