| Systemic therapy is the main treatment for advanced hepatocellular carcinoma.In 2010,the first-line treatment of advanced hepatocellular carcinoma with the FOLFOX4 regimen containing oxaliplatin was successful,making systemic chemotherapy one of the options for the treatment of advanced hepatocellular carcinoma.But the overall benefit of chemotherapy in hepatocellular carcinoma system is limited.Chemotherapy resistance is a bottleneck that is difficult to break through in the process of chemotherapy in hepatocellular carcinoma system.Autophagy(autophagy)has been confirmed by a large number of studies to participate in the treatment resistance of hepatocellular carcinoma.RNA binding motif protein 8A(RNA binding motif protein 8a,RBM8A)is the core component of the exon junction complex,and it is also one of the proteins necessary for nonsense-mediated m RNA decay.It is associated with RNA localization,transcription and transport,Translation and degradation,and also closely related to cell cycle regulation and apoptosis regulation.Existing studies have confirmed that RBM8 A is widely involved in the occurrence and development of neurological diseases and malignant tumors.Our previous research found that: RBM8 A is highly expressed in hepatocellular carcinoma tissues.High expression of RBM8 A in hepatoma cell lines in vitro can inhibit apoptosis and promote the proliferation,migration and invasion of hepatoma cells.Increased expression.However,the mechanism by which RBM8 A participates in the drug resistance of liver cancer cells is unclear.In order to further understand the relationship between RBM8 A and autophagy and the role of the two in hepatocellular carcinoma chemotherapy resistance process,this study analyzed the RBM8 A and The relationship between cell autophagy;through the silencing/overexpression of RBM8 A in vitro hepatocellular carcinoma cell model resistant to oxaliplatin,combined with autophagy inducers and autophagy inhibitors,to detect changes in the level of autophagy resistance to liver cancer cells To analyze the effects of RBM8 A and autophagy on oxaliplatin resistance in liver cancer cells.This topic will be studied from the following three chapters.Part I The correlation between RBM8 A and autophagy marker protein LC3 B,clinicopathological characteristics and prognosis in hepatocellular carcinoma patients.Objective: To study the relationship between the expression level of RBM8 A and the autophagy marker protein LC3 B,clinicopathological characteristics and survival prognosis in patients with hepatocellular carcinoma at tissue level.Methods: Retrospectively collected 60 patients who were surgically removed from the Cancer Hospital of Guangxi Medical University from October to December 2016 and confirmed by pathological results as hepatocellular carcinoma.Expression,analysis of the correlation between RBM8 A and LC3 B and the relationship with clinicopathological characteristics,survival analysis of patients with complete follow-up data.Results: In 60 cases of hepatocellular carcinoma,27 cases(45%)had high expression of RBM8 A,and 25 cases(41.7%)had high expression of LC3B;the expression level of RBM8 A and portal tumor thrombus(P<0.001),tumor diameter(P=0.004),Edmondson classification(P=0.017),tumor TNM stage(P<0.001),BCLC stage(P=0.002),and LC3 B expression(P=0.048)have significant correlations.RBM8 A expression level was positively correlated with LC3 B expression level(P=0.007,correlation coefficient r=0.341).Univariate analysis showed whether portal vein tumor thrombus(P=0.001),tumor diameter(P=0.012),serum alpha-fetoprotein(AFP)level(P=0.004),pathological Edmondson grade(P=0.043),tumor TNM stage(P=0.001),BCLC stage(P=0.001),recurrence(P=0.013),RBM8 A expression level(P=0.021),LC3 B expression level(P=0.023)are all factors that affect the prognosis.Multivariate survival analysis showed that portal tumor thrombosis(P=0.032),tumor TNM stage(P=0.035),BCLC stage(P=0.002),and RBM8 A expression level(P=0.049)were the patients with hepatocellular carcinoma.Independent factors of prognosis.Conclusion: The expression levels of RBM8 A and autophagy marker protein LC3 B in hepatocellular carcinoma tissue were positively correlated.The expression level of RBM8 A is a factor affecting the prognosis of patients with hepatocellular carcinoma.Part II Preliminary study on the effect of RBM8 A to autophagy in hepatocellular carcinoma.Objective: Based on the preliminary study of the correlation between the expression level of RBM8 A and the expression of autophagy marker protein LC3 B,the RBM8 A interference and overexpression cell lines were constructed to detect the changes of hepatocellular carcinoma autophagy level after RBM8A interference or overexpression,and further explore RBM8 A The effect of genes on autophagy in hepatocellular carcinoma.Methods: 1.Using q RT-PCR and Western blot methods to detect liver cancer cell lines in vitro,select the cell line with the highest expression level of RBM8 A,design and screen RBM8 A interference sequences,construct knockdown lentivirus and control virus,and infect target cells;verified by q RT-PCR RBM8 A knocks down the efficiency,establishes a stable transfected RBM8A-KD cell line,and establishes an unloaded RBM8A-NC cell line as a control;select the cell line with the lowest RBM8 A expression level,and retrieve the coding sequence(CDS)of RBM8 A gene Sequence,constructed on lentiviral overexpression vector;using packaged overexpression lentivirus and control lentivirus to infect target cells;Western blot to verify RBM8 A overexpression efficiency;obtain stable RBM8 A high expression RBM8A-OE cell line,and establish empty load RBM8A-NC cell line as a control;2.q RT-PCR and Western blot detection of RBM8 A overexpression and interference with LC3A/B,RBM8 A,p62 expression levels in liver cancer cells after 2 hours of normal culture and EBSS starvation stimulation;3.Immunofluorescence detection of RBM8 A overexpression and interference with the distribution and expression of LC3 in liver cancer cells after 2 hours of normal culture and EBSS starvation stimulation;4.Immunofluorescence was used to detect sublocalization changes in RBM8 A cells after 2 hours of starvation stimulation.Results: 1.The screening results showed that Bel-7404 cell line had the highest RBM8 A expression level,which was used for the subsequent construction of RBM8 A knockdown Bel7404-RBM8A-KD cell line;while MHCC97 cell line RBM8A had the lowest expression level,which was used for the subsequent construction of RBM8 A overexpression MHCC97H-RBM8A-OE cell line.2.The results of q RT-PCR showed that the m RNA expressions of autophagy-related markers LC3 A and LC3 B of MHCC97H-RBM8A-OE liver cancer cell line were significantly higher than that of the no-load control group(P< 0.05),but there was no significant change in p62 expression level;after 2hours of normal culture and EBSS starvation stimulation,m RNA expression of autophagy-related markers LC3 A and LC3 B of Bel7404-RBM8A-KD hepatoma cell line were significantly lower than that of the empty control group(P<0.05),but no significant change in p62 expression level;3.Western blot test results showed that under normal culture conditions,the expression of autophagy markers LC3 A and LC3 B in MHCC97H-RBM8A-OE was higher than that of RBM8 A interfering cell line Bel7404-RBM8A-KD,and no significant change in p62 protein expression.After 2 hours of EBSS starvation stimulation,the expression of autophagy markers LC3 A and LC3 B in MHCC97H-RBM8A-OE was significantly higher than that of RBM8 A interfering cell line Bel7404-RBM8A-KD,and the expression of p62 protein was lower in MHCC97H-RBM8A-OE than RBM8 A Interfering cell line Bel7404-RBM8A-KD.4.The results of immunofluorescence experiments showed that under normal culture conditions,the RBM8 A interfering cell lines Bel7404-RBM8A-KD,RBM8 A overexpressing cell lines MHCC97H-RBM8A-OE and the unloaded Bel7404-NC,MHCC97H-NC cell lines LC3 fluorescent markers were more Weak,no obvious autophagy was observed.After being stimulated by EBSS starvation for 2 hours,the number of LC3-labeled spots increased in the RBM8 A overexpressing cell line MHCC97H-RBM8A-OE compared to the empty group MHCC97H-NC(P=0.0152),while RBM8 A interfered with the cell line Bel7404-RBM8A-KD compared with the unloaded group Bel7404-NC,the number of LC3 labeled spots in the cells was reduced(P=0.0059).Compared with RBM8 A over-expressing cell line MHCC97H-RBM8A-OE and RBM8 A interfering cell line Bel7404-RBM8A-KD,the number of LC3-marked spots increased significantly(P=0.0473).5.Autophagy flow monitoring results showed that after 2 hours of EBSS starvation stimulation,compared with the empty cell lines MHCC97H-NC,MHCC97H-RBM8A-OE,the dual fluorescence m RFP-e GFP-LC3 method showed that the red and green bright spots increased,and the red and green The yellow bright spots(autophagosomes)and red bright spots(autophagolysosomes)in the synthesized images increased,confirming that RBM8 A induced autophagy activation;while in the RBM8 A interfering cell line Bel-7404-RBM8A-KD,it can be observed The yellow fluorescent bright spot increased after starvation stimulation,and the red fluorescent bright spot remained unchanged,indicating that the inhibition of autophagy caused the degradation of autophagosomes,which confirmed that the interference of RBM8 A expression caused the inhibition of autophagy.6.Immunofluorescence staining showed that after EBSS starvation: after 2 hours,there was no significant change in intracellular sublocalization of RBM8 A in MHCC97 H cell line with low expression of RBM8 A and Bel-7404 cell line with high expression of RBM8 A.Conclusion: Up-regulation of RBM8 A gene can induce the activation of autophagy in hepatocellular carcinoma cells.Part III RBM8 A affects hepatocellular carcinoma resistance to oxaliplatin through autophagy.Objective: Through the construction of RBM8 A interference and overexpression hepatocellular carcinoma oxaliplatin(OXA)drug resistance model,to detect the effect of RBM8 A changes on cell autophagy level,cell proliferation ability and drug resistance,and study whether the change of autophagy level Reverse drug resistance,and initially explore the correlation between RBM8 A,cell autophagy and the occurrence and development of liver cancer OXA resistance.Methods: 1.Using OXA concentration increasing method,intermittent effect induced the establishment of drug resistant hepatocellular carcinoma models BEL7404/OXA and MHCC97H/OXA,using transfection overexpression technology and RNA interference technology to establish stable parental hepatocellular carcinoma models MHCC97H-RBM8A-OE,BEL7404,respectively-RBM8A-KD and OXA-resistant hepatocellular carcinoma models MHCC97H/OXA-RBM8A-OE,BEL7404/OXA-RBM8A-KD;2.q RT-PCR and Western Blot to detect the expression of LC3 B in parental/drug resistant hepatocellular carcinoma;3.CCK8 method was used to detect the proliferative ability and IC50 of parental/drug resistant hepatocellular carcinoma cell lines;4.Combined with autophagy agonists or autophagy inhibitors,IC50 was detected after OXA treatment to evaluate the oxaliplatin resistance of each group of cells.Results: 1.The results of q RT-PCR and Western blot showed that the expression of RBM8 A in drug-resistant hepatocellular carcinoma cell lines MHCC-7H/OXA and BEL7404/OXA was significantly higher than that in parental hepatocellular carcinoma cell lines MHCC97H-NC and BEL7404-NC.2.The expression level of LC3 B in oxaliplatin-resistant cell lines was significantly higher than that of parental liver cancer cell lines;3.The IC50 value of BEL7404/OXA-RBM8A-KD group decreased,and the tolerance of hepatocellular carcinoma to OXA was significantly reduced.The IC50 value of MHCC97H/OXA-RBM8A-OE group increased,and the tolerance of hepatocellular carcinoma to OXA was significantly improved.4.After co-cultivation with the autophagy agonist rapamycin,the IC50 values??of the above liver cancer cell lines were reduced,and the drug resistance of the liver cancer cells was partially reversed.The IC50 value of the BEL7404/OXA-RBM8A-KD group was the lowest.5.After co-cultivation with the autophagy inhibitor chloroquine,the IC50 value of the above hepatoma cell lines increased,and the tolerance of the hepatocellular carcinoma cells to OXA increased.Among them,the BEL-7404/OXA-NC empty load group combined with the autophagy inhibitor chloroquine showed the highest IC50.Conclusion: The expression of RBM8 A is increased during the induction of OXA resistance in HCC.Overexpression of RBM8 A promotes the proliferation of parental and drug-resistant hepatocellular carcinoma in vitro.Autophagy is involved in the resistance process of hepatocellular carcinoma to OXA. |
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