LncRNA GUSBP11/miR-28-5p/PAQR7 axis regulates the phosphorylation of E-cadherin and N-cadherin on the migration of papillary thyroid carcinoma cells

doi:10.21203/rs.3.rs-4952662/v1

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LncRNA GUSBP11/miR-28-5p/PAQR7 axis regulates the phosphorylation of E-cadherin and N-cadherin on the migration of papillary thyroid carcinoma cells

https://doi.org/10.21203/rs.3.rs-4952662/v1

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Papillary thyroid carcinoma represents the most prevalent form of thyroid cancer, exhibiting the lowest degree of malignancy. It constitutes approximately 85% of all thyroid cancer cases. This cancer type can manifest at any age but is predominantly observed in children or young women under the age of 40. The aberrant expression of long non-coding RNA (lncRNA) and messenger RNA (mRNA) has been identified as a crucial factor in the pathogenesis of this disease. Our analysis of existing databases revealed that lncRNA GUSBP11 and the membrane progesterone receptor PAQR7 are significantly overexpressed in thyroid cancer, demonstrating a notable positive correlation between them. However, RNA sequencing analysis indicated the absence of a direct relationship between these molecules. Through a comprehensive pooled analysis across multiple studies, we discovered a direct link between miR-28-5p and both lncRNA GUSBP11 and mRNA PAQR7, which were found to be significantly downregulated in thyroid cancer cases. This research endeavors to lay a novel foundation for the development of targeted therapy for thyroid cancer, focusing on the interaction between lncRNA, miRNA, and mRNA.

Biological sciences/Cancer

Biological sciences/Molecular biology

Papillary Thyroid Carcinoma (PTC)

Cell Migration

Phosphorylation

Globally, thyroid cancer (TC) has been documented in approximately 586,000 cases ¹. The American Cancer Society reports an estimate of 43,800 new TC cases in the United States for the year 2022, comprising 11,860 males and 31,940 females, alongside approximately 2,230 deaths (1,070 males and 1,160 females) from these tumors ². This data underscores a significant gender disparity in TC prevalence and prognosis, with women experiencing a higher incidence but more favorable outcomes, whereas men have a lower incidence but poorer prognosis. TC is a malignant tumor originating from thyroid gland tissue, with various cell types within the thyroid leading to different TC categories: (1) Differentiated TCs (DTCs), which include papillary (PTC), follicular (FTC), and Huerthle cell tumors; (2) Medullary TCs (MTCs); and (3) Anaplastic TCs. The long-term survival rate for patients with DTCs is approximately 90%, contrasting starkly with less than 10% for those with poorly differentiated TC, attributed to resistance to standard treatment modalities ³. Our research is particularly focused on differentiated papillary thyroid carcinoma (PTC).

Membrane progesterone receptor mPRα (also known as PAQR7) is identified as a 40kDa transmembrane protein localized on the cell membrane, functioning to recognize and mediate progesterone (P4) signaling ⁴. Additional receptors, mPRβ (PAQR8) and mPRγ (PAQR5), part of the larger progesterone and adiponectin receptor (PAQR) gene family ⁵, have been discovered in various species. The nuclear progesterone receptor (nPR) has been detected in cervical cancer specimens ⁶, alongside membrane components such as PGRMC1 and PGRMC2, which have been implicated in supporting the survival of human breast cancer cells and promoting xenograft tumor growth ⁷.

Long non-coding RNAs (lncRNAs), with a length exceeding 200 nucleotides ⁸, were initially considered transcriptional by-products devoid of physiological function⁹. However, advancements in sequencing technology have revealed the significant role of lncRNAs in cellular molecular regulation, including transcription, and their impact on a variety of human diseases ¹⁰. They are particularly influential in the regulation of tumor cell migration, proliferation, apoptosis, autophagy, and drug resistance ¹¹. This study investigates lncRNA GUSBP11, which promotes PTC cell migration and tumor growth by indirectly regulating PAQR7. Aberrant expression of GUSBP11 has been linked to immune cell dysfunction and the onset of diseases such as periodontitis ¹², with its presence in serum and plasma being a potential biomarker for gastric cancer diagnosis and prognosis ¹³. Furthermore, lncRNAs are known to interact with miRNAs in processes leading to tumorigenesis ^14,15.

In this investigation, we identified significant overexpression of lncRNA GUSBP11 and membrane progesterone receptor PAQR7 in thyroid cancer patient tissues. Database analyses revealed a significant positive correlation between the two. Gene alignment indicated complementary sequences between these molecules and miR-28-5p, suggesting a reciprocal regulatory mechanism. Abnormal expression of miR-28-5p in various cancers indicates its potential role as an oncogenic factor ^16,17. Additionally, the study examines the effects of phosphorylation at S838/840 of E-cadherin and Y820 of N-cadherin on the migration of PTC cells, offering new insights into the molecular dynamics influencing thyroid cancer progression.

Cell lines

Human papillary thyroid carcinoma (PTC) cell lines TPC-1, K1, BCPAP, human thyroid epithelial cell line Nthy-ori3-1 and human embryonic renal epithelial cell line 293T were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). They were cultured in DMEM or 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37℃ in an incubator with 5% CO₂.

Sample Collection

Carcinogenic and normal thyroid tissue samples were collected from patients diagnosed with papillary thyroid carcinoma (PTC) undergoing surgery at Tongji Hospital, Wuhan, China, from January 2017 to October 2020. Specimens were either immediately fixed in formalin and embedded in paraffin or snap-frozen in liquid nitrogen for subsequent analyses. Prior to surgery, these patients had not undergone any medical treatment or radioactive iodine therapy. Informed consent was secured from all participants, and the study's protocol received approval from the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.

Quantitative Real-Time PCR (qRT-PCR)

Total RNA, including mRNA and miRNA, was isolated from cell or tissue samples using CWBIO's RNA and miRNA extraction kits (China). The isolated RNA was then reverse-transcribed to cDNA using a reverse transcription kit from Vazyme (China). The qRT-PCR was performed on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (USA) employing qPCR SYBR Green Master Mix (YEASEN, China) for fluorescence detection. The qRT-PCR conditions were as follows: initial denaturation at 95°C for 5 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 20 seconds, and extension at 72°C for 20 seconds. For normalization, U6 and GAPDH were employed as internal controls for miRNA and mRNA quantification, respectively. Primer sequences used in this study are listed in Table S1.

Western Blot Analysis

Total protein was extracted using RIPA buffer (Meilunbio, China), and its concentration was determined with a BCA Protein Assay Kit (Meilunbio, China). Subsequently, 30 µg of protein per sample was resolved on 10% SDS-PAGE gels and electrotransferred to PVDF membranes at 210 mA. Membranes were blocked with 5% skim milk in TBST for 1 hour at room temperature, followed by overnight incubation at 4°C with primary antibodies. After washing thrice with TBST, membranes were incubated with secondary antibodies for 1 hour at room temperature. Protein bands were visualized using a ChemiDoc Imaging System (Bio-Rad, USA), and quantification was performed with Image Lab software (version 6.0, Bio-Rad, USA).

Plasmid Construction

The sequences of the 3′-untranslated region (3′-UTR) of PAQR7 and the full-length sequence of LncRNA GUSBP11 were amplified from the human genome. These fragments were subsequently ligated into the pmirGLO vector (Addgene, USA) to generate wild-type luciferase reporter plasmids (WT-pmirGLO-PAQR7 and WT-pmirGLO-GUSBP11). These wild-type plasmids served as templates to introduce mutations at the miR-28-5p binding sites within the 3′-UTR of PAQR7 and GUSBP11, resulting in mutant luciferase reporter plasmids (MUT-pmirGLO-PAQR7 and MUT-pmirGLO-GUSBP11). Additionally, fragments of GUSBP11 were amplified and cloned into the pcDNA3.1 vector and pLVX-EF1α-IRES-puro vector (Addgene, USA) for overexpression studies. To achieve lentivirus-mediated knockdown, four distinct short hairpin RNA (shRNA) constructs targeting PAQR7 and GUSBP11 were designed, alongside non-targeting control shRNAs. These constructs were cloned into the pLKO.1 vector (DynaBio, China).

For the engineering of CDH1 and CDH2 expression plasmids, coding sequence fragments were amplified from human cDNA and cloned into the pcDNA3.1 vector (Addgene, USA), resulting in CDH1-CDS and CDH2-CDS plasmids. Subsequent mutations were introduced to alter specific amino acid residues within these coding sequences, generating CDH1-S838/840F and CDH2-Y820F plasmids, aimed at studying the functional consequences of these mutations.

Wound Healing Assay

Approximately 1 × 10^6 thyroid cancer cells were seeded in 6-well plates. Upon reaching 90% confluence, a sterile pipette tip was used to create a scratch wound, aided by a sterilized steel ruler for uniformity. The medium was then replaced with a low-serum medium containing 0.2% fetal bovine serum (FBS) to restrict cell proliferation while maintaining viability. Wound closure was monitored and imaged at 0, 24, and 48 hours post-wounding using an inverted microscope (Olympus, Japan), and the area of the wound gap was quantified using Image J software (NIH, USA). This assay was performed in triplicate.

Transwell Invasion Assay

The transwell assay was conducted using chambers with an 8 µm pore size (Corning, USA), with the bottom coated with Matrigel (BD, USA) at a 1:4 dilution. Cells were serum-starved for 12 hours prior to the assay. Then, 3 × 10^4 cells in 150 µL of 2% FBS medium were placed in the upper chamber, while the lower chamber was filled with 600 µL of medium containing 20% FBS. After 24 hours, non-invading cells were removed, and cells that had invaded through the Matrigel were fixed and stained with 0.1% crystal violet. Invasion was assessed under an inverted microscope (Olympus, Japan), counting cells in five random fields per chamber at 10× magnification. This procedure was replicated three times to ensure reproducibility.

Ethics approval

The study was approved and reviewed by the Medical Ethics Committee of Wuhan University of Science and Technology and was conducted with the consent of the subjects and in accordance with the Declaration of Helsinki. All animal experiments were performed in compliance with the ARRIVE guidelines. The study was approved by the Animal Ethics Committee of Wuhan University of Science and Technology. All methods were carried out in accordance with relevant guidelines and regulations.

Statistical Analysis

All experiments in this article were repeated at 3 times. These values are expressed as mean ± standard deviation. Differences between various groups were evaluated using a two-tailed Student’s t-test. Two-sided P values < 0.05 were considered statistically significant.

Upregulation of LncRNA GUSBP11 and Membrane Progesterone Receptor PAQR7 in Thyroid Cancer

The StarBase database predicted an elevated expression of LncRNA GUSBP11 and the membrane progesterone receptor PAQR7 in thyroid cancer (Fig. 1A). To validate this prediction, the study analyzed three groups of PTC patients and detected the protein expression of PAQR7 in their tumors and adjacent normal tissues. The results showed that the protein expression of PAQR7 was significantly high in the tumor tissues of the patients(Fig. 1C). Furthermore, RNA was extracted from thyroid cancer tissues and adjacent non-cancerous tissues of six PTC patients, and RT-QPCR analysis confirmed the upregulation of both GUSBP11 and PAQR7 in the cancerous tissues of all six patients (Fig. 1B). A positive correlation between the expressions of PAQR7 and GUSBP11 in thyroid cancer was also predicted by the StarBase database (Fig. 1D). Additionally, an analysis of 131 thyroid cancer patients from the TCGA database regarding their survival outcomes (Fig. 1E) indicated that patients with high levels of membrane progesterone receptor expression exhibited a lower survival rate compared to those with lower expression levels.

Suppression of Membrane Progesterone Receptor PAQR7 Attenuates Papillary Thyroid Cancer Cell Migration and the Phosphorylation of E-cadherin and N-cadherin

Investigations into the potential activation of signaling pathways by LncRNA and the membrane progesterone receptor PAQR7 in thyroid cancer, utilizing FUNRICH software, indicated that GUSBP11 and PAQR7 could activate the PI3k/Akt signaling pathway and are closely related to cadherin in thyroid cancer (Fig. 2A). To delve deeper into this matter, papillary thyroid carcinoma (PTC) cell lines TPC-1, K1, BCPAP, along with the thyroid follicular epithelial cell line Nthy-ori3-1, were employed for protein and RNA extraction, with PAQR7 detection carried out via Western blot and RT-qPCR for both GUSBP11 and PAQR7 (Fig. 2B). Notably, GUSBP11 and PAQR7 exhibited significant overexpression in PTC cell lines, particularly in TPC-1 and BCPAP. Immunofluorescence assays further corroborated the localization of the membrane progesterone receptor PAQR7 on the cell membrane of TPC-1 cells (Fig. 2C).Subsequently, four sh-PAQR7 plasmids were introduced into TPC-1 and BCPAP cells to assess the knockdown efficiency of sh-PAQR7 (Figs. 2D-F), revealing plasmid sh-PAQR7-2 as the most efficacious in both cell types. To explore the influence of PAQR7 knockdown on PTC cell migration, wound healing and transwell assays were conducted (Figs. 2G-H), demonstrating that PAQR7 knockdown significantly inhibited thyroid cancer cell migration. Western blot analysis of E-cadherin, phosphorylated E-cadherin (S838/840), N-cadherin, and phosphorylated N-cadherin (Y820) levels (Fig. 2I) indicated that PAQR7 knockdown not only reduced N-cadherin levels and increased E-cadherin levels but also inhibited their phosphorylation.

To verify the impact of PAQR7 knockdown on in vivo tumor growth of PTC cells, tumorigenic assays in nude mice were performed (Fig. 2J), showing significantly reduced tumor growth upon PAQR7 knockdown. Immunohistochemistry and Western blot analyses of the tumors revealed a notable increase in E-Cadherin expression and a decrease in N-cadherin expression (Fig. 2K). These findings collectively suggest that PAQR7 knockdown impedes PTC cell migration and tumor growth, an effect linked to alterations in cadherin protein expression and phosphorylation.

Knockdown of LncRNA GUSBP11 Reduces Migration and Phosphorylation of Cadherins in Papillary Thyroid Cancer Cells

The study evaluated the expression of LncRNA GUSBP11 in three papillary thyroid carcinoma (PTC) cell lines (TPC-1, K1, BCPAP) and the thyroid epithelial cell line NTHY-ori3-1, with RT-qPCR revealing significantly higher GUSBP11 levels in TPC-1 and BCPAP cells compared to NTHY-ori3-1 (Fig. 3A). A functional enrichment analysis of LncRNA GUSBP11, conducted via FUNRICH, aimed to elucidate its role in thyroid cancer (Fig. 3B). Subsequent transfection of four sh-GUSBP11 plasmids into PTC cells demonstrated that the sh-GUSBP11-4 plasmid achieved the most significant knockdown of GUSBP11, alongside notable effects on PAQR7 levels (Figs. 3C-D). This knockdown efficacy prompted further assays to confirm that reducing GUSBP11 expression could indeed impede the migration of PTC cells, as shown through transwell and wound healing assays (Figs. 3E-F).

To delve deeper into the mechanistic impact of GUSBP11 knockdown in PTC, Western blot analyses were performed to assess the expression of membrane progesterone receptor PAQR7, E-cadherin, N-cadherin, phosphorylated E-cadherin (S838/840), and phosphorylated N-cadherin (Y820) in BCPAP and TPC-1 cells (Fig. 3G). The results indicated that GUSBP11 knockdown not only reduced the phosphorylation levels of E-cadherin and N-cadherin but also led to an upregulation of E-cadherin and a downregulation of N-cadherin, suggesting a reversal of epithelial-mesenchymal transition (EMT) markers.

The potential of LncRNA GUSBP11 as a tumor-associated LncRNA was further evaluated in vivo through tumorigenic assays in nude mice, showing significantly slower tumor growth following GUSBP11 knockdown (Fig. 3H). Additional analyses of the tumors via Western blot, immunohistochemistry, and RT-qPCR confirmed the upregulation of E-cadherin and downregulation of N-cadherin, alongside a decrease in PAQR7 expression in the GUSBP11 knockdown group (Figs. 3I-J).

These findings collectively suggest that LncRNA GUSBP11 plays a critical role in promoting the migration and potentially the invasiveness of PTC cells, acting through modulation of cadherin expression and phosphorylation, and possibly through effects on the membrane progesterone receptor PAQR7. Knockdown of GUSBP11 thus emerges as a promising therapeutic strategy to inhibit PTC progression by reversing EMT and reducing tumor growth.

LncRNA GUSBP11 and Membrane Progesterone Receptor PAQR7 Co-regulation in PTC Cell Migration, Cadherin Phosphorylation, and Tumor Growth

This study delves into the synergistic effects of LncRNA GUSBP11 and membrane progesterone receptor PAQR7 on the behavior of papillary thyroid carcinoma (PTC) cells, particularly focusing on cell migration, changes in cadherin expression and phosphorylation, and tumor growth dynamics. Overexpression of LncRNA GUSBP11 in PTC cells (BCPAP and TPC-1) with concurrent knockdown of PAQR7 (using sh-PAQR7-2) revealed that GUSBP11 positively regulates PAQR7 levels, promotes E-cadherin expression, and inhibits N-cadherin along with their phosphorylated forms (E-cadherin S838/840 and N-cadherin Y820) as shown by Western blot analysis (Fig. 4A). Interestingly, the simultaneous manipulation of GUSBP11 overexpression and PAQR7 knockdown demonstrated that these molecules might work in concert, where the effects of GUSBP11 overexpression could be modulated or reversed by PAQR7 knockdown.

The investigation extended to examining the impact of these manipulations on PTC cell migration through transwell and wound healing assays. The assays indicated that GUSBP11 overexpression alone enhances the migration of PTC cells. However, when GUSBP11 overexpression is paired with PAQR7 knockdown, this migration is notably inhibited, suggesting a complex interaction between GUSBP11 and PAQR7 in regulating cell motility (Figs. 4B-C).

Further exploration into the in vivo effects of these genetic manipulations on tumor growth was conducted using tumor formation experiments in nude mice. The outcomes demonstrated that GUSBP11 overexpression alone accelerated tumor growth, whereas the concurrent overexpression of GUSBP11 and knockdown of PAQR7 significantly slowed tumor progression compared to controls (Fig. 4D). Western blot analysis of tumors extracted from mice supported these findings, showing an increase in PAQR7 and N-cadherin and a decrease in E-cadherin in the GUSBP11 overexpression group. In contrast, tumors from the group with both GUSBP11 overexpression and PAQR7 knockdown exhibited downregulation of PAQR7 and N-cadherin and upregulation of E-cadherin, indicating a reversal of the effects seen with GUSBP11 overexpression alone (Fig. 4E).

These results highlight a complex interplay between LncRNA GUSBP11 and PAQR7 in PTC cells, with evidence pointing towards a scenario where GUSBP11 may directly or indirectly influence PAQR7 expression to modulate cell migration, cadherin protein expression and phosphorylation, and tumor growth. The co-regulation of these molecules suggests potential therapeutic targets for inhibiting PTC progression, where strategies aimed at disrupting this regulatory axis could offer new avenues for treatment.

The study explores the regulatory relationship between miR-28-5p and the expression of membrane progesterone receptor PAQR7, with a focus on its implications for papillary thyroid carcinoma (PTC) cell migration. Utilizing bioinformatic tools from four different databases (TargetScan, miRDIP, StarBase, and miRWalk), researchers identified potential microRNA (miRNA) binding sites within the 3' untranslated regions (3'UTRs) of PAQR7. Among these, miR-28-5p emerged as a significant candidate, particularly noted for its under-expression in thyroid cancer and its negative correlation with disease presence, as identified in the StarBase database (Fig. 5A-B).

To validate the interaction between miR-28-5p and the 3'UTR of PAQR7, luciferase reporter assays were conducted in 293T cells. These assays compared the luciferase activity in cells transfected with wild-type (WT) or mutant (MUT) versions of the PAQR7 3'UTR attached to the pmirGlo vector, alongside miR-28-5p mimics. The results demonstrated a significant decrease in luciferase activity in cells containing the WT 3'UTR construct upon miR-28-5p mimic introduction, confirming direct binding between miR-28-5p and the 3'UTR of PAQR7. No significant change was observed in the mutant construct, further supporting the specificity of this interaction (Fig. 5C).

Further investigations into the functional role of miR-28-5p in PTC revealed its potential involvement in activating specific signaling pathways relevant to thyroid cancer, including the PI3K/Akt and cadherin protein signaling pathways, as suggested by FUNRICH analysis (Fig. 5D). RT-qPCR analysis of miR-28-5p expression in three PTC cell lines (TPC-1, K1, BCPAP) and normal thyroid epithelial cells (NTHY) indicated a significant under-expression of miR-28-5p in the PTC cell lines (Fig. 5E).

Overexpression studies of miR-28-5p in BCPAP and TPC-1 cell lines demonstrated a marked inhibition of PTC cell migration, as evidenced by transwell and wound healing assays (Fig. 5G). Western blot analysis further elucidated the downstream effects of miR-28-5p overexpression, showing downregulation of PAQR7, N-cadherin, phosphorylated E-cadherin (S838/840), and phosphorylated N-cadherin (Y820), alongside upregulation of E-cadherin (Fig. 5H). These results collectively underscore the inhibitory role of miR-28-5p on PTC cell migration, mediated through its direct targeting of the PAQR7 3'UTR, and its subsequent influence on key molecular pathways and cadherin dynamics in PTC cells. The findings highlight the therapeutic potential of miR-28-5p as a target for managing PTC, particularly by modulating cell migration and invasion characteristics critical to cancer progression.

This study investigates the interaction between long non-coding RNA (LncRNA) GUSBP11 and microRNA-28-5p (miR-28-5p) in the context of papillary thyroid carcinoma (PTC) and elucidates their roles in regulating cell migration. The research leverages the StarBase database to uncover base pairing between LncRNA GUSBP11 and miR-28-5p, along with evidence of a negative correlation between the two in thyroid cancer, suggesting a competitive endogenous RNA (ceRNA) sponge mechanism (Fig. 6A).

To confirm the direct interaction between GUSBP11 and miR-28-5p, luciferase reporter assays were conducted in 293T cells transfected with wild-type (WT) or mutant (MUT) GUSBP11 constructs along with miR-28-5p mimics. The results indicated a significant reduction in luciferase activity for the WT construct upon miR-28-5p mimic co-transfection, whereas the MUT construct showed no significant change in luciferase activity, confirming the specific binding of miR-28-5p to GUSBP11 (Fig. 6B).

Further validation of the GUSBP11-miR-28-5p interaction in PTC cells was achieved through RNA-binding protein immunoprecipitation (RIP) and fluorescence in situ hybridization (FISH) assays. RIP assays demonstrated that both GUSBP11 and miR-28-5p were enriched in the AGO2-containing complexes compared to the IgG control in TPC-1 cells, indicating their association in the RNA-induced silencing complex (RISC) (Fig. 6C). FISH experiments provided visual evidence of co-localization in the cytoplasm of BCPAP cells, further supporting the interaction between GUSBP11 and miR-28-5p within cells (Fig. 6D).

The functional implications of this interaction were explored by overexpressing GUSBP11 and miR-28-5p in BCPAP and TPC-1 cells. Migration assays revealed that GUSBP11 overexpression promoted cell migration, an effect that was reversed by the simultaneous overexpression of miR-28-5p, suggesting that miR-28-5p can counteract the pro-migratory influence of GUSBP11 on PTC cells (Fig. 6E-F).

This study's findings highlight the complex regulatory network involving LncRNA GUSBP11 and miR-28-5p in PTC. GUSBP11 acts as a ceRNA or "sponge" for miR-28-5p, sequestering it away from its target mRNAs. This interaction not only elucidates a novel mechanism of post-transcriptional regulation involving LncRNA and miRNA in thyroid cancer but also points to potential therapeutic targets for controlling PTC cell migration and invasion. The manipulation of this LncRNA-miRNA axis could offer new avenues for PTC treatment, emphasizing the importance of understanding the intricate molecular interactions within cancer cells.

To elucidate the impact of phosphorylation at serines 838 and 840 on E-cadherin and tyrosine 820 on N-cadherin, we engineered sequences for the coding region (CDS) of both E-cadherin and N-cadherin. Specifically, serines 838 and 840 in the CDS of E-cadherin and tyrosine 820 in the CDS of N-cadherin were altered to aspartic acid, a residue less susceptible to phosphorylation, and subsequently cloned into the pcDNA3.1 vector to produce CDH1(S838/840)D and CDH2(Y820)D constructs, respectively (as depicted in Fig. 7A). These mutant constructs, alongside the wild-type CDS plasmids for CDH1 and CDH2, were transfected into BCPAP and TPC-1 cell lines. The phosphorylation status was assessed through Western blot analysis (referenced in Fig. 7B), revealing that the phosphorylation levels of E-cadherin and N-cadherin in cells transfected with the mutant plasmids did not exhibit significant alterations or reductions compared to the control group. The influence of these mutations on cell migration was determined using wound healing and transwell migration assays (illustrated in Figs. 7C-D). The findings indicated that mutations disrupting the phosphorylation sites on E-cadherin and N-cadherin effectively reduced the migratory capacity of BCPAP and TPC-1 cells. Moreover, transfecting cells with the wild-type CDS of E-cadherin enhanced their migratory behavior, whereas the introduction of the E-cadherin (S838/840)D mutation suppressed this activity. Similarly, while the wild-type N-cadherin CDS diminished cell migration, the N-cadherin (Y820)D mutation led to a further decrease in migratory potential.

An increasing number of studies have demonstrated that long non-coding RNAs (lncRNAs) serve as critical oncogenes or tumor suppressors, significantly influencing the regulation of gene expression associated with cancer and the functionality of pathways implicated in tumor progression. Investigations across various cancer types, including esophageal, lung, gastric, and breast cancers, have highlighted the pivotal roles of lncRNAs. For instance, the downregulation of LncRNA PVT1 in esophageal cancer cells hampers cell migration and invasion while promoting apoptosis through the microRNA-145-mediated suppression of FSCN1 ¹⁸. Similarly, LncRNA FAM83H-AS1 has been implicated in the pathogenesis of non-small cell lung cancer ¹⁹, LncRNA TUG1 in the enhancement of gastric cancer malignancy, and LncRNA SNHG1 in the progression of breast cancer and its resistance to cisplatin ²⁰. Within the context of papillary thyroid carcinoma (PTC), LncRNAs such as OIP5-AS1 ²¹, MPEG1-1 ²², FOXD2-AS1 ²³, and XIST ²⁴ have been identified as having significant roles.

This study primarily explores the role and underlying molecular mechanisms of LncRNA GUSBP11 in PTC. Notably, in liver cancer, LncRNA GUSBP11's expression has been associated with a poor prognosis, characterized by high expression levels correlating with reduced survival rates ²⁵. Moreover, the membrane progesterone receptor (PAQR7) has been shown to activate several signaling pathways, including MAPK, Akt/PI3K, RhoA/ROCK, JNK1/2, and p38 signaling pathways ^26–28, which contribute to the proliferation, migration, and neurotrophic factor expression of Schwann cell-like adipose stem cells by activating the PI3K/AKT and Src protein kinase signaling pathways ²⁹.

In this study, through the functional enrichment analysis of LncRNA-miRNA-mRNA interactions, it was identified that these interactions could activate the PI3K/AKT and cadherin signaling pathways in thyroid cancer. The study specifically investigated the phosphorylation of serines 838 and 840 on E-cadherin, revealing that such phosphorylation could impair E-cadherin's function and thereby promote the migration of PTC cells. Additionally, tyrosine 860 phosphorylation on N-cadherin was found to enhance N-cadherin's function, thus facilitating melanoma cell migration ³⁰.

The findings also underscore the notion that the upregulation of N-cadherin and downregulation of E-cadherin are instrumental in promoting the epithelial-mesenchymal transition (EMT) pathway in tumors ^31–33. Integrating the outcomes of this study suggests further research into the effects of LncRNA GUSBP11 and PAQR7 on the EMT pathway in PTC could be valuable. The EMT pathway's involvement in regulating matrix metalloproteinases 2 (MMP2) and 9 (MMP9) further substantiates this point ³⁴.

Additionally, miR-28-5p, found to be underexpressed in various cancers, plays a crucial role. In breast cancer, its low expression allows it to target the 3’ UTR of WSB2, inhibiting the migration of cancer cells ³⁵. In prostate cancer, miR-28-5p impedes cell migration by targeting SREBF2 ³⁶, and in colon cancer, it inhibits carcinogenesis and is involved in ferroptosis by targeting N4BP1 ³⁷. This study identifies miR-28-5p as a mediator in the relationship between LncRNA GUSBP11 and the membrane progesterone receptor PAQR7, suggesting the GUSBP11/miR-28-5p/PAQR7 axis as a potential therapeutic pathway for PTC and potentially other cancers.

This research elucidates the pivotal role of LncRNA GUSBP11 and the membrane progesterone receptor PAQR7 in modulating the expression and phosphorylation states of E-cadherin and N-cadherin in papillary thyroid carcinoma (PTC) cells. Furthermore, it establishes a direct association with miR-28-5p in the regulatory mechanisms involving GUSBP11 and PAQR7. The findings demonstrate that in PTC cells, LncRNA GUSBP11, miR-28-5p, and PAQR7 collectively govern cell migration, suggesting a synergistic regulatory mechanism. This study not only advances our understanding of the molecular intricacies associated with PTC progression but also unveils a potential avenue for molecular-targeted therapy in the treatment of papillary thyroid carcinoma, offering new insights into therapeutic strategies aimed at mitigating tumor spread and improving patient outcomes.

Funding

This work was supported by grants from the National Natural and Science Foundation of China (82273331, 82103621, 82203392, 82203497), Department of Science and Technology of Hubei Province (2023BCB090)，the Hubei Natural Science Foundation (2022CFB026), Hubei Province Health and Family Planning Scientific Research Project (WJ2021Q051), Hubei Province Key R&D Program (No. 2022BCA007), and “The 14^th-Five Year Plan” Hubei Provincial advantaged characteristic disciplines (gropus) project of Wuhan University of Science and Technology (2023C0303), Hunan Natural Science Foundation (2023JJ50310), Wuhan University of Science and Technology 2022 Graduate Innovation Venture Fund Project (JCX2022044).

Authors' information

Ji Shi, Yuan Xiang,Yi-Meng Liu and Hong-Shan Guo contributed equally to this work.

Corresponding authors: Correspondence to Xing-Hua Liao and Han-Ning Li

Authors' contributions

Conceptualization: X.H.L.and H.N.L. Experiment lead: J.S.and Y.X. Analytic lead: Y.M.L. Assistance: Y.M.L.and H.S.G. Manuscript prep: J.S.and Y.X. Supervision: X.H.L. Research funding: X.H.L.and H.N.L.

Consent for publication

Consent for publication has been obtained from the patients.

Competing interests

The authors declare that there is no conflict of interest.

Data Availability

All data generated or analysed during this study are included in this published article.

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No competing interests reported.

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LncRNA GUSBP11/miR-28-5p/PAQR7 axis regulates the phosphorylation of E-cadherin and N-cadherin on the migration of papillary thyroid carcinoma cells

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Version 1

Abstract

Figures

Introduction

Materials and Methods

Cell lines

Sample Collection

Quantitative Real-Time PCR (qRT-PCR)

Western Blot Analysis

Plasmid Construction

Wound Healing Assay

Transwell Invasion Assay

Ethics approval

Statistical Analysis

Results

Upregulation of LncRNA GUSBP11 and Membrane Progesterone Receptor PAQR7 in Thyroid Cancer

Knockdown of LncRNA GUSBP11 Reduces Migration and Phosphorylation of Cadherins in Papillary Thyroid Cancer Cells

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1