| Hepatocellular carcinoma(HCC),named as “liver cancer”,accounts for85%-90% of primary liver cancer,and its annual incidence will exceed 1million by 2025.Currently,it is the fourth most common malignant tumor and the second leading cause of death in China,which is a serious threat to human health.More than 60% of patients with HCC are already in the middle and advanced stages when diagnosed,and systemic drug therapy is the most important treatment.However,drug resistance is currently the most prominent and thorny problem,and it is also the most fundamental reason for treatment failure.Like many other cancers,HCC is also highly heterogeneous.Even if patients have similar disease phenotypes,they may also have different molecular types,making treatment responses different.Stratification of patients at the molecular level helps to formulate individualized treatment,improve drug efficacy,reduce drug resistance rates,and improve patient prognosis.Therefore,it is urgent to clarify the molecular mechanism of HCC drug resistance,seek molecular markers that can predict drug resistance,and find new targets to control it.Oxaliplatin(OXA)and sorafenib are currently the most commonly used first-line drugs for systemic chemotherapy and targeted therapy of HCC.OXA belongs to the third generation of platinum drugs.As a bifunctional alkylating agent,OXA can bind DNA to form a platinum-DNA complex,which will block DNA replication and transcription,then kill tumor cells.It has been reported that chemoresistance of platinum drugs is an important cause of poor clinical chemotherapy,and different changes in the same gene may lead to different drug effect.Gene level can reflect the efficacy of drug,and some of them can be used as biomarkers to predict response of drugs.Personalized drugs for cancer patients based on the target gene can provide the most effective chemotherapy.With the development of non-coding RNA(lnc RNA)research,we found they play important role in regulating gene transcription,translation and function,and it can be used as an important molecule to assist the diagnosis and treatment of HCC.Therefore,screening OXA resistance-related lnc RNA and evaluating its mechanism is of great importance in clinical.Sorafenib is the first tyrosine kinase inhibitor(TKI)for systemic therapy in advanced HCC.At present,sorafenib resistance has become an important limitation in clinical,and it is also a major challenge in advanced HCC patients.However,the mechanism of sorafenib resistance is unclear.Therefore,it is urgent to explore new molecule and mechanism of sorafenib resistance to provide a new direction for the control of sorafenib resistance.Part 1 A novel OXA-resistance related molecule LINC13 in HCC and its clinical significanceObjective: To identify new molecules related to OXA resistance through differentially expressed molecules in OXA resistance HepG2 cells,and to explore its clinical significance.Methods:1.Combining with the current status of the high drug resistance rate of OXA in HCC patients,we first established OXA-resistant(OXA-R)HepG2 cells through high-dose drug stimulation.Then clone formation experiment and flow cytometry were used to analyze the characteristic of OXA-R HepG2 cells.The total RNA of OXA-sensitive(OXA-S)and OXA-R HepG2 cells was extracted,and transcriptome sequencing(RNA-Seq)was performed,focusing on the differential level of lnc RNAs that caused by OXA resistance.Combined with Reverse Transcription Polymerase Chain Reaction(RT-PCR),lnc RNA with the highest correlation with OXA resistance was preliminarily identified.2.The 342 patients with pathological diagnosis of HCC were screened from the cancer genome atlas(TCGA).LINC13 data of HCC patients in the database was used to draw the ROC curve and then we determine the cutoff value of LINC13.We divided HCC patients into high and low levels of LINC13,and analyzed the clinical and pathological characteristics(age,gender,Child-pugh stage,tumor size,pathological stage,clinical stage,vascular infiltration and clinical prognosis)of two groups.Finally,we analyzed the relationship between LINC13 and clinicopathological features and prognosis of HCC patients.3.Gene Expression Profiling Interactive Analysis(GEPIA)was used to analyze the relationship between LINC13 and overall survival(OS)and Diesase free survival(DFS)in 364 liver cancer patients.Then Kaplan-Meier survival curve was drawn.4.A total of 153 tumor tissue samples were collected from the General Hospital of the Chinese people's Liberation Army and the Cancer Hospital Affiliated to Guangzhou Medical University,including 82 patients with OXA chemotherapy and 71 patients of intravenous chemotherapy without OXA.All of the patients had complete prognostic information.The total RNA of 153 tissues was extracted,and then the level of LINC13 was detected by RT-PCR.The relationship between LINC13 and the patient's prognosis(Progression-free survival,PFS)was analyzed,and the Kaplan-Meier survival curve was drawn.The diagnosis of HCC must meet the diagnostic criteria of2019 “Primary Liver Cancer Diagnosis and Treatment Guidelines”.The project was approved by the ethics committee of the Chinese People's Liberation Army Hospital and the Affiliated Tumor Hospital of Guangzhou Medical University.Related experiments meet the requirements of the Declaration of Helsinki and had been approved by the Ethics Committee of the Third Hospital of Hebei Medical University.5.According to the OXA effect,82 patients who received OXA chemotherapy were divided into OXA-S and OXA-R group.Among them,10 tumor tissues were selected from each group.Fluorescent in situ hybridization(FISH)was performed to detect the level of LINC13 of two groups for analyzing the relationship between LINC13 and OXA resistance.Results:1.Find new molecules related to OXA resistance of HCC cellsWe successfully established OXA-R HepG2 cell line(resistance index was 3.36).Compared with OXA-S HepG2 cells,OXA-R cells have stronger proliferation ability(P<0.01)and lower apoptosis rate(P<0.01)when treated with OXA.We confirmed that LINC13 is the most up-regulated lnc RNA in OXA-R cells by RNA-Seq and RT-PCR(P<0.01).2.The relationship between LINC13 and the prognosis of HCCAnalysis of the level of LINC13 of 364 HCC patients in the TCGA database,we found that LINC13 was related to the size,clinical stage,vascular invasion and prognosis of HCC(P<0.05),which was an independent risk factor for the prognosis of HCC(P=0.003).Through GEPIA website we found HCC patients with higher level LINC13 had a worse clinical prognosis,manifested with shorter OS(P=1.3e-05)and DFS(P=0.003)).3.The relationship between LINC13 and the prognosis of patients with different chemotherapyWe collected 82 HCC patients with OXA treatment and found that 43 patients had high level of LINC13,39 patients had low level LINC13.Among71 HCC patients receiving chemotherapy without OXA,48 patients had high level of LINC13,and 23 patients had low level of LINC13.Analysis of the relationship between LINC13 and PFS of each group,we found that higher level of LINC13 had shorter PFS than low level of LINC13 among receiving OXA patients(P=0.001).LINC13 were not associated with PFS in HCC patients receiving OXA free intravenous chemotherapy(P=0.306).4.LINC13 level in OXA-sensitive and resistant HCC tissuesFluorescence probe in situ hybridization(FISH)was used to detect the level of LINC13 in 10 cases of OXA-S and OXA-R HCC tissues.It was found that compared with OXA-S HCC tissues,the level of LINC13 in OXA-R HCC tissues was higher(P<0.01).Conclusion:1.LINC13 is a new molecule related to OXA resistance in HCC.2.LINC13 is related to the HCC progression and it is an independent risk factor of HCC prognosis.Part 2 The possible mechanism of LINC13 regulating SM1 mediated OXA resistance in HCCObjective: To analyze the signal pathway of OXA resistance related gene enrichment by RNA-Seq,and to further elucidate the possible mechanism of OXA resistance induced by LINC13 regulating target genes.Methods:1.The signal pathway of OXA resistance genes were analyzed by differential m RNA in RNA-Seq,and the relationship between LINC13 and OXA resistance genes enrichment was explored by Gene Set Enrichment Analysis(GSEA)enrichment analysis.Flow cytometry was used to explore the ROS level of OXA-S and OXA-S cells.2.The levels of LINC13 and drug resistance related genes of possible pathway of OXA resistance(SM1,KEAP1,NQO1,BLVRB and GPX2)in364 HCC patients were extracted from TCGA database.The correlation between LINC13 and antioxidant stress genes was analyzed,and then the possible gene regulated by LINC13 was determined.3.After determining the most relevant gene of LINC13 among the antioxidative stress pathway,we analyzed the relationship between SM1 and OS and DFS in HCC patients through GEPIA website.RT-PCR was used to detect the level of SM1 in HCC tissues to analyze the effect of SM1 level on PFS of 82 HCC patients who received OXA chemotherapy and 71 patients who received non-OXA chemotherapy.4.The total RNA and protein of OXA-S and OXA-R HepG2 cells were extracted,and the transcription and protein level of SM1 were detected.Then the expression of SM1 of 10 OXA-S and OXA-R HCC tissues were detected by immunohistochemical staining.5.The total RNA and protein of LO2,HepG2,HCC-LM3,Hu H-7,Hep3 B and MHCC97 H cells were extracted,and the transcription level of LINC13 and SM1 protein level were detected.Then we analyzed the correlation of LINC13 and SM1,also the relationship between SM1 and OXA resistance in HCC was analyzed.6.The recombinant plasmids of LINC13 and SM1 were constructed by molecular cloning technique,and the regulation of LINC13 on SM1 was analyzed by cell transfection,RT-PCR and western blot.The survival rate of HepG2 and MHCC97 H cells treated with different concentrations of OXA was detected by CCK-8 method,also IC50 of two HCC cell lines were determined.Results:1.OXA resistance pathways and the relationship between LINC13 and the most possible pathway of OXA resistanceOXA resistance-related signal pathway was analyzed via RNA-Seq data,and we found that antioxidant stress pathway was one of the main pathways for the enrichment of OXA resistance-related genes.Also,GSEA analysis showed that LINC13 was positively correlated with the enrichment of antioxidant stress-related genes.Flow cytometry showed that the level of ROS in OXA-R cells was lower than that in OXA-R cells(P < 0.01).2.Analysis of the correlation between LINC13 and some antioxidative stress genes of drug resistance pathwayThe correlation between LINC13 and SM1,KEAP1,NQO1 and GPX2 of antioxidant stress pathways was confirmed in 364 HCC patients of TCGA database.The correlation between LINC13 and SM1 was the highest(P <0.0001).3.The relationship between SM1 and the prognosis of HCC patients and the effect of OXACombined with GEPIA website,we analyzed the relationship between SM1 level and prognosis of 364 HCC patients,and found that the OS and DFS of patients with higher level of SM1 were shorter but not statistically significant.Then we alanlyzed the relationship between the level of SM1 and prognosis of patients with intravenous chemotherapy of OXA,and found that the level of PFS of patients with higher level of SM1 was shorter than that of patients with lower level of SM1(p=0.007).The level of SM1 of HCC patients who received intravenous chemotherapy without OXA had no effect on PFS.4.Expression level of SM1 in OXA-S and OXA-R HCC tissuesThe level of SM1 in OXA-R cells was higher than that in OXA-S HepG2 cells,and the expression level of SM1 in OXA-R HCC tissues was higher than that in OXA-S tissues(P < 0.01).5.The relationship and regulation mechanism between LINC13 and SM1Compared with liver LO2 cells,transcription level of LINC13 and protein level of SM1 in HCC cells were higher.Correlation analysis showed that there was a significant positive correlation between LINC13 transcription level and SM1 protein level(R = 0.972,P = 0.0012).Compared with the control,the level of SM1 in HepG2 cells increased significantly after overexpression of LINC13,and the level of SM1 decreased significantly in MHCC97 H cells after silencing LINC13.The IC50 of OXA in HepG2 cells was 9.14 ?M by CCK-8 method,and the IC50 of OXA in MHCC97 H cells was 14.87 ?M.Conclusion:1.LINC13 is positively correlated with antioxidant stress response,which may lead to OXA resistance by promoting antioxidant stress response.2.LINC13 targeted regulation of SM1,which may mediate OXA resistance through antioxidant stress pathway.Part 3 The molecular mechanism of LINC13 regulating SM1Objective: To analyze how LINC13 regulates SM1 and the role of OXA in LINC13-SM1 pathway.Methods:1.The localization of LINC13 in HepG2 and MHCC97 H was observed by FISH,and then we determined whether LINC13 regulated SM1 through post-transcriptional translation or transcription level.2.Try to find the promoter region of SM1 from NCBI website,and combine PROMO,Gene Cards and UCSC sites to predict transcription factors of SM1.Then venn diagram was used to predict the intersection of transcription factors.Combined with dual luciferase reporter gene assay,the effects of transcription factors(NRF2,STAT3,ATF3,KLF5,SF1)co-transfected with LINC13 on SM1 promoter were detected,and SF1 was determined to be the most effective transcription factor of SM1 upon LINC13 overexpression.3.According to the JASPAR website,the binding sequence of SF1 and SM1 was predicted,and then the SM1 promoter and its mutant plasmid were constructed.The effect and binding site of LINC13 and SF1 on SM1 transcription were determined by cell transfection technique and dual luciferase reporter gene experiment.4.Dual luciferase reporter gene assay was used to analyze the effect of LINC13 and OXA on SM1 promoter,and Ch IP assay was used to analyze the effect of LINC13 and OXA on SF1 transcriptional binding SM1.This would clarify the transcriptional regulation mechanism of LINC13 and OXA on SM1.5.The HepG2 and MHCC97 H cells were divided into control group,LINC13 transfection group,SF1 inhibitor mithramycin a(MMA)group and LINC13 transfection plus MMA group.Transfection efficiency of LINC13 was determined by RT-PCR.The protein level of SM1 was analyzed by western blot.Then we analyze the mechanism of LINC13 regulating SM1 translation.6.10 HCC cases with higher level of LINC13,10 cases with lower level of LINC13 and 20 cases of paracancerous tissues were taken for immunohistochemical staining and FISH to detect the levels of LINC13,SM1 and SP1 in HCC tissues,analyzing the correlation between LINC13 level and the expression of SM1 and SF1 in HCC tissues.Results:1.Localization of LINC13 in HCC cellsThe localization of LINC13 in HepG2 and MHCC97 H cell lines was observed,and it was confirmed that LINC13 was expressed in both nucleus and cytoplasm,but mainly located in nucleus.2.Screening of SM1 transcription factors and possible locations of binding promotersPROMO,Gene Cards and UCSC websites were used to predict transcription factor of SM1.We confirmed that SF1 could significantly increase SM1 promoter activity when LINC13 was co-transfected with NRF2,STAT3,ATF3,KLF5 and SF1 by dual luciferase reporter gene assay.The binding site of transcription factor SF1 and SM1 was in the second part of SM1 promoter region.3.LINC13 or OXA can regulate the activity of SM1 promoterThe promoter activity of SM1 could be increased by LINC13 or OXA via dual luciferase reporter gene assay.We further confirmed that LINC13 and OXA played a role in transcriptional regulation of SM1 by enhancing SF1 recruitment to SM1 promoter by Ch IP assay.4.LINC13 regulates SM1 protein expression through SF1LINC13 could promote the expression of SM1,and MMA could inhibit the expression of SM1.LINC13 could not reverse the inhibitory effect of MMA on SM1.This proved that LINC13 regulated the expression of SM1 through SF1.5.Analysis of the correlation between LINC13 and SF1 and SM1in clinical sampleThe expression of SF1 and SM1 in HCC tissue with higher level of LICN13,and the levels of LINC13,SF1 and SM1 in HCC tissue were significantly higher than those in paracancerous tissues through correlation analysis of LINC13 level,SF1 and SM1 expression in clinical tissue.Conclusion:1.LINC13 plays a role in regulating SM1 at the transcriptional level.2.LINC13 or OXA can play a transcriptional regulatory role by enhancing the recruitment of SF to the SM1 promoter region.3.LINC13 was positively correlated with SF1 and SM1 expression,and LINC13 regulated SM1 expression through SF1.Part 4 LINC13-SF1-SM1 axis promotes oxidative stress and leads to OXA resistanceObjective: To explore the regulation of reactive oxygen species and glutathione levels in HCC cells by LINC13-SF1-SM1 axis and to analyze the role of redox homeostasis in OXA resistance.Methods: HepG2 cells were divided into control group,overexpression LINC13 group,overexpression SF1 group,SM1 knockout(KO)cell with overexpression of LINC13 group,SM1 KO cell with overexpression of SF1 group.MHCC97H cells were divided into control group,silencing LINC13 group,silencing LINC13 and then re-transfected with SM1 group.HepG2 cells were divided into control group,overexpression LINC13 group,MMA group,overexpression of LINC13 togeteher with MMA group.After adding different concentrations of OXA,the cell survival rate was detected by CCK-8method,and cell proliferation was detected by colony formation assay.The reactive oxygen species(ROS)level was analyzed by flow cytometry,and the GSH/GSSG ratio was detected by glutathione/glutathione disulfide(GSH/GSSG)kit.Result:1.Effect of LINC13-SF1-SM1 on the survival rate of HCC cells when treated with OXASilencing LINC13,MMA or SM1 KO could enhance the killing effect of OXA on HepG2 cells when compared with the control group(P < 0.01).Overexpression of LINC13 or SF1 could weaken the killing effect of OXA in HepG2 cells.Re-transfected with SM1 could reverse the killing effect of OXA on MHCC97 H cells after silencing LINC13.Overexpression of SF1 or LINC13 could not reverse the killing effect of OXA in SM1 KO HepG2 cells.Overexpression of LINC13 could not reduce the killing effect of MMA combined with OXA in HepG2 cells.2.Effect of LINC13-SF1-SM1 on ROS level of HCC cells when treated with OXAOXA could increase the level of ROS in HCC cells.Compared with OXA group,overexpression of LINC13 or SF1 could weaken the stimulating effect of OXA and decrease the level of ROS.Silencing LINC13,MMA or SM1 KO could enhance the stimulating effect of OXA and increase the level of ROS.SM1 could reverse the effect of LINC13 silencing;LINC13 and SF1 could not reverse the effect of SM1 KO,and LINC13 could not reverse the effect of MMA.(P < 0.05)3.Effect of LINC13-SF1-SM1 on glutathione level of HCC cells induced by OXAOXA could reduce the proportion of GSH/GSSG of HCC cells.Overexpression of LINC13 or SF1 could weaken the stimulating effect of OXA,and GSH/GSSG was higher than that of OXA group.Silencing LINC13,MMA or SM1 KO could enhance the stimulating effect of OXA,while GSH/GSSG was significantly lower than that of OXA group.SM1 could reverse the silencing effect of LINC13.LINC13 or SF1 could not reverse the effect of SM1 KO,and LINC13 could not reverse the effect of MMA.(P <0.05)Conclusion:1.LINC13 regulates SF1-SM1 pathway and inhibits the killing effect of OXA on HCC cells.2.LINC13-SF1-SM1 axis promotes the antioxidant stress response of HCC cells and leads to OXA resistance.Part 5 ACTR regulates glycolysis and mediates sorafenib resistance in HCCObjective: To screen new molecules related to sorafenib resistance and clarify its mechanism.Methods:1.We searched sorafenib sensitive and resistant RNA-Seq data to find sorafenib resistance related genes through gene expression database(Gene Expression Omnibus,GEO).The nude mice were inoculated with sorafenib sensitive and resistant HepG2 cells.After the xenograft tumor was formed,total RNA of tumor tissue was extracted to identify the level of sorafenib resistance related genes,and then new molecules related to sorafenib resistance was identified.2.Combined with the 364 HCC cases of TCGA database,the relationship between ACTR level and OS in HCC patients was analyzed,and the correlation between ACTR and glycolysis genes(GLUT1,PKM2 and LDHA)was analyzed through GEPIA website.3.HepG2 cells were divided into control group,glycolysis inhibitor2-deoxy-D-glucose(2-DG)group,ACTR KO group,ACTR KO plus 2-DG group,ACTR KO and ACTR re-transfected group,ACTR KO cell plus 2-DG followed by ACTR re-transfected group.Hu H-7 cells were divided into control group,2-DG group,silencing ACTR group,silencing ACTR plus2-DG group,silencing ACTR and then ACTR re-transfected group,silencing ACTR plus 2-DG and then ACTR re-transfected group.The cell survival and proliferation were detected by CCK-8 method and colony formation assay,and the extracellular acidification rate(ECAR)and oxygen consumption rate(OCR)were detected by Seahorse XFe96.4.A retrospective study included 126 patients with HCC tissues without any initial treatment.The patients were from the General Hospital of the Chinese people's Liberation Army.The project had been approved by the ethics committee of the Third Hospital of Hebei Medical University and the General Hospital of the Chinese people's Liberation Army.The expressions of ACTR,LDHA and PKM2 were analyzed by immunohistochemical staining in HCC tissues of 126 patients,and the correlation was analyzed.Combined with oncomine database,the correlation between ACTR and glycolysis genes was further analyzed.Result:1.Screening new molecules related to sorafenib resistanceThe differential genes of sorafenib-sensitive and drug-resistant xenografts were screened from GEO database.It was found that ACTR was significantly up-regulated in sorafenib-resistant xenografts.The differential expression of sorafenib-resistant genes in sensitive and resistant xenografts was analyzed by tumor formation of nude mice.ACTR was the most significantly up-regulated gene of sorafenib resistance.2.The relationship between ACTR and HCC prognosis and glycolysis geneThe HCC data in GEPIA website were from TCGA HCC patients.Among the 364 HCC patients,there were 182 cases had higher level of ACTR and 182 patients had lower level of ACTR.Compared with lower level of ACTR,patients with higher level of ACTR had shorter OS.We found that ACTR was significantly positively correlated with glycolysis genes GLUT1(R = 0.32),PKM2(R = 0.38)and LDHA(R = 0.40)through correlation analysis(P < 0.0001).3.Effect of ACTR-glycolysis on cell viability and proliferation upon sorafenib treatmentCompared with the control,the growth and proliferation of Hu H-7 and MHCC97 H cells were slowed down and the sorafenib sensitivity was enhanced after silencing or knockout ACTR.2-DG can enhance the anti-tumor effect of sorafenib.ACTR re-transfection could reverse the effect of silencing or ACTR KO on the sensitization of sorafenib,but could not reverse the effect of 2-DG.4.Effect of ACTR-glycolysis on ECAR and OCR upon sorafenib treatmentSilencing ACTR or sorafenib could inhibit the level of ECAR and HCC glycolysis when compared with control.Silencing ACTR could enhance the inhibitory effect of sorafenib on ECAR which reflected inhibition of glycolysis fucntion.After ACTR re-transfection,it could reverse this silencing effect and restore glycolysis function.Silencing ACTR or sorafenib could enhance the aerobic respiration ability of cells,which was shown by the enhancement of oxidative phosphorylation function and the increase of OCR when compared with the control.While,the effect of silencing ACTR could be reversed after ACTR re-transfection,which would weaken the aerobic respiration of cells and decreased the level of OCR.(P < 0.01)5.Analysis of the correlation between ACTR and glycolysis genes in clinical samplesImmunohistochemical staining showed that ACTR was positively correlated with the expression of LDHA and PKM2 in HCC patients.Through Oncomine website,we found that there was a positive correlation between ACTR and glycolysis-related gene GLUT1/PKM2/LHDA/PFKL/ENO1(P <0.05).Conclusion:1.ACTR is a new molecule significantly up-regulated in sorafenib resistance,which is related to glycolysis.2.ACTR leads to sorafenib resistance by regulating glycolysis.Conclusion:1.LINC13 is a new molecule related to OXA resistance in HCC.2.LINC13 regulates the SF1-SM1 pathway to enhance the anti-oxidative stress response and leads to OXA resistance.3.ACTR is a new molecule related to sorafenib resistance in HCC.4.ACTR leads to sorafenib resistance by regulating glycolysis. |
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