ssc-miR-185 targets cell division cycle 42 and promotes the proliferation of intestinal porcine epithelial cell

Objective microRNAs (miRNAs) can play a role in a variety of physiological and pathological processes, and their role is achieved by regulating the expression of target genes. Our previous high-throughput sequencing found that ssc-miR-185 plays an important regulatory role in piglet diarrhea, but its specific target genes and functions in intestinal porcine epithelial cell (IPEC-J2) are still unclear. We intended to verify the target relationship between porcine miR-185 and cell division cycle 42 (CDC42) gene in IPEC-J2 and to explore the effect of miR-185 on the proliferation of IPEC-J2 cells. Methods The TargetScan, miRDB, and miRanda software were used to predict the target genes of porcine miR-185, and CDC42 was selected as a candidate target gene. The CDC42-3′ UTR-wild type (WT) and CDC42-3′UTR-mutant type (MUT) segments were successfully cloned into pmirGLO luciferase vector, and the luciferase activity was detected after co-transfection with miR-185 mimics and pmirGLO-CDC42-3′UTR. The expression level of CDC42 was analyzed using quantitative polymerase chain reaction and Western blot. The proliferation of IPEC-J2 was detected using cell counting kit-8 (CCK-8), methylthiazolyldiphenyl-tetrazolium bromide (MTT), and 5-ethynyl-2′-deoxyuridine (EdU) assays. Results Double enzyme digestion and sequencing confirmed that CDC42-3′UTR-WT and CDC42-3′UTR-MUT were successfully cloned into pmirGLO luciferase reporter vector, and the luciferase activity was significantly reduced after co-transfection with miR-185 mimics and CDC42-3′UTR-WT. Further we found that the mRNA and protein expression level of CDC42 were down-regulated after transfection with miR-185 mimics, while the opposite trend was observed after transfection with miR-185 inhibitor (p<0.01). In addition, the CCK-8, MTT, and EdU results demonstrated that miR-185 promotes IPEC-J2 cells proliferation by targeting CDC42. Conclusion These findings indicate that porcine miR-185 can directly target CDC42 and promote the proliferation of IPEC-J2 cells. However, the detailed regulatory mechanism of miR-185/CDC42 axis in piglets’ resistance to diarrhea is yet to be elucidated in further investigation.


INTRODUCTION
Diarrhea is the main cause of death of newborn and suckling piglets, which brings enor mous economic loss to the pig industry [1]. The occurrence of piglet diarrhea is related to a combination of genetic and improper management factors, especially the infection of pathogenic microorganisms, such as Escherichia coli [2], Salmonella [3], Clostridium perfringens [46], porcine epidemic diarrhea virus [7], etc. Therefore, it is necessary to search for molecular markers of diarrhea resistance and carry out porcine diseaseresistant breeding. MicroRNAs (miRNAs), a class of small and endogenous non coding RNA molecules with 1925 nucleotides, can play a key role in posttranscription by binding to the 3'untrans lated region (3'UTR) of target mRNA [8,9]. It can play roles in multiple biological processes, such as the cell proliferation [10], apoptosis [11], tumorigenesis [12], and immune in flammation [13] by suppressing the expression of its target genes.
In our previous study, we researched the expression pro files of the ileum miRNAs of 7 days piglets infected with Clostridium perfringens type C using small RNASeq, and found that sscmiR185 was differentially expressed between the resistant group and susceptible group of diarrhea piglets [14]. It is reported that miR185 can play important roles in a variety of cancers, covering pancreatic cancer [15], bladder cancer [16], nonsmall cell lung cancer [17], prostate carcinoma [18], gastric cancer [19], breast cancer [20], hepatocellular carcinoma [21], and colorectal cancer [22]. In addition, miR 185 can also play a regulatory role in response the immune inflammatory. Liu et al [23] analyzed microRNAs in alco holic liver diseases using microarrays and found that miR 185 can participate in immune response, inflammatory response and glutathione metabolism. Ma et al [24] found that the CCAT1/miR1853p/MLCK signaling pathway damages intestinal barrier function and promotes the dete rioration of inflammatory bowel disease. Based on this, we speculate that miR185 also plays an important role in the resistance of piglets to diarrhea infection.
Cell division cycle 42 (CDC42) is one of the members of Rho GTPase family [25]. It is reported that CDC42 regulates cell cytoskeleton and adhesion, cell functions, which are cru cial in the development of various cancer diseases [26]. The research reported that miR137 may directly target CDC42, inducing G1 cell cycle arrest and inhibiting the proliferation and invasion activities of colorectal cancer cells [27]. More over, miR185 is a negative regulator of RhoA and CDC42, and could inhibit the proliferation and invasion of human colorectal cancer cells [28]. However, the function of miR 185/CDC42 in intestinal porcine epithelial cell (IPECJ2) remains to be determined.
In our current study, the relationship between porcine miR185 and CDC42 was investigated in IPECJ2. We pre dicted the target relationship between miR185 and CDC42 using bioinformatics software. The mRNA and protein ex pression level of CDC42 in IPECJ2 were detected after transfection with miR185. The luciferase activity of recom binant plasmids was also detected. In addition, the effects of overexpression miR185 or knockdown CDC42 on pro liferation activity of IPECJ2 were explored. In this study, our results show that porcine miR185 can directly target CDC42 and promote the proliferation of IPECJ2 cells.

Ethics statement
All animal experiments were conducted according to the Regulations and Guidelines for Experimental Animals es tablished by the Ministry of Science and Technology (Beijing, China, revised in 2004) and approved by the Committee for Animal Ethics of the College of Animal Science and Tech nology, Gansu Agricultural University (approval number 2006398).

Sample collection and cell culture
The liver tissue samples were collected from three male landrace at six months and stored at -80°C until RNA extrac tion and as a template for CDC42 gene 3'UTR amplification. The 293T cells and IPECJ2 were purchased from BeNa Culture Collection (BNCC, Beijing, China). The cells were cultured in DMEM/F12 medium (HyClone, New York, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Inc., New York, USA), and 1% penicillinstreptomycin at 37°C and 5% CO 2 . When cell confluence reached 70% to 80%, the transfection is carried out.

Total RNA extraction and cDNA synthesis
Total RNA was extracted from porcine liver tissues and IPEC J2 cells using TransZol Up reagent (TransGen Biotech, Beijing, China) according to the manufacturer's instructions. Sub sequently, the cDNA was synthesized by reverse transcription using PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China) and stored at -20°C.

Bioinformatic analysis
Since the miR185 is highly conserved among different spe cies, the miRNA databases: TargetScan [29] (http://www. targetscan.org/vert_72/), miRDB [30] (http://www.mirdb. org/), and miRanda [31] (http://www.microrna.org/microrna/ home.do) online software were used to predict the target genes for porcine miR185. Based on predictive criteria, be ing bound to targeted sequences with low free energy of binding and having good complementarity with targeted se quences, CDC42 was selected as a candidate mRNA for followup studies.

Plasmid construction and dual-luciferase reporter assay
To verify the targeting relationship between miR185 and CDC42, a partial segment of the CDC42 mRNA 3'UTR (WT) containing the miR185 bindingsequence was poly merase chain reaction (PCR) amplified using specific primers (Table 1). A mutated segment of the CDC42 mRNA 3'UTR (MUT) in which the miR185 binding sequence TCTCTCC was converted to AGAGAGG was obtained using gene syn thesis and subcloning (GENEWIZ, Suzhou, China). The PCR products were cloned into the pmirGLO (7,350 bp) dual lu ciferase reporter vector (Promega, Madison, WI, USA). The recombinant plasmids were confirmed by double enzyme digestion with Xho I and Sal I (TaKaRa, China) and sequenc ing.
For transfection, the 293T cells reached 70% to 80% conflu ences, cells were incubated in 24well plates. The recombinant plasmids were cotransfected with miR185 mimics (50 nM) and inhibitor (100 nM) using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manu facturer's protocol, respectively. The miR185 mimics and inhibitor were designed and synthesized by RiboBio Biotech Co., Ltd. (RiboBio, Guangzhou, China). After 48 h post transfection, the luciferase activity was detected using the Dual Luciferase Reporter Assay System (Promega, USA). In this experiment, the pmirGLO vector was considered as a blank control, mimics NC and inhibitor NC were consid ered as a negative control. All reactions were performed in triplicate.

Quantitative polymerase chain reaction
The IPECJ2 cells were collected after transfection with miR 185 mimics and inhibitor. The quantitative polymerase chain reaction (qPCR) reaction was analyzed using TB Green Pre mix Ex Taq II (Tli RNaseH Plus) quantitative kit (TaKaRa, China) in Roche LightCycler 480 II instrument (Roche, Penz berg, Germany). The primer sequences are shown in Table 1. The thermal cycle for PCR was performed at 95°C for 30 sec onds, 40 cycles at 95°C for 5 seconds and 60°C for 30 seconds. The relative mRNA expression of CDC42 gene was normal ized with βactin (ACTB) gene, and the results were calculated using the 2 -ΔΔCt method [32].

Western blotting
After cell transfection 48 h, total proteins were collected from the treated cells by RIPA buffer (Solarbio, Beijing, China) and quantified using the BCA protein assay kit (Solarbio, Beijing, China). The each group of denatured proteins were loaded into 10% sodium dodecyl sulfatepolyacrylamide gelelec trophoresis, and transferred onto polyvinylidene fluoride membrane. Then, the membranes were blocked in Trisbuff ered saline with Tween20 and incubated with 5% skim milk at room temperature for 1 h. Next the membranes were in cubated with primary antibodies (antiCDC42, bs3555R, 1:1,000; antiβactin, bsm33036M, 1:1,500, Bioss, Beijing, China) at 4°C overnight. The membranes were then incu bated with secondary antibodies (HRP, goat antirabbit IgG, bs0295GHRP, 1:2,000, Bioss, Beijing, China) for 2 h at room temperature. The final protein bands were visualized by enhanced chemiluminescence, and the gray level of the protein bands was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Interference RNA synthesis and overexpression vector construction
The interference RNAs used in this experiment were de signed and synthesized by GenePharma Company (Shanghai, China). The siNC was regarded as a negative control. The interference sequences were shown in Table 2. The CDC42 gene was cloned into pcDNA3.1 (+) vector with Nhe I and BmaH I restriction sites. The pcDNACDC42 overexpression vector was constructed by GENEWIZ Company (Suzhou, China).

Methylthiazolyldiphenyl-tetrazolium bromide assay
The methylthiazolyldiphenyltetrazolium bromide (MTT, Beyotime, Shanghai, China) was also used to examine cell viability. The 5×10 3 cells per well were cultured for 24 h in 96well plates before treatment with miR185 mimics, mim ics NC, miR185 inhibitor and inhibitor NC. Then they were incubated for 24 h at 37°C containing 5% CO 2 . Followed by 10 μL of MTT reagent (5 mg/mL) added to per well for an other 4 h. The medium was discarded after 4 h of treatment and the formazan crystals were dissolved using 110 μL of di methyl sulfoxide. The wavelength at 490 nm was selected, and the OD was determined using SkanIt microplate reader (Thermo Fisher Scientific Inc., USA).

5-ethynyl-2'-deoxyuridine assay
The BeyoClick EdU Cell Proliferation Kit with Alexa Fluor 555 (EdU, Beyotime, Shanghai, China) was used to detect cell proliferation. After seeding in 24well plates (5×10 3 cells per well) for 24 h, the IPECJ2 cells were transfected. After transfected 24 h, the cells were incubated with 10 μM EdU solution in growth medium for 2 h. Then, the cells stained with Azide 555 solution (red) and Hoechst 33342 (blue). Finally, the results were observed under a fluorescence microscope (Olympus IX71, Tokyo, Japan) with 200× magnification. The EdU positive cells were analyzed with the ImageJ software.

Statistical analysis
The IBM SPSS Statistics software (version 21.0; IBM, Armonk, NY, USA) was used to analyze the data, all experiments were repeated at least three times. A Student's ttest was applied to compare two groups and oneway analysis of variance (ANOVA) was performed for multiple groups. All values in this study were expressed as the mean±standard deviation, a p value of less than 0.05 was indicated statisti cal significance.

Predicting targeted mRNA
To explore the potential mechanism of sscmiR185, we per formed a multisequence alignment analysis of the mature miR185 sequences in different species and found that the mature sequences of miR185 was highly conserved in ver tebrates ( Figure 1A). The targeting mRNAs of miR185 were predicted using TargetScan, miRDB and miRanda software, and 385, 1,137, and 1,225 target genes were obtained respec tively, and 100 common target genes were obtained by the intersection ( Figure 1B). It was found that CDC42 gene 3'UTR can complement and bind to the seed region of miR185 ( Figure 1C). The CDC42 gene 3'UTR partial sequences con tain miR185 binding sites as showed in Figure 1D.

Recombinant plasmids identification and luciferase activity detection
Double enzyme digestion and sequencing confirmed that CDC423'UTRWT and CDC423'UTRMUT were suc cessfully cloned into pmirGLO luciferase reporter vector ( Figure 2A2D). The TCTCTCC sequences were successful ly mutated to AGAGAGG, without changes to other bases. In order to confirm the role of miR185 in regulating CDC42 3'UTR, the luciferase activity was detected using the Dual Luciferase Reporter Assay System according to specification. We found that miR185 mimics remarkably reduced the luci ferase activity of the pmirGLOCDC42WT (p<0.01), but not that of the pmirGLO and pmirGLOCDC42MUT (p> 0.05) (Figure 3).

Effects of miR-185 on CDC42 expression level in IPEC-J2
To further confirm the effects of miR185 on CDC42, qPCR and Western blot analyses were used to examine the mRNA and protein expression in IPECJ2 cells after transfected with miR185 mimics, mimics NC, miR185 inhibitor and inhibitor NC, respectively. The results showed that the mRNA and protein expression level of CDC42 was dramatically decreased when transfected with miR185 mimics than in transfected with mimics NC (p<0.01), however, the CDC42 expression level both mRNA and protein were significantly increased when transfected with miR185 inhibitor than in transfected with inhibitor NC (p<0.01) ( Figure 4A4C). These results demonstrate that miR185 directly regulates CDC42 expres sion.

miR-185 promotes the IPEC-J2 cell proliferation
In order to explore the effect of miR185 on IPECJ2 cell proliferation, CCK8 assay and MTT assay were used to de tect the cell viability after transfected with miR185 mimics, mimics NC, miR185 inhibitor and inhibitor NC. We found that overexpression miR185 enhanced cell viability, while knockdown miR185 can inhibit cell viability of IPECJ2 cells. The CCK8 and MTT assays showed similar expression trends ( Figure 5A, 5B). The EdU assay was used to detected cell proliferation, and the results showed that the EdU positive cells were significantly increased after transfected with miR 185 mimics. On the contrary, after transfected with miR185 inhibitor, the EdU positive cells were significantly reduced. Therefore, we conclude that miR185 can promote IPECJ2 cells proliferation.

Knockdown CDC42 promotes the proliferation of IPEC-J2 cells
To confirm whether miR185 directly promotes proliferation of IPECJ2 cells by targeting CDC42, we compared knock down and overexpression of CDC42 in IPECJ2 cells. After transfected with siCDC421 and siCDC422, the expres sion of CDC42 was downregulated by 0.828 and 0.238 fold. So, the siCDC422 was used in subsequent experiments. However, after transfected with pcDNACDC42 plasmid, the expression of CDC42 was significantly upregulated (Fig  ure 6A). The CCK8, MTT, and EdU assays were used to detect cell vitality and cell proliferation, these results showed that knockdown CDC42 promoted cell proliferation, while over expression CDC42 inhibited proliferation of IPECJ2 cells ( Figure 6B, 6C, 6D, and 6E). Therefore, we hypothesized that miR185 might directly target CDC42 to promote IPECJ2 cell proliferation.

DISCUSSION
Diarrhea is a common disease in pig industry, especially harm ful to piglets. Our previous study found that sscmiR185 was upregulated in the resistance group of diarrhea piglets [14]. We speculated that it may play an important role in re sisting diarrhea, but the specific target gene is unknown. It is well known that bioinformatics prediction combined with experimental validation is an effective method for screening    miRNA target genes. In this study, three softwares: TargetScan, miRDB, and miRanda were used to predict the target genes for miR185, which could effectively reduce the false positive rate. By finding the intersection, CDC42 was selected as a candidate target gene. Previous research has shown that CDC42 is a potential target of miR185. For example, Zhang et al [21] confirmed that CDC42 is a direct target of miR185 in human hepato cellular carcinoma using luciferase reporter assays. Liu et al [28] showed that miR185 expression significantly suppressed the RhoA and CDC42 3'UTR activities using a luciferasere porter assay, and could inhibit the proliferation and invasion of human colorectal cancer cells. Notably, the miR185 and CDC42 gene sequence are highly conserved between pig and human. Hence, we assumed that sscmiR185 could be bind ing to the conserved sites of CDC42. In our present study, we found that CDC423'UTR contained miR185 binding site according to the bioinformatics software. The luciferase activity is remarkably suppressed in pmirGLOCDC42WT group after transfection with miR185 mimics. These results indicated that CDC42 was a target gene of porcine miR185. As a chemokine that mediates tumors, CDC42 can partici pate in the migration and invasion of various cancer cells [33]. Previous research reported that microRNA384 inhibits proliferation, migration and invasion of glioma by targeting at CDC42 [34]. Yang et al [35] confirmed that downregula tion of miR25 markedly inhibited A549 cell proliferation, induced G1 cell cycle arrest, by targeting CDC42. In addi tion, miR330 regulates the proliferation of colorectal cancer cells by targeting CDC42 [36].
More and more studies have confirmed that miRNA can negatively regulate the expression of target genes. Niu et al [37] demonstrated that ROCK2 was negatively associated with miR1855p and promoted hepatocellular carcinoma cell migration and invasion. Fang et al [38] revealed that the expression level of miR185 and STIM1 were negatively cor related as detected by qRTPCR and Western blot assays. In this study, the mRNA and protein expression level of CDC42 were dramatically decreased after overexpression of miR185, which further confirms the targeting relationship between the porcine miR185 and CDC42. Functionally, miR185 has been reported to inhibit the proliferation of cancer cells and promote apoptosis. For example, upregulation of miR 185 promotes apoptosis of the human gastric cancer cell line MGC803 [39]. Zou et al [40] found that RKIP through upreg ulation of miR185 suppresses the proliferation and metastasis of breast cancer cell lines. Furthermore, miR185 can inhibit virus infection through the regulation of immunometabolic pathways [41]. In our present research, we detected the exact function of miR185 for proliferation and proved that miR185 promoted the proliferation of normal IPECJ2 cells. However, whether miR185 can resist piglet diarrhea caused by patho genic bacteria infection and inhibit intestinal cell apoptosis requires further research. In summary, our results may pro vide new insights into the screening of miR185/CDC42 molecular markers.

CONCLUSION
In conclusion, luciferase activity, qPCR and Western blot assays displayed that porcine miR185 can directly target CDC42 gene. In addition, overexpression miR185 and knock down CDC42 can promote cell proliferation of IPECJ2. However, the detailed regulatory mechanism of miR185/ CDC42 axis in piglets' resistance to diarrhea requires fur ther investigation.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.