MiR-139-5p/SLC7A11 Inhibits the Proliferation and Migration of Pancreatic Carcinoma via PI3K/Akt Signaling Pathway

Objective: Pancreatic carcinoma (PANC) is one of the important aggressive cancers, with deficiency in effective therapeutics. Studies have unveiled that miR-139-5p expression is significantly downregulated in other types of cancers. However, the functions and mechanisms of miR-139-5p in PANC remain unclear. Methods: Bioinformatic analysis was performed to analyze the differentially expressed genes in the TCGA database. PANC cell line with overexpressed miR-139-5p and Solute Carrier Family 7, Member 11 (SLC7A11) was established, and has been used to detect cell proliferation, migration, invasion and colony formation in PANC. Subsequently, bioinformatic analysis and luciferase assay were performed to confirm that SLC7A11 was a target gene of miR-139-5p. Xenograft mouse model was used to investigate the role of miR-139-5p in PANC tumorigenicity. Results: Through bioinformatic analysis, miR-139-5p was predicted to regulate phosphatidylinositol signaling pathway by targeting SLC7A11. MiR-139-5p was found to be lowly expressed in PANC tissues, while SLC7A11 was highly expressed. Low expression of miR-139-5p and high expression of SLC7A11 were positively associated with poor clinical outcomes. PANC cell proliferation, migration, and invasion could be inhibited by miR-139-5p overexpression and could be promoted by SLC7A11 overexpression. MiR-139-5p could regulate the protein expression level of PI3K and Akt associated with phosphatidylinositol signaling pathway could be by inhibiting the expression of SLC7A11. MiR-139-5p overexpression could suppress PANC tumor growth and the expression of SLC7A11, p-PI3K, p-Akt in tumor tissues. Therefore, the inhibiting effect of miR-139-5p to PANC cell proliferation, invasion and migration, at least, was partly due to its inhibiting effect on SLC7A11 expression. Conclusion: These results demonstrated a novel role of miR-139-5p/SLC7A11 in PANC and provided potential prognostic predictors for PANC patients. signaling pathway. The results can help people to learn more about the role of miR-139-5p in PANC, and to find novel targeted therapies. overexpression of miR-139-5p cloning formation and cells proliferated miR-139-5p overexpressed G,


Introduction
Pancreatic cancer (PANC) is the seventh leading cause of cancer-related deaths in China, while it's the fourth cause in the United States 1 with over 2,500,000 deaths every year. Because of the little progress of PANC diagnosis and therapeutic methods made in the past several decades, poor prognosis has become the leading cause to PANC as one of the most aggressive cancers 2 . Therefore, the development of novel methods of early diagnosis and treatments for PANC are of great important both in the study and clinic.
MicroRNA (miRNA) is a type of small RNA with about 22 nucleotides in length and widely distributed in eukaryotic cells, characterized with highly conservativity in evolution progress and regulatory functions in cell differentiation, proliferation and apoptosis 3,4 . Studies have shown that miRNA expression is abnormal in tissues of various human tumors, which is significantly different in normal tissues, with distinct tissue specificity that could be used to determine the origin of tumors 5 . Therefore, miRNA plays an important role in differentiation and migration of tumor. In the study of tumor miRNA, miR-139-5p has been widely regarded as an important member of miRNA family 6,7 . MiR-139-5p is located in the second intron of the phosphodiesterase 2A gene on chromosome 11q13.4 with 23nt in size 6,8 .It is a common type of mature miRNA generated from a miR-139 precursor ,and has been found to be lowly expressed in various cancers, such as malignant glioblastoma 9 , bladder cancer 10 , hepatic carcinoma 11 and breast cancer 12 . Moreover, overexpression of miR-139-5p could inhibit cancer cell proliferation, invasion and migration, but the functions and mechanisms of miR-139-5p in PANC cells are little studied.
MiRNA generally regulates tumor growth and migration by regulating target genes. Solute Carrier Family 7, Member 11 (SLC7A11), a subunit of amino acid transport system Xc, mainly functions in regulating cystine into cells during glutamate exchange 13,14 . The raw material of glutathione is synthesized in the intracellular of cysteine, and the reduce of glutathione can regulate cycytoxicity 15,16 .
Previous studies have shown that SLC7A11 is related to cancer lesions and occurrence, and its expression has been found to be correlated with poor prognosis in various malignancies. Okuno, et al. found that Xc system could strengthen the resistance of ovarian cancer on platinum compound 15 . Huang, et al. found that SLC7A11 expression can be used as a predictor of cells on the drug resistance of geldanamycin mediated by L-alanosine and glutathione, which provides potential targets for improving the chemosensitivity of various drugs 17 . Therefore, SLC7A11 plays an important role in cancer cell proliferation and migration.
In the present study, we investigated the target relationship between miR-139-5p and SLC7A11. Then we studied the regulatory effect of miR-139-5p/SLC7A11 on the expression of PI3K/Akt. We also identify the role of miR-139-5p/SLC7A11 in the proliferation and migration of PANC cell line. These results can help researchers develop novel therapeutic approaches.

Bioinformatic Analysis
The miRNA and mRNA expression data of TCGA-PAAD were downloaded from the TCGA database, and the difference analysis (|logFC|>2, padj<0.05) was performed by edgeR. The clinical information of the sample was used to analyze the survival of the differential miRNA to determine the target miRNA of the study. The target miRNA was targeted predicted using the starBase database, and the differential mRNA was taken intersection to obtain the target gene. Pathological enrichment analysis of target miRNAs and their target genes was performed using GSEA software.

Clinical data and organizational sources
We collected 45 pancreatic cancer tissues and adjacent nontumour tissues tissues that were acquired from July 2018 to July 2019. All registered patients met the following criteria: (a) clear pathological diagnosis of pancreatic cancer, (b) Have signed a pre-operative informed letter. The clinical and pathological data of patients are shown in Schedule 1.

Cell and cell culture
The cell line information used in this study is shown in The synthesized primer sequences are shown in Table 3.

Real-time PCR(qRT-PCR)
Total RNA was extracted from frozen tissue using Trizol (Invitrogen) according to the manufacturer's protocol. cDNA was synthesized using a reverse transcription system kit (Invitrogen). qRT-PCR was performed on an ABI 7900HT instrument (Applied Biosystems, USA). Quantitative PCR was performed using the miScript SYBR Green PCR Kit (Qiagen, Germany) under the following thermal cycler conditions: 95 °C for 2 min, 95 °C for 5 s and 60 °C for 30 s for 40 cycles. SLC7A11 and miR-139a-5p was normalized using GAPDH and U6 as internal reference respectively. The primers used are shown in Table 3. The 2 -ΔΔCt value was used to compare the difference in mRNA expression of the target gene between the control group and the experimental group. The experiment was repeated 3 times. anti-goat anti-rabbit IgG was added and hybridized at room temperature. After 120 min, TBST was washed 3 times. After 20 min, the luminescence reaction was carried out with an ECL kit (Solarbio, Beijing, China) and the protein imprint was photographed. The experiment was repeated three times. The antibody information used in the experiment is shown in Table 4.
HEK293T cells (Thermo fisher, USA) were seeded in 48-well plates and cultured for 24 h. The miR-139-5p/NC and psiCHECK wt/mut plasmids were co-transfected into the cells. Finally, luciferase activity was measured by a luciferase assay reagent (Promega, Fitchburg, WI, USA).
The tumor weight was measured and photographed.

Statistical analysis
All data were processed by SPSS 22.0 statistical software. The measurement data were presented as mean±standard deviation. The comparison between the two groups was t test. The comparison between multiple groups should be analyzed by one-way ANOVA. P < 0.05 indicates that the difference is statistically significant, and P < 0.01 indicates an extremely significant difference.

Bioinformatics predicted miR-139-5p regulate SLC7A11 related pathways
Six differentially expressed miRNAs and 301 differentially expressed mRNAs were obtained by edgeR differential analysis ( Figure 1A). Survival analysis of differential miRNA showed that the low expression of miR-139 in tumor tissues had a significant impact on prognosis, and the survival time of patients with low expression of miR-139 was significantly lower than that of patients with high expression of miR-139 ( Figure 1B). Targeting genes of miR-139-5p, which has been identified mature sites in miR-139 and is most important for prognosis, were predicted by starBase database. After intersection with 26 up-regulated differentially expressed mRNAs, 4 differentially expressed mRNAs (SLC7A11, B3GNT3, SH3TC2, CHRNA5) with binding sites of miR-139-5p were obtained ( Figure 1C). SLC7A11, which has a significant effect on prognosis and plays an important role in cancer prognosis, was selected for in-depth analysis as the targeting gene of miR-139-5p in this study ( Figure 1D). The GSEA pathway enrichment analysis of miR-139 and its target genes found that miR-139 was mainly concentrated in the phosphatidylinositol signaling system, calcium signaling pathway and cell adhesion molecule CAMS signaling pathway (Attached map 1A). Excessive activation of phosphatidylinositol will lead to abnormal cell proliferation, metastasis, endocytosis and exocytosis, and even tumor occurrence. However, SLC7A11 mainly concentrated on pathways such as endocytosis and tight junctions (Attached map 1B). Therefore, miR-139-5p is likely to regulate the phosphatidylinositol signaling system to inhibit tumor proliferation and metastasis by down-regulating SLC7A11.

In clinical tissues and PANC cells of PANC patients, the expression of miR-139-5p is low while expression of SLC7A11 is high
From the survival curve, it was found that miR-139-5p expression was positively correlated with patient survival, while SLC7A11 was negatively correlated. We studied from tissue and cell levels to further explore the relationship between the expression of miR-139-5p and SLC7A11 and PANC. The expression of miR-139-5p and SLC7A11 in PANC tissue samples and adjacent tissues as well as the protein expression of SLC7A11 were detected at the tissue level. The results showed that the expression level of miR-139-5p in PANC tissues was lower than that in adjacent tissues (Figure 2A), and the mRNA and protein expression levels of SLC7A11 in PANC tissues were higher than that in adjacent tissues (Figure 2B, C). To study the relationship between the expression of miR-139-5p and clinicopathology, the relative expression level of miR-139-5p in pancreatic cancer tissues was ranked from high to low, and the median was taken as the cutoff point to divide 45 patients into two groups: high and low expression. The results showed that there was no significant correlation between miR-139-5p and clinical parameters such as gender, age, tumor size, tumor site, differentiation degree, TNM staging and lymphatic metastasis (P>0.05) ( Table 1). The correlation analysis of the expression of miR-139-5p and SLC7A11 tissues showed that the expression of miR-139-5p and SLC7A11 was significantly negatively correlated (P<0.01) ( Figure 2D). The expression of miR-139-5p and SLC7A11 in human pancreatic duct epithelial cells HPDE and two pancreatic cancer cells PANC-1 and Bx-PC3 were compared at the cellular level. The results showed that the expression of miR-139-5p in the two kinds of PANC cells was significantly lower than that in HPDE (Figure 2E), while the mRNA and protein expression of SLC7A11 in the two kinds of PANC cells was significantly higher than that in HPDE (Figure 2F, G).

Overexpression of miR-139-5p inhibited proliferation, migration and invasion of PANC cells
The expression of miR-139-5p in PANC clinical tissues and cells shows that there is a negative correlation. And we infer that overexpression of miR-139-5p in PANC cells will inhibit cell proliferation, migration and invasion. To verify the possibility, we examined the effects of overexpression of miR-139-5p on proliferation, migration and invasion of PANC-1 and Bx-PC3 cancer cells. MTT results showed that the overexpression of miR-139-5p significantly reduced the viability of both types of cells (Figure 3A, B). Cloning formation experiments also showed that overexpression of miR-139-5p significantly inhibited the growth ability of PANC cells (Figure 3C,   D). In addition, the EdU assay showed that miR-139-5p overexpression reduced the reproductive activity of cancer cells (Figure 3E, F). We further measured the effect of miR-139-5p on PANC cell migration and invasion, and found that the migration and invasion ability of PANC cells were significantly inhibited after the overexpression of miR-139-5p (Figure 3G-J). These results indicated that miR-139-5p as a tumor suppressor could inhibit the proliferation, migration and invasion of PANC cells.

MiR-139-5p regulates PANC cell proliferation, migration and invasion by targeting SLC7A11
After confirming the anti-cancer function of miR-139-5p, we further studied the potential target genes of miR-139-5p. Online miRNA data analysis software (starBase) showed that SLC7A11 was a direct target gene of miR-139-5p according to the 3 'UTR predicted target binding site of SLC7A11 ( Figure 4A). Results of Western blot and qRT-PCR showed that overexpression of miR-139-5p inhibited the expression of SLC7A11 (Figure 4B, C). In addition, luciferase assay results also showed that overexpression of miR-139-5p inhibited the luciferase activity of wild-type SLC7A113 'UTR without affecting the luciferase activity of mutated SLC7A113' UTR ( Figure 4D). These results showed that miR-139-5p directly targets SLC7A11 and inhibits the expression of SLC7A11.
In the study of the effect of SLC7A11 targeted by miR-139-5p on PANC cell proliferation, migration and invasion, the MTT results showed that the overexpression of SLC7A11 significantly increased the survival activity of the two cancer cells (P<0.05), but when SLC7A11 and miR-139-5p were overexpressed simultaneously, the survival activity of the two cancer cells was significantly decreased (Figure 5A,   B). The results of cell cloning formation experiment also showed that overexpression of SLC7A11 significantly increased the number of clone formation of the two cancer cells (P<0.05), but when SLC7A11 and miR-139-5p were overexpressed simultaneously, the number of clone formation of the two cancer cells was significantly decreased (Figure 5C, D). We further measured the migration and invasion of the two PANC cancer cells and found that the migration and invasion of PANC-1 and Bx-PC3 cells significantly increased after SLC7A11 overexpression (P<0.05), but the migration and invasion of the two cancer cells was significantly inhibited when SLC7A11 and miR-139-5p were overexpressed simultaneously ( Figure 5E-H). These results suggest that miR-139-5p regulates cell proliferation, migration, and invasion by inhibiting SLC7A11 expression. and SLC7A11 were overexpressed simultaneously, the expression levels of p-PI3K and p-Akt were not significantly different from the blank group (Figure 6A and B).

MiR-139-5p overexpression suppressed PANC tumour proliferation in vivo
In order to study the effect of miR-139-5p on the growth of PANC cells in vivo,

we selected PANC-1 cells as in vivo injection cells to construct lentivirus-coated
overexpressed miR-139-5p cell lines and injected them into the subcutaneous of mice.
The blank control group was the blank lentivirus PANC-1 cells. The results showed that tumor growth was significantly inhibited in the miR-139-5p overexpression treatment group compared with the blank group (P<0.05) (Figure 7B). Compared with the blank group, the tumor volume and weight of the miR-139-5p overexpression group were significantly smaller in nude mice (Figure 7A, C). These results showed that miR-139-5p had a significant inhibitory effect on the growth of PANC tumors.
However, SLC7A11, p-PI3K and p-Akt signal related proteins were significantly inhibited in tumor tissues of nude mice after nc-mimic or overexpression of miR-139-5p (Figure 7D), which was consistent with the results in PANC cells. In view of the findings, miR-139-5p plays an important role in cell growth and migration of PANC acting as a tumor suppressor.

Discussion
Through bioinformatic analysis and luciferase assay, miR-139-5p is confirmed to targeted regulate SLC7A11. In addition, considering the expression of miR-139-5p and SLC7A11 in tissues and cells, their expressions were negatively correlated in PANC. Transport system Xc, combination of SLC7A11 and SLC3A2, plays an important role in cell growth and drug resistance of PANC 13, 26 . In this study, we found that overexpression of SLC7A11 could significantly promote PANC cell proliferation and migration, which is consistent with the previous studies in other cancers 5 .
Moreover, when miR-139-5p and SLC7A11 were overexpressed simultaneously, the promotion effect of SLC7A11 on PANC cell proliferation and migration was significantly inhibited. Results above indicated that the regulatory effect of miR-139-5p on PANC cells is partly processed by targeting SLC7A11. involves in various pathways, including PI3K/Akt, in breast cancer 8 . In our study, the enrichment results of GSEA pathway for miR-139-5p and SLC7A11 revealed that both miR-139-5p and SLC7A11 enriched in PI3K/Akt-associated signaling pathways.
In addition, the protein expression of PI3K/Akt after overexpressing miR-139-5p and SLC7A11 showed that miR-139-5p negatively regulated PI3K/Akt in PANC, which was partly due to the targeting action on SLC7A11.

Attached map 1 Enrichment pathways of miR-139 and SLC7A11
A: GSEA pathway enrichment analysis results of miR-139; B: GSEA pathway enrichment analysis results of SLC7A11.