GOLPH3 promotes tumor malignancy via inhibition of ferroptosis by upregulating SLC7A11 in cholangiocarcinoma.

Golgi phosphoprotein 3 (GOLPH3) has been reported as an oncogene in various tumors; however, the role and function of GOLPH3 and its relevant molecular mechanism in cholangiocarcinoma (CCA) are unclear. Herein, GOLPH3 expression in CCA tissues was observed to be significantly higher than that in paired adjacent noncancerous tissues. Clinicopathological analysis showed that GOLPH3 expression correlated positively with the tumor-node-metastasis stage. In addition, GOLPH3 expression correlated inversely with the overall survival of patients with CCA. Multivariate analysis showed that GOLPH3 was an independent prognostic factor for patients with CCA. Transcriptome analysis (RNA sequencing) of GOLPH3 knockdown cells showed that the expression levels of nine ferroptosis-related genes were significantly changed, indicating the important biological function of GOLPH3 in ferroptosis in CCA cells. Furthermore, GOLPH3 knockdown could significantly promote Erastin-induced ferroptosis in vitro and suppress tumor growth in vivo. Overexpression of GOLPH3 had the opposite effect on this phenotype. Further studies revealed that GOLPH3 knockdown was significantly associated with a decrease in cysteine content, an accumulation of the lipid peroxidation product malondialdehyde, an increase in reactive oxygen species, and sensitized CCA cells to Erastin-induced ferroptosis. Moreover, changes in GOLPH3 expression were found to be consistent with the expression of light chain subunit solute carrier family 7 member 11 (SLC7A11). Thus, our study suggested that GOLPH3 functions as an oncoprotein in CCA and may suppress ferroptosis by facilitating SLC7A11 expression, suggesting that GOLPH3 could serve as a therapeutic target for CCA treatment.

A rare CHD5 haplotype and its interactions with environmental factors predicting hepatocellular carcinoma risk.

BACKGROUND: CHD5 is a conventional tumour-suppressing gene in many tumours. The aim of this study was to determine whether CHD5 variants contribute to the risk of hepatocellular carcinoma (HCC). METHODS: Gene variants were identified using next-generation sequencing targeted on referenced mutations followed by TaqMan genotyping in two case-control studies. RESULTS: We discovered a rare variant (haplotype AG) in CHD5 (rs12564469-rs9434711) that was markedly associated with the risk of HCC in a Chinese population. A logistical regression model and permutation test confirmed the association. Indeed, the association quality increased in a gene dose-dependent manner as the number of samples increased. In the stratified analysis, this haplotype risk effect was statistically significant in a subgroup of alcohol drinkers. The false-positive report probability and multifactor dimensionality reduction further supported the finding. CONCLUSIONS: Our results suggest that the rare CHD5 gene haplotype and alcohol intake contribute to the risk of HCC. Our findings can be valuable to researchers of cancer precision medicine looking to improve diagnosis and treatment of HCC.

miR-17-5p suppresses cell proliferation and invasion by targeting ETV1 in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is the malignancy with the worst outcome among all breast cancer subtypes. We reported that ETV1 is a significant oncogene in TNBC tumourigenesis. Consequently, investigating the critical regulatory microRNAs (miRNAs) of ETV1 may be beneficial for TNBC targeted therapy. METHODS: We performed in situ hybridization (ISH) and immunohistochemistry (IHC) to detect the location of miR-17-5p and ETV1 in TNBC patient samples, respectively. miR-17-5p expression in TNBC tissues and cell lines was assessed by quantitative real-time PCR (qRT-PCR). ETV1 expression was evaluated by qRT-PCR, western blotting and IHC. Cell Counting Kit-8 (CCK-8), colony formation, Transwell and wound closure assays were utilized to determine the TNBC cell proliferation and migration capabilities. In vivo tumour metastatic assays were performed in a zebra fish model. RESULTS: The abundance of miR-17-5p was significantly decreased in TNBC cell lines and clinical TNBC tissues. The miR-17-5p expression levels were closely correlated with tumour size (P < 0.05) and TNM stage (P < 0.05). By contrast, the expression of ETV1 was significantly up-regulated in TNBC cell lines and tissues. There is an inverse correlation between the expression status of miR-17-5p and ETV1 (r = -0.28, P = 3.88 × 10-3). Luciferase reporter assay confirmed that ETV1 was a direct target of miR-17-5p. Forced expression of miR-17-5p in MDA-MB-231 or BT549 cells significantly decreased ETV1 expression and suppressed cell proliferation, migration in vitro and tumour metastasis in vivo. However, rescuing the expression of ETV1 in the presence of miR-17-5p significantly recovered the cell phenotype. High miR-17-5p expression was associated with a significantly favourable prognosis, in either the ETV1-positive or ETV1-negative groups (log-rank test, P < 0.001; P < 0.001). Both univariate and multivariate analyses showed that miR-17-5p and ETV1 were independent risk factors in the prognosis of TNBC patient. CONCLUSIONS: Our data indicate that miR-17-5p acts as a tumour suppressor in TNBC by targeting ETV1, and a low-abundance of miR-17-5p may be involved in the pathogenesis of TNBC. These findings indicate that miR-17-5p may be a therapeutic target for TNBC.

MicroRNA-129-5p inhibits hepatocellular carcinoma cell metastasis and invasion via targeting ETS1.

MiR-129-5p is deregulated in various human cancers and has been associated with hepatocellular carcinoma (HCC) progression. However, the underlying mechanisms of miR-129-5p involvement in the development and progression of HCC and the effects of miR-129-5p deregulation on the clinical characteristics observed in HCC patients remain poorly understood. We therefore investigated the correlation between low miR-129-5p expression and vascular invasion, intrahepatic metastasis, and poor patient survival. Ectopic restoration of miR-129-5p expression in HCC cells suppressed cellular migration and invasion and the expression of v-ets erythroblastosis virus E26 oncogene homolog 1 (ETS1), while inhibition of endogenous miR-129-5p caused an increase in these parameters. We identified the ETS1 gene as a novel direct target of miR-129-5p. SiRNA-mediated ETS1 knockdown rescued the effects of anti-miR-129-5p inhibitor in HCC cell lines, while the effects of miR-129-5p overexpression were partially phenocopied in the knockdown model. In addition, miR-129-5p levels inversely correlated with those of ETS1 in HCC cells and tissues. Taken together, our findings indicate an important role for miR-129-5p in the molecular etiology of invasive HCC and suggest that miR-129-5p could have potential therapeutic applications in HCC.

MicroRNA-362 induces cell proliferation and apoptosis resistance in gastric cancer by activation of NF-κB signaling.

BACKGROUND: According to cancer-related microRNA (miRNA) expression microarray research available in public databases, miR-362 expression is elevated in gastric cancer. However, the expression and biological role of miR-362 in gastric progression remain unclear. METHODS: miR-362 expression levels in gastric cancer tissues and cell lines were determined using real-time PCR. The roles of miR-362, in promoting gastric cancer cell proliferation and apoptosis resistance, were assessed by different biological assays, such as colony assay, flow cytometry and TUNEL assay. The effect of miR-362 on NF-κB activation was investigated using the luciferase reporter assay, fluorescent immunostaining. RESULTS: MiR-362 overexpression induced cell proliferation, colony formation, and resistance to cisplatin-induced apoptosis in BGC-823 and SGC-7901 gastric cancer cells. MiR-362 increased NF-κB activity and relative mRNA expression of NF-κB-regulated genes, and induced nuclear translocation of p65. Expression of the tumor suppressor CYLD was inhibited by miR-362 in gastric cancer cells; miR-362 levels were inversely correlated with CYLD expression in gastric cancer tissue. MiR-362 downregulated CYLD expression by binding its 3' untranslated region. NF-κB activation was mechanistically associated with siRNA-mediated downregulation of CYLD. MiR-362 inhibitor reversed all the effects of miR-362. CONCLUSION: The results suggest that miR-362 plays an important role in repressing the tumor suppressor CYLD and present a novel mechanism of miRNA-mediated NF-κB activation in gastric cancer.

Overexpression of GOLPH3 is associated with poor prognosis and clinical progression in pancreatic ductal adenocarcinoma.

BACKGROUND: Golgi phosphoprotein 3 (GOLPH3) has been identified as an oncoprotein in various human cancers; however, its role in pancreatic ductal adenocarcinoma (PDAC) is unknown. We examined GOLPH3 expression levels and relationship with survival in patients with PDAC to establish the significance of GOLPH3 in the development and progression of PDAC. METHODS: Real-time qPCR and Western blotting were performed to analyze the expression levels of GOLPH3 mRNA and protein in paired PDAC tumor and adjacent non-tumor tissues. Immunohistochemistry was used to analyze the expression levels of GOLPH3 protein in paraffin-embedded tissues from 109 cases of PDAC. Univariate and multivariate analyses were performed to identify correlations between the immunohistochemical data for GOLPH3 expression and the clinicopathologic characteristics in PDAC. RESULTS: Expression levels of GOLPH3 mRNA and protein were upregulated in PDAC lesions compared to paired adjacent noncancerous tissues. Expression of GOLPH3 was significantly correlated with clinical stage (P = 0.006), T classification (P = 0.021), N classification (P = 0.049) and liver metastasis (P = 0.035). Patients with high GOLPH3 expression had shorter overall survival times compared to those with low GOLPH3 expression (P = 0.007). Multivariate analysis revealed that GOLPH3 overexpression was an independent prognostic factor in PDAC. CONCLUSIONS: Our findings suggest that GOLPH3 expression status may be a potential prognostic biomarker and therapeutic target in PCAC.

miR-145 suppresses cell invasion in hepatocellular carcinoma cells: miR-145 targets ADAM17.

AIM: miR-145 is a candidate tumor suppressor miRNA. However, it is unknown whether miR-145 is involved in the invasion of hepatocellular carcinoma (HCC). Therefore, we aimed to explore the effect and mechanism of miR-145 in the control of HCC cell invasion. METHODS: HCC cell invasion was evaluated by transwell assays after transfection with pre-miR-145 or anti-miR-145. A luciferase reporter assay was used to determine whether a disintegrin and metalloprotease 17 (ADAM17) were a target of miR-145. The levels of miR-145 and ADAM17 mRNA were detected by a real-time polymerase chain reaction assay, and the level of ADAM17 protein was measured by western blot analysis. Pearson's correlation test was used to assess the correlation between ADAM17 mRNA expression and miR-145 expression in 20 HCC tissue samples. RESULTS: miR-145 was significantly downregulated in HCC tissues and cell lines. The loss of miR-145 expression was associated with the tumor-node-metastasis stage, vascular invasion and intrahepatic metastasis. The overexpression of miR-145 was able to suppress tumor MHCC-97H cell invasion, whereas the knockdown of miR-145 expression induced SMMC-7721 cell invasion. We demonstrated that miR-145 bound directly to the 3'-untranslated region of ADAM17 and inhibited the expression of ADAM17. The knockdown of ADAM17 in SMMC-7721 cells could partially reverse the effects of anti-miR-145. miR-145 expression was inversely associated with ADAM17 expression in 20 HCC tissue specimens. CONCLUSION: Our findings indicate that miR-145 could inhibit HCC cell invasion by regulating the expression of ADAM17.

Identification and Functional Characterization of PI3K/Akt/mTOR Pathway-Related lncRNAs in Lung Adenocarcinoma: A Retrospective Study.

OBJECTIVE: This paper aimed to investigate the PI3K/Akt/mTOR signal-pathway regulator factor-related lncRNA signatures (PAM-SRFLncSigs), associated with regulators of the indicated signaling pathway in patients with lung adenocarcinoma (LUAD) undergoing immunotherapy. MATERIALS AND METHODS: In this retrospective study, we employed univariate Cox, multivariate Cox, and least absolute shrinkage and selection operator (LASSO) regression analyses to identify prognostically relevant long non-coding RNAs (lncRNAs), construct prognostic models, and perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Subsequently, immunoassay and chemotherapy drug screening were conducted. Finally, the prognostic model was validated using the Imvigor210 cohort, and tumor stem cells were analyzed. RESULTS: We identified seven prognosis-related lncRNAs (AC084757.3, AC010999.2, LINC02802, AC026979.2, AC024896.1, LINC00941 and LINC01312). We also developed prognostic models to predict survival in patients with LUAD. KEGG enrichment analysis confirmed association of LUAD with the PI3K/Akt/mTOR signaling pathway. In the analysis of immune function pathways, we discovered three good prognostic pathways (Cytolytic_activity, Inflammation-promoting, T_cell_co-inhibition) in LUAD. Additionally, we screened 73 oncology chemotherapy drugs using the "pRRophetic" algorithm. CONCLUSION: Identification of seven lncRNAs linked to regulators of the PI3K/Akt/mTOR signaling pathway provided valuable insights into predicting the prognosis of LUAD, understanding the immune microenvironment and optimizing immunotherapy strategies.

miR-124 inhibits cell proliferation in gastric cancer through down-regulation of SPHK1.

SPHK1 expression is elevated in gastric cancer and is associated with shorter survival times for patients. However, the molecular mechanism of SPHK1 up-regulation in gastric cancer remains unclear. In the present study, we report that miR-124 down-regulated SPHK1 expression by directly targeting its 3'-untranslated region (3'-UTR) and that miR-124 expression was inversely correlated with SPHK1 expression in gastric cancer samples. Furthermore, we demonstrated that, similar to the effect of silencing SPHK1, up-regulation of miR-124 markedly inhibited proliferation and tumourigenicity of gastric cancer cells both in vitro and in vivo. This was found to be mechanistically associated with induction of cyclin-dependent kinase inhibitors p21$^{{\rm Cip1}}$ and p27$^{{\rm Kip1}}$, enhancement of the transcriptional activity of FOXO1 and suppression of AKT activity. Moreover, we showed that the re-introduction of SPHK1 (without the 3'-UTR), but not with the 3'-UTR, could abrogate the miR-124-mediated induction of p21$^{{\rm Cip1}}$ and p27$^{{\rm Kip1}}$, as well as rescue the miR-124-induced proliferation inhibition. Together, these results suggest that miR-124 has an important role in the suppression of gastric cancer and presents a novel mechanism of miRNA-mediated SPHK1 expression in cancer cells.

Double-positive expression of high-mobility group box 1 and vascular endothelial growth factor C indicates a poorer prognosis in gastric cancer patients.

BACKGROUND: Although many studies have indicated that high-mobility group box 1 protein (HMGB1) is associated with oncogenesis and a worse prognosis, the prognostic value of HMGB1 in gastric cancer (GC) remains unclear. In the present work, we aimed to evaluate the role of HMGB1 in GC and examined whether aberrant expression of both HMGB1 and vascular endothelial growth factor C (VEGF-C) increased the malignant potential of GC. METHODS: A total of 166 GC patients and 32 normal subjects were enrolled. HMGB1 and VEGF-C expression was detected by tissue microarrays (TMAs) and immunohistochemical staining. The correlation between HMGB1 and VEGF-C expression and their relationships with clinicopathological GC variables were examined. Univariate and multivariate analyses were performed using the Cox proportional hazard model to predict the factors related to the patients' overall survival rates. RESULTS: HMGB1 and VEGF-C expression were observed in 81 (48.80%) and 88 (53.01%) tumors, respectively, significantly higher than the rates among the corresponding controls. In addition, HMGB1 and VEGF-C expression were positively correlated (R2 = 0.972). HMGB1 expression was also closely associated with tumor size, pT stage, nodal status, metastasis status, TNM stage, and poor prognosis. Multivariate survival analysis indicated that patients with HMGB1 and VEGF-C coexpression had the worst prognoses and survival rates (hazard ratio, 2.78; log rank P<0.001). CONCLUSIONS: HMGB1 is commonly expressed in GC. Combined evaluation of HMGB1 and VEGF-C may serve as a valuable independent prognostic factor for GC patients.

Exploring the Prognostic Significance and Immunotherapeutic Potential of Single-Cell Sequencing-Identified Long Noncoding RNA (LncRNA) in Patients With Non-small Cell Lung Cancer.

BACKGROUND: Single-cell RNA sequencing technology can provide insight into lung cancer. The purpose of this study was to analyze the relationship between long noncoding RNA (lncRNA) discovered by RNA sequencing and immunotherapy in patients with non-small cell lung cancer (NSCLC). METHODS: In this study, we utilized data from The Cancer Genome Atlas (TCGA) to extract gene expression data and prognostic information from patients with NSCLC. We employed univariate, least absolute shrinkage and selection operator (LASSO), multivariate Cox regression analyses to construct risk models, and Kaplan-Meier (KM) analysis to compare survival differences between high- and low-risk groups. To evaluate the accuracy of our risk model predictions, we utilized a nomogram, calibration curve, correlation index curve (C-index), and receiver operating characteristic (ROC). Additionally, we conducted Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to investigate the differential expression of lncRNA genes. We also used the tumor immune dysfunction and exclusivity (TIDE) algorithm and the R package "pRRophetic" to analyze the tumor microenvironment. Finally, we utilized stem cell indices based on mRNA expression-based stemness index (mRNAsi) expression to better assess patient prognosis. RESULTS: Our analysis identified a set of 28 lncRNAs with prognostic risk profiles in patients with lung adenocarcinoma. Notably, patients in the low-risk group exhibited significantly better overall survival (OS) compared to those in the high-risk group. Kaplan-Meier (KM) survival curves revealed that these prognostic risk markers accurately predicted survival outcomes in non-small cell lung cancer (NSCLC) patients. MerCK18 and myeloid-derived suppressor cells (MDSC) were strongly associated with immune escape and immunotherapy in high- and low-risk subgroups. In our investigation of potential chemotherapeutic agents for the treatment of NSCLC, we screened a total of 60 agents and found that PPM1D was more effective in the low-risk group. However, we did not observe a strong correlation between the stem cell index mRNAsi and OS. CONCLUSION: Our study highlights the close association between lncRNAs and prognostic risk profiles and the prognosis of patients with non-small cell lung cancer, offering a promising avenue for the clinical implementation of immunotherapy.

Primate-specific microRNA-637 inhibits tumorigenesis in hepatocellular carcinoma by disrupting signal transducer and activator of transcription 3 signaling.

UNLABELLED: MiR-637 (microRNA-637) is a primate-specific miRNA belonging to the small noncoding RNA family, which represses gene regulation at the post-transcriptional expression level. Although it was discovered approximately 5 years ago, its biomedical significance and regulatory mechanism remain obscure. Our preliminary data showed that miR-637 was significantly suppressed in four HCC cell lines and, also, in most of the hepatocellular carcinoma (HCC) specimens, thereby suggesting that miR-637 would be a tumor suppressor in HCC. Simultaneously, the enforced overexpression of miR-637 dramatically inhibited cell growth and induced the apoptosis of HCC cells. The transcription factor, signal transducer and activator of transcription 3 (Stat3), is constitutively activated in multiple tumors, and aberrant Stat3 activation is linked to the promotion of growth and desensitization of apoptosis. Our study showed that Stat3 tyrosine 705 phosphorylation and several Stat3-regulated antiapoptotic genes were down-regulated in miR-637 mimics-transfected and Lv-miR637-infected HCC cells. In addition, miR-637 overexpression negatively regulated Stat3 phosphorylation by suppressing autocrine leukemia inhibitory factor (LIF) expression and exogenous LIF-triggered Stat3 activation and rescued cell growth in these cells. A nude mice model also demonstrated the above-described results, which were obtained from the cell model. Furthermore, we found that LIF was highly expressed in a large proportion of HCC specimens, and its expression was inversely associated with miR-637 expression. CONCLUSION: Our data indicate that miR-637 acted as a tumor suppressor in HCC, and the suppressive effect was mediated, at least in part, by the disruption of Stat3 activation.

miR-338-3p suppresses invasion of liver cancer cell by targeting smoothened.

MicroRNAs are involved in human carcinogenesis and cancer progression. Our previous study has shown that loss of miR-338-3p expression is associated with clinical aggressiveness of hepatocellular carcinoma (HCC). However, the exact roles and mechanisms of miR-338-3p remain unknown in HCC. To determine whether and how miR-338-3p influences liver cancer cell invasion, we studied miR-338-3p in the liver cancer cell lines, and we found that miR-338-3p is down-regulated in treated cells. Forced expression of miR-338-3p in SK-HEP-1 cells suppressed cell migration and invasion, whereas inhibition of miR-338-3p in SMMC-7721 cells induced cell migration and invasion. Furthermore, smoothened (SMO) was identified as a direct target of miR-338-3p. Forced expression of miR-338-3p down-regulated SMO and matrix metalloproteinase (MMP)-9 expression, but inhibition of miR-338-3p up-regulated SMO and MMP9 expression. However, small interfering RNA targeted SMO reversed the effects induced by blockade of miR-338-3p. SMO and MMP9 were overexpressed and associated with invasion and metastasis in HCC tissues. These data indicate that miR-338-3p suppresses cell invasion by targeting the smoothened gene in liver cancer in vitro and miR-338-3p might be a novel potential strategy for liver cancer treatment.

Golgi Phosphoprotein 3 Promotes Colon Cancer Cell Metastasis Through STAT3 and Integrin α3 Pathways.

Background: Golgi phosphoprotein 3 (GOLPH3) overexpression was recently reported to be associated with a poor clinical outcome in patients with colorectal cancer (CRC). However, the underlying molecular mechanism through which GOLPH3 promotes CRC metastasis remains poorly understood. Methods: In vitro genetic ablation of GOLPH3 was performed using siRNA transfection, and a stably overexpressed GOLPH3 colon cancer cell line was constructed using the lentivirus system. Cell invasion and migration assays were conducted with or without Matrigel. Immunoblotting, qRT-PCR, immunofluorescence and immunohistochemistry were utilized to study the expression level of GOLPH3, ZEB1, integrin α3 and phosphorylation level of STAT3, AKT/mTOR and Raf/MEK/ERK pathways. Co-immunoprecipitation was used to investigate the interaction between GOLPH3 and p-STAT3 (Tyr705) or total STAT3. Results: Overexpression of GOLPH3 was found in CRC tissues and colon cancer cell lines. Knockdown of GOLPH3 using siRNAs significantly suppressed the invasion and migration of HCT116 and HCT8 cells. In contrast, the overexpression of GOLPH3 promoted the migratory and invasive ability of colon cancer cells. The phosphorylation level of STAT3 as well as the protein and mRNA levels of ZEB1 and integrin α3, were significantly decreased after GOLPH3 knockdown. Moreover, Integrin α3 expression was correlated with GOLPH3 expression in CRC tissues. Co-immunoprecipitation assay revealed that GOLPH3 interacted with pSTAT3 (Tyr705) and total STAT3. Our further experiments suggested that GOLPH3 facilitated IL-6 induced STAT3 activation and subsequently induced transcription of integrin α3 and ZEB1, which promoted the metastasis and progression of CRC. Conclusion: Our current work demonstrates that GOLPH3 facilitates STAT3 activation and regulates the expression of EMT transcription factor ZEB1 and Integrin α3 in colon cancer cells. These findings indicate that GOLPH3 plays a critical role in CRC metastasis and might be a new therapeutic target for CRC treatment.

A PDX model combined with CD-DST assay to evaluate the antitumor properties of KRpep-2d and oxaliplatin in KRAS (G12D) mutant colorectal cancer.

Patient-derived xenograft (PDX) models are more faithful in maintaining the characteristics of human tumors than cell lines and are widely used in drug development, although they have some disadvantages, including their relative low success rate, long turn-around time, and high costs. The collagen gel droplet embedded culture drug sensitivity test (CD-DST) has been used as an in-vitro drug sensitivity test for patients with cancer because of its high success rate of primary cell culture, high sensitivity, and good clinical relevance, but it is based on an in-vitro cell culture and may not simulate the tumor microenvironment accurately. This study aims to combine a PDX model with CD-DST to evaluate the efficiency of antitumor agents. KRpep-2d, a small peptide targeting KRAS (G12D), and oxaliplatin were used to verify the feasibility of this approach. Whole-exome sequencing and Sanger sequencing were first applied to test and validate the KRAS mutation status of a panel of colorectal cancer PDX tissues. One PDX model was verified to carry KRAS (G12D) mutation and was used for in-vivo and the CD-DST drug tests. We then established the PDX mouse model from the patient with the KRAS (G12D) mutation and obtained viable cancer cells derived from the same PDX model. Next, the antitumor abilities of KRpep-2d and oxaliplatin were estimated in the PDX model and the CD-DST. We found that KRpep-2d showed no significant antitumor effect on the xenograft model or on cancer cells derived from the same PDX model. In contrast, oxaliplatin showed significant inhibitory effects in both tests. In conclusion, the PDX model in combination with the CD-DST assay is a comprehensive and feasible method of evaluating the antitumor properties of compounds and could be applied for new drug discovery.

Clinical significance of miR-221 and its inverse correlation with p27Kip¹ in hepatocellular carcinoma.

The aim of the present study is to explore possible role of miR-221 in the pathogenesis of HCC. Matched HCC and adjacent non-cancerous samples were assayed for the expression of miR-221 and three G1/S transition inhibitors: p27(Kip1), p21(WAF1/Cip1)and TGF-ß1 by in situ hybridization and immunohistochemistry respectively. p27(Kip1) is one of miR-221's proven targets. Real time qRT-PCR was used to investigate miR-221 and p27(Kip1) transcripts in different clinical stages. Western blotting was used to analyze the expression levels of p27(Kip1) protein in different clinical stages. In result, miR-221 and TGF-ß1 are frequently up-regulated in HCC, while p27(Kip1) and p21(WAF1/Cip1) proteins are frequently down-regulated. Moreover, miR-221 and p27(Kip1)'s expression correlated with metastasis and miR-221's expression also correlated with tumor size. Both of p21(WAF1/Cip1)and TGF-ß1's expression correlated with tumor differentiations. miR-221's upregulation and p27(Kip1)'s downregulation were significantly associated with tumor stages and metastasis. In conclusion, miR-221 is important in tumorigenesis of HCC, possibly by specifically down-regulating p27(Kip1), a cell-cycle inhibitor. These results indicate miR-221 as a new therapeutic target in HCC.

No association between the haplotypic block in the 3' UTR of GRP78 and risk of hepatocellular carcinoma.

BACKGROUND/AIMS: Glucose-regulated protein 78 (GRP78), being a major chaperone, is implicated in the progression of hepatocellular carcinoma (HCC), and elevated GRP78 levels in tissues had been known to be related with poor prognosis in patients with HCC. In the present study, we investigated the possible association between the polymorphisms of haplotypic block in the 3' untranslated region (UTR) of GRP78 and HCC risk in a Han Chinese population. METHODOLOGY: DNA from 576 unrelated patients with HCC and 539 sex- and age-matched healthy controls was typed for rs16927997, rs1140763 and rs12009 in the GRP78 gene by TaqMan assays. Polymorphism distributions were computed by logistic regression to test the association of certain alleles, genotypes, haplotypes and diplotypes with HCC risk. RESULTS: Linkage disequilibrium (LD) analysis identified a total of 3 haplotypes and 6 diplotypes in this region. The distributions of allelotype, genotype, haplotype and diplotype in HCC patients were not significantly different from that in controls. CONCLUSION: The present study suggested that polymorphisms in the 3' UTR of GRP78 were not useful diagnostic markers to predict the HCC risk.

Involvement of PI3K/PTEN/AKT/mTOR pathway in invasion and metastasis in hepatocellular carcinoma: Association with MMP-9.

AIM: To investigate the status of Phosphatidylinositol 3-kinase (PI3K)/PTEN/AKT/mammalian target of rapamycin (mTOR) pathway and its correlation with clinicopathological features and matrix metalloproteinase-2, -9 (MMP-2, 9) in human hepatocellular carcinoma (HCC). METHODS: PTEN, Phosphorylated AKT (p-AKT), Phosphorylated mTOR (p-mTOR), MMP-2, MMP-9 and Ki-67 expression levels were evaluated by immunohistochemistry on tissue microarrays containing 200 HCCs with paired adjacent non-cancerous liver tissues. PTEN, MMP-2 and MMP-9 mRNA levels were determined by real-time RT-PCR in 36 HCCs. The relationships between PI3K/PTEN/AKT/mTOR pathway and clinicopathological factors and MMP-2, 9 were analyzed in HCC. RESULTS: In HCC, PTEN loss and overexpression of p-AKT and p-mTOR were associated with tumor grade, intrahepatic metastasis, vascular invasion, TNM stage and high Ki-67 labeling index (P < 0.05). PTEN loss was correlated with p-AKT, p-mTOR and MMP-9 overexpression. Furthermore, PTEN and MMP-2, 9 mRNA levels were down-regulated and up-regulated in HCC compared with paired non-cancerous liver tissues, respectively (P < 0.01). PTEN, MMP-2 and MMP-9 mRNA levels were correlated with tumor stage and metastasis. There was an inverse correlation between PTEN and MMP-9 mRNA expression. However, PI3K/PTEN/AKT/mTOR pathway was not correlated with MMP-2. CONCLUSIONS: PI3K/PTEN/AKT/mTOR pathway, which is activated in HCC, is involved in invasion and metastasis through up-regulating MMP-9 in HCC.

Bead-based microarray analysis of microRNA expression in hepatocellular carcinoma: miR-338 is downregulated.

AIM: Recent studies have underlined causative links between microRNA (miRNA) deregulation and cancer development. However, the relevance of abnormally expressed miRNA to tumor biology has not been well understood in hepatocellular carcinoma (HCC). METHODS: A bead-based miRNA expression profiling method was performed on 20 pairs of surgically removed HCC and adjacent non-tumorous tissue (NT). Special miR-338 downregulations and miR-338 associated with clinical characteristics was validated in an extended samples set of 36 paired HCC and adjacent non-tumorous liver tissues by real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. RESULTS: Out of our bead-based microarray data, 12 upregulated and 19 downregulated miRNA were found to be associated with HCC. Further characterization of miRNA-338, in which 20 pairs of the samples were clustered clearly into two groups according to expression of miR-338, revealed that the level of miR-338 expression can be associated with clinical aggressiveness, such as, tumor size, tumor-node-metastasis stage, vascular invasion and intrahepatic metastasis. These results were validated by real-time RT-PCR analysis. CONCLUSION: Our study suggests that miRNA expression could have relevance to the clinical behavior of HCC and that the bead-based miRNA expression profiling method might be a suitable system to assay miRNA expression in large-scale diagnostic trails.

Expression patterns and polymorphisms of PTCH in Chinese hepatocellular carcinoma patients.

Aberrant activation of Hedgehog signaling pathway leads to pathological consequences in a variety of human tumors. PTCH (PTCH1), the receptor of Hedgehog pathway, is reported to function as a gatekeeper in tumor formation. Here we report, by semi-quantitative RT-PCR, PTCH expression was found in 38 hepatocellular carcinoma (HCC) patients (66%). Evidences from real time quantitative RT-PCR further indicate that compared to their matched nontumorous liver tissue, PTCH exhibit a higher expression in well and moderate differentiated tumor, but a lower expression in poorly differentiated tumor. Immunohistochemical staining showed PTCH protein was detected in the cytoplasm of 56.3% HCC samples (9/16). For the first time, we investigate the polymorphisms of PTCH in HCC. First we sequenced the recognized mutation hot spots regions of PTCH of 38 HCC samples. Two previously reported single nucleotide polymorphisms (SNPs) and a novel SNP A1056G were identified. Then we examined these three SNPs in 171 HCC samples and 162 normal liver samples. However, statistic analysis showed none of these SNPs was statistically significant for association with HCC. In conclusion, our data suggest that PTCH is involved in early stage tumor development and the Hh pathway in Chinese HCC is activated by ligand expression but not by mutation.