We have elucidated for the first time that the PBMCs miRNA profile of HIV patients is specifically dysregulated according to the HCV exposure, being different for chronic hepatitis C (CHC) infection, acute infection followed by spontaneous clearance or those never infected by HCV. Moreover, here we report potential miRNAs and target genes whose expression may be compromising, and thus the biological processes in which they are involved.
Analysis of clinic and metabolic characteristics showed significant deregulated levels of lipid parameters of the three groups of HIV patients with respect to HC, while no differences were found within HIV patients. The AIP, which is an index of dyslipidaemia that accurately correlates with the size of HDL-C and LDL-C particles [19
], was higher for all HIV groups, especially for HIV/HCV+. Accordingly, we found dysregulation of key lipid metabolism miRNAs, such as hsa-miR-33a, hsa-miR-122-5p and hsa-miR-125a-5p [20
] that were downregulated in all HIV patients. Both HIV and HCV infections disturb host lipid metabolism generating abnormalities that could lead to severe pathologies [22
]. Thus, the miRNA profile of PBMCs reflects common comorbidities in HIV/HCV patients regarding lipid metabolism, such as an increased cardiometabolic risk, dyslipidaemia, insulin resistance and hepatic steatosis [23
Furthermore, all HIV patients showed a strong downregulation of miR-1248, miR-4508 and miR-3960. This feature gives us a common pattern between HIV groups highlighting a shared signature of miRNA profile deregulation, probably due to immune response against viral infections. Previously, the miR-1248 was positively correlated with immune system-dependent processes, such as inflammation [24
]. Thus far, little is known about these miRNAs, and their relevance in HIV infection needs to be further explored.
4.1. HIV/HCV+ miRNA Profile
HIV/HCV coinfection leaves a fingerprint of 153 dysregulated miRNAs in PBMCs. We found that these miRNAs might modulate the expression of genes involved in the PI3K-Akt signaling pathway, which have been previously associated with the HCV infection and immune response against other pathogens [25
]. Among these putative miRNAs, we highlight the miR-126-5p, hsa-miR-34a-5p and hsa-miR-148b-3p, whose expression levels were significantly higher in HIV/HCV coinfected PBMCs compared to HC. The miR-126-5p hepatic expression has been associated with HCV viral load [26
], the circulating hsa-miR-34a-5p promotes liver fibrosis in CHC [27
], and the hsa-miR-148b-3p has been previously identified as dysregulated in PBMCs of HIV patients [28
]. Although these miRNAs were also associated with the regulation of carcinogenesis and antitumor responses [29
], we found them involved in the PI3K-Akt signaling pathway. Both HIV and HCV infections promote the activation of the PI3K-Akt signaling pathway, often resulting in other pathogenesis, such as increasing the risk of viral carcinogenesis, and triggering immune-dependent processes, such as inflammation [31
]. Therefore, the enhanced expression that we have observed of miR-126-5p, miR-34a-5p and miR-148b-3p in HIV/HCV patients—and their putative target genes HOTAIR
—could be associated with a higher immune pro-inflammatory responses and deregulation of carcinogenic genes [30
], which is common in HIV/HCV patients.
Additionally, we also identified the downregulation of the well-known miR-125a/b-3p and miR-122-5p in HIV/HCV+ patients, which were also previously observed in the PBMCS of HCV chronic patients and HIV/HCV- patients, compared to HC [10
]. The miR-122 is a specific and highly abundant liver miRNA that favors HCV replication in hepatocytes and other non-hepatic target cells due to HCV viral hijacking [34
]. According to our data set, miR-122-5p is downregulated in HIV/HCV coinfection and modulates the expression of COPA
. Both genes are positively associated with the mechanism of replication of HCV within their target cells through the Golgi apparatus mechanism allowing virogenic organelle formation and HCV release [35
]. Therefore, miR-122-5p downregulation might allow COPA
expression in HIV/HCV patients, promoting HCV replication in PBMCs. In line with our results, Moghoofei et al. 2021 also reported that hsa-miR-122-5p was differentially expressed in PBMCs of both HIV, HCV and HIV/HCV patients [17
]. These outcomes indicate that both HCV and HIV influence the expression of hsa-miR-122-5p not only in liver cells but also in PBMCs.
On the one hand, miR-125a-5p has a dual role in blocking the expression of genes involved in the lipid uptake and modulating anti-inflammatory processes [36
], while miR-125b-5p is mostly related to lipogenesis by targeting SCD-1
]. Therefore, hsa-miR-125a/b-5p downregulation in the PBMCs of HIV/HCV+ may contribute to excessive lipid accumulation, highlighting a worse prognosis of HIV/HCV patients.
On the other hand, we explored the specific HIV/HCV+ signature of PBMCs. Functional analysis for up and downregulated miRNAs were separately performed to maximize the identification of regulatory events related to phenotypic differences among a group of patients [38
]. The upregulation of hsa-miR-34a, hsa-miR-9, hsa-miR-299 and hsa-miR-582 suggest that they may be involved in the suppression of key target genes associated with HCV-host interactions as the ATG5
, whose inhibition may help to block the HCV replication [39
]. Upregulated miRNAs potentially target genes related to liver-disease events, such as PRKAA1
]. Interestingly, we also observed a potential target of the PDGFRbeta
, highly expressed by hepatic stellate cells (HSCs). The PDGFRbeta
is a key mediator in liver injury and fibrogenesis, contributing to human cirrhosis [42
]. Therefore, PBMCs, similar to HSCs, are susceptible to suffer alterations at the transcriptome level in response to HCV infection. Thus, although most miRNAs involved in HCV infection have been characterized in hepatocytes, in this study, we showed dysregulated miRNAs in HIV patients chronically infected by HCV involved in liver-related diseases, thereby indicating a similar response between PBMCs and hepatic cells against HCV infection.
Similarly, we also identified specific miRNAs exclusively downregulated in HIV/HCV+ patients (hsa-miR-589-3p, hsa-miR-423-3p and hsa-miR-331-3p), all of them being putative modulators of SPTBN1
expression. This gene is involved in the TGF-β pathway, among others, where it interacts with SMAD3
. Within the liver, the loss or downregulation of SPTBN1
expression promotes hepatocellular carcinoma—and other tumor progression—by interrupting the TGF-β pathway and enhancing the Wnt signaling pathway in mice [43
] and humans [44
]. Since both liver cells and PBMCs are host cells for HCV, they might converge in the dysregulation of miRNA, gene or functional response [45
4.2. HIV/HCV- Spontaneous Clearance Profile
We explored the long-term effects of HCV spontaneous clarification in PBMCs of HIV patients, where 169 SDE miRNAs were observed. The top downregulated miRNAs (hsa-miR-3960, hsa-miR-1248 and hsa-miR-4508) were previously identified in the HIV/HCV+ group. Additionally, we found that the expression levels of hsa-miR-1246 were also lower in HIV/HCV- patients compared to healthy individuals. Hsa-miR-1246 upregulation in serum has been considered a potential biomarker for the early detection of several cancers, such as hepatocellular carcinoma, common in chronic HCV patients [46
]. Hsa-miR-1246 was also deeply downregulated in the PBMCs of HIV/HCV- patients, as previously observed by our group in PBMCs after spontaneous clarification of HCV in HCV-monoinfected patients [10
]. The downregulation of hsa-miR-1246 may be due to HIV infection [47
], but further studies are required to decipher whether a clear association between the spontaneous resolution of HCV and deregulation of hsa-miR-1246 exists.
Despite more cancer-related diseases being reported during chronic HCV infection, our results indicate that several cancer-related routes are highly represented for the 169 SDE miRNAs identified in HIV/HCV- patients. Among these, we report 21 deregulated miRNAs in HIV/HCV- patients as potential targets of the Programmed Cell Death Ligand 1
) pathway in cancer. The PD-L1
(and its receptor PD-1) is an immune inhibitory factor that blocks cytokine secretion, and therefore, tumor cell apoptosis is the leading cause for cancer escape. The blockage of PD-L1 and PD-1 is closely associated with immune activation and HCV viremia reduction [48
]. Therefore, our results indicate that miRNA profile dysregulation highlights the molecular consequences due to HCV resolution, although further investigations are needed to decipher the long-term consequences of HCV spontaneous clearance and cancer-related events.
By exploring the specific footprint of HIV/HCV- patients, we exclusively found 24 SDE miRNAs. Regarding the upregulated miRNAs, we observed that hsa-miR-376c-3p, hsa-miR-1304-3p and hsa-miR-337-3p are putative regulators of CLDN1
. This gene encodes for a key cellular co-receptor for HCV entry, which increases susceptibility to HCV infection in the target cells where it is expressed [49
]. The upregulation of the mentioned miRNAs in HIV/HCV- patients will suppress CLDN1
expression, and thus, reduce PBMCs’ susceptibility to HCV infection.
On the other hand, we also analyzed those miRNAs specifically downregulated in HIV/HCV- patients. We observed a specific downregulation of hsa-miR-627-3p and hsa-miR-629-3p. These miRNAs are putative targets of ABRAXAS2
, which is a part of the deubiquitinating enzyme complex BRISC, whose deficiency resulted in the strong attenuation of IFN response [50
], which is stimulated by HCV infection [51
]. The IFN-signaling pathway has an essential role during the early stages of HCV infection by enhancing and regulating the antiviral immune response [52
], where hundreds of genes are activated. Thus, hsa-miR-627-3p and hsa-miR-629-3p may be potential biomarkers that highlight molecular alterations specifically due to HCV resolution in HIV-1 patients.
In addition, roughly 75% of HIV/HCV- patients enrolled in this study presented the favorable interleukin-28B polymorphism (rs12979860) (CC genotype) at IFNL4,
which is part of type 1 IFNs that exhibit antiviral activity since it has been associated with a high rate of HCV spontaneous resolution [7
4.3. HIV miRNA Profile
Finally, we explored the miRNA expression profile of the HIV group without previous HCV infection, where a total of 153 miRNAs were found dysregulated. Thus, the miRNA profile in PBMCs of HIV patients lacks normalization, thereby indicating persistent host immune activation and molecular pathway alterations in cellular metabolism, several pathways related to host immune response against several pathogens stand out, such as HIV, EBV, Chagas, toxoplasma and measles [53
]. Although ART therapy has enormously improved HIV suppression, it fails to eradicate HIV latently in target cells [54
]. The HIF-1 signaling pathway was the most significant targeted pathway by the downregulated of hsa-miR-143-3p (putative gene targets: BCL2
) among others [55
]. The HIF-1 pathway regulates the cellular response to low oxygen through HIFs that control the expression of a wide range of genes involved in energy metabolism and inflammation [56
]. Hypoxia directly influences viral proliferation, promoting or suppressing infectivity depending on the virus [57
]. Similarly, Santanu et al. 2020 also reported an alteration of the HIF-1 signaling pathway in the PBMCs of HIV-1 patients [28
Moreover, we explored those specific miRNAs exclusively altered in HIV infection compared to HC. Among the 13 HIV-specific upregulated miRNAs, we found the characteristic miR-223. This microRNA is an important inhibitor of viral replication since it binds to the 3’ UTR end of HIV RNA, inducing viral latency [13
]. Thus, the suppression of this miRNA increases the susceptibility of mononuclear cells to HIV-1 infection [13
Additionally, we explored the specific downregulated miRNAs. Our results showed a panel of three downregulated miRNAs (hsa-miR-4516, hsa-miR-494-3p and hsa-miR-542-3p) involved in the regulation of genes associated with viral latency (SUPT16H
], HIV reactivation (PIM1
] and persistent metabolic and immune disorders (ZADH2
). Taken all together, SDE miRNAs in suppressed HIV patients may indicate ongoing abnormalities in PBMCs of HIV suppressed patients and potential biomarkers for HIV latency. With respect to fatty acid metabolism, we also observed that the downregulated miRNAs hsa-miR-494-3p and hsa-miR-4516 also targeted the ZADH2
gene, a negative regulator of fat cell differentiation. In this setting, HIV-infected patients showed significant alterations of lipid profile (HDL, AI and AIP) and liver transaminases with respect to non-infected patients. Even though HIV-infected individuals on ART reduced AIDS-associated morbidities, one might expect a complete recovery of the metabolic equilibrium; however, HIV-1 latency induces ongoing abnormalities in the lipid profile giving rise to non-AIDS-associated comorbidities [60
In conclusion, our study offers key insights into the molecular mechanisms of pathogenesis of HCV infection in HIV patients, as well as the opportunity to define a characteristic molecular fingerprint that can be a useful clinical tool for personalized patient management and treatment.
To summarize, roughly 75% (109 SDE miRNAs) of deregulated miRNAs were shared among the different HIV-1 groups (HIV/HCV+, HIV/HCV- and HIV+ vs. HC). HIV/HCV+ showed higher deregulation of infection and cancer-related pathways, HIV/HCV- mainly displayed alteration of cancer-related pathways and HIV+ showed a lack of normalization with remained dysregulation of infection-related pathways, such as HIV and HIF-signaling, among others. Moreover, our findings show a specific miRNA profile dysregulation in the PBMCs of HIV+, HIV/HCV+ and HIV patients after HCV spontaneous clearance. All this together indicates that miRNAs are promising targets for biomarker analysis and may also be a basic pillar of host immune response against viral infections by targeting and regulating protein-coding genes expression.