Research On The Mechanism Of MiRNA-23b-3p Regulating The Proliferation And Invasion Of Hepatocellular Carcinoma Through Targeting UBA6

Background and Objective:Primary liver cancer has the fifth-ranked incidence and the second-ranked mortality in China.Hepatocellular carcinoma(HCC)has a relatively high incidence and poor prognosis,which accounts for approximately 90% of primary liver cancers.The 5-year survival rate is only 10% ～ 20%.The metastasis of HCC,which is affected by the proliferating,migrating and invading ability of tumor cells,significantly compromises all temptation to cure it.In-depth research on the molecular mechanism of the proliferation,migration,and invasion of HCC cells can provide new theoretical support for the diagnosis and treatment of HCC.Micro RNA(miRNA)is a type of small RNA molecule that can regulate gene expression activity.It can perform biological functions by inhibiting the expression of target genes or promoting the degradation of target genes.We found that miRNA-23b-3p is negatively related to the5-year survival and incidence of metastasis in HCC.The molecular mechanism of its effect on HCC is still unclear.An in-depth analysis of the impacts of miRNA-23b-3p on cell proliferation,migration,and invasion in the malignant progression of HCC,as well as the study of its downstream target genes,will help clarify the regulatory mechanism of HCC metastasis and explore new targets for the treatment of HCC.The endogenous proteins of eukaryotes are mainly degraded through ubiquitination.Ubiquitination is a process in which ubiquitin is covalently bound to target proteins under the catalysis of a series of enzymes,regulating protein stability,localization,activity,and interaction.Ubiquitination is widely involved in many crucial Physiological and pathological processes,including transcription regulation,DNA damage repair,cell proliferation,apoptosis,immune response,etc.,plays an essential role in the genesis and development of tumors.Ubiquitin-like modifier-activating enzyme 6(UBA6)is a crucial ubiquitin-activating enzyme,a key factor that regulates intracellular signal transduction,cell growth,antigen presentation,etc.The role of UBA6 in HCC and its mechanism are still unclear.Studying the regulation mechanism of miRNAs on UBA6 and its role in HCC is helpful for the development of proteasome inhibitors for HCC treatment.The study of the biological functions of miRNA-23b-3p and UBA6 in HCC tissues and their interaction mechanism will help provide new solutions for diagnosing and treating hepatocellular carcinoma.Methods:1.Excavate the data of HCC cancer tissue and adjacent tissues from the TCGA database,analyze the expression of miRNA-23b-3p,and analyze the expression of miRNA-23b-3p and clinical characteristics in 75 HCC specimens from our hospital,and analyze the relationship between miRNA-23b-3p and clinical features.CCK-8,cell clone formation experiment,cell apoptosis experiment,Transwell migration,and invasion experiment were used to detect the effect of miRNA-23b-3p on the function of SMMC7721 and Huh7 cells.2.Bioinformatics analysis of the targeting relationship between miRNA-23b-3p and UBA6 was performed based on the TCGA database to analyze the correlation between miRNA-23b-3p and UBA6 expression in HCC cancer tissues.The dual-luciferase reporter gene experiment was used to analyze the targeted binding relationship between miRNA-23b-3p and UBA6.The effect of overexpression or underexpression of miRNA-23b-3p on the expression of UBA6 m RNA is tested.UBA6 was detected by q PCR and Western blot.3.UBA6 interference plasmid and overexpression plasmid was constructed.HCC cells SMMC7721 and Huh7 were cultured and co-transfected by miR-23b-3p mimics and UBA6 overexpression plasmid,or miR-23b-3p inhibitor and UBA6 interference plasmid.q PCR and Western blot were used to detect the expression of UBA6,using CCK8 and cell clone formation,flow cytometry,Transwell to analyze UBA6 in miR-23-3p regulation of HCC cell proliferation,apoptosis,migration,and invasion.4.An SMMC7721 cell line that was stably transfected with miR-23b-3p mimics and the negative control NC were subcutaneously injected into the nude mice to form tumors.The tumor growth curve was drawn,and the tumor tissues were collected.q PCR was used to detect the expression of miR-23b-3p in the tumor,Western blot and immunohistochemistry were used to detect the expression of UBA6 in the tumor,Ki-67 was used to detect the proliferation ability of tumor cells,and Tunel was used to determine the apoptosis of tumor cells.Results:1.TCGA database analysis and clinical specimen testing have shown that miRNA-23b-3p is low expressed in HCC tissues and is related to clinicopathological characteristics,such as tumor size,microvascular infiltration,and BCLC staging.The lower the expression of miRNA-23b-3p,the worse the prognosis of the patient.Overexpression of miRNA-23b-3p can inhibit HCC cell proliferation and cloning,promote HCC cell apoptosis,and inhibit HCC cell migration and invasion.The low expression of miRNA-23b-3p promotes HCC cell proliferation and cloning,inhibits HCC cell apoptosis,and enhances HCC cell migration and invasion.2.There is a negative correlation between miRNA-23b-3p and UBA6 expression.Dual-luciferase gene experiments showed that miRNA-23b-3p could specifically bind to UBA6.Up-regulation of miRNA-23b-3p expression can inhibit UBA6 m RNA and UBA6 expression.Down-regulation of miRNA-23b-3p will increase the expression of UBA6 m RNA and UBA6.The above results indicate that UBA6 is a specific target gene of miRNA-23b-3p.3.UBA6 overexpression can reverse the inhibitory effect of miR-23b-3p mimics on the proliferation,migration,and invasion of HCC cells.Knockdown of UBA6 can reverse the impact of the miR-23b-3p inhibitor on the proliferation,migration,and invasion of HCC cells.4.In vivo experiments have shown that miR-23b-3p mimics can inhibit subcutaneous tumor formation in nude mice,resulting in high expression of miR-23b-3p and low expression of UBA6 in tumor tissues,reducing tumor cell proliferation and increasing the proportion of apoptotic cells.Conclusion:1.miRNA-23b-3p is low-expressed in HCC tissues and is closely related to clinical features such as tumor growth and vascular infiltration and the prognosis of HCC patients.2.miRNA-23b-3p reduces the expression of UBA6 by targeting,inhibits the proliferation of HCC cells,promotes the apoptosis of HCC cells,and reduces the migration and invasion ability of HCC cells.3.In the nude mouse tumor-bearing model,overexpression of miRNA-23b-3p can down-regulate UBA6,inhibit tumor cell proliferation and promote tumor cell apoptosis,thereby inhibiting tumor growth of HCC.Therefore,the miRNA-23b-3p/UBA6 axis plays an essential role in the occurrence and development of hepatocellular carcinoma and may become a new pathway for treating hepatocellular carcinoma.