Abstract

   Aberrant expression of miR-30d is associated with the development and
   progression of several human cancers. However, its biological roles and
   underlying mechanisms in pancreatic cancer are largely unknown. The
   expression of miR-30d in pancreatic cancer was evaluated in public
   databases and further valuated by real-time quantitative PCR, western
   blot, and immunohistochemistry in a cohort of pancreatic cancer
   patients. The role of miR-30d in the proliferation and metastasis of
   pancreatic cancer cells was determined using in vitro and in vivo
   assays. Bioinformatics analyses were performed to examine potential
   target genes of miR-30d. Luciferase reporter assay and functional
   rescue experiments were used to elucidate the mechanisms of miR-30d.
   miR-30d was found frequently decreased in pancreatic cancer compared
   with nontumor tissues, and downregulation of miR-30d predicted poor
   prognosis and early relapse of pancreatic cancer patients.
   Overexpression of miR-30d significantly repressed the growth and
   metastasis of pancreatic cancer cells both in vitro and in vivo.
   Bioinformatics analyses identified sex-determining region Y-box 4
   (SOX4) as a target gene of miR-30d. Mechanically, miR-30d exerted its
   tumor suppressive effect by directly targeting SOX4, which caused
   inhibition of the PI3K-AKT signaling pathway. Overexpression of SOX4
   partially antagonized the inhibitory effects of miR-30d. Our study
   demonstrated that dysregulation of the miR-30d/SOX4/PI3K-AKT axis
   promotes the development and progression of pancreatic cancer. These
   findings suggest miR-30d as a promising and reliable therapeutic target
   for pancreatic cancer.

   graphic file with name 41419_2021_3576_Figa_HTML.jpg

   Subject terms: Oncogenes, Oncogenesis

Background

   Pancreatic ductal adenocarcinoma (PDAC) is one of the most common
   malignancies worldwide and characterized by a high lethality
   rate^[36]1. Although progress has been made in the treatment of
   pancreatic cancer, the survival rate of pancreatic cancer is still very
   poor^[37]2–[38]4. Therefore, better understanding of the underlying
   molecular mechanisms in PDAC with the aim of identifying novel
   therapeutic targets is critical.

   MicroRNAs (miRNAs) are small non-coding RNAs that regulate mRNA
   expression by a post-transcriptional mechanism^[39]5. Accumulating
   studies have showed that miRNAs are key players in cancer pathogenesis
   through their functions in regulating cell motility, apoptosis, cell
   cycle, proliferation, and stress response in various
   cancers^[40]6,[41]7. Several studies showed that miRNAs are of
   functional significance and might be promising prognostic biomarkers in
   pancreatic cancer. For example, miR-92b-3p suppresses pancreatic cancer
   formation by targeting Gabra3 both in vitro and in vivo^[42]8.
   miRNA-141 inhibits the growth and invasion of pancreatic cancer cells
   by targeting MAP4K4^[43]9. miR-1181 inhibits cancer stem cell-like
   characteristics of pancreatic cancer cells by suppressing both SOX2 and
   STAT3^[44]10.

   Previous studies showed that miR-30d, a member of the miR-30 family, is
   dysregulated in multiple cancers and acts as an oncogene or tumor
   suppressor in different tumors. For instance, upregulation of miR-30d
   promotes invasion and migration of breast cancer cells by targeting
   KLF-11 and pSTAT3^[45]11. In contrast, miR-30d inhibits cell
   proliferation and motility in non-small cell lung cancer by directly
   targeting CCNE2^[46]12. miR-30d targets beclin-1 to impact the
   sensitivity of anaplastic thyroid carcinoma cells to cisplatin^[47]13.
   However, the expression and potential function of miR-30d in the
   development and progression of pancreatic cancer is still unknown.

   SOX4 is a member of the highly conserved SOX family of transcription
   factors^[48]14, which play a vital role in embryonic development and
   cell fate determination^[49]15–[50]17. Accumulating evidence indicates
   that SOX4 is an oncoprotein in numerous cancers including breast,
   liver, colorectal, and esophageal cancers^[51]18–[52]21.

   In the present study, we integrated public expression datasets and
   bioinformatics predictions and determined the expression and prognostic
   value of miR-30d in pancreatic cancer. Gain and loss of function
   experiments were performed to determine the biological roles of
   miR-30d. We found that miR-30d is downregulated in pancreatic cancer
   tissues and cell lines. Furthermore, miR-30d inhibited pancreatic cell
   growth and invasion by targeting SOX4 via inactivating the
   PI3K-AKTsignaling pathway. These findings demonstrated that
   dysregulation of the miR-30d–SOX4–PI3K-AKT axis is of great importance
   for the carcinogenesis of pancreatic cancer and might be a potential
   therapeutic target.

Results

miR-30d is downregulated in pancreatic cancer and predicts poor prognosis

   We mined public GEO datasets and found that miR-30d was significantly
   downregulated in pancreatic cancer compared with non-tumor tissues
   (Fig. [53]1a, b). We then examined the expression level of miR-30d in
   35 pairs of pancreatic cancer tissues and adjacent non-tumor tissues
   from The First Hospital of Zhengzhou University (ZZU cohort) by RT-qPCR
   and found downregulation of miR-30d in pancreatic cancer tissues (Fig.
   [54]1c). We also examined the expression level of miR-30d in pancreatic
   cancer cell lines by RT-qPCR, and the results showed that miR-30d
   expression was lower in all five pancreatic cancer cell lines compared
   with the normal HPDE cell line (Fig. [55]1d). To study the clinical
   significance of miR-30d in pancreatic cancer, we mined the TCGA PAAD
   dataset and performed Kaplan–Meier analysis. Compared with patients in
   the low miR-30d expression group (n = 86), patients in the high miR-30d
   expression group (n = 85) showed longer overall survival (OS) (median:
   23.06 months vs. 19.45 months; Fig. [56]1e) and longer relapse-free
   survival (RFS) (median: 20.27 months vs. 14.03 months; Fig. [57]1f).
   Univariate and multivariate Cox regression analyses showed that miR-30d
   was an independent prognostic factor for both prognosis and recurrence
   (Supplementary Tables [58]1 and [59]2). These results suggest that
   miR-30d is downregulated in pancreatic cancer tissues and cell lines
   and predicts poor prognosis and early recurrence in pancreatic cancer
   patients.

Fig. 1. miR-30d is downregulated in pancreatic cancer.

   [60]Fig. 1
   [61]Open in a new tab

   a Expression of miR-30d in the [62]GSE24279 dataset (n = 136). b
   miR-30d expression in the [63]GSE60978 dataset (n = 51). c miR-30d
   expression was detected in pancreatic cancer tissues compared with
   matched adjacent non-tumor tissues from the ZZU cohort by RT-qPCR
   analysis (n = 35). d miR-30d expression was examined in five pancreatic
   cancer cell lines and HPDE cells by RT-qPCR. U6 served as the internal
   reference for miR-30d. e, f Kaplan–Meier analysis of the OS (n = 171)
   and RFS (n = 132) of pancreatic cancer patients from the TCGA cohort.
   Patients were grouped according to median miR-30d expression.
   *P < 0.05, **P < 0.01, ***P < 0.001.

miR-30d inhibits pancreatic cancer cell growth and invasion in vitro

   Given the findings that downregulation of miR-30d was associated with a
   worse outcome in pancreatic cancer patients, we hypothesized that
   miR-30d might function as a tumor suppressor in pancreatic cancer. We
   investigated the role of miR-30d in pancreatic cancer by transfecting
   miR-30d mimics or control sequences into Mia PaCa-2 and Panc-1 cells;
   these have low endogenous expression of miR-30d, and knocked down
   miR-30d expression by transfection of miR-30d inhibitors or control
   sequences into Capan-2 cells, which have relatively high endogenous
   expression of miR-30d. Knockdown and overexpression of miR-30d were
   confirmed by RT-qPCR (Supplementary Fig. [64]S1). CCK-8 and colony
   formation assays showed that re-expression of miR-30d significantly
   inhibited the proliferation of both Mia PaCa-2 and Panc-1 cells
   compared with the control group (Fig. [65]2a, b and Supplementary Fig.
   [66]S2). Conversely, knockdown of miR-30d expression promoted the
   growth of Capan-2 cells compared with the control group (Fig. [67]2c
   and Supplementary Fig. [68]S2). Flow cytometry was used to investigate
   the effect of miR-30d on cell apoptosis and cell cycle progression.
   Compared with the control group, cells overexpressing miR-30d showed an
   increased proportion of cell apoptosis (Fig. [69]2d) and cell cycle
   arrest at G1/S phase (Fig. [70]2f and Supplementary Fig. [71]S3). In
   Capan-2 cells, inhibition of miR-30d expression reduced the apoptosis
   and promoted cell cycle progression (Fig. [72]2e, g and Supplementary
   Fig. [73]S3). Since our previous data suggested that downregulation of
   miR-30d predicted early recurrence of pancreatic cancer patients, we
   next studied the influence of miR-30d on PDAC cell motility and
   invasiveness by transwell assays. Both the migratory and invasive
   capacities of Mia PaCa-2 and Panc-1 cells were suppressed after
   transfection of miR-30d mimics compared with controls (Fig. [74]2h, i).
   Conversely, inhibition of miR-30d promoted the migration and invasion
   of Mia PaCa-2 cells (Supplementary Fig. [75]S4). Changes of cell
   apoptosis, cell cycle, and cell invasion were also confirmed by western
   blotting analysis of the key regulators in these processes (Fig.
   [76]2j, k). Taken together, these results indicate that miR-30d
   suppresses the proliferation and invasion of pancreatic cancer cells in
   vitro.

Fig. 2. miR-30d inhibits pancreatic cancer cell growth and invasion in vitro.

   [77]Fig. 2
   [78]Open in a new tab

   a–c CCK8 assays of pancreatic cancer cells after transient transfection
   with lipofectamine. d Cell apoptosis analysis by flow cytometry after
   transfection of miR-30d mimics in Mia PaCa-2 (left) and Panc-1 cells
   (right). e Cell apoptosis analysis by flow cytometry after transfection
   of miR-30d inhibitors in Capan-2 cells. f Cell cycle analysis by flow
   cytometry after transfection of miR-30d mimics in Mia PaCa-2 (left) and
   Panc-1 cells (right). g Cell cycle analysis by flow cytometry after
   transfection of miR-30d inhibitors in Capan-2 cells. h, i Transwell
   assays without or with matrigel coating to assess cell migration and
   invasion activities, respectively, in Mia PaCa-2 (left) and Panc-1
   cells (right). Scale bar, 50 μm (red line). j, k Western blot analysis
   of key cell apoptosis, cell cycle, and cell invasion markers.
   *P < 0.05, **P < 0.01, ***P < 0.001.

SOX4 is a direct target of miR-30d

   To identify potential target genes of miR-30d, bioinformatics analyses
   were performed using two algorithms of miRDB and miRTarBase (Fig.
   [79]3a). The predicted target gene should also be upregulated in
   [80]GSE62165. Among the 21 candidate targets (Supplementary Table
   [81]3), we selected SOX4 as one of the likely candidates. We identified
   a putative miR-30d binding site in its 3′-untranslated region (UTR)
   (Fig. [82]3b). Overexpression of miR-30d resulted in dramatic
   downregulation of SOX4 mRNA and protein levels in both cells (Fig.
   [83]3c–e). To verify whether SOX4 is directly targeted by miR-30d, a
   dual-luciferase reporter assay was performed using a luciferase
   reporter vector containing the 3′-UTR of SOX4 mRNA. miR-30d mimics
   significantly downregulated the luciferase activity of the reporter
   compared with controls (Fig. [84]3f, g). However, no effect was
   observed on luciferase activity when miR-30d mimic was transfected with
   a reporter vector containing a mutation in the putative miR-30d-binding
   site. These findings indicate that miR-30d regulates SOX4 by directly
   interacting with the 3′-UTR of SOX4 mRNA. We further observed an
   inverse expression correlation between miR-30d and SOX4 in pancreatic
   cancer tissues of ZZU cohort (Fig. [85]3h). We examined the TCGA
   dataset and also found a negative expression correlation between SOX4
   and miR-30d in pancreatic cancer tissues (Fig. [86]3i). We divided
   patients in the TCGA PAAD into four groups according to the median mRNA
   expression levels of miR-30d and SOX4: miR-30d^low&SOX4^high,
   miR-30d^low&SOX4^low, miR-30d^high&SOX4^high, and
   miR-30d^high&SOX4^low. Patients in the miR-30d^high&SOX4^low group
   showed significantly better OS (median: 71.68 months vs. 20.6 months,
   respectively; log-rank test, P < 0.05; Fig. [87]3j) and RFS (median:
   32.42 months vs. 13.53 months, respectively; log-rank test, P < 0.01;
   Fig. [88]3k) than patients in the miR-30d^low&SOX4^high group. These
   results show that the combination of miR-30d and SOX4 was more
   effective in predicting clinical outcome in pancreatic cancer patients
   than miR-30d alone.

Fig. 3. SOX4 is a direct target of miR-30d.

   [89]Fig. 3
   [90]Open in a new tab

   a Venn diagram analysis to determine potential miR-30d targets. b
   Predicted binding sequences between miR-30d and the 3′-UTR of SOX4. c–e
   Levels of SOX4 mRNA and protein in pancreatic cancer cells after
   transient transfection with lipofectamine by RT-qPCR and western blot,
   respectively. f, g Relative luciferase activity in Mia PaCa-2 and
   Panc-1 cells after transient transfection with lipofectamine. h
   Correlations between miR-30d and SOX4 mRNA expression in pancreatic
   cancer tissues (n = 35). i Correlations between miR-30d and SOX4 mRNA
   expression in patients in the TCGA dataset (n = 171). j OS status
   analysis of pancreatic cancer patients in TCGA based on SOX4 and
   miR-30d expression (n = 171). k RFS status analysis of pancreatic
   cancer patients in TCGA based on SOX4 and miR-30d expression (n = 131).
   *P < 0.05, **P < 0.01, ***P < 0.001.

miR-30d suppresses the growth and invasion of PDAC by targeting SOX4 in vitro

   To explore whether the tumor suppressive effect in pancreatic cancer of
   miR-30d was mediated by SOX4, we first constructed
   miR-30d-overexpressing Mia PaCa-2 and Panc-1 cells by transfection
   lentivirus of Lv-miR-30d or control vectors, then overexpressed SOX4 by
   transfection of Lv-SOX4 lentivirus vector. The expressions of miR-30d
   and SOX4 were confirmed by RT-qPCR (Fig. [91]4a) and western blot
   (Supplementary Fig. [92]S5a, b). Both CCK-8 and colony formation assays
   showed that while overexpression of miR-30d inhibited the proliferation
   abilities of pancreatic cancer cells, these effects were partially
   reversed after SOX4 overexpression (Fig. [93]4b–d). In addition,
   transwell assays showed that SOX4 reversed the inhibition of metastasis
   and invasion induced by miR-30d overexpression (Fig. [94]4e, f). These
   data confirmed that the anti-cancer activities of miR-30d in pancreatic
   cancer are mediated by its suppression of SOX4.

Fig. 4. Re-expression of SOX4 attenuated the anti-cancer activities of
miR-30d.

   [95]Fig. 4
   [96]Open in a new tab

   a Expression of SOX4 in cells transfected as indicated. b–d CCK8 and
   colony formation assays in Mia PaCa-2 and Panc-1 cells transfected as
   indicated. e, f Transwell assays assessing cell migration and invasion
   of Mia PaCa-2 and Panc-1 cells transfected as indicated. Scale bar,
   50 μm (red line). *P < 0.05, **P < 0.01, ***P < 0.001.

miR-30d suppresses the SOX4/PI3K-AKT signaling pathway

   To identify the mechanism of SOX4 in pancreatic cancer, gene set
   enrichment analysis (GSEA) with the GEO dataset [97]GSE62165 was
   performed. The results revealed that gene sets associated with
   regulation of transcription and cell migration were significantly
   enriched in cases with high expression of SOX4 (Fig. [98]5a, b). To
   investigate the key signaling pathways involved in the effects of SOX4,
   we explored the TCGA dataset (n = 171) and obtained 213 genes with high
   correlation values (above 0.4) with SOX4, which is an activating
   transcription factor. The 10 most enriched signaling pathways from GO
   enrichment analysis are shown in Fig. [99]5c. We focused on the
   PI3K-AKT pathway, which participates in the development and progression
   of many cancers^[100]22. Knockdown of SOX4 by transfection of two
   independent siRNAs significantly decreased the levels of phosphorylated
   Akt(S473) in pancreatic cancer cells, while total Akt expression was
   not affected (Fig. [101]5d, e and Supplementary Fig. [102]S5c, d). We
   next investigated whether the tumor suppressive effect of miR-30d in
   pancreatic cancer was mediated by the SOX4–PI3K-AKTsignaling pathway.
   Overexpression of miR-30d by lentivirus vector transfection reduced the
   levels of phosphorylated AKT(S473) in pancreatic cancer cells, which
   was partially counteracted by SOX4 re-expression by lentivirus vector
   transfection (Fig. [103]5f, g). Accordingly, changes of cell apoptosis,
   cell cycle and cell invasion regulators were also confirmed by western
   blotting analysis. Taken together, our results indicate that miR-30d
   inhibited the growth and invasion of PDAC by suppressing the
   SOX4–PI3K-AKTsignaling pathway.

Fig. 5. miR-30d suppresses the SOX4/PI3K-AKT-signaling pathway.

   [104]Fig. 5
   [105]Open in a new tab

   a, b GSEA plots showing gene sets which are positively correlated with
   high SOX4 expression samples (left red) in the dataset [106]GSE62165. c
   KEGG pathway enrichment analysis based on SOX4 expression in the TCGA
   dataset. d, e Western blot analysis in cells transfected as indicated.
   f, g Western blot analysis in cells transfected as indicated.
   *P < 0.05, **P < 0.01, ***P < 0.001.

miR-30d/SOX4/PI3K-AKT axis suppresses the growth and invasion of PDAC in vivo

   To validate the in vitro results of miR-30d on tumor growth, we next
   established a subcutaneous xenograft tumor model. Tumor weight and rate
   of tumor growth were significantly reduced in the miR-30d
   overexpression group compared with the control group (Fig. [107]6a, b).
   In contrast, tumor growth was enhanced in the miR-30d downregulated
   group. These observations were confirmed by hematoxylin and eosin (H&E)
   staining (Fig. [108]6c). Immunohistochemistry (IHC) analyses also
   showed that tumors from the miR-30d overexpression group showed reduced
   staining of Ki67, SOX4, and p-AKT compared with controls, while tumors
   from the miR-30d knockdown group showed increased staining of Ki67,
   SOX4, and p-AKT. To evaluate the metastatic effect of miR-30d in vitro,
   we then established a liver metastasis model. Fewer and smaller
   metastatic nodules were observed in livers from the miR-30d
   overexpression group compared with controls, while the miR-30d
   knockdown group had higher numbers and larger metastatic nodules in the
   liver (Fig. [109]6d and Supplementary Table [110]4). We also observed
   weak staining of Ki67, SOX4, and p-AKT in tumors in the miR-30d
   overexpression group compared with control group (Fig. [111]6e). Taken
   together, these results indicate that miR-30d/SOX4/PI3K-AKT axis
   suppresses the growth and invasion of pancreatic cancer cells in vivo.

Fig. 6. miR-30d suppresses the growth and invasion of PDAC in vivo.

   [112]Fig. 6
   [113]Open in a new tab

   a Subcutaneous xenograft tumors in the indicated groups after stable
   transfection with lentivirus vectors. b Tumor weight of the indicated
   groups. c H&E and IHC staining of xenograft tumors of the indicated
   groups. Scale bar, 50 μm (red line). d Representative
   immunofluorescence images of live metastasis in the indicated groups. e
   H&E and IHC staining of nodules formed in the livers. Scale bar, 50 μm
   (red line). *P < 0.05, **P < 0.01, ***P < 0.001.

SOX4 is upregulated and associated with survival and progression in
pancreatic cancer

   We first mined the GEO datasets of pancreatic cancer and found that
   SOX4 was increased in tumor samples compared with matched non-tumor
   counterparts (Supplementary Fig. [114]S6). Overexpression of SOX4 mRNA
   and protein in pancreatic tissues compared with non-tumor tissues was
   observed in RT-qPCR (Fig. [115]7a) and western blot assays (Fig.
   [116]7b, c). Kaplan–Meier survival analysis of patients in
   [117]GSE62165 showed that pancreatic cancer patients with high SOX4
   expression had worse OS compared with patients with low SOX4 expression
   (Supplementary Fig. [118]S6). Analysis of the patients in the TCGA
   dataset also showed that patients with high SOX4 expression had worse
   prognosis and early relapse (Fig. [119]7d, e). To further explore the
   expression and clinical significance of SOX4 in pancreatic cancer, IHC
   staining was performed on in 80 pairs of PDAC tissues of ZZU cohort,
   and the results showed that SOX4 was significantly upregulated in PDAC
   tissues compared with matched non-tumor tissues (Fig. [120]7f, g).
   Correlation analysis showed that SOX4 expression was related to tumor
   size and tumor TNM stage (Supplementary Table [121]5). Survival
   analysis revealed that high SOX4 expression was associated with a poor
   clinical outcome in the ZZU cohort (Fig. [122]7h). IHC staining of
   p-AKT in pancreatic cancer tissues of the same ZZU cohort revealed a
   positive expression correlation between SOX4 and p-AKT (n = 80;
   Supplementary Fig. [123]S7). These observations suggest that the
   miR-30d/SOX4/PI3K-AKT axis might play important roles in the
   tumorigenesis and metastasis of pancreatic cancer.

Fig. 7. High SOX4 expression predicts the prognosis and progression of
pancreatic cancer patients.

   [124]Fig. 7
   [125]Open in a new tab

   a Detection of SOX4 mRNA expression in pancreatic cancer tissues in the
   ZZU cohort by RT-qPCR (n = 35). b, c Detection of SOX4 protein levels
   in pancreatic cancer tissues in the ZZU cohort by western blot (n = 6).
   d, e Kaplan–Meier analysis of the OS (n = 171) and RFS (n = 132) status
   of pancreatic cancer patients in TCGA based on SOX4 expression
   (n = 171). f, g IHC staining of SOX4 in pancreatic cancer tissues
   (n = 80). Scale bar, 50 μm (red line). h Kaplan–Meier analysis of the
   OS status of pancreatic cancer patients in the ZZU cohort based on SOX4
   expression. *P < 0.05, **P < 0.01, ***P < 0.001.

Discussion

   The elucidation of the molecular mechanisms underlying pancreatic
   cancer and identifying reliable therapeutic targets is critical in
   order to improve outcome of PDAC patients. Here we found that miR-30d
   is downregulated in pancreatic cancer and is strongly associated with
   PDAC progression. miR-30d inhibits pancreatic cancer cell
   proliferation, migration, and invasion in association with the PI3K-AKT
   signaling pathway by targeting SOX4. Taken together, these findings
   indicate that miR-30d functions as a tumor suppressor in the
   carcinogenesis and metastasis of pancreatic cancer.

   Several studies have showed that the expression, role, and targets of
   miR-30d differ according to the cancer type. In non-small cell lung
   cancer, miR-30d is decreased and functions as a tumor suppressor by
   targeting CCNE2^[126]12. Downregulation of miR-30d promoted autophagic
   survival of anaplastic thyroid carcinoma cells in response to
   cisplatin^[127]13. Other studies have shown that miR-30d functions as
   an oncogene in different cancers. miR-30d promotes prostate cancer
   progression by targeting both ARID4A and ARID4B^[128]23. Another report
   showed that miR-30d promotes angiogenesis and tumor growth of prostate
   cancer cells via the MYPT1/c-JUN/VEGFA pathway^[129]24. In cervical
   squamous cell carcinoma, upregulation of miR-30d due to genomic
   amplification was associated with disease progression^[130]25. In our
   study, we found that miR-30d is decreased in pancreatic cancer and
   might act as a potential prognostic marker for pancreatic cancer
   patients. Downregulation of miR-30d was significantly correlated with a
   poor prognosis in PDAC. Consistent with these findings, overexpression
   of miR-30d significantly inhibited pancreatic cancer cell
   proliferation, induced G1/S cell cycle arrest and apoptosis and
   repressed cell migration and invasion by inducing EMT. The
   dysregulation of miRNAs in cancer were usually caused by genomic
   alteration, DNA methylation, transcription, synthesis process, nuclear
   export, and stability in cells. miR-30d loss was frequent in cancers,
   which means miR-30d might act as a vital driver gene in cancer and be a
   promising therapeutic target for pancreatic cancer. As K-Ras and p53
   mutations are common and vital in the development and progression of
   PDAC, additional studies are required to elucidate the relations
   between them. Taken together, further studies are required to clarify
   the mechanisms for the loss and action mechanisms of miR-30d in PDAC.

   To investigate the underlying mechanisms underlying the tumor
   suppressive function of miR-30d, we examined potential targets of
   miR-30d by bioinformatics analyses and identified SOX4 as a target gene
   of miR-30d in pancreatic cancer. We identified a miR-30d binding site
   in the 3′ UTR of SOX4. Functional assays showed that overexpression of
   SOX4 could partially reverse the anti-cancer effects of miR-30d. These
   results demonstrate that SOX4 is a direct functional target of miR-30d.
   The median OS of miR-30d high, SOX4 low and miR-30d^high&SOX4^low
   groups were 23.06, 22.83, and 71.68 months, respectively. The median
   DFS of miR-30d high, SOX4 low, and miR-30d^high&SOX4^low groups were
   20.27, 20.27, and 34.42 months, respectively. Both miR-30d and SOX4 are
   good markers for PDAC. But a combination of miR-30d and SOX4 are better
   than each separate one to identify a small group of people who had the
   highest or the lowest risk of death or recurrence. Our findings suggest
   that a combination of miR-30d and SOX4 may help to identify pancreatic
   cancer patients who need intensive care and early intervention at early
   stages.

   SOX4 is a member of the HMG box superfamily and participates in various
   development processes as a transcriptional activator^[131]26,[132]27.
   Several studies have shown that SOX4 is upregulated and promotes the
   development of cancers^[133]28. SOX4 is increased in lung cancer due to
   genomic amplification and contributes to the transforming ability of
   lung cancer cells^[134]29. Multiple studies have demonstrated the
   critical role of dysregulation of the PI3K-AKT-signaling pathway in
   carcinogenesis^[135]30,[136]31. However, how the PI3K-AKT pathway is
   regulated in cancer is largely under-characterized. Previous studies
   showed that amplification of SOX4 modulates PI3K-AKT signaling in
   breast cancer by regulating AKT phosphorylation^[137]18. In
   cholangiocarcinoma, SOX4 is overexpressed and predicts poor
   prognosis^[138]32. In cervical cancer, SOX4 was reported to promote the
   progression and chemotherapeutic resistance by upregulating ABCG2
   transcriptionally^[139]33. Here we found that SOX4 activated the
   PI3K-AKT pathway to promote the tumorigenesis and metastasis of
   pancreatic cancer. However, whether SOX4 regulate the growth and
   invasion abilities of pancreatic cancer cells dependent or independent
   on PI3K-AKT, and the detailed action mechanism are still needed to be
   clarified.

   Here we demonstrated that miR-30d inhibits pancreatic cancer cell
   growth and metastasis via targeting the SOX4/PI3K-AKT-signaling
   pathway. Our findings further show that miR-30d and SOX4 may be
   valuable diagnostic markers for predicting the prognosis and recurrence
   of pancreatic cancer patients. This newly identified
   miR-30d/SOX4/PI3K-AKT axis might represent new promising therapeutic
   targets for pancreatic cancer.

Materials and methods

Pancreatic cancer tissue samples

   Eighty pairs of paraffin-embedded pancreatic cancer tissues and matched
   non-tumor tissues were obtained at The First Hospital of Zhengzhou
   University between February 2011 and February 2012 after informed
   consent was obtained. All patients were histopathologically diagnosed
   as adenocarcinoma. All patients received no treatments before surgery.

Cell culture and transfections

   BxPC-3, Capan-2, Mia PaCa-2, Panc-1, and SW-1990 PDAC cells were
   obtained from the American Type Culture Collection (ATCC, Manassas, VA,
   USA). The normal human pancreatic duct epithelial (HPDE) cell line was
   purchased from the Cell Repository of Chinese Academy of Sciences
   (Shanghai, China). All cell lines were authenticated by short tandem
   repeat DNA profiling within 3 months and cultured according to the
   manufacturer’s protocol as described in our previous study^[140]3. All
   cell lines were tested for mycoplasma contamination. Inhibitors and
   mimics of miR-30d, two independent siRNAs against SOX4 and control
   mimics were purchased from RiboBio (Guangzhou, China). Transient
   transfection of cells was performed using Lipofectamine 3000
   (Invitrogen, Carlsbad, CA, USA) at a final concentration of 50 nM.
   Human miR-30d knockdown, miR-30d-overexpressing or SOX4-overexpressing
   lentiviruses and control vectors were purchased from Genechem
   (Shanghai, China). Lentivirus vector transfection was performed
   according to the manufacturer’s instructions.

RT-qPCR

   Total RNA preparation and PCR were performed as described in our
   previous study^[141]3. Total RNA was prepared from tissues and cells
   using TRIZOL reagent (Invitrogen). A NanoDropND-2000 spectrophotometer
   (NanoDropTech, Wilmington, DE, USA) was used to evaluate the
   concentration and quality of total RNA. cDNA was reverse-transcribed
   using the Prime Script™ RT reagent Kit with gDNA Eraser (Takara,
   Dalian, China). RT-qPCR was carried out using the SYBR Premix Ex Taq™
   II kit (Takara). GAPDH and U6 were used as the internal references for