miR-4668-5p is up-regulated in serum of patients with pancreatic cancer

Backgroud: Pancreatic cancer is the most common of gastrointestinal tumor, with a high malignancy and poor prognosis,and early and effective diagnosis is the key for prolonging patient’s overall survival, especially for uid biopsy. Methods In this study, expression prole array was downloaded from GEO database, and differentially expressed miRNAs in serum were screened from patients with pancreatic cancer and healthy individuals, then co-expressed serum miRNAs in pancreatic cacner patients was analyzed by R software with weighted correlation network analysis(WGCNA) package. Finally, key differentially expressed miRNA was veried in serum of 16 patients with pancreatic cancer and 10 healthy individuals by quantitative real-time PCR (qRT-PCR). Results found that there were eleven differentially expressed miRNAs(miR-155-5p, miR-4668-5p, miR-3613-3p, -miR-3201, miR-548ac, miR-486-5p, miR-548a-3p, miR-8084, miR-455-3p, miR-6068, and miR-1246) between pancreatic cancer and healthy individuals. WGCNA results further found that miR-4668-5p has a higher associated modules compared to other differentially expressed miRNAs. Then, the expression level of miR-4668-5p was further veried, and results found that serum miR-4668-5p expression level was signicantly higher in patients with pancreatic cancer than that of healthy individuals. results rstly miR-4668-5p up-regulated


Conclusion
Our results rstly substantiated that miR-4668-5p was signi cantly up-regulated in serum of patients with pancreatic cancer, which may be a potential biomarker for pancreatic cancer diagnosis.

Background
With the development of socioeconomic and population ageing, pancreatic cancer is becoming the most common malignant tumor in China, and usually with a poor prognosis 1 . The most profound issue surrounding poor prognosis is wildly considered as lack of early diagnostic method and effective treatment 2 .
In order to improve patients' prognosis, a majority of methods associated with diagnosis, therapy, and risk strati cation are constantly exploring. Number of novel molecular biomarkers, associated with pancreatic cancer, was increasing gradually, including mutation status and abnormal expression of genes 3 . Recently, DNA methylation 4 , cell-free DNA 5 , exosomes 6 are becoming research hotpot. But there are very limited markers can be really used in clinic.
As we known, it is arduous to discover a novel biomarkers with high sensitivity and speci city 7 .
Consequently, it is indispensable to explor more novel biomarkers associated with occurrence and development of pancreatic cancer. Here, a combination analysis of expression pro le array and WGCNA was performed, which aimed to found and identify key molecular biomarkers related to pancreatic cancer diagnosis.

Materials And Methods
Download and analysis of miRNAs expression pro le. miRNAs expression pro le array(GSE85589) was downloaded from GEO databse(https://www.ncbi.nlm.nih.gov/gds/), and serum miRNAs expression data was obtained from 88 patients with pancreatic cancer and 19 healthy control. Differentially expressed miRNAs were screened by R software. Compared with healthy control, those miRNAs that absolute value of log 2 FoldChange(FC) >1 and FDR<0.05 were considered as differentially expressed miRNAs.
Serum miRNAs expression data from 88 patients with pancreatic cancer was obtained from GSE85589, WGCNA analysis was performed according to previous report 8 .
cDNA synthesis and quantitative real-time PCR .
Serum(2 ml) was collected from 10 healthy control and 16 pancreatic cancer patients from our hospital, and total RNA was extracted referring to previous report 9 . First-Strand cDNA synthesis kit (Agilent Technologies Inc.)was used for cDNA synthesis, and SYBR® Green PCR master mix (Applied Biosystems, NY, USA) was used to qRT-PCR. U6 as an internal control, and 1 µl of a universal reverse primer (Agilent Technologies Inc., CA, USA) were used in this experiment. The relative miRNAs levels were determined in terms of their FC between patients with pancreatic cancer and healthy controls using 2 −ΔΔCt , and this experiment was performed in triplicate 10  IBM SPSS Statistics 21.0 software was used to statistical analysis. miR-4668-5p expression was compared between pancreatic cancer patients and healthy individuals via non-paired t test. P<0.05 was considered as statistically signi cant.

Results
Eleven dysregulated miRNAs were screened in serum of patients with pancreatic cancer.
According to GEO and WGCNA analysis results, miR-4668-5p was considered as a potential key molecular in pancreatic cancer. Then, expression level of miR-4668-5p was further investigated by database and qRT-PCR assay. In GSE85589,up-regulated miR-4668-5p was observed in serum of patients with pancreatic cancer(n = 88) compared to healthy control(n = 19)(P<0.0001; Fig. 2A).To further identify whether miR-4668-5p was also up-regulated in our serum sample, its expression was detected in patients and healthy individuals by qRT-PCR. And results suggested that miR-4668-5p expression was consistently up-regulated in serum of patients with pancreatic cancer (n = 16) compared to those healthy control(n = 10) (P<0.05; Fig. 2B).

Discussion
In this study, eleven differentially expressed miRNAs in serum of patients with pancreatic cancer were rstly screened by GEO database. Meanwhile, co-expressed miRNAs in serum of patients with pancreatic cancer was further analyzed by WGCNA package. Among these differentially expressed miRNAs, modules correlated with miR-4668-5p was higher than other miRNAs, which may indicate miR-4668-5p was a key molecular biomarkers. Then, up-regulated miR-4668-5p were further investigated in pancreatic cancer patients.
Recent studies indicated that patients with incidentally diagnosed pancreatic cancer have better prognosis than those with symptoms 11 , which explain the bene ts of early diagnosis in improving prognosis. In many of the diagnosis methods, liquid biopsy is a potential non-invasive diagnosis method, and with convenient, economical, and minimally traumatic features 12 . To explore whether there were potential biomarkers existed inserum of patients with pancreatic cancer, non-coding RNA pro le was download from GEO database, and followed by the screening of differentially expressed miRNAs, eleven differentially expressed miRNAs were observed. However, it is indispensable to further identify key miRNA among these differentially expressed miRNAs. Different from other studies, here, a combimation of weighted correlation network analysis (WGCNA) and GEO database was used to constructed coexpression network and modules, which aimed to increase our understanding of the host-pathogen relationship. WGCNA has been gradually used to construct coexpression network 13 , especially for miRNAs. As an example, hub miRNAs related to prognosis were identi ed in colorectal cancer (CRC) based on WGCNA analysis 14,15 . Here, WGCNA was used to constructed modules and co-expressed network,and combined with differentially expressed miRNAs, which indicated miR-4668-5p was a key molecular among these differentially expressed miRNAs.
In fact, dysregulated miR-4668-5p has been observed in blood and tissues of many diseases. For example, miR-4668-5p level was higher in serum of hepatocellular carcinoma patients compared to healthy control 16 . Similarly, dysregulated miR-4668-5p was also observed in gastric cancer tissues 17 , and dedifferentiated liposarcoma tissues 18 compared to healthy controls. Not only in malignant tumor, dysregulated miR-4668-5p was also associated with nervous system diseases, for example AD 10 and mesial temporal lobe epilepsy with hippocampal sclerosis 19 . However, it is limit that studies related to miR-4668-5p in pancreatic cancer. In this study, we observed and con rmed miR-4668-5p was obviously up-regulated in serum of patients with pancreatic cancer. Moveover, a combination analysis of GEO and WGCNA suggested that miR-4668-5p may indicate a key molecular biomarker.
Nevertheless, there are still some limitations in this study. For example, it is better to combine miRNAs expression data with clinical data, such as overall survival, clinical stage, ect. Besides, the potential mechanism of miR-4668-5p regulating target genes and its functions should be deeply explored in future.
At last, it is necessary to detect miR-4668-5p expression in a large number of samples.

Conclusions
Our study rstly found that miR-4668-5p was a key differentially expressed miRNAs, and consistently upregulated in serum of patients with pancreatic cancer compared to healthy control, which may be a potential early diagnosis marker for pancreatic cancer.

Declarations
Consent for publication We con rmed that all subjects participated in this study signed written informed consent for publication their data and relevant information.
Acknowledgements Not applicable.

Funding
No funding was received.
Authors' contributions PL performed the primary bioinformatics analysis, experiments, and writed the manuscript; ZH and HZ made substantial contributions to data analysis and technical support. JL reviewed and approved this manuscript.

Ethics approval and consent to participate
The study has been approved by the ethics committee of our hospital, and the patient signed the informed consent form.

Competing interests
The authors declare that they have no competing interests.  Figure 1