A Study On Correlation Of LAPTM4B Gene With Hepatocellular Carcinoma And Its Clinical Significance

Objective:To investigate the expression of LAPTM4B gene in HCC and its effect on thebiological behaviour of hepatocellular carcinoma cells by in vitro interferenceexperiment. To analyze the relationship between LAPTM4B and the occurrence anddevelopment of HCC and its clinical significance.Methods:1.The expressions of LAPTM4BmRNA were determined by Real-time PCR,immunohistochemical staining method and Western Blot in HCC samples, carcinomaadjacent liver tissue samples and hemangioma adjacent normal liver tissue samples.The correlation of LAPTM4B mRNA to clinicopathologic parameters of HCC wasalso analyzed.2.LAPTM4B-siRNA sequences were synthesized and screened. The mosteffective LAPTM4B-siRNA sequence was transiently transfected into Huh7cells andto interfere the expression of LAPTM4B mRNA and pretein. The effects ofLAPTM4B-siRNA on proliferation, apoptosis, migration and invasion of Huh7cellswere detected by MTT assays, flow cytometry（FCM）and transwell technique, and theexpression of genes related with LAPTM4B was also detected.Results:1.The expression of LAPTM4B mRNA and LAPTM4B-35protein weresignificantly higher in HCCs than carcinoma adjacent liver tissues, far-carcinomaliver tissues and normal liver tissues（P<0.05）.2.The expression of LAPTM4B mRNA in HCCs was associated with patients’age, preoperative AFP level, BCLC staging and the related factors to HBsAg(P<0.05), not associated with sex, tumor diameter, tumor numbers and thebackground of liver cirrhosis(P>0.05). LAPTM4B mRNA expression level wasnegatively associated with patients’ age(P<0.01), positively associated with patients’ preoperative AFP level(P<0.05). Furthermore, LAPTM4B mRNA expression levelwas higher in the BCLC stage c period than0~b period(P<0.05), and higher inpostive HBsAg group than negative HBsAg group(P<0.05).3.The most effective LAPTM4B-siRNA sequence screened and its interferenceefficiency was69.11%. The expression of LAPTM4B mRNA and LAPTM4B-35protein decreased significantly after transient transfection, detected by Real timePCR、Western Blot and immunocytochemistry staining method.4. The MTT results showed that the proliferation of the LAPTM4B-siRNA groupin Huh7cells significantly decreased, compared with FAM-NC-siRNA group(P<0.01).5.The percentage of Huh7cells at S phase decreased and the apoptosis ratio ofHuh7cells increased after LAPTM4B interference, analyzing by FCM.6.The motility of the cells in the LAPTM4B-siRNA group was significantlyretarded, compared with the FAM-NC-siRNA group(P<0.01). The invasion assayrevealed that the invasion of cells in the LAPTM4B-siRNA group was significantlyretarded, compared with the FAM-NC-siRNA group(P<0.01).7.The expression of cyclinE significantly decreased and the expression ofCdkn1B, P53and P16significantly increased after LAPTM4B interference in Huh7cells, detected by Real time PCR.Conclusion:1.The expression of LAPTM4B was very low in normal liver tissues, but muchhigher in HCCs，and was associated with patients’ age, preoperative AFP levels,BCLC staging and the related factors to HBsAg. LAPTM4B might be a novel markerin HCC clinical diagnosis and prognosis evaluation.2. LAPTM4B was associated with proliferation, migration, invasion and apoptosisof hepatocellular carcinoma, and might be a new target in HCC gene treatment.3.The molecular mechanisms of LAPTM4B promoting the proliferation of tumorcells might be associated with cyclinE, Cdkn1b, P53, P16.