Upregulation of lncRNA CALML3-AS1 promotes cell proliferation and metastasis in cervical cancer via activation of the Wnt/ß-catenin pathway.

OBJECTIVE: It is well verified that lncRNA are emerging as imperative regulators in various tumors. LncRNA CALML3-AS1 (CALML3-AS1), a freshly discovered lncRNA, has been confirmed as a tumor promoter in bladder cancer. This present study aimed to explore the biological functions and molecular mechanisms of CALML3-AS1 in cervical cancer (CC). PATIENTS AND METHODS: We analyzed RNA sequencing data from The Cancer Genome Atlas (TCGA) datasets to determine dysregulated lncRNAs in CC. Real Time-Polymerase Chain Reaction (RT-PCR) was applied for the assays of CALML3-AS1 amplification in CC samples and cell lines. Kaplan-Meier analysis and multivariate assays were carried out for determination of the prognostic values. The functions of CALML3-AS1 on cell proliferation, invasion, migration, and apoptosis were determined by a series of cells experiments by knocking down CALML3-AS1. MRNA and protein expressions of signaling pathways were examined using Western blot. RESULTS: We found that CALML3-AS1 was upregulated in CC tissues and this upregulation was associated with FIGO stage, histological grade, and reduced overall survival. Multivariate assays indicated that high CALML3-AS1 expression was an independent prognostic parameter indicating poorer clinical outcome for CC patients. Functional assays suggested that knockdown of CALML3-AS1 suppressed the proliferation, migration, and invasion of CC cells, and induced apoptosis. Mechanistic investigations revealed that inhibiting the expression of CALML3-AS1 decreased the levels of ß-catenin, cyclin D1, and c-myc via Western blot. CONCLUSIONS: Our study revealed that CALML3-AS1 could be an oncogenic lncRNA promoting the growth and metastasis of CC by modulating Wnt/ß-catenin pathway, suggesting that CALML3-AS1 may be an important contributor to CC progression.

An in vivo model for the study of ovarian cancer and the persistence of characteristic mutations in xenografts.

OBJECTIVE: To identify factors affecting xenograft growth of epithelial ovarian cancer (EOC) cells in nude mice and to detect characteristic mutations occurring in the xenografts following serial passage. MATERIALS AND METHODS: A total of 64 human EOCs were subcutaneously inoculated in Balb/c nude mice in order to obtain a series of xenografts. Whole-exome sequencing was analyzed with Agilent SureSelect targeted enrichment capture system and Illumina Solexa Hiseq 2000 sequencing platform. Mutations were confirmed by comparison against the reference genome build 37.3. RESULTS: The tumor take rate was 50% (32/64). TP53 mutation was detected in nine often Type II tumors. BRAF and CTNNB1 were not mutated in any of the samples, and PTEN mutation occurred in only one sample. The present data indicate that advanced stage serous EOCs and early stage non-serous EOCs were easy to grow in nude mice, and xenografts maintained the characteristic mutations. CONCLUSIONS: Advanced stage serous EOCs and early stage non-serous EOCs were easy to grow in nude mice, and xenografts maintained the characteristic mutations. Xenografts in nude mice are useful in vivo models for the study of human EOCs.

The prognostic significance of APC gene mutation and miR-21 expression in advanced-stage colorectal cancer.

AIM: Colorectal cancer (CRC) is the second commonest cause of cancer death in Taiwan. Although numerous genes have been associated with tumorigenesis in colorectal cancer, only a few have been validated and used as biomarkers for predicting clinical outcome. The aim of this study was to analyse the association of APC gene mutation and miR-21 expression with clinical outcome in CRC patients. METHOD: In total, 195 colorectal cancer patients were enrolled in a single medical centre between 2003 and 2007. APC gene mutation and expression of APC and miR-21 were analysed by direct DNA sequencing and real-time reverse transcription polymerase chain reaction. The primary outcome included 5-year overall survival and univariate (Kaplan-Meier) and multivariate (Cox regression) analyses of prognostic factors. RESULTS: The results showed that 66 (33.8%) of 195 tumour tissues contained an APC mutation. The predominant APC gene variations were deletion mutations (50.0%). APC gene expression was low in CRC and negatively correlated with miR-21 expression and gene mutation. In advanced-stage cancer, patients with APC mutation/high miR-21 had poorer overall survival rates than those with APC mutation/low miR-21, APC wild-type/high miR-21 and APC wild-type/low miR-21. CONCLUSION: In Taiwan, downregulation of the APC gene in CRC correlated with gene mutation and miR-21 upregulation. APC mutation and miR-21 expression could be used to predict the clinical outcome of CRC, especially in patients with advanced disease.

Hyperexcitability in sensory neurons of rats selected for high versus low neuropathic pain phenotype.

Selection line rats congenitally high or low for autotomy in the neuroma model of neuropathic pain (HA and LA rats) were found to be correspondingly high and low in a second type of neuropathic pain, the Chung model, which employs an alternative phenotypic endpoint, tactile allodynia. It has been proposed that both phenotypes reflect ectopic hyperexcitability in axotomized primary sensory neurons. To test this hypothesis we made in vitro recordings from sensory neurons in the L4 and 5 dorsal root ganglia. Baseline excitability was similar in HA and LA rats, and axotomy caused an increase in both lines. However, in the one neuronal subclass previously linked to neuropathic pain in these models the increase was significantly greater in HA than LA rats, and only at the time when pain scores in the two lines were diverging. Heritable differences in electrical response to axotomy in a specific afferent cell type appear to be a fundamental determinant of neuropathic pain.

Effects of nicotine on spontaneous and amphetamine-induced motor behaviors: the differences between nicotine tolerant and nontolerant rats.

This study was designed to investigate the effect of nicotine on spontaneous and amphetamine (AMP)-induced motor activity in rats with or without tolerance to nicotine. Tolerance were induced by treating the rats with nicotine (0.3 mg/kg, s.c.) 2 hr before receiving challenge doses. Motor activity including locomotion and stereotypy was monitored automatically by videocamera every 15 min for 90 min. The results indicated that: (1) Nicotine increased spontaneous locomotion at 0.15 or 0.3 mg/kg (s.c.) in naive rats and at 0.6 mg/kg in tolerant rats. Nicotine also slightly affected AMP-induced locomotion at 0.15, 0.3 or 0.6 mg/kg in both naive and tolerant rats, and (2) Nicotine increased spontaneous stereotypy at 0.3 or 0.6 mg/kg in naive rats only and showed no effect on AMP-induced stereotypy in either naive or tolerant rats. Comparing the results of spontaneous motor activity between naive and tolerant rats, it revealed behavioral desensitization in locomotion at low doses (0.15 or 0.3 mg/kg) and hyperlocomotion at higher dose (0.6 mg/kg), and revealed desensitization in stereotypy at 0.3 or 0.6 mg/kg. Moreover, nicotine had temporary (at 0-15 min interval) attenuating effect on AMP-induced locomotion in naive rats but showed a potentiating effect on AMP-induced locomotion in tolerant rats. The present results indicated that acute tolerance modified the action of nicotine in both spontaneous and AMP-induced locomotion, while stereotypy was changed only in the spontaneous one but not in the AMP-induced one. In other words, acute tolerance modified the effect of nicotine on locomotion-related dopaminergic system, and it affected the stereotypy-related dopaminergic system only in the spontaneous one but not in the AMP-induced one.

[Cultivation of urologic and male genital tumor cells].

Thirty specimens from 30 patients with urologic and male genital system tumors were cultured. Eighteen (60%) showed initial cell growth. Four (13%) were successful in primary culture. In a renal cell carcinoma and a bladder transitional cell carcinoma, the subcultures were successful after the primary culture. In eight months, having transferred the former for twelve generations and the latter for twenty-seven generations, these two cell lines are established. The morphological characters of the cultured cells, eradication of fibroblasts, conditions of culture and prevention of contamination are discussed.