MicroRNA-363-3p inhibits colorectal cancer progression by targeting interferon-induced transmembrane protein 1.

BACKGROUND: The molecular mechanisms of colorectal cancer development and progression are far from being elucidated. AIM: To investigate the role of microRNA-363-3p (miR-363-3p) in the progression of colorectal cancer. METHODS: Real-time polymerase chain reaction was performed to detect miRNA expression in human colorectal cancer tissues and paired normal colorectal tissues. PITA 6 was utilized to predict the targets of miR-363-3p. Dual-luciferase reporter system was used to validate the target of miR-363-3p. Plate colony formation assay and wound-healing assay were performed to evaluate cancer cells' clonogenic survival ability and migration ability, respectively. Cell proliferation was examined by cell counting kit-8 assay. Immunohistochemical staining was used to determine the expression level of interferon-induced transmembrane protein 1 (IFITM1) in colorectal cancer tissues and adjacent tissues. The TCGA and GTEx databases were used to compare the expression levels of IFITM1 mRNA in colorectal cancer tissues and normal colorectal tissues and analyze the correlation between the expression levels of IFITM1 mRNA and overall survival and disease-free survival of patients. A colorectal cancer cell line with a deficiency of IFITM1 was constructed, and the regulation effect of IFITM1 on the clonogenic growth of colorectal cancer cells was clarified. RESULTS: MiR-363-3p was decreased in colorectal cancer tissues compared to normal colorectal tissues. IFITM1 was characterized as a direct target of miR-363-3p. Overexpression of miR-363-3p led to decreased clonogenic survival, proliferation, and migration of colorectal cancer cells, which could be reversed by forced IFITM1 expression. CONCLUSION: MiR-363-3p can constrain clonogenic survival, proliferation, and migration of colorectal cancer cells via targeting IFITM1.

Hyperthermic intrathoracic/intraperitoneal chemotherapy versus conventional intrapleural/intraperitoneal chemotherapy for the malignant effusion: a multi-center randomized clinical trial.

OBJECTIVE: To compare the efficacy and safety of hyperthermic intrathoracic/intraperitoneal chemotherapy versus conventional intrapleural/intraperitoneal chemotherapy in the treatment of malignant pleural or peritoneal effusion. METHODS: A randomized clinical trial was carried out in 8 cancer centers across China. Patients with malignant pleural or peritoneal effusion were randomly assigned to the study group or control group. Patients in the study group were treated with cisplatin-based hyperthermic intrathoracic chemotherapy (HITHOC) or hyperthermic intraperitoneal chemotherapy (HIPEC), while the control group was treated with conventional intrapleural or intraperitoneal chemotherapy using same chemotherapeutic regime as the study group. The objective response rate (ORR) was analyzed as primary outcome. Quality-of-life (QOL) score was recorded as secondary outcome using the questionnaire 30 (QLQ-C30) of the European Organization for Research and Treatment of Cancer (EORTC). The efficacy and safety of the two treatments were compared. RESULTS: Total 135 patients were recruited and randomized in this study, with 67 patients in the study group and 68 patients in the control group. The ORR in the study group (80.70%) was significantly higher than that in the control group (31.03%, p < 0.001). However, neither changes of QOL scores, nor incidence rates of adverse events were significantly different between the two groups (p = 0.076 and 0.197, respectively). CONCLUSION: Efficacy of HITHOC or HIPEC is superior to that of conventional modality for the treatment of malignant effusion with comparable side effects.

Combining a CDK4/6 Inhibitor With Pemetrexed Inhibits Cell Proliferation and Metastasis in Human Lung Adenocarcinoma.

Background: Recent clinical trials of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) in human lung adenocarcinoma (LUAD) have not achieved satisfactory results. The disappointing results of single-drug treatments have prompted studies about synergistic therapies of CDK4/6i with other drugs. We aimed to test the anti-tumor effect of ribociclib (a CDK4/6i) combined with pemetrexed on LUAD and the potential mechanisms. Methods: Cell lines were exposed to ribociclib and pemetrexed at different doses. Antitumor effects were measured using growth inhibition. Cell cycle distribution and apoptosis were evaluated using flow cytometry. Cell migration and invasion were measured using wound healing and transwell invasion assays, respectively. The expression levels of proteins were analyzed using western blotting. Mice xenograft models were used for validation in vivo. Results: Synergism was associated with a combination of cell cycle effects from both agents. Cell cycle analysis revealed that pemetrexed blocked cells in the S phase, whereas ribociclib arrested cells in the G1 phase. Concomitant treatment with pemetrexed and ribociclib resulted in a significantly stronger antitumor ability than treatment alone. We also found that ribociclib strongly enhanced the pro-apoptotic activity of pemetrexed via the caspase/bcl-2 signaling pathway. In addition, we report for the first time that combination treatment with ribociclib and pemetrexed significantly inhibits the migration and invasion of LUAD cells. Conclusions: Combining ribociclib and pemetrexed showed a powerful ability to inhibit cancer proliferation, invasion, and metastasis, and it holds potential as a novel effective combinative therapy for patients with LUAD.

Active demethylation upregulates CD147 expression promoting non-small cell lung cancer invasion and metastasis.

Non-small cell lung cancer (NSCLC) is a fatal disease, and its metastatic process is poorly understood. Although aberrant methylation is involved in tumor progression, the mechanisms underlying dynamic DNA methylation remain to be elucidated. It is significant to study the molecular mechanism of NSCLC metastasis and identify new biomarkers for NSCLC early diagnosis. Here, we performed MeDIP-seq and hMeDIP-seq analyses to detect the genes regulated by dynamic DNA methylation. Comparison of the 5mC and 5hmC sites revealed that the CD147 gene underwent active demethylation in NSCLC tissues compared with normal tissues, and this demethylation upregulated CD147 expression. Significantly high levels of CD147 expression and low levels of promoter methylation were observed in NSCLC tissues. Then, we identified the CD147 promoter as a target of KLF6, MeCP2, and DNMT3A. Treatment of cells with TGF-ß triggered active demethylation involving loss of KLF6/MeCP2/DNMT3A and recruitment of Sp1, Tet1, TDG, and SMAD2/3 transcription complexes. A dCas9-SunTag-DNMAT3A-sgCD147-targeted methylation system was constructed to reverse CD147 expression. The targeted methylation system downregulated CD147 expression and inhibited NSCLC proliferation and metastasis in vitro and in vivo. Accordingly, we used cfDNA to detect the levels of CD147 methylation in NSCLC tissues and found that the CD147 methylation levels exhibited an inverse relationship with tumor size, lymphatic metastasis, and TNM stage. In conclusion, this study clarified the mechanism of active demethylation of CD147 and suggested that the targeted methylation of CD147 could inhibit NSCLC invasion and metastasis, providing a highly promising therapeutic target for NSCLC.

A novel method of bedside hyperthermic intraperitoneal chemotherapy as adjuvant therapy for stage-III gastric cancer.

OBJECTIVE: To investigate the efficacy and safety of a novel method of hyperthermic intraperitoneal chemotherapy (HIPEC) as adjuvant therapy for stage-III gastric cancer. METHODS: Patients with stage-III gastric cancer who underwent D2 radical gastrectomy were randomly assigned to the HIPEC or control group four weeks after surgery. The HIPEC group was treated with cisplatin (60 mg/m2) administered with a HIPEC device on days 1 and 3 (30 mg/m2 each time), along with oral S-1, 40-60 mg, twice daily, for 14 days. The control group was treated with cisplatin (60 mg/m2) administered intravenously plus oral S-1 (40-60 mg, 2/d for 14 days). The primary outcome of the study was disease-free survival (DFS). RESULTS: Total 114 patients were included in the study, with 57 patients in each group. The median DFS was 29.0 months in the HIPEC group, which was significantly longer than that in the control group (15.0 months, p = 0.006). The two-year DFS rate in the HIPEC group was higher than that in the control group (50.4% vs. 25.5%). Median OS was 42.0 month in the HIPEC group and 31.0 month in the control (p = 0.042). Peritoneal metastasis occurred in six patients in the HIPEC group (10.5%) and 12 patients in the control (21.1%, p = 0.198). No significant difference in the incidence of adverse event except for thrombocytopenia. CONCLUSION: HIPEC with cisplatin plus oral S-1 is a safe and effective adjuvant therapy for patients with advanced gastric cancer following D2 radical gastrectomy. Trial registration: This study was registered at ClinicalTrials.gov with the identifier (NCT number): NCT02396498.

MicroRNA-10b regulates epithelial-mesenchymal transition by modulating KLF4/KLF11/Smads in hepatocellular carcinoma.

BACKGROUND: Our previous work showed that miR-10b was overexpressed in hepatocellular carcinoma (HCC) and promoted HCC cell migration and invasion. Epithelial-mesenchymal transition (EMT) is involved in HCC metastasis. So, we suspected that miR-10b might participate in the HCC EMT. METHODS: We performed morphological analysis and immunofluorescence to observe the roles of miR-10b in HCC EMT. The expression of KLF11 and EMT markers were detected by real-time RT-PCR and western blot. The regulation roles of miR-10b on KLF11 and KLF4 were determined by luciferase reporter assay. The chromatin immunoprecipitation revealed the binding relationship between KLF4 and KLF11. RESULTS: We found that overexpression of miR-10b could promote HCC EMT. miR-10b could upregulated KLF11 expression. The upregulation of KLF11 reduced the downstream molecular Smad7 expression, which upregulated the Smad3 expression to promote EMT development. Furthermore, the induction role of miR-10b in HCC EMT could be blocked by KLF11 siRNA. But our results showed that there was no direct regulation of miR-10b in KLF11 expression. Specifically, miR-10b could bind to the 3'UTR of KLF4 and inhibit KLF4 expression. KLF4 could directly bind to KLF11 promoter and downregulate KLF11 transcription. CONCLUSION: Our results reveal that miR-10b downregulates KLF4, the inhibitory transcriptional factor of KLF11, which induces Smads signaling activity to promote HCC EMT. Our study presents the regulation mechanism of miR-10b in EMT through the KLF4/KLF11/Smads pathway for the first time and implicates miR-10b as a potential target for HCC therapies.

Interferon-α adjuvant therapy decreases the recurrence of early clear cell renal cell carcinoma and improves the prognosis of Chinese patients.

The survival time of patients with early clear cell renal cell carcinoma (ccRCC) is fairly long, but 20% to 30% of patients with localized tumors experience relapse, and the effect of IFN-α on survival has not been well studied in patients with early ccRCC. In this study, 208 patients with early ccRCC were treated with surgery, and 54 of the patients received IFN-α as adjuvant therapy. The remaining 115 patients were treated with surgery but not with IFN-α therapy. The primary endpoint was the recurrence rate, 20.37% (11/54) and 33.04% (38/115) in the IFN-α and surgery-only group, respectively. The secondary endpoint was progression-free survival (PFS), which was 123.70 (95% CI: 107.18-140.22) months for the IFN-α group, and 95.80 (95% CI: 82.18-109.42) months for the non-IFN-α group; this difference was significant (P < 0.05). The main side effects were pyrexia (61.11%), muscle pain (24.07%), malaise (9.26%), anorexia (5.56%), hepatic dysfunction (3.70%) and renal dysfunction (1.85%). Moreover, a multivariate regression identified older age, higher BMI index and smoking as significant and independent predictors of decreased PFS (P < 0.05). Overall, IFN-α therapy significantly improved PFS in Chinese patients with early ccRCC and was an independent prognostic factor (P < 0.05). In conclusion, our study showed that adjuvant IFN-α therapy decreased the recurrence rate and prolonged PFS in patients with ccRCC. Thus, this treatment may help clinicians to select a better treatment modality and better predict survival in these patients.

Interaction of KLF6 and Sp1 regulates basigin-2 expression mediated proliferation, invasion and metastasis in hepatocellular carcinoma.

Accumulating evidence suggests that the tumor suppressor gene Krüppel-like factor 6 (KLF6) plays important roles in both development and progression of cancer. However, the role of KLF6 in hepatocellular carcinoma (HCC) remains unclear. Cancer-related molecule basigin-2 plays an important role in HCC progression and metastasis. Sp1, one of Sp/KLFs family members, regulates basigin-2 expression in HCC. The involvement of KLFs in basigin-2 regulation and HCC progression and metastasis has not been investigated. We first measured KLF6 expression levels in 50 pairs of HCC and adjacent normal tissues (ANTs) by immunohistochemistry. Specifically, low KLF6 expression but high Sp1 and basigin-2 expression were found in HCC tissues. By contrast, the ANTs showed high KLF6 expression but low Sp1 and basigin-2 expression. Kaplan-Meier analysis showed that higher expression of KLF6 was associated with better overall survival. The survival rate of KLF6-negative patients was lower than that of KLF6-positive patients (P = 0.015). We also found that KLF6 binds to the basigin-2 and Sp1 promoters and decreases their expression. Thus, we identified a microcircuitry mechanism in which KLF6 can repress basigin-2 expression directly by binding to its promoter or indirectly by inhibiting the expression of the transcription factor Sp1 to block gene expression. Additionally, overexpression of KLF6 suppressed the invasion, metastasis and proliferation of HCC cells in vitro and in vivo by targeting basigin-2. Our study provides new evidence that interaction of KLF6 and Sp1 regulates basigin-2 expression in HCC and that KLF6 represses the invasive and metastatic capacities of HCC through basigin-2.

Basigin-2 upregulated by receptor activator of NF-κB ligand enhances lung cancer-induced osteolytic lesions.

BACKGROUND: Lung cancer bone metastasis causes poor prognosis. Basigin-2, a novel cancer-associated biomarker, is upregulated in lung cancer and has been linked with tumor progression. But little is known about the role of basigin-2 in lung cancer bone metastasis and osteolytic lesion. METHODS: Basigin-2 expression was evaluated in biopsy tissue specimens of 20 lung cancer patients with bone metastases via immunohistochemistry. Invasion assay and MTT proliferation assay were performed to test the invasion and proliferation of lung cancer cell after modulated basigin-2 expression. The osteoclastic activity of basigin-2 was detected in tibia cancer model by injected of lung cancer cells. The regulation role of receptor activator of NF-κB ligand (RANKL) on basigin-2 and its downstream molecules were measured by real-time quantitative RT-PCR, gelatin zymography and western blot analysis. RESULTS: We found that basigin-2 was highly expressed in lung cancer bone metastases. Then, we demonstrated that basigin-2 could promote lung cancer cells invasion, metastasis and proliferation through upregulating metalloproteinases-2 (MMP-2), MMP-9 and vascular endothelial growth factor (VEGF) expression. The lung cancer cells overexpressing basigin-2 strongly induced the osteolytic lesions in immunodeficient mice, which were reduced by treatment with basigin-2 blocking antibody. Furthermore, we explored the enhanced basigin-2 molecular mechanism in lung cancer bone metastasis. Our results indicated the RANKL, pivotal for the control of bone resorption, could increase basigin-2 and its downstream molecules MMP-2, MMP-9 and VEGF expression in vitro. CONCLUSIONS: Basigin-2 upregulated by RANKL induces MMPs and VEGF, which may increase lung cancer cell metastasis ability and support osteoclastic activity. Thus, our data suggest important roles for basigin-2 in lung cancer-induced osteolytic lesion and implicate this protein potential application as a target for lung cancer bone metastasis therapy.

CD147 overexpression promotes tumorigenicity in Chinese hamster ovary cells.

CD147 overexpresses in many epithelium-originated tumors and plays an important role in tumor migration and invasion. Most studies aim at the role of CD147 in tumor progression using tumor cell models. However, the influence of abnormal overexpression of CD147 on neoplastic transformation of normal cells is unknown. Here, the role of CD147 in malignant phenotype transformation in CHO cells was investigated. Three CHO cell lines that stably overexpressed CD147 (CHO-CD147), EGFP-CD147 (CHO-EGFP-CD147), and EGFP (CHO-EGFP) were generated by transfection of plasmids containing human CD147, EGFP-human CD147, and EGFP genes into CHO cells. Cell migration and invasion were detected by wound healing and transwell matrix penetration assay. Trypan blue exclusion, MTT, cell cycle analysis, and BrdU cell proliferation assay were used to detect cell viability and cell proliferation. Annexin V-FITC analysis was performed to detect apoptosis. We found that CD147 overexpression promoted the migration and invasion of CHO cells. CD147 accelerated the G1 to S phase transition and enhanced the CHO cell proliferation. Overexpression of CD147 inhibited both early- and late-stages of apoptosis of CHO-CD147 cells, which is caused by serum deprivation. CHO-EGFP-CD147 cells showed an increased anchorage-independent growth compared with CHO-EGFP cells as detected by soft-agar colony formation assay. The tumors formed by CHO-CD147 cells in nude mice were larger and coupled with higher expression of proliferating cell nuclear antigen and Ki-67 than that of CHO cells. In conclusion, human CD147 overexpression induces malignant phenotype in CHO cells.

miR-10b is overexpressed in hepatocellular carcinoma and promotes cell proliferation, migration and invasion through RhoC, uPAR and MMPs.

BACKGROUND: Recently, miR-10b is identified as a miRNA highly expressed in many human cancers, promoting cell migration and invasion. However, the specific function of miR-10b in hepatocellular carcinoma (HCC) is unclear at this point. METHODS: The miR-10b expression levels in 60 paired different TNM Stage HCC tumor tissues compared with adjacent non-tumor (ANT) tissues, normal tissue control (8 benign tumor and 7 normal liver tissues), 3 normal liver and 7 HCC cell lines were measured by real-time quantitative RT-PCR and to evaluate their association with HCC clinicopathologic features. Invasion assay, MTT proliferation assay and wound-healing assay were performed to test the invasion and proliferation of HCC cell after transfection. The effect of miR-10b on HCC in vivo was validated by murine xenograft model. RESULTS: We found that miR-10b expression was increased in human HCC tissues and cell lines compared with normal control, respectively. The expression of miR-10b was correlated with HCC metastatic ability. Overexpression of miR-10b in MHCC-97L cells increased cell motility and invasiveness, whereas inhibition of miR-10b in MHCC-97H cells reduced cell motility and invasiveness in vitro and in vivo. We also showed that HOXD10 was negatively regulated by miR-10b at the posttranscriptional level, via a specific target site within the 3'UTR by luciferase reporter assay. Furthermore, we found that miR-10b induced HCC cell invasion and migration by modulating the HOXD10 target gene RhoC, uPAR, MMP-2 and MMP-9 expression. CONCLUSIONS: Our results suggested that miR-10b was overexpressed in HCC and promoted HCC cell migration and invasion through the HOXD10/ RhoC/ uPAR/ MMPs pathway which may provide a novel bio-target for HCC therapy.

A regulatory loop involving miR-22, Sp1, and c-Myc modulates CD147 expression in breast cancer invasion and metastasis.

Breast cancer is the most common cancer in women for which the metastatic process is still poorly understood. CD147 is upregulated in breast cancer and has been associated with tumor progression, but little is known about its regulatory mechanisms. In this study, we demonstrated that CD147 was overexpressed in breast cancer tissues and cell lines, and the high expression correlated with tumor invasion and metastasis. We also found that the transcription factors Sp1 and c-Myc could bind to the CD147 promoter and enhance its expression. The CD147 mRNA has a 748-bp 3'-untranslated region (UTR) with many miRNA target sites, suggesting possible regulation by miRNAs. We discovered that miR-22 repressed CD147 expression by directly targeting the CD147 3'UTR. We also determined that miR-22 could indirectly participate in CD147 modulation by downregulating Sp1 expression. miR-22 could form an autoregulatory loop with Sp1, which repressed miR-22 transcription by binding to the miR-22 promoter. Together with the c-Myc-mediated inhibition of miR-22 expression, our investigation identified a miR-22/Sp1/c-Myc network that regulates CD147 gene transcription. In addition, miR-22 overexpression suppressed breast cancer cell invasion, metastasis, and proliferation by targeting CD147 in vitro and in vivo. Furthermore, we found that miR-22 was significantly downregulated in breast cancer tissues and that its expression was inversely correlated with the tumor-node-metastasis stage and lymphatic metastasis in patients. Our study provides the first evidence that an miR-22/Sp1/c-Myc network regulates CD147 upregulation in breast cancer and that miR-22 represses breast cancer invasive and metastatic capacities.

miR-22 is down-regulated in gastric cancer, and its overexpression inhibits cell migration and invasion via targeting transcription factor Sp1.

Accumulating evidence has shown that microRNAs are involved in multiple processes in cancer development and progression. Recently, miR-22 has been identified as a tumor-suppressing microRNA in many human cancers. However, the specific function of miR-22 in gastric cancer is unclear at this point. In this study, we first measured miR-22 expression level in 30 pairs of gastric cancer and matched normal tissues, two normal and six gastric cancer cell lines by real-time quantitative RT-PCR. We found that the expression of miR-22 in gastric cancer tissues and cell lines was much lower than that in normal control, respectively. Transfection of miR-22 expression plasmid could significantly inhibit the cell migration and invasion in SGC-7901 and NCL-N87 gastric cancer cell lines. Moreover, we also showed that Sp1 was negatively regulated by miR-22 at the posttranscriptional level, via a specific target site within the 3'UTR by luciferase reporter assay. The expression of Sp1 was inversely correlated with miR-22 expression in gastric cancer tissues, and knockdown of Sp1 by siRNA inhibited cell malignant behaviors. Thus, our findings suggest that miR-22 acts as tumor suppressor by targeting the Sp1 gene and inhibiting gastric cancer cell migration and invasion. The findings of this study contribute to current understanding of the functions of miR-22 in gastric cancer.

Suppression of MMP-9 activity by NDRG2 expression inhibits clear cell renal cell carcinoma invasion.

Members of the NDRG (N-Myc downstream-regulated) gene family have been shown to play a variety of roles in human malignancies. Recently, it was shown decreased expression in clear cell renal cell carcinoma (CCRCC) and inhibited cell proliferation, but the role of the NDRG2 in CCRCC invasion has not been described. We examined the expression of NDRG2 protein in CCRCC samples and the association between NDRG2 expression and CCRCC patients survival. Real-time RT-PCR and immunohistochemical analysis were used to measure NDRG2 expression in 60 paired CCRCC and adjacent normal tissues. Changes in cell invasion were detected by up- or down-regulating NDRG2 by adenovirus or siRNA. We found that NDRG2 expression is significantly down-regulated in CCRCC at mRNA and protein levels in a manner negatively associated with aggressive tumor behaviors, such as TNM stage (P = 0.003), Fuhrman's grade (P = 0.024), tumor invasion (P = 0.001) and tumor recurrence (P = 0.004), as well as shorter patient survival rates (P = 0.0041). Furthermore, NDRG2 could suppress CCRCC cell invasion through regulating MMP-9 expression and activity. So, these results suggest that NDRG2 can inhibit extracellular matrix-based tumor cell invasion and thereby play important roles in suppressing tumor metastasis in CCRCC. NDRG2 expression may also be a significant prognostic indicator for CCRCC.

Characterization of basigin isoforms and the inhibitory function of basigin-3 in human hepatocellular carcinoma proliferation and invasion.

Basigin, which has four isoforms, plays an important role in invasion of hepatocellular carcinoma (HCC). Detailed transcriptional regulation and functions of the basigin isoforms have not been reported except in the case of the predominant isoform basigin-2, which act as inducer of matrix metalloproteinases (MMPs). Here we determined that basigin-2, basigin-3, and basigin-4 were the most abundant transcript variants in human cell lines. GeneRacer PCR and luciferase reporter assays showed that basigin-3 and basigin-4 were initiated from an alternative promoter. Basigin-3 and basigin-4 were widely expressed in various normal human tissues at the mRNA level and were upregulated in HCC tissues compared to in normal tissues. Western blotting and confocal imaging showed that glycosylated basigin-3 and basigin-4 were expressed and localized to the plasma membrane. However, in cultured cell lines, only native basigin-3, and not basigin-4, was detected at protein level. Overexpression of basigin-3 inhibited HCC cell proliferation, MMP induction, and cell invasion in vitro and in vivo. Bimolecular fluorescence complementation assays and nuclear magnetic resonance (NMR) analysis indicated that basigin-3 interacted with basigin-2 to form hetero-oligomers. In conclusion, we systematically investigated the alternative splicing of basigin and found that basigin-3 could inhibit HCC proliferation and invasion, probably through interaction with basigin-2 as an endogenous inhibitor via hetero-oligomerization.

Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma.

CD147 is a transmembrane glycoprotein overexpressed in human hepatocellular carcinoma (HCC) which could promote HCC progression and metastasis. Promoter methylation is one of the most important processes in gene regulation. In this study, we aim to investigate CD147 promoter methylation status and the correlation with clinicopathological features and prognosis in HCC. CD147 promoter methylation statuses and expression levels in normal and HCC cell lines and 54 paired HCC and adjacent non-tumour (ANT) tissues were, respectively, examined by bisulphite genomic sequencing, methylation-specific PCR, real-time RT-PCR, Western blot and immunohistochemistry. The correlations of promoter methylation statuses with CD147 expression level and the clinicopathological features were statistically analysed in HCC patients. Significantly higher expression of CD147 and significantly lower promoter methylation level were observed in HCC cell lines compared to normal cell lines and tissues control. In vivo and in vitro analysis indicated that demethylation with 5-Aza-2'-deoxycytidine led to increased CD147 expression through enhancing Sp1 binding affinity, and methylation with methyltransferase reduced CD147 transcriptional activity through interfering Sp1 binding. CD147 promoter methylation level in HCC tissues (22.22%) was lower than that in ANT tissues (46.30%; P < 0.05). Within HCC tissues, a significant inverse correlation was observed between CD147 expression and methylation level (r=-0.615). Moreover, HCC patients with unmethylated CD147 promoter had a significantly higher recurrence rate (88.1%versus 58.3%; P < 0.05) and death rate (83.3%versus 50.0%; P < 0.05) than patients with methylated CD147 promoter. In conclusions, promoter hypomethylation up-regulates CD147 expression primarily through increasing Sp1 binding and associates with poor prognosis in HCC patients.

NDRG2 suppresses the proliferation of clear cell renal cell carcinoma cell A-498.

BACKGROUND: Recently, the anti-tumor activity of N-myc downstream-regulated gene 2 (NDRG2) was shown decreased expression in clear cell renal cell carcinoma (CCRCC), but the role of the down-expression of NDRG2 has not been described. METHODS: The NDRG2 recombinant adenovirus plasmid was constructed. The proliferation rate and NDRG2 expression of cell infected with recombinant plasmid were mesured by MTT, Flow cytometry analysis and western blot. RESULTS: The CCRCC cell A-498 re-expressed NDRG2 when infected by NDRG2 recombinant adenovirus and significantly decreased the proliferation rate. Fluorescence activated cell sorter analysis showed that 25.00% of cells expressed NDRG2 were in S-phase compared to 40.67% of control cells, whereas 62.08% of cells expressed NDRG2 were in G1-phase compared to 54.39% of control cells (P < 0.05). In addition, there were much more apoptotic cells in NDRG2-expressing cells than in the controls (P < 0.05). Moreover, upregulation of NDRG2 protein was associated with a reduction in cyclin D1, cyclin E, whereas cyclinD2, cyclinD3 and cdk2 were not affected examined by western blot. Furthermore, we found that p53 could upregulate NDRG2 expression in A-498 cell. CONCLUSIONS: We found that NDRG2 can inhibit the proliferation of the renal carcinoma cells and induce arrest at G1 phase. p53 can up-regulate the expression of NDRG2. Our results showed that NDRG2 may function as a tumor suppressor in CCRCC.

Transcription factor Sp1 regulates expression of cancer-associated molecule CD147 in human lung cancer.

CD147 is a novel cancer-associated biomarker that plays an important role in the invasion and metastasis of human lung cancer. In spite of its many known functions, little is known about CD147 transcriptional regulation. In this study, we explored the regulation of CD147 in human lung cancer tissues. Over 60% of the human lung cancer tissues expressed differential high levels of CD147. We then cloned the 5'-flanking region of the human CD147 gene and identified a critical promoter region at -108 to -42 which contained one binding site for Sp1, which was essential in up-regulating CD147 promoter activity. These results were proven by blocking Sp1 using RNAi or mithramycin A treatment and up-regulating Sp1 using transfection with eukaryotic expression vector. Consistent with the CD147 transcription activation, a high level of Sp1 expression was detected in lung cancer cell lines overexpressing CD147. Chromatin immunoprecipitation assay showed that much more Sp1 could bind to the CD147 promoter in 95-D with CD147 high expression than in SK-MES-1 with CD147 low expression. There was a significant positive correlation between CD147 expression and Sp1 expression level detected by immunohistochemistry (r = 0.831). Collectively, our results suggest that Sp1 is essential for regulating the CD147 gene expression in human lung cancer.

Regulated upon activation normal T-cell expressed and secreted originating from the epididymis differentially associates with viable and defective spermatozoa.

OBJECTIVE: To investigate the expression and cellular distribution of regulated upon activation normal T-cell expressed and secreted (RANTES) in the male reproductive system. DESIGN: Basic research. SETTING: University academic medical center. PATIENT(S): Three adult male organ donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, immunohistochemical staining, and immunofluorescence staining were used to examine the distribution of RANTES in human and mouse epididymis. Western blot was used to quantitate the levels of RANTES expression in mouse epididymis on postnatal days. Immunofluorescence staining was applied to detect RANTES association with spermatozoa from mouse epididymis. RESULT(S): The location of RANTES was restricted to ciliated cells of the efferent duct and apical, narrow, and basal cells of the epididymal ducts, in both humans and mouse. RANTES-positive basal cells were only identified in the epididymal ducts in humans. The signals of RANTES were first detected on day 28 and increased during mouse sexual maturation. We also observed that RANTES was bound on both normal and defective epididymal sperm, but in different patterns. CONCLUSION(S): RANTES is constitutively expressed in the epididymis and secreted into the lumen of epididymis throughout sexual maturity, and differentially associates with viable and defective spermatozoa.

HAb18G (CD147), a cancer-associated biomarker and its role in cancer detection.

AIMS: To evaluate HAb18G/CD147 as a cancer-associated biomarker using its monoclonal antibody HAb18. METHODS AND RESULTS: On immunohistochemical analysis of 28 tissue microarrays and pathological sections of 1117 breast tissue samples, HAb18G/CD147 was expressed in carcinoma with an overall positivity rate of 67.76%, which was significantly higher than that in sarcomas (27.34%, P < 0.0001) and normal epithelial (5.18%, P < 0.0001) and fetal (2.67%, P < 0.0001) tissues. In epithelial tissues from 14 organs, the difference in HAb18G/CD147 expression between normal epithelium and the corresponding carcinoma was also significant (P < 0.05 for each pair). This expression in carcinoma was also found at the mRNA level, suggesting transcriptional level regulation of HAb18G/CD147 expression. In a retrospective study of 106 patients with infiltrating ductal carcinoma of the breast, the level of HAb18G/CD147 expression was positively correlated with tumour recurrence/metastasis (P = 0.0003) and negatively correlated with survival of breast cancer patients (P = 0.002). Multivariable Cox regression analysis showed that HAb18G/CD147 was an independent prognostic factor. CONCLUSIONS: HAb18G/CD147 is significantly expressed in various cancers and appears to have prognostic significance, rendering it a possible cancer-associated biomarker for pathological diagnosis, prognostic evaluation, targeted therapy and radioimmunoimaging of a broad range of cancer types.