Vírus in human cells alters the expression profile of miRNA-15 and activation of apoptotic caspases

Objective: Evaluate the miRNA-15 expression profile involved in cellular apoptotic regulation factors. Methodology: We used the H818308 Asian strain of ZIKV without neurological damage. The inoculations occurred in human embryonic kidney cells (HEK-293). After inoculation, samples were extracted for RT-qPCR quantification of viral RNA and miR-15. The level of activation of caspases 1, 3/7 and 8 of cells was performed using chemofluorescence. Results: The ZIKV infection alters the expression of genes and their regulators, affecting several cellular physiological processes such as apoptosis. Conclusion: Therefore, it is important to emphasize that renal progenitor cells (HEK-293) are susceptible to VZIK infection. The genetic deregulation resulting from infection directly affects important cellular processes such as apoptosis from the disordered miRNA-15 expression during the infection period.


expression of cellular miRNAs, closely related to viral infection, both in vertebrates and
invertebrates, indicating that some viruses might have the ability to deregulate the miRNA profile. Thus, there is a variation in expression levels of these miRNAs, resulting from the host's immune response to viral infection or by the interference of the viral replication cycle in the host cell (Asgary, 2014). It is also established the relationship of miRNAs in apoptosis, as well as in studies that promote the debate on the interaction in metabolism and cellular defense (Pfeffer, 2007). In this perspective, some specific miRNAs, such as mIR-15, may undergo changes in their expression levels during viral infection, since they are involved in the host's immune response, making it essential to study their expression and the cellular responses, such as apoptosis (Kozaki et al, 2008;Umbach & Cullen, 2009).
Apoptosis, or programmed cell death, is an essential physiological process for cell maintenance, although this mechanism is also involved in pathological conditions, as seen in viral infections (Luchs & Pantaleão, 2010). The main effector proteins activated during apoptosis are caspases, or aspartate-specific cysteine-dependent proteases, whose function is to cleave cellular substrates, resulting in the amplification of the apoptotic process and the beginning cell breakdown (Cuconati & White, 2002). In order to occur, the target protein must have a caspase cleavage site containing aspartic acid residue, in addition to being recognized by the cysteine side chain enzyme that catalyzes the cleavage of the peptide bond. Caspases can exist in the cell in inactive pro-caspases or zymogens, cleaved and activated in response to apoptotic stimuli, activated via a proteolytic cascade (apoptotic caspases), or as caspase initiators (activated first, containing a longer pro-domain -caspases 2, 8, 9 and 10) (Cuconati & White, 2002;Best & Bloom, 2003). These caspases, cleave and activate a shorter prodomain, called effector caspases (caspases 3, 6 and 7). Caspase 3 cleaves most cell substrates during apoptosis and is also responsible for cleaving viral proteins. Caspases 7, 6 and 2 are also important in the cleavage of viral protein substrates (Ricarte & Kimura, 2006).
In this perspective, several discoveries related to ZIKV tropism by neural progenitor cells infer the susceptibility of an apoptosis trigger, which, in a systematic spectrum, introduces the ability to contribute to a neurological impact, although it is possible to establish a direct relationship the tropic multiplicity of VZIK with cells from different tissues (Dang, et al, 2016;Tang, et al, 2016;Miner, Diamond, 2017). Human studies have detected viral RNA in maternal and fetal tissue, originating in the placental matrix, umbilical cord, amniotic fluid and during the fetal and neonatal development of the human brain, thus revealing a broad tissue tropism by progenitor cells (Onorati, et al, 2016;Garcez et al, 2016). It is then noted, in function analogous to tissues, the need for studies that promote the search for analysis of apoptotic Research, Society and Development, v. 9, n. 12, e3991210699, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10699 6 activation in renal progenitor cells infected by VZIK, in order to demonstrate significant results in embryonic kidney cells (Miner & Diamond, 2017) .

Methodology
Experimental, cross-sectional, quantitative study (Pereira, 2018). The strain used in this work is registered H818308 and is stored in cell culture of Aedes albopictus (Clone C6 / 36), stored at -70ºC, belonging to the Section of Arbovirology and Hemorrhagic Fevers of the Evandro Chagas Institute. The original sample comes from a patient who died of ZIKV infection in the state of Maranhão (Brazil).

Viral stock in Aedes albopictus cell culture, clone C6 / 36
The viral stock was obtained from the inoculation and cell culture of Aedes albopictus clone C6 / 36 cells. For the maintenance of cell culture, the L-15 medium was used, keeping the cells at 28ºC with weekly passages of the confluent monolayers in 25cm² culture bottles containing 10mL of growth medium. Subsequently, the cells were transferred to 175 cm² bottles after the formation of the monolayer, approximately on the third day, the cell cultures were subjected to infection in the proportion of 1: 100. On the tenth day after inoculation, aliquots of infected cell suspension were collected for the indirect immunofluorescence test (IFI).
Subsequently, the stock was stored at -70oC until the moment of use.  Graphical representation of miR-15 expression and viral load during days 1, 2, 3, 4, and 5 after ZIKV infection in HEK-293 cells. The value of p <0.05 (***) was considered to determine the statistical significance of the results. The viral load changes during the infection period, with its peak at 2 DPI and 3 DPI respectively. However, there is a significant decline from the 3 DPI determining the final stage in the infection period. It is noticeable that the infection caused by ZIKV alters the level of expression of miR-15, this microRNA aims to regulate genes involved in apoptosis activation. MiR-15 is positively expressed during the infection period and has its peak expression between 3 DPI and 4 DPI, and may be closely related to viral load, as it is suggestive that the host needs apoptotic activation to ease ZIKV infection, resulting in the disordered miR-15 expression. The 5 DPI shows that with the decrease in viral load, there is also a considerable decline in the expression of miR-15, determining that infection by ZIKV causes changes in apoptotic gene regulators according to their level of replication in the host. Furthermore, it was related to ZIKV infection with the activation of pro-apoptotic and effector caspases during the infection period, which are mediators of the activation of the apoptosis process by extrinsic and intrinsic via the chemofluorescence method as shown in Figure 2. Research, Society and Development, v. 9, n. 12, e3991210699, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10699 It is observed that infection by ZIKV in HEK-293 cells is also responsible for causing the change in caspase activity, which are mediators of apoptosis and inflammasome, such as caspase-1. It is possible to observe in graph A, that caspase-1 is less active during 3DPI and 4DPI, with an apparent decline from 2 DPI, a phase where the highest viremia peak occurs, and although the values corroborate and have overlapping parallelism between 2-4 DPI. The demonstrated products suggest that the infection may interfere by inhibiting the action of this caspase to maintain the viral replication cycle, since caspase-1 it is also related to inflammasome, could signal the activation of inflammatory cytokines and generate a more accentuated response to infection. Research, Society andDevelopment, v. 9, n. 12, e3991210699, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10699