Predictive power of circulating miRNAs in detecting colorectal cancer.

Many studies indicate that circulating microRNAs (miRNAs) could play important roles in screening human cancers, including colorectal cancer (CRC). However, the conflicting results on the accuracy of miRNA detection lead us to conduct this meta-analysis to access the predictive value of miRNAs for predicting CRC. Eligible studies were identified from the Medline, Embase, CNKI, and Web of Science by the search strategies and screening criterion. We used random effects models to calculate the pooled results from studies. The summary receiver operator characteristic curve (SROC) and the area under the SROC curve (AUC) were used to estimate the predictive accuracy. Subgroup analyses and meta-regression were used to analyze potential sources of heterogeneity. We used Deeks' funnel plot asymmetry test to test publication bias. This meta-analysis included a total of 24 studies from 19 articles, including 1558 CRC patients and 1085 controls. The overall pooled results from the meta-analysis were as follows: sensitivity was 0.81 (95% confidence interval (CI) 0.77-0.85), specificity was 0.84 (95% CI 0.78-0.88), PLR was 5.0 (95% CI 3.5-6.9), NLR was 0.22 (95% CI 0.18-0.28), DOR was 23 (95% CI 14-37), and AUC was 0.89 (95% CI 0.86-0.91). Subgroup and meta-regression analyses demonstrated that multiple miRNAs (AUC, sensitivity, and specificity of 0.92, 0.84, and 0.87, respectively) had a higher predictive accuracy than single miRNA (AUC, sensitivity, and specificity of 0.84, 0.78, and 0.78, respectively). In addition, we found that serum can be a better matrix for miRNA assays in screening CRC compared with plasma. In summary, our data suggests that circulating miRNAs, particularly multiple miRNAs, which have higher accuracy than single miRNAs, are excellent biomarker for screening CRC with good sensitivity and noninvasive nature.

Serine threonine kinase Pim-3 regulates STAT3 pathway to inhibit proliferation of human liver cancers.

OBJECTIVE: This study aimed to investigate the effects of serine threonine kinase Pim-3 on the growth of HepG2 cells and to explore the role of STAT3 signaling pathway. METHODS: Synthetic Pim-3shRNA and negative control shRNA were independently transfected into HepG2 cells in the presence of Lipofectamine(TM) 2000. Cells were divided into 4 groups: Pim-3 shRNA group, negative control group, liposome control group, and blank control group. Flow cytometry was performed to detect the apoptosis of these cells; RT-PCR was employed to detect the mRNA expression of Pim-3; Western blot assay was done to measure the protein expression of Pim-3, STAT3, pSTAT3(Tyr705), Bcl-Xl, Bad and pBad(Ser112). RESULTS: When compared with blank control group, liposome group and negative control group, the apoptosis index increased and the protein expression of Pim-3, pSTAT3(Tyr705), Bcl-Xl and pBad(Ser112) and the Pim-3 mRNA expression reduced in the Pim-3 shRNA group, but the protein expression of STAT3 and Bad was comparable among groups. CONCLUSION: Pim-3 shRNA may down-regulate pSTAT3(Tyr705) and pBad(Ser112) protein expression to inhibit the proliferation of liver cancer cells and Pim-3 may serve as a target for the treatment of liver cancer.

Human equilibrative nucleoside transporter 1 predicts survival in patients with pancreatic cancer treated with gemcitabine: a meta-analysis.

CONTEXT: Increasing scientific evidence suggests that human equilibrative nucleoside transporter 1 (hENT1) may be a powerful predictor of survival in patients with pancreatic cancer treated with adjuvant gemcitabine-based chemotherapy after operative resection, but many existing studies have yielded inconclusive results. OBJECTIVE: This meta-analysis aims to assess the prognostic role of hENT1 in predicting survival in patients with pancreatic cancer treated with gemcitabine. METHODS: An extensive literature search for relevant studies was conducted on PubMed, Embase, Web of Science, Cochrane Library, and CBM databases from their inception through May 1, 2013. This meta-analysis was performed using the STATA 12.0 software. The crude hazard ratio (HR) with 95% confidence interval (CI) was calculated. RESULTS: Eleven clinical studies were included in this meta-analysis with a total of 851 pancreatic cancer patients, including 478 patients in the high hENT1 expression group and 373 patients in the low hENT1 expression group. Our meta-analysis revealed that high hENT1 expression was associated with improved overall survival (OS) of pancreatic cancer patients (HR=2.61, 95% CI=2.02-3.34). Pancreatic cancer patients with high hENT1 expression also had a longer disease-free survival (DFS) than those with low hENT1 expression (HR=2.62, 95% CI=1.94-3.54). Further, high hENT1 mRNA showed significant association with improved OS and DFS of pancreatic cancer patients (HR=2.65, 95% CI=1.75-4.00; HR=3.29, 95% CI=1.85-5.84; respectively). CONCLUSION: In conclusion, our meta-analysis suggests that high hENT1 expression may be associated with improved OS and DFS of pancreatic cancer patients treated with gemcitabine. Detection of hENT1 expression may be a promising biomarker for gemcitabine response and prognosis in pancreatic cancer patients.

Cannabinoid CB1 receptor facilitation of substance P release in the rat spinal cord, measured as neurokinin 1 receptor internalization.

The contribution of CB1 receptors in the spinal cord to cannabinoid analgesia is still unclear. The objective of this study was to investigate the effect of CB1 receptors on substance P release from primary afferent terminals in the spinal cord. Substance P release was measured as neurokinin 1 (NK1) receptor internalization in lamina I neurons. It was induced in spinal cord slices by dorsal root stimulation and in live rats by a noxious stimulus. In spinal cord slices, the CB1 receptor antagonists AM251, AM281 and rimonabant partially but potently inhibited NK1 receptor internalization induced by electrical stimulation of the dorsal root. This was due to an inhibition of substance P release and not of NK1 receptor internalization itself, because AM251 and AM281 did not inhibit NK1 receptor internalization induced by exogenous substance P. The CB1 receptor agonist ACEA increased NK1 receptor internalization evoked by dorsal root stimulation. The effects of AM251 and ACEA cancelled each other. In vivo, AM251 injected intrathecally decreased NK1 receptor internalization in spinal segments L5 and L6 induced by noxious hind paw clamp. Intrathecal AM251 also produced analgesia to radiant heat stimulation of the paw. The inhibition by AM251 of NK1 receptor internalization was reversed by antagonists of mu-opioid and GABA(B) receptors. This indicates that CB1 receptors facilitate substance P release by inhibiting the release of GABA and opioids next to primary afferent terminals, producing disinhibition. This results in a pronociceptive effect of CB1 receptors in the spinal cord.

Involvement of vasopressin 3 receptors in chronic psychological stress-induced visceral hyperalgesia in rats.

Visceral hypersensitivity and stress have been implicated in the pathophysiology of functional gastrointestinal disorders. We used a selective vasopressin 3 (V(3)) receptor antagonist SSR149415 to investigate the involvement of the vasopressin (AVP)/V(3) signaling system in the development of stress-induced visceral hyperalgesia in rats. Rats were exposed to a daily 1-h session of water avoidance stress (WAS) or sham WAS for 10 consecutive days. The visceromotor response to phasic colorectal distension (CRD, 10-60 mmHg) was assessed before and after stress. Animals were treated daily with SSR149415 (0.3, 1, or 3 mg/kg ip 30 min before each WAS or sham WAS session), with a single dose of SSR149415 (1 mg/kg ip), or the selective corticotropin-releasing factor 1 (CRF(1)) antagonist DMP-696 (30 mg/kg po) before CRD at day 11. Effects of a single dose of SSR149415 (10 mg/kg iv) on acute mechanical sensitization during repetitive CRD (12 distensions at 80 mmHg) were also assessed. In vehicle-treated rats, repeated WAS increased the response to CRD, indicating visceral hypersensitivity. Repeated administration of SSR149415 at 1 or 3 mg/kg completely prevented stress-induced visceral hyperalgesia. Similarly, a single dose of DMP-696 or SSR149415 completely blocked hyperalgesic responses during CRD. In contrast, a single dose of SSR149415 did not affect the acute hyperalgesic responses induced by repeated, noxious distension. These data support a major role for V(3) receptors in repeated psychological stress-induced visceral hyperalgesia and suggest that pharmacological manipulation of the AVP/V(3) pathway might represent an attractive alternative to the CRF/CRF(1) pathway for the treatment of chronic stress-related gastrointestinal disorders.

Noxious mechanical stimulation evokes the segmental release of opioid peptides that induce mu-opioid receptor internalization in the presence of peptidase inhibitors.

The internalization of mu-opioid receptors (MORs) provides an ideal way to locate areas of opioid peptide release. We used this method to study opioid release in the spinal cord evoked by noxious stimuli in anesthetized rats. Previous studies have shown that opioids released in the spinal cord produce MOR internalization only when they are protected from peptidase degradation. Accordingly, rats were implanted with chronic intrathecal catheters that were used to inject a mixture of peptidase inhibitors (amastatin, captopril and phosphoramidon) onto the lumbar spinal cord. Five minutes later, a noxious stimulus was delivered to the paw. Lumbar spinal segments were double-stained with antibodies against MORs and neurokinin 1 receptors (NK1Rs) using immunofluorescence. Mechanical stimulation of the hindpaw consisted of repeated 10 s clamps with a hemostat for 10 min. In the ipsilateral dorsal horn, the stimulus produced abundant NK1R internalization in segments L3-L6, and a more modest but significant MOR internalization in segments L5 and L6. In the contralateral dorsal horn, NK1R was substantially lower and MOR internalization was negligible. The same mechanical stimulus applied to a forepaw did not produce NK1R or MOR internalization in the lumbar spinal cord. Thermal stimulation consisted of immersing a hindpaw in water at 52 degrees C for 2 min. It produced substantial NK1R internalization ipsilaterally in segment L6, but no MOR internalization. These results show that mechanical stimulation induces segmental opioid release, i.e., in the dorsal horn receiving the noxious signals and not in other spinal segments.

Comparing analgesia and mu-opioid receptor internalization produced by intrathecal enkephalin: requirement for peptidase inhibition.

Opioid receptors in the spinal cord produce strong analgesia, but the mechanisms controlling their activation by endogenous opioids remain unclear. We have previously shown in spinal cord slices that peptidases preclude mu-opioid receptor (MOR) internalization by opioids. Our present goals were to investigate whether enkephalin-induced analgesia is also precluded by peptidases, and whether it is mediated by MORs or delta-opioid receptors (DORs). Tail-flick analgesia and MOR internalization were measured in rats injected intrathecally with Leu-enkephalin and peptidase inhibitors. Without peptidase inhibitors, Leu-enkephalin produced neither analgesia nor MOR internalization at doses up to 100 nmol, whereas with peptidase inhibitors it produced analgesia at 0.3 nmol and MOR internalization at 1 nmol. Leu-enkephalin was 10 times more potent to produce analgesia than to produce MOR internalization, suggesting that DORs were involved. Selective MOR or DOR antagonists completely blocked the analgesia elicited by 0.3 nmol Leu-enkephalin (a dose that produced little MOR internalization), indicating that it involved these two receptors, possibly by an additive or synergistic interaction. The selective MOR agonist endomorphin-2 produced analgesia even in the presence of a DOR antagonist, but at doses substantially higher than Leu-enkephalin. Unlike Leu-enkephalin, endomorphin-2 had the same potencies to induce analgesia and MOR internalization. We concluded that low doses of enkephalins produce analgesia by activating both MORs and DORs. Analgesia can also be produced exclusively by MORs at higher agonist doses. Since peptidases prevent the activation of spinal opioid receptors by enkephalins, the coincident release of opioids and endogenous peptidase inhibitors may be required for analgesia.

Dual role of 5-HT3 receptors in a rat model of delayed stress-induced visceral hyperalgesia.

Despite its beneficial effect in IBS patients, the mechanism of action of the 5-HT3 receptor (5-HT3R) antagonist alosetron is still incompletely understood. We aimed to characterize the effect and site(s) of action in a model of stress-induced sensitization of visceral nociception in rats. Adult male Wistar rats were equipped for recording of visceromotor response (VMR) to phasic colorectal distension (CRD; 10-60 mmHg). VMR to CRD was recorded 24 h after an acute session of water avoidance (WA) stress (post-WA). Baseline and post-WA responses were measured in rats exposed to WA or sham-WA, treated with alosetron at 0.3 mg/kg subcutaneously (s.c.) 25 nmol intrathecally (i.t.) or vehicle before post-WA CRD. Some rats were treated with capsaicin/vehicle on the cervical vagus nerve and received alosetron (0.3 mg/kg, s.c.) 15 min before post-WA CRD. WA stress led to visceral hyperalgesia 24 h later. Alosetron (0.3 mg/kg, s.c.), failed to inhibit WA-induced exacerbation of VMR to CRD. Stress-induced visceral hyperalgesia was abolished when alosetron was injected intrathecally (P<0.05) in intact rats or subcutaneously (0.3 mg/kg) in capsaicin-pretreated animals (P<0.05). Capsaicin-pretreatment did not affect the exacerbating effect of stress on visceral sensitivity. Alosetron had no inhibitory effect on normal visceral pain responses when administered subcutaneously or intrathecally. We demonstrated that 5-HT3Rs on central terminals of spinal afferents are engaged in the facilitatory effect of stress on visceral sensory information processing. In addition, we showed that stress-induced sensitization of visceral nociception is independent of 5-HT3R activation on vagal afferents.

Effect of peptidases on the ability of exogenous and endogenous neurokinins to produce neurokinin 1 receptor internalization in the rat spinal cord.

The ability of peptidases to restrict neurokinin 1 receptor (NK1R) activation by exogenously applied or endogenously released neurokinins was investigated by measuring NK1R internalization in rat spinal cord slices. Concentration-response curves for substance P and neurokinin A were obtained in the presence and absence of 10 microm thiorphan, an inhibitor of neutral endopeptidase (EC 3.4.24.11), plus 10 microm captopril, an inhibitor of dipeptidyl carboxypeptidase (EC 3.4.15.1). These inhibitors significantly decreased the EC50 of substance P to produce NK1R internalization from 32 to 9 nm, and the EC50 of neurokinin A from 170 to 60 nm. Substance P was significantly more potent than neurokinin A, both with and without these peptidase inhibitors. In the presence of peptidase inhibitors, neurokinin B was 10 times less potent than neurokinin A and 64 times less potent than substance P (EC50=573 nm). Several aminopeptidase inhibitors (actinonin, amastatin, bacitracin, bestatin and puromycin) failed to further increase the effect of thiorphan plus captopril on the NK1R internalization produced by 10 nm substance P. Electrical stimulation of the dorsal root produced NK1R internalization by releasing endogenous neurokinins. Thiorphan plus captopril increased NK1R internalization produced by 1 Hz stimulation, but not by 30 Hz stimulation. Therefore, NEN and DCP restrict NK1R activation by endogenous neurokinins when they are gradually released by low-frequency firing of primary afferents, but become saturated or inhibited when primary afferents fire at a high frequency.

Adenosine inhibits excitatory transmission to substantia gelatinosa neurons of the adult rat spinal cord through the activation of presynaptic A(1) adenosine receptor.

Although intrathecal administration of adenosine analogues or A(1) adenosine receptor agonists is known to result in antinociception, this has not been examined yet at the cellular level. In the present study, we examined in pharmacology an action of adenosine on glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in substantia gelatinosa (SG) neurons of an adult rat spinal cord slice; this was done under the condition where a postsynaptic action of adenosine was blocked. In 65% of the neurons examined (n=72), adenosine at a concentration of 100 microM depressed the frequency of mEPSC in a reversible manner; the remaining neurons exhibited an inhibition followed by potentiation of the frequency. When examined quantitatively in extent in some cells (n=25), the inhibition was 40+/-3% (n=25) while the potentiation was 42+/-8% (n=6). These actions were not accompanied by a change in mEPSC amplitude. The inhibitory action on mEPSC frequency was dose-dependent in a range of 10-500 microM with an EC(50) value of 277 microM. The inhibitory action of adenosine was mimicked by a selective A(1) adenosine receptor agonist, CPA (1 microM; depression: 54+/-9%, n=4); this action of adenosine (100 microM) was not observed in the presence of a specific A(1) adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (1 microM; 94+/-4% of control, n=3). The facilitatory action of adenosine (100 microM) was unaffected by an A(2a) antagonist, ZM 241385 (0.1 microM, n=3); an A(2a) agonist, CGS 21680 (0.1-10 microM; n=6), was without actions on mEPSC frequency. It is concluded that adenosine inhibits excitatory transmission to SG neurons through the activation of presynaptic A(1) adenosine receptor and that some of the inhibition is followed by a potentiation of the transmission. It remains to be examined which subtypes of adenosine receptors except for the A(1)- and A(2a)-subtypes are involved in the potentiating action. Considering that adenosine-like immunoreactivity and adenosine receptors are expressed at a high density in the SG, which is thought to play an important role in modulating nociceptive transmission from the periphery to the central nervous system, this inhibitory action of adenosine could contribute to a negative modulation of pain transmission.