Melatonin Regulates Endoplasmic Reticulum Stress-Related Apoptotic Resistance By Inhibiting BMAL1 Expression In Hepatocellular Carcinoma

Background Liver cancer is the sixth most common malignant tumor in the world,with the third highest mortality rate.It is a serious threat to human health.Hepatocellular carcinoma（HCC）is the prominent histological type of liver cancer.Due to its insidious onset,many HCC patients are often diagnosed in advanced stage,and the clinical prognosis is generally poor.Therefore,there is an emergency to further understand the molecular mechanism of HCC tumor growth and metastasis.Circadian rhythms are biological processes with ～24-hour oscillations,which help organisms better adapt to the environment.Disrupted circadian rhythms have been linked to a number of human diseases,including cancer.CLOCK and BMAL1 exist as heterodimers and are regulated by multiple feedback loops,and they have been identified to be required for the maintenance of circadian rhythms.Studies have shown that circadian rhythm and its biological clock genes play a crucial role in the development of cancer.Some previous researches have indicated that BMAL1,as an oncogenic gene,has an increased expression in thyroid cancer,pleural mesothelioma and prostate cancer.However,we still do not understand the role of BMAL1 in HCC and its molecular mechanism.Unfolded or misfolded proteins cumulate in the ER lumen is a phenomenon known as endoplasmic reticulum stress（ERS）.ERS triggers a series of protective cascade responses called unfolded protein response（UPR）.Endoplasmic reticulum transmembrane proteins IRE1α,PERK and ATF6 mediate three classic unfolded protein pathways,which play important roles in alleviating ER stress and maintaining homeostasis.ERS and circadian clockwork signaling pathways have similar physiological roles in cell regulation,suggesting that the two pathways interact with each other.Studies have shown that circadian genes regulate ERS and aging through the transcription of clock regulation control gene Pdia3,and the activation of ERS also damages the expression of circadian clock and clock control genes in NIH3T3 cells through an ATF4-dependent mechanism.However,the interaction between BMAL1 and ERS in liver cancer remains unclear.Melatonin（N-acetyl-5-methoxy-tryptophan）is the main pineal gland emanated hormone and a key regulator of circadian rhythm which has a wide range of physiological functions.Many studies have shown that as an effective cancer suppressor,melatonin has anti-cancer activities.Our previous studies have demonstrated that melatonin has anti-proliferation and pro-apoptosis effects in liver cancer by reversing ERS-induced apoptosis resistance.In addition,studies have shown that melatonin resynchronizes the dysregulated circadian circuits of human prostate cancer cells.However,there is few research on the relationship between melatonin and BMAL1 in liver cancer.Objective（1）To investigate the expression level of BMAL1 in hepatocellular carcinoma and its relationship with clinicopathological features and overall survival（OS）of patients with hepatocellular carcinoma.（2）To explore the effects of BMAL1 on the proliferation,migration and apoptosis of HCC cells.（3）To explore the relationship between BMAL1 and ERS.（4）To further explore the effect of melatonin on BMAL1 expression and hepatoma cell apoptosis.Methods（1）A total of 133 cases of primary liver cancer tissue and normal adjacent liver tissue samples were collected and made into tissue microarrays.Immunohistochemistry（IHC）was used to study the expression of BMAL1 in liver cancer tissue and liver tissue of HCC patients,and the relationship between BMAL1 and relevant clinicopathological features and overall survival of HCC patients was analyzed.Western Blot was used to analyse BMAL1 protein expression in 7 pairs of fresh liver cancer tissues and normal liver tissues from HCC patients.（2）Western blot was used to detect the expression of BMAL1 in liver cell lines and different hepatoma cell lines.Transient transfection technique was used to transfect small interfering RNA to knock down BMAL1 in Hep G2,Hep3 B,and transfection of overexpressed plasmid in MHCC-97 H increased the expression of BMAL1.（3）The effects of BMAL1 on the proliferation of HCC cells were detected by CCK8 experimental and clonogenesis assay;the effects of BMAL1 on apoptosis of HCC cells were detected by Western blot and flow cytometry;and the effects of BMAL1 on the migration of HCC cells were detected by Transwell and scratch assay.（4）The relationship between BMAL1 and ERS-related proteins were analyzed by IHC,western blot,RT-q PCR,TCGA database analysis.（5）Flow cytometry and western blot were used to detect the effect of melatonin on BMAL1 expression and hepatoma cell apoptosis.Results（1）IHC results showed that 73 of 133 samples had high expression of BMAL1 in liver cancer tissues,and the positive rate was 54.89%.In addition,the expression level of BMAL1 was correlated with tumor size and degree of differentiation,but not with age,gender,history of hepatitis and cirrhosis and AFP level.Survival analysis showed that patients with low BMAL1 expression had a longer overall survival.Western blot results also showed that BMAL1 protein was highly expressed in fresh liver cancer tissues compared with normal liver tissues.（2）Western blot results showed that the expression of BMAL1 in 5 liver cancer cell lines was higher than that in liver cell lines,and the efficiency of knockdown and overexpression of BMAL1 in HCC cell lines were verified.（3）CCK8 and clone formation assay showed that BMAL1 promoted the proliferation of HCC cells.The results of flow cytometry and western blot showed that BMAL1 inhibited the apoptosis of HCC cells.Wound-healing assay and Transwell assay showed that BMAL1 promoted the migration of HCC cells.（4）Immunohistochemical results showed that the the upregulation of BMAL1 was related to the high expression of ERS-related protein GRP78 and IRE1ɑ.Western blot results showed that BMAL1 was positively correlated with the expression of ERS-related protein GRP78,IRE1ɑ,PERK and XBP1 s,but not with ATF6 and ATF4.Correlation analysis of TCGA database showed that BMAL1 was positively correlated with the expression of these ERS-related proteins.（5）ERS was promoted by different concentrations of Tunicamycin（TM）in HCC cells.Western blot and RT-q PCR showed that low concentration of ERS increased the expression of BMAL1,and inhibited the expression of BMAL1 when the concentration increased to a certain level.（6）Flow cytometry and western blot results showed that melatonin inhibited the expression of BMAL1 in a concentration gradient and promoted apoptosis of HCC cells.Conclusions（1）BMAL1 is highly expressed in HCC,and its expression is related to the progression and poor prognosis of HCC.（2）BMAL1 promote the proliferation and migration of HCC cells and inhibit cell apoptosis.（3）The pro-survival effect of BMAL1 in HCC may be related to its interaction with ERS.（4）Melatonin regulates ERS-related apoptotic resistance of HCC cells by inhibiting BMAL1 expression.