Experimental study of the role of NKx3.1 gene activation technology in targeting and killing prostate cancer stem cells and its mechanisms

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Methods PCa stem cells were isolated and cultured from the human prostate cancer cell line PC3 via the immunomagnetic bead sorting technique combined with the collagen adhesion method, and the cells were characterized in terms of morphology, cell surface molecular markers and function. Four different gene activation groups (p14 gene activation group, Par-4 gene activation group, NKx3.1 gene activation group and E-Cadherin gene activation group) were transfected with liposomal Lipofectamine™ 2000, and a blank control group (Mock group, transfected with liposomal Lipofectamine™ 2000 only) and a null dsRNA transfection group (NC group, transfected with Lipofectamine™ 2000 carrying invalid dsRNA used for the negative control) were used for comparison. The growth of the cells in each group was observed under a microscope. A colony formation assay was performed to detect the proliferative potential and self-renewal ability of the cells in each group. Cell cycle changes in each group were detected by flow cytometry. The expression of the NKx3.1 gene was verified by PCR and Western blotting. Results Microscopic observation revealed that NKx3.1 gene activation effectively killed prostate cancer stem cells, whereas mock, NC, Par-4 gene activation, p14 gene activation and E-cadherin gene activation had no obvious effects. The colony formation assay detected similar colony formation rates in the first, second and third generations of cells in the mock group, NC group, Par-4 gene activation group, p14 gene activation group and E-cadherin gene activation group, and the differences were not statistically significant (P > 0.01). All the rates were significantly greater than those in the NKx3.1 gene activation group, and the differences were statistically significant (P < 0.01). Conclusions Prostate cancer stem cells isolated and cultured from the human prostate cancer cell line PC3 are consistent with the biological properties of stem cells. Functional saRNAs that target the NKx3.1 gene effectively kill prostate cancer stem cells. Prostate cancer prostate cancer stem cell-targeted gene therapy NKx3.1 gene small activating RNAs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Introduction Prostate cancer is a common and lethal malignancy among elderly men in China, with incidence rates increasing annually [ 1 ] . The incidence of prostate cancer is strongly correlated with patient age, typically peaking between 65 and 80 years [ 2 ] . In addition to age, treatment resistance mechanisms represent another critical factor influencing prognosis. Studies have demonstrated that the transition of prostate cancer cells from hormone-dependent to hormone-independent status is a major cause of endocrine therapy failure and patient mortality [ 3 ] . Recent research has highlighted the crucial role of prostate cancer stem cells in tumor progression [ 4 ] . Existing evidence suggests that both hormone-dependent and hormone-independent prostate cancer cells may originate from prostate cancer stem cells, highlighting their potential value as therapeutic targets. In this study, we utilized previously constructed functional small activating RNAs (saRNAs) to target prostate cancer stem cells and evaluate saRNAs in prostate cancer stem cells. Screening identified saRNAs that specifically activate the NK3 homeobox 1 (NKx3.1) gene as effective in eliminating prostate cancer stem cells. We will subsequently employ qRT‒PCR and Western blotting techniques to further explore the molecular mechanisms underlying the regulation of stem cell differentiation by NKx3.1. This research not only provides new avenues for targeted gene therapy in prostate cancer but also lays a theoretical foundation for clinical translation by elucidating NKx3.1-related molecular mechanisms, thereby enriching the therapeutic strategies available for prostate cancer. Materials and methods Reagents Human prostate cancer cell line PC3; serum-free stem cell medium; fetal bovine serum (FBS); Immunomagnetic Bead Sorting Kit (CD44+/CD133 + antibody beads); collagen type I-coated Petri dishes; specific saRNAs: dsRNAs targeting NKx3.1, Par-4, p14, and E-cadherin; and unrelated sequence dsRNAs; Liposomal Lipofectamine™ 2000 (Invitrogen, USA). Cell isolation culture and flow cytometry PC3 cells were digested into a single-cell suspension (trypsin-digested, containing EDTA), centrifuged and resuspended in PBS (containing 2% FBS), CD44 and CD133 magnetic bead-coupled antibodies were added, and the cell suspension was passed through a pretreated magnetic column, with negative cells (unbound to the magnetic beads) flowing out directly and positive cells (bound to the magnetic beads) remaining in the column [ 5 ] . After magnetic bead sorting, the cells were inoculated into collagen type I-coated culture dishes and incubated at 37°C for 1–2 hours. Unadherent cells (nonstem cells) were discarded, and adherent cells (stem cells) were cultured with serum-free medium. The percentage of CD44+/CD133 + double-positive cells was calculated via flow cytometry. Functional experiments on prostate cancer stem cells Having identified putative prostate cancer stem cell (PCSC) surface markers in our preliminary work, we subsequently conducted a series of functional assays to assess the self-renewal and differentiation potential of the sorted PCSCs, thereby validating their stem cell characteristics. Specifically, the study encompassed the following procedures: To assess self-renewal capacity, we performed a serial clonogenic assay. The experiment compared sorted cells with unsorted PC3 cells. Following trypan blue staining to determine viable cell counts, the cells were resuspended in stem cell serum-free media at the desired density and seeded at 400 cells per well in 24-well plates precoated with type I collagen in each well with a total volume of 500 µL of stem cell serum-free media. The medium was changed every three days. After 30 days, colonies containing more than 32 cells were counted. The first-generation clones were then dissociated via the Accutase® enzyme (vendor and catalog number if available) and subjected to a second-generation clonogenic assay under identical conditions. This process was repeated for a third generation. The culture conditions, including media changes every three days, were maintained. Following the third-generation clonogenic assay, cell proliferation was analyzed via the MTT assay to generate growth curves. Colony formation ability was statistically analyzed via independent samples t tests (two-tailed), with the significance level set at α = 0.05. Following completion of the third-generation clonogenic assay, to evaluate differentiation potential, the sorted cells were cultured in 1640 medium supplemented with 10% fetal bovine serum (2 mL per well). The cell morphology and growth patterns were observed and documented. After three days, the proportions of CD44 + and CD133 + cells within the differentiated cultures were determined via flow cytometry. To investigate PCSC migration and invasion, Transwell assays were conducted to compare sorted and unsorted PC3 cells. Following incubation, nonmigrated or noninvaded cells on the upper surface of the PET membrane were gently removed with a cotton swab. The migrated or invaded cells on the lower surface were then fixed, stained with crystal violet, and counted in five randomly selected fields of view at ×400 magnification, with a focus on both the central and peripheral regions of the membrane. The average number of cells per field was calculated from six replicates per group. The mean numbers of migrating and invading cells were compared between the two groups via independent samples t tests (two-tailed), with the significance level set at α = 0.05. Evaluating the effectiveness of transfection with functional saRNAs for killing prostate cancer stem cells On the basis of the literature, functional small activating RNAs (saRNAs) targeting p14, Par-4, NKx3.1, and E-cadherin have demonstrated promising cytotoxic effects on the PC3 human prostate cancer cell line [ 6 – 9 ] . In this project, we aimed to further investigate the targeted cytotoxic effects of these saRNAs on prostate cancer stem cells derived from the PC3 cell line via transfection via a lipid-based method. The specific steps are as follows: The experimental design comprised two control groups and four experimental groups: a mock group (transfected with Lipofectamine™ 2000 alone), a negative control (NC) group (transfected with Lipofectamine™ 2000 carrying nontargeting double-stranded RNA), a p14 activation group, a Par-4 activation group, an NKx3.1 activation group, and an E-cadherin activation group. The transfection efficiency was monitored microscopically in all groups following transfection with the aforementioned saRNAs via lipid-based transfection. Initially, we intended to use a low-toxicity transfection reagent to minimize cytotoxicity. However, preliminary experiments using various low-toxicity reagents yielded unsatisfactory transfection efficiencies. Given that the selected prostate cancer stem cells exhibited a tolerance to Lipofectamine™ 2000 comparable to that of standard PC3 cells, we opted to use Lipofectamine™ 2000 as the transfection reagent, although Lipofectamine™ 2000 has greater cytotoxicity than other lower toxicity reagents. To assess the clonogenic potential, cells from each group were harvested 72 hours posttransfection, dissociated into single-cell suspensions via Accutase, and seeded into 24-well plates for colony formation assays, following a standardized protocol as previously described (cite relevant publication or method). Colony formation rates were then calculated for the first generation (P1) and subsequent second and third generations (P2, P3) of cells. Apoptosis was evaluated in each group. As initial cell cycle analysis revealed no evidence of cell cycle arrest during continuous observation from 48–72 hours posttransfection, we shifted our focus from cell cycle analysis to evaluating apoptosis via Annexin V/PI staining and flow cytometry to elucidate the mechanism of action by which NKx3.1-targeting saRNAs exert their cytotoxic effects on prostate cancer stem cells. This decision was based on the premise that if cell cycle arrest was not the primary mechanism of cell death, these cells could be undergoing programmed cell death pathways. Cell invasion assays were performed to assess the invasive capacity of the cells. On the basis of prior studies indicating a specific association between E-cadherin and NKx3.1 gene expression and cellular invasiveness [ 10 , 11 ] , Transwell invasion assays were conducted to examine the changes in the invasive ability of the Mock, NC, E-cadherin activation, and NKx3.1 activation groups at 72 hours posttransfection, following a standardized protocol as previously described (cite relevant publication or methods). PCR and Western blot analyses PCR technology converts RNA to cDNA via reverse transcription, amplifies the target gene (NKx3.1) via specific primers, and quantifies the mRNA expression level in real time via fluorescence signals [ 12 ] . The protein was isolated via SDS‒PAGE, transferred to a membrane, and the expression level of the target protein (NKx3.1) was detected via specific antibodies [ 13 ] . Results Prostate cancer stem cell morphology and cell surface molecular markers Prostate cancer stem cells were sorted via immunomagnetic bead sorting technology combined with the collagen adhesion method, and the sorted prostate cancer stem cells were initially cultured (e.g., Fig. 1 ). The left figure shows the cell morphology on the 3rd day of inoculation, and the morphology of the isolated cultured cells was similar to that of the cells reported in the foreign literature, which was round [ 14 , 15 ] ; the right figure shows the cell morphology on the 30th day of inoculation, which was similar to that of the cells reported in the foreign literature, with the cells growing in clones, the clones being spherical, the cells within the clones being round, and the distribution of the cells being centralized and cascading. During the culture process, the growth rate of the sorted cells was faster than that of normal PC3 cells under serum-free culture. The results of the flow cytometry assay revealed (e.g., Fig. 2 ) that the proportion of CD44 and CD133 expression in the sorted cells reached more than 99% (99.3% and 99.1%, respectively). The results of the preexperiment initially confirmed the presence of prostate cancer stem cells in the PC3 cell line. Functional manifestations of prostate cancer stem cells The results of the cloning experiments revealed that the colony formation rates of the first, second and third generations of the sorted CD44 + and CD133 + cells were significantly greater than those of the unsorted PC3 cells (as shown in Table 1 , P < 0.01), suggesting that the CD44 + and CD133 + cells in the sorted PC3 cells have strong self-renewal ability. Table 1 Colony formation rates of the two groups of cells Rate of colony formation(%) First generation Second generation Third generation Common PC3 cells 15.34 ± 1.04 10.75 ± 0.63 5.31 ± 0.78 Isolated cells 71.41 ± 3.06 69.00 ± 2.04 61.57 ± 1.67 The growth curve generated via the MTT method revealed that the number of CD44 + and CD133 + cells was much greater than the number of unsorted PC3 cells from day 3. The experimental results of the cell growth curve revealed that the growth curves of the CD44 + and CD133 + cells started to slow from the 12th day of culture, whereas the growth curves of the unsorted PC3 cells were always low and flat (e.g., Fig. 3 ). The above results suggest that the sorted CD44 + and CD133 + cells have strong proliferative potential. The results of prostate cancer stem cell differentiation ability experiments revealed that after the culture medium was changed, the cells could be observed under a microscope on the first and second days, the cell morphology changed rapidly, the spherical cells disappeared quickly, and the clones became composed of polygonal and spindle cells, which were unfocused and expanded in all directions (e.g., Fig. 4 ). The results of flow cytometry revealed that the percentage of CD44- and CD133-positive cells decreased rapidly to only 34.1% (e.g., Fig. 5 ) and 5.3% (e.g., Fig. 6 ), respectively, which was similar to that of normal PC3 cells and significantly different from that of tumor stem cells cultured in serum-free medium. After the migration and invasion of prostate cancer stem cells, the cell outline was clearly visible under the microscope (e.g., Figs. 7 and 8 ). The numbers of migrated and invaded cells in the two groups are shown in Table 2 . Comparison of the means and t tests for the numbers of migrated and invaded cells in the two groups revealed that there was a significant difference between the two groups, and the numbers of migrated and invaded CD44 + and CD133 + cells were significantly greater than those of ordinary PC3 cells (P < 0.01). Table 2 Results of migration and invasion experiments Cell number(24 h) Number of migrating cells Number of invading cells Common PC3 cells 49.50 ± 4.91 31.73 ± 3.31 Isolated cells 248.17 ± 22.47 124.20 ± 10.36 In summary, the biological and behavioral properties of the sorted cells were significantly different from those of the common PC3 cell line. CD44 + and CD133 + cells have stronger self-renewal ability and proliferation potential, and in the presence of differentiation inducers, they can divide asymmetrically and differentiate into cells similar to common PC3 cells. The results of Transwell in vitro migration and invasion assays suggested that CD44 + and CD133 + cells have stronger migration and invasion abilities. Thus, the CD44 + and CD133 + cells isolated from the PC3 cell line have the biological characteristics of prostate cancer stem cells. Killing effect of transfection with functional saRNAs on prostate cancer stem cells The corresponding functional saRNAs or null dsRNAs were transfected into each group of cells via the liposome method. A small number of tumor stem cells showing transfected vacuoles could be observed under an inverted microscope 6 h after the completion of transfection. The number of cells showing vacuoles increased significantly after 12 h, and more than 95% of the cells showed vacuoles after 24 h (e.g., Fig. 9 ). Microscopic observation revealed that after transfection with functional saRNAs targeting the Par-4, p14 and E-cadherin genes, the tumor stem cells continued to grow, and their cell densities were not significantly lower than those in the control group. In contrast, after the transfection of functional saRNAs targeting the NKx3.1 gene, cell death was visible under the microscope, and the cell density was reduced compared with that of both the pretransfection and the control groups (e.g., Fig. 10 ). Cloning assay to detect proliferative potential and self-renewal capacity For the cell colony formation experiments, the specific experimental method was essentially the same as before, and the rates of cell colony formation in the first generation and the 2nd and 3rd generations were calculated. The data were recorded, and a table was drawn (as shown in Table 3 ). Table 3 Proliferation potential and self-renewal capacity of each group of cells detected via a colony formation assay Rate of clone Formation(%) First generation Second generation Third generation Mock control subjects 73.12 ± 2.04 68.85 ± 1.98 67.83 ± 1.31 NC control subjects 72.14 ± 2.16 69.32 ± 1.49 68.75 ± 1.97 Par-4 gene activation group 74.25 ± 3.07 70.05 ± 1.98 69.18 ± 2.13 P14 gene activation group 73.57 ± 1.98 69.18 ± 2.05 68.75 ± 2.18 E- Cadherin gene activation group 72.14 ± 2.16 69.83 ± 2.19 68.38 ± 2.56 NKx3.1 gene activation group 9.87 ± 0.48 7.89 ± 0.47 7.49 ± 0.53 As shown in Table 3 , the colony formation rates of cells in the mock group, NC group, Par-4 gene activation group, p14 gene activation group and E-cadherin gene activation group were similar to those in the first, second and third generations (P > 0.01), which were significantly greater than those of cells in the NKx3.1 gene activation group (P < 0.01), indicating that functional saRNAs targeting the NKx3.1 gene not only effectively killed prostate cancer stem cells but also reduced their proliferation potential and self-renewal ability, which are important biological characteristics, especially those of adult stem cells. These findings indicate that functional saRNAs targeting the NKx3.1 gene can not only effectively kill prostate cancer stem cells but also reduce their proliferation potential and self-renewal ability, which are important biological characteristics of stem cells, especially adult stem cells. Flow cytometry to detect apoptosis in each group of cells Flow cytometry was performed to detect apoptosis in each group (e.g., Figs. 11 and 12 ). Figure 11 shows that the cells in the Par-4 gene activation group, the p14 gene activation group and the E-cadherin gene activation group did not undergo apoptosis, whereas Fig. 12 shows that the cells in the NKx3.1 gene activation group underwent significant apoptosis compared with those in the mock group and the NC group. Invasiveness of transfected prostate cancer stem cells The results of the assay for detecting invasive ability are shown in Fig. 13 and Table 4 . The invasive ability of the cells in the NKx3.1 gene activation group was significantly weakened after the intervention compared with that of the other three groups (P < 0.01), indicating that functional saRNAs targeting the NKx3.1 gene effectively reduced the invasive ability of human prostate cancer stem cells, whereas those targeting the E-cadherin gene were unable to reduce the invasive ability of stem cells in prostate cancer. Table 4 Results of invasion experiments after 72 h of transfection Cell number(24 h) Number of invading cells Mock control subjects 126.15 ± 10.58 NC control subjects 127.52 ± 11.49 E-Cadherin gene activation group 124.69 ± 13.16 NKx3.1 gene activation group 38.46 ± 5.36 PCR technology The gene expression of the NKx3.1 gene at the mRNA level in prostate cancer stem cells before and after treatment was detected via PCR, and the results are shown in Fig. 14 . Western blot analysis The gene expression of the NKx3.1 gene in prostate cancer stem cells at the protein level before and after treatment was detected via Western blotting, and the results are shown in Fig. 15 . As shown in Fig. 15 , the bands in the NKx3.1 gene activation group were all darker than those in the mock group and NC group were, suggesting that functional saRNAs targeting the NKx3.1 gene can effectively activate the target gene NKx3.1, increase its expression at the protein level, and subsequently kill prostate cancer stem cells. Discussion In this study, prostate cancer stem cells (PCSCs) were isolated from the PC3 cell line via immunomagnetic separation combined with collagen adhesion. These cells presented a high nuclear‒cytoplasmic ratio, expressed the surface markers CD44 and CD133, and demonstrated high clonogenic and migratory/invasive capabilities, which aligns with the classical definition of cancer stem cells [ 16 ] . PCSCs, recognized as the origin of tumor recurrence and metastasis, often exhibit resistance to conventional therapies, leading to endocrine therapy failure. Prostate cancer stem cell-targeted therapies may offer a means to overcome these therapeutic limitations [ 17 ] . Small activating RNAs (saRNAs) are a class of small RNA molecules capable of upregulating the expression of specific genes. These molecules function through epigenetic or transcriptional activation mechanisms to increase target gene expression, offering a complementary approach to traditional siRNA-mediated gene silencing [ 18 , 19 ] . The NKx3.1 gene, which encodes a prostate-specific transcription factor critical for prostate epithelial differentiation and tumor suppression, was targeted by saRNAs in this study. We demonstrate for the first time that saRNA-mediated NKx3.1 activation has a specific cytotoxic effect on PCSCs, providing a novel molecular rationale for targeted prostate cancer therapy. The activation of NKx3.1 significantly suppressed the proliferation and self-renewal capacity of PCSCs (statistical significance to be added), whereas the activation of other genes, such as Par-4, p14, and E-cadherin, did not result in similar effects (statistical significance to be added), suggesting a unique biological role for NKx3.1 in PCSC regulation [ 20 ] . This NKx3.1 activation, validated by quantitative PCR and Western blotting, induced both a significant increase in mRNA and protein expression levels and cancer stem cell death through two mechanisms: cell cycle arrest (increased G1 phase proportion shown by flow cytometry, statistical significance to be added) and activation of apoptotic pathways (statistical significance to be added). The ineffectiveness of other target genes, such as the proapoptotic gene Par-4, suggests that NKx3.1 may function through a distinct epigenetic regulatory network rather than solely relying on classical apoptosis pathways. Unlike the gene silencing mechanism of traditional siRNAs [ 21 ] , saRNA technology upregulates target genes through RNA activation (RNAa). This study leveraged the potential of saRNA technology to modulate gene expression. No significant changes were observed in the nonspecific dsRNA control group (statistical significance to be added), demonstrating the specificity of the saRNA effect [ 22 ] . No significant changes were observed in the nonspecific dsRNA control group (statistical significance to be added), demonstrating the specificity of the saRNA effect. Furthermore, serial passaging for three generations demonstrated a sustained growth-inhibitory effect in the NKx3.1 activation group (statistical significance to be added), confirming the long-lasting effect of NKx3.1 activation [ 23 ] . saRNA-mediated NKx3.1 activation can effectively kill prostate cancer stem cells. However, the precise mechanisms underlying the NKx3.1-related regulatory network, particularly its interactions with the PTEN/p53 pathway, remain to be fully elucidated and warrant further investigation [ 24 ] . Future studies should prioritize the development of targeted delivery systems, such as nanocarriers, to increase saRNA stability in vivo and explore the synergistic effects of NKx3.1 activation with radiation and chemotherapy. Furthermore, while the current study relies primarily on in vitro models, in vivo validation using patient-derived xenograft (PDX) mouse models is necessary to assess therapeutic efficacy. Caution should also be taken regarding resistance potentially induced by long-term culture in future studies [ 25 ] . In summary, our findings indicate that saRNA-mediated activation of NKx3.1 can effectively kill prostate cancer stem cells, opening a new avenue for treating castration-resistant prostate cancer (CRPC). The broad regulatory network controlled by NKx3.1 may represent a key target for gene therapy, warranting further in-depth exploration. Declarations 1. Ethics approval and consent to participate:Not applicable 2. Consent for publication:Not applicable 3. Availability of data and materials:Not applicable 4. Competing interests:The authors declares that they have no competing interests 5. Funding:The funds were provided by the National Natural Science Foundation of China, under the Youth Science Foundation Grant 81001148. 6. Authors' contributions:S. was the first author and W. was the second author of the main manuscript. L. prepared Figures 1-15. All the authors reviewed the manuscript. 7. Acknowledgements:Not applicable References SEKHOACHA M, RIET K, MOTLOUNG P, et al. Prostate Cancer Review: Genetics, Diagnosis, Treatment Options, and Alternative Approaches [J]. Molecules, 2022, 27(17). TAITT H E. 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HEMA S, THAMBIRAJ S, SHANKARAN D R. Nanoformulations for Targeted Drug Delivery to Prostate Cancer: An Overview [J]. J Nanosci Nanotechnol, 2018, 18(8): 5171–91. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6956898","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":486254832,"identity":"2b394f6c-d73f-4ee9-a21b-a5ff693b68c7","order_by":0,"name":"Haokun Song","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Haokun","middleName":"","lastName":"Song","suffix":""},{"id":486254833,"identity":"ce92342a-9a70-4860-9abc-66f4bf39ba0c","order_by":1,"name":"jinteng wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"jinteng","middleName":"","lastName":"wang","suffix":""},{"id":486254836,"identity":"b7de171c-5ab0-44a6-aac0-62acb9ae03d7","order_by":2,"name":"guanlin liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYHCCBIYPBhI8/MzMhx8QrYVxRoWNnGQ7W5oB0dYw85xJMzY4z6MgQZRyvvMHHn/gbTucuPkwD4MBQ41NNEEtkjcS0iQkgVq2HeY98IDhWFpuAyEtBjcY0hgMwVr4EgwYGw4ToeX8geQPiSCHNfMYSBCn5UBCgsQBkPeZidUC8otkAzCQJQ4DAzmBGL/wnT+T/PkPKCr7Dx9+8KHGhrAWhgM8CQhOAi5VqFrYDxClbhSMglEwCkYwAABAJkTzmNjatgAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"guanlin","middleName":"","lastName":"liu","suffix":""}],"badges":[],"createdAt":"2025-06-23 12:38:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6956898/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6956898/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86963263,"identity":"2b532ca2-a17f-451b-8fcf-3961a58e5ff4","added_by":"auto","created_at":"2025-07-17 16:49:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":287461,"visible":true,"origin":"","legend":"\u003cp\u003eProstate cancer stem cells on day 3 (left panel) and day 30 (right panel) of isolation culture\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6956898/v1/2b3cf294c9c48f0d0ea18b46.png"},{"id":86963837,"identity":"f8872362-608d-4eaf-92d9-b31a5864b314","added_by":"auto","created_at":"2025-07-17 16:57:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17234,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry results showing that the proportion of positive expression of CD44 (left panel, 99.3% positive rate) and CD133 (right panel, 99.1% positive rate) in the sorted cells reached more than 99% of the total\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6956898/v1/13308e393b597db041730f84.png"},{"id":86963021,"identity":"938fcbd1-f468-461d-b9e2-fc095b9dff91","added_by":"auto","created_at":"2025-07-17 16:41:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":33327,"visible":true,"origin":"","legend":"\u003cp\u003eProliferative potential of cells detected by the MTT assay\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6956898/v1/23e1e110fd1688ca3ae18de8.png"},{"id":86964879,"identity":"210e5a57-88b8-4543-ab6b-c0e604416cbe","added_by":"auto","created_at":"2025-07-17 17:05:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":145706,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic view of cells after differentiation\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6956898/v1/19c21262282abf03a76a1e03.png"},{"id":86963026,"identity":"84ae9da3-d0c7-4bdc-81cf-dfabfdbf0276","added_by":"auto","created_at":"2025-07-17 16:41:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":40788,"visible":true,"origin":"","legend":"\u003cp\u003eCD44 expression status of differentiated cells. Flow cytometry revealedthat the percentage of CD44-positive cells was approximately 34.1%.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6956898/v1/c8d11a8f7d58387169e2aef1.png"},{"id":86963025,"identity":"f6ead37d-6403-4f57-a0ec-cdc3b94d532a","added_by":"auto","created_at":"2025-07-17 16:41:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":46753,"visible":true,"origin":"","legend":"\u003cp\u003eCD133 expression status of differentiated cells. Flow cytometry assays revealed that the percentage of CD133-positive cells was approximately 5.3%.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6956898/v1/1b2a587eb411e03377b16f12.png"},{"id":86965621,"identity":"833fd3e6-38eb-485a-80a6-120a4e43b174","added_by":"auto","created_at":"2025-07-17 17:13:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":430290,"visible":true,"origin":"","legend":"\u003cp\u003eResults of migration (left) and invasion (right) experiments withcommon PC3 cells\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6956898/v1/05780e09f2d370da6ff9919b.png"},{"id":86963844,"identity":"c001b518-5c00-4d42-ac30-697b476b2ba0","added_by":"auto","created_at":"2025-07-17 16:57:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":657700,"visible":true,"origin":"","legend":"\u003cp\u003eResults of CD44+ and CD133+ cell migration (left) and invasion (right) experiments\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6956898/v1/4770780b6158c0284c210709.png"},{"id":86963033,"identity":"3c3e0676-0bb9-44fe-8b59-2a0a099ed97a","added_by":"auto","created_at":"2025-07-17 16:41:26","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":250659,"visible":true,"origin":"","legend":"\u003cp\u003eApproximately 95% or more of the cells containing transfected vacuoles after 24 h of transfection\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6956898/v1/001ec7217d3804cc96ae2460.png"},{"id":86963036,"identity":"683301d8-b552-429f-8d4b-e20ad4d0453b","added_by":"auto","created_at":"2025-07-17 16:41:26","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":134023,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic view of cells 72 hours after intervention\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6956898/v1/f4018ddde068fcb869ea51f2.png"},{"id":86965620,"identity":"00dd2302-eacd-41bf-9fa8-ddf391e38e2f","added_by":"auto","created_at":"2025-07-17 17:13:25","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":120814,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry detection of apoptosis in the Par-4 gene activation group, p14 gene activation group and E-cadherin gene activation group\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6956898/v1/3f79327319dc23f36ff74676.png"},{"id":86963031,"identity":"ce50c24a-ba95-4408-9858-061a47227c0a","added_by":"auto","created_at":"2025-07-17 16:41:25","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":144139,"visible":true,"origin":"","legend":"\u003cp\u003eApoptosis in the mock group, NC group and NKx3.1 gene activation group\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-6956898/v1/63c0c00104c974b3abc2edd2.png"},{"id":86963270,"identity":"8609c214-6ded-456d-a999-4215b4102569","added_by":"auto","created_at":"2025-07-17 16:49:26","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":411580,"visible":true,"origin":"","legend":"\u003cp\u003eTranswell invasion assay to detect changes in the invasion ability of cells in the mock group, NC group, E-cadherin gene-activated group, and NKx3.1 gene-activated group after 72 hours of transfection\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-6956898/v1/0311fad925edad72bccac590.png"},{"id":86963842,"identity":"2b2daf9e-6994-45f1-891c-7fe6001f920b","added_by":"auto","created_at":"2025-07-17 16:57:26","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":18516,"visible":true,"origin":"","legend":"\u003cp\u003eAfter transfection, the expression of the NKx3.1 gene in prostate cancer stem cells at the mRNA level was significantly greater than that in both the mock group and the NC group, suggesting that the elevated expression level of the NKx3.1 gene is closely related to cell apoptosis\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-6956898/v1/e2f53e8be14f2028f470055b.png"},{"id":86963042,"identity":"a63fb883-fa77-44d6-979c-39563f1382ee","added_by":"auto","created_at":"2025-07-17 16:41:26","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":37249,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot technique for detecting the expression of the NKx3.1 gene at the protein level\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-6956898/v1/c2dce3254ed6a5e950453f9a.png"},{"id":93657885,"identity":"24e24c68-7c81-4c19-a3a3-0516c1223056","added_by":"auto","created_at":"2025-10-16 07:24:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4085793,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6956898/v1/7f3cbabf-e5b0-4236-b2c5-b186a4ef4bc8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Experimental study of the role of NKx3.1 gene activation technology in targeting and killing prostate cancer stem cells and its mechanisms","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProstate cancer is a common and lethal malignancy among elderly men in China, with incidence rates increasing annually\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The incidence of prostate cancer is strongly correlated with patient age, typically peaking between 65 and 80 years\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. In addition to age, treatment resistance mechanisms represent another critical factor influencing prognosis. Studies have demonstrated that the transition of prostate cancer cells from hormone-dependent to hormone-independent status is a major cause of endocrine therapy failure and patient mortality\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Recent research has highlighted the crucial role of prostate cancer stem cells in tumor progression\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Existing evidence suggests that both hormone-dependent and hormone-independent prostate cancer cells may originate from prostate cancer stem cells, highlighting their potential value as therapeutic targets. In this study, we utilized previously constructed functional small activating RNAs (saRNAs) to target prostate cancer stem cells and evaluate saRNAs in prostate cancer stem cells. Screening identified saRNAs that specifically activate the NK3 homeobox 1 (NKx3.1) gene as effective in eliminating prostate cancer stem cells. We will subsequently employ qRT‒PCR and Western blotting techniques to further explore the molecular mechanisms underlying the regulation of stem cell differentiation by NKx3.1. This research not only provides new avenues for targeted gene therapy in prostate cancer but also lays a theoretical foundation for clinical translation by elucidating NKx3.1-related molecular mechanisms, thereby enriching the therapeutic strategies available for prostate cancer.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eReagents\u003c/h2\u003e\u003cp\u003eHuman prostate cancer cell line PC3; serum-free stem cell medium; fetal bovine serum (FBS); Immunomagnetic Bead Sorting Kit (CD44+/CD133\u0026thinsp;+\u0026thinsp;antibody beads); collagen type I-coated Petri dishes; specific saRNAs: dsRNAs targeting NKx3.1, Par-4, p14, and E-cadherin; and unrelated sequence dsRNAs; Liposomal Lipofectamine\u0026trade; 2000 (Invitrogen, USA).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell isolation culture and flow cytometry\u003c/h3\u003e\n\u003cp\u003ePC3 cells were digested into a single-cell suspension (trypsin-digested, containing EDTA), centrifuged and resuspended in PBS (containing 2% FBS), CD44 and CD133 magnetic bead-coupled antibodies were added, and the cell suspension was passed through a pretreated magnetic column, with negative cells (unbound to the magnetic beads) flowing out directly and positive cells (bound to the magnetic beads) remaining in the column\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. After magnetic bead sorting, the cells were inoculated into collagen type I-coated culture dishes and incubated at 37\u0026deg;C for 1\u0026ndash;2 hours. Unadherent cells (nonstem cells) were discarded, and adherent cells (stem cells) were cultured with serum-free medium. The percentage of CD44+/CD133\u0026thinsp;+\u0026thinsp;double-positive cells was calculated via flow cytometry.\u003c/p\u003e\n\u003ch3\u003eFunctional experiments on prostate cancer stem cells\u003c/h3\u003e\n\u003cp\u003eHaving identified putative prostate cancer stem cell (PCSC) surface markers in our preliminary work, we subsequently conducted a series of functional assays to assess the self-renewal and differentiation potential of the sorted PCSCs, thereby validating their stem cell characteristics. Specifically, the study encompassed the following procedures:\u003c/p\u003e\u003cp\u003eTo assess self-renewal capacity, we performed a serial clonogenic assay. The experiment compared sorted cells with unsorted PC3 cells. Following trypan blue staining to determine viable cell counts, the cells were resuspended in stem cell serum-free media at the desired density and seeded at 400 cells per well in 24-well plates precoated with type I collagen in each well with a total volume of 500 \u0026micro;L of stem cell serum-free media. The medium was changed every three days. After 30 days, colonies containing more than 32 cells were counted. The first-generation clones were then dissociated via the Accutase\u0026reg; enzyme (vendor and catalog number if available) and subjected to a second-generation clonogenic assay under identical conditions. This process was repeated for a third generation. The culture conditions, including media changes every three days, were maintained. Following the third-generation clonogenic assay, cell proliferation was analyzed via the MTT assay to generate growth curves. Colony formation ability was statistically analyzed via independent samples t tests (two-tailed), with the significance level set at α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003eFollowing completion of the third-generation clonogenic assay, to evaluate differentiation potential, the sorted cells were cultured in 1640 medium supplemented with 10% fetal bovine serum (2 mL per well). The cell morphology and growth patterns were observed and documented. After three days, the proportions of CD44\u0026thinsp;+\u0026thinsp;and CD133\u0026thinsp;+\u0026thinsp;cells within the differentiated cultures were determined via flow cytometry.\u003c/p\u003e\u003cp\u003eTo investigate PCSC migration and invasion, Transwell assays were conducted to compare sorted and unsorted PC3 cells. Following incubation, nonmigrated or noninvaded cells on the upper surface of the PET membrane were gently removed with a cotton swab. The migrated or invaded cells on the lower surface were then fixed, stained with crystal violet, and counted in five randomly selected fields of view at \u0026times;400 magnification, with a focus on both the central and peripheral regions of the membrane. The average number of cells per field was calculated from six replicates per group. The mean numbers of migrating and invading cells were compared between the two groups via independent samples t tests (two-tailed), with the significance level set at α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e\n\u003ch3\u003eEvaluating the effectiveness of transfection with functional saRNAs for killing prostate cancer stem cells\u003c/h3\u003e\n\u003cp\u003eOn the basis of the literature, functional small activating RNAs (saRNAs) targeting p14, Par-4, NKx3.1, and E-cadherin have demonstrated promising cytotoxic effects on the PC3 human prostate cancer cell line\u003csup\u003e[\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. In this project, we aimed to further investigate the targeted cytotoxic effects of these saRNAs on prostate cancer stem cells derived from the PC3 cell line via transfection via a lipid-based method. The specific steps are as follows:\u003c/p\u003e\u003cp\u003eThe experimental design comprised two control groups and four experimental groups: a mock group (transfected with Lipofectamine\u0026trade; 2000 alone), a negative control (NC) group (transfected with Lipofectamine\u0026trade; 2000 carrying nontargeting double-stranded RNA), a p14 activation group, a Par-4 activation group, an NKx3.1 activation group, and an E-cadherin activation group.\u003c/p\u003e\u003cp\u003eThe transfection efficiency was monitored microscopically in all groups following transfection with the aforementioned saRNAs via lipid-based transfection. Initially, we intended to use a low-toxicity transfection reagent to minimize cytotoxicity. However, preliminary experiments using various low-toxicity reagents yielded unsatisfactory transfection efficiencies. Given that the selected prostate cancer stem cells exhibited a tolerance to Lipofectamine\u0026trade; 2000 comparable to that of standard PC3 cells, we opted to use Lipofectamine\u0026trade; 2000 as the transfection reagent, although Lipofectamine\u0026trade; 2000 has greater cytotoxicity than other lower toxicity reagents.\u003c/p\u003e\u003cp\u003eTo assess the clonogenic potential, cells from each group were harvested 72 hours posttransfection, dissociated into single-cell suspensions via Accutase, and seeded into 24-well plates for colony formation assays, following a standardized protocol as previously described (cite relevant publication or method). Colony formation rates were then calculated for the first generation (P1) and subsequent second and third generations (P2, P3) of cells.\u003c/p\u003e\u003cp\u003eApoptosis was evaluated in each group. As initial cell cycle analysis revealed no evidence of cell cycle arrest during continuous observation from 48\u0026ndash;72 hours posttransfection, we shifted our focus from cell cycle analysis to evaluating apoptosis via Annexin V/PI staining and flow cytometry to elucidate the mechanism of action by which NKx3.1-targeting saRNAs exert their cytotoxic effects on prostate cancer stem cells. This decision was based on the premise that if cell cycle arrest was not the primary mechanism of cell death, these cells could be undergoing programmed cell death pathways.\u003c/p\u003e\u003cp\u003eCell invasion assays were performed to assess the invasive capacity of the cells. On the basis of prior studies indicating a specific association between E-cadherin and NKx3.1 gene expression and cellular invasiveness\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, Transwell invasion assays were conducted to examine the changes in the invasive ability of the Mock, NC, E-cadherin activation, and NKx3.1 activation groups at 72 hours posttransfection, following a standardized protocol as previously described (cite relevant publication or methods).\u003c/p\u003e\n\u003ch3\u003ePCR and Western blot analyses\u003c/h3\u003e\n\u003cp\u003ePCR technology converts RNA to cDNA via reverse transcription, amplifies the target gene (NKx3.1) via specific primers, and quantifies the mRNA expression level in real time via fluorescence signals\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. The protein was isolated via SDS‒PAGE, transferred to a membrane, and the expression level of the target protein (NKx3.1) was detected via specific antibodies\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eProstate cancer stem cell morphology and cell surface molecular markers\u003c/h2\u003e\u003cp\u003eProstate cancer stem cells were sorted via immunomagnetic bead sorting technology combined with the collagen adhesion method, and the sorted prostate cancer stem cells were initially cultured (e.g., Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The left figure shows the cell morphology on the 3rd day of inoculation, and the morphology of the isolated cultured cells was similar to that of the cells reported in the foreign literature, which was round\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e; the right figure shows the cell morphology on the 30th day of inoculation, which was similar to that of the cells reported in the foreign literature, with the cells growing in clones, the clones being spherical, the cells within the clones being round, and the distribution of the cells being centralized and cascading. During the culture process, the growth rate of the sorted cells was faster than that of normal PC3 cells under serum-free culture. The results of the flow cytometry assay revealed (e.g., Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) that the proportion of CD44 and CD133 expression in the sorted cells reached more than 99% (99.3% and 99.1%, respectively). The results of the preexperiment initially confirmed the presence of prostate cancer stem cells in the PC3 cell line.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eFunctional manifestations of prostate cancer stem cells\u003c/h3\u003e\n\u003cp\u003eThe results of the cloning experiments revealed that the colony formation rates of the first, second and third generations of the sorted CD44\u0026thinsp;+\u0026thinsp;and CD133\u0026thinsp;+\u0026thinsp;cells were significantly greater than those of the unsorted PC3 cells (as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting that the CD44\u0026thinsp;+\u0026thinsp;and CD133\u0026thinsp;+\u0026thinsp;cells in the sorted PC3 cells have strong self-renewal ability.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eColony formation rates of the two groups of cells\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRate of colony formation(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFirst generation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSecond generation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThird generation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCommon PC3 cells\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e15.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e10.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e5.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsolated cells\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e71.41\u0026thinsp;\u0026plusmn;\u0026thinsp;3.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e69.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e61.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe growth curve generated via the MTT method revealed that the number of CD44\u0026thinsp;+\u0026thinsp;and CD133\u0026thinsp;+\u0026thinsp;cells was much greater than the number of unsorted PC3 cells from day 3. The experimental results of the cell growth curve revealed that the growth curves of the CD44\u0026thinsp;+\u0026thinsp;and CD133\u0026thinsp;+\u0026thinsp;cells started to slow from the 12th day of culture, whereas the growth curves of the unsorted PC3 cells were always low and flat (e.g., Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The above results suggest that the sorted CD44\u0026thinsp;+\u0026thinsp;and CD133\u0026thinsp;+\u0026thinsp;cells have strong proliferative potential.\u003c/p\u003e\u003cp\u003eThe results of prostate cancer stem cell differentiation ability experiments revealed that after the culture medium was changed, the cells could be observed under a microscope on the first and second days, the cell morphology changed rapidly, the spherical cells disappeared quickly, and the clones became composed of polygonal and spindle cells, which were unfocused and expanded in all directions (e.g., Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The results of flow cytometry revealed that the percentage of CD44- and CD133-positive cells decreased rapidly to only 34.1% (e.g., Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and 5.3% (e.g., Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), respectively, which was similar to that of normal PC3 cells and significantly different from that of tumor stem cells cultured in serum-free medium.\u003c/p\u003e\u003cp\u003eAfter the migration and invasion of prostate cancer stem cells, the cell outline was clearly visible under the microscope (e.g., Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The numbers of migrated and invaded cells in the two groups are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Comparison of the means and t tests for the numbers of migrated and invaded cells in the two groups revealed that there was a significant difference between the two groups, and the numbers of migrated and invaded CD44\u0026thinsp;+\u0026thinsp;and CD133\u0026thinsp;+\u0026thinsp;cells were significantly greater than those of ordinary PC3 cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResults of migration and invasion experiments\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCell number(24 h)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNumber of migrating cells\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNumber of invading cells\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCommon PC3 cells\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e49.50\u0026thinsp;\u0026plusmn;\u0026thinsp;4.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e31.73\u0026thinsp;\u0026plusmn;\u0026thinsp;3.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsolated cells\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e248.17\u0026thinsp;\u0026plusmn;\u0026thinsp;22.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e124.20\u0026thinsp;\u0026plusmn;\u0026thinsp;10.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn summary, the biological and behavioral properties of the sorted cells were significantly different from those of the common PC3 cell line. CD44\u0026thinsp;+\u0026thinsp;and CD133\u0026thinsp;+\u0026thinsp;cells have stronger self-renewal ability and proliferation potential, and in the presence of differentiation inducers, they can divide asymmetrically and differentiate into cells similar to common PC3 cells. The results of Transwell in vitro migration and invasion assays suggested that CD44\u0026thinsp;+\u0026thinsp;and CD133\u0026thinsp;+\u0026thinsp;cells have stronger migration and invasion abilities. Thus, the CD44\u0026thinsp;+\u0026thinsp;and CD133\u0026thinsp;+\u0026thinsp;cells isolated from the PC3 cell line have the biological characteristics of prostate cancer stem cells.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eKilling effect of transfection with functional saRNAs on prostate cancer stem cells\u003c/h2\u003e\u003cp\u003eThe corresponding functional saRNAs or null dsRNAs were transfected into each group of cells via the liposome method. A small number of tumor stem cells showing transfected vacuoles could be observed under an inverted microscope 6 h after the completion of transfection. The number of cells showing vacuoles increased significantly after 12 h, and more than 95% of the cells showed vacuoles after 24 h (e.g., Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMicroscopic observation revealed that after transfection with functional saRNAs targeting the Par-4, p14 and E-cadherin genes, the tumor stem cells continued to grow, and their cell densities were not significantly lower than those in the control group. In contrast, after the transfection of functional saRNAs targeting the NKx3.1 gene, cell death was visible under the microscope, and the cell density was reduced compared with that of both the pretransfection and the control groups (e.g., Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCloning assay to detect proliferative potential and self-renewal capacity\u003c/h2\u003e\u003cp\u003eFor the cell colony formation experiments, the specific experimental method was essentially the same as before, and the rates of cell colony formation in the first generation and the 2nd and 3rd generations were calculated. The data were recorded, and a table was drawn (as shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eProliferation potential and self-renewal capacity of each group of cells detected via a colony formation assay\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRate of clone\u003c/p\u003e\u003cp\u003eFormation(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFirst generation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSecond generation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThird generation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMock control subjects\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e73.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e68.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e67.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNC control subjects\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e72.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e69.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e68.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePar-4 gene activation group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e74.25\u0026thinsp;\u0026plusmn;\u0026thinsp;3.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e70.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e69.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP14 gene activation group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e73.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e69.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e68.75\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eE- Cadherin gene\u003c/p\u003e\u003cp\u003eactivation group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e72.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e69.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e68.38\u0026thinsp;\u0026plusmn;\u0026thinsp;2.56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNKx3.1 gene\u003c/p\u003e\u003cp\u003eactivation group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e7.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e7.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the colony formation rates of cells in the mock group, NC group, Par-4 gene activation group, p14 gene activation group and E-cadherin gene activation group were similar to those in the first, second and third generations (P\u0026thinsp;\u0026gt;\u0026thinsp;0.01), which were significantly greater than those of cells in the NKx3.1 gene activation group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating that functional saRNAs targeting the NKx3.1 gene not only effectively killed prostate cancer stem cells but also reduced their proliferation potential and self-renewal ability, which are important biological characteristics, especially those of adult stem cells. These findings indicate that functional saRNAs targeting the NKx3.1 gene can not only effectively kill prostate cancer stem cells but also reduce their proliferation potential and self-renewal ability, which are important biological characteristics of stem cells, especially adult stem cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometry to detect apoptosis in each group of cells\u003c/h2\u003e\u003cp\u003eFlow cytometry was performed to detect apoptosis in each group (e.g., Figs.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e and \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e shows that the cells in the Par-4 gene activation group, the p14 gene activation group and the E-cadherin gene activation group did not undergo apoptosis, whereas Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e shows that the cells in the NKx3.1 gene activation group underwent significant apoptosis compared with those in the mock group and the NC group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eInvasiveness of transfected prostate cancer stem cells\u003c/h2\u003e\u003cp\u003eThe results of the assay for detecting invasive ability are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The invasive ability of the cells in the NKx3.1 gene activation group was significantly weakened after the intervention compared with that of the other three groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating that functional saRNAs targeting the NKx3.1 gene effectively reduced the invasive ability of human prostate cancer stem cells, whereas those targeting the E-cadherin gene were unable to reduce the invasive ability of stem cells in prostate cancer.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResults of invasion experiments after 72 h of transfection\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCell number(24 h)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNumber of invading cells\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMock control subjects\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e126.15\u0026thinsp;\u0026plusmn;\u0026thinsp;10.58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNC control subjects\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e127.52\u0026thinsp;\u0026plusmn;\u0026thinsp;11.49\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eE-Cadherin gene\u003c/p\u003e\u003cp\u003eactivation group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e124.69\u0026thinsp;\u0026plusmn;\u0026thinsp;13.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNKx3.1 gene\u003c/p\u003e\u003cp\u003eactivation group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e38.46\u0026thinsp;\u0026plusmn;\u0026thinsp;5.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003ePCR technology\u003c/h2\u003e\u003cp\u003eThe gene expression of the NKx3.1 gene at the mRNA level in prostate cancer stem cells before and after treatment was detected via PCR, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot analysis\u003c/h2\u003e\u003cp\u003eThe gene expression of the NKx3.1 gene in prostate cancer stem cells at the protein level before and after treatment was detected via Western blotting, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003e, the bands in the NKx3.1 gene activation group were all darker than those in the mock group and NC group were, suggesting that functional saRNAs targeting the NKx3.1 gene can effectively activate the target gene NKx3.1, increase its expression at the protein level, and subsequently kill prostate cancer stem cells.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, prostate cancer stem cells (PCSCs) were isolated from the PC3 cell line via immunomagnetic separation combined with collagen adhesion. These cells presented a high nuclear‒cytoplasmic ratio, expressed the surface markers CD44 and CD133, and demonstrated high clonogenic and migratory/invasive capabilities, which aligns with the classical definition of cancer stem cells\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. PCSCs, recognized as the origin of tumor recurrence and metastasis, often exhibit resistance to conventional therapies, leading to endocrine therapy failure. Prostate cancer stem cell-targeted therapies may offer a means to overcome these therapeutic limitations\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSmall activating RNAs (saRNAs) are a class of small RNA molecules capable of upregulating the expression of specific genes. These molecules function through epigenetic or transcriptional activation mechanisms to increase target gene expression, offering a complementary approach to traditional siRNA-mediated gene silencing\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. The NKx3.1 gene, which encodes a prostate-specific transcription factor critical for prostate epithelial differentiation and tumor suppression, was targeted by saRNAs in this study. We demonstrate for the first time that saRNA-mediated NKx3.1 activation has a specific cytotoxic effect on PCSCs, providing a novel molecular rationale for targeted prostate cancer therapy. The activation of NKx3.1 significantly suppressed the proliferation and self-renewal capacity of PCSCs (statistical significance to be added), whereas the activation of other genes, such as Par-4, p14, and E-cadherin, did not result in similar effects (statistical significance to be added), suggesting a unique biological role for NKx3.1 in PCSC regulation\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. This NKx3.1 activation, validated by quantitative PCR and Western blotting, induced both a significant increase in mRNA and protein expression levels and cancer stem cell death through two mechanisms: cell cycle arrest (increased G1 phase proportion shown by flow cytometry, statistical significance to be added) and activation of apoptotic pathways (statistical significance to be added). The ineffectiveness of other target genes, such as the proapoptotic gene Par-4, suggests that NKx3.1 may function through a distinct epigenetic regulatory network rather than solely relying on classical apoptosis pathways.\u003c/p\u003e\u003cp\u003eUnlike the gene silencing mechanism of traditional siRNAs\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, saRNA technology upregulates target genes through RNA activation (RNAa). This study leveraged the potential of saRNA technology to modulate gene expression. No significant changes were observed in the nonspecific dsRNA control group (statistical significance to be added), demonstrating the specificity of the saRNA effect\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. No significant changes were observed in the nonspecific dsRNA control group (statistical significance to be added), demonstrating the specificity of the saRNA effect. Furthermore, serial passaging for three generations demonstrated a sustained growth-inhibitory effect in the NKx3.1 activation group (statistical significance to be added), confirming the long-lasting effect of NKx3.1 activation\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003esaRNA-mediated NKx3.1 activation can effectively kill prostate cancer stem cells. However, the precise mechanisms underlying the NKx3.1-related regulatory network, particularly its interactions with the PTEN/p53 pathway, remain to be fully elucidated and warrant further investigation\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Future studies should prioritize the development of targeted delivery systems, such as nanocarriers, to increase saRNA stability in vivo and explore the synergistic effects of NKx3.1 activation with radiation and chemotherapy. Furthermore, while the current study relies primarily on in vitro models, in vivo validation using patient-derived xenograft (PDX) mouse models is necessary to assess therapeutic efficacy. Caution should also be taken regarding resistance potentially induced by long-term culture in future studies\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn summary, our findings indicate that saRNA-mediated activation of NKx3.1 can effectively kill prostate cancer stem cells, opening a new avenue for treating castration-resistant prostate cancer (CRPC). The broad regulatory network controlled by NKx3.1 may represent a key target for gene therapy, warranting further in-depth exploration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e1. Ethics approval and consent to participate:Not applicable\u003c/p\u003e\n\u003cp\u003e2. Consent for publication:Not applicable\u003c/p\u003e\n\u003cp\u003e3. Availability of data and materials:Not applicable\u003c/p\u003e\n\u003cp\u003e4. Competing interests:The authors declares that they have no competing interests\u003c/p\u003e\n\u003cp\u003e5. Funding:The funds were provided by the National Natural Science Foundation of China, under the Youth Science Foundation Grant 81001148.\u003c/p\u003e\n\u003cp\u003e6. Authors\u0026apos; contributions:S. was the first author and W. was the second author of the main manuscript. L. prepared Figures 1-15. All the authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e7. Acknowledgements:Not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSEKHOACHA M, RIET K, MOTLOUNG P, et al. Prostate Cancer Review: Genetics, Diagnosis, Treatment Options, and Alternative Approaches [J]. Molecules, 2022, 27(17).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTAITT H E. Global Trends and Prostate Cancer: A Review of Incidence, Detection, and Mortality as Influenced by Race, Ethnicity, and Geographic Location [J]. Am J Mens Health, 2018, 12(6): 1807\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSHELLEY M, HARRISON C, COLES B, et al. Chemotherapy for hormone-refractory prostate cancer [J]. Cochrane Database Syst Rev, 2006, (4): Cd005247.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTU S M, LIN S H. Prostate cancer stem cells [J]. Clin Genitourin Cancer, 2012, 10(2): 69\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDEPONTE S, STEINGROEWER J, L\u0026ouml;SER C, et al. Biomagnetic separation of \u003cem\u003eEscherichia coli\u003c/em\u003e by use of anion-exchange beads: measurement and modeling of the kinetics of cell-bead interactions [J]. Anal Bioanal Chem, 2004, 379(3): 419\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHEMMATI P G, G\u0026uuml;NER D, GILLISSEN B, et al. Bak functionally complements for loss of Bax during p14ARF-induced mitochondrial apoptosis in human cancer cells [J]. Oncogene, 2006, 25(50): 6582\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGAO S, WANG H, LEE P, et al. Androgen receptor and prostate apoptosis response factor-4 target the c-FLIP gene to determine survival and apoptosis in the prostate gland [J]. J Mol Endocrinol, 2006, 36(3): 463\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePADMANABHAN A, RAO V, DE MARZO A M, et al. Regulating NKX3.1 stability and function: Posttranslational modifications and structural determinants [J]. Prostate, 2016, 76(6): 523\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDHAR S, KUMAR A, GOMEZ C R, et al. MTA1-activated Epi-microRNA-22 regulates E-cadherin and prostate cancer invasiveness [J]. FEBS Lett, 2017, 591(6): 924\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMAO Q, ZHENG X, YANG K, et al. Suppression of migration and invasion of PC3 prostate cancer cell line by activating E-cadherin expression by small activating RNA [J]. Cancer Invest, 2010, 28(10): 1013\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHUANG V, QIN Y, WANG J, et al. RNAa is conserved in mammalian cells [J]. PLoS One, 2010, 5(1): e8848.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWATERS D L, SHAPTER F M. The polymerase chain reaction (PCR): general methods [J]. Methods Mol Biol, 2014, 1099: 65\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBEGUM H, MURUGESAN P, TANGUTUR A D. Western blotting: a powerful staple in scientific and biomedical research [J]. Biotechniques, 2022, 73(1): 58\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCOLLINS A T, BERRY P A, HYDE C, et al. Prospective identification of tumorigenic prostate cancer stem cells [J]. Cancer Res, 2005, 65(23): 10946\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWEI C, GUOMIN W, YUJUN L, et al. Cancer stem-like cells in human prostate carcinoma cells DU145: the seeds of the cell line? [J]. 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Genet Mol Res, 2015, 14(4): 15769\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSOORESHJANI M A, KAMRA M, ZOUBEIDI A, et al. Reciprocal deregulation of NKX3.1 and AURKA axis in castration-resistant prostate cancer and NEPC models [J]. J Biomed Sci, 2021, 28(1): 68.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLI M, BAI G, CEN Y, et al. Silencing HOXC13 exerts anti-prostate cancer effects by inducing DNA damage and activating cGAS/STING/IRF3 pathway [J]. J Transl Med, 2023, 21(1): 884.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYANG K, SHEN J, CHEN S W, et al. Upregulation of PAWR by small activating RNAs induces cell apoptosis in human prostate cancer cells [J]. Oncol Rep, 2016, 35(4): 2487\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eREN S, KANG M R, WANG J, et al. Targeted induction of endogenous NKX3-1 by small activating RNA inhibits prostate tumor growth [J]. Prostate, 2013, 73(14): 1591\u0026ndash;601.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLABB\u0026eacute; D P, BROWN M. Transcriptional Regulation in Prostate Cancer [J]. Cold Spring Harb Perspect Med, 2018, 8(11).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHEMA S, THAMBIRAJ S, SHANKARAN D R. Nanoformulations for Targeted Drug Delivery to Prostate Cancer: An Overview [J]. J Nanosci Nanotechnol, 2018, 18(8): 5171\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Prostate cancer, prostate cancer stem cell-targeted gene therapy, NKx3.1 gene, small activating RNAs","lastPublishedDoi":"10.21203/rs.3.rs-6956898/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6956898/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eTo explore the killing effect of functional saRNAs that target the NKx3.1 gene to intervene in prostate cancer stem cells sorted from PC3 cells.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003ePCa stem cells were isolated and cultured from the human prostate cancer cell line PC3 via the immunomagnetic bead sorting technique combined with the collagen adhesion method, and the cells were characterized in terms of morphology, cell surface molecular markers and function. Four different gene activation groups (p14 gene activation group, Par-4 gene activation group, NKx3.1 gene activation group and E-Cadherin gene activation group) were transfected with liposomal Lipofectamine\u0026trade; 2000, and a blank control group (Mock group, transfected with liposomal Lipofectamine\u0026trade; 2000 only) and a null dsRNA transfection group (NC group, transfected with Lipofectamine\u0026trade; 2000 carrying invalid dsRNA used for the negative control) were used for comparison. The growth of the cells in each group was observed under a microscope. A colony formation assay was performed to detect the proliferative potential and self-renewal ability of the cells in each group. Cell cycle changes in each group were detected by flow cytometry. The expression of the NKx3.1 gene was verified by PCR and Western blotting.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eMicroscopic observation revealed that NKx3.1 gene activation effectively killed prostate cancer stem cells, whereas mock, NC, Par-4 gene activation, p14 gene activation and E-cadherin gene activation had no obvious effects. The colony formation assay detected similar colony formation rates in the first, second and third generations of cells in the mock group, NC group, Par-4 gene activation group, p14 gene activation group and E-cadherin gene activation group, and the differences were not statistically significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.01). All the rates were significantly greater than those in the NKx3.1 gene activation group, and the differences were statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eProstate cancer stem cells isolated and cultured from the human prostate cancer cell line PC3 are consistent with the biological properties of stem cells. Functional saRNAs that target the NKx3.1 gene effectively kill prostate cancer stem cells.\u003c/p\u003e","manuscriptTitle":"Experimental study of the role of NKx3.1 gene activation technology in targeting and killing prostate cancer stem cells and its mechanisms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-17 16:41:21","doi":"10.21203/rs.3.rs-6956898/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8ac98241-e455-4e0a-9e97-b40c7358288a","owner":[],"postedDate":"July 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-16T07:23:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-17 16:41:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6956898","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6956898","identity":"rs-6956898","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}