Mechanism Of MiR-4461 Regulating Liver Cancer Stem Cells And Inhibiting Drug Resistance And Its Clinical Relevance

Background:Hepatocellular carcinoma(HCC)is a common malignant tumor in China with a high incidence rate.It is a major disease that seriously threatens the health of people and is expected to be the third leading cause of cancer death in the world by 2030.Although there are several diagnostic and therapeutic methods available for HCC,effective clinical treatments for patients with different stages of HCC are still lacking due to the incomplete understanding of its pathogenesis.Liver cancer is characterized by strong invasion,rapid proliferation,and high recurrence rate.These features are caused by various factors,including the tumor microenvironment,complex pathogenesis,and frequent drug resistance.Liver cancer stem cells are one of the significant contributors to the rapid progression and high recurrence rate of liver cancer.Liver cancer stem cells(LCSCs)are a subset of cancer cells that possess selfrenewal and unlimited proliferation abilities.They are characterized by their abundant sources,wide distribution,strong tumorigenic potential,and multi-drug resistance.Consequently,CSCs are significant contributors to tumor development and treatment resistance.The abundance,accessibility,and proliferative capacity of LCSCs have made them a prominent focus of research in the field of oncology in recent years.Micro RNAs(miRNAs)have been found to play a crucial role in the regulation of tumorigenesis,progression,metastasis,recurrence,and chemo-resistance in various cancers.miR-4461,a newly discovered mitochondrial-related miRNA,has not been fully studied in terms of its role in biological processes and pathogenesis.Previous studies have shown that miR-4461 inhibits tumorigenesis in renal cell carcinoma by targeting PPP1R3 C.Additionally,Chen and colleagues found that miR-4461 inhibits tumorigenesis by downregulating COPB2 expression in colorectal cancer.However,the function of miR-4461 in liver CSCs remains unclear.Liver cancer stem cells are known for their multidrug resistance,and many miRNAs have been found to regulate cancer drug resistance.For instance,miR-122 promotes apoptosis and sensitizes drug-resistant liver cancer cells to sorafenib,while miR-221 inhibits caspase-3 and enhances drug resistance.Therefore,we sought to determine whether miR-4461 also plays a role in regulating drug resistance in HCC.In conclusion,this study aims to investigate the role of miR-4461 in liver cancer stem cells and drug resistance,and its potential clinical significance.Methods:Part Ⅰ1.The expression of miR-4461 in liver cancer stem cells.Realtime PCR was used to detect the expression of miR-4461 in primary liver cancer stem cells derived from liver cancer lesions and human liver cancer cell lines.Changes in miR-4461 expression during the differentiation process of liver cancer stem cells were also examined.2.miR-4461 regulates the malignant phenotype of the self-renewal ability and tumorigenicity of liver cancer stem cells.miR-4461 overexpression and knockdown liver cancer cell lines were constructed.The expression of liver cancer stemness-related molecules Ep CAM and CD24 were detected using low adhesion sphere assay,flow cytometry analysis and Real-time PCR.The study also examined the expression of transcription factors SOX2 and OCT4 related to liver cancer stem cells using Real-time PCR and Western Blot to confirm the effect of miR-4461 on the self-renewal ability of liver cancer stem cells.Additionally,the effect of miR-4461 on the tumorigenic ability of liver cancer stem cells was explored by limiting dilution experiments in vitro and in vivo and histochemical staining of tumor-bearing tissues.1.Exploration of the mechanism of miR-4461 regulating the self-renewal ability and tumorigenicity of liver cancer stem cells.Through bioinformatics analysis,it was discovered that miR-4461 could bind to SIRT1.Further experiments using Real-time PCR and Western Blot detection showed that the expression of SIRT1 increased in liver cancer cells when miR-4461 was knocked down.Luciferase reporter assay verified that miR-4461 directly targets SIRT1.The negative correlation between the expression levels of miR-4461 and SIRT1 in mouse subcutaneous tumor-bearing tumors formed by xenografting of human tumor cell line and clinical liver cancer samples further confirmed the binding effect of miR-4461 and SIRT1.Finally,SIRT1 si RNA was transfected into miR-4461-knockdown liver cancer cells and control cells,and the spheroid formation assay,and limiting dilution experiments in vivo and in vitro proved that miR-4461 could inhibit the self-renewal ability and tumorigenicity of liver cancer cells by affecting SIRT1.Part Ⅱ1.This study aims to determine the expression level of miR-4461 in drug-resistant liver cancer tissues and investigate its relationship with drug resistance in liver cancer.2.A cell line with miR-4461 overexpression will be constructed to determine whether it affects cisplatin-induced apoptosis of liver cancer cells through measuring cisplatin IC50,plate cloning,and cell apoptosis experiments.3.The study also use the HCC-PDX model to compare the effect of miR-4461 expression on the responsiveness of the liver cancer model to cisplatin.Part Ⅲ1.The correlation between miR-4461 levels and the prognosis of patients after surgery.In order to explore the correlation between miR-4461 levels and the prognosis of patients after surgery,Kaplan-Meier analysis was used to evaluate the relationship between miR-4461 levels and patient prognosis after surgery,so as to determine whether miR-4461 could serve as a clinical predictor of liver cancer surgery outcomes.2.The correlation between miR-4461 levels and the prognosis of patients receiving TACE after surgery.Clinical samples were collected,and the Kaplan-Meier method was used to evaluate the guiding value of miR-4461 on the prognosis of patients receiving TACE after surgery.Results:Part IThe study reveals that miR-4461 expression is reduced in liver cancer stem cells,which is linked to their differentiation.Additionally,miR-4461 expression is low in recurrent and metastatic HCC tissues.Knockdown of miR-4461 promotes the selfrenewal ability and tumorigenicity of liver cancer stem cells,while overexpression of miR-4461 inhibits these processes.The study also demonstrates that miR-4461 regulates the function of liver cancer stem cells by targeting sirtuin 1(SIRT1).Part ⅡThe study found that the expression level of miR-4461 is associated with drug resistance in HCC.Overexpression of miR-4461 in liver cancer cells enhanced the drug sensitivity to cisplatin therapy.PDXs experiments showed that high miR-4461 expression in human liver cancer xenografts resulted in higher sensitivity to cisplatin treatment.Part ⅢAccording to Kaplan-Meier analysis,low expression of miR-4461 can indicate a poor prognosis for liver cancer patients post-surgery.Furthermore,clinical cohorts have shown that patients with high miR-4461 expression who undergo transcatheter arterial chemotherapy after surgery acquire more benefits from TACE.Conclusions:Our study demonstrates the significant role of miR-4461 in suppressing the malignant properties of liver cancer stem cells.This highlights the potential of miR-4461 as a promising target for the prevention and treatment of liver cancer.Additionally,the prognostic value of miR-4461 in surgical outcomes and response to TACE therapy underscores its clinical relevance in guiding treatment decisions for liver cancer patients.