Alpha-fetoprotein mRNA Research Articles

AUF1 promotes hepatocellular carcinoma progression and chemo-resistance by post-transcriptionally upregulating alpha-fetoprotein expression.

The target genes of AU-rich Element RNA-binding Protein 1 (AUF1), which is an RNA binding protein, and its role in the progression of hepatocellular carcinoma (HCC) is still elusive. This study aims to investigate the biological function and the underlying target genes of AUF1 in HCC. RNA sequencing data and the Liver Cancer Institute (LCI) database were used to screen candidate targets of AUF1. LCI database, TCGA database, and a retrospective HCC cohort were used to investigate the correlation between AUF1 and alpha-fetoprotein (AFP) and their prognostic values in HCC patients. Huh-7, HepG2, and HepAD38 cell lines were used to investigate the underlying mechanism of AUF1 regulating the AFP expression. Cell Counting Kit-8, colony formation, EdU incorporation, and flow cytometry assays were performed to detect the effect of AUF1-AFP axis on the progression and doxorubicin resistance of HCC cells. A combined analysis of the transcriptome data from Huh-7 cells after knockdown of AUF1 and gene expression data from LCI database revealed that AFP was the most significantly downregulated gene after AUF1 depletion. AUF1 expression was positively associated with AFP expression in HCC tissues and the high expression of both AUF1 and AFP were correlated with a worse prognosis in HCC patients of LCI and TCGA databases, as well as our retrospective cohort. Mechanistically, AUF1 bound to the 3' untranslation region (UTR) of AFP mRNA to enhance the mRNA stability of AFP, thereby upregulating AFP. Functional tests showed that AFP knockdown inhibited tumor growth and doxorubicin resistance of HCC cells induced by AUF1. AFP may be an important target gene of AUF1. AUF1 promoted HCC progression and doxorubicin resistance by upregulating AFP expression via increasing its mRNA stability.

Xiaoaiping injection affects the invasion and metastasis of hepatocellular carcinoma by controlling AFP expression

Abstract Objective Xiaoaiping (XAP) is a traditional Chinese medicine that is a commonly used as an anticancer drug in clinical practice owing to its high efficiency and low toxicity. Specifically, XAP can effectively inhibit the growth of hepatocellular carcinoma (HCC). Alpha-fetoprotein (AFP) is a key HCC diagnostic marker and is closely related to certain malignant cytological behaviors of HCC. However, whether AFP expression and XAP treatment are related to the invasion and metastasis of HCC remains unclear. In the present study, we aimed to evaluate the effects and underlying mechanism of XAP on the invasion and metastasis of HCC.. Methods Using a cell scratch assay, Transwell technology, and western blotting we detected the different invasion and metastatic abilities of Hep3B cells in XAP treatment and blank control groups. This allowed comparison of the invasion and metastatic abilities of Hep3B cells with differing levels of AFP expression. AFP mRNA sequencing technology was used to analyze the mechanism of tumor invasion and metastasis associated with AFP and XAP treatment. Results Cell invasion and metastasis abilities in the XAP group were significantly lower than those in the control group (P < 0.05). Additionally, compared to the control group, the expression of AFP significantly decreased after XAP treatment (P < 0.05). The ability of Hep3B cells to invade and metastasize was promoted when AFP expression was up-regulated, whereas it was inhibited when AFP was silenced. XAP injection and AFP regulate the invasion and metastatic ability of HCC by affecting matrix metalloproteinases (MMPs). Conclusion XAP injection inhibits the invasion and metastatic ability of HCC by influencing the expression of AFP; additionally, this inhibition of AFP is achieved by affecting MMPs.

Induction of peripheral blood mononuclear cells to hepatocyte-like cells and preliminary study of cell response to injury under the effect of acetaminophen

Objective: To establish a method for the induction of peripheral blood mononuclear cells to hepatocyte-like cells, and preliminarily investigate cell response to injury under the effect of acetaminophen (APAP). Methods: The surface marker CD45 of peripheral blood mononuclear cells wase detected cells by using flow cytometry and immunofluorescence methods. The cellular morphology of induced hepatocyte-like cells was observed under an inverted microscope. Real-time fluorescent quantitative PCR (RT-PCR) was used to detect the expression level of hepatocyte-specific genes, such as cytochrome (CY) P1A2, CYP3A4, CYP2C9, albumin (ALB), alpha-fetoprotein (AFP), and hepatocyte nuclear factor (HNF)4α mRNA. Immunofluorescence method was used to detect intracellular hepatocyte markers AFP, HNF4α, and ALB expression at the protein level. Biochemical analyzer was used to detect hepatocyte-specific secretory functions of AFP, ALB, and urea. Luciferase chemiluminescence method was used to detect the activity of key drug metabolizing enzyme CYP3A4. Colorimetric assay was used to detect the effect of the drug acetaminophen on hepatocyte-like cells, and alanine aminotransferase (ALT) was used as an indicator of liver cell injury. The statistical differences between the data were compared with t-test and rank-sum test. Results: The positive expression rate of CD45 cell surface markers isolated from peripheral blood mononuclear cells was about 98%, and hepatocyte-like cell morphology changes appeared on 15th day of induction. Compared with isolated mononuclear cells, CYP1A2, CYP3A4, CYP2C9, ALB, AFP and HNF4α mRNA was markedly elevated. The expression level of AFP, ALB and HNF4α protein were equally increased, and the secretory function of AFP, ALB and urea were enhanced. Compared with primary hepatocytes, CYP1A2, CYP2C9, AFP, HNF4α mRNA, and CYP3A4 mRNA did not decrease. The expression levels of AFP, ALB, and HNF4α proteins in the cells did not decrease, and the secretory function of AFP, ALB, and urea did not decrease. In addition, the CYP3A4 enzyme activity produced by hepatocyte-like cells was similar to that of primary hepatocytes. Compared with hepatocyte-like cells incubated without APAP, hepatocyte-like cells incubated with APAP had higher ALT level. Under the effect of APAP, the ALT level of hepatocyte-like cells was higher than isolated mononuclear cells. Conclusion: Peripheral blood mononuclear cells can be induced into hepatocyte-like cells with partial characteristics of hepatocytes, including the activity of CYP3A4, a key enzyme of hepatocyte drug metabolism. Additionally, preliminarily ALT secretory features reflect the hepatocytes injury under the effect of acetaminophen.

Relationship between Protein kinase C isoforms, Telomerase and Alpha-fetoprotein through PI3K/AKT/mTOR pathway in Hepatocellular carcinoma

Protein kinase C (PKC) family has been an alluring objective for new cancer drug discovery. It has been reported to regulate telomerase in several cancer types. Our team had previously used telomerase to elucidate alpha-fetoprotein (AFP) modulation in hepatocellular carcinoma (HCC). The aim of this study was to investigate the interrelationships among PKC isoforms, telomerase and AFP in HCC. PKCα and PKCδ were the most expressed isoforms in HepG2/C3A, PLC/PRF/5, SNU-387 and SKOV-3 cells. Following the upregulation of AFP using pCMV3-AFP and the human telomerase reverse transcriptase (hTERT) using a construct expressing a wild-type hTERT, and after their inhibition with all-trans retinoic acid and hTERT siRNA each respectively, we found that the expression of PKCα, PKCβI, PKCβII and PKCδ was affected by the variation of AFP and hTERT mRNA levels. An increase in AFP expression and secretion was observed after gene silencing of PKCα, PKCβ, PKCδ, and PKCε in HepG2/C3A. A similar pattern was observed in transfected PLC/PRF/5 cells, however PKCδ isoform silencing decreased AFP expression. Furthermore, telomerase activity was quantified using quantitative telomeric repeat amplification protocol. The variations in hTERT expression and telomerase activity were similar to those of AFP. Further investigation showed that PKC isoforms regulate AFP and hTERT expression levels through PI3K/AKT/mTOR pathway in HepG2/C3A and PLC/PRF/5 cells. Thus, these results show for the first time a possible interrelationship that links PKC isoforms to both AFP and hTERT via PI3K/AKT/mTOR pathway in HCC.

Antioxidant and anti-apoptotic prophylactic effect of silymarin against lead-induced hepatorenal toxicity in rats.

This study assessed prophylactic potentials of silymarin against lead-induced hepatorenal toxicity in rats with the respect to its antioxidant and anti-apoptotic activities. Forty male albino rats were distributed into four groups. Control group is provided with distilled water. Lead acetate group was given lead acetate (100 mg/kg bwt) orally for 10 weeks. The third and fourth groups administered silymarin at doses of 50 or 100 mg/kg bwt, respectively, 1 h before administration of lead acetate for 10 weeks. Lead acetate altered liver structure and function that represented by significant elevation of the activities of serum aspartate and alanine aminotransferases and serum levels of urea and creatinine. Hepatic and renal tissues' malondialdehyde concentrations were increased, while reduced glutathione content and superoxide dismutase and catalase activities were reduced in the lead acetate group. Also, lead acetate increased caspase-3 mRNA expression and inhibited alpha-fetoprotein mRNA expression in hepatic tissues, as well as it altered liver and kidney tissues' architectures. In contrast, silymarin ameliorated in a dose dependent mannarthe toxic effects of lead acetate on the liver and kidneys through modulation of lead acetate which altered liver and kidney function and structures via reducing lipid oxidation and pathological changes of hepatic and renal tissue structure, improving antioxidant defense system of liver and kidneys, and decreasing pro-apoptotic gene expression in hepatic tissue. This study indicated that silymarin ameliorated lead acetate-induced hepatorenal toxicity via its antioxidant and cytoprotective potentials.

Alpha-fetoprotein/endoplasmic reticulum stress signaling mitigates injury in hepatoma cells.

Alpha-fetoprotein (AFP) and endoplasmic reticulum (ER) stress play multiple roles in hepatocellular carcinoma. Here, we analyzed the crosstalk between AFP and ER stress in human hepatoma cells. We induced ER stress in human hepatoma cell lines (HepG2 and SK-Hep1 cells) with thapsigargin (TG, an ER stress inducer), and mitigated ER stress with 4-phenylbutyrate acid (4-PBA, an ER stress inhibitor). AFP expression was knocked down by AFP short hairpin RNA and rescued by the pCI-AFP vector. AFP expression and ER stress were examined, and their roles in apoptosis, necroptosis, and proliferation were analyzed. TG significantly induced ER stress, apoptosis, necroptosis, and intracellular AFP protein levels, and reduced proliferation and AFP mRNA expression as well as supernatant AFP protein levels in HepG2 and SK-Hep1 cells. 4-PBA pretreatment partially reversed those changes in HepG2 cells. By contrast to AFP overexpression, knockdown of AFP significantly exacerbated TG-induced ER stress, apoptosis, and necroptosis, and decreased proliferation and the expression of activating transcription factor 6 alpha. In conclusion, ER stress causes the accumulation of AFP protein, which may be related to the reduction of AFP secretion. Accumulated AFP mitigates apoptosis and necroptosis and restores the proliferation of hepatoma cells by reducing ER stress.

Toll-like receptor 7 mRNA is reduced in hepatitis C-based liver cirrhosis and hepatocellular carcinoma, out-performs alpha-fetoprotein levels, and with age and serum aspartate aminotransferase is a new diagnostic index

ABSTRACT Background Hepatitis B and C viruses are leading causes of liver cirrhosis and hepatocellular carcinoma (HCC). Toll-like receptor 7 (TLR-7) has been implicated in the pathogenesis of HCC linked to hepatitis B. We hypothesised a role of leukocyte TLR-7 mRNA in hepatitis C related liver cirrhosis and HCC, using alpha-fetoprotein (AFP) and liver function tests as comparators. Methods We recruited 102 patients with HCV-related HCC, 97 with HCV-related liver cirrhosis and 60 healthy controls. Quantification of TLR-7 mRNA was performed using real-time PCR, AFP and routine LFTs by standard techniques. Results TLR-7 mRNA levels were significantly lower in HCC patients compared to cirrhotic patients and lower again in healthy controls (p < 0.001 for trend). In multivariate analysis, age, aspartate transaminase (AST), AFP, and TLR-7 mRNA were significant predictors of HCC. The ROCC/AUC for age, AST and TLR-7 mRNA were all between 0.64 and 0.78 (all P < 0.01), but for AFP was 0.57 (95% CI 0.48–0.65, P = 0.09). We derived an index score using age, AST and TLR-7 mRNA for the diagnosis of HCC. The ROCC/AUC for the index was superior to all three root indices in the prediction of HCC. The index linked significantly with the Tokyo and Vienna liver cancer staging systems, but not with those of the CLIP and Okuda systems, in distinguishing HCC from liver cirrhosis. Conclusion The combination of TLR-7 mRNA levels with age and AST improves the performance of TLR-7 in HCC diagnosis, out-performs alpha-fetoprotein and predicts early HCC.

Differential analysis of gene expression profiles in hepatocellular carcinoma patients with high and low levels of alpha-fetoprotein

Objective: To investigate the differential gene expression profiles of alpha-fetoprotein (AFP) high- and low-expressing hepatocellular carcinoma (HCC), and to provide a theoretical basis for the molecular mechanism and prognosis analysis of HCC. Methods: The transcriptome data and related clinical information from 368 HCC cases were obtained from the Cancer Gene Atlas (TCGA) public database. The samples were divided into AFP high expression (AFP(high)) group and low expression (AFP(low)) group according to the quartile of AFP mRNA expression, with 92 cases in each group. The differential gene analysis was carried out using the DEseq2 package in the R software. The functional and KEGG pathway enrichment analysis of the differential genes was performed using ClusterProfiler package. The protein-protein interaction network was constructed to screen hub genes using the String database and Cytoscape software. The single-sample GSEA analysis was performed to enrich and score signature gene sets using the GSVA package. And then RNAseq data and real-time quantitative polymerase chain reaction (RT-qPCR) were used for independent dataset validation and tissue validation. Results: The clinical analysis showed that high expression of AFP was significantly associated with poor pathological differentiation and ethnicity (P<0.05 for both). A total of 1 382 differential genes were obtained by bioinformatics analysis, of which 931 genes were up-regulated and 451 genes were down-regulated in AFP(high) group. GO enrichment analysis showed that the highly expressed genes were mainly correlated with the processes of appendage development, limb development, and skeletal system development, while lowly expressed genes were related to metabolic-related processes such as xenobiotic metabolism, steroid metabolism, and cellular response to xenobiotic stimuli. KEGG pathway enrichment analysis revealed that highly expressed genes were mainly involved in primary immunodeficiency, neuroactive ligand-receptor interaction, and cytokine-cytokine receptor interaction, while lowly expressed genes were mainly involved in retinol metabolism, chemical carcinogenesis, steroid hormone biosynthesis and other pathways. A prognostic related gene set that was consisted of AURKB, TTK, CENPA, UBE2C, HJURP, and KIF15 was identified. And the high expression of this gene set was related to the shorter recurrence-free survival and overall survival time in HCC patients, and its enrichment score was positively correlated with AFP expression (r=0.475, P<0.001). The validation results of RNAseq data were basically consistent with the TCGA data. The RT-qPCR results showed that AURKB, KIF15, and UBE2C were significantly overexpressed in HCC tissues with high AFP expression. Although the expression of AURKB, TTK, KIF15, and UBE2C was not related to recurrence-free survival and overall survival of HCC patients, there was a tendency that the patients with high AFP levels showed relatively shorter recurrence-free survival time and overall survival time. Conclusions: There is a large difference in gene expression profiles between AFP(high) and AFP(low) HCC. The prognostic signature may cooperate with AFP to promote the initiation and development of HCC. It also may explain the tumorigenesis in HCC with different AFP levels, and provide new clues for the prognosis of HCC.

TUG1 Is a Regulator of AFP and Serves as Prognostic Marker in Non-Hepatitis B Non-Hepatitis C Hepatocellular Carcinoma.

Thyroid hormone (T3) and its receptor (TR) are involved in cell metabolism and cancer progression. Hypothyroidism is associated with significantly elevated risk of hepatocellular carcinoma (HCC). Levels of the glycoprotein alpha-fetoprotein (AFP) are increased in the majority of patients with HCC and may be useful in diagnosis and follow-up. However, the relationship between T3/TR and AFP levels in HCC is currently unclear. The expression profiles of long non-coding RNAs (lncRNAs) were compared in microarrays of HepG2-TRα1 cells treated with/without T3 and HCC specimens. The effects of T3 on taurine upregulated gene 1 (TUG1) and AFP expression were validated using qRT-PCR. A correlation between TUG1 and AFP was confirmed via RNAi and clustered regularly interspaced short palindromic repeats (CRISPR) strategies. Finally, overall and recurrence-free survival rates were analyzed using the Kaplan–Meier method and confirmed in online datasets. T3/TR treatment reduced TUG1 expression in vitro, resulting in the downregulation of AFP mRNA. Knockdown of TUG1 suppressed cell cycle progression and soft agar colony formation and induced cellular senescence. Our data support the involvement of TUG1 in the T3/TR-mediated suppression of cell growth. AFP mRNA levels showed strong positive correlations with TUG1 and unfavorable prognosis in patients with non-hepatitis B/non-hepatitis C HCC (NBNC-HCC). T3/TR, TUG1, and AFP may potentially serve as effective prognostic markers for NBNC-HCC.

Aberrant AFP expression characterizes a subset of hepatocellular carcinoma with distinct gene expression patterns and inferior prognosis.

Background Serum tumor markers are ubiquitously used in the clinic for cancer screening. However, the mechanisms accounting for the elevated levels of the serum tumor markers remain to be explored.Methods We performed a pan-cancer analysis of serum alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and prostate-specific antigen (PSA). The relation between concentration of serum tumor markers and the expression of their coding genes was assessed. Then the expression of AFP and its genomic background in hepatocellular carcinoma (liver cancer) was studied.Results High expression of AFP mRNA was found mainly in liver cancer. In gastric cancer, breast cancer and lung cancer, high AFP mRNA expression was also discovered occasionally. In liver cancer patients, serum AFP levels correlated significantly with AFP mRNA expression in cancer tissues (r = 0.72, p = 1.6e-45). Whole transcriptome analysis revealed that serum AFP levels clearly separated liver cancer into two classes with distinct expression profiles according to PCA analysis. Gene co-expression analysis revealed that AFP expression was connected to a module enriched with genes accounting for cell cycle and cell proliferation regulation. In addition, high AFP expression was associated with the molecular classification of liver cancer, including iCluster (Chi-square: 16.86, P = 0.0002). Methylation analysis revealed de-methylation of AFP promoter occurred in some liver cancer tissues, which was significantly related to AFP mRNA expression. Survival analysis indicated high serum AFP levels was prognostic of poorer survival of the liver cancer patients (Log-rank test: p = 0.046). This was confirmed by an independent dataset in which liver cancer patients with high serum AFP also had poorer survival (Log-rank test: p = 0.024).Conclusion High expression of AFP defined a subtype of liver cancer with distinct gene expression profiles and clinical features. De-methylation of cytosine from CpG di-nucleotides in AFP promoter may be the cause of AFP re-expression in adult human liver cancer tissue.

Hepatitis B virus X protein upregulates alpha-fetoprotein to promote hepatocellular carcinoma by targeting miR-1236 and miR-329.

Hepatitis B virus (HBV) infection is the most common cause of hepatocellular carcinoma (HCC) worldwide, wherein the expression of alpha-fetoprotein (AFP) is reactivated to promote tumorgenesis. Hepatitis B virus X protein (HBx) protein encoded by the HBV virus X gene has been considered to be oncogenic and implicated in hepatocarcinogenesis. However, the relationship between HBx and abnormal AFP expression in HCC is yet to be fully understood. To explore the potential regulation of HBx on AFP re-expression in HCC, 97 HCC samples of different etiologies were analyzed, and extremely higher serum AFP levels were found in patients with HBsAg+ . Analyses of HBV-related HCC specimens showed that the expression of AFP was negatively correlated with the levels of miR-1236 and miR-329. Further analyses indicated that HBx promotes the expression of AFP by orchestrating the levels of miR-1236 and miR-329 both in vitro and in vivo. Specifically, miR-1236 and miR-329 bind to the potential target sequences in AFP mRNA 3'-untranslated region to suppress its expression. HBx transfection resulted in the significant decrement of these microRNAs and increment of AFP expression. Moreover, AFP promotes the proliferation of hepatoma cells and attenuates the proapoptotic effect of chemotherapy agents. These findings revealed a novel regulatory mechanism of HBx on the abnormal AFP expression in HCC, which may provide a therapeutic approach for combating HBV-related HCC by targeting the regulation of AFP expression.

Abstract 1810: LncCGU1 and AFP serve as important prognostic marker in non-hepatitis B non-hepatitis C hepatocellular carcinoma

Abstract Background: Thyroid hormone (T3) and its receptor (TR) are involved in cell metabolism and cancer progression. Hypothyroidism is a significantly elevated risk factor for hepatocellular carcinoma (HCC). Alpha-fetoprotein (AFP) is increased in the majority of patients with HCC and may be useful in the diagnosis and follow-up of cases of HCC. The relationship between T3/TR and AFP levels in HCC is still unclear. Design: We used microarrays to compare expression profiles of lncRNAs in HepG2-TRα1 cells treated with/without T3 and HCC specimens. The profiling results were validated using qRT-PCR. The overall survival and recurrence-free survival were analyzed using Kaplan-Meier method and further confirmed by online available datasets. Results: In the current study, long non-coding RNA CGU1 was downregulated by T3/TR in vitro. Notably, α-fetoprotein (AFP) mRNA levels were downregulated by T3/TR through repression of CGU1. Notably, AFP mRNA is strongly positively correlated with CGU1 and unfavorable prognosis in non-hepatitis B/non-hepatitis C HCC (NBNC-HCC) group patients. Conclusions: our data collectively suggest that T3/TR/ CGU1/AFP act as a potential prognostic marker in NBNC-HCC. Citation Format: Kwang-Huei Lin, Yang-Hsiang Lin, Yu-Chin Liu. LncCGU1 and AFP serve as important prognostic marker in non-hepatitis B non-hepatitis C hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1810.

PO-041 TNF pathway in metastatic colorectal cancer according to RAS status and implication of potassium channels

IntroductionDespite its role as a diagnostic and prognosis marker for Hepatocellular carcinoma (HCC), alpha fetoprotein (AFP) plays a key role in advancing tumorigenesis and tumour expansion. Telomerase, an enzyme elongating telomere length, is upregulated in 80% of cancers including HCC. Our team had elucidated a modulation of AFP by telomerase and protein kinase C (PKC). PKC family is formed of several isoforms, each with a specific cellular function and expression. The aim of this study is to investigate the interrelation between PKC isoforms, telomerase and AFP in HCC.Material and methodsPKC isoforms were quantified by RT-qPCR in two AFP secretory cell lines, HepG2/C3A and PLC/PRF/5 and two non-secretory AFP cell lines SNU-387 (hTERT+) and SKOV-3 (hTERT-). According to the results, the expression of four isoforms was suppressed by si-RNAs in HepG2/C3A and PLC cells. AFP and telomerase mRNA levels were quantified in transfected cells by q-PCR, and AFP secretion by ELISA. Toxicity and cell proliferation were assessed by WST-1. In order to examine the effect of the dual presence of AFP and hTERT on PKC isoforms, SNU-387 and SKOV-3 were transfected with AFP expression plasmid pCMV3-AFP, then PKC isoforms mRNA was assessed by qPCR.Results and discussionsFour PKC isoforms, alpha, beta, delta and epsilon exhibited the highest expression levels in all cells compared to the remaining isoforms. An increase in telomerase expression, AFP expression and secretion and cell proliferation reaching a maximum of two folds was observed after individual gene silencing of the four isoforms in HepG2/C3A cells; however, an inverse pattern was noticed when using PKC pan inhibitor Go6983. Similar results were observed in PLC cell line. The expression of the four isoforms increased in SNU-387 and SKOV-3 cells after 24 hour transfection. The effect persisted after 48 hour in SNU-387, contrary to SKOV-3 where the levels decreased returning to the controls expression levels.ConclusionTaken together, decreased individual PKC isoforms rise telomerase expression and AFP secretion. However, PKC isoforms overexpression requires the presence of hTERT in AFP secretory cells. Thus, these results show for the first time the possible inter relation linking PKC isoforms to both AFP and hTERT in HCC.

Alpha-Fetoprotein in Retina and Lens of the Human Eye at Early Stages of Prenatal Development

The presence of alpha-fetoprotein (AFP) was shown in the retina and lens of the human fetal eye at different stages of prenatal development. PCR analysis revealed AFP mRNA neither in the retina nor in the lens, whereas in the fetal liver (control) AFP mRNA was found to be expressed. The data obtained indicate that AFP is not synthesized in retinal and lens cells of the human fetal eye but is imported from elsewhere to be taken up by these cells. The presence of AFP in the retina and lens implies its involvement in early morphogenesis and differentiation of these ocular tissues during prenatal human development.

Effect of porcine small intestinal submucosa extracellular matrix in promoting vitality and functional gene expression of hepatocyte

To investigate the effect of porcine small intestinal submucosa extracellular matrix (PSISM) on the vitality and gene regulation of hepatocyte so as to lay the experimental foundation for the application of PSISM in liver tissue engineering. The experiment was divided into two parts: ① BRL cells were cultured with 50, 100, and 200 μg/mL PSISM-medium which were prepared by adding PSISM into the H-DMEM-medium containing 10%FBS in groups A1, B1, and C1, and simple H-DMEM-medium served as a control (group D1); ② BRL cells were seeded on 1%, 2%, and 3% PSISM hydrogel which were prepared by dissolving PSISM in sterile PBS solution containing 0.1 mol/L NaOH in groups A2, B2, and C2, and collagen type I gel served as a control (group D2). At 1, 3, and 5 days after culture, the morphology and survival of liver cells were detected by the Live/Dead fluorescent staining. The cell vitality was tested by cell counting kit-8 (CCK-8) assay. And the relative expressions of albumin (ALB), cytokeratin 18 (CK18), and alpha-fetoprotein (AFP) in hepatocytes were determined by real-time fluorescent quantitative PCR (RT-qPCR). The Live/Dead fluorescent staining showed the cells survived well in all groups. CCK-8 results displayed that the absorbance ( A) value of group C1 was significantly higher than that of group D1 at 5 days after culture with PSISM-medium, and there was no significant difference between groups at other time points ( P>0.05). After cultured with PSISM hydrogels, the A values of groups A2, B2, and C2 were significantly higher than those of group D2 at 3 and 5 days ( P<0.05), the A value of group A2 was significantly higher than that of groups B2 and C2 at 5 days ( P<0.05), but there was no significant difference between groups at other time points ( P>0.05). RT-qPCR showed that the relative expressions of ALB and CK18 mRNA significantly increased and the relative expression of AFP mRNA significantly decreased in groups A1, B1, and C1 when compared with group D1 ( P<0.05). The relative expression of CK18 mRNA in group C1 was significantly lower than that in groups A1 and B1 ( P<0.05). The relative expressions of ALB and CK18 mRNA were significantly higher and the relative expression of AFP mRNA was significantly lower in groups A2, B2, and C2 than group D2 ( P<0.05); the relative expression of CK18 mRNA in group A2 was significantly higher than that in group B2 ( P<0.05), and the relative expression of AFP mRNA in group A2 was significantly lower than that in group C2 ( P<0.05), but no significant difference was found between other groups ( P>0.05). PSISM has good compatibility with hepatocyte and can promote the vitality and functional gene expression of hepatocyte. PSISM is expected to be used as culture medium supplement or cell carrier for liver tissue engineering.

AFP anti‐sense transcripts in mouse liver and their potential role in gene regulation

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, ranking the sixth most common cancer and third most common cause of cancer mortality worldwide. We have been studying mouse liver gene regulation to better understand mechanisms by which changes in gene expression contribute to liver development, homeostasis and disease. We identified Zinc Fingers and Homeoboxes 2 (Zhx2) as a regulator of alpha‐fetoprotein (AFP), a plasma protein that is highly expressed in the fetal liver but is shut off after birth. AFP expression is elevated in regenerating adult liver and hepatocellular carcinoma (HCC) and has been used extensively as a diagnostic marker of liver cancer. Interestingly, all genes that are misregulated in the absence of Zhx2 are also misregulated in HCC. Thus, to better understand gene regulation during HCC, we have been studying of the mechanism by which Zhx2 regulates gene expression. While studying AFP mRNA regulation by Zhx2, we identified previously unannotated antisense transcripts (asAFP) that partially overlap the 3′ half of the mouse AFP gene. asAFP RNAs are ~5kb alternatively spliced, mainly cytoplasmic, transcripts containing 2–4 exons that are likely to be non‐coding RNAs. These antisense transcripts were also detected in mouse liver RNA‐seq data. The abundance of asAFP RNA inversely correlates with AFP mRNA levels during postnatal development. Normally, asAFP RNA levels are high and AFP mRNA levels are low in the adult mouse liver. However, in the absence of Zhx2, AFP mRNA levels are higher and asAFP RNA levels are reduced. When portions of the asAFP RNA are over‐expressed in a liver cell line, endogenous AFP mRNA was reduced, suggesting asAFP transcripts repress AFP mRNA expression in trans. We will also knock‐down asAFP RNA and predict that AFP mRNA levels will increase. My central hypothesis is that asAFP RNA contributes to the post‐transcriptional regulation of AFP mRNA through an RNA‐RNA interaction. We are designing experiments to test this hypothesis and to determine at what level the regulation may occur.Support or Funding InformationMCB‐1158234 from the National Science Foundation, R01‐DK59866 from the National Institutes of Health

Zhx2 and the Regulation of AFP Anti‐sense RNAs in Mouse Liver

Although the genomes of eukaryotes are mostly transcribed, only a very small percentage of the transcripts code for proteins. The majority of the transcripts do not have known functions. In recent years, growing evidence has suggested that these non‐coding transcripts may play important roles in gene regulation. In the past two years, our lab has identified and characterized long non‐coding RNAs that are transcribed from the anti‐sense strand of, and partially overlap with, the alpha‐fetoprotein (AFP) gene. AFP is highly expressed in fetal liver, but its expression decreases to a very low level after birth. In adult liver, AFP expression increases during regeneration due to liver damage or during the onset of hepatocellular carcinoma. Our study has shown that anti‐sense AFP (asAFP) transcripts are alternatively spliced and their abundance is inversely correlated with AFP mRNA during liver development. While these asAFP RNAs had not been annotated, RNA‐seq data from mouse liver confirmed and extended our characterization and ENCODE data for histone modifications and DNase I hypersensitivity show signals near the asAFP exons we have identified. Expression levels of AFP mRNA and asAFP RNA also are inversely correlated in the presence or absence of the Zinc fingers and homeoboxes 2 (Zhx2) gene. Zhx2, a putative transcription factor, was shown previously to contribute to the developmental repression of AFP; in the absence of Zhx2, AFP mRNA is high while asAFP RNA is low, compared to wild‐type. Transiently transfecting asAFP into a liver cell line causes a decrease in endogenous AFP, showing asAFP contributes to AFP regulation in trans. We have shown that the asAFP transcripts localize mainly in the poly(A)+ fraction of cytoplasmic RNA, but this does not identify where the asAFP RNA functions. To determine whether the asAFP RNA represses AFP mRNA in the nucleus or cytoplasm, we have been analyzing the abundance of these two RNAs in nuclear and cytoplasmic RNA from liver of Zhx2 wild‐type or knock‐out animals. To better understand the role of Zhx2 in modulating AFP and asAFP RNA expression, future studies will use chromatin IP to determine whether Zhx2 interacts directly with these genes. To identify which of the genes that are mis‐regulated in the absence of Zhx2 are direct targets, we plan to do Zhx2 ChIP‐seq experiments.Support or Funding InformationMCB‐1158234 from the National Science Foundation, R01‐DK59866 from the National Institute of Health

The Clinical Significance of Alpha-Fetoprotein mRNAs in Patients with Hepatocellular Carcinoma

Background/Aims: Alpha-fetoprotein (AFP) mRNA-expressing cells are candidates for circulating tumor cells in hepatocellular carcinoma. We analyzed portal vein blood, peripheral blood, and peritoneal lavage samples to detect the presence of AFP mRNA-expressing cells, and explored their relationship with metastasis. Methods: We measured the AFP mRNA expression in 112 sets of portal vein and peripheral blood samples and 61 peritoneal lavage samples that had been obtained during surgery. We estimated the change in the positive ratio of patients with AFP mRNA, the associated background factors, and the rate of recurrence. Results: The change in AFP mRNA positivity in the peripheral blood specimens was remarkable, while that in the portal vein blood and peritoneal lavage samples was similar during hepatectomy. Tumor location was the only factor associated with AFP mRNA positivity. The rate of recurrence was higher in the patients who were positive for AFP mRNA than in those who were negative 9-24 months after hepatectomy. During this limited period, the recurrence rate in the AFP mRNA-positive cases was significantly higher than that in the AFP mRNA-negative cases (p = 0.0472). Postoperative AFP mRNA positivity was not related to very early recurrence (0-9 months) or multicentric liver carcinogenesis (>24 months). Conclusion: AFP mRNA positivity in the peripheral blood was elevated after hepatectomy, and the elevation depended on the tumor location. AFP mRNA positivity might contribute to recurrence-free survival 9-24 months after hepatectomy.

Telomerase inhibition decreases alpha-fetoprotein expression and secretion by hepatocellular carcinoma cell lines: in vitro and in vivo study.

Alpha-fetoprotein (AFP) is a diagnostic marker for hepatocellular carcinoma (HCC). A direct relationship between poor prognosis and the concentration of serum AFP has been observed. Telomerase, an enzyme that stabilizes the telomere length, is expressed by 90% of HCC. The aim of this study was to investigate the effect of telomerase inhibition on AFP secretion and the involvement of the PI3K/Akt/mTOR signaling pathway. Proliferation and viability tests were performed using tetrazolium salt. Apoptosis was determined through the Annexin V assay using flow cytometry. The concentrations of AFP were measured using ELISA kits. The AFP mRNA expression was evaluated using RT-PCR, and cell migration was evaluated using a Boyden chamber assay. The in vivo effect of costunolide on AFP production was tested in NSG mice. Telomerase inhibition by costunolide and BIBR 1532 at 5 and 10 μM decreased AFP mRNA expression and protein secretion by HepG2/C3A cells. The same pattern was obtained with cells treated with hTERT siRNA. This treatment exhibited no apoptotic effect. The AFP mRNA expression and protein secretion by PLC/PRF/5 was decreased after treatment with BIBR1532 at 10 μM. In contrast, no effect was obtained for PLC/PRF/5 cells treated with costunolide at 5 or 10 μM. Inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP concentration. In contrast, the MAPK/ERK pathway appeared to not be involved in HepG2/C3A cells, whereas ERK inhibition decreased the AFP concentration in PLC/PRF/5 cells. Modulation of the AFP concentration was also obtained after the inhibition or activation of PKC. Costunolide (30 mg/kg) significantly decreased the AFP serum concentration of NSG mice bearing HepG2/C3A cells. Both the inhibition of telomerase and the inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP production of HepG2/C3A and PLC/PRF/5 cells, suggesting a relationship between telomerase and AFP expression through the PI3K/Akt/mTOR pathway

Effects of culturing media on hepatocytes differentiation using Volvox sphere as co-culturing vehicle

Volvox sphere is a unique design to mimic natural volvox consists of a large outer-sphere that contains smaller inner-spheres, which provide three-dimensional (3D) environment to culture cells. The purpose of this study is to co-culture mesenchymal stem cells (MSCs) and AML12 liver cells in Volvox spheres and to evaluate the effects of two media, DMEM and DMEM/F12 on the cultured cells. The results of this study shows that the 3D Volvox sphere can successfully be applied for co-culture of MSCs and AML12 liver cells, and the MSCs are able to differentiate into hepatocyte-like cells expressing hepatocyte-specific markers including albumin (ALB), alpha feto-protein (AFP) and cytokeratin 18 (CK18) mRNA expressions and producing CK18 and ALB proteins. Interestingly, the MSCs expressed higher ALB, AFP and CK18 mRNA expression at the initial 7-day culture by using DMEM, whereas, the MSCs expressed more mRNA expressions from 7-day to 14-day by the usage of DMEM/F12. The result demonstrated that DMEM and DMEM/F12 media could affect MSCs behaviors during a 14-day culture.