HCV infection associated hepatocellular carcinoma

Editor's Notes

• #3: Earlier reports of molecular markers of liver cancer in a mouse model were examined by ex vivo manipulation of progenitor cells followed by their transplantation into recipient mice While these studies initiated with progenitor cells harboring cancer-predisposing lesions identified valuable markers of liver cancer, they do not model HCV infection-associated HCC. To identify molecular markers of HCV infection-associated HCC, we compared expression levels of oncoproteins and tumor suppressor proteins from liver tissues of HCV-infected HCC with the HCV-infected but HCC-negative mice. Results suggest that loss of PTEN tumor suppressor protein is an early indicator of HCV infection-associated HCC. By contrast, induction of c-Myc and inflammatory response molecules, correlate with HCC progression. Micro-RNAs have been studied as independent markers of oncogenic progression [8]. In comparison with miRNAs reported from liver cancer of unspecified origin, our analysis suggests that induction of oncomiRs (miR-21, miR-221 and miR-141) and tumor suppressor miR-26a constitute signature miRNA markers of HCV infection-associated HCC. Overall, the results indicate that human hepatocyte engrafted MUP-uPA/SCID/Bg mice are a suitable small animal model for studying HCV-infected HCC and the role of tumor-promoting factors in liver cancer.
• #4: Twenty-five μL of reaction mixture containing 200 ng of genomic DNA extracted from a tail snip was subjected to the following conditions: 1) 92°C for 2 minutes; 2) 35 cycles of: 45 seconds at 92°C, 1 minute at 60°C, and 1 minute at 72°C; and 3minutes at 72°C for 5 minutes. An amplified product from the uPA transgene showed a 300 bp band on a 2% agarose gel. Primary human hepatocytes were transplanted immediately upon arrival within 12-16 hour after isolation. Viable cell counts were determined by trypan blue exclusion. Initially, mice were injected with less than 1 x 106 cells, but as we optimized the system, the number of cells was increased to 4-6 x 106 hepatocytes and suspended in 200 uL of normal saline solution (Vedco, St. Joseph, MO). An approximate 1 cm skin incision was made in the upper left quadrant of the mouse abdomen leaving the peritoneum intact. By moving the incision around over the peritoneal membrane, the spleen could be visualized. Using a 27-gauge needle, 200 uL of the cell suspension were injected directly into the spleen. The incision was then closed with a drop of Vetbond tissue adhesive Similarly, control mice were injected intra-splenically with 200 uL of normal saline solution. Human albumin measurement Small amounts of blood were collected weekly from the tail vein and the serum was collected after centrifugation. After 1,000 and 10,000 dilutions of the serum with Tris-buffered saline, human albumin concentrations were measured with the ELISA Quantitation Kit
• #5: Liver samples were stored at -80 °C until analysis. Liver tissues were lysed with RIPA buffer supplemented with protease inhibitor cocktail (Roche) as described [9]. Cell fractionation kit (Thermo Pierce) was used for the fractionation of nuclear and cytoplasmic proteins. HRP conjugated anti-rabbit or anti-goat antibodies (Abcam; SuperSignal West Dura Chemiluminescent Substrate) were visualized with BIO-RAD ChemiDoc™ XRS+ System. Estimates of relative protein levels were based on β-actin as internal (loading) control. Western blot images were quantified
• #6: Liver tissues from control animals HCC-negative mice (engrafted and HCV infected mice that did not develop HCC), and HCC positive mice (engrafted and HCV infected) were examined by Western blotting Panels (a) and (c) show representative Western blots of nuclear protein fraction (with Lamin B1 as loading control); and panel (b) is representative Western blot of corresponding cytoplasmic fraction (with GAPDH as loading control) observed a consistent decline of both nuclear (Fig. 1a and c) and cytoplasmic (Fig. 1b) PTEN protein in HCV-infected HCC. Interestingly, PTEN protein in HCV-infected but HCC-negative liver (N) also declined to similar extent (Fig. 1d), suggesting that loss of PTEN may be necessary but insufficient to promote HCC. Oncoproteins: Western blots of the control, HCC-negative and liver tumor tissues (as is shown in Fig. 1) were probed with antibodies against c-Myc, DLC-1 or p21 proteins (panels a, b and b). Panel (d) represents quantitative analysis (based on the loading controls) of liver tissues from uninfected control, HCC negative and HCC positive mice (as in Fig. 1) observed increased c-Myc protein levels in HCV-infected liver tumors compared to the control (Fig. 2a). By contrast, induction of c-Myc in HCC-negative liver was modest (Fig. 2d), suggesting that induction of c-Myc oncoprotein is a relatively late event in the development of HCV-infection associated HCC. We observed the loss of DLC1 protein in HCV-infected liver tumors and less so in HCV-infected but HCC-negative liver tissue ( it was important to determine if HCV infection of humanized mice modulated p53 to promote HCC. We assessed the modulation of p53 function in HCV-infected chimeric mice on the basis of p21 expression, a direct target of p53 transcriptional regulatory function. We observed a marked decline of p21 protein in HCV-infected liver tumor (Fig. 2c and d), and less so in HCC-negative mice (N, Fig. 2d), suggesting that HCC progression is correlated with the loss of function of p53 tumor suppressor. Persistent viral infection is an underlying cause of inflammation-induced cancer, including HCC. More than 90 % of HCCs arise in the context of hepatic injury and inflammation. Inflammation-associated oncogenic response is mediated by STAT proteins; in particular, activated STAT3 [27, 28]. To ascertain if HCV infection-associated HCC in humanized mice mimics the natural inflammatory response, we assayed activated STAT3 levels in the liver tumors and in HCC-negative as compared to the uninfected control mice. As shown (Fig. 3), there is a marked induction of activated (phosphorylated) STAT3 in HCV infection-associated liver tumors as compared to HCV-infected but HCC-negative liver. Quantitative assessment of β-Catenin, STAT-3 and IL-6R (from 7 uninfected control, 8 HCC negative and 7 HCC positive mice) was based on B-Actin internal controls analyzed by three independent SDS-PAGE runs MicroRNAs can function as tumor suppressors or oncogenes (oncomiRs) [32]. Altered expression levels of miRNAs have been reported in a number of human cancers [8, 32–34]. In this study we sought to identify miRNAs that would serve as distinguishing markers of HCV infection-associated HCC. MicroRNA 141 (miR-141) is induced in HCV-infected human primary hepatocytes. Importantly, miR-141 directly targets DLC1 tumor suppressor protein expression [10], attesting to its role as bona fide oncomiR. Here we compared expression levels of miR-141 along with other known oncomiRs (miR-21 and miR-221) in HCV infection-induced HCC (Fig. 4). Results suggest that expression of miR-141 and oncomiRs miR-21 and miR-221 that target cell cycle inhibitors [34, 35] is coordinately induced in HCV infection-associated HCC.
• #7: Liver tissues from control animals HCC-negative mice (engrafted and HCV infected mice that did not develop HCC), and HCC positive mice (engrafted and HCV infected) were examined by Western blotting Panels (a) and (c) show representative Western blots of nuclear protein fraction (with Lamin B1 as loading control); and panel (b) is representative Western blot of corresponding cytoplasmic fraction (with GAPDH as loading control) observed a consistent decline of both nuclear (Fig. 1a and c) and cytoplasmic (Fig. 1b) PTEN protein in HCV-infected HCC. Interestingly, PTEN protein in HCV-infected but HCC-negative liver (N) also declined to similar extent (Fig. 1d), suggesting that loss of PTEN may be necessary but insufficient to promote HCC. Oncoproteins: Western blots of the control, HCC-negative and liver tumor tissues (as is shown in Fig. 1) were probed with antibodies against c-Myc, DLC-1 or p21 proteins (panels a, b and b). Panel (d) represents quantitative analysis (based on the loading controls) of liver tissues from uninfected control, HCC negative and HCC positive mice (as in Fig. 1) observed increased c-Myc protein levels in HCV-infected liver tumors compared to the control (Fig. 2a). By contrast, induction of c-Myc in HCC-negative liver was modest (Fig. 2d), suggesting that induction of c-Myc oncoprotein is a relatively late event in the development of HCV-infection associated HCC. We observed the loss of DLC1 protein in HCV-infected liver tumors and less so in HCV-infected but HCC-negative liver tissue ( it was important to determine if HCV infection of humanized mice modulated p53 to promote HCC. We assessed the modulation of p53 function in HCV-infected chimeric mice on the basis of p21 expression, a direct target of p53 transcriptional regulatory function. We observed a marked decline of p21 protein in HCV-infected liver tumor (Fig. 2c and d), and less so in HCC-negative mice (N, Fig. 2d), suggesting that HCC progression is correlated with the loss of function of p53 tumor suppressor. Persistent viral infection is an underlying cause of inflammation-induced cancer, including HCC. More than 90 % of HCCs arise in the context of hepatic injury and inflammation. Inflammation-associated oncogenic response is mediated by STAT proteins; in particular, activated STAT3 [27, 28]. To ascertain if HCV infection-associated HCC in humanized mice mimics the natural inflammatory response, we assayed activated STAT3 levels in the liver tumors and in HCC-negative as compared to the uninfected control mice. As shown (Fig. 3), there is a marked induction of activated (phosphorylated) STAT3 in HCV infection-associated liver tumors as compared to HCV-infected but HCC-negative liver. Quantitative assessment of β-Catenin, STAT-3 and IL-6R (from 7 uninfected control, 8 HCC negative and 7 HCC positive mice) was based on B-Actin internal controls analyzed by three independent SDS-PAGE runs MicroRNAs can function as tumor suppressors or oncogenes (oncomiRs) [32]. Altered expression levels of miRNAs have been reported in a number of human cancers [8, 32–34]. In this study we sought to identify miRNAs that would serve as distinguishing markers of HCV infection-associated HCC. MicroRNA 141 (miR-141) is induced in HCV-infected human primary hepatocytes. Importantly, miR-141 directly targets DLC1 tumor suppressor protein expression [10], attesting to its role as bona fide oncomiR. Here we compared expression levels of miR-141 along with other known oncomiRs (miR-21 and miR-221) in HCV infection-induced HCC (Fig. 4). Results suggest that expression of miR-141 and oncomiRs miR-21 and miR-221 that target cell cycle inhibitors [34, 35] is coordinately induced in HCV infection-associated HCC.
• #8: Liver tissues from control animals HCC-negative mice (engrafted and HCV infected mice that did not develop HCC), and HCC positive mice (engrafted and HCV infected) were examined by Western blotting Panels (a) and (c) show representative Western blots of nuclear protein fraction (with Lamin B1 as loading control); and panel (b) is representative Western blot of corresponding cytoplasmic fraction (with GAPDH as loading control) observed a consistent decline of both nuclear (Fig. 1a and c) and cytoplasmic (Fig. 1b) PTEN protein in HCV-infected HCC. Interestingly, PTEN protein in HCV-infected but HCC-negative liver (N) also declined to similar extent (Fig. 1d), suggesting that loss of PTEN may be necessary but insufficient to promote HCC. Oncoproteins: Western blots of the control, HCC-negative and liver tumor tissues (as is shown in Fig. 1) were probed with antibodies against c-Myc, DLC-1 or p21 proteins (panels a, b and b). Panel (d) represents quantitative analysis (based on the loading controls) of liver tissues from uninfected control, HCC negative and HCC positive mice (as in Fig. 1) observed increased c-Myc protein levels in HCV-infected liver tumors compared to the control (Fig. 2a). By contrast, induction of c-Myc in HCC-negative liver was modest (Fig. 2d), suggesting that induction of c-Myc oncoprotein is a relatively late event in the development of HCV-infection associated HCC. We observed the loss of DLC1 protein in HCV-infected liver tumors and less so in HCV-infected but HCC-negative liver tissue ( it was important to determine if HCV infection of humanized mice modulated p53 to promote HCC. We assessed the modulation of p53 function in HCV-infected chimeric mice on the basis of p21 expression, a direct target of p53 transcriptional regulatory function. We observed a marked decline of p21 protein in HCV-infected liver tumor (Fig. 2c and d), and less so in HCC-negative mice (N, Fig. 2d), suggesting that HCC progression is correlated with the loss of function of p53 tumor suppressor. Persistent viral infection is an underlying cause of inflammation-induced cancer, including HCC. More than 90 % of HCCs arise in the context of hepatic injury and inflammation. Inflammation-associated oncogenic response is mediated by STAT proteins; in particular, activated STAT3 [27, 28]. To ascertain if HCV infection-associated HCC in humanized mice mimics the natural inflammatory response, we assayed activated STAT3 levels in the liver tumors and in HCC-negative as compared to the uninfected control mice. As shown (Fig. 3), there is a marked induction of activated (phosphorylated) STAT3 in HCV infection-associated liver tumors as compared to HCV-infected but HCC-negative liver. Quantitative assessment of β-Catenin, STAT-3 and IL-6R (from 7 uninfected control, 8 HCC negative and 7 HCC positive mice) was based on B-Actin internal controls analyzed by three independent SDS-PAGE runs MicroRNAs can function as tumor suppressors or oncogenes (oncomiRs) [32]. Altered expression levels of miRNAs have been reported in a number of human cancers [8, 32–34]. In this study we sought to identify miRNAs that would serve as distinguishing markers of HCV infection-associated HCC. MicroRNA 141 (miR-141) is induced in HCV-infected human primary hepatocytes. Importantly, miR-141 directly targets DLC1 tumor suppressor protein expression [10], attesting to its role as bona fide oncomiR. Here we compared expression levels of miR-141 along with other known oncomiRs (miR-21 and miR-221) in HCV infection-induced HCC (Fig. 4). Results suggest that expression of miR-141 and oncomiRs miR-21 and miR-221 that target cell cycle inhibitors [34, 35] is coordinately induced in HCV infection-associated HCC.
• #9: Liver tissues from control animals HCC-negative mice (engrafted and HCV infected mice that did not develop HCC), and HCC positive mice (engrafted and HCV infected) were examined by Western blotting Panels (a) and (c) show representative Western blots of nuclear protein fraction (with Lamin B1 as loading control); and panel (b) is representative Western blot of corresponding cytoplasmic fraction (with GAPDH as loading control) observed a consistent decline of both nuclear (Fig. 1a and c) and cytoplasmic (Fig. 1b) PTEN protein in HCV-infected HCC. Interestingly, PTEN protein in HCV-infected but HCC-negative liver (N) also declined to similar extent (Fig. 1d), suggesting that loss of PTEN may be necessary but insufficient to promote HCC. Oncoproteins: Western blots of the control, HCC-negative and liver tumor tissues (as is shown in Fig. 1) were probed with antibodies against c-Myc, DLC-1 or p21 proteins (panels a, b and b). Panel (d) represents quantitative analysis (based on the loading controls) of liver tissues from uninfected control, HCC negative and HCC positive mice (as in Fig. 1) observed increased c-Myc protein levels in HCV-infected liver tumors compared to the control (Fig. 2a). By contrast, induction of c-Myc in HCC-negative liver was modest (Fig. 2d), suggesting that induction of c-Myc oncoprotein is a relatively late event in the development of HCV-infection associated HCC. We observed the loss of DLC1 protein in HCV-infected liver tumors and less so in HCV-infected but HCC-negative liver tissue ( it was important to determine if HCV infection of humanized mice modulated p53 to promote HCC. We assessed the modulation of p53 function in HCV-infected chimeric mice on the basis of p21 expression, a direct target of p53 transcriptional regulatory function. We observed a marked decline of p21 protein in HCV-infected liver tumor (Fig. 2c and d), and less so in HCC-negative mice (N, Fig. 2d), suggesting that HCC progression is correlated with the loss of function of p53 tumor suppressor. Persistent viral infection is an underlying cause of inflammation-induced cancer, including HCC. More than 90 % of HCCs arise in the context of hepatic injury and inflammation. Inflammation-associated oncogenic response is mediated by STAT proteins; in particular, activated STAT3 [27, 28]. To ascertain if HCV infection-associated HCC in humanized mice mimics the natural inflammatory response, we assayed activated STAT3 levels in the liver tumors and in HCC-negative as compared to the uninfected control mice. As shown (Fig. 3), there is a marked induction of activated (phosphorylated) STAT3 in HCV infection-associated liver tumors as compared to HCV-infected but HCC-negative liver. Quantitative assessment of β-Catenin, STAT-3 and IL-6R (from 7 uninfected control, 8 HCC negative and 7 HCC positive mice) was based on B-Actin internal controls analyzed by three independent SDS-PAGE runs MicroRNAs can function as tumor suppressors or oncogenes (oncomiRs) [32]. Altered expression levels of miRNAs have been reported in a number of human cancers [8, 32–34]. In this study we sought to identify miRNAs that would serve as distinguishing markers of HCV infection-associated HCC. MicroRNA 141 (miR-141) is induced in HCV-infected human primary hepatocytes. Importantly, miR-141 directly targets DLC1 tumor suppressor protein expression [10], attesting to its role as bona fide oncomiR. Here we compared expression levels of miR-141 along with other known oncomiRs (miR-21 and miR-221) in HCV infection-induced HCC (Fig. 4). Results suggest that expression of miR-141 and oncomiRs miR-21 and miR-221 that target cell cycle inhibitors [34, 35] is coordinately induced in HCV infection-associated HCC.
• #10: Liver tissues from control animals HCC-negative mice (engrafted and HCV infected mice that did not develop HCC), and HCC positive mice (engrafted and HCV infected) were examined by Western blotting Panels (a) and (c) show representative Western blots of nuclear protein fraction (with Lamin B1 as loading control); and panel (b) is representative Western blot of corresponding cytoplasmic fraction (with GAPDH as loading control) observed a consistent decline of both nuclear (Fig. 1a and c) and cytoplasmic (Fig. 1b) PTEN protein in HCV-infected HCC. Interestingly, PTEN protein in HCV-infected but HCC-negative liver (N) also declined to similar extent (Fig. 1d), suggesting that loss of PTEN may be necessary but insufficient to promote HCC. Oncoproteins: Western blots of the control, HCC-negative and liver tumor tissues (as is shown in Fig. 1) were probed with antibodies against c-Myc, DLC-1 or p21 proteins (panels a, b and b). Panel (d) represents quantitative analysis (based on the loading controls) of liver tissues from uninfected control, HCC negative and HCC positive mice (as in Fig. 1) observed increased c-Myc protein levels in HCV-infected liver tumors compared to the control (Fig. 2a). By contrast, induction of c-Myc in HCC-negative liver was modest (Fig. 2d), suggesting that induction of c-Myc oncoprotein is a relatively late event in the development of HCV-infection associated HCC. We observed the loss of DLC1 protein in HCV-infected liver tumors and less so in HCV-infected but HCC-negative liver tissue ( it was important to determine if HCV infection of humanized mice modulated p53 to promote HCC. We assessed the modulation of p53 function in HCV-infected chimeric mice on the basis of p21 expression, a direct target of p53 transcriptional regulatory function. We observed a marked decline of p21 protein in HCV-infected liver tumor (Fig. 2c and d), and less so in HCC-negative mice (N, Fig. 2d), suggesting that HCC progression is correlated with the loss of function of p53 tumor suppressor. Persistent viral infection is an underlying cause of inflammation-induced cancer, including HCC. More than 90 % of HCCs arise in the context of hepatic injury and inflammation. Inflammation-associated oncogenic response is mediated by STAT proteins; in particular, activated STAT3 [27, 28]. To ascertain if HCV infection-associated HCC in humanized mice mimics the natural inflammatory response, we assayed activated STAT3 levels in the liver tumors and in HCC-negative as compared to the uninfected control mice. As shown (Fig. 3), there is a marked induction of activated (phosphorylated) STAT3 in HCV infection-associated liver tumors as compared to HCV-infected but HCC-negative liver. Quantitative assessment of β-Catenin, STAT-3 and IL-6R (from 7 uninfected control, 8 HCC negative and 7 HCC positive mice) was based on B-Actin internal controls analyzed by three independent SDS-PAGE runs MicroRNAs can function as tumor suppressors or oncogenes (oncomiRs) [32]. Altered expression levels of miRNAs have been reported in a number of human cancers [8, 32–34]. In this study we sought to identify miRNAs that would serve as distinguishing markers of HCV infection-associated HCC. MicroRNA 141 (miR-141) is induced in HCV-infected human primary hepatocytes. Importantly, miR-141 directly targets DLC1 tumor suppressor protein expression [10], attesting to its role as bona fide oncomiR. Here we compared expression levels of miR-141 along with other known oncomiRs (miR-21 and miR-221) in HCV infection-induced HCC (Fig. 4). Results suggest that expression of miR-141 and oncomiRs miR-21 and miR-221 that target cell cycle inhibitors [34, 35] is coordinately induced in HCV infection-associated HCC.
• #11: Liver tissues from control animals HCC-negative mice (engrafted and HCV infected mice that did not develop HCC), and HCC positive mice (engrafted and HCV infected) were examined by Western blotting Panels (a) and (c) show representative Western blots of nuclear protein fraction (with Lamin B1 as loading control); and panel (b) is representative Western blot of corresponding cytoplasmic fraction (with GAPDH as loading control) observed a consistent decline of both nuclear (Fig. 1a and c) and cytoplasmic (Fig. 1b) PTEN protein in HCV-infected HCC. Interestingly, PTEN protein in HCV-infected but HCC-negative liver (N) also declined to similar extent (Fig. 1d), suggesting that loss of PTEN may be necessary but insufficient to promote HCC. Oncoproteins: Western blots of the control, HCC-negative and liver tumor tissues (as is shown in Fig. 1) were probed with antibodies against c-Myc, DLC-1 or p21 proteins (panels a, b and b). Panel (d) represents quantitative analysis (based on the loading controls) of liver tissues from uninfected control, HCC negative and HCC positive mice (as in Fig. 1) observed increased c-Myc protein levels in HCV-infected liver tumors compared to the control (Fig. 2a). By contrast, induction of c-Myc in HCC-negative liver was modest (Fig. 2d), suggesting that induction of c-Myc oncoprotein is a relatively late event in the development of HCV-infection associated HCC. We observed the loss of DLC1 protein in HCV-infected liver tumors and less so in HCV-infected but HCC-negative liver tissue ( it was important to determine if HCV infection of humanized mice modulated p53 to promote HCC. We assessed the modulation of p53 function in HCV-infected chimeric mice on the basis of p21 expression, a direct target of p53 transcriptional regulatory function. We observed a marked decline of p21 protein in HCV-infected liver tumor (Fig. 2c and d), and less so in HCC-negative mice (N, Fig. 2d), suggesting that HCC progression is correlated with the loss of function of p53 tumor suppressor. Persistent viral infection is an underlying cause of inflammation-induced cancer, including HCC. More than 90 % of HCCs arise in the context of hepatic injury and inflammation. Inflammation-associated oncogenic response is mediated by STAT proteins; in particular, activated STAT3 [27, 28]. To ascertain if HCV infection-associated HCC in humanized mice mimics the natural inflammatory response, we assayed activated STAT3 levels in the liver tumors and in HCC-negative as compared to the uninfected control mice. As shown (Fig. 3), there is a marked induction of activated (phosphorylated) STAT3 in HCV infection-associated liver tumors as compared to HCV-infected but HCC-negative liver. Quantitative assessment of β-Catenin, STAT-3 and IL-6R (from 7 uninfected control, 8 HCC negative and 7 HCC positive mice) was based on B-Actin internal controls analyzed by three independent SDS-PAGE runs MicroRNAs can function as tumor suppressors or oncogenes (oncomiRs) [32]. Altered expression levels of miRNAs have been reported in a number of human cancers [8, 32–34]. In this study we sought to identify miRNAs that would serve as distinguishing markers of HCV infection-associated HCC. MicroRNA 141 (miR-141) is induced in HCV-infected human primary hepatocytes. Importantly, miR-141 directly targets DLC1 tumor suppressor protein expression [10], attesting to its role as bona fide oncomiR. Here we compared expression levels of miR-141 along with other known oncomiRs (miR-21 and miR-221) in HCV infection-induced HCC (Fig. 4). Results suggest that expression of miR-141 and oncomiRs miR-21 and miR-221 that target cell cycle inhibitors [34, 35] is coordinately induced in HCV infection-associated HCC.
• #12: Liver tissues from control animals HCC-negative mice (engrafted and HCV infected mice that did not develop HCC), and HCC positive mice (engrafted and HCV infected) were examined by Western blotting Panels (a) and (c) show representative Western blots of nuclear protein fraction (with Lamin B1 as loading control); and panel (b) is representative Western blot of corresponding cytoplasmic fraction (with GAPDH as loading control) observed a consistent decline of both nuclear (Fig. 1a and c) and cytoplasmic (Fig. 1b) PTEN protein in HCV-infected HCC. Interestingly, PTEN protein in HCV-infected but HCC-negative liver (N) also declined to similar extent (Fig. 1d), suggesting that loss of PTEN may be necessary but insufficient to promote HCC. Oncoproteins: Western blots of the control, HCC-negative and liver tumor tissues (as is shown in Fig. 1) were probed with antibodies against c-Myc, DLC-1 or p21 proteins (panels a, b and b). Panel (d) represents quantitative analysis (based on the loading controls) of liver tissues from uninfected control, HCC negative and HCC positive mice (as in Fig. 1) observed increased c-Myc protein levels in HCV-infected liver tumors compared to the control (Fig. 2a). By contrast, induction of c-Myc in HCC-negative liver was modest (Fig. 2d), suggesting that induction of c-Myc oncoprotein is a relatively late event in the development of HCV-infection associated HCC. We observed the loss of DLC1 protein in HCV-infected liver tumors and less so in HCV-infected but HCC-negative liver tissue ( it was important to determine if HCV infection of humanized mice modulated p53 to promote HCC. We assessed the modulation of p53 function in HCV-infected chimeric mice on the basis of p21 expression, a direct target of p53 transcriptional regulatory function. We observed a marked decline of p21 protein in HCV-infected liver tumor (Fig. 2c and d), and less so in HCC-negative mice (N, Fig. 2d), suggesting that HCC progression is correlated with the loss of function of p53 tumor suppressor. Persistent viral infection is an underlying cause of inflammation-induced cancer, including HCC. More than 90 % of HCCs arise in the context of hepatic injury and inflammation. Inflammation-associated oncogenic response is mediated by STAT proteins; in particular, activated STAT3 [27, 28]. To ascertain if HCV infection-associated HCC in humanized mice mimics the natural inflammatory response, we assayed activated STAT3 levels in the liver tumors and in HCC-negative as compared to the uninfected control mice. As shown (Fig. 3), there is a marked induction of activated (phosphorylated) STAT3 in HCV infection-associated liver tumors as compared to HCV-infected but HCC-negative liver. Quantitative assessment of β-Catenin, STAT-3 and IL-6R (from 7 uninfected control, 8 HCC negative and 7 HCC positive mice) was based on B-Actin internal controls analyzed by three independent SDS-PAGE runs MicroRNAs can function as tumor suppressors or oncogenes (oncomiRs) [32]. Altered expression levels of miRNAs have been reported in a number of human cancers [8, 32–34]. In this study we sought to identify miRNAs that would serve as distinguishing markers of HCV infection-associated HCC. MicroRNA 141 (miR-141) is induced in HCV-infected human primary hepatocytes. Importantly, miR-141 directly targets DLC1 tumor suppressor protein expression [10], attesting to its role as bona fide oncomiR. Here we compared expression levels of miR-141 along with other known oncomiRs (miR-21 and miR-221) in HCV infection-induced HCC (Fig. 4). Results suggest that expression of miR-141 and oncomiRs miR-21 and miR-221 that target cell cycle inhibitors [34, 35] is coordinately induced in HCV infection-associated HCC.
• #13: Liver tissues from control animals HCC-negative mice (engrafted and HCV infected mice that did not develop HCC), and HCC positive mice (engrafted and HCV infected) were examined by Western blotting Panels (a) and (c) show representative Western blots of nuclear protein fraction (with Lamin B1 as loading control); and panel (b) is representative Western blot of corresponding cytoplasmic fraction (with GAPDH as loading control) observed a consistent decline of both nuclear (Fig. 1a and c) and cytoplasmic (Fig. 1b) PTEN protein in HCV-infected HCC. Interestingly, PTEN protein in HCV-infected but HCC-negative liver (N) also declined to similar extent (Fig. 1d), suggesting that loss of PTEN may be necessary but insufficient to promote HCC. Oncoproteins: Western blots of the control, HCC-negative and liver tumor tissues (as is shown in Fig. 1) were probed with antibodies against c-Myc, DLC-1 or p21 proteins (panels a, b and b). Panel (d) represents quantitative analysis (based on the loading controls) of liver tissues from uninfected control, HCC negative and HCC positive mice (as in Fig. 1) observed increased c-Myc protein levels in HCV-infected liver tumors compared to the control (Fig. 2a). By contrast, induction of c-Myc in HCC-negative liver was modest (Fig. 2d), suggesting that induction of c-Myc oncoprotein is a relatively late event in the development of HCV-infection associated HCC. We observed the loss of DLC1 protein in HCV-infected liver tumors and less so in HCV-infected but HCC-negative liver tissue ( it was important to determine if HCV infection of humanized mice modulated p53 to promote HCC. We assessed the modulation of p53 function in HCV-infected chimeric mice on the basis of p21 expression, a direct target of p53 transcriptional regulatory function. We observed a marked decline of p21 protein in HCV-infected liver tumor (Fig. 2c and d), and less so in HCC-negative mice (N, Fig. 2d), suggesting that HCC progression is correlated with the loss of function of p53 tumor suppressor. Persistent viral infection is an underlying cause of inflammation-induced cancer, including HCC. More than 90 % of HCCs arise in the context of hepatic injury and inflammation. Inflammation-associated oncogenic response is mediated by STAT proteins; in particular, activated STAT3 [27, 28]. To ascertain if HCV infection-associated HCC in humanized mice mimics the natural inflammatory response, we assayed activated STAT3 levels in the liver tumors and in HCC-negative as compared to the uninfected control mice. As shown (Fig. 3), there is a marked induction of activated (phosphorylated) STAT3 in HCV infection-associated liver tumors as compared to HCV-infected but HCC-negative liver. Quantitative assessment of β-Catenin, STAT-3 and IL-6R (from 7 uninfected control, 8 HCC negative and 7 HCC positive mice) was based on B-Actin internal controls analyzed by three independent SDS-PAGE runs MicroRNAs can function as tumor suppressors or oncogenes (oncomiRs) [32]. Altered expression levels of miRNAs have been reported in a number of human cancers [8, 32–34]. In this study we sought to identify miRNAs that would serve as distinguishing markers of HCV infection-associated HCC. MicroRNA 141 (miR-141) is induced in HCV-infected human primary hepatocytes. Importantly, miR-141 directly targets DLC1 tumor suppressor protein expression [10], attesting to its role as bona fide oncomiR. Here we compared expression levels of miR-141 along with other known oncomiRs (miR-21 and miR-221) in HCV infection-induced HCC (Fig. 4). Results suggest that expression of miR-141 and oncomiRs miR-21 and miR-221 that target cell cycle inhibitors [34, 35] is coordinately induced in HCV infection-associated HCC.
• #14: While the loss of PTEN is important for the initiation of HCV infection-associated HCC, PTEN depletion by itself is insufficient for tumor progression. Induction cMyc oncoprotein is more pronounced in HCV-infected liver tumors as compared to HCC-negative liver (Fig. 2a and ​andd),d), suggesting that the induction of cMyc oncoprotein is a relatively late event in the development of hepatocellular carcinoma. By contrast, down-regulation of tumor suppressor proteins PTEN, DLC-1 and p21 appears to be initiated early in HCV infection-associated HCC.
• #15: While the loss of PTEN is important for the initiation of HCV infection-associated HCC, PTEN depletion by itself is insufficient for tumor progression. Induction cMyc oncoprotein is more pronounced in HCV-infected liver tumors as compared to HCC-negative liver (Fig. 2a and ​andd),d), suggesting that the induction of cMyc oncoprotein is a relatively late event in the development of hepatocellular carcinoma. By contrast, down-regulation of tumor suppressor proteins PTEN, DLC-1 and p21 appears to be initiated early in HCV infection-associated HCC.