Simultaneous and Sensitive Sensing of Intracellular MicroRNA and mRNA for the Detection of the PI3K/AKT Signaling Pathway in Live Cells

Simultaneous detection of the concentration variations of microRNA-221 (miRNA-221) and PTEN mRNA molecules in the PI3K/AKT signaling pathway is of significance to elucidate cancer cell migration and invasion, which is useful for cancer diagnosis and therapy. In this work, we show the biodegradable MnO2 nanosheet-assisted and target-triggered DNAzyme recycling signal amplification cascaded approach for the specific detection of the PI3K/AKT signaling pathway in live cells via simultaneous and sensitive monitoring of the variation of intracellular miRNA-221 and PTEN mRNA. Our nanoprobes enable highly sensitive and multiplexed sensing of miRNA-221 and PTEN mRNA with low detection limits of 23.6 and 0.59 pM in vitro, respectively, due to the signal amplification cascades. Importantly, the nanoprobes can be readily delivered into cancer cells and the MnO2 nanosheets can be degraded by intracellular glutathione to release the Mn2+ cofactors to trigger multiple DNAzyme recycling cycles to show highly enhanced fluorescence at different wavelengths to realize sensitive and multiplexed imaging of PTEN mRNA and miRNA-221 for detecting the PI3K/AKT signaling pathway. Moreover, the regulation of PTEN mRNA expression by miRNA-221 upon stimulation by various drugs can also be verified by our method, indicating its promising potentials for both disease diagnosis and drug screening

Cycling of maternal behaviors that affected offspring mental health during lactation were monitored at 24 hours nest video.

Cycling of maternal behaviors that affected offspring mental health during lactation were monitored at 24 hours nest video.</p

Grooming.

This is a maternal behavior video captured by infrared camera in continuous home cage video monitoring: maternal rat is licking offspring’s feather and perineum to clean. (MP4)</p

Concept graphs of circadian rhythms of control mothers and stressed mothers.

Images of rats were collected on the home cage infrared monitoring device which were different maternal behaviors described in the Result section. Mothers in the CM tend to more breast-feeding and less grooming in light cycle compared with IM. Mothers in the IM showed a tend who are less off-the-pups and more breast-feeding in dark cycle. According to the definition and observation of these behaviors, breast-feeding was considered to be a manifestation of inactivity, and off-the-pups and grooming was activity.</p

Durations of maternal behaviors which affected offspring during lactation was monitored by 24 h nest video.

The mean time (seconds ± SEM) engaged in a maternal behavior for each hour over a 24 h period. Rats were housed in a 12:12 light: dark cycle, shaded areas indicate dark cycle and white areas indicate periods of light. Comparison of the maternal behavior in IM group and CM group: (a) Off-the-pups; (b) breast-feeding; (c) grooming.</p

Off-the-pups.

This is a maternal behavior video captured by infrared camera in continuous home cage video monitoring: maternal rat is doing any behavior without contacting with pups. (MP4)</p

Breastfeeding.

This is a maternal behavior video captured by infrared camera in continuous home cage video monitoring: maternal rat is nursing in any position. (MP4)</p

Frequencies of maternal behaviors which affect offspring during lactation was monitored by 24 h nest video.

The frequency (number± SEM) engaged in a maternal behavior within one hour over a 24 h period. Rats were housed in a 12:12 light: dark phase, shaded areas indicate dark phase and white areas indicate periods of light. Comparisons of maternal behaviors in IM group and CM group: (a) Off-the-pups; (b) breast-feeding; (c) grooming.</p

S1 Data -

This study investigated the potential impacts of depressive rats’ maternal behavior as an early life stress on the outcome of offspring as an adulthood. Offspring from the same mother were divided into two groups, half of them were fostered or remained by a depressive mother, and the other half remained or fostered by a control mother, respectively. The results showed that offspring fostered by depressive mothers presented significant depressive behaviors. Meanwhile, depressive mothers engaged in more grooming during the light cycle, but less off-the-pup behavior during the dark phase. In conclusion, offspring exposed to a postnatal depressive maternal environment developed a depressive-like behavior. Contrarily, postpartum maternal behaviors play an essential role, which might determine the outcome of the next generation. Furthermore, the appropriate timing of postpartum maternal caring sequences, which might eliminate prenatal stressful influences, was recognized and might be a promising approach for reducing children’s predisposition to mental disorders in their life time.</div

Depressive behavior of offspring tests.

(a) sucrose preference test; (b) open field test inactivity; (c) standing frequency; (d) forced swim test; (e) serum cortisol. The four offspring groups were compared: (1) Intervention-Origin group (IM-IM’o); (2) Intervention-Foster group (IM-CM’o); (3) Control-Origin group (CM-CM’o); (4) Control-Foster group (CM-IM’o). Significant main effects of tests using a 2-tailed t-test, *P < 0.05, **P < 0.01.</p