Discovering Innate Driver Variants for Risk Assessment of Early Colorectal Cancer Metastasis.

Metastasis is the main fatal cause of colorectal cancer (CRC). Although enormous efforts have been made to date to identify biomarkers associated with metastasis, there is still a huge gap to translate these efforts into effective clinical applications due to the poor consistency of biomarkers in dealing with the genetic heterogeneity of CRCs. In this study, a small cohort of eight CRC patients was recruited, from whom we collected cancer, paracancer, and normal tissues simultaneously and performed whole-exome sequencing. Given the exomes, a novel statistical parameter LIP was introduced to quantitatively measure the local invasion power for every somatic and germline mutation, whereby we affirmed that the innate germline mutations instead of somatic mutations might serve as the major driving force in promoting local invasion. Furthermore, via bioinformatic analyses of big data derived from the public zone, we identified ten potential driver variants that likely urged the local invasion of tumor cells into nearby tissue. Of them, six corresponding genes were new to CRC metastasis. In addition, a metastasis resister variant was also identified. Based on these eleven variants, we constructed a logistic regression model for rapid risk assessment of early metastasis, which was also deployed as an online server, AmetaRisk (http://www.bio-add.org/AmetaRisk). In summary, we made a valuable attempt in this study to exome-wide explore the genetic driving force to local invasion, which provides new insights into the mechanistic understanding of metastasis. Furthermore, the risk assessment model can assist in prioritizing therapeutic regimens in clinics and discovering new drug targets, and thus substantially increase the survival rate of CRC patients.

Silencing of DHX32 increases the proliferation of liver cancer cells.

BACKGROUND: Liver cancer ranks fifth in malignancy incidence globally and is the second leading cause of cancer-related death in China. Chronic hepatitis B or C infection and alcohol abuse have been identified to be the major risk factors for liver cancer development. Some evidence implicates DHX32 as being critically involved in tumor progression. The role of DHX32 in liver cancer specifically, however, remains unclear. METHODS: Fifty-three liver cancer tissue and paracancerous tissue samples were surgically resected from 53 patients who were admitted to Zhongshan Hospital between 2006 and 2008. We used immunohistochemistry (IHC) to analyze the expressions of DHX32, established liver cancer cells with stable DHX32 knockdown, and investigated the proliferation of these cells with methyl thiazolyl tetrazolium (MTT) and 5-ethynyl-2'-deoxyuridine (EdU) data. RESULT: Baseline characteristics of enrolled liver cancer patients (53 patients) were summarized, and the IHC results firstly showed that 88.7% (47/53) of paracancerous tissues exhibited a high expression of DHX32, while only 43.4% (23/53) of liver cancer tissues showed similar expression. We then established liver cancer cells with the stable knockdown of DHX32. MTT and EdU data demonstrated that DHX32 knockdown in liver cancer cells enhanced the proliferative potential of liver cancer cells. Furthermore, phosphorylated levels of extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) were upregulated in liver cancer cells with DHX32 knockdown. We also found the level of cyclin-dependent kinases 6 (CDK6) to be increased in liver cancer cells with DHX32 knockdown. CONCLUSIONS: DHX32 showed a lower expression in liver cancer tissues than in paracancerous tissues and could harbor a proliferation-suppressing property in liver cancer. DHX32 may thus be a possible target for gene therapy.

Plasma microRNA-19a as a potential biomarker for esophageal squamous cell carcinoma diagnosis and prognosis.

AIM: To investigate whether plasma miR-19a can serve as a biomarker for esophageal squamous cell carcinoma (ESCC) diagnosis and prognosis. MATERIALS & METHODS: Plasma samples from 89 ESCC, 45 benign lesion patients and 80 healthy controls were subjected to RT-qPCR analyses for miR-19a. In addition, plasma samples from 30 patients were collected before and after surgery for the same analyses. RESULTS: Plasma miR-19a was significantly increased in ESCC patients compared with healthy controls. The sensitivity of miR-19a for early stages of ESCC was 68.09%. Combination of miR-19a and cytokeratin 19 fragment 21-1 (Cyfra21-1) further improved the sensitivity to 78.70%. Moreover, plasma miR-19a level was decreased in patients after surgery. CONCLUSION: Plasma miR-19a may serve as a potential biomarker that complements Cyfra21-1 in detecting early stages of ESCC.

DHX32 Promotes Angiogenesis in Colorectal Cancer Through Augmenting ß-catenin Signaling to Induce Expression of VEGFA.

We previously reported that overexpression of DHX32 contributes to the growth and metastasis of colorectal cancer (CRC). However, the underlying mechanism is not largely characterized. Herein, we reported that DHX32 in CRC cells upregulated expression of vascular endothelial growth factor A (VEGFA) at the transcription level through interacting with and stabilizing ß-catenin. This promoted the recruitment of host endothelial cells to the tumor, and therefore, formation of microvessel in the tumor. Xenograft model revealed that depletion of DHX32 in CRC cells significantly reduced the microvessel density in the grafts and suppressed the growth of grafts. Furthermore, the expression level of DHX32 was positively associated with microvessel density in human CRC and poor outcome of CRC patients. Therefore, the report demonstrates that DHX32 is a pro-angiogenic factor, that inhibition of DHX32-ß-catenin pathway can provide a strategy for CRC treatment, and that the expression level of DHX32 has the potential to serve as a biomarker for CRC diagnosis and prognosis.

Depletion of Cks1 and Cks2 expression compromises cell proliferation and enhance chemotherapy-induced apoptosis in HepG2 cells.

The present study explored the oncogenic roles of overexpressed Cks1 and Cks2 in human hepatocellular carcinoma cells. Gene expression of Cks1 and Cks2 in HepG2 cells was disrupted by siRNA or increased by cDNA transfection. Cell proliferation was assayed by CCK-8 analysis and cell counting. Cisplatin-induced apoptosis after transfection was measured by flow cytometry using Annexin V/propidium iodide (PI) double staining. Cell cycle changes after transfection were determined by flow cytometry with PI staining. Protein levels of Akt and GSK-3ß were measured after transfection. The results revealed that HepG2 proliferation was decreased by depletion of endogenous Cks1 or Cks2, and increased by overexpression of Cks1 or Cks2. HepG2 apoptosis increased concordantly with the decline of Cks1 or Cks2 expression. Overexpression of Cks1 or Cks2 prevented cell apoptosis. Protein levels of p­Akt and p­GSK-3ß were downregulated after RNA interference of Cks1 or Cks2. In conclusion, Cks1 and Cks2 promoted proliferation and prevented apoptosis of HepG2 cells. The Akt/GSK-3ß-related PI3K/Akt signaling pathway may be a key signaling pathway that is involved in the regulation of cell growth and cell death.

Plasma levels of microRNA-24, microRNA-320a, and microRNA-423-5p are potential biomarkers for colorectal carcinoma.

BACKGROUND: MicroRNAs are stable and easy to detect in plasma. The plasma levels of microRNAs are often changed in disease conditions, including cancer. This makes circulating microRNAs a novel class of biomarkers for cancer diagnosis. Analyses of online microRNA data base revealed that expression level of three microRNAs, microRNA-24 (miR-24), microRNA-320a (miR-320a), and microRNA-423-5p (miR-423-5p) were down-regulated in colorectal cancer (CRC). However, whether the plasma level of these three microRNAs can serve as biomarkers for CRC diagnosis and prognosis is not determined. METHODS: Plasma samples from 223 patients with colorectal related diseases (111 cancer carcinoma, 59 adenoma, 24 colorectal polyps and 29 inflammatory bowel disease) and 130 healthy controls were collected and subjected to reverse transcription-quantitative real time PCR (RT-qPCR) analyses for the three microRNAs. In addition, plasma samples from 43 patients were collected before and after surgical treatment for the same RT-qPCR analyses. RESULTS: The concentrations of plasma miR-24, miR-320a and miR-423-5p were all decreased in patients with CRC and benign lesions (polyps and adenoma) compared with healthy controls, but increased in inflammatory bowel disease (IBD). The sensitivity of miR-24, miR-320a and miR-423-5p for early stage of CRC were 77.78 %, 90.74 %, and 88.89 %, respectively. Moreover, the plasma concentration of the three microRNAs was increased in patients after the surgery who had clinical improvement. CONCLUSIONS: The plasma levels of miR-24, miR-320a, and miR-423-5p have promising potential to serve as novel biomarkers for CRC detection, especially for early stage of CRC, which are superior to the currently used clinical biomarkers for CRC detection, such as CEA and CA19-9. Further efforts to develop the three microRNAs as biomarkers for early CRC diagnosis and prediction of surgical treatment outcomes are warrant.

Overexpression of DHX32 contributes to the growth and metastasis of colorectal cancer.

Our previous work demonstrates that DHX32 is upregulated in colorectal cancer (CRC) compared to its adjacent normal tissues. However, how overexpressed DHX32 contributes to CRC remains largely unknown. In this study, we reported that DHX32 was overexpressed in human colon cancer cells. Overexpressed DHX32 promoted SW480 cancer cells proliferation, migration, and invasion, as well as decreased the susceptibility to chemotherapy agent 5-Fluorouracil. Furthermore, PCR array analyses revealed that depleting DHX32 in SW480 colon cancer cells suppressed expression of WISP1, MMP7 and VEGFA in the Wnt pathway, and anti-apoptotic gene BCL2 and CA9, however, elevated expression of pro-apoptotic gene ACSL5. The findings suggested that overexpressed DHX32 played an important role in CRC progression and metastasis and that DHX32 has the potential to serve as a biomarker and a novel therapeutic target for CRC.

Overexpression of DEK gene is correlated with poor prognosis in hepatocellular carcinoma.

The oncogene DEK was originally identified as one of the parts of the DEK­CAN fusion gene, arising from the translocation (6;9) in a subtype of acute myeloid leukemia. Since then, DEK has been shown to promote tumorigenesis in a variety of cancer cell types through its roles in inhibiting cell differentiation, senescence and apoptosis. Certain studies have established that DEK is dysregulated in several types of cancer, including hepatocellular carcinoma (HCC). However, its clinical significance in human HCC remains unknown. In this study, the expression of DEK mRNA and protein was examined in 55 surgical HCC specimens and matched non­tumorous tissues. In addition, the correlation between DEK expression and clinicopathological characteristics and prognosis was analyzed. mRNA and protein levels of DEK were found to be significantly overexpressed in the majority of HCC tumors when compared with matched normal hepatic tissues (P<0.05). In addition, the expression pattern of DEK was closely correlated with differentiation status, portal venous invasion and tumor size (P<0.05). Kaplan­Meier curves demonstrated that patients with higher DEK expression levels had significantly poorer survival than those with lower DEK expression levels (P=0.003). In addition, Cox regression analysis demonstrated that the level of DEK expression may be a valuable prognostic factor (P<0.05). These results suggested that DEK may play a significant role in hepatocyte differentiation and may serve as a useful prognostic marker and biomarker for the staging of HCC.

Clinical significance of combined testing of YKL-40 with CEA in Chinese colorectal cancer patients.

BACKGROUND: To investigate the practical value of individual and combined testing of plasma levels of YKL-40, CEA, and CA199 for auxiliary diagnosis and detection of recurrence of colorectal cancer. METHODS: ELISA and ECLIA were used to evaluate levels of YKL-40, CEA, and CA199 in 120 colorectal cancer patients (56 initial-diagnosis, 42 post-operative, and 22 recurrent cases). Forty-three patients with benign colorectal disease and 36 healthy patients were enrolled as controls. The relationship between YKL-40 and clinical indicators of tumor pathology was analyzed. The positive rate and diagnostic efficacy of single and combined YKL-40, CEA, and CA199 testing were assessed in patients with colorectal cancer. RESULTS: Plasma YKL-40 in the cancer group was significantly higher than in the benign control and healthy control group, and the mean values were 145.4 ng/mL, 107.7 ng/mL, and 51.3 ng/mL (p < 0.05), respectively. With 72 ng/mL as the diagnostic threshold, the sensitivity and specificity of YKL-40 in colorectal cancer diagnosis were found to be 73.2% and 66.7%, respectively. Early-stage colorectal cancer patients showed a YKL-40 positive rate (73%) significantly higher than those of CEA and CA199 (50% and 32%, respectively; p < 0.05). When YKL-40 testing was combined with CEA or CA199, the positive rate increased to 82.1% and 80.3%, respectively. Through ROC curve analysis of the post-operative recurrent group against the non-recurrent group, the areas under the curve for YKL-40, CEA, and CA199 were found to be 0.907, 0.714, and 0.759, respectively. Based on the Dukes classification, the mean YKL-40 value for stages A/B, C, and D were 120.1 ng/mL, 131.7 ng/mL, and 226.8 ng/mL (p = 0.008), respectively. The plasma YKL-40 level gradually increased as the disease progressed. Lower degrees of tumor differentiation were correlated with higher YKL-40 levels. The mean YKL-40 values of high, medium, and low tumor differentiation groups were 96.8 ng/mL, 127.5 ng/mL, and 225.7 ng/mL (p = 0.004), respectively. CONCLUSIONS: The benefits of using YKL-40 testing are higher than CEA and CA199 for the monitoring of colorectal cancer recurrence. Combined testing of both YKL-40 and CEA was found to be optimal for auxiliary diagnosis of colorectal cancer. Plasma YKL-40 was found to be suitable for auxiliary diagnosis of colorectal cancer.

Effects of silencing cyclooxygenase-2 expression via RNA interference on the tumorigenicity of the SMMC-7721 human hepatocarcinoma cell line.

We constructed a vector carrying a shRNA sequence against cyclooxygenase-2 (COX-2) that was subsequently transfected into the human hepatocarcinoma cell line SMMC­7721. Furthermore, we established a COX-2-deficient stable cell line and a model of tumor-shRNA transplantation in nude mice. Negative shRNA was used as the control. The tumor volume in the experimental group was smaller compared to that in the control group. Hematoxylin and eosin staining indicated that the cells in the experimental group differentiated better than those in the control group. The COX-2 mRNA level in the tumor tissues injected with SMMC-7721/COX-2i was markedly downregulated compared to that in the tumor tissues injected with SMMC-7721/negative shRNA. The inhibition rate reached 68.6%. Immunohistological study showed a significantly strong COX-2 expression in the control group tumor cells, whereas the experimental group exhibited moderate expression, indicating the inhibition of COX-2 expression after transfection of cells with shRNA against COX-2. Western blot analysis further proved the inhibition of COX-2 expression. In conclusion, RNAi-mediated regulation of COX-2 expression could efficiently inhibit liver-transplanted tumor growth in BALB/c nude mice.

Optimization of conditions for transfection with the Sofast gene vector.

We previously reported the synthesis and characterization of a novel cationic polymer gene vector. The present article further explored and optimized the working conditions of the Sofast gene vector both in vitro and in vivo, and improved its performance. The transfection conditions of Sofast, such as cell type, cell density, transfection time, N/P values and analysis time after transfection, were further explored. Moreover, the effects of the fusion peptide diINF-7 on transfection efficiency were examined. Sofast was successfully applied for the transfection of exogenous genes into more than 40 types of cell lines derived from humans, mice, monkeys and other species. When the cells were 50-80% confluent, Sofast possessed a better transfection efficiency. In most cases, Sofast also had a higher transfection efficiency when it was used to transfect cells that were seeded for several hours and had adhered to the substrate. The results from in vitro experiments indicate that the recommended Sofast to DNA mass ratio is 16:1, and the optimum analysis time after transfection is 48 h. The salt concentration in the Sofast working solution markedly affected the transfection efficiency. When conducting in vivo transfection, the working solution should be salt-free, whereas for in vitro transfection, it is more appropriate for the working solution to include certain salt concentrations. Finally, the results confirm that diINF-7 significantly promotes the transfection efficiency of Sofast. In conclusion, the present research not only established the optimal conditions for Sofast in the transfection of commonly used cells, but also built the foundations for in vivo and in vitro applications of Sofast, as well as its use in clinical practice.

Further evaluation of a novel nano-scale gene vector for in vivo transfection of siRNA.

In this research, a lipid-cationic polymer (LCP) containing the side-chain branching of brassidic acid was synthesized using chemical methods. As a gene vector for small interfering ribonucleic acid (siRNA) transfection, the efficiency and biosafety of LCP were preliminarily evaluated to investigate its possible application on tumor gene therapy. The toxicity, side-effects, and biosafety of LCP were investigated in animals based on the results of in vitro experiments. The siRNA against cyclooxygenase-2 (COX-2) was transfected by LCP to interfere with the COX-2 expression in nude-transplanted tumors. Hematoxylin and eosin stains, immunohistochemistry, reverse transcription-polymerase chain reaction, and Western blot were performed to evaluate the efficiency of LCP for siRNA transfection. The animal toxicity experiment showed that a high concentration of LCP had a low toxic effect on animals and did not induce allergic or pyrogenic reactions. The results from the in vivo transfection indicated that LCP could efficiently transfect siRNA and silence the target gene expression. The LCP gene vector for siRNA transfection is highly efficient during in vivo transfection and had low toxicity. From all aspects of tumor gene therapy and basic research, LCP is valuable for scientific research and medical applications.

Clinical significance and expression of cyclin kinase subunits 1 and 2 in hepatocellular carcinoma.

BACKGROUND: The mammalian cyclin kinase subunit (Cks) family has two members, Cks1 and Cks2, which were identified based on the protein sequence homology to yeast Cks. Overexpression of Cks1 and Cks2 has been reported to be associated with high aggressiveness and a poor prognosis in various malignancies, including gastric, breast and prostate carcinomas. Yet, whether Cks1 and Cks2 are overexpressed in hepatocellular carcinoma (HCC) remains uncharacterized. AIMS: To investigate whether overexpression of the Cks family is clinically relevant to HCC, and whether expression patterns of Cks1 and Cks2 in HCC have diagnostic and prognostic value. METHODS: Real-time quantitative reverse transcriptase polymerase chain reaction, immunostaining and Western blot analyses were used to detect the expression of Cks1 and Cks2 at the mRNA and protein levels respectively. The associations between Cks1 and Cks2 expressions and clinical features, as well as the association between Cks1 or Cks2 and p27(kip1) expressions in HCC, were analysed. RESULTS: Expressions of Cks1 and Cks2 at both mRNA and protein levels were significantly higher in HCC than those in the adjacent noncancerous tissues (including chronic hepatitis and cirrhosis) and normal liver tissues. Overexpressions of Cks1 and Cks2 in HCC were closely associated with poor differentiation features. The expressions of both Cks1 and Cks2 were negatively associated with p27(kip1) at the protein level. CONCLUSIONS: Overexpression of Cks1 and Cks2 is associated with the aggressive tumour behaviours of HCC, and thus has diagnostic and prognostic value. Further efforts are needed to develop novel biomarkers for HCC based on CKs1 and Cks2 expressions.