The Role And Mechanism Of LDLR Downregulation In Malignant Progression Of Hepatocellular Carcinoma

Background and ObjectiveMulti-level pathological process of tumor development puts higher demands on energy metabolism and nutrient supply,in order to meet the demand of proliferation and metastasis,and to resist toxic stresses such as radiotherapy and chemotherapy.Adaptive changes of multiple metabolic modalities such as glucose and lipid metabolism together constitute the metabolic reprogramming of tumor cells.As one of the important nutrients in human body,lipids are involved in many life activities such as cell composition,energy supply and signal transduction.Cholesterol is an essential component involved in cell membrane formation,as well as precursor substance for steroid hormones,bile acids and specific vitamins.More studies are focusing on the role of altered cholesterol metabolism on cancer progression.The liver is the main organ of lipid metabolism in the body.Several liver-related diseases have been shown to be directly related to energy metabolism disturbance.The liver is the main organ for both the de novo synthesis of cholesterol and the metabolism of lipoprotein-related cholesterol.Studies have shown that the liver plays an important role in maintaining cholesterol homeostasis in the body’s internal environment by mediating approximately 70% of LDL degradation via the low-density lipoprotein receptor(LDLR)pathway,which has been shown to be the major form of cholesterol presence,transport,and metabolism in the body’s circulation.It has been reported that aberrant LDLR expression is closely associated with malignant progression in several cancer types,like breast cancer and cervical cancer;However,its specific role in tumor progression remains controversial.In hepatocellular carcinoma(HCC),LDLR expression has been found to be reduced in HCC with paraneoplastic hypercholesterolemia;High external cholesterol levels have been found significantly inhibit the invasive and metastatic ability of HCC.However,there is a lack of direct studies to confirm whether aberrant LDLR expression mediates the malignant progression of HCC by affecting cholesterol metabolism.We plan to verify the expression of LDLR in HCC with database analyses and clinical samples,explore the effect of LDLR abnormal expression on the malignant progression of HCC and the alteration of cholesterol metabolism pattern by functional assays and orthotopic liver transplantation model.Furthermore,we also intend to reveal the possible mechanism through transcriptome sequencing.We expect to clarify the expression of LDLR in HCC,and initially demonstrate the alteration of cholesterol metabolism behavior in HCC and finally explore new strategies for HCC treatment under abnormal LDLR expression.Research methods1.TCGA database and ONCOMINE database with LDLR m RNA data of HCC patients were applied to analyze the difference of LDLR expression in cancerous and paraneoplastic tissues of HCC patients;Kaplan-Meier Plotter online analysis website was used to analyze the survival difference of liver cancer patients with different expression levels of LDLR.2.Clinical tissue samples of HCC patients were collected to detect the difference in LDLR expression in paired cancerous and paraneoplastic samples and to analyze the prognosis significance.3.Select HCC cell lines with different malignancy MHCC 97 H and HLE to construct LDLR down-regulated cell lines with lentiviral transfection technology;Detect the effects of LDLR downregulation on the proliferation and motility of HCC cells by CCK-8,colony formation,cell cycle detection,migration assay and invasion assay.4.Analyze the effect of LDLR downregulation on the cholesterol metabolism mode in HCC cells by transcriptome sequencing;Detect intracellular free cholesterol levels by Filipin staining under different expression levels of LDLR;Detect the expression levels of genes related to cholesterol homeostasis by q RT-PCR in HCC cells;Detect the expression of cholesterol homeostasis-related genes in different pathological types of liver tissues by GEO database.5.Suppress intracellular cholesterol compensatory regulatory behaviors with small molecule inhibitors simvastatin in LDLR down-regulated HCC cells and detect the the change of intracellular cholesterol content and malignant behavioral changes in HCC cells by Filipin staining and cell functional assays.6.The GO enrichment analysis was applied to further mine the transcriptome sequencing data to search for significantly altered signaling pathways in LDLR low-expressing HCC cells,to explore their interrelationship with compensatory alterations in cholesterol metabolism,and to detect changes in malignant behaviors of HCC cells with small molecule inhibitors targeting related pathways.7.Perform the orthotopic liver transplantation model and the lung metastasis model to verify the effect of LDLR downregulation on malignant behaviors of HCC cells in vivo;The immunohistochemistry was applied to verify the expression of related pathways and cholesterol metabolism-related markers at the tissue level.Results1.Database analysis suggested LDLR expression was significantly lower in HCC tissues than in paired noncancerous regions;LDLR expression level decreases from normal liver to primary HCC foci to distal metastases of HCC;Patients with lower LDLR expression levels have poorer overall survival.2.Analysis of clinical samples confirmed that lower LDLR expression was associated with BCLC B&C stage,positive satellite nodules,higher Ki67 intensity and poorer disease-free survival and overall survival.3.Reduced expression of LDLR led to LDL uptake impairment and facilitated the proliferation,cell cycle progression and metastasis abilities of HCC cells in vitro.4.Upregulation of multiple cholesterol synthase m RNA levels and increased intracellular cholesterol content were revealed by transcriptome sequencing and filipin staining in LDLR downregulation HCC cells.The q RT-PCR was applied to verify the upregulation of m RNA levels of cholesterol synthesis enzymes in LDLR downregulating MHCC 97 H and HLE cells;The GEO database analysis verified that m RNA levels of cholesterol synthesis enzymes increased with worsening liver lesions.5.Simvastatin effectively inhibited the cholesterol synthesis ability in LDLR downregulating HCC cells,which efficiently reduced the intracellular cholesterol content and furthermore inhibited the proliferation,cell cycle progression and motility of HCC cells under LDLR downregulation.6.Activation of MEK/ERK pathway under LDLR downregulation was involved in regulating intracellular cholesterol synthesis.Inhibition of MEK/ERK pathway activity by PD98059 significantly reduced the m RNA levels of various cholesterol synthases and the intracellular free cholesterol accumulation caused by LDLR reduction.7.LDLR downregulation effectively promoted HCC proliferation and lung metastasis in vivo.The MEK/ERK pathway was activated in LDLR downregulating HCC tissues.The staining intensity of the rate-limiting enzyme of cholesterol synthesis HMGCR was higher in the LDLR downregulation group.Simvastatin treatment significantly reduced HCC proliferation and inhibited HMGCR expression in mice,which had no influence on the activation of MEK/ERK pathway.ConclusionLDLR expression was significantly lower in HCC tissues than in paired noncancerous regions and was identified as a negative prognostic factor in HCC.Downregulation of LDLR led to LDL uptake impairment of HCC cells in vitro.Intracellular cholesterol synthesis was elevated partially attributed to the stimulation of the MEK/ERK signaling pathway to accelerate the proliferation and motility of HCC cells.Simvastatin,by suppressing the cholesterol synthesis process,efficiently decreased the intracellular cholesterol level and,in turn,blocked the malignant phenotypes of HCC cells.Blocking the MEK/ERK signaling pathway at lower LDLR expression could inhibit the cholesterol synthesis ability of HCC cells and thus the malignant behavior of hepatocellular carcinoma.This study initially elucidated the altered cholesterol metabolism pattern and relating regulatory pathways in LDLR lowexpressing HCC patients,which provided new strategies for the clinical treatment of HCC patients.