Xuedong Fang

miR-10b promotes cell invasion through RhoC-AKT signaling pathway by targeting HOXD10 in gastric cancer

MicroRNAs play critical roles in tumorigenesis as either oncogenes or tumor suppressors. As a microRNA induced by Twist, miR-10b function as a metastasis driver in different types of cancer, in which the downstream target gene HOXD10 is the main mediator. In gastric tumor species, miR-10b levels were dramatically elevated in lymphoma node metastasis-positive tumor tissues compared with lymphoma node metastasis-free tumor tissues, and were correlated to dowregulation of HOXD10 expression. In gastric cell lines with distinct degrees of differentiation, miR-10b was highly expressed in the cell line with strong metastatic ability. In MNK45 cells, inhibition of miR-10b led to abrogation of cell invasion. While in GES-1 cells, miR-10 overexpression resulted in enhancement of invasiveness through translational inhibition of HOXD10, and constitutive expression of HOXD10 reversed the effects of miR-10b on cell invasion. Furthermore, either knockdown of RhoC or inhibition of AKT activation interfered miR-10-induced invasiveness in GES-1 cells. In summary, these observations suggest that miR-10b can stimulate the upregulation of RhoC and AKT phosphorylation through targeting HOXD10, thus promoting cell invasion in gastric tumors.

miR-133b acts as a tumor suppressor and negatively regulates FGFR1 in gastric cancer.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that negatively regulate protein expression by binding protein-coding mRNAs and repressing translation. Accumulating evidence suggests that miRNAs are involved in cancer development and progression, acting as either tumor suppressors or oncogenes. Intriguingly, it has been shown that miR-133b was significantly downregulated in several types of cancers. However, its role and relevance in gastric cancer are still largely unknown. We showed that miR-133b was downregulated in human gastric cancer tissues and cell lines compared with nontumor counterparts by quantitative RT-PCR analysis. Overexpression of miR-133b could inhibit cell proliferation and colony formation of the gastric cancer cell lines MKN-45 and SGC-7901. Bioinformatics analysis indicated two putative miR-133b binding sites in the 3′-untranslated region of fibroblast growth factor receptor 1 (FGFR1) mRNA. In dual-luciferase reporter assay, miR-133b reduced the luciferase activity of Luc-FGFR1-wt, and mutation of miR-133b binding sites abolished the inhibitory effect of miR-133b. In this study, we found that miR-133b reduced the protein but not the mRNA levels of endogenous FGFR1. Furthermore, FGFR1 expression was upregulated in gastric cancer tissues and inversely correlated with miR-133b expression. Finally, knockdown of FGFR1 inhibited the growth of MKN-45 cells in a dose-dependent manner and overexpression of FGFR1 promoted the growth of GES-1 cells. These results indicate that miR-133b targets FGFR1 and inhibits gastric cancer cell growth, suggesting that it may serve as a tumor suppressive target in gastric cancer therapy.

Decreased CD27 on B lymphocytes in patients with primary hepatocellular carcinoma.

OBJECTIVES: Hepatitis B virus (HBV) replicates in the liver and can lead to hepatocellular carcinoma (HCC). The B lymphocytes may provide a means for HBV to persist although the mechanism remains unknown. This study aimed to characterize B lymphocyte subset phenotypes and measure levels of B lymphocyte-related cytokines in HCC patients. METHODS: The study population included 38 HCC patients and 30 healthy control subjects. Phenotyping of B lymphocytes was performed by flow cytometry. Serum cytokine levels were measured using a cytometric bead array immunoassay. RESULTS: The ratio of naïve (CD29+CD27−) to memory (CD19+CD27+) B lymphocytes was significantly higher in HCC patients compared with healthy controls. The percentage of memory B lymphocytes decreased with the progression of HCC. Levels of interleukin (IL)-6 and IL-10 were significantly increased in HCC patients compared with healthy controls. CONCLUSIONS: The depletion of memory B lymphocytes may contribute to unresponsiveness to HBV or to HCC. This humoral defect might be related to raised production of IL-6 and IL-10.

Overexpression of miR-203 sensitizes paclitaxel (Taxol)-resistant colorectal cancer cells through targeting the salt-inducible kinase 2 (SIK2)

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression through the endogenous RNA interference machinery. Treatments with combination of chemotherapy with surgery are essential for advanced-stage colorectal cancer. However, the development of chemoresistance is a major obstacle for clinical application of anticancer drugs. In this study, we report a miR-203-SIK2 axis that involves in the regulation of Taxol sensitivity in colon cancer cells. MiR-203 is downregulated in human colon tumor specimens and cell lines compared with their normal counterparts. We report miR-203 is correlated with Taxol sensitivity: overexpression of miR-203 sensitizes colon cancer cells and the Taxol-resistant cells display downregulated miR-203 compared with Taxol-sensitive cells. We identify SIK2 as a direct target of miR-203 in colorectal cancer cells. Overexpression of miR-203 complementary pairs to the 3′ untranslated region (UTR) of SIK2, leading to the sensitization of Taxol resistant cells. In addition, miR-203 and the salt-inducible kinase 2 (SIK2) are reverse expressed in human colorectal tumors. Finally, we demonstrate recovery of SIK2 by overexpression of SIK2-desensitized Taxol-resistant cells, supporting the miR-203-mediated sensitization to Taxol, is through the inhibition of SIK2. In general, our study will provide mechanisms of the microRNA-based anti-tumor therapy to develop anti-chemoresistance drugs.

p38MAPK Signaling Enhances Glycolysis Through the Up-Regulation of the Glucose Transporter GLUT-4 in Gastric Cancer Cells.

Background/Aims: Previous studies have shown that p38MAPK is involved in gastric cancer, yet the underlying mechanism remains unclear. Methods: q-PCR, Western blot and immunohistochemistry were used to explore the expression of PP2A and the phosphorylation of p38MAPK in gastric cancer tissues and normal gastric tissues. Activated p38MAPK in the gastric cancer cell line MKN45 using activator, then q-PCR, glucose uptake assay and colony formation assay were performed to determine whether p38MAPK promotes gastric cancer through the enhancement of glycolysis. After transfection of p38MAPK dominant negative mutation (p38DN) into MKN45 cells or MKN45 cells treated with an inhibitor of p38MAPK, Western blot was performed to detect the expression of GLUT-4. The knock down of MEF2α in MKN45 cells by siRNA was followed by Western blot and luciferase reporter assay to investigate the underlying mechanism of the role of p38MAPK in the promotion of gastric cancer. Finally, q-PCR, Western blot and immunohistochemistry were performed to examine GLUT-4 expression in gastric cancer tissues and normal gastric tissues. Results: We found that p38MAPK activation significantly increases GLUT-4 expression and promotes glucose uptake and cell growth in gastric cancer cells. Inhibition of p38MAPK abrogates the up-regulation of GLUT-4. MEF2α knockdown abolishes p38MAPK-mediated GLUT-4 up-regulation. PP2A, an inhibitor of p38MAPK, is down-regulated in gastric cancer tissues, which might contribute to the activation of p38MAPK. Conclusions: Our data indicate that the abnormal activation of p38MAPK promotes glycolysis within gastric cancer cells through the upregulation of GLUT-4 in a MEF2a-dependent manner.

MicroRNA-143 replenishment re-sensitizes colorectal cancer cells harboring mutant, but not wild-type, KRAS to paclitaxel treatment.

Colorectal cancer (CRC) global incidence is one of the highest among cancers. The KRAS gene has been shown as a robust biomarker for poor prognosis and drug resistance. MicroRNA-143 (miR-143) and let-7 are families of tumor suppressor microRNAs that are often downregulated in CRC, especially with coexistent KRAS mutations. In order to evaluate if miR-143 and/or let-7b replenishment would re-sensitize CRC cells to paclitaxel treatment, we investigated in effect of miR-143 and let-7b replenishments on sensitivity to paclitaxel treatment in KRAS mutant LoVo and wild-type SW48 CRC cell lines. Our results showed that miR-143, but not let-7b, increased sensitization of KRAS mutant tumor cells to paclitaxel. Furthermore, transfection of miR-143, but not let-7b, mimic negatively regulated the expression of mutant but not wild-type KRAS. Combination of miR-143 mimic and paclitaxel induced the onset of apoptosis, and reverted in vitro metastatic properties (migration and invasion) in KRAS mutant tumor cells. MiR-143 thus can be used as a chemosensitizer for the treatment of KRAS mutant tumors and warrants further investigations in in vitro and pre-clinical in vivo models.

Fos-like antigen 2 (FOSL2) promotes metastasis in colon cancer

&NA; Among different cancers, incidence and mortality of colorectal cancer (CRC) is one of the highest. KRAS mutation is one of the underlying features in the pathogenesis of CRC with CRC tumors harboring mutant KRAS exhibiting a more aggressive behavior compared to CRC tumors with wild type KRAS. We had earlier shown that the microRNA‐143 (miR‐143) replenishment not only chemosensitizers CRC cell line with mutant KRAS instead of wild‐type KRAS gene, to paclitaxel‐mediated cytotoxicity, but also inhibits cell migration and invasion ability. Hence, the study aimed to determine how miR‐143 replenishment is inhibiting pre‐metastatic behavior in CRC cells with mutant KRAS. Top ten mRNA targets of miR‐143 as predicted by TargetScan were evaluated by qRT‐PCR in LoVo cells which were performed mock transfection or miR‐143 mimic transfection. Evaluation of the changes in cognate mRNA target(s) was done in 30 paired CRC tissue and tumor adjacent normal tissue specimens and in LoVo cells by western blot. Effect of the mRNA target on pro‐metastatic behavior was assayed by gain‐ and loss‐of‐function studies using a combination of western blotting and in vitro cell proliferation and transwell migration/invasion assay in LoVo cells and in the normal colonic epithelium cell line FHC. In vivo effect of the cognate mRNA target on CRC metastasis was assayed by xenograft assay. Of the 10 predicted mRNA targets, FOSL2 (P < 0.05) and IGFBP5 (P > 0.05) was down regulated in LoVo cells transfected with the miR‐143 mimic. FOSL2 mRNA levels were significantly downregulated in CRC tissue specimens compared with adjacent normal tissue (P < 0.05). Immunoblot analysis showed that FOSL2, but not IGFBP5, protein expression is down regulated in LoVo cells after the miR‐143 mimic transfection. FOSL2 overexpression in the normal colonic epithelial cell line FHC or siRNA‐mediated silencing in LoVo cells induced and repressed, respectively, pro‐mesenchymal cell features. Whereas manipulation of FOSL2 expression did not have any effect on cell proliferation rates, silencing its expression inhibited cell migration and invasion ability in vitro. In addition, silencing of FOSL2 expression in the LoVo cells can significantly inhibited invasion of hepatic, while no effect was found for tumorigenic potential. Our results suggest that FOSL2 is a critical regulator of CRC metastasis and might be an important marker for prognostic in CRC patients.

Clinical research of totally laparoscopic modified Roux-en-Y reconstruction

Background: To evaluate the clinical efficacy of improved Roux-en-Y reconstruction after Totally laparoscopic total gastrectomy (LTG) for gastric cancer. Methods: Clinical data of 36 patients who underwent totally laparoscopic total gastrectomy with intracorporeal Roux-en-Y reconstruction for gastric cancer with complete follow-up data between January 2014 and December 2014 in the Second Hospital of Jilin University. Patients were divided into modified Roux-en-Y group (MRY 20 cases), classic Roux-en-Y group (CRY 16 cases) according to reconstructive methods. Results: All cases were successfully completed, without conversion to laparotomy. There were no significant differences in lymph nodes harvest, time to flatus, hospital stay and postoperative complications between the two groups. However, the MRY group had shorter mean operative time [(260.9±21.2) vs . (287.9±19.0) min, P=0.000], shorter mean reconstruction duration [(32.4±9.2) vs . (45.4±13.2) min, P=0.001] and less intraoperative bleeding [(50.9±23.5 vs . (67.0±20.5) mL, P=0.000]. The dissection of the mesentery of the jejunum and the jejunum resection were not needed in the MRY group. Conclusions: The Modified Roux-en-Y reconstruction (MRY) is feasible and safe. It can short the mean operative time, simplify the surgical procedures.