| Background and Objective:Hepatocellular carcinoma(HCC)is a malignant tumor with a rich blood supply,of which 90% of the blood supply comes from the hepatic artery.Recently,transarterial chemoembolization(TACE)is one of the most widely used minimally invasive surgical treatments for liver cancer,which can effectively improve the overall survival rate.However,liver cancer usually develops from chronic liver disease,and its genetic background is complex and highly heterogeneous.Existing clinical staging methods,such as TNM staging or Barcelona clinical staging of liver cancer,can predict the prognosis of patients to a certain extent,but patients with the same staging of liver cancer may have completely different outcomes.In recent years,the development of genomics provides strong support for the accurate diagnosis and treatment of liver cancer.Ideally,after TACE is selectively or super selectively implanted into the blood supply target artery of liver cancer tissue,combined with embolic agents and chemotherapeutic drugs will eventually cause tumor tissue necrosis.However,the hypoxic microenvironment after TACE may promote the residual tumor cells to obtain epithelial-mesenchymal transformation(EMT),which increases the adaptability of tumor cells to the environment and enhances the proliferation and metastatic potential of HCC.Therefore,it is particularly important to study the specific molecular mechanism of EMT after liver cancer TACE,to find the optimal scheme for postoperative tumor invasiveness evaluation,and to provide the basis for follow-up time after interventional embolization of liver cancer.FOXM1 is a specific proliferating protein in the Forkhead Box Protein(Fox)transcription factor family,which exists in all mammalian cells and mainly regulates the process of cell differentiation,proliferation,and apoptosis.At the same time,the imbalance of FoxM1 expression is closely related to the occurrence and development of cancer.FoxM1 has been identified to be highly expressed in a variety of malignant tumors and plays an important role in promoting tumor angiogenesis,invasion,and metastasis.In hepatocellular carcinoma,related studies have shown that the expression of FoxM1 is the highest in tumor tissues with poor prognosis,and its expression level is positively correlated with genomic instability,tumor index of proliferation,and the density of tumor tissue microvessel,and negatively correlated with apoptosis.Therefore,further understanding of the regulatory mechanism of FoxM1 expression in HCC can provide a new theoretical basis for recurrence and metastasis of HCC.After the fusion of intracellular poly-vesicles and cell membranes,the membranous vesicles,exosomes are released to outside the cells which are about 50-150 nm.Almost all cells in the body can secrete exocrine bodies.A variety of substances,including DNA and non-coding RNA(ncRNA),proteins,are carried in the exocrine body.Exocrine bodies and their contents play an important role in a variety of physiological and pathological processes,such as cell-to-cell communication,immune response,antigen presentation,and so on.The exocrine is produced in specific tissues and cells.Compared with the free substance in the circulatory system,the tissue specificity of the content carried by the exocrine is increased.At the same time,the substances contained in the exocrine body are more stable than those in the peripheral blood.Long non-coding RNAs(lncRNAs)is a transcript with a length of more than 200 nucleotides,which is usually an RNA without the function of protein-coding.LncRNAs play an important role in cell proliferation,differentiation,and metabolism.More and more evidence shows that lncRNAs affect the proliferation,survival,invasion,metastasis,and drug resistance of tumor cells by regulating the process of tumor proliferation,differentiation,and EMT.The abnormal expression of lncRNAs in digestive system tumors has a great impact on the occurrence and development of tumors.Exocrine lncRNAs can be used not only as a biomarker for diagnosis and prognosis evaluation but also as a potential target for tumor therapy.The objective of this research is to find a potential biomarker that can be used to evaluate the prognosis of patients with liver cancer after TACE,and to provide a theoretical basis for the strategy of TACE operation and postoperative follow-up of patients with liver cancer.Materials and Methods:1.Population cohort study.The differentially expressed lncRNAs related to hypoxia and EMT in hepatocellular carcinoma were screened according to the database.The peripheral blood of patients with liver cancer before and after TACE was collected,and the expression level of plasma exocrine LncRNA-PVT1 was detected by qPCR.Combined with clinical data,Cox multivariate regression analysis was used to construct the prognosis evaluation model after TACE,and the effectiveness of the model was evaluated by the ROC curve.2.In vitro experiment.The proliferation activity of cells was observed by MTT,the invasiveness of cells was observed by the Transwell test,and the ability of cell migration was observed by the scratch test.The highly invasive hepatoma cell model induced by hypoxia was established by Co Cl2,and the expression of high invasive exosome LncRNA-PVT1 was detected by qPCR.The exosomes of highly invasive hepatocellular carcinoma cells were extracted and co-cultured with low invasive hepatocellular carcinoma cells to observe the effects of exosomes derived from highly invasive hepatocellular carcinoma cells on the proliferation,invasion,and migration of low invasive hepatoma cells.The whole gene was synthesized and overexpressed LncRNA-PVT1 plasmid and Si-lncRNA PVT1 were transfected into low invasive hepatocellular carcinoma cells.The effects of LncRNA-PVT1 on the proliferation,invasion,and migration,and apoptosis of low invasive cells were observed.Double luciferase reporter gene assay was used to determine the miRNA-345-5p site and mechanism of LncRNA-PVT1-FoxM1 binding.miRNA mimic and ASO were transfected into low invasive hepatoma cells,and the effects of miRNA-345-5p on proliferation,invasion,and migration,and apoptosis of low invasive hepatoma cells were observed.Salvage experiments were conducted to observe the effects of miRNA-345-5p on the proliferation,invasion and migration,and apoptosis of low invasive cells co-cultured with highly invasive exosomes induced by hypoxia.3.In vivo experiment.Experimental groups: low invasive cells,low invasive cells co-cultured with hypoxia-induced highly invasive exosomes,and low invasive cells cocultured with normal hepatocyte exosomes.Different groups of liver cancer cells were inoculated subcutaneously in nude mice,and the body weight,tumor formation,and tumor size of nude mice were observed after inoculation with different treatments of liver cancer cells.Immunohistochemical staining was used to observe the expression of FoxM1,invasive markers in tumor tissues.Western Blot(WB)was used to detect the level of EMT markers.Results:1.Population cohort study.According to the results of the differentially expressed lncRNAs related to hypoxia and EMT in liver cancer of the database,we found that compared with adjacent tissues,there were 144 differentially expressed lncRNAs in tumor tissues.Combined with literature analysis,there were 25 hypoxiarelated lncRNAs and 45 lncRNAs related to liver cancer EMT.After the intersection,we confirmed that 2 lncRNAs(LncRNA-PVT1 and GAPLINC)were associated with EMT in the hypoxic microenvironment of liver cancer.Among them,the positive feedback regulation of LncRNA-PVT1 and FoxM1 participates in the process of tumor proliferation,differentiation,and EMT,and promotes the proliferation,survival,invasion,metastasis,and drug resistance of liver cancer.According to the median PFS,the patients were divided into early recurrence groups and late recurrence groups(non-recurrence patients were the late recurrence group).Between the general data of patients and the results of preoperative laboratory examination between the median PFS groups,there was no statistical difference.The prognostic evaluation model established by Cox multivariate regression analysis was exosomal PVT*1.05+ALB*(-0.16).According to the median score of the model,the survival analysis showed that the PFS of the high-risk group was lower than that of the low-risk group,and between the two groups,there was a significant difference(P < 0.001).The median PFS of the high-risk group was about 50 days.The ROC results show that the cutoff value is-5.267 and the area under the curve is 0.799.2.In vitro experiment.The results of cell line identification showed that the proliferative activity,invasion,and migration ability of HepG2 were higher than those of other hepatocellular carcinoma cell lines.The proliferative activity,invasion,and migration ability of Huh7 were the lowest in all hepatocellular carcinoma cell lines and low invasive hepatocellular carcinoma cells.The expression of HIF-1 α was up-regulated after hypoxia in HepG2,which was significantly different from that before hypoxia(P = 0.009).The expression of exosome lncRNA-PVT1 was upregulated,and there was no significant difference between the two groups(P = 0.923)due to the large dispersion of exosome lncRNA-PVT1 expression in HepG2 without hypoxia treatment.After co-culture with Huh7,the results of fluorescence staining showed that Huh7 was the hypoxia-induced HepG2 exosome > HepG2 exosome > Lo-2exosome at the same time.Compared with un intervened Huh7 cells and Huh7 cells co-cultured with hypoxia-induced HepG2 exosomes,the expression of EMT marker E-cadherin in Huh7 cells co-cultured with hypoxia-induced HepG2 exosomes decreased,while the expression of Ncadherin and vimentin increased,and there were significant differences between the two groups(P < 0.05).Further experiments showed that after coculture of Huh7 cells with exosomes,the proliferation activity of hypoxiainduced HepG2 exosomes was the highest,and the ability of invasion and migration was the strongest,followed by Huh7 cells co-cultured with hypoxia-induced HepG2 exosomes.It is suggested that the exocrine secreted by highly invasive hepatocellular carcinoma cells can promote the EMT process of low invasive hepatocellular carcinoma cells.Compared with the blank control,the expression of E-cadherin was down-regulated after overexpression of LncRNA-PVT1,while the expression of vimentin and FoxM1 was up-regulated after overexpression of si-NC.Compared with the control group transfected with LncRNA-PVT1,the expression of E-cadherin was up-regulated and the expression of vimentin and FoxM1 was down-regulated after silencing cadherin.The proliferative activity,invasion,and migration ability of Huh7 cells were the highest in the over-expressed LncRNA-PVT1 cells and the lowest in the over-expressed LncRNA-PVT1 cells.The apoptosis was the least in the over-expressed LncRNA-PVT1 cells and the most in the LncRNA-PVT1-suppressed cells.The double luciferase reporter gene experiment miR-345-5p could directly target the 3 ’UTR of LncRNA-PVT1-5:1 and FoxM1,but the targeting effect disappeared after the seed sequence was mutated.The expression of miR-345-5p was the lowest in HepG2 cells and the highest in Lo-2 cells.When Huh7 cells were treated with miR-345 mimics overexpressing miR-345,the expression of miR-345-5p was increased,while the expression of downstream target molecule FoxM1 was decreased compared with the control group(P=0.005 and P=0.038),and the expression of N-cadherin was down-regulated(P=0.004),vimentin down-regulated,there was a significant difference between the two groups(P=0.011).The expression of E-cadherin was up-regulated,and the difference was statistically significant(P=0.021).The proliferative activity,invasion,and migration ability decreased and apoptosis increased.Compared with the control group ASO NC,the expression of miR-345-5p was down-regulated and the expression of downstream target molecule FoxM1 was up-regulated in Huh7 cells treated with miR-345 ASO which suppressed the expression of miR-345,and the difference was statistically significant.The expression of E-cadherin was down-regulated,the expression of N-cadherin was up-regulated,and the expression of vimentin was up-regulated,and the proliferative activity,invasion,and migration ability were increased,and apoptosis was decreased.Compared with hypoxia-induced exosome + mimics NC-treated Huh7 cells,hypoxia-induced HepG2 exosome + miR-345 mimics interfered with Huh7 cells up-regulated miR-345-5p expression,up-regulated E-cadherin expression,down-regulated FoxM1,N-cadherin,and vimentin expression(P< 0.001).3.In vivo experiment.Compared with the untreated low invasive hepatoma cells,the tumor volume of the low invasive hepatocellular carcinoma cells co-cultured with highly invasive exosomes was the largest and grew the fastest in nude mice,and the expression of LncRNA-PVT1 and FoxM1 was up-regulated in tumor tissues,and the expression of miR-345-5p was down-regulated(P=0.018,P< 0.001).The results of immunohistochemistry showed that the expression of FoxM1,Ki67,and MMP9 was up-regulated,and there were significant differences(P=0.005,P <0.001,P=0.003).Conclusion:Plasma exosomal lncRNA-PVT1 can be used as a potential biomarker to evaluate the prognosis of patients with liver cancer after TACE.After hypoxia induction,the expression of exosome lncRNA-PVT1 of highly invasive cells is up-regulated.The expression of downstream FoxM1 in low invasive cells is positively regulated by the miRNA-345-5p-mediated ce RNA mechanism,which makes low invasive cells acquire EMT ability,enhance their proliferative activity,invasion and migration ability,and inhibit apoptosis,which is related to the recurrence and metastasis after HCC interventional embolization. |
