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miR-630 as a therapeutic target in pancreatic cancer stem cells: modulation of the PRKCI-Hedgehog signaling axis

Biology Direct volume 19, Article number: 109 (2024) Cite this article

Abstract

Background

MicroRNAs (miRNAs) are critical regulators of cancer progression, prompting our investigation into the specific function of miR-630 in pancreatic cancer stem cells (PCSCs). Analysis of miRNA and mRNA expression data in PCSCs revealed downregulation of miR-630 and upregulation of PRKCI, implying a potential role for miR-630 in PCSC function and tumorigenicity.

Results

Functional assays confirmed that miR-630 directly targets PRKCI, leading to the suppression of the Hedgehog signaling pathway and consequent inhibition of PCSC self-renewal and tumorigenicity in murine models. This study unveiled the modulation of the PRKCI-Hedgehog signaling axis by miR-630, highlighting its promising therapeutic potential for pancreatic cancer (PC) treatment.

Conclusions

MiR-630 emerges as a pivotal regulator in PCSC biology, opening up new avenues for targeted interventions in PC. The inhibitory effect of miR-630 on PCSC behavior underscores its potential as a valuable therapeutic target, offering insights into innovative treatment strategies for this challenging disease.

Introduction

Pancreatic cancer (PC) encompasses a diverse group of diseases, each with varying levels of lethality. For instance, pancreatic neuroendocrine tumors, which are relatively rare, have a 10-year survival rate of approximately 80%. In contrast, pancreatic adenocarcinoma, which is more common, is associated with a 5-year survival rate of less than 5% [1]. It is reported that the highly invasive and metastatic potential of PC cells contributed to the high mortality [2]. In the early stage, surgical resection and adjuvant chemotherapy are two common treatments for PC [3]. The chemotherapy and radiation therapy are not so effective because of the features of late presentation and early metastasis [4, 5]. Notably, cancer stem cells (CSCs) exert appealing potential in cancer initiation and development [6]. Importantly, it is of great value to study the characteristics of collected CSCs and exploit CSC-specific treatment modalities for improving the prognosis of PC [7]. Consequently, identifying related markers associated with CSCs is critical for PC treatment.

microRNAs (miRNAs), a group of small non-coding RNA molecules, can negatively modulate gene expression by binding to the 3’ untranslated region (3’-UTR) of the targets and play essential roles in a range of biological processes involving cell development, proliferation, differentiation, apoptosis and metabolism [2, 8]. The overexpression of miR-630 has been reported to promote apoptosis, postpone the cell progression and suppress cell proliferation of breast cancer cells [9]. However, the clinical and prognostic value of miR-630 in PC has not been explored up to now. In our study, the bioinformatics website and dual luciferase reporter gene assay are used to verify the targeting relationship between miR-630 and protein kinase C iota (PRKCI). PRKCI is part of the 3q26 amplicon of the protein kinase C iota, and is frequently turned into an oncogene by CNA in some tumors [10,11,12]. PRKCI encodes PKCι, an atypical subclass of the PKC gene family. PKCι is overexpressed in many tumors and is associated with poor prognosis, especially in advanced malignancies [13,14,15]. PKCι overexpression can promote the transformation of normal cervical epithelial cells to invasive cancer [16, 17]. PKCι affects the polarity and fate of epithelial cells and the tissue integrity of untransformed cells through subcellular localization [18].

Previous evidence reported that PRKCI was highly expressed in clear cell ovarian cancer [19]. As a tumor-promoting protein, PRKCI enhances lung cancer cell proliferation and activates the Hedgehog signaling pathway [20]. Furthermore, the activation of the Hedgehog signaling pathway promotes proliferation and tumorigenesis in cancers such as skin basal cell carcinoma, gastrointestinal cancer, prostate cancer, breast cancer, acute myeloid leukemia, lung cancer, and PC [21,22,23]. Based on the above evidence, we hypothesized that miR-630 could inhibit the development of PC, and the underlying mechanism involved the regulation of PRKCI and the Hedgehog signaling pathway by miR-630.

Materials and methods

Ethics statement

The study protocol was approved by the ethics committee of Jiangxi Cancer Hospital. The written informed consents were provided from all the patients. Mice were treated humanely, and all experiment procedures were permitted by the Institutional Animal Care and Use Committee of Jiangxi Cancer Hospital (Approval no. 2024ky110). Efforts have made to minimize the mice suffering.

Microarray data analysis

The differentially expressed miRNA and genes were retrieved from the PC-related microarray data obtained from the Gene Expression Omnibus (GEO) database. The PC-related miRNA expression chip was GSE41369, and the microarray data included GSE91035, GSE71989 and GSE32676. The detailed information is displayed in Table S1. The affy package in R language software was adopted for background correction and standardization pre-processing [24], and the limma package was used for screening differentially expressed miRNAs or genes with the screening threshold of | log2FolfChange | > 2.0 and adj.P.Val (the corrected p value) < 0.05 [25], and the heat maps of differentially expressed miRNAs or genes were drawn. The target genes of the differentially expressed miRNA were predicted using the online software on the websites TargetScan, mischemia-reperfusion DIP and microDB. The comparison of the 6 data sets was analyzed by jvenn, and the Venn diagram was drawn [26]. Jvenn was also used to compare the differences between the three miRNA target genes and the differential genes of the three chips and to screen the differentially expressed miRNAs and genes with targeted binding relationships. Gene Expression Profiling Interactive Analysis (GEPIA) was a database used to analyze cancer-related gene expression and their interaction, and the data was also obtained from the Cancer Genome Atlas Cancer Genome (TCGA) and Genotype-Tissue Expression (GTEx) [27]. Finally, the GEPIA was applied to verify the differentially expressed genes (DEGs) and to analyze the association between the DEGs and the overall survival (OS) of patients with PC.

Cell culture

Human PC cell line PANC-1 and the normal pancreatic cell HPDE were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in a 5% CO2 incubator with RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and mycillin at 37oC. The medium was changed every 24 to 48 h. The cells were detached with 0.25% trypsin and passaged. The cells in the logarithmic growth phase were selected for the experiments.

Flow cytometry

A total of 2 × 105 PANC-1 cells were transferred to a 5 mL centrifuge tube and washed twice with phosphate buffer saline (PBS), counted and re-suspended in PBS. Then, the cells were probed with the antibodies to CD44 (ab264539, Abcam, UK, 1:1200), CD24 (ab202073, Abcam, UK, 1:500) and ESA (347200, Becton Dickinson, USA, 1:40) on ice for 20 min avoiding exposure to light, and the respective isotype control antibodies were applied according to the instructions. The cells were washed and re-suspended in 500 µL PBS and analyzed by flow cytometry (FACSAria II, BD Biosciences, Franklin Lakes, NJ, USA), and the lateral scattering and forward scattering were used to eliminate cell doublets. The cells were sorted twice conventionally and the collected PCSCs were used for the subsequent experiments.

Cell treatment

The cells were assigned into the following groups: the mimic-negative control (NC) group (introduced with miR-630 NC sequence), the miR-630 mimic group (introduced with miR-630 mimic), the inhibitor-NC (introduced with inhibitor-NC), the miR-630 inhibitor group (introduced with miR-630 inhibitor), the sh-PRKCI group (introduced with shRNA-PRKCI), the sh-NC group (introduced with shRNA-PRKCI-NC), the miR-630 inhibitor + sh-PRKCI group (introduced with miR-630 inhibitor and sh-PRKCI), the inhibitor-NC + sh-NC group (introduced with inhibitor-NC and shRNA-PRKCI-NC) (the above plasmids were purchased from Shanghai GenePharma Co., Ltd., Shanghai, China), the miR-630 inhibitor + DMSO group (treated with DMSO and introduced with miR-630 inhibitor), the miR-630 inhibitor + cyclopamine (treated with 3 µM cyclopamine and introduced with miR-630 inhibitor). Cyclopamine is the inhibitor of the Hedgehog signaling pathway and was purchased from ApexBio, Taiwan, China. The cells were plated in a six-well plate 24 h before transfection. When the cell confluence reached 50%, the cells were transiently transfected with the reagents mentioned above by lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), respectively. After 6 h, the medium was renewed. Subsequently, the cells were cultured for another 48 h and collected for the following experiments.

Agomirs are specially labeled and chemically modified double-stranded small RNAs that modulate the biological functions of target genes by mimicking endogenous miRNAs. Antagomir is a single-stranded small RNA specially labeled and chemically modified according to the sequence of the mature microRNA. It is a highly effective blocker specially designed to inhibit endogenous microRNA. miR-630 agomir and miR-630 antagomir are agonists and inhibitors of miR-630, respectively.

5-ethynyl-2’-deoxyuridine (EdU) staining

The cells in the culture plate were incubated with the EdU solution at room temperature for 2 h and washed with PBS. The cell culture medium and the EdU solution were mixed at a ratio of 1000: 1. The cells were fixed by 4% paraformaldehyde for 30 min, incubated in glycine solution for 8 min, washed with PBS, and re-suspended with 0.5% Triton X-100. The cells were stained with the Apollo® staining reaction solution for 30 min at room temperature avoiding exposure to light, and washed two times with methanol and PBS, respectively. Afterwards, the cells were reacted with the Hoechst 3334 reaction solution for 20 min at room temperature avoiding exposure to light, and observed under the fluorescence microscope.

Cell colony formation assay

The LB culture plate was prepared, and then the cells were detached, centrifuged, counted and re-suspended in RPMI-1640 medium. After that, cells were seeded at a density of 500 cells per dish (10 cm) and incubated with 5% CO2 for 2 weeks at 37 Â°C. After the removal of the medium, the cells were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 20 min, and finally stained with crystal violet for 60 min. Following the removal of the staining solution, the culture plate was dried in air and observed under the microscope to count the number of colonies.

Sphere formation assay

The microsphere cells in each cell line were made into the single cell suspension. The cells were seeded into the 24-well ultra-low adhesion culture plate at a density of 1000 cells per well, cultured in serum-free stem cell medium for 5 days, and observed under the Nikon Eclipse TE2000-S microscope. The tumorspheres were quantified, and the sphere formation rate was calculated on the basis of the following formula: the sphere formation rate (%) = the average number of tumor spheres per well / the number of cells per well × 100%. As the tumor sphere formation rate of the solvent control group was 100%, the relative tumor sphere formation rate of each experimental group was normalized.

Dual-luciferase reporter gene assay

To confirm the binding relationship between miR-630 and PRKCI, and whether PRKCI was a direct target e of miR-630, the fragment of 3’-UTR of PRKCI gene was synthesized and inserted into the vector pMIR-reporter (Beijing Huayueyang Biotechnology Co., Ltd Beijing, China) via the endonuclease sites Spel and Hind III. The mutant (MUT) fragment was designed based on the predicted binding sites between miR-630 and the wide type (WT) 3’-UTR of PRKCI which was also inserted into the pMIR-reporter. The WT and MUT luciferase reporter plasmids were respectively co-transfected with miR-630 into the HEK-293T cells (Shanghai Beinuo Biological Technology Co. Ltd., Shanghai, China). After 48 h, the cells were harvested and lysed. The luciferase activity was determined using the Luciferase Assay Kit (K801-200, BioVision, Mountain View, CA, USA) and the Glomax20/20 Luminometer Fluorescence Detector (Promega, Madison, WI, USA). The experiment was repeated three times.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

The total RNA was extracted by the RNA extraction kit (Invitrogen Inc., Carlsbad, CA, USA). The primers of miR-630, PRKCI, Oct4, Nanog, U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were synthesized by Takara Biotechnology Co., Ltd. (Dalian, China) (Table S2). RNA was reversely transcribed into cDNA using the PrimeScript RT Kit. The reverse transcription system (10 µL) was set in strict accordance with the instructions. The cDNA was subjected to the fluorescence quantitative PCR based on the instructions of the SYBR® Premix Ex TaqTMII kit. The reaction was conducted on the ABI PRISM® 7300 system. The level of U6 snRNA expression was used as the loading control for miR-630, and GAPDH for other genes. The relative transcription level of the genes was calculated by the 2−△△Ct method [28].

Western blot analysis

The tissue samples in each group were added with liquid nitrogen, and ground into uniform fine powder. Then the samples were centrifuged at 25,764 ×g at 4oC with protein lysis buffer for 20 min and the supernatant was obtained. The protein concentration was subsequently tested and adjusted with deionized water to ensure a consistent quantity of loading sample. The 10% sodium dodecyl sulfate separation gel and concentrated gel were prepared. The sample was boiled at 100oC with a loading buffer for 5 min, ice-bathed, centrifuged, and loaded to each lane using a micropipette for separation. Then, the protein was transferred onto the nitrocellulose membrane. The membrane was sealed with 5% skim milk at 4oC overnight, probed with the primary antibodies to Smo (1 : 500, ab5694), Gli1 (1 : 10000, ab49314), Gli2 (1 : 1000, ab167389) overnight, and washed with PBS for 3 times (5 min each time) at room temperature. The membrane was then incubated with horseradish peroxidase (HRP)-labeled IgG secondary antibody (1 : 1000) (Wuhan Boster Biological Technology Co., Ltd., Wuhan, Hubei, China) for 1 h at 37oC and washed 3 times with PBS (5 min for each) at room temperature. After that, the membrane was developed by the ECL reaction solution (Pierce, Rockford, IL, USA) at room temperature for 1 min and observed. The relative protein levels were expressed by the gray value of the target protein band to that of the GAPDH or proliferating cell nuclear antigen (PCNA) protein band using the ImageJ2x software. The protein marker was purchased from Piercenet (#84785).

Immunofluorescence staining

At the 48 h after transfection, the cell slides were washed 3 times with PBS (5 min each time), and fixed by 4% paraformaldehyde at room temperature for 30 min. After being washed 3 times with PBS, the cells were treated with 0.2% TritonX-100 for 15 min at room temperature, blocked by 3% BSA at 4oC for 30 min, and probed with primary antibodies to Gli1 (1 : 10000, ab49314, Abcam, UK) and Gli2 (1:100, A16864, ABclonal, China). Next, the cells were washed 3 times with PBS, and probed with the fluorescent-labeled secondary antibody (1 : 500) at room temperature for 2 h avoiding exposure to light. Later, the cells were washed 3 times with PBS, and stained with DAPI (ab104139, 1 : 100, Abcam, Shanghai China) for 10 min at room temperature avoiding exposure to light. After being washed with PBS 3 times, the cells were mounted, observed and photographed under the inverted fluorescence microscope.

In vivo limiting-dilution assay

The miR-630 agomir and antagomir, lentiviral interference plasmid vectors (sh-PRKCI) and their corresponding NC vectors were purchased from Invitrogen (Carlsbad, California, USA) and co-transfected into 293T cells when the cell density reached to 50%. The medium was changed 6 h after transfection. After cultured for 24 h, the cell supernatant was collected, and the virus was concentrated by ultracentrifugation. The cells were grouped into: the agomir-NC group, the miR-630 agomir group, the antagomir-NC group, the miR-630 antagomir group, the sh-PRKCI group, the sh-NC group, the miR-630 antagomir + sh-PRKCI group, and the miR-630 antagomir + sh-NC group. The stable PC stem cell lines were constructed and the cells were seeded into the low-adherent culture plates. After culturing for 7 days, the PCSC spheres were collected. The cells were prepared into the single cell suspension and then counted. Different numbers of cells (1 × 103, 1 × 104, 1 × 105, 1 × 106) were re-suspended in 50 µL of normal saline, mixed with 50 µL Matrigel Matrix, and subcutaneously inoculated into the NOD-SCID (4–6 weeks, male, Beijing Vitalriver) mice. A total of 192 mice were used, with 6 mice in each group. Two weeks after the inoculation, the tumor growth was observed and recorded.

Xenograft tumor in NOD-SCID mice

The cells were seeded into the low-adherent culture plate. After culturing for 7 days, the PCSC spheres were collected into a 10 mL glass centrifuge tube, centrifuged and washed once with normal saline. The single cell suspension was prepared and the cells were counted. A total of 2 × 106 cells was re-suspended in 50 µL normal saline, then mixed with 50 µL Matrigel Matrix, and inoculated subcutaneously into NOD-SCID mice (8 in each group). The mice were kept in the same environment and observed once every 7 days. The length and width of the tumor were recorded, followed by the calculation of the tumor volume according to the formula: volume = length × width² / 2. On the 35th day, the NOD-SCID mice were sacrificed, and the tumors were collected.

Statistical analysis

All data were processed using SPSS 21.0 statistical software (SPSS, Inc., Chicago, IL, USA), and graphics were plotted using GraphPad Prism. Measurement data were expressed as mean ± standard deviation. For comparisons between two groups, an independent sample t-test was used if the data followed a normal distribution; otherwise, the Mann-Whitney U test was applied. For comparisons among multiple groups, one-way analysis of variance (ANOVA) followed by Tucky’s post hoc test was used. A P-value of < 0.05 was considered statistically significant.

Results

miR-630 was reduced in PCSCs

To study the regulatory role of miRNA in PC, initially, we retrieved the PC-related microRNA microarray data (GSE41369) to screen out the differentially expressed miRNA and genes, respectively. Twenty-nine differentially expressed miRNAs were screened, and the heatmap was drawn (Fig. 1A). It was identified that in PC tissues, hsa-miR-630 was the most significantly downregulated miRNA. The PCSCs were sorted by flow cytometry (Fig. 1B). In the PANC-1 cell line, the CD44+/CD24+ cells occupied 0.9 − 2% of the total cells, and the CD44+/CD24+/ESA+ cells occupied 0.1 − 0.9%. The CD44+/CD24+/ESA+ cells in PC tissues were sorted and considered as PCSCs by flow cytometry. Compared with PANC-1 cells, the shape of CD44+/CD24+/ESA+ cells was spheroid (Fig. 1C). Since the roles of Oct4 and Nanog were essential in maintaining stem cell characteristics, the Oct4 and Nanog expression was determined by RT-qPCR in PANC-1 cells and CD44+/CD24+/ESA+ cells (Fig. 1D). The expression of Oct4 and Nanog in CD44+/CD24+/ESA+ cells was revealed to be enhanced versus that in the PANC-1 cells. The above experiments indicated the successful isolation of PCSCs. The miR-630 expression in different types of cells was evaluated by RT-qPCR (Fig. 1E). Relative to the normal pancreatic cells HPDE, the miR-630 expression in PANC-1 cells was remarkably diminished (p < 0.05), and the decline of that was more pronounced in the CD44+/CD24+/ESA+ cells (p < 0.01). Therefore, it was concluded that miR630 may affect tumor stem cell function.

Fig. 1
figure 1

miR-630 was lowly expressed in PCSCs. A, Heat map of differential miRNA expression in PC miRNA expression chip GSE41369. The x axis represents the sample numbers, and the y axis represents differentially expressed miRNAs. The histogram in the upper right is the color gradation, and each rectangle corresponds to the expression of each miRNA in the sample; B, Detection of CD44+/CD24+ cells and CD44+/CD24+/ESA+ cells in PANC-1 cell line by flow cytometry; C, Morphology of PANC-1 cells and CD44+/CD24+/ESA+ cells (Scar bar = 100 Î¼m); D, Expression of Oct4 and Nanog in PANC-1 cells and CD44+/CD24+/ESA+ cells were determined by RT-qPCR; * p < 0.05 vs. PANC-1 cells; E, Expression of miR-630 in normal pancreatic cells HPDE, PANC-1 cells and CD44+/CD24+/ESA+ cells were determined by RT-qPCR; * p < 0.05 vs. HPDE cells. RT-qPCR, Reverse transcription quantitative polymerase chain reaction; PC, pancreatic cancer. Measurement data was expressed by mean ± standard deviation. Comparison between the two groups was analyzed by independent sample t-test, and comparison between multiple groups was analyzed by one-way analysis of variance, followed by Tucky’s post hoc test. The experiment was repeated three times

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miR-630 inhibits the proliferation, colony formation, and sphere formation abilities of PCSCs

To further analyze the effect of miR-630 on the self-renewal and maintenance of PCSCs, we overexpressed and suppressed the expression of miR-630 in PCSCs (Fig. 2A). The proliferation of cells was explored by the EdU staining assay. As shown in Fig. 2B and Fig. S1A, compared with the mimic-NC group, the miR-630 mimic group presented a significant decrease in the EdU-positive expression rate, indicating that the ability of cell proliferation was inhibited. Conversely, down-regulated miR-630 caused an opposite trend. Determination of colony formation and sphere formation revealed that the number of clones and the ability of sphere formation in the miR-630 mimic group were markedly decreased while those in the miR-630 inhibitor group were increased (Fig. 2C, D, Fig. S1B, C). Moreover, the expression of Nanog and Oct4 was found to be remarkably decreased in the miR-630 mimic group, whereas that in the miR-630 inhibitor group was increased (Fig. 2E, F). The above results showed that miR-630 could reduce the self-proliferation and stem maintenance ability of PCSC cells in vitro.

Fig. 2
figure 2

miR-630 inhibits the self-renewal and maintenance of PCSCs. A, Expression of miR-630 in PCSCs after the treatment of miR-630 mimic/inhibitor were determined by RT-qPCR; B, Detection of cell proliferation by EdU and the positive expression rate in each group; C, The cell colony formation and the number of cell clones in each group; D, The sphere formation in each group; E, The expression of Nanog and Oct4 of cells in each group were determined by RT-qPCR; F, The protein expression of Nanog and Oct4 of cells in each group were determined by western blot analysis; * p < 0.05 vs. the mimic-NC group, # p < 0.05 vs. the inhibitor-NC group; miR-630, microRNA-630; PCSCs, pancreatic cancer stem cells; EDU, 5-ethynyl-2’-deoxyuridine; RT-qPCR, Reverse transcription quantitative polymerase chain reaction; NC, negative control. Measurement data was expressed by mean ± standard deviation. Comparison between two groups was analyzed by independent sample t-test. The experiment was repeated for three times

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Overexpression of miR-630 inhibits the formation and growth of tumors in vivo

To further investigate the effect of miR-630 on PCSCs in vivo, the abilities of tumorigenicity of PCSCs with different treatments were assessed. As shown in Table S3, the minimum number of tumor sphere cells that could cause tumors was 1 × 105 in the miR-630 agomir group while that in the miR-630 antagomir group was 1 × 103. The tumor ability formation of PCSCs in each group was appraised by the xenograft tumor assay in NOD-SCID mice (Fig. 3A, B). The volume and mass of subcutaneous tumors after miR-630 agomir injection decreased significantly, while increasing upon miR-630 antagomir. Detection of miR-630 expression in tumors also showed that miR-630 agomir increased the expression of miR-630, while miR-630 antagomir decreased miR-630 expression (Fig. 3C). It could be seen that miR-630 could also affect the tumorigenic ability of tumor stem cells in vivo.

Fig. 3
figure 3

miR-630 inhibits the tumor formation and growth of PCSCs in vivo. A, The xenograft tumor in NOD-SCID mice was performed to detect the tumor size in mice of each group; B, Tumor volume curve and weight chart of the mice in each group; C, Detection of the expression of miR-630 by RT-qPCR; * p < 0.05 vs. the agomir-NC group, #p < 0.05 vs. the antagomir-NC group. miR-630, microRNA-630; PCSCs, pancreatic cancer stem cells; NC, negative control. Measurement data was expressed by mean ± standard deviation. Comparison between two groups at different time points was analyzed by repeated measurement of variance, followed by Tucky’s post hoc test. Comparison between two groups was analyzed by independent sample t-test. The experiment was repeated for three times. N = 8 per group

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PRKCI is a direct target of miR-630

We then focused on the potential molecular mechanism behind miR-630 in PC. The target genes of miR-630 were predicted in TargetScan, mirDIP and miRDB websites, and 182, 366 and 797 target genes were obtained, respectively. Meanwhile, 1599, 1307 and 381 DEGs were screened from the microarray data GSE91035, GSE71989 and GSE32676, respectively. The differences between the differentially expressed genes (DEGs) of the 3 chips and between the target genes of miR-630 were compared by jvenn (Fig. 4A). The Venn diagram showed that there was only one intersection gene PRKCI. The heat map of the top 50 DEGs of GSE32676 showed that PRKCI was overexpressed in PC tissues (Fig. 4B), which was consistent with the results of the GSE91035 and GSE71989 datasets (Fig. 4C, D). The overexpression of PRKCI in PC tissues was also verified by GEPIA (Fig. 4E), and the results of the survival curve presented that the PRKCI expression was positively correlated with the OS rate of patients with PC (Fig. 4F). The specific binding sites were identified between the sequences of miR-630 and 3’-UTR of PRKCI (Fig. 4G). The targeting relationship was further verified by the dual-luciferase reporter gene assay (Fig. 4H). The luciferase activity in the cells co-transfected with miR-630 mimic and PRKCI-wt was reduced compared with that in the mimic-NC group (p < 0.05). Nevertheless, no significant difference was visible in luciferase activity in PRKCI-Mut either co-transfected with mimic-NC or miR-630 (p > 0.05), indicating that miR-630 could specifically target the PRKCI gene. The targeting relationship was also validated in PCSCs (Fig. 4I), and we found that the mRNA expression of PRKCI in the miR-630 mimic group was significantly decreased, while elevated in the miR-630 inhibitor group. For the results mentioned above, it was concluded that miR-630 negatively mediated the expression of PRKCI.

Fig. 4
figure 4

PRKCI is the direct target gene of miR-630. A, The target gene of miR-630 and PC-related differentially expressed gene predicted by TargetScan, mirDIP and microDB; B, Heatmap of the top 50 differentially expressed genes of the PC-related gene expression chip (GSE32676). The x axis represents sample numbers, and the y axis represents differentially expressed genes. The histogram in the upper right is the color gradation, and each rectangle corresponds to the expression of each gene in the sample; C & D, The expression changes of PRKCI in PC-related chips GSE91035 and GSE71989, respectively; E, The expression of PRKCI in PC identified by GEPIA database; F, The correlation between the expression of PRKCI and the survival rate of PC patients in GEPIA database; G, Predictive binding sites between miR-630 and PRKCI 3’-UTR; H, Luciferase activity was detected by dual luciferase reporter assay, * p < 0.05 compared with the mimic-NC group; I, PRKCI expression was detected by RT-qPCR, * p < 0.05 vs. the mimic-NC group, #p < 0.05 vs. the inhibitor-NC group; PRKCI, protein kinase C iota; miR-630, microRNA-630; PC, pancreatic cancer; RT-qPCR, Reverse transcription quantitative polymerase chain reaction; NC, negative control; GEPIA, Gene Expression Profiling Interactive Analysis. Measurement data was expressed by mean ± standard deviation. Comparison between two groups was analyzed by independent sample t-test. The experiment was repeated three times

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MiR-630 inhibits the self-renewal and maintenance of PCSCs via down-regulating PRKCI

In order to verify the effect of PRKCI on PCSCs, the expression of PRKCI was knocked down in PCSCs (Fig. 5A). It was found that there was no change of miR-630 expression in the sh-PRKCI + inhibitor-NC group compared with the sh-NC + inhibitor-NC group, but the PRKCI expression decreased; the miR-630 expression decreased, and the PRKCI expression increased in the miR-630 inhibitor + sh-NC group. Relative to the miR-630 inhibitor + sh-NC group, PRKCI expression was reduced in the miR-630 inhibitor + sh-PRKCI group (Fig. 5B-C). Functional assays demonstrated that sh-PRKCI treatment led to suppressed EdU-positive expression rate, colony formation ability, and cell sphere formation ability while miR-630 inhibitor treatment caused contrary findings; relative to the miR-630 inhibitor + sh-NC group, the miR-630 inhibitor + sh-PRKCI group showed limited EdU-positive expression rate, colony formation ability, and cell sphere formation ability (Fig. 5D-F). As evaluated by RT-qPCR and western blot analysis, the expression of Nanog and Oct4 was found to be repressed upon sh-PRKCI but higher in the presence of miR-630 inhibitor; relative to miR-630 inhibitor + sh-NC group, low Nanog and Oct4 expression was found in the miR-630 inhibitor + sh-PRKCI group (Fig. 5G, H). The results suggested that miR-630 could inhibit the self-renewal and maintenance of PCSCs through PRKCI.

Fig. 5
figure 5

miR-630 inhibits the self-renewal and maintenance of PC stem cells via inhibiting PRKCI. A, Western blot was used to detect the interference effect of sh-PRKCI on PRKCI in PCSCs, and the one with the highest efficiency was selected for subsequent experiments; * p < 0.05 vs. the sh-NC group; B, RT-qPCR detection of miR-630 expression after sh-PRKCI or miR-630 inhibitor; C: Western blot detection of PRKCI expression after sh-PRKCI or miR-630 inhibitor; D, EdU incorporation experiment was employed to detect the cell proliferation (25 Î¼m); E, The cell colony formation and the number of cell clones after miR-630 inhibitor or sh-PRKCI treatment; F, The sphere formation after miR-630 inhibitor or sh-PRKCI treatment (100 Î¼m); G, RT-qPCR was performed for the detection of Nanog and Oct4 expression after miR-630 inhibitor or sh-PRKCI treatment; H, Western blot analysis was used to detect the protein expression of Nanog and Oct4 expression after miR-630 inhibitor or sh-PRKCI treatment; * p < 0.05 vs. the sh-NC + inhibitor-NC group, #p < 0.05 vs. the miR-630 inhibitor + sh-NC group. PRKCI, protein kinase C iota; miR-630, microRNA-630; PC, pancreatic cancer; RT-qPCR, Reverse transcription quantitative polymerase chain reaction; NC, negative control; EdU, 5-ethynyl-2’-deoxyuridine. Measurement data was expressed by mean ± standard deviation. Comparison between two groups was analyzed by independent sample t-test. The experiment was repeated for three times

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miR-630 inhibits tumor formation and growth in vivo via down-regulating PRKCI

To verify the effect of miR-630-mediated PRKCI on PC in vivo, the abilities of tumorigenicity were evaluated. As shown in Table S4, the minimum number of tumor sphere cells that could cause tumors was 1 × 105 in the sh-PRKCI + antagomir-NC group when compared with the sh-NC + antagomir-NC group. Compared with the miR-630 antagomir + sh-NC group, the minimum number of tumor sphere cells that could cause tumors was 1 × 104 in the miR-630 antagomir + sh- PRKCI group. In addition, compared with the sh-NC + antagomir-NC group, the volume and mass of subcutaneous tumors in the sh-PRKCI + antagomir-NC group decreased significantly. Compared with the miR-630 antagomir + sh-NC group, the volume and mass of subcutaneous tumors in the miR-630 antagomir + sh-PRKCI group decreased (Fig. 6A, B). Detection of miR-630 and PRKCI expression in tumor tissues was also found that compared with the sh-NC + antagomir-NC group, the expression of miR-630 had no change in the sh-PRKCI + antagomir-NC group, but the expression of PRKCI decreased; in addition, the miR-630 expression decreased while PRKCI increased in miR-630 antagomir + sh-NC group. It is worth noting that PRKCI expression was significantly reduced in the miR-630 antagomir + sh-PRKCI group compared with miR-630 antagomir + sh-NC group (Fig. 6C, D). The experimental results also showed that the inhibition of PRKCI could alleviate the effect of miR-630 inhibition on the tumorigenic ability of PCSCs in vivo.

Fig. 6
figure 6

miR-630 inhibits tumor growth via PRKCI. A, The xenograft tumor in NOD-SCID mice was performed to detect the tumor size in mice of each group; B, Tumor volume curve and weight chart of the mice after miR-630 antagomir or sh-PRKCI injection; C, RT-qPCR detection of miR-630 expression after miR-630 antagomir or sh-PRKCI injection; D, Western blot detection of PRKCI expression after miR-630 antagomir or sh-PRKCI injection; * p < 0.05 vs. the sh-NC + antagomir-NC group, #p < 0.05 vs. the miR-630 antagomir + sh-NC group. PRKCI, protein kinase C iota; miR-630, microRNA-630; NC, negative control. Comparison between two groups at different time points was analyzed by repeated measurement of variance, followed by Tucky’s post hoc test. Comparison between two groups was analyzed by independent sample t-test. The experiment was repeated for three times. N = 8 per group

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Overexpression of miR-630 inhibits activation of the hedgehog signaling pathway via down-regulating PRKCI

The Hedgehog signaling pathway has been implicated in the development of PC [29, 30], while PRKCI activates the Hedgehog signaling pathway [20]. We may speculate that miR-630 targets and regulates PRKCI through the Hedgehog signaling pathway in PC. Immunofluorescence staining revealed that the intranuclear expression of Gli1 and Gli2 upon miR-630 inhibitor was significantly increased while decreased after further addition of sh-PRKCI (Fig. 7A). As quantified by western blot analysis, the protein expression of Smo was increased upon miR-630 inhibitor while diminished after further addition of sh-PRKCI (Fig. 7B).

Fig. 7
figure 7

miR-630 inhibits the Hedgehog signaling pathway through down-regulating PRKCI. A, Immunofluorescence staining was used to detect the intranuclear transfer of Gli1 and Gli2 in cells after miR-630 inhibitor or sh-PRKCI treatment; B, Western blot analysis was employed to detect the Smo protein expression in cells after miR-630 inhibitor or sh-PRKCI treatment; * p < 0.05 vs. the inhibitor-NC + sh-NC group, #p < 0.05 vs. the miR-630 inhibitor + sh-NC group. C, RT-qPCR analysis was used to detect the expression of miR-630 after miR-630 inhibitor or cyclopamine treatment; D, Western blot analysis was used to detect the protein expression of Smo after miR-630 inhibitor or cyclopamine treatment; E, Western blot analysis was used to detect the intranuclear protein expression of Gli1 and Gli2 after miR-630 inhibitor or cyclopamine treatment; F, RT-qPCR analysis was used to detect the expression of Nanog and Oct4 after miR-630 inhibitor or cyclopamine treatment; G, Western blot analysis was used to detect the protein expression of Nanog and Oct4 after miR-630 inhibitor or cyclopamine treatment, * p < 0.05 vs. the inhibitor-NC + DMSO group, #p < 0.05 vs. the miR-630 inhibitor + DMSO group. PRKCI, protein kinase C iota; miR-630, microRNA-630; NC, negative control. Measurement data was expressed by mean ± standard deviation. Comparison between two groups was analyzed by independent sample t-test. The experiment was repeated three times

Full size image

In order to verify the function of miR-630-regulated PRKCI in PCSCs through the Hedgehog signaling pathway, cells were treated with cyclopamine, an inhibitor of Hedghog signaling pathway. It was found that compared with the inhibitor NC + DMSO group, the expression of miR-630 decreased, the expression of PRKCI, Smo, Nanog, and Oct4as well as the intranuclear expression of Gli1 and Gli2 increased in the miR-630 inhibitor + DMSO group; relative to the miR-630 inhibitor + DMSO group, the miR-630 inhibitor + cyclopamine group showed reduced Smo, Nanog and Oct4 expression and intranuclear expression of Gli1 and Gli2 (Fig. 7C-G). The above results indicated that miR-630 affected the stemness of PCSCs through the Hedgehog signaling pathway by regulating PRKCI.

Discussion

PC is a highly malignant disease that has a very high resistance to treatment, and many patients cannot survive for more than one year due to the poor efficacy of some treatments [31]. Hence, there is an urgency to develop new therapies. It is well known that small non-coding miRs are related to several characteristics of cancer cells, including proliferation, invasion, and migration [32]. The important role of miR-630 in cancer progression has been previously highlighted [8]. Our study therefore aimed to investigate the functional role of miR-630 in PC development. The results suggested that over-expressed miR-630 weaken self-renewal ability and stemness of PCSCs through inhibiting PRKCI-mediated Hedgehog signaling pathway.

It was determined that the miR-630 was poorly expressed while PRKCI was highly expressed in PC cells. It is reported that miR-630 is lowly expressed in advanced breast cancer tissues and its elevation could induce apoptosis and suppress cell proliferation [9]. In addition, adamantyl retinoid-related (ARR) molecule 3-Cl-AHPC-dependent apoptosis in PC cells can be regulated by miR-630 [33]. It is proved that PRKCI is highly expressed in ovarian cancer, lung and liver cancers [12, 34]. In our study, PRKCI was verified to be the target gene of miR-630. miR-630 promotion or PRKCI silencing could impede cell proliferation, colony formation, and tumor cell sphere formation in addition to reducing the expression of Nanog and Oct4. miR-630 was expressed at a low level in esophageal squamous cell carcinoma (ESCC), and over-expressed miR-630 could significantly constrain ESCC cell proliferation and invasion [35]. In addition, the suppression of cell growth and metastasis in lung cancer through targeting LMO3 by miR-630 was also clarified [36]. As a critical oncogene, PRKCI has been reported to play multifunctional roles in cell maintenance, cell proliferation, survival, differentiation and apoptosis [37]. The high expression of Oct4 and Nanog in metaplastic ducts indicates that they are involved in the early stage of PC carcinogenesis [38]. Another report reveals that the low expression of Oct4 and Nanog inhibits the stemness of PC cells [39]. From the above results, it was concluded that miR-630 negatively targeted PRKCI, and down-regulated miR-630 and up-regulated PRKCI are closely related to the progression and deterioration of PC.

Importantly, the involvement of the Hedgehog signaling pathway in the process of PC has been emphasized in the current study, and the induction of the pathway was modulated by miR-630 and PRKCI. The mRNA and protein expression of Smo, Gli1, and Gli2 was observed to be increased in PC. As a typical G protein-coupled receptor, Smo could positively regulate cell proliferation and differentiation in insects and vertebrates, and the over-expression of Smo may be responsible for the development of various cancers, including PC [40]. Gli1 and Gli2 are transcription factors promoting the PC cell proliferation [41, 42]. The expression of Smo, Gli1 and Gli2 are down-regulated when the Hedgehog signaling pathway is inhibited. Abnormality of the Hedgehog signaling pathway was closely associated with various developmental defects and human cancers, PC included [43, 44]. Previous studies have shown that PRKCI participates in the signal transduction involved in the Hedgehog signaling pathway. Gli1 is phosphorylated by PRKCI and the downstream protein Smo is activated, which inhibits the signal transduction as well as the growth of basal cell carcinoma cell lines [45]. It is elucidated that miR-212 can also promote cell growth and invasion of PC cells by binding to PTCH1, a well-known factor in the Hedgehog signaling pathway [46]. Accordingly, it is concluded that up-regulation of miR-630 inhibits the expression of Smo, Gli1, and Gli2 in the Hedgehog signaling pathway by down-regulating PRKCI.

However, our study has some limitations. First, our research was conducted in PCSCs rather than clinical patient-derived tissue samples, so further validation in clinical samples or patient-derived models is needed before this study can be applied in clinical settings. Additionally, our results indicate that treatment with miR-630 antagomir significantly delayed tumor growth, but whether it can prevent further tumor progression remains to be investigated. Previous studies have shown that specific targeting of upstream molecules in the Hedgehog signaling pathway did not yield the expected clinical benefits [47], suggesting that miR-630-targeted therapy alone may also have limited efficacy. Furthermore, since miRNAs can target multiple mRNAs, unexpected consequences could arise in clinical applications. In future studies, miR-630 therapy could be combined with chemotherapy as a potential combination therapy for clinical use.

Conclusions

In summary, our study shows that overexpression of miR-630 inhibits the characteristics of PCSCs by inhibiting the activation of PRKCI-mediated Hedgehog signaling pathway (Fig. 8). These results suggest that supplementary understanding of the molecular mechanisms of PC may have promising clinical applications for the PC treatment.

Fig. 8
figure 8

The regulatory mechanism of miR-630 in PC. Overexpression of miR-630 inhibits the characteristics of PCSCs by inhibiting the activation of the PRKCI-mediated Hedgehog signaling pathway

Full size image

Data availability

The data that supports the findings of this study are available on request from the corresponding author.

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Acknowledgements

We acknowledge and appreciate our colleagues for their valuable efforts and comments on this paper.

Funding

This study was supported by Jiaxing Key Discipiline of Medicine-Surgery Med (Hepatobiliary pancreatology) (2023-ZC-005).

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Authors and Affiliations

  1. Department of Abdominal Oncology Surgery, Jiangxi Cancer Hospital, Nanchang, Jiangxi Province, China

    Jun Zou & Bangran Xu

  2. Department of Nursing, Jiangxi College of Traditional Chinese Medicine, Fuzhou, Jiangxi Province, China

    Sha Yang

  3. Department of Breast Surgery, Jiangxi Cancer Hospital, Nanchang, Jiangxi Province, China

    Chongwu He

  4. Department of Medical Oncology, Jiangxi Cancer Hospital, Nanchang, Jiangxi Province, China

    Lei Deng

  5. Department of General Surgery, The Affiliated Hospital of Jiaxing University, No. 1882, Southern Zhonghuan Road, Jiaxing, Zhejiang, 314001, China

    Shuai Chen

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Contributions

Jun Zou and Shuai Chen conceived and designed the study. Shuai Chen and Lei Deng performed the experiments and collected data. Chongwu He and Bangran Xu analyzed the data and contributed to data interpretation. Jun Zou and Shuai Chen drafted the manuscript. Sha Yang provided critical revisions and supervised the project. All authors read and approved the final manuscript.

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The study protocol was approved by the ethics committee of Jiangxi Cancer Hospital. The written informed consents were provided from all the patients. Mice were treated humanely, and all experiment procedures were permitted by the Institutional Animal Care and Use Committee of Jiangxi Cancer Hospital (Approval no. 2024ky110). Efforts have made to minimize the mice suffering.

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Supplementary Material 1: Fig. S1 A, Representative image of Figure 2B (25 μm); B, Representative image of Figure 2C; C, Representative image of Figure 2D (100 μm).

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Zou, J., Yang, S., He, C. et al. miR-630 as a therapeutic target in pancreatic cancer stem cells: modulation of the PRKCI-Hedgehog signaling axis. Biol Direct 19, 109 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13062-024-00539-1

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