Expression And Clinical Significance Of Beta-Lactamase In Hepatocellular Carcinoma

BackgroundHepatocellular Carcinoma(HCC)is also referred to as liver cancer and is most commonly seen tumor in primary malignant tumors of the digestive system.Related studies have showed that about 1 million new cases of HCC are detected each year,of which more than two-thirds of new cases are distributed in the western Pacific [1].The mortality rate of HCC ranks is second in the world.The latest research data show that HCC accounts for the highest proportion of deaths caused by malignant tumors worldwide [2].HCC is characterized by insidious onset,high malignancy and rapid progression,and early diagnosis of HCC patients remains a huge challenge that has not yet been overcome.Most patients have final diagnoses when the tumors have developed into advanced stage,and the common treatments for hepatocellular carcinoma such as surgical resection,liver transplantation,radiofrequency ablation,etc.have many contraindications.Therefore,the probability that advanced HCC patients achieve the goal of eradicating HCC by the above surgical treatment is very low [3,4].Studies have shown that the 1-year overall survival rate after HCC treatment is relatively considerable(66.1%),while the 3-year and 5-year overall survival rates are reduced to 39.7% and 32.5%,respectively,which in early HCC patients are 93.5%,70.1% and 59.1% [5].However,due to the lack of early diagnosis,the mortality rate of HCC is still high.Given the limitations of the current HCC treatments that have not been overcome,it is important to determine the molecular mechanisms of HCC and to develop novel HCC early diagnosis and prognostic strategies.A growing body of evidence suggests that metabolic disorders,especially abnormal lipid metabolism,play an important role in tumor progression,including HCC [6-9].Beta-lactamase(LACTB)is mainly located in the mitochondria of mammalian nucleated cells and is ubiquitous in different tissues of the human body,especially in bone,heart,skeletal muscle and liver.As a mammalian active site serine protein,LACTB is evolutionarily related to the bacterial penicillin binding protein.LACTB can be involved in the regulation of lipid metabolism pathways as a regulator of mitochondrial inner membrane tissue and energy homeostasis,which in turn inhibits tumor progression and acts as a novel protease homologue of cellular metabolic processes [10].In addition,studies have shown that LACTB is expressed lower in some malignant tumors such as breast cancer [11],glioma [12],gastric cancer [13],and colorectal cancer [14,15],and overexpressed LACTB can inhibit the cancer cells proliferation.Therefore,the above studies indicate that LACTB can be used as a novel epigenetic silencing tumor suppressor and plays an important role in tumor progression.However,the expression and function of LACTB in HCC remains largely unexplored.Objective1.To investigate the expression levels of LACTB in HCC tissues:(1)To analyze whether LACTB mRNA in TCGA database has different transcriptional levels in HCC and paracancerous tissues,as well as detect the correlation of the rate LACTB mRNA transcription level and TNM staging,pathological grading and overall survival rate(OSR)of HCC patients.(2)To investigate whether the expression levels of LACTB in Zhengzhou University Cohort HCC tissues were different,and explore the correlation between LACTB expression level and TNM stage,pathological grade and overall survival rate of liver cancer patients.2.To investigate the effects of LACTB on the phenotype of HCC cells:(1)the effect of overexpression of LACTB on the biological behaviors of HCC cells proliferation,migration and invasion in vitro.(2)In vivo experiments to verify the effect of overexpression of LACTB on the tumorigensis of nude mice.Methods1.Download the clinical information of HCC patients in the TCGA(the cancer genome atlas)database,and the LACTB mRNA transcription level data of patients to screen and integrate,analyze the transcription levels of LACTB mRNA in HCC and paired adjacent tissues.Additionally,the correlation between the LACTB mRNA transcription levels and TNM stage,pathological grade and overall survival rate of HCC patients was analyzed.2.Tissue samples from 341 patients with HCC were selected.These patients were admitted to the First Affiliated Hospital of Zhengzhou University from 2011 to 2015,and were all diagnosed as HCC by pathological diagnosis.These specimens were used to construct tissue microarray(TMA)spares.3.Immunohistochemical(IHC)staining was used to detect the expression level of LACTB protein in clinical tissue samples,and the correlation between LACTB expression level and clinicopathological features of HCC tissues and its prognostic significance for HCC patients were analyzed.4.Overexpression of LACTB by lentiviral transfection technique and confirmation by Western blotting,CCK-8 assay,EdU staining assay and colony formation assay were used to detect the proliferative capacity of HCC cells.Wound healing assay and transwell assay were used to detect the migration and invasive capabilities of HCC cells.5.The HCC cells that overexpressed LACTB were injected subcutaneously into nude mice,and the growth rate of subcutaneous tumors was recorded by a living imager machine.After 6 weeks,the mice were sacrificed and the expression of LACTB and Ki-67 was detected by IHC method.6.Analyze statistical data using GraphPad Prism software(version 7.0)or SPSS software(version 23.0).The chi-square test was used to detect the correlation between LACTB expression and clinicopathological features,and Student's t-test was adopted to analyze the statistical difference among groups.Kaplan-Meier survival analysis was used to investigate the effect of different LACTB expression on prognosis.Results1.The results of TCGA analysis showed that the expression level of LACTB mRNA in HCC tissues was significantly lower than that in normal tissues.The expression level of LACTB was significantly lower in patients with high pathological grade(III-IV grades),and there was no significant difference between the expression of LACTB in patients with different TNM stages.Moreover,2.Kaplan-Meier analysis showed that overall survival(OS)and Relapse-free survival(RFS)of HCC patients with low expression of LACTB were significantly lower than those with high expression of LACTB.Additionally,GSEA analysis results showed that the genes negatively correlated with the survival of HCC patients were significantly enriched in the low expression group of LACTB.On the contrary,the genes positively associated with the survival of HCC were significantly enriched in the LACTB high expression group.3.Immunohistochemical staining results showed that the expression level of LACTB protein in HCC tissues was significantly lower than that in paired adjacent non-tumor tissues.The expression of LACTB was significantly different between TNM I-II stages and III-IV stages patients.Furthermore,the expression of LACTB protein was much lower in TNM III-IV stages.4.The overall survival(OS)rate of patients with low LACTB expression was significantly lower than that of patients with high expression of LACTB.Univariate and multivariate analyses results showed that low expression of LACTB and advanced TNM stages were independent prognostic risk factors for HCC patients.5.The results of in vitro cellular experiments showed that overexpression of LACTB significantly inhibited the HCC cell lines proliferation,migration and invasion.6.In vivo experiments showed that overexpression of LACTB had a remarkable inhibitory effect on the tumorigensis of HCC cells.ConclusionLow expression of LACTB was closely related to the clinical prognosis of patients with HCC.In vitro experiments have demonstrated that overexpression of LACTB could inhibit HCC cell proliferation,migration,invasion and tumorigenesis in vivo.LACTB was expected to be a biomarker for the early diagnosis of HCC and to judge the prognosis of patients with HCC,and LACTB also suggested a novel method for targeted therapy of HCC patients.