Study On The Role Of MiR-138-5p/EZH2 Axis In The Radiosensitivity Of Hepatocellular Carcinoma (HCC) Cells

Hepatocellular carcinoma（HCC）is the most common type of primary liver cancer and one of the most common malignant tumors in our country.Radiotherapy,as one of the main treatment methods for HCC,has been widely used in clinical application.However,in actual treatment,the radiation sensitivity is often affected by the large tumor size and the presence of hypoxic tissue.Micro RNA（miRNA,miRNA）regulates the expression of target genes by binding to the 3’UTR of target genes,and then participates in a variety of biological processes,including cell proliferation,apoptosis,metastasis,invasion,radiotherapy or chemotherapy resistance,etc.Studies have reported that miR-138-5p is underexpressed in different malignant tumors such as hepatoma,cervical cancer,osteosarcoma,and non-small cell lung cancer,and plays a tumor suppressor role by regulating the expression of different target genes.However,the effect of miR-138-5p on the radiation sensitivity of HCC has not been reported yet.Enhancer of zeste homolog 2（EZH2）,as a histone methylase,inhibits target gene expression by catalyzing H3K27me3.Bioinformatics analysis showed that EZH2 was one of the target genes of miR-138-5p.As two different epigenetic regulation pathways,the effects of miR-138-5p and EZH2 on the radiosensitivity of hepatocellular carcinoma cells are still worthy of further investigation.Therefore,this study intends to explore the regulatory role of miR-138-5p/EZH2 axis in the radiosensitivity of hepatoma cells,which will provide experimental basis and ideas for improving the radiotherapy efficacy of HCC.Objective:To observe the effects of miR-138-5p and EZH2 on the ability of colony formation,proliferation,apoptosis,cell cycle arrest,migration and invasion of hepatoma cells after radiation;to verify the targeting regulation relationship between miR-138-5p and EZH2;to explore the effect and mechanism of miR-138-5p/EZH2regulatory axis on the radiosensitivity of hepatocellular carcinoma cells.Methods:1.Using bioinformatics methods to analyze the expression of miR-138-5p and EZH2 in different stages of liver cancer tissues,and their effects on the survival of HCC patients.2.The miR-138-5p overexpression cell model was constructed by transfecting miR-138-5p mimic,and the Hep G2-EZH2^LOW cell model was constructed by lentiviral plasmid transfection.RT-q PCR and Western blot were used to verify whether the model was successfully constructed.Radiosensitivity was detected by colony formation assay,cell proliferation ability was detected by MTT assay,cell cycle arrest and apoptosis were detected by flow cytometry,cell migration and invasion ability was detected by wound healing experiment and Transwell method.Western blot method was used to detect related protein expression changes.3.The bidirectional regulatory relationship between miR-138-5p and EZH2 was demonstrated by dual luciferase reporter assay and chromatin immunoprecipitation assay.Results:1.Relationship between miR-138-5p and disease stage and survival in HCC patientsThe results of bioinformatics analysis on the online analysis website showed that miR-138-1,the precursor of miR-138-5p,was lowly expressed in liver cancer tissues,and the expression levels were lower in hepatoma patients with different stages.Patients with higher basal expression levels of miR-138-5p had significantly better survival than those with lower basal expression levels（P<0.01）.Subsequent cell-level verification results showed that the level of miR-138-5p in HCC cells was significantly lower than that in normal hepatocytes L-02（P<0.01）.2.The effect of miR-138-5p on the post-radiation response of Hep G2 and Hep3B cells（1）The effect of miR-138-5p on the radiosensitivity of Hep G2 and Hep3B cellsThe results of cell clone formation experiments showed that the survival fraction（SF）of both hepatoma cells decreased with the increase of radiation dose.Compared with the control group,at the same dose,the SF value of the cells in the overexpression miR-138-5p group was significantly decreased（P<0.05）.Compared with the negative control group,the mean lethal dose D₀ value and SF₂ value of the miR-138-5p overexpression group were both lower,indicating that miR-138-5p could enhance the radiosensitivity of Hep G2 and Hep3B cells.（2）The effect of miR-138-5p on the proliferation of Hep G2 and Hep3B cells after radiationThe results of MTT showed that in Hep G2 and Hep3B cells,compared with the control group,the ability of cell proliferation in the miR-138-5p overexpression group was significantly inhibited with 0Gy or 8Gy radiation for 48h and 72h（P<0.05）.It is suggested that miR-138-5p can reduce the proliferation ability of the two types of hepatoma cells after radiation.（3）The effect of miR-138-5p on the cycle progression of Hep G2 and Hep3B cells after radiationThe detection results of cell cycle progression by flow cytometry showed that,compared with cells in the control group,the percentage of G₀/G₁ phase was significantly increased and the percentage of S phase was decreased in the miR-138-5p overexpression group 24h after 0Gy treatment（P<0.01）;24h after 8 Gy radiation of the two types of hepatoma cells,compared with the control group,the percentage of cells in the G₀/G₁ phase in the miR-138-5p overexpression group was significantly increased（P<0.001）,suggesting that G₀/G₁ phase arrest occurred.（4）The effect of miR-138-5p on apoptosis of Hep G2 and Hep3B cells after radiationFlow cytometry was used to detect the apoptosis of two kinds of hepatoma cells after radiation and miR-138-5p overexpression treatment.The apoptosis rate of cells in the miR-138-5p overexpression group increased significantly at 24h after 0Gy or8Gy treatment（P<0.05）.It is suggested that miR-138-5p can promote the apoptosis of hepatoma cells induced by radiation.（5）The effect of miR-138-5p on the migration and invasion ability of Hep G2 and Hep3B cells after radiationThe wound healing experiment was used to detect the changes in the migration ability of the two types of hepatoma cells after radiation.The results showed that the migration ability of the miR-138-5p overexpression group was significantly inhibited compared with the control group at 24h or 48h after 0Gy treatment（P<0.05）;24h and48h after 8Gy treatment,compared with the control group,the migration ability of the overexpression miR-138-5p group was also significantly inhibited（P<0.05）.It suggested that the migration ability of Hep G2 and Hep3B cells with 0Gy or 8Gy radiation was weakened after overexpression of miR-138-5p.The changes of cell invasion ability were observed by Transwell method.The results showed that after0Gy or 8Gy treatment,compared with the control group,the number of invasive cells in the overexpression miR-138-5p group was significantly reduced（P<0.05）,suggesting that miR-138-5p overexpression can inhibit the invasive ability of hepatoma cells after radiation.（6）The effect of miR-138-5p on EMT markers after radiation of Hep G2 and Hep3B cellsEpithelial to mesenchymal transition（EMT）related markers E-cadherin,N-cadherin,Vimentin and SNAIL（snail family transcriptional repressor 1）protein expression were detected by Western Blot.The results showed that in hepatoma cells,after 0 Gy or 8 Gy treatment,the expressions of N-cadherin,Vimentin,and SNAIL in the miR-138-5p overexpression group were decreased compared with the control group.In Hep3B cells treated with 0Gy or 8Gy,the protein level of E-cadherin in the miR-138-5p overexpression group increased,and in Hep G2 cells,the protein level of E-cadherin in the miR-138-5p overexpression group also increased after 0Gy treatment,but no obvious change after 8Gy treatment.The results suggest that overexpression of miR-138-5p can inhibit the EMT process of the two hepatoma cells after radiation.3.Exploration of miR-138-5p on EZH2 targeting regulationThe target gene of miR-138-5p was predicted by the online website,and it was screened that miR-138-5p may have a targeting relationship with EZH2,and a dual-luciferase reporter gene assay was carried out.The results showed that compared with the control group,co-transfection of miR-138-5p mimic with psi CHECK2-EZH2-3′UTR could significantly inhibit the luciferase activity（P<0.05）.At the same time,the luciferase activity did not change significantly after co-transfection of miR-138-5p mimic or the corresponding negative control with the mutant plasmid psi CHECK2-EZH2-3’UTR mut.Further verification in hepatoma cells showed that the m RNA level of EZH2 in the two hepatoma cells was significantly decreased（P<0.01）after overexpression of miR-138-5p,and the protein level was also inhibited.It is suggested that miR-138-5p has a targeted regulatory relationship with EZH2.4.Relationship between EZH2 and disease stage and survival in HCC patientsThe results of bioinformatics analysis on the online analysis website showed that EZH2 was highly expressed in hepatocellular carcinoma tissue（P<0.001）,and with the increase in the stage of liver cancer patients,the expression level of EZH2gradually increased,and the basal expression level of EZH2 was higher than that of HCC.The survival time of patients with high expression of EZH2 was significantly worse than that of patients with low basal expression（P<0.001）.Subsequent verification results at the cellular level showed that the m RNA level of EZH2 in five types of hepatoma cells was significantly higher than that of L-02（P<0.01）,and the protein expression level was also higher than that of L-02.It is suggested that EZH2 is significantly highly expressed in hepatoma tissues,which is opposite to that of miR-138-5p.5.The effect of silencing EZH2 on the post-radiation response of Hep G2 cells（1）The effect of silencing EZH2 on the radiosensitivity of Hep G2 cellsThe results of cell clone formation experiments showed that the SF of Hep G2cells after silencing EZH2 decreased gradually with the increase of radiation dose.Compared with the transfection vector plasmid group,under the same dose,the SF value of the silenced EZH2 group was significantly decreased（P<0.05）.Compared with the transfection vector plasmid group,the average lethal dose D₀ value and SF₂value of the silencing EZH2 group were both lower,indicating that silencing EZH2expression can enhance the radiation sensitivity of Hep G2 cells,and the trend of the effect achieved by increasing the expression of miR-138-5p is the same.（2）The effect of silencing EZH2 on the proliferation of Hep G2 cells after radiationAccording to the results of MTT experiment,in Hep G2 cells,compared with the transfection vector plasmid group,the cell proliferation in the silenced EZH2 group was inhibited after 48h treatment with 0Gy（P<0.05）.Seventy-two hours after 8Gy radiation,the EZH2 silenced cells had a significant inhibitory effect on proliferation（P<0.05）.It suggested that silencing EZH2 could reduce the proliferation ability of Hep G2 after radiation.（3）The effect of silencing EZH2 on the post-radiation cycle progression and apoptosis of Hep G2 cellsThe detection results of cell cycle progression showed that compared with the transfection vector plasmid group,the percentage of G₀/G₁ phase decreased and the percentage of G₂/M phase increased.Twenty-four hours after 0Gy treatment of cells in the silenced EZH2 group（P<0.05）;After 24h,compared with the transfection vector plasmid group,the percentage of cells in the G₀/G₁ phase in the silenced EZH2group was significantly increased（P<0.05）,suggesting that G₀/G₁ phase arrest occurred.The results of detecting apoptosis showed that compared with the 0Gy group,after 8Gy radiation,the apoptosis rate of each group increased.Compared with the transfection vector plasmid group,the apoptosis rate of the silenced EZH2 group was significantly increased at 24h after 0Gy or 8Gy treatment（P<0.05）.It is suggested that silencing EZH2 group can promote the apoptosis of hepatoma cells induced by radiation,which is consistent with the effect of overexpression of miR-138-5p.（4）The effect of silencing EZH2 on the migration,invasion ability and EMT markers of Hep G2 cells after radiationThe results of wound healing experiment showed that the migration ability of cells in the silenced EZH2 group was significantly inhibited（P<0.05）compared with the transfection vector plasmid group at 24h and 48h after 0Gy or 8Gy treatment.The results of Transwell experiment showed that after 0Gy or 8Gy treatment,compared with the transfection vector plasmid group,the number of invasive cells in the silenced EZH2 group was significantly reduced（P<0.001）,suggesting that silencing EZH2 and overexpression of miR-138-5p have a significant effect on hepatocellular carcinoma.The effect of cell migration ability with or without radiation was consistent.Subsequent detection of EMT-related marker protein expression levels showed that after 0Gy or 8Gy treatment,the expression of N-cadherin,Vimentin,and SNAIL in the silenced EZH2 group was reduced compared with the transfection vector plasmid group.The above results suggest that in hepatoma cells,miR-138-5p can enhance the radiosensitivity and inhibit migration and invasion of HCC cells by targeting EZH2.6.The effect of EZH2 on the expression of miR-138-5p via H3K27me3Our results showed that after silencing EZH2 expression in Hep G2 cells,the expression level of miR-138-5p was significantly increased（P<0.01）,while the expression level of miR-138-5p was significantly increased after EZH2 was overexpressed in Hep3B cells（P<0.01）.The results of chromatin immunoprecipitation（Ch IP）analysis showed that compared with the transfected vector plasmid group,the expression level of H3K27me3 in the partial region of the miR-138-5p promoter in cells after Hep G2 silencing of EZH2 was significantly reduced.It is suggested that EZH2 can regulate the expression of miR-138-5p by mediating H3K27me3modification in the promoter region of miR-138-5p in hepatoma cells,suggesting that there is a feedback regulation mechanism of mutual inhibition between miR-138-5p and EZH2.Conclusion:1.miR-138-5p can enhance the radiation sensitivity of hepatoma cells,inhibit the proliferation after radiation,promote apoptosis,and affect the migration and invasion ability of hepatoma cells after radiation by affecting the EMT process.2.Inhibition of EZH2 can increase the radiation sensitivity of hepatoma cells,increase the apoptosis of hepatoma cells after radiation,reduce the proliferation ability,and at the same time,inhibit the migration and invasion ability of hepatoma cells after radiation.3.A feedback axis is formed between miR-138-5p and EZH2 through a mutual inhibitory regulatory mechanism,which jointly regulates the radiosensitivity,migration and invasion of hepatoma cells.