The Expression And Its Clinical Significance Of MiR-375 In Disease Progression For HBV-related Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma, one of the most common malignant tumor, has some characteristics of high malignant degree, rapid progression, and case fatality rate increased year by year. HBV infection is the dominating pathogenesis that results to HCC in China. The poor clinical outcome of HCC is due to symptom occult in early, diagnosis in late stage and limited treatment options. Many biomarks have been used widely for HCC diagnosis at present. Nevertheless, most of them still lack of sensitivity or specificity in common. Although a number of new serological markers were explored, it is too limited to apply in clinic, and also difficult to the prediction of disease progression of HBV related HCC. Therefore, a serological marker is an urgent need to HCC, which is not only closely related to HBV infection developing liver cancer, but also higher specificity and sensitivity for diagnosis. We try to explore for a biological marker, which will benefit to diagnosis and treatment of HCC.MicroRNAs(miRNAs) are endogenous and single-stranded non-coding RNAs of about 22 nucleotides in length. That regulated gene expression by post-transcription. miR-375 plays an important role and have been identified to downregulated expression in a variety of malignant tumors. miR-375 inhibited proliferation, migration and invasion in hepatocarcinoma cell lines. However, there still lack of further epidemiological field data about the mi R-375 expressions in chronic asymptomatic HBV carriers, patients with chronic HBV infection and HBV-related HCC. The studies try to detect the expression levels of miR-375 in the serum of the HBV carries, the patients of HBV infection and HBV related HCC, and to analyze the correlation between the miR-375 levels and disease progression and clinical features. At the same time, we upregulate the miR-375 expression in HepG2 cells to observe the effect of miR-375 on the profiferation, migration and invasion. The study also try to clarify the mechanism of generation and development in HCC, and to find a new strategy for prediction of disease progression of HBV-related HCC. Methods1. Tumor tissue study in HCC patients: we recruited the HBsAg positive HCC patients who underwent curative liver resection for HCC in Xijing Hospital from August 2014 to August 2015. Tumor tissues and adjacent tissues samples were collected from each patient. A standard questionnaire was developed to collect demographic information and clinical data. Using qRT- PCR was used to detect the miR-375 expression level in tissue sample. Hepatits B virus serological markers were detected by enzyme linked immunosorbent assay(ELISA), and to be analyzed its correlation with miR-375 levels, tumor stage, size, number, and tumour markers for HCC.2. Serological study in different populations: From October 2014 to February 2015, we recruited patients with HBV infection in Tangdu Hospital, and healthy control after physical examination enrolled from outpatient department of the Fourth Military Medical University. mi R-375 and HBV DNA expression profiles were detected by using qRT-PCR. The receiver operating characteristic curve(ROC) analysis was applied to assess the diagnostic accuracy of miR-375.3. Mechanism study in cell lines: HepG2.2.15 and and its parental HepG2 were selected for experiment. Using qRT-PCR detected the miR-375 expression level in cell lines. miR-375 mimic overexpression was tested by transient transfection, and to analyze the effects of miR-375 overexpression on proliferation, migration and invasion in vitro by CCK-8, cell scratches and transwell experiment.4. Statistical analysis: The data process was used Epidata3.1 software, and the statistical analyses were carried out with SPSS19.0 software. Differences between experimental groups were statistically evaluated using Student's t test, ANOVA analysis and Mann-Whitney U test. The chi-square test was used to analyze categorical data.The Spearman correlation analysis was used to test association between different variables. P value of less than 0.05 was considered to be statistically significant. Results1. Tumor tissue study in HCC patients: The study enrolled 43 participants. The mean size of tumor was 5.99 ± 2.58 cm, and the average number of tumors was 1.24 ± 0.58.(1) miR-375 expression in tumor tissues were significantly lower than adjacent tissues for HBV-related HCC(P<0.001).(2)To analyze correlation between the tumor tissues of miR-375 expression and HBV serological markers, the miR-375 expression was significantly downregulated in HBV DNA positive patients than that in HBV DNA negative patients(P<0.05). The miR-375 expression was significantly lower in anti-HBc positive patients than that in anti-HBc negative patients(P < 0.001).(3) To analyze correlation between the tumor tissues of miR-375 expression and clinic features of the patient, the miR-375 expression was significantly down-expression in male patients than that in famale patients(P<0.05). The miR-375 expression was significantly lower in patients with cirrhosis than patients with non-cirrhosis(P<0.05). The mi R-375 expression was down-expression in CA19-9 raised patients than patients with normal(P<0.05).2. Serological study in different populations: A total of 63 HCC patients, 74 of HBV infection patients and 40 healthy controls were enrolled in this study. There are 143 male participants(80.8%) and 34 females participants(19.2%).(1) A decreased expression of serum miR-375 was distinctly observed in HBV infection patients, compared with healthy controls. The expression of serum miR-375 were descended in order in chronic hepatitis B patients without cirrhosis, patients with cirrhosis, and HBV-related HCC patients.(2)To analyze correlation between the serum of mi R-375 expression and HBV serological markers, the miR-375 expression was significantly downregulated in HBeAg positive patients than that in HBeAg negative patients(P<0.05). The miR-375 expression was significantly lower in anti-HBe negative patients than that in anti-HBc positive patients(P<0.05). The miR-375 expression was significantly downregulated in HBV DNA positive patients than that in HBV DNA negative patients(P<0.05).(3)To analyze correlation between the serum of miR-375 expression and clinic features of the patients, there were correlation between miR-375 expression levels and AFP and CEA of the patient(P<0.05).(4)The ROC curve was analyzed to verify the diagnostic accuracy of serum miR-375. The results displayed that serum miR-375 could serve as valuable biomarkers for different HCC patients from healthy controls with the AUC of 0.838(95%CI:0.780~0.897). And its sensitivity and specificity were 73.9% and 93.0%, respectively. Subsequently, in respect to AFP, the sensitivity was 75.0%, and the specificity was 65.5% to identify HCC from the HBV infection subjects with the AUC of 0.584(95%CI:0.456~0.713). miR-375 could differentiate HCC from the HBV infection subjects with the AUC of 0.768(95%CI:0.644~0.891). The sensitivity was 93.8% and the specificity was 63.9%.3. Mechanism study in cell lines:(1)miR-375 was obviously reduced in HepG2.2.15 cells compared with HepG2 cells(P < 0.05).(2)miR-375 cells proliferation rate was less than without transfection after expressing cells. The proliferation of HepG2.2.15 cells transfected with miR-375 was suppressed significantly than in HepG2 cells.(3) Compared with the transfected cells, untransfected cells showed more migration. There were morphological changes in Hep G2.2.15 with transfection and the adherent ability weaker than HepG2 cells with transfection.(4)The number of the overexpression of miR-375 significantly inhibited less than the cellular invasion. And overexpression of miR-375 was obviously reduced the number of invasion in HepG2.2.15 cells compared with HepG2 cells of overexpression miR-375. Conclusions1. MiR-375 was significantly downregulated in tumor tissues than adjacent tissues for HBV-related HCC patients. There were negative correlation between miR-375 expression levels of tumor tissues and anti-HBc and HBV DNA.2. Serum miR-375 levels were reduced significantly in groups of chronic hepatitis B patients without cirrhosis, cirrhosis, and HBV-related HCC compared normal group(P < 0.05). The results provided a new strategy to predict progression for HCC. There were correlation between serum miR-375 expression levels and HBeAg, anti-HBe, HBV DNA, AFP and CEA.3. Serum miR-375 could serve as valuable biomarkers for finding HCC patients with high sensitivity and specificity.4. miR-375 was obviously reduced in HepG2.2.15 cells compared with HepG2 cells. And overexpression of miR-375 was obviously inhibited proliferation, migration and invasion in vitro.