| Objective: Globally,liver cancer is the second most common cause of cancer death,accounting for more than 700,000 deaths every year.It is usually an aggressive malignancy associated with poor prognosis,and the five-year survival rate is estimated to be less than 9%.As an important epigenetic regulation mechanism,histone methylation plays an important role in many biological processes.Retinoblastoma protein-interacting zinc finger gene 1(RIZ1)interacts with retinoblastoma protein.It is a methyltransferase,RIZ1 can methylate H3K9 of histone into H3K9me1.Long non-coding RNA(lnc RNA)is a set of non-protein-coding RNA with a length greater than 200 nucleotides.More and more evidences indicate that lnc RNA,which may be an oncogene or tumor suppressor gene,plays a vital role in the pathophysiology of human diseases,especially in the occurrence and development of tumors.However,the regulatory mechanism of Lnc RNA in liver cancer has not been fully elucidated.In view of this,the purpose of this study is to explore whether histone methyltransferase RIZ1 can mediate the enrichment of H3K9me1 on the lnc RNA HOTAIRM1 promoter,and regulate the growth and metastasis of hepatocellular carcinoma by targeting miR-125 b.To study the change of its expression and its possible molecular mechanism,and then explain the mechanism of RIZ1 and Lnc RNA HOTAIRM1 in hepatocellular carcinoma from a new perspective.Methods:1.To study the expression of RIZ1 and lnc RNA HOTAIRM1 in hepatocel lular carcinoma and its clinical significance.(1)Using TCGA database and GEPIA database to analyze the expression levels of RIZ1 and lnc RNA HOTAIRM1 in hepatocellular carcinoma and their clinical significance;(2)qRT-PCR was used to detect the expression and clinical significance of RIZ1 and HOTAIRM1 in tissue samples,blood samples and cell cultures of hepatocellular carcinoma patients collected in the undergraduate room.(3)Western Blot and immunohistochemical staining methods were used to determine the expression level of RIZ1 protein in tissue samples from patients with hepatocellular carcinoma.2.In order to study the effect of histone methyltransferase RIZ1 mediated H3K9me1 enrichment on the HOTAI RM1 promoter on the biological behavior of hepatocellular carcinoma cells(1)Co nstruction of RIZ1 and HOTAIRM1 interfering lentivirus: LV-RIZ1-RNAi(97334)-1),LV-RIZ1-RNAi(97335-1),LV-RIZ1-RNAi(97336-1);LV-HOTAIRM1-RNAi(97354-1),LV-HOTAIRM1-RNAi(97355-1),LV-HOTAIRM1-RNAi(97356-1);and negative control virus: KLLVCONO77(hu6-MCS-ubiquitin-EGFP-IRES-puromycin);HOTAIRM1 overexpression lentivirus: LV-HOTAIRM1(65150-1),negative control virus is CON238(ubi-MCS-SV40-EGFP-IRES-pur-omycin),to find out the MOI and optimal infection conditions for lentivirus infection of hepatocellular carcinoma cells.(2)After transfecting hepatocellular carcinoma cells with LV-RIZ1-RNAi lentivirus,study the effects of RIZ1 interference on the proliferation,invasion and migration of hepatocellular carcinoma cells;use PI staining to detect the effects of RIZ1 inte rference on the cycle of hepatocellular carcinoma cells The effect of RIZ1 interference on the apoptosis of hepatocellular carcinoma cells was detected by Annexin V-PE staining method.(3)We used chromatin immunoprecipitation(ChIP)experim ental analysis to evaluate the binding of H3K9me1 to the HOTAIRM1 promoter.(4)The Western Blot method was used to determine the expression of H3K9me1 in hepatocellular carcinoma cells after LV-RIZ1-RNAi lentivirus transfection;qRT-PCR method was used to detect the hepatocellular carcinoma cells HOTAIRM1 after LV-RIZ1-RNAi lentivirus transfection The level of expression.(5)Co-transfect hepatocellular carcinoma cells with LV-RIZ1-RNAi+LV-HOTAIRM1-RNAi or LV-RI Z1-RNAi+LV-HOTAIRM1,and analyze the proliferation,invasion,migration and a poptosis of hepatocellular carcinoma cells.3.To study the effect of the interaction between lncRNA HOTAIRM1 and miR-125 b on the biological behavior of hepatocellular carcinoma and its clinical significance.(1)Use bioinformatics software to predict the targeted miRNA of lncRNA HOTAIRM1.(2)Through the dual lucifera se reporter gene experiment,we verified whether HOTAIRM1 can directly bind to miR-125 b.We constructed HOTAIRM1-wt luciferase reporter gene vector and H -mut 3’-UTR luciferase reporter gene vector,and carried Luciferase reporter gene analysis.(3)Determine the expression level of miR-125 b after transfecting hepatocellular carcinoma cells with LV-HOTAIRM1-RNAi or LV-HOTAIRM1 lentivirus by qRT-PCR.(4)After transfection with LV-RIZ1-RNAi lentivirus,the expression level of miR-125 b in hepatocellular carcinoma cells was determined by qRT-PCR.(5)In order to study the effect of miR-125 b on the biological behavior of hepatocellular carcinoma cells,we constructed LV-hsa-mir-125b-1 lentivirus.(6)Transfect hepatocellular carcinoma with lentivirus LV-hsa-mir-125b-1 or LV-HOTAIR M1+LV-hsa-mir-125b-1 or LV-HOTAIRM1-RNAi+LV-hsa-mir-125b-1 After cells,analyze the proliferation,invasion,migration and apoptosis of hepatocellular carcinoma cells.(7)Detect the expression of miR-125 b in tissue samples,blood samples and liver cell lines collected from patients with hepatocellular carcinoma by q RTPCR.Results:(1)Use the TCGA database and GEPIA database to analyze the expressi on levels of RIZ1 and lnc RNA HOTAIRM1 in the tissues of patients with hepato cellular carcinoma.Through the analysis,it can be seen that the expression of RIZ1 is increased and the expression of HOTAIRM1 in hepatocellular carcinoma patients.The level was significantly down-regulated;analysis found that the expression of RIZ1 had nothing to do with age and TNM stage,but was related to gender.The expression of RIZ1 in cancer tissue samples of female hepatocellular carcinoma patients was higher than that of men;Kaplan-Meier analysis showed that high expression of RIZ1 was associated with overall survival There is no significant difference between the expression of HOAIRRM1 and the stage classification of hepatocellular carcinoma;Kaplan-Meier analysis shows that there is no significant difference between the high expression of HOTARM1 in patients with hepatocellular carcinoma and prolonged overall survival.(2)The real-time fluorescent quantitative PCR test results show that in the tissue samples of hepatocellular carcinoma patients,RIZ1 is highly expressed in cancer tissues;RIZ1 expression in blood samples of hepatocellular carcinoma patients is higher than that of normal patients;H CC-LM3,HEGP2 In the LO2 liver cell line,RIZ1 is highly expressed in HCC-L M3 cells.In tissue samples from hepatocellular carcinoma patients,HOTAIRM1 is highly expressed in normal tissues;HOTAIRM1 expression in blood samples from hepatocellular carcinoma patients is lower than that of normal patients;in HCC-LM3,HEGP2,LO2 hepatocyte cell lines,HOTAIRM1 in LO2 cells High expression.(3)The results of Western Blot and immunohistochemical staining methods show that RIZ1 is highly expressed in cancer tissues of patients with hepatocellular carcinoma.(4)The analysis found that the expression of RIZ1 and HOTAIRM1 was closely related to the patient’s tumor size and number of tumors,but it was not related to the patient’s age,gender,hepatitis B,liver cirrhosis,AFP,portal vein tumor thrombus,tumor differentiation,and TNM staging.2.(1)The optimal infection conditions for LV-RIZ1-RNAi lentivirus transfection of HEPG2 are: LV-RIZ1-RNAi(97336-1)lentivirus + Hi Trans G P group,MOI=10;LV-RIZ1-RNAi lentivir us transfection The best infection condition for HCC-LM3 is LV-RIZ1-RNAi(97334-1)lentivirus + Hi Trans G P group,MOI=20;LV-RIZ1-RNAi+LV-HOTAIRM1-RN Ai co-transfects HEPG2,the best infection The conditions are: LV-RIZ1-RNAi(97336-1)+LV-HOTAIRM1-RNAi(97355-1)lentivirus+Hi Trans G A group,MOI=10;LVRIZ1-RNAi+LV-HOTAIRM1 co-transfects HCC-LM3 The best infection condition is LV-RIZ1-RNAi(97334-1)+LV-HOTAIRM1(65150-1)lentivirus+Hi Trans GP group,MOI=20.(2)ChIP analysis can evaluate the binding of H3K9me1 to the HOTAIR M1 promoter,and we observed an increase in the binding between H3K9me1 and HOTAIRM1 promoter in HEPG2 and HCC-LM3 cells.(3)The results of Western Blot method showed that the expression of H3K9me1 in HEPG2 and HCC-LM3 cells was inhibited after LV-RIZ1-RNAi lentivirus transfection.(4)After transfecti on with LV-RIZ1-RNAi lentivirus,analysis of HEPG2 and HCC-LM3 cells showed that compared with the control group,inhibiting the expression of RIZ1 reduced the proliferation and invasion of HEPG2 and HCC-LM3 cells,And migration,enhanced cell apoptosis;cell cycle analysis results showed that the inhibition of RI Z1 increased the proportion of HEPG2 and HCC-LM3 S-phase cells,combined with the cell proliferation test results,indicating that RIZ1 interference can promote hepatocellular carcinoma arrest With S phase.(5)LV-RIZ1-RNAi+LV-HOTAIRM1-RNAi lentivirus co-transfects HEPG2 cells.The combined effect of down-regulated expression of RIZ1 and HOTAIRM1 inhibition in HEPG2 cells can reverse the proliferation,invasion and migration of RIZ1 silencing on hepatocellular carcinoma cells And apoptosis.(6)LV-RIZ1-RNAi+LV-HOTAIRM1 co-transfected HCC-LM3 cells.The combined effect of RIZ1 inhibition and HOTAIRM1 overexpression further inhibited the cell proliferation,invasion and migration of HCC-LM3,and enhanced cell apoptosis.3.(1)We used the Star Base 2.0 database to predict the target miRNA of HOTAIRM1 and found that miR-125 b is the target miRNA of HOTA IRM1.(2)We constructed HOTAIRM1-wtluciferase reporter gene vector and HO TAIRM1-mut3’UTR luciferase reporter gene vector,and carried out luciferase reporter gene analysis.The results showed that the overexpression of miR-125 b caused a significant decrease in the luciferase activity of HOTAIRM1-WT,while HOTAI RM1-MUT did not.These results indicate that HOTAIRM1 can directly bind miR-125 b.(3)Transfect HEPG2 cells and HCC-LM3 cells with LV-HOTAIRM1-RNAi or LV-HOTAIRM1 lentivirus.The results showed that the overexpression of HOTAI RM1 significantly inhibited the expression of miR-125 b,while the downregulation of HOTAIRM1 reversely supported the expression of miR-125 b in liver cancer cells.(4)HEPG2 and HCC-LM3 cells were transfected with LV-RIZ1-RNAi lentivir us.The results showed that the silencing of RIZ1 inhibited the expression level of miR-125 b in HEPG2 and HCC-LM3 cells.(5)Transfect HEPG2 cells with LVHOTAIRM1-RNAi or LV-HOTAIRM1-RNAi+LV-hsa-mir-125b-1 lentivirus.The down-regulated expression of HOTAIRM1 in HEPG2 cells promotes cell proliferation,invasion and migration;Inhibit cell apoptosis.Together with miR-125 b overexpression,it further promoted the proliferation,invasion and migration of HEPG2 cells,and inhibited cell apoptosis.(6)HCC-LM3 cells were transfected with LV-HOTA IRM1 or LV-HOTAIRM1+LV-hsa-mir-125b-1 lentivirus.The results showed that the overexpression of HOTAIRM1 inhibited the proliferation,invasion and migration of HCC-LM3 cells.Promotes cell apoptosis and completely reverses the co-transfection results of overexpression of HOTAIRM1 and miR-125 b.(7)miR-125 b is hi ghly expressed in cancer tissues and blood samples of hepatocellular carcinoma patients,and miR-125 b is highly expressed in HCC-LM3 cells in cell culture samples.(8)The analysis found that the expression of miR-125 b is closely related to the patient’s hepatitis B,tumor size and tumor number,and has nothing to do with t he patient’s age,gender,liver cirrhosis,AFP,portal vein tumor thrombosis,tumor differentiation,and TNM staging.Conclusion: 1.Through the analysis of the TCGA and GEPIA databases and the samples we collected,the results showed that the expression level of RIZ1 in the tissue and blood samples of patients with hepatocellular carcinoma was up-regulated,while the expression of HOTAIRM1 was down-regulated.In the liver cell line,RIZ1 is highly expressed in HCC-LM3 cells,and HOTAIRM1 is highly expressed in LO2 cells.2.Kaplan-Meier survival curve analysis shows that the high expression of RIZ1 is related to the decrease of overall survival,and the high expression of HOTAIRM1 is not significantly different from the prolonged overall survival of patients with hepatocellular carcinoma.3.The expression of RIZ1 and HOT AIRM1 is closely related to the patient’s tumor size and number of tumors.Compared with hepatocellular carcinoma tissues with high RIZ1 expression and low RI Z1 expression,the tumor volume of hepatocellular carcinoma tissues with high RI Z1 expression is relatively larger.The number is relatively large;compared with hepatocellular carcinoma tissues with high HOTAIRM1 expression and low expressi on of HOTAIRM1,the tumor volume of hepatocellular carcinoma tissues with high HOTAIRM1 expression is relatively small,and the number of tumors is relative ly small.4.The binding between H3K9me1 and HOTAIRM1 promoter in hepatocellular carcinoma cells is increased;down-regulation of RIZ1 can effectively inhibit the proliferation,invasion,migration,and apoptosis of hepatocellular carcinoma cells;after the combined effect of down-regulation of RIZ1 and overexpression of HOTAIRM1,Further inhibits the proliferation,invasion,migration,and apoptosis of hepatocellular carcinoma cells;the combined effect of down-regulation of RIZ1 and HOTAIRM1 inhibition can reverse the inhibition of down-regulation of RIZ1 on the proliferation,invasion,migration,and apoptosis of hepatocellular carcinoma cells;these results It shows that histone methyltransferase RIZ1 can mediate the enrichment of H3K9me1 on the HOTAIRM1 promoter,thereby regulating the proliferation,invasion,migration and apoptosis of hepatocellular carcinoma cells.5.Bioinformatics prediction and dual luciferase reporter gene experiment proved that miR-125 b can interact with HOTAIRM1 through direct binding.miR-125 b is highly expressed in cancer tissues and blood samples of hepatocellular carcinoma patients,and highly expressed in HCC-LM3 cell culture samples.The down-regulated expression of HOTAIRM1 in HEPG2 cells promotes cell proliferation,invasion and migration,and inhibits cell apoptosis.Together with miR-125 b overexpression,it further promotes the proliferation,invasion and migration of HEPG2 cells,and inhibits cell apoptosis.Overexpression of HOTAIRM1 inhibited the proliferation,invasion and migration of HCC-LM3 cells,promoted cell apoptosis,and completely reversed the co-transfection results of overexpression of HOTAIRM1 and miR-125 b.The expression of miR-125 b is closely related to the patient’s hepatitis B,tumor size and number of tumors.That is,hepatocellular carcinoma tissues with high expression of miR-125 b have relatively larger tumor volume compared with hepatocellular carcinoma tissues with low expression of miR-125 b.The number of tumorsis relatively large;miR-125 b is highly expressed in hepatitis B-positive patients,an d miR-125 b is low in hepatitis B-negative patients.6.In short,our research found that histone methyltransferase RIZ1 can mediate the H3K9me1 enrichment on the lnc RNA HOTAIRM1 promoter,and regulate the growth and metastasis of hepatocellular carcinoma by targeting miR-125 b,which can further affect hepatocellular carcinoma The occurrence and development of So as to provide new targets and ideas for the treatment of liver cancer. |
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