Exosomes Mediated Transfer of Circ_0000337 Contributes to Cisplatin (CDDP) Resistance of Esophageal Cancer by Regulating JAK2 via miR-377-3p.

Background: Chemoresistance remains a major obstacle to the treatment of esophageal cancer patients. Exosome-mediated transfer of circular RNAs (circRNAs) has been reported to be related to drug resistance in esophageal cancer. This study is designed to explore the role and mechanism of exosomal circ_0000337 on CDDP resistance in esophageal cancer. Methods: Cell viability, proliferation, colony number, apoptosis, migration, and invasion were assessed by Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry, and transwell assays. Circ_0000337, microRNA-377 (miR-377-3p), and Janus kinase 2 (JAK2) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Exosomes were isolated and detected by differential centrifugation and a transmission electron microscope. Protein levels of CD9, CD63, and JAK2 were tested by Western blot assay. The binding relationship between miR-377-3p and circ_0000337 or JAK2 was predicted by circinteractome or Starbase and then verified by dual-luciferase reporter assay and RNA pull-down assay. The biological role of exosomal circ_0000337 and CDDP on esophageal cancer cell growth was examined by the xenograft tumor model in vivo. Results: Circ_0000337 and JAK2 were highly expressed, and miR-377-3p was decreased in CDDP-resistant esophageal cancer tissues and cells. Moreover, circ_0000337-containing exosomes secreted by CDDP-resistant esophageal cancer cells could promote CDDP resistance, cell growth, and metastasis in CDDP-sensitive esophageal cancer cells in vitro. The mechanical analysis discovered that circ_0000337 functioned as a sponge of miR-377-3p to regulate JAK2 expression. Exosomal circ_0000337 increased the drug resistance of esophageal cancer in vivo. Conclusion: Exosomal circ_0000337 accelerated CDDP resistance of esophageal cancer cells partly by regulating the miR-377-3p/JAK2 axis, hinting a promising therapeutic target for the esophageal cancer treatment.

ZFPM2-AS1 transcriptionally mediated by STAT1 regulates thyroid cancer cell growth, migration and invasion via miR-515-5p/TUSC3.

Objective: Our purpose was to study the roles and molecular mechanisms of long non-coding RNA (lncRNA) ZFPM2 Antisense RNA 1 (ZFPM2-AS1) in thyroid cancer. Methods: Firstly, the expression of ZFPM2-AS1, miR-515-5p and TUSC3 was detected in thyroid cancer tissues and cells. Secondary, their biological functions (proliferation, apoptosis, migration and invasion) were analyzed by a serious of functional experiments including cell counting kit-8 (CCK-8), clone formation, 5-Ethynyl-2'-deoxyuridine (EdU), enzyme-linked immunosorbent assay (ELISA), wound healing and Transwell assays. Thirdly, the mechanisms of STAT1/ZFPM2-AS1 and ZFPM2-AS1/miR-515-5p/TUSC were validated using chromatin immunoprecipitation (CHIP), pull-down and luciferase reporter assays. Results: ZFPM2-AS1 and TUSC were both highly expressed and miR-515-5p was down-regulated in thyroid cancer tissues as well as cells. Their knockdown weakened thyroid cancer cell growth, migration, and invasion. ZFPM2-AS1 was mainly distributed in the nucleus and cytoplasm of thyroid cancer cells. Mechanistically, up-regulation of ZFPM2-AS1 was induced by transcription factor STAT1 in line with CHIP and luciferase reporter assays. Furthermore, as a sponge of miR-515-5p, ZFPM2-AS1 decreased the ability of miR-515-5p to inhibit TUSC3 expression by pull-down, luciferase reporter and gain-and-loss assays, thereby promoting malignant progression of thyroid cancer. Conclusion: ZFPM2-AS1 acted as an oncogene in thyroid cancer, which was transcriptionally mediated by STAT1. Furthermore, ZFPM2-AS1 weakened the inhibitory effect of miR-515-5p on TUSC3. Thus, ZFPM2-AS1 could be an underlying biomarker for thyroid cancer.

Association of High Expression of Mitochondrial Fission Regulator 2 with Poor Survival of Patients with Esophageal Squamous Cell Carcinoma.

Mitochondrial fission regulator 2 (MTFR2) is associated with mitochondrial fission, while few studies have assessed the associations between MTFR2 expression and clinical characteristics or prognosis of esophageal squamous cell carcinoma (ESCC). In this study, we compared the expression of MTFR2 in 6 ESCC tumors and relative normal tissues by immunohistochemistry (IHC). To assess the effect of MTFR2 expression on clinicopathologic characteristics and survival, 115 paraffin embedded ESCC tissue samples were assessed by IHC staining. Furthermore, the association between clinicopathological properties and MTFR2 expression in patients with ESCC was examined. The survival analysis was performed using the Cox regression models. We found that MTFR2 expression was significantly increased in ESCC tumors compared with normal esophageal epithelial cells. IHC analysis of 115 paraffin embedded ESCC tumor specimens of the patients showed that the expression of MTFR2 was significantly associated with clinical stage (P < 0.001), tumor classification (P < 0.001), histological grade (P < 0.001), and other clinicopathological characteristics. Both univariate and multivariate analyses showed that MTFR2 expression was inversely correlated with the survival of ESCC patients. In conclusion, the expression of MTFR2 is significantly associated with clinicopathologic characteristics and prognosis of ESCC. Thus, MTFR2 expression could serve as a potentially important prognostic biomarker and clinical target for patients with ESCC.

Overexpression of MTFR2 Predicts Poor Prognosis of Breast Cancer.

BACKGROUND: Mitochondrial fission regulator 2 (MTFR2) has been reported to promote proliferation, migration and invasion in tumors; however, little is known about its function in breast cancer. Thus, we investigated the effect of MTFR2 expression on prognosis of breast cancer. METHODS: The expression of MTFR2 in breast cancer tissues was detected by immunohistochemistry, and overall survival (OS) and recurrence free survival (RFS) were evaluated by the Log rank test and Cox model. RESULTS: We found that MTFR2 expression was significantly associated with clinical stage (P<0.001), T classification (P=0.005), N classification (P=0.001), M classification (P=0.041), HER2 expression (P= 0.001), and molecular subtypes (P=0.002), respectively. Compared with low MTFR2 expression, the patients with higher expression of MTFR2 exhibited significantly shorter OS and RFS (All P < 0.001). Both univariate and multivariate analyses showed that MTFR2 was an independent prognostic factor for OS (HR, 2.8, 95% CI 1.1-6.8, P = 0.023) and RFS (HR, 2.8, 95% CI 1.2-6.4, P = 0.015) in breast cancer patients. Moreover, in HER2 positive and TNBC subtype, the associations between high MTFR2 expression and poor OS and RFS were more pronounced. CONCLUSION: Taken together, our results demonstrated that high MTFR2 expression was associated with poor prognosis of breast cancer patients, and such an association was more pronounced in the patients with aggressive tumors. Therefore, MTFR2 expression might be a potentially important prognostic biomarker and clinical target for patients with breast cancer.

Highly expressed microRNA-124 inhibits migration and promotes apoptosis of esophageal cancer cells by degrading PDCD6.

PURPOSE: To investigate whether microRNA (miR)-124 could affect the proliferation, migration and apoptosis of esophageal cancer cells and participate in the occurrence of esophageal cancer through regulating PDCD6 expression. METHODS: The expression of miR-124 in cancer tissues and adjacent tissues of esophageal cancer patients were detected by quantitative real time-polymerase chain reaction (qRT-PCR). Esophageal cancer cells TE-1 and SKYE30 were cultured. The effects of miR-124 on tumor size and metastasis were analyzed. The functions of miR-124 in cell proliferation, migration and apoptosis were detected by cell counting kit-8 (CCK-8), transwell and flow cytometry, respectively. Rescue experiments were performed to assess whether miR-124 could regulate the proliferation, migration and apoptosis of esophageal cancer cells by inhibiting PDCD6 expression. RESULTS: The expression of miR-124 in cancer tissues of esophageal cancer patients were lower than that of the control group. In addition, its expression in patients with stage III-IV esophageal cancer was remarkably lower than that in patients with stage I-II. Based on the level of miR-124, we divided the patients into high and low expression group, and found significant differences in tumor size, tumor metastasis and lymph node metastasis between the two groups. Also, overexpression of miR-124 reduced the proliferation and migration of TE-1 and SKYSE30 cells and increased the apoptosis, and vice versa. Luciferase reporting assay results confirmed that PDCD6 was one of the target genes of miR-124. A further mechanism study demonstrated that overexpression of PDCD6 in TE-1 and SKYSE30 cells could partially reverse the effect of miR-124 on cell apoptosis. CONCLUSIONS: The low expression of miR-124 can promote the proliferation as well as migration of esophageal cancer cells and inhibit cell apoptosis. The mechanism may be that miR-124 can regulate the expression of PDCD6.

Involved-field irradiation for elderly bladder cancer patients.

OBJECTIVES: Considering that adjuvant radiation therapy is one of the most common treatment methods and the influence of the clinical target volume to treatment-related toxicity, this study aimed to observe the differences in treatment failures about involved-field irradiation (IFI) without lymph node areas versus elective nodal irradiation (ENI) with lymph node areas in elderly patients with bladder cancer. MATERIAL AND METHODS: Ninety-two elderly bladder cancer patients were analyzed from January 2010 to December 2014 in one institution. The primary inclusion criteria were previous after transurethral resection of bladder tumor or partial cystectomy with adjuvant radiotherapy, and the radiation techniques included IFI or ENI. The study required that elderly patients did not received radiotherapy before treatment. We observed treatment-related toxicity and tumor failures, evaluated local progression-free survival, estimated the 3-year overall survival, and analyzed prognostic factors, after IFI and ENI in elderly bladder cancer patients. The outcomes were determined by chi square tests, Kaplan-Meier method and Cox multiple factors analysis. RESULTS: In the experimental group, 42 patients (45.65%) received IFI, and a matched group of 50 patients (54.35%) received ENI. With a median follow-up of 31.47 months (range 4.00-86.00 months), the Kaplan-Meier analysis with a log-rank test demonstrated a statistical difference between the IFI group and the ENI group in acute toxicity (45.23% vs 72.00%, P = 0.008). However, there were no statistical differences in the 3-year overall survival rate (45.20% vs 48.00%, P = 0.860) or the duration of local progression-free survival (24.98 vs 34.30, P =0.729). CONCLUSIONS: IFI is feasible in elderly bladder cancer patients, as shown by a decrease in acute toxicity and no increase in local failure. We need a large number of clinical trials and data to further confirm these results.

Individualized Radiation Dose Escalation Based on the Decrease in Tumor FDG Uptake and Normal Tissue Constraints Improve Survival in Patients With Esophageal Carcinoma.

BACKGROUND: To determine whether individualized radiation dose escalation after planned chemoradiation based on the decrease in tumor and normal tissue constraints can improve survival in patients with esophageal carcinoma. METHODS: From August 2005 to December 2010, 112 patients with squamous esophageal carcinoma were treated with radical concurrent chemoradiation. Patients received positron emission tomography-computer tomography scan twice, before radiation and after radiation dose of 50.4 Gy. All patients were noncomplete metabolic response groups according to the Response Evaluation Criteria in solid tumors. Only 52 patients with noncomplete metabolic response received individualized dose escalation based on tumor and normal tissue constraints. Survival and treatment failure were observed and analyzed using SPSS (13.0). RESULTS: The rate of complete metabolic response for patients with noncomplete metabolic response after dose escalation reached 17.3% (9 of 52). The 2-year overall survival rates for patients with noncomplete metabolic response in the conventional and dose-escalation groups were 20.5% and 42.8%, respectively( P = .001). The 2-year local control rates for patients were 35.7% and 76.2%, respectively ( P = .002). When patients were classified into partial metabolic response and no metabolic response, 2-year overall survival rates for patients with partial metabolic response were significantly different in conventional and dose-escalation groups (33.8% vs 78.4%; P = .000). The 2-year overall survival rates for patients with no metabolic response in two groups (8.6% vs 15.1%) did not significantly differ ( P = .917). CONCLUSION: Individualized radiation dose escalation has the potential to improve survival in patients with esophageal carcinoma according to increased rate of complete metabolic response. However, further trials are needed to confirm this and to identify patients who may benefit from dose escalation.

Efficacy and safety of late-course hypofractionated radiation therapy for muscle-invasive bladder carcinoma after bladder-conserving surgery.

AIM: To evaluate the efficacy and safety of late-course hypofractionated radiation treatment of muscle-invasive bladder carcinoma after bladder-conserving surgery. METHODS: Seventy-six patients with transitional cell bladder carcinoma, stage II (T2-4N0M0), after transurethral resection, were enrolled. Pirarubicin was given at 30 mg/m2 and 100 mL physiological saline once weekly (QW) for 12 weeks through and after intravesical instillation postoperatively. Radiation schedule delivered 46 Gy in 20 fractions for planning target volume, with an additional 20 Gy in five fractions for gross tumor volume as late-course radiation. Chemotherapy was stopped if Radiation Therapy Oncology Group grade 3 or higher bladder or bowel toxicity occurred. The primary end points were acute toxicity, local control and patients' survival. RESULTS: One-, three- and five-year overall survival rates were 98, 78 and 69.5%, respectively. Mean survival time was 58.4 months (95% CI: 52.6, 64.2). In addition, 1-, 3- and 5-year local control rates were 100, 80.5 and 76.1%, respectively. Mean local control time was 60.7 months (95% CI: 55.1, 66.3). The cumulative incidence of local/regional failure and distant failure was 28.9%. The rate of single local/regional failure was 13.2%, but distant failure rate was 21.1%. CONCLUSIONS: Concurrent pirarubicin-based late-course hypofractionated radiation therapy showed desirable local control rate and acceptable toxicity. It could be used after bladder-conserving surgery to allow patients to preserve their bladder.

A genetic variant in CHRNB3-CHRNA6 increases risk of esophageal squamous cell carcinoma in Chinese populations.

Nicotinic acetylcholine receptors are important regulators of smoking behavior and tobacco carcinogenesis. We studied the association of the CHRNB3-A6 variant rs13280604 in relation to esophageal squamous cell carcinoma (ESCC) in Chinese populations. Two independent case-control studies were conducted. The first case-control study, consisted of 866 ESCC patients and 1621 healthy controls from Northern China, and the second case-control study consisted of 853 ESCC patients and 860 unrelated controls from Southern China. A logistic regression model was used to evaluate the associations of rs13280604 with cancer risk. We found that Rs13280604 GG/AG genotypes were significantly associated with increased risk for ESCC in both case-control studies from Northern [odds ratio (OR), 1.42, 95% confidence interval (CI), 1.19-1.70, P = 1.1×10(-4)], Southern China (OR, 1.56, 95% CI, 1.26-1.93, P = 5.2×10(-5)), and the combined population of both studies (OR, 1.44, 95% CI, 1.26-1.65, P = 8.7×10(-8)), respectively. Our results suggest that this CHRNB3-A6 variant confers susceptibility to ESCC risk. However, future larger studies are needed to validate our finding.

A clinical study of shrinking field radiation therapy based on (18)F-FDG PET/CT for stage III non-small cell lung cancer.

The aim is to investigate the feasibility of shrinking field technique after 40 Gy for stage III non-small cell lung cancer (NSCLC) during radiation therapy. Eighty-seven consecutive patients treated with intensity-modulated radiation therapy or three-dimensional conformal radiation therapy were enrolled in this study. (18)F-fluorodeoxyglucose positron emission tomography/computed tomography ((18)F-FDG PET/CT) scanning was performed prior to treatment and repeated after 40 Gy, and the delineation of target volume was based on fused images of PET and CT. After 40 Gy of conventional fractionated radiotherapy to the initial planning target volume (PTV), a boost of 19.6-39.2 Gy was delivered to the shrunken PTV through late course accelerated hyperfractionated radiotherapy, and the median total dose was 66.0 Gy (range, 59.6-79.2 Gy). Gross tumor volume (GTV) and PTV regressions were recorded, and prescription doses with or without shrinking field were calculated. Local recurrence patterns were investigated through follow-up. The tumor volumes regressed in 84 (96.6%) patients and increased in 3 (3.4%) patients after 40 Gy. The mean GTV and PTV reduction was 38% (range, -13-95%) and 30% (range, -5-95%). Mean total prescription dose escalated from 62.0 Gy to 68.5 Gy through shrinking field technique. The median follow-up was 17 months, ranging from 5 to 46 months, and the 1- and 2-year overall survival rates in our study were 74.7% and 34.6%. The response rate was 79.5%, and radiation toxicity was acceptable. Tumor progression occurred in 67.8% (59/87) patients. Numbers of patients who had outfield, infield and both infield and outfield recurrences were 3 (3.4%), 26 (29.5%), and 3 (3.4%), respectively. In conclusion, significant tumor regression was observed after 40 Gy, and radiation dose escalated after shrinking field with acceptable toxicity and outfield relapse. Shrinking field radiotherapy based on (18)F-FDG PET/CT after 40 Gy was safe and feasible for stage III NSCLC.

Application of serial section method to determine the radiotherapy target volume for esophageal squamous carcinoma.

The three-dimensional conformal radiotherapy (3D CRT) plays an important role in the combination treatment of esophageal carcinoma. However, an accurate estimation of the clinical target volume (CTV) of esophageal squamous carcinoma (ESCC) by 3D CRT is still problematic. This study aimed to provide reference values for CTV estimation. The serial section method was applied to observe the range of the microscopic spread proximally and distally from the tumor. Further, relationships between clinicopathological features and the microscopic spread were analyzed. The positive ratio of the proximal microscopic spread was significantly higher than in the distal spread, especially in the specimens sampled 1 and 2 cm away from the tumor (p < 0.05). Probability of infiltration and metastases was still high in the proximal "3 cm" and distal "4 cm" groups, and became much lower in more distant specimens. Further, ESCC tended to exhibit stronger ascending invasion ability. A single factor analysis showed that tumors with the length of longer than 5 cm, poorer differentiation, lymph nodes metastasis, and more aggressive phase had significantly higher microscopic spread ratio (p < 0.05). A multiple factors analysis showed that differentiation degree and tumor length were the major factors affecting the microscopic spread of ESCC. In conclusion, to cover 95 % of the microscopic spread, a proximal margin of 3.0 cm and a distal margin of 4.0 cm are needed. In order to cover 90 %, proximal and distal 3-cm margins are needed. Clinicopathological features of patients can affect the range of the microscopic spread.