Study On The Increased Expression Of The MiR-545/374a Cluster Encoded In The Ftx LncRNA In Human HBV-Related Hepatocellular Carcinoma

Background and ObjectiveHepatocellular carcinoma (HCC) is one of the most frequently occurring cancers worldwide. A variety of risk factors are associated with HCC development, including the following most common risk factors:hepatitis B virus (HBV) infection, hepatitis C virus (HCV) infection, consumption of food contaminated with aflatoxin B1, heavy alcohol intake, and nonalcoholic fatty liver disease. Due to the growing incidence of HCC, intense research efforts are currently being undertaken to understand the cellular and molecular mechanisms of the disease so that novel diagnostic markers and therapeutic strategies can be developed.The mechanism of HBV induced oncogenesis may involve integration of viral genomic fragments into the human genome. An alternative hypothesis is that HBV produces regulators which participate in cellular pathways that lead to carcinogenesis. However, the exact mechanism by which HBV infection leads to HCC still remains obscure. In this study, we provide evidence that overexpression of a microRNA cluster promotes tumorigenesis and tumor progression in HBV-related HCC.RNA has become widely suspected as the culprit behind almost every case of epigenetic regulation in a variety of diseases. Only 1% of the mammalian genome carries protein-coding potential, yet 70 to 90% is transcribed to produce a large transcriptome of long noncoding RNA (lncRNA, defined as RNA> 100 nucleotides in length). Clear roles have emerged for several lncRNAs which participate in HCC disease progression. Mechanistically, lncRNAs target a site-specific pathway, and may directly interact with chromatin/mRNAs and recruit histone-modifying enzymes to induce epigenetic silencing of target genes. Interestingly, few studies have reported that some of these self-functional lncRNAs are also acting as microRNA (miRNA) precursors. The lack of experimental data on these miRNAs leaves open questions about their possible relation to malignant tumors.The Ftx transcript is a conserved functional lncRNA encoded within the X-inactivation center (Xic). Ftx encodes 4 microRNAs in its introns. Intron 12 encodes 1 cluster of 2 microRNAs (miR-374b and miR-421), which is well conserved in different mammalian species. Intron b encodes 1 related cluster of 2 microRNAs (miR-374a and miR-545), which is absent in mouse and rat due to mutational changes. A recent study showed that miR-374a is upregulated in breast cancer tumors and promotes breast cancer metastasis by activating the Wnt/β-catenin signaling pathway. It remains unknown whether the altered expression of miR-374a occurs independently from the other 3 microRNAs or whether the Ftx microRNA clusters are regulated as a group. No studies have explored the expression of the other 3 microRNAs and their potential relationship with malignant tumors. This raises a critical question that our research addresses:does Ftx microRNA clusters play a role in cancer progression?Another example of altered microRNA expression occurs in hand-foot-and-mouth disease (HFMD). In this case miR-545 expression is upregulated, upon enterovirus 71 (EV71) or coxsackievirus 16 (CAV16) infection. Since viral infections can alter miR-545 expression, we explored the possible relationship between Ftx microRNA cluster expression and HBV infection.In this study, the expression profiles of the miR-545/374a cluster and the miR-421/374b cluster derived from the Ftx transcript were examined in HBV-related HCC. Based upon correlation analysis with clinical features, the microRNAs were assayed for their ability to regulate tumor progression. In vitro cell culture assays were established to confirm the biological function of the microRNAs and potential regulating mechanism(s). Next, we followed a "tissue to serum, preoperative to postoperative" pattern to investigate the diagnostic value of corresponding serum microRNAs.PART ⅠThe increased expression of miR-545/374a cluster encoded in the Ftx IncRNA is associated with the prognosis of HBV-related HCC patients.Objective1. To detect whether miR-545, miR-374a, miR-421 and miR-474b is aberrantly expressed in HBV-related HCC patients.2. To explore the potential relationship between miR-545/374a expression and prognosis-related clinical features.Methods1. Sixty-six HBV-related HCC patients and 11 hepatic hemangioma patients (negative control) were recruited from February 2012 to January 2013 at the Provincial Hospital Affiliated to Shandong University. For each HBV-related HCC subject, HCC tissue and matched distal non-cancerous liver tissue are collected. The tissue specimens are kept in ultra-low temperature.2. Real-time PCR was used to measure the expression of miR-545, miR-374a, miR-421 and miR-474b in tissue specimens.3. The samples obtained from HBV-related HCC subjects after surgery were analysed by pathologists and features including histological grade, metastasis, tumor capsule and cirrhosis were reported.4.The one-Sample Kolmogorov-Smirnov Test was used to confirm the normal distribution of experimental data. To evaluate significant differences between matched HBV-HCC tissue and nontumor tissue, paired t tests were performed. Unmatched continuous data were compared using an independent 2-tailed t test. Pearson’s correlation (r) was used to measure correlation. P<0.05 was considered statistically significant.Results1. The miR-545/374a cluster encoded in the Ftx transcript is upregulated in HBV-related HCC tissue.The expression of individual microRNAs was measured by quantitative RT-PCR. The result showed that, the expression of the miR-545/374a cluster was lower than the miR-421/374b cluster, but showed clear variation in different groups. MicroRNA 545 was significantly upregulated in HCC tissue compared with non-cancerous liver tissue (NT) (HCC=6.60×10-5 vs. NT=3.27×10-5; p=0.003, paired t test), as well as in NT compared with negative control (NC) (NC=2.06×10-5; p=0.034, independent t test). Likewise, miR-374a expression was significantly higher in HCC tissue compared with NT (HCC=9.73×10-6 vs. NT=1.05×10-6; p=0.000, paired t test), while no significant difference was found between NT and NC regarding miR-374a expression (NC=0.73×10-6; p=0.584, independent t test). On the other hand, the comparatively conserved cluster of miR-374b and miR-421 were highly expressed with large individual difference, but surprisingly showed no significant difference between 66 pairs of matched HCC tissue and corresponding NT. Furthermore, the expression levels of miR-374b and miR-421 did not show any differences between 66 HCC tissues and 11 NC.2.The expression of miR-545/374a is significantly correlated with prognosis-related clinical features.To investigate the potential role of miR-545/374a in HBV-related HCC, the expression levels of these microRNAs were analyzed with key clinical features associated with tumor progression and disease prognosis. The histological differentiation analysis showed no restrict grade-by-grade variation. However, the expression of miR-545 is significantly higher in the poor differentiated group compared to the good differentiated group (p=0.019), while the expression of miR-374a is significantly higher in the moderate differentiated group compared to the good differentiated group (p=0.005). Patients with an incomplete tumor capsule had higher levels of both miR-545 and miR-374a than patients with a complete tumor capsule (p=0.04, p=0.013). Furthermore, the patients with distal metastasis had significantly higher miR-374a expression than patients without distal metastasis (p=0.002). No significant differences were observed in the miR-545 and miR-374a expression levels with respect to cirrhosis. Likewise, no significant differences were observed for the levels of miR-545 and miR-374a with respect to age.Conclusion1.The miR-545/374a cluster encoded in the Ftx transcript is upregulated in HBV-related HCC tissue.2.The unregulated expression of miR-545/374a is significantly correlated with tumor histological differentiation, metastasis, and tumor capsule, indicating miR-545/374a may promote tumor invasion and metastasis, and finally lead to poor prognosis.1.The miR-545/374a cluster encoded in the Ftx transcript is upregulated in HBV-related HCC tissue.2.The unregulated expression of miR-545/374a is significantly correlated with tumor histological differentiation, metastasis, and tumor capsule, indicating miR-545/374a may promote tumor invasion and metastasis, and finally lead to poor prognosis.PART ⅡHBV infection may induce the overexpression of miR-545/374a, and therefore promote tumor progression.Objective1. To analysis the relationship between miR-545/374a and HBV infection related clinical indicators, including HBsAg and HBV-DNA.2. Study on the influence of HBV infection on miR-545/374a expression.3. Study on the regulating effect of HBx protein on miR-545/374a expression.4. To explore whether miR-545/374a can influence tumor proliferation, migration and invasion.Methods1.Human HCC cell lines Bel-7402, HepG2 and previously constructed stable HBV positive HepG2.2.15 cells were cultured, and Bel-7402 are seeded at a density of 2.0×105 cells/mL and starved for 24 hours before transfection.2.The wild-type HBV genome-containing plasmid (pCDNA3.1-HBV), and the plasmid encoding full-length HBx protein (pIRSE-HBx) were transfected using LipofectamineTM 2000 according to the manufacturer’s protocol. Empty expression vectors were transfected as a negative control. Expression of plasmids in Bel-7402 cell lines was confirmed by high-sensitivity fluorescent quantitative PCR and western blotting.3. MicroRNA 545 mimics, miR-545 inhibitor, miR-374a mimics, miR-374a inhibitor, and negative control duplex (designated as NC) were all synthesized by GenePharma. Oligonucleotide transfection was performed using Lipofectamine 2000.4. Cell proliferation was assayed using MTT assays. Cell migration and invasion were assayed using a transwell chamber with and without Matrigel, respectively. For the invasion assay, a transwell chamber was placed into a 24-well plate and was coated with 30ul of Matrigel.5.Data were analyzed with SPSS 16.0 statistical software. The Data were compared using an independent 2-tailed t test. P<0.05 was considered statistically significant.Results1.Study on the influence of HBV infection on miR-545/374a expression.In light of previous evidence that HBeAg positive patients showed a significantly higher level of miR-374a than HbeAg negative patients, and patients with a positive HBV-DNA level had higher expression of both miR-545 and miR-374a compared to patients with a negative HBV-DNA level. To confirm our hypothesis that miR-545/374a expression might be related to HBV infection, we monitored the expression of miR-545/374a in HBV positive and negative cell lines. A statistically significant increase in miR-545 expression was observed in the HepG2.2.15 cell line compared to the HepG2 cell line (p=0.003), but the data obtained for miR-374a was not statistically significant (p=0.057). Next, the HCC cell line Bel-7402 was transfected with the HBV-genome-containing plasmid (pIRSE-HBV) and empty pIRSE plasmid was utilized as a negative control. The levels of miR-545/374a were then monitored by quantitative RT-PCR. As hypothesized, both miR-545 and miR-374a were significantly increased after transfection of the HBV genome encoding plasmid (p=0.001, p=0.032 respectively).2. Study on the regulating effect of HBx protein on miR-545/374a expression.HBx protein is a key regulator of HBV infection. Therefore, we monitored the expression of this protein in 66 HBV-HCC subjects and compared it to the levels of miR-545/374a. The levels of miR-545/374a and HBx mRNA were measured by quantitative RT-PCR. Our result showed that, HBx expression is significantly correlated with miR-545 expression (r=0.312, p=0.011, Pearson’s correlation). To investigate whether HBx acts as a potential regulator of the Ftx miR-545/374a cluster, an overexpression strategy was conducted via transfection of a HBx expressing plasmid (pCDNA3.1-HBx) into Bel-7402 cells. The HBx protein was overexpressed in pCDNA3.1-HBx transfected cells. Furthermore, the expression level of miR-545/374a was increased in HBx expressing cells compared to negative control cells transfected with empty plasmid (p=0.046, p=0.008).3.The miR-545/374a cluster promotes HCC cell proliferation, cell migration and invasion.To gain a mechanistic understanding of the biological significance of the upregulated miR-545/374a cluster, the Bel-7402 cell line was transfected with individual microRNA mimics and inhibitors. Nonsense oligonucleotide was transfected as a negative control. The microRNA expression status at 72h after transfection was monitored by quantitative RT-PCR. The inhibition rates for miR-545 and miR-374a were 58.6% and 31.3%, respectively.To determine whether miR-545 and miR-374a have an impact on tumor cell proliferation, we transfected cells with various microRNA mimics and inhibitors and performed a MTT assay to monitor cell viability. The proliferation of HCC cells was significantly decreased after transfection with miR-545/374a inhibitors compared to negative control. Overexpression of miR-545/374a (via transfection of the mimics) in the HCC cell line promoted cell proliferation to a small degree. Next, transwell assays were performed using transfected cells to investigate the effect of miR-545/374a expression on cell migration and invasion. Both microRNAs enhanced the migration and invasion ability of HCC cell lines, except that inhibition of miR-374a had no statistically significant influence in cell migration assays (P=0.102).Conclusion1. Patients with a positive HBeAg or HBV-DNA level had higher expression of both miR-545 and miR-374a compared to patients with a negative HBeAg or HBV-DNA level.2.Expression of miR-545/374a is positively regulated by HBV infection and may be induced by HBx expression.3. The overexertion of miR-545/374a promotes HCC cell proliferation, cell migration and invasion.PART ⅢStudy on the sera miR-545/374a expression and its clinical application.Objective1.To explore the correspondence on miR-545/374a expression between in serum and in tissue.2. To detect whether sera miR-545, miR-374a, miR-421 and miR-474b is aberrantly expressed in HBV-related HCC patients, and to explore its possible clinical application in diagnosing tumorigenesis.3. To explore the changing tendency of sera miR-545/374a expression after the tumor resection.4.To analysis the relationship between sera miR-545/374a expression before the surgery and prognosis-related clinical indicators.Methods1. For Sixty-six HBV-related HCC patients, collect 5ml venous peripheral blood before surgery, and 5ml venous peripheral blood 20 days after surgery. The tissue specimens are kept in ultra-low temperature.2.Features including patients’ age, sex, HBV-DNA, α-fetoprotein (AFP), carcino-embryonic (CEA) and y-glutamyltransferase (y-GT) were collected to analyze their relationship with sera microRNA expression.3.To evaluate significant differences between matched serum specimens before and after surgery, paired t tests were performed. Unmatched continuous data were compared using an independent 2-tailed t test. Pearson’s correlation (r) was used to measure correlation. P<0.05 was considered statistically significant.Result1. Study on sera miR-545/374a expression in HBV-related HCC patients, and its correspondence with expression in tissue.Tissue to serum analysis represents the process from mechanism to application. As a first step, the changing tendency of serum microRNAs should be tested to determine if their presence is consistent with their presence in tissue. Our study showed that, in 66 HBV-HCC subjects, the expression of miR-545/374a in preoperative period serum is significantly correlated with miR-545/374a expression in HCC tissue (r=0.294, p=0.017; r=0.365, p=0.002; Pearson’s correlation). In addition, the expression of miR-545 and miR-374a both showed a statistically significant distinction between sera of HCC patients and sera of negative control patients (p=0.030, p=0.000, independent t test). These data demonstrated that detection of miR-545/374a in serum may indicate HBV-related HCC occurrence.2. The changing tendency of sera miR-545/374a expression before and after the tumor resection.To determine the levels of miR-545/374a in sera following surgical excision of tumor tissue, we collected 4 patients’sera repeatedly at different time points after surgery. The miR-545/374a levels were determined by quantitative RT-PCR. The dynamic tendency of individual microRNA levels in the sera samples were recorded as line charts. The sera miR-545 levels show a significant decrease at about 25 days after surgery, while the sera miR-374a levels decrease at approximately 15 days after surgery. Based on these data, we collected the postoperative sera more than 25 days after surgery. The miR-545/374a levels in the sera samples were determined by quantitative RT-PCR. Next, a paired t test was used to compare the microRNA levels between preoperative sera and postoperative sera. The results showed that the miR-545 concentration decreased by 47.3%(p=0.001), while miR-374a similarly decreased by 45.8%(p=0.042), which further confirmed that serum miR-545/374a is tumor-derived. As expected, nearly all patients show a reduction in miR-545/374a expression following surgery.3.Correlation analysis of preoperative serum miR-545 and miR-374a expression with prognosis/diagnosis related clinical features.The serum levels of miR-545 and miR-374a were examined by quantitative RT-PCR of samples obtained from 66 HBV-HCC subjects and were correlated with prognosis/diagnosis related clinical features. Even though miR-545 and miR-374a in each histological grade show significant differences compared to the negative control, there were no statistically significant differences between any two histological grades. The miR-545 and miR-374a showed no significant differences between patients with or without distal metastasis. Next,3 previously established clinical features forecasting HCC recurrence (AFP, CEA, and γ-GT) were analyzed along with serum miR-545/374a expression. AFP positive patients (≥20ng/ml) had a significantly higher miR-545/374a level than AFP negative patients (p=0.012, p=0.009, independent t test). However, no significant differences in the levels of miR-545/374a were observed for patients expressing CEA or γ-GT.Conclusion1.In HBV-related HCC patients, the sera miR-545/374a expression is significantly unregulated, and their expression is coincident with expression in tissue, indicating the sera miR-545/374a expression has potential value in diagnosing HBV-HCC tumorigenesis.2. After the tumor section surgery, miR-545/374a expression in serum is decreased, showing miR-545/374a are tumor derived.3. The expression of miR-545/374a is higher in AFP positive patients than negative patients, but have no relationship with other prognosis/diagnosis related clinical features.