PTOV1-AS1 desensitizes colorectal cancer cells to 5-FU through depressing miR-149-5p to activate the positive feedback loop with Wnt/ß-catenin pathway.

5-Fluorouracil is a commonly used chemotherapy drug for colorectal cancer. Resistance to 5-Fluorouracil remains a challenge. This research aimed to explore the mechanism of 5-Fluorouracil resistance in colorectal cancer. RT-qPCR and Western blot were used to determine the RNA and protein expression in both cells and exosome. Assays in vitro and in vivo were performed to measure the role of miR-149-5p in colorectal cancer cells. RIP, luciferase activity report, and RNA pulldown assay were applied to detect the association of PTOV1-AS1, SUV39H1, miR-149-5p, and FOXM1. MiR-149-5p was down-expressed in 5-Fluorouracil-resistant cells. MiR-149-5p enhanced the effectiveness of 5-Fluorouracil both in vitro and in vivo. Sensitive colorectal cancer cells released exosomal miR-149-5p to sensitize resistant cells to chemotherapy. Mechanistically, miR-149-5p targeted the FOXM1 to inactivate Wnt/ß-catenin pathway, and PTOV1-AS1 recruited SUV39H1 to suppress miR-149-5p transcription, in turn activating Wnt/ß-catenin pathway, and forming a positive feedback loop with FOXM1. PTOV1-AS1 inhibits miR-149-5p by a positive feedback loop with FOXM1-mediated Wnt/ß-catenin pathway, which provides insights into a potential novel target for enhancing the effectiveness of chemotherapy in colorectal cancer patients.

MicroRNA-149 Increases the Sensitivity of Colorectal Cancer Cells to 5-Fluorouracil by Targeting Forkhead Box Transcription Factor FOXM1.

BACKGROUND/AIMS: Previously, we have shown that microRNA (miR)-149 suppresses the migration and invasion of colorectal cancer (CRC) cells by targeting forkhead box transcription factor (FOXM1). However, the roles of miR-149 in the chemoresistance of CRC cells to 5-Fluorouracil (5-FU) is unclear. The aim of this study is to investigate whether miR-149 targets FOXM1 to regulate the 5-FU resistance of CRC. METHODS: The qRT-PCR assay was performed to detect the expression of miR-149 in 5-FU-resistant CRC cells (HCT-8/5-FU and LoVo/5-FU) and their parental CRC cells (HCT-8 and LoVo). Also, the effects of miR-149 expression on the sensitivity of CRC cells to 5-FU were determined by gain- and loss-of-function assays. Finally, whether miR-149 regulates the 5-FU resistance of CRC cells by targeting the mammalian Forkhead Box M1 (FOXM1) was investigated. RESULTS: The expression of miR-149 was significantly downregulated in 5-FU-resistant CRC cells in comparison with their parental CRC cells. Re-expression of miR-149 could enhance the 5-FU sensitivity of 5-FU-resistant CRC cells by increasing 5-FU-inducing apoptosis, while downregulation of miR-149 could decrease the 5-FU sensitivity of parental CRC cells by decreasing 5-FU-inducing apoptosis. In addition, the luciferase assay indicated that miR-149 could bind to the 3'-UTR sequence of FOXM1 mRNA. The silencing of FOXM1 could mimic the effect of miR-149 upregulation on the 5-FU resistance of 5-FU-resistant CRC cells. Furthermore, the expression of miR-149 in the 5-FU-responding CRC tissues was significantly higher than that in the non-responding tissues and inversely correlated with FOXM1 mRNA level. CONCLUSIONS: MiR-149 reverses the resistance of CRC cells to 5-FU by directly targeting FOXM1. Thus, targeting miR-149/FOXM1 signaling will be a potential strategy in the treatment of 5-FU-chemoresistant CRC.

MicroRNA-149 suppresses colorectal cancer cell migration and invasion by directly targeting forkhead box transcription factor FOXM1.

BACKGROUND/AIMS: The aim of this study is to investigate the clinicopathological and prognostic values of miR-149 expression and its roles in colorectal cancer (CRC) progression. METHODS: qRT-PCR was performed to detect miR-149 expression in CRC cell lines or tissues. Also, the clinical significance of miR-149 expression was investigated. The study further explored whether miR-149 inhibits migration and invasion of CRC cells by targeting the mammalian Forkhead Box M1 (FOXM1). RESULTS: miR-149 was significantly downregulated in CRC tissues, and low miR-149 expression was observed to be significantly correlated with lymph node or distant metastasis and advanced TNM stage of CRC patients. Patients with low miR-149 expression showed poorer prognosis than those with high miR-149 expression, and multivariate analyses indicated that status of miR-149 expression might be an independent prognostic factor. Gain- and loss - of - function assays indicated that miR-149 significantly inhibited growth, migration and invasion of CRC cells by targeting FOXM1. Furthermore, FOXM1 was significantly uiregulated in CRC tissues and inversely correlated with miR-149 expression. CONCLUSIONS: mR-149 was an independent prognostic factor and could inhibit migration and invasion of CRC cells, at least partially by targeting FOXM1.

The incidence of second primary cancer in male and female patients with initial colorectal cancer: a SEER population-based study.

BACKGROUND: Second primary cancer (SPC) after primary colorectal cancer (CRC), emerges as a novel challenge for cancer prevention with pronounced differences between female and male patients. METHODS: This was a retrospective study of 140 907 CRC survivors from the surveillance, epidemiology, and end results program database. Competing risk models and nomograms were constructed to predict the risk of SPCs, which were assessed with the C-Index, calibration and decision curve analysis. RESULTS: The 10-year cumulative incidence of SPC was higher in male than in female CRC survivors. The top five common SPCs in female CRC survivors were colorectal, breast, lung and bronchus, corpus and uterus and pancreatic cancers, while in male were prostate, colorectal, lung and bronchus, urinary cancer and melanoma of the skin. Breast and prostate were the most common sites for the development of SPCs after CRC. Older age, stage I and surgery were common risk factors for SPCs in both female and male. The nomogram for predicting the risk of developing SPC-breast cancer in female patients included age, race, site, histology grade, surgery, chemotherapy and stage. However, the model of predicting SPC-prostate cancer in male patients included age, race, site, size, surgery, chemotherapy, radiation and stage. Notably, the nomograms were validated to have a precise discriminative ability, accuracy and clinical effectiveness. CONCLUSIONS: The study surveyed the characteristics of CRC survivors with a particular focus on the incidence of SPC. The models could help supervise the development of a second breast or prostate cancer in female or male CRC survivors.

Lymph node status and its impact on the prognosis of left-sided and right-sided colon cancer: A SEER population-based study.

BACKGROUND: Some significant differences exist between the outcomes of left- and right-sided colon cancer patients. The presence of nodal metastases is a critical prognostic factor, especially in the absence of distant metastasis. Our research studied the lymph nodes status of left- and right-sided colon cancer patients to determine the influence of this factor on prognosis. METHODS: Our data were obtained from the Surveillance, Epidemiology and End Results (SEER) database. We used the chi-square test to analyze the clinicopathological characteristics. The X-tile program was adopted to acquire optimal cutoff points of lymph node index. Kaplan-Meier curves were used to analyze prognosis and multivariate Cox regression models were performed to identify the independent factors associated with survival. Nomograms were built to predict the overall survival of patients, Harrell's C-index and calibration plots were used to validate the nomograms. RESULTS: The study included 189,941 patients with colon cancer without metastasis (left 69,885, right 120,056) between 2004 and 2015. There are more patients with adequate examined lymph nodes in right-sided. Lymph node status in patients with right colon cancer has a more significant impact on the risk of death. LODDS (C-index: 0.583; AIC: 6875.4) was used to assess lymph node status. The nomograms showed that lymph node status was the main factor to predict the outcome in right-sided colon patients. CONCLUSIONS: The influence of lymph node status on predicting prognosis is significantly different between patients with left and right colon cancer without metastasis. The tumor site needs to be considered when lymph node status is used to assess the outcome of patients.

Activation of CD3+ T cells by Helicobacter pylori DNA vaccines in potential immunotherapy of gastric carcinoma.

Most of gastric carcinoma (GC) is attributed to infection by Helicobacter pylori (H. pylori) but there is increasing evidence that the positive H. pylori status correlates with better prognosis in GC. The H. pylori-induced cellular immune response may suppress cancer and in this work, recombinant pcDNA3 plasmids encoding various fragments of H. pylori virulence genes of cagA, vacA and babA are constructed and combined into groups to immunize BALB/c mice. The activated splenic CD3+ T cells are purified and the anticancer effects are investigated in vitro and in vivo. The H. pylori DNA vaccines induce a shift in the response from Th1 to Th2 that mimicks the immune status in patients of GC with chronic H. pylori infection. The stimulated CD3+ T cells inhibit the growth of human GC cells in vitro and adoptive transfusions of the CD3+ T cells suppress the growth of GC xenograft in vivo. The effects may be caused by the larger ratios of infiltrated CD8+/CD4+ T cells, reduced infiltration of regulatory FOXP3+ T cells, and enhanced apoptosis induced by upregulation of Caspase-9/Caspase-3 and downregulation of Survivin. Our results reveal the potential immunotherapeutic value of H. pylori vaccine-activated CD3+ T cells in those with advanced GC.

Dysregulation of miR-6868-5p/FOXM1 circuit contributes to colorectal cancer angiogenesis.

BACKGROUND: Transcription factor forkhead box M1 (FOXM1) is a crucial regulator in colorectal cancer (CRC) progression. However, the regulatory mechanisms causing dysregulation of FOXM1 in CRC remain unclear. METHODS: Dual-luciferase reporter assay was conducted to determine FOXM1 as miR-6868-5p target. The function of miR-6868-5p and FOXM1 in CRC angiogenesis was verified in vitro. Intratumoral injection model was constructed to explore the effect of miR-6868-5p on angiogenesis in vivo. Chromatin immunoprecipitation assays were used to assess direct binding of H3K27me3 to the miR-6868 promoter. RESULTS: Through integrated analysis, we identified miR-6868-5p as the potent regulator of FOXM1. Overexpression of miR-6868-5p in CRC cells inhibited the angiogenic properties of co-cultured endothelial cells, whereas silencing of miR-6868-5p had opposite effects. In vivo delivery of miR-6868-5p blocked tumor angiogenesis in nude mice, resulting in tumor growth inhibition. Rescue of FOXM1 reversed the effect of miR-6868-5p on tumor angiogenesis. Further mechanistic study revealed that FOXM1 promoted the production of IL-8, which was responsible for the miR-6868-5p/FOXM1 axis-regulated angiogenesis. Reciprocally, FOXM1 inhibited miR-6868-5p expression through EZH2-mediated H3K27me3 on miR-6868-5p promoter, thus forming a feedback circuit. Clinically, the level of miR-6868-5p was downregulated in CRC tissues and inversely correlated with microvessel density as well as levels of FOXM1 and IL-8 in tumor specimens. CONCLUSIONS: Together, these data identify miR-6868-5p as a novel determinant of FOXM1 expression and establish a miR-6868-5p/FOXM1 regulatory circuit for CRC angiogenesis, providing potential target for CRC treatment.

Inhibition of CXCL12/CXCR4 axis as a potential targeted therapy of advanced gastric carcinoma.

The whole outcome for patients with gastric carcinoma (GC) is very poor because most of them remain metastatic disease during survival even at diagnosis or after surgery. Despite many improvements in multiple strategies of chemotherapy, immunotherapy, and targeted therapy, exploration of novel alternative therapeutic targets is still warranted. Chemokine receptor 4 (CXCR4) and its chemokine ligand 12 (CXCL12) have been identified with significantly elevated levels in various malignancies including GC, which correlates with the survival, proliferation, angiogenesis, and metastasis of tumor cells. Increasing experimental evidence suggests an implication of inhibition of CXCL12/CXCR4 axis as a promising targeted therapy, although there are rare trials focused on the therapeutic efficacy of CXCR4 inhibitors in GC until recently. Therefore, it is reasonable to infer that specific antagonists or antibodies targeting CXCL12/CXCR4 axis alone or combined with chemotherapy will be effective and worthy of further translational studies as a potential treatment strategy in advanced GC.

Rare Gingival Metastasis by Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) uncommonly metastasizes to the gingiva, which always means a poor outcome. We reported a rare HCC case with multiple metastases to gingiva, lungs, and brain. A 60-year-old man was initially diagnosed as HCC with metastases to double lungs. He was subjected to a transarterial chemoembolization (TACE) (5-fluorouracil, 750 mg) and two cycles of intravenous chemotherapy (gemcitabine 1.8 g at days 1 and 8, oxaliplatin 200 mg at day 2, every 4 weeks). However, the volume of liver tumor still increased. A bean-size gingival nodule growing with occasional bleeding was also found. TACE (5-fluorouracil 750 mg, perarubicin 40 mg, cisplatin 20 mg) was performed again and an oral sorafenib therapy (400 mg, twice per day) was adopted. The disease maintained relatively stable for about 6 months until a second obvious progress. The gingival nodule was then palliatively excised and identified as a poorly differentiated metastatic HCC by histopathological examination. Best supportive treatments were made since the performance score was too bad. Finally, cerebral metastases occurred and the patient died of systemic failure. Upon review of previous reports, we discussed risk factors, clinical and pathological characteristics, treatments, and prognosis of gingival metastasis by HCC.

FOXM1 evokes 5-fluorouracil resistance in colorectal cancer depending on ABCC10.

5-Fluorouracil (5-FU) is the most commonly used chemotherapeutic agent for colorectal cancer (CRC). However, frequently occurred 5-FU resistance poses a great challenge in the clinic. Elucidating the underlying mechanisms and developing effective strategies against 5-FU resistance are highly desired. Here we identified the upregulation of FOXM1 in 5-FU nonresponsive CRC patients by gene expression profile analysis and 5-FU-resistant CRC cells by qRT-PCR assay. Silencing of FOXM1 promoted the sensitivity of CRC cells to 5-FU by enhancing cell apoptosis, while overexpression of FOXM1 conferred CRC cells with 5-FU resistance both in vitro and in vivo. Furthermore, we showed that genetic and pharmacological inhibition of FOXM1 resensitized resistant CRC cells to 5-FU treatment. Mechanistically, FOXM1 promoted the transcription of ABCC10 by directly binding to its promoter region. Notably, treatment with ABCC10 inhibitor reversed FOXM1-induced resistance to 5-FU in vivo. Clinical investigation revealed that the levels of FOXM1 and ABCC10 were positively correlated in CRC tissues. Therefore, FOXM1 promotes 5-FU resistance by upregulating ABCC10, suggesting that FOXM1/ABCC10 axis may serve as a potential therapeutic target for 5-FU resistance in CRC patients.

Expression of chemokine receptor CXCR7 in colorectal carcinoma and its prognostic significance.

Previous studies have shown that chemokine receptor CXCR7 plays critical roles in tumor development. However, the clinicopathological and prognostic significance of CXCR7 in colorectal carcinoma (CRC) has not been fully understood. The aim of our study is to investigate the expression of CXCR7 and its clinical significance in CRC. First, quantitative RT-PCR and Western blot assays were performed to determine the expression of CXCR7 mRNA and protein in 20 paired of CRC tissues and corresponding adjacent non-tumor tissues. Next, immunohistochemistry was performed to detect the expression of CXCR7 protein in another 96 cases of CRC tissues, and analyze its correlation with clinicopathological factors of patients. Finally, the correlation of CXCR7 with 5-year overall survival (OS) and progression free survival (PFS) was statistically analyzed by the Kaplan-Meier method and Cox proportional hazards model. Results showed that the expression levels of CXCR7 mRNA and protein were significantly higher in CRC tissues than in normal tissues. Positive CXCR7 expression was observed to be significantly correlated with lymph nodal metastasis (P < 0.001), distant metastasis (P = 0.017), and advanced TNM stage (P < 0.001). Patients with positive expression of CXCR7 were demonstrated to be associated with worse OS and PFS (P < 0.001, P < 0.001, respectively). Moreover, multivariate survival analysis revealed that CXCR7 expression level might be an independent predictive factor for OS and PFS of CRC patients. Collectively, positive CXCR7 expression in CRC was correlated with tumor development and poor prognosis of patients.

[Genome-wide screening of CpG island methylation changes induced by low-dose X-ray radiation in mice].

OBJECTIVE: To study the methylation changes in promoter CpG islands induced by low-dose X-ray radiation (LDR). METHODS: Twenty male BALB/c mice were randomly divided into control and fractionated radiation group exposed to 6 MV X-ray for 10 days (0.05 Gy/day). All the mice were sacrificed 2 h after the last radiation on day 10, and blood samples were collected for detecting DNA methylation changes using Roche-NimbleGen mouse DNA methylation 3×720K Promoter Plus CpG Island Array. MeDIP-qPCR was used to further validate the methylation status of specific genes. RESULTS: A total of 811 genes were found to show specific hypermethylation in fractional radiation group as compared with the control group, involving almost all the main biological processes by GO analysis. Eight candidate genes (Rad23b, Tdg, Ccnd1, Ddit3, Llgl1, Rasl11a, Tbx2, and Slc6a15) were confirmed to be hypermethylated in LDR samples by MeDIP-qPCR, consistent with the results of the methylation chip study. CONCLUSION: LDR induces promoter hypermethylation on specific genes, which may contribute to radiation-induced pathogenesis.

Genome-wide screen of DNA methylation changes induced by low dose X-ray radiation in mice.

Epigenetic mechanisms play a key role in non-targeted effects of radiation. The purpose of this study was to investigate global hypomethylation and promoter hypermethylation of particular genes induced by low dose radiation (LDR). Thirty male BALB/c mice were divided into 3 groups: control, acutely exposed (0.5 Gy X-rays), and chronic exposure for 10 days (0.05Gy/d×10d). High-performance liquid chromatography (HPLC) and MeDIP-quantitative polymerase chain reaction (qPCR) were used to study methylation profiles. DNMT1 and MBD2 expression was determined by qPCR and western blot assays. Methylation and expression of Rad23b and Ddit3 were determined by bisulfate sequencing primers (BSP) and qPCR, respectively. The results show that LDR induced genomic hypomethylation in blood 2 h postirraditaion, but was not retained at 1-month. DNMT1 and MBD2 were downregulated in a tissue-specific manner but did not persist. Specific hypermethylation was observed for 811 regions in the group receiving chronic exposure, which covered almost all key biological processes as indicated by GO and KEGG pathway analysis. Eight hypermethylated genes (Rad23b, Tdg, Ccnd1, Ddit3, Llgl1, Rasl11a, Tbx2, Scl6a15) were verified by MeDIP-qPCR. Among them, Rad23b and Ddit3 gene displayed tissue-specific methylation and downregulation, which persisted for 1-month postirradiation. Thus, LDR induced global hypomethylation and tissue-specific promoter hypermethylation of particular genes. Promoter hypermethylation, rather than global hypomethylation, was relatively stable. Dysregulation of methylation might be correlated with down-regulation of DNMT1 and MBD2, but much better understanding the molecular mechanisms involved in this process will require further study.

Expression and clinical significance of CD73 and hypoxia-inducible factor-1α in gastric carcinoma.

AIM: To investigate the expression of CD73 and hypoxia-inducible factor-1α (HIF-1α) in human gastric carcinoma, and explore their clinical significance and prognostic value. METHODS: CD73 and HIF-1α expressions were detected by immunohistochemistry in consecutive sections of tissue samples from 68 gastric carcinoma patients. The peritumor tissues 2 cm away from the tumor were obtained and served as controls. The presence of CD73 and HIF-1α was analyzed by immunohistochemistry using the Envision technique. RESULTS: CD73 and HIF-1α expressions in gastric carcinoma were significantly higher than those in gastric mucosal tissues as control (P < 0.001) and showed a close correlation (Spearman r = 0.390, P = 0.001). Overexpression of CD73 was positively correlated with differentiation of tumor (P = 0.000), histopathology (P = 0.041), depth of invasion (P < 0.001), nodal status (P = 0.003), metastasis (P = 0.013), and the American Joint Committee on Cancer (AJCC) stage (P < 0.001). High expression of HIF-1α was positively correlated with tumor diameter (P = 0.031), depth of invasion (P = 0.022), and AJCC stage (P = 0.035). The overall survival rate was low in the patients with high expression of CD73 (P < 0.001). Moreover, CD73+/HIF-1α+ patients had the worst prognosis (P < 0.001). CD73 expression was proven to be an independent predictor for patients with gastric carcinoma by both multivariate Cox regression analysis (P = 0.021) and receiver operating characteristic curves (P = 0.001). CONCLUSION: CD73 expression correlates closely with HIF-1α expression in gastric carcinoma. CD73 could be an independent prognostic indicator for gastric carcinoma.

Rapid Remission in Peripheral T-Cell Lymphoma of the Nasal Type by the Bortezomib plus CHOP Therapy.

Peripheral T-cell lymphoma (PTCL) is rare and difficult to treat for its high relapse rate. The authors report a case of PTCL of the skin, regarding which clinical and pathological features, treatment, and prognosis were discussed. A 66-year-old woman was admitted with complaints of enlarging erythematous noduloplaques on the right anterior tibial skin for one year and similar lesions on the left for 6 months. Surgical resection of right leg lesion and biopsy of enlarged inguinal lymph nodes histologically indicated a PTCL of the nasal type. The patient was treated by CHOP plus bortezomib, reached complete remission just after two courses of chemotherapy and then received another two as consolidation. The patient remained in remission for 11 months until local relapse. As for cutaneous lesions, detailed lymph node examination and prompt tissue biopsy are judicious choices prior to any medical management. The chemotherapy consisting of bortezomib and CHOP is safe and efficient in PTCL of the skin.

[Measurement of serum free light chains and its clinical significance in 20 newly diagnosed patients of multiple myeloma].

The objective of this study was to explore the clinical significance of measuring serum free light chains (sFLC) and to compare with serum total light chains (free and binded) in multiple myeloma (MM). sFLC in 20 newly diagnosed MM patients and 20 cases of healthy people as control were measured by immuno-nephelometric assays; the serum light chains and kappa/lambda ratio were measured in all patients, while immunofixation electrophoresis (IFE) tests were carried out at the same time in 18 out of 20 patients. The results showed that the abnormality of serum free light chains and kappa/lambda ratio were found in all of the 20 newly diagnosed MM patients (p < 0.01). The measurement of sFLC showed higher sensitivity than the total serum chains (p < 0.01). It is concluded that the method testing sFLC by immuno-nephelometric assay combined with kappa/lambda ratio is valuable for MM diagnosis, and the measurement of sFLC can be used as one of indicators for MM diagnosis.