| Objective:Hepatocellular Carcinoma（HCC）accounts for 75% of primary liver cancers.Because of the increasingly incidence（18.3/100,000 people）and the huge population base in China,the current number of HCC ranks first in the world.In China the occurrence of HCC is mainly related to chronic HBV infection and aflatoxin exposure.At present,the therapies of HCC mainly include liver resection,liver transplantation,local radiofrequency ablation therapy,transarterial chemoembolization,radiotherapy,systemic treatment（including molecular targeted drugs and systemic chemotherapy,immunotherapy）,traditional Chinese medicine and others.Surgical resection,local ablation therapy,or liver transplantation are effective in early-stage patients（Barcelona stage 0 and A）.In experienced medical centers,the patients with Child-Pugh A and no apparent portal hypertension have a 5-year survival about 59 to 68 % after surgery.However,due to the influence of proliferation,invasion,microvascular invasion and inflammatory environment of HCC,there is still the possibility of recurrence after surgical resection,local radiofrequency ablation therapy or liver transplantation.Studies have shown that after surgical resection and local ablation,more than 60% of patients still have HCC recurrence within 5 years.The severe shortage of allogeneic livers greatly restricts liver transplantation for HCC [9].Therefore,for patients with recurrence of HCC and unresectable HCC,systemic treatment is of great significance as a method to delay the development of the tumor,prolong the survival period of the patient,and improve the quality of life of the patient.The FOLFOX4（fluorouracil,leucovorin,oxaliplatin）regimen is recommended as the only first-line systemic chemotherapy regimen by China’s National Health and Family Planning Commission in 2022.In a randomized multicenter clinical trial,the FOLFOX4 regimen improved the progression-free survival and overall survival rate of HCC patients compared with doxorubicin.However,there are still studies finding that the occurrence of drug resistance limits the efficacy of platinum drugs in the treatment of HCC.Therefore,improving the sensitivity of HCC to chemotherapy drugs is of great significance to patients with advanced HCC.In this study,we obtained differentially expressed mi RNAs through microarray data screening analysis of cisplatin-treated and untreated HCC cells in the GEO database,and confirmed that mi R-27a-3p could enhance sensitivity of hepatocellular carcinoma to cisplatin in vitro and in vivo by transfection and MTT.And it was determined by flow cytometry and Western blotting that mi R-27a-3p enhanced the sensitivity of hepatocellular carcinoma cells to cisplatin by enhancing the apoptosis of hepatocellular carcinoma cells.Then,by RNA high-throughput sequencing technology,we speculated that there is a regulatory pathway of mi R-27a-3p/JUN in HCC cells,and we performed cytological verification by q PCR and western blotting,which proved that mi R-27a-3p can inhibit the JNK pathway activation.Finally,we inhibited the expression of JUN by si RNA,and proved by MTT and Western blotting experiments that mi R-27a-3p enhanced the sensitivity of HCC cells to cisplatin through the JUN pathway.Method（1）Screening differentially expressed micro RNAs through the GEO database.（2）To construct mi R-27a-3p overexpression and low expression HCC cells by transfection,and then determine the effect of differentially expressed micro RNAs on the sensitivity of HCC cells to cisplatin by MTT assay.The mechanism of HCC cell death induced by cisplatin and key protein were determined by flow cytometry and Western blotting.（3）Combined with TCGA database and RNA high-throughput sequencing technology,the possible downstream targets of mi R-27a-3p were speculated through gene set enrichment analysis of KEGG and GO.（4）The effect of mi R-27a-3p on the expression level of the downstream target gene JUN was verified by RT-q PCR and Western blotting.（5）The expression of the downstream target JUN gene was inhibited by si RNA technology,and the knockout effect was verified by RT-q PCR and western blotting.MTT was used to explore its function in regulating cisplatin-induced apoptosis of HCC cells.（6）Animal model with high expression of mi R-27a-3p was constructed by subcutaneous injection of mi R-27a-3p agomir.After subcutaneous tumor formation,cisplatin was injected into the tail vein once every 3 days for a total of 3 times,and the body weight and tumor volume of nude mice were calculated.Result:（1）Differential expression analysis was performed on the GSE55806 data set through the Limma function package,and the differentially expressed micro RNAs were screened.Among them,the differential expression of mi R-27a-3p was significantly different and the Log FC was in the forefront,suggesting that mi R-27a-3p may be closely related to cisplatin sensitivity of HCC cells.（2）HCC cell models with mi R-27a-3p overexpression and low expression were constructed by Lipofection 3000 technology,and MTT assay found that mi R-27a-3p enhanced the sensitivity of HCC cells to cisplatin.And by flow cytometry,it was found that mi R-27a-3p enhanced the sensitivity of HCC cells to cisplatin by promoting the apoptosis of HCC cells.Further western blot results showed that mi R-27a-3p promoted the expression of apoptotic protein caspase 3.（3）Combined with TCGA database and RNA high-throughput sequencing technology,the possible downstream targets of mi R-27a-3p were speculated through gene set enrichment analysis of KEGG and GO.（4）Mi R-27a-3p inhibited the expression level of JUN by real-time quantitative PCR and western blotting.（5）The expression of the downstream target JUN gene was inhibited by si RNA technology,and the knockout effect was verified by RT-q PCR and western blotting.Inhibition of JUN can enhance the sensitivity of HCC cells to cisplatin by MTT assay.（6）Animal model with high expression of mi R-27a-3p was constructed by subcutaneous injection of mi R-27a-3p agomir.After subcutaneous tumor formation,cisplatin was administered through the tail vein and the tumor volume was counted.It was found that mi R-27a-3p could enhance liver function in vivo.Sensitivity of cancer cells to cisplatin.Conclusion:（1）In this study,the differential expression analysis of the micro RNAs related to the anti-tumor effect of cisplatin in the GEO database found that micro RNA-27a-3p may be related to the cisplatin sensitivity of HCC cells.In vivo and vitro,it was shown that mi R-27a-3p enhanced the sensitivity of HCC to cisplatin.In vitro experiments it was found that micro RNA-27a-3p promoted the sensitivity of HCC to cisplatin by enhancing the high expression of caspase3 in Huh7 cell line.（2）In this study,a comprehensive biological analysis of HCC tissues with high and low expression of micro RNA-27a-3p in the TCGA database combined with RNAsequencing of Huh7 cells with high and low expression of micro RNA-27a-3p was complied.It is speculated that mi R-27a-3p may regulate the sensitivity of HCC cells to cisplatin by regulating the Jun signaling pathway.In vitro,it was found that micro RNA-27a-3p enhanced the sensitivity of liver cancer to cisplatin by inhibiting the activation of Jun signaling pathway in Huh7 cell line. |
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