Ubiquitin-specific protease 44 inhibits cell proliferation and migration via inhibition of JNK pathway in clear cell renal cell cancer

Background: Clear cell renal cell carcinoma (ccRCC) is one of the most common malignancies. USP44 has been reported to be involved in various cancers. This study aimed to investigate the function role and molecular mechanism of USP44 in ccRCC. Methods: Data obtained from TCGA data portal and GSO database were analyzed to uncover the clinical relevance of USP44 expression and tumor development. The function of USP44 in cell proliferation and migration was assessed by cellular and molecular analysis. Results: USP44 was lowly expressed in the ccRCC cancer tissues compared to the normal tissue. Further, USP44 expression was negatively correlated with tumor stage, tumor grade, and patient survival . USP44 overexpression significantly inhibited tumor cell proliferation and migration of 786-O cell as well as Caki-1 cell. In addition, USP44 overexpression also prohibited cell proliferation by up-regulating P21, down-regulating Cyclin D1 expression, and inhibited cell migration by up-regulating MMP2 and MMP9 expression. In contrast, USP44 knockdown enhances ccRCC cell proliferation and migration. Furthermore, the USP44 function in inhibiting ccRCC cell proliferation and migration is associated with the phosphorylation level of JNK. Conclusion: In summary, this study showed that USP44 may be a marker in predicting the ccRCC progression and USP44 inhibits ccRCC cell proliferation and migration dependent on the JNK pathway.

and reproduce. While feeding, they can transmit viruses, bacteria, protozoa and helminths that may subsequently infect the host [2]. Globally, the incidence/prevalence of tick-borne diseases is rising [3,4], mostly due to increased interactions between pathogens, vectors and hosts. Some of the most important factors that account for the increasing incidence include urbanization and human population growth, behavioral changes such as human encroachment into natural environments, climate and habitat changes, and increased wildlife populations in urban and peri-urban areas [5,6].
Tick-borne pathogens (TBPs) able to cause disease in humans are overwhelmingly zoonotic [7]. Domestic dogs may be infected with TBPs of sylvatic origin and are also competent reservoirs for human tick-transmitted infectious agents, such as Ehrlichia chaffeensis, Ehrlichia ewingii, and Rickettsia conorii [8]. Wild animals are usually considered the main reservoir hosts of TBPs like Borrelia burgdorferi sensu lato (s.l.), Anaplasma phagocytophilum, Babesia venatorum and B. microti [9][10][11][12]. Dogs provide a means by which infected ticks can be carried into domestic settings, thus enhancing the risk of human infection, and can act as "sentinels" for monitoring the risk of human disease in an endemic area [13,14].
Several country-wide studies have been made in Europe to assess ticks and TBPs presence and distribution in companion animals [15][16][17][18][19][20]. In Italy, several efforts have been made to evaluate the prevalence of circulating tick-borne pathogens in ticks collected from dogs [21,22], although limited to certain areas. In order to better understand the distribution of TBPs in Italy, we propose the first large-scale nationwide survey on ticks collected from privately-owned dogs [23]. In particular, the aim is to evaluate the presence of the protozoa Babesia and Theileria, and of bacteria of the family Anaplasmataceae and to the Borrelia burgdorferi s.l. complex, chosen for their importance in human and/or animal health.

Results
A total of 2681 Ixodidae ticks grouped into 1578 homogeneous pools were included ( Table   1  Although dogs treated with collars (χ 2 = 53.60, p<0.05; OR = 6.99; 95% CI = 3.89-12.55%) and spot-on products (χ 2 = 119.29, p<0.05; OR = 7.75; 95% CI = 5.18-11.59%) were more likely to be parasitized than those treated with oral formulations. Sequencing determined the presence of at least 9 species of the genus Babesia and 5 species belonging to the genus Theileria, as reported in Table 2. For 37 PCR-positive samples, sequencing was not possible due to low-quality DNA. The zoonotic B. venatorum was the most prevalent species (MIR = 7.54%; 95% CI = 6.34-8.95%), followed by unspecified attached to asymptomatic human patients from the same part of Italy [31]. Table 2 reports the overall sequencing results for Anaplasma/Ehrlichia related to tick species.  Table 2). Geographical distribution at NUTS3 level of B. burgdorferi s.l. is reported in Figure 1 (cf also Supplementary material S3). B. burgdorferi s.l. was detected in 11.54% of the sampled NUTS3 provinces (95% CI = 6.19-20.50%).

Discussion
Ticks and tick-borne diseases have shown patterns of "general emergence" over the past few decades [32]. When pets like domestic dogs are involved, they are perceived by public opinion as a significant threat to both animal and human health. Protozoa of the genera Babesia/Theileria were detected in 27.57% of the examined tick pools, with a higher prevalence in I. ricinus, which is the second most frequently reported tick affecting Italian dogs [23]. The importance of I. ricinus in relation to the epidemiology of Babesia and Theileria is confirmed by the large variety of species infecting this tick species. Piroplasms for which wild animals are the definitive reservoir hosts were detected with a higher prevalence in Ixodes species, especially the zoonotic B. venatorum. Given its widespread distribution, feeding habits and anthropophagic behavior, I. ricinus can transmit a wide variety of pathogens, linking together sylvatic, rural and peri-urban environments [33].
Notably, other zoonotic Babesia species, i.e. B. microti and B. microti "Munich-type", were detected not only in I. ricinus but also in R. sanguineus group, I. hexagonus and D.
marginatus. Isolates of B. vulpes n. sp.34]were detected with a higher prevalence in I.
platys and E. canis were reported homogeneously in tick pools from both northern and southern provinces, in contrast with previous reports of higher seroprevalence levels in dogs from southern Italy [51,52] and Sardinia [53]. Notably, E. canis DNA was detected in R. sanguineus group, which is its main tick vector in Mediterranean areas [54], but also with higher MIR in I. ricinus and I. hexagonus.
Borrelia burgdorferi s.l. DNA was detected with low prevalence across the country, in both I. ricinus and R. sanguineus group.The geographical distribution of ticks infected with B.
burgdorferi s.l. shows isolated infected tick pools from 8 of the 78 examined NUTS3 provinces, while in the province of Oristano (Sardinia) 2 tick pools from 2 different dogs were infected with B. burgdorferi s.l. A cross-sectional seroepidemiological study carried out in Sardinia [55] reported a seroprevalence of 6.1% in teen-agers but showed no association between seropositivity and pet ownership. In other Italian regions, anti-B.
burgdorferi antibodies are present in the human population with a prevalence that varies considerably between geographical areas (from 0% to 23.2%) [56]. The results of our study confirm the localized distribution of B. burgdorferi, while the low number of ticks submitted from the northeastern regions of Italy (traditionally highly endemic for B. burgdorferi s.l.) [56] did not allow a detailed assessment of the epidemiological situation of dog-infesting ticks from this area.
B. burgdorferi s.l. DNA was detected in ticks infesting dogs exposed not only to rural and sylvatic environments, but also in ticks of dogs exposed to urban environments.

Conclusions
The results obtained from this study highlight the high variability of piroplasms, Anaplasmatacea and Spirochaetae in dog-infesting ticks in Italy. Our data confirm that the emergence of TBPs, which have mainly wild reservoir hosts (i.e. roe deer for B. venatorum and wild rodents for A. phagocytophilum and B. burgdorferi s.s.) [9,57], are not limited or confined to sylvatic and rural environments but are increasingly reported in anthropic biological communities (human, pet and, as in the present work, the ectoparasites of owned/pet dogs). The overall high prevalence of TBPs in ticks of privately-owned dogs reflects the importance of an in-depth understanding of ticks and TBPs by veterinary practitioners and veterinary authorities, which must duly inform pet owners and assist them in accessing preventive care through ectoparasitic treatments.

Sample collection and pathogen identification
A nationwide survey of ticks collected from privately-owned dogs in Italy was carried out over 20 months, from February 2016 to September 2017. The project involved 153 veterinary practices from 64 Italian provinces. Veterinarians were asked to check five randomly chosen dogs per month for ticks, and to complete a questionnaire for each dog.
All collected ticks were morphologically identified at species level, and epidemiological risk factors as well as the owners' habits regarding antiparasitic drug usage were evaluated, as reported by Maurelli et al. [23].
Results of morphological and molecular identification of the ticks analyzed in the present study has been previously reported [23]. We included in the present work only those tick species that are commonly reported to feed on dogs (Table 1). Identified ticks were divided into pools comprised of specimens collected from the same dog and homogeneous for species, developmental stage, sex and engorgement status, then ginned with a sterile scalpel. The resulting material was homogenized in TRI-Reagent® (Sigma-Aldrich, Italy) and total DNA was extracted according to the manufacturer's instructions with additional overnight incubation in Proteinase K (0.8 mg) and 500 µl of TRI-Reagent.
Borrelia burgdorferi s.l. was detected using the primers FlaF (5'-AGAGCAACTTACAGACGAAATTAAT-3') and FlaR (5'-CAAGTCTATTTTGGAAAGCACCTAA-3'), targeting a conserved region of the fla gene [26]. Positive and negative controls were included in each PCR reaction and all necessary measures were taken to minimize the risk of contamination. The PCR results were expressed as a minimum infection rate (MIR) or the minimum percentage of ticks in a pool with detectable DNA for each specific pathogen. This calculation was based on the assumption that a PCR-positive pool contains only one positive tick [27]. PCR-positive amplicons were purified using a commercial kit (Nucleospin Extract II Kit, Macherey-Nagel, Düren, Germany) and sequenced on both strands (Macrogen Europe, Spain) for species identification. The resulting nucleotide sequences were analyzed using MEGA X software [28] and compared to those available in GenBank (www.ncbi.nlm.nih.gov/genbank).

Mapping and statistical analysis
Distributions of tick samples were geo-referenced using QGis [29], entering the owner's hometown or, if missing, the location of the veterinary practice that enrolled the dog.
Chi-square tests, logistic regressions and confidence intervals at 95% were calculated using R 3.4.4 [30]. Differences were considered significant at p<0.05.  Veterinarians did not receive any payment for their participation. dog recruitment at veterinary premises, LR, GC and EZ conceived the study, revised data analysis and finalized the manuscript, MPM coordinated data analysis and laboratory work at Naples University.

Availability of data and materials
All data generated and analyzed during this study are included in this published article and supplementary tables.  Zoonotic and dog-related Babesia spp. geographical distribution at NUTS3 level.

Figure 3
Zoonotic and dog-related Anaplasma and Ehrlichia spp. geographical distribution at NUTS3 level.

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to download. SupplMat_BMC.doc