Published online Apr 19, 2024.
https://doi.org/10.3802/jgo.2024.35.e97
LncRNA STARD7-AS1 suppresses cervical cancer cell proliferation while promoting autophagy by regulating miR-31-5p/TXNIP axis to inactivate the mTOR signaling
Abstract
Objective
Cervical cancer (CC) is a serious gynecologic health issue for women worldwide. Long non-coding RNA (lncRNA) has been well-documented in controlling malignant behavior of various cancer cells. The role of lncRNA STARD7-AS1 in regulating CC cell proliferation and autophagy and its possible mechanism were investigated in this work.
Methods
RNA expression and protein levels were quantified by reverse transcription quantitative polymerase chain reaction and western blotting. The location of STARD7-AS1 in CC cells was examined using subcellular fraction assays. Cell Counting Kit-8 assays and colony forming assays were performed to measure CC cell viability and proliferation. Autophagy in CC cells was evaluated using macrophage-derived chemokine (MDC) staining and transmission electron microscopy. The binding between microRNA (miR)-31-5p and STARD7-AS1 (or thioredoxin-interacting protein [TXNIP]) was determined by performing luciferase reporter, RNA pull-down or RNA immunoprecipitation assays.
Results
STARD7-AS1 overexpression significantly suppressed CC cell viability and proliferation while notably inducing autophagy. STARD7-AS1 upregulated TXNIP expression via interaction with miR-31-5p. In addition, the effects of STARD7-AS1 on CC cell proliferation and autophagy were reversed by TXNIP silencing. The suppressive effect of STARD7-AS1 overexpression on phosphorylated levels of mTOR and S6K1 was countervailed by TXNIP deficiency.
Conclusion
In conclusion, lncRNA STARD7-AS1 inhibits CC cell proliferation and promotes cell autophagy by targeting the miR-31-5p/TXNIP axis to inactivate the mTOR signaling.
Synopsis
STARD7-AS1 is downregulated in cervical cancer (CC) cells. STARD7-AS1 overexpression inhibits CC cell proliferation while inducing autophagy. STARD7-AS1 interacts with microRNA-31-5p to alter thioredoxin-interacting protein (TXNIP) expression. STARD7-AS1 affects cell proliferation and autophagy by upregulating TXNIP and inactivating mTOR signaling.
INTRODUCTION
Cervical cancer (CC) is a prevalent gynecological carcinoma worldwide, occupying approximately 10%–15% of tumor-related mortality in females [1]. Infection of high-risk human papillomavirus is the major risk factor for CC [2]. Poor prognosis is observed in CC patients at advanced stages with the 5-year survival rate less than 40% [3]. Hence, there is an urgent requirement to pay more attention to the pathogenesis of CC and identify specific and sensitive biomarkers for CC.
Long non-coding RNA (lncRNA) refers to noncoding RNA with exceeding 200 nts in length, which has various biological function including epigenetic modulation, splicing, translation, transcription, and chromatin modification [4]. LncRNA could act as competing endogenous RNA (ceRNA) to compete with cancer driver genes for microRNA (miR/miRNA) response elements [5]. According to recent studies, STARD7-AS1 has been identified as an autophagy-related lncRNA and has the potential to be an independent prognostic indicator in CC, as evidenced by the significant correlation between low STARD7-AS1 expression and poor survival outcome in patients with CC based on bioinformatics analysis (data from The Cancer Genome Atlas database) [6, 7, 8]. Moreover, STARD7, as the adjacent gene of STARD7-AS1, has been frequently explored in cancer development [9, 10]. Since STARD7 exerted a prominent role in various types of cancer, the function of its adjacent gene STARD7-AS1 in cancer development deserved further investigation. Despite STARD7-AS1, DBH-AS1 is also suspected as a lncRNA related to autophagy in CC [6, 7, 8]. DBH-AS1 has been widely investigated in various types of cancer. For example, DBH-AS1 is downregulated in pancreatic cancer tissues, and DBH-AS1 suppresses cell growth while increasing the sensitivity of cancer cells to gemcitabine [11]. However, DBH-AS1 is upregulated in diffuse large B-cell lymphoma, and the silencing of DBH-AS1 inhibits cancer cell proliferation, migration, and invasion [12]. Thus, DBH-AS1 was demonstrated to play an oncogenic or tumor-suppressive role in different types of cancer. Bioinformatics analysis reveals that low DBH-AS1 expression significantly correlates to poor prognosis of patients with CC, suggesting the potential anti-tumor property of DBH-AS1 in CC. Hence, the current study compared STARD7-AS1 and DBH-AS1 in CC from the aspects of expression, overall survival, and cellular distribution.
Autophagy exerts an essential role in cell homeostasis [13]. The double-edged functions of autophagy in cancer development has been demonstrated by previous studies [14]. Autophagy provides nutrition for cancer cells by digesting and decomposing normal cells, thus facilitating tumor development at late stages; while in the early stages, autophagy elicits a suppressive function [15]. Importantly, there exists close association between autophagy and CC development [16]. The mTOR signaling is the main pathway in autophagy regulation [17], which exerts vital functions in tumorigenesis and it can determine the death and survival of cancer cells [18]. Nonetheless, whether STARD7-AS1 or DBH-AS1 can modulate CC cell autophagy via regulating the mTOR pathway is unclear.
In the present work, we aimed to reveal STARD7-AS1 and DBH-AS1 expression status, the functions of STARD7-AS1 in regulating CC cell proliferation and autophagy and the underlying mechanism. It is hypothesized that STARD7-AS1 may play as a ceRNA to regulate CC cell autophagy via the mTOR signaling. This study may help to understand CC pathogenesis in depth and develop effective targeted therapies for CC treatment in the future.
MATERIALS AND METHODS
1. Cell culture
Human CC cell line HeLa (#MY-K5046), CasKi (#SY4779), and C-33A (#SY4717) and human immortal keratinocyte cell line (HaCaT) (#MY-K5266) were obtained from Shanghai Biological Technology enzyme research (Shanghai, China). Cells were maintained in DMEM medium (YT8231, YITA Biotechnology, Beijing, China) supplemented with 10% fetal bovine serum (76294-180; AVANTOR, Perth, Australia) and 100 mg/mL streptomycin/penicillin at 37°C in an incubator with 5% carbon dioxide.
2. Bioinformatics methods
The bioinformatics tool, ENCORI (https://rnasysu.com/encori/), was used to predict the overall survival of STARD7-AS1 and DBH-AS1 in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC). ENCORI was also used to predict downstream miRNAs that can bind with STARD7-AS1 and predict target genes of miR-31-5p. Another bioinformatics tool, GEPIA (http://gepia.cancer-
3. Cell transfection
The pcDNA3.1 vector containing STARD7-AS1 sequence (pcDNA3.1/STARD7-AS1) was used to induce STARD7-AS1 overexpression. Short hairpin (sh) RNAs targeting STARD7-AS1 were employed to silence STARD7-AS1 expression. CC cells were plated in 6-well plates (3×105 cells/well). Upon reaching 90% confluence, CC cells were transfected with pcDNA3.1/STARD7-AS1, miR-31-5p inhibitor, sh-STARD7-AS1#1/2, sh-TXNIP#1/2 and negative control (NC) plasmids using lipofectamine™ 2000 reagent (11668019; Invitrogen, Carlsbad, CA, USA) for 48 hours. The abovementioned oligonucleotides were provided by Genepharma (Shanghai, China).
4. Subcellular fraction assay
Cytoplasmic and nuclear RNA was isolated from CC cells using the PARIS™ kit (AM1556; Invitrogen). As per the product manuals, HeLa and CaSki cells were seeded to a 10-cm Petri dish, rinsed by phosphate buffered saline (PBS), resuspended in cell fractionation buffer (300 μL) and incubated on ice for 5–10 minutes. Then, cell centrifugation at 500 × g for 3 minutes at 4°C was performed followed by isolation of cytoplasmic fraction from the nuclear pellet. U6 and GAPDH functioned as cytoplasmic and nuclear control, respectively. Cellular localization of STARD7-AS1 was determined by analyzing its expression in cytoplasmic and nuclear parts using reverse transcription quantitative polymerase chain reaction (RT-qPCR).
5. RT-qPCR analysis
Isolation of total RNA from CC cells was performed using the RNeasy Mini Kit (74104; Qiagen, Valencia, CA, USA). After RNA quantification utilizing the Qubit 2.0 Fluorometer (Q32867; Invitrogen), a cDNA Reverse Transcription Kit (70-MK0601-100; MultiSciences Biotech Co., Ltd., Hangzhou, China) was utilized to transcribe RNA samples into cDNA. Quantitative PCR was conducted on the ABI PRISM 7900HT Fast PCR System (Applied Biosystems, Waltham, MA, USA) with SYBR Premix Ex Taq (Takara, Kusatsu, Japan) as per the product manuals. Relative gene expression was calculated using the 2-ΔΔCt method. Expression levels of miRNAs were calculated with normalization to U6 while the expression of rest genes was normalized to GAPDH. Sequences of primers are presented in Table S1.
6. Colony formation assay
The resuspended cells (1×103 cells/well) were plated in 10-cm culture plates post transfection. After cells were grown in culture medium for 2 weeks, colonies formed were harvested and rinsed with PBS. During the 2 weeks, the culture medium was replaced every three days. Next, cells were fixed with 5% glutaraldehyde followed by staining with crystal violet (Sigma Aldrich, St. Louis, MO, USA). After washed with PBS, colonies were observed and imaged under an optical microscope. The number of colonies containing more than 50 cells was counted.
7. Cell Counting Kit-8 (CCK-8) assay
Cells were inoculated in 96-well plates (3×103 cells/well) and grown for specific time periods (24, 48, and 72 hours). CCK-8 solution (A311-02; Vazyme Biotech Co., Ltd., Nanjing, China) was supplemented to each well at each timepoint for further 4-hour of cell incubation. A microplate reader (Bio-Rad, Hercules, CA, USA) was utilized to detect the absorbance at the wavelength of 450 nm.
8. Macrophage-derived chemokine (MDC)
Cells were cultured on small round slides coated with polylysine. Fluorescent dye MDC (20 μM) was added for cell incubation for 30 minutes. PBS was added and the cells were centrifuged at 179 g twice (3 minutes each time). The supernatant was discarded post centrifugation. Then, the cell samples were subjected to 3-minute fixation with 40 μL formaldehyde and 1-hour incubation in 40 μL MDC (0.1 mmol/L) at room temperature. Cells were centrifuged again prior to adjusting cell concentration using 1% paraformaldehyde. Flow cytometry was used for autophagy detection.
9. Transmission electron microscopy
CaSki and HeLa cells with indicated treatment were washed twice with PBS and then subjected to centrifugation (800 ×g, 5 minutes). Next, cell pellets were collected and supplemented with 2% preheated agarose (#75510019; Thermo Fisher Scientific, Waltham, MA, USA). The agar was cut into small sections (1 mm3) and fixed with 2.5% glutaraldehyde/0.1 M PBS (4°C, overnight). After that, the sections were washed twice with PBS followed by fixation with osmium tetroxide (1%, 4°C, 1 hour). Then, cell samples were dehydrated in graded ethanol solutions, embedded into araldite, and cut into semi-thin sections. At last, the cell samples were stained with 1% uranyl acetate. A transmission electron microscope (Olympus, Tokyo, Japan) was used to observe cellular autophagy.
10. Western blotting
Proteins were extracted from CC cells using radio immunoprecipitation lysis buffer (R0020-100; Solarbio, Beijing, China). Protein concentration was determined using a bicinchoninic acid protein assay kit (orb90441, biobyt; Booute Biotechnology, Wuhan, China). The protein contents were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to PVDF membranes (1620177; Bio-Rad Laboratories, Shanghai, China). The membranes were sealed with 5% nonfat milk at 37°C for 1 hour followed by incubation with primary antibodies of anti-ATF2 (ab32160, 1:10,000; Abcam Inc., Waltham, MA, USA), anti-light chain 3 (LC3) (ab62721, 2 µg/mL, Abcam), anti-TXNIP (ab210826, 5 µg/mL, Abcam), anti-mTOR (ab134903, 1:10,000, Abcam), anti-p-mTOR (ab109268, 1:5,000, Abcam), anti-S6K1 (ab186753, 1:1,000, Abcam), anti-p-S6K1 (ab59208, 1:500, Abcam), and anti-β-actin (ab115777, 1:200, Abcam) overnight at 4°C. Then, the membranes were incubated with secondary antibody (ab288151, 1:2,000, Abcam) at room temperature for 1 hour. The enhanced chemiluminescent reagent (Pierce; Thermo Fisher Scientific) was employed for protein band visualization. B-actin acts as the loading control. Relative protein levels were quantitated using ImageJ software.
11. RNA pull-down assay
Pierce magnetic RNA-protein pull-down Kit (Thermo Fisher Scientific) was utilized for the assay. Bio-NC, bio-miR-31-5p wild type (Wt)/mutated (Mut) were designed by Sangon (Shanghai, China) and transfected into CC cells. After transfection, cells were lysed and harvested. M-280 streptavidin magnetic beads (Invitrogen) were then incubated with the lysate at 4°C for 1 hour. The bound RNA was then collected and subjected to RT-qPCR.
12. Luciferase reporter assay
The sequences of STARD7-AS1 Wt or its Mut sequences with miR-31-5p were inserted into pmirGLO vectors (Promega, Madison, WI, USA) to construct STARD7-AS1-Wt/Mut reporters. TXNIP-Wt/Mut was generated by inserting TXNIP Wt or Mut sequences with miR-31-5p binding site into pmirGLO vectors. HeLa and CaSki cells were transfected with miR-31-5p-Wt/Mut and pcDNA3.1 or pcDNA3.1/STARD7-AS1 using lipofectamine™ 2000. TXNIP-Wt/Mut was transfected with NC inhibitor, miR-31-5p inhibitor, or miR-31-5p inhibitor + sh-STARD7-AS1#1 into CC cells. After transfection for 48 hours, luciferase activities were measured utilizing a luciferase reporter assay kit (Promega).
13. RNA immunoprecipitation (RIP) assay
EZ-Magna RIP Kit (17-701) was utilized to perform the RIP assay as instructed. Briefly, cells were lysed in lysis buffer containing RNase inhibitor and protease inhibitor cocktail. Then, cell incubation with RIP buffer and magnetic bead precoated with Ago2 antibody was performed (2 hours, 4°C). Immunoglobulin G acts as the control. The immunoprecipitated RNAs were isolated form beads, purified, and subjected to RT-qPCR.
14. Statistical analysis
SPSS (version 17.0; SPSS, Chicago, IL, USA) software was used for data analysis, and these data are shown as the mean ± standard deviation. Each experiment was executed as least in triplicate. The differences between two groups were compared using Student’s t test. The comparison among three groups was conducted using one-way ANOVA and Tukey’s multiple comparisons test. The value of p<0.05 was deemed as statistically significant.
RESULTS
1. STARD7-AS1 is downregulated in CC cells
According to results of RNA sequencing in previous articles, STARD7-AS1 and DBH-AS1 are autophagy-related lncRNAs in CC [6, 7, 8]. In this study, we compared the expression of two lncRNAs in CC cells and immortal keratinocyte cells HaCaT. RT-qPCR analysis displayed that STARD7-AS1 was significantly lowly expressed in CC cell lines, especially in HeLa and CaSki cells (0.32±0.03, 0.37±0.04), which is more significant than DBH-AS1 expression in CC cells (0.42±0.04, 0.45±0.05) (Fig. 1A). Bioinformatics analysis shows that the low expression of the two lncRNAs are significantly related to poor prognosis in CC patients (Fig. 1B and C). The p value in the overall survival plot for STARD7-AS1 is 0.0043, and that for DBH-AS1 is 0.014 (Fig. 1B and C). A bioinformatic tool, lncLocator, was used to predict the cellular distribution of STARD7-AS1 and DBH-AS1. The results showed that the distribution of STARD7-AS1 in cytosol is slightly higher than that of DBH-AS1 (Fig. 1D). Considering the abovementioned comparison, we decided to further explore the role of STARD7-AS1 in CC cells. The above finding also reveals the promising role of DBH-AS1 in CC development, which deserves further investigation in the future. Subcellular fractionation assay further validated that STARD7-AS1 was predominantly located in the cytoplasm of HeLa and CaSki cells (Fig. 1B). The results suggested that STARD7-AS1 may play a role in CC cells at post-transcriptional level.
Fig. 1
*p<0.05.
Downregulation of long non-coding RNA STARD7-AS1 in CC cells. (A) Reverse transcription quantitative polymerase chain reaction was performed to measure STARD7-AS1 and DBH-AS1 expression in CC cells and immortal keratinocyte cells HaCaT. (B-C) ENCORI was used for prediction of the prognosis of CESC patients with low and high STARD7-AS1 (or DBH-AS1) expression. (D) The bioinformatic tool, lncLocator, was used to predict the cellular distribution of STARD7-AS1 or DBH-AS1. (E) A subcellular fraction assay was performed to measure the distribution of STARD7-AS1 in nucleus or cytoplasm of CC cells.
CC, cervical cancer; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma.
2. STARD7-AS1 upregulation inhibits CC cell proliferation while inducing autophagy
To probe into the role of STARD7-AS1 in regulating CC cell growth, STARD7-AS1 expression was amplified in HeLa and CaSki cells through transfection of pcDNA3.1/STARD7-AS1. As a result, relatively high STARD7-AS1 expression was observed in CC cells post transfection (Fig. 2A). Cell viability was notably inhibited after elevation of STARD7-AS1 expression when compared to the viability of cells containing control pcDNA vectors (Fig. 2B). A significant decrease in the number of cell colonies was seen in the pcDNA3.1/STARD7-AS1 group (Fig. 2C), implying that overexpressed STARD7-AS1 impaired CC cell proliferation. Since STARD7-AS1 was analyzed to be an autophagy-related lncRNA in CC [6, 7, 8], the influence of STARD7-AS1 on autophagy of CC cells was then explored. As indicated by western blot analysis and the MDC assay, CC cell autophagy was significantly promoted upon STARD7-AS1 overexpression, as manifested by the significantly increased ATF2 protein expression, LC3 I/II ratio, and MDC positive rate (Fig. 2D and E). Transmission electron microscopy was conducted to observe cellular autophagy, and representative images showed that STARD7-AS1 led to an increased number of the double membrane and autophagosome-like structures in CC cells (Fig. 2F). Overall, STARD7-AS1 upregulation suppresses CC cell proliferation while enhancing cell autophagy.
Fig. 2
*p<0.05, **p<0.01, ***p<0.001.
Effects of STARD7-AS1 overexpression on the proliferation and autophagy of CC cells. (A) Overexpression efficiency of STARD7-AS1 in CC cells. (B-C) The impacts of upregulated STARD7-AS1 on the viability and proliferation of CC cells were measured by CCK-8 assays and colony formation assays, respectively. (D) The levels of autophagy-related proteins (ATF2 and LC3) were quantified by western blot analysis. (E) Due to autophagy, MDC fluorescence changed from scattered distribution to puncta distribution in cytoplasm. MDC staining was performed to examine cell autophagy. (F) Transmission electron microscopy was used to observe cellular autophagy which can be reflected by the number of the double membrane and autophagosome-like structures.
CC, cervical cancer; MDC, macrophage-derived chemokine.
3. STARD7-AS1 interacts with miR-31-5p
After confirming the post-transcriptional regulatory potential and the regulatory role of STARD7-AS1 in CC cell proliferation and autophagy, we subsequently explored the ceRNA mechanism mediated by STARD7-AS1. Two candidate miRNAs (miR-31-5p and miR-212-3p) that have binding site with STARD7-AS1 were identified from ENCORI online website (Fig. 3A). MiR-31-5p expression was significantly reduced in CC cells with pcDNA3.1/STARD7-AS1 transfection, while miR-212-3p level displayed no significant change between pcDNA3.1 and pcDNA3.1/STARD7-AS1 groups (Fig. 3B). Therefore, miR-31-5p was deduced as a potential target miRNA regulated by STARD7-AS1. To verify this speculation, we examined the luciferase activity of STARD7-AS1-Wt/Mut reporters in HeLa and CaSki cells. The results depicted that inhibition of miR-31-5p led to a surge in STARD7-AS1-Wt activity, but STARD7-AS1-Mut activity failed to be significantly altered (Fig. 3C). RNA pull-down assays also confirmed the interaction between STARD7-AS1 and miR-31-5p, as shown by the abundant enrichment of STARD7-AS1 in bio-miR-31-5p WT group compared to STARD7-AS1 level in bio-NC and bio-miR-31-5p MUT groups (Fig. 3D). Moreover, miR-31-5p was upregulated in CC cells, including HeLa, CaSki, and C-33A, compared to that in immortal keratinocyte cells HaCaT (Fig. 3E). The successful knockdown efficiency of miR-31-5p inhibitors was shown in Fig. 3F, as evidenced by the significantly reduced miR-31-5p expression in the miR-31-5p inhibitor group.
Fig. 3
**p<0.01, ***p<0.001.
STARD7-AS1 sponges miR-31-5p. (A) ENCORI online website was used for predicting the miRNAs having binding area with STARD7-AS1 (screening condition: Pan-cancer ≥6 cancer types). (B) Relative expression of miR-212-3p and miR-31-5p in CC cells overexpressing STARD7-AS1 was measured by PCR analysis. (C) Relative luciferase activity of STARD7-AS1-Wt/Mut in the context of miR-31-5p inhibition in HeLa and CaSki cells. (D) RNA pull-down assays were performed to assess relative enrichment of STARD7-AS1 in bio-NC group, bio-miR-31-5p Wt group, and bio-miR-31-5p Mut group. (E) MiR-31-5p expression in CC cells and HaCaT cells was evaluated by RT-qPCR analysis. (F) Knockdown efficiency of miR-31-5p inhibitor in CC cells by was evaluated by RT-qPCR.
CC, cervical cancer; miR, microRNA; Mut, mutated; PCR, polymerase chain reaction; RT-qPCR, reverse transcription quantitative polymerase chain reaction; Wt, wild type.
4. MiR-31-5p targets TXNIP
ENCORI website was employed for the predication of target genes that may regulated by miR-31-5p. Under the screening condition of Pan-cancer ≥6 cancer types, 5 potential downstream targets of miR-31-5p were identified. Among which, TXNIP attracted our attention due to its significantly increased expression in CC cells silencing miR-31-5p (Fig. 4A). The binding sequences between TXNIP and miR-31-5p were displayed in Fig. 4B. For the subsequent luciferase reporter assay, STARD7-AS1 expression was knocked out through transfection of shRNAs targeting STARD7-AS1 (Fig. 4C). Fig. 4D delineated that inhibition of miR-31-5p increased the luciferase activity of TXNIP-Wt, and the trend was reversed by STARD7-AS1 deficiency. In addition, TXNIP-Mut activity was not obviously altered among NC inhibitor, miR-31-5p inhibitor, and miR-31-5p inhibitor + sh-STARD7-AS1#1 groups (Fig. 4D). RIP assays showed that STARD7-AS1, miR-31-5p, and TXNIP were enriched in RNA-induced silencing complexes, further validating the regulatory relationship among the three factors (Fig. 4E).
Fig. 4
**p<0.01, ***p<0.001.
TXNIP is a direct target of miR-31-5p. (A) ENCORI was used to seek downstream targets of miR-31-5p, and the top five genes with the highest combined scores were selected. The mRNA expression of these genes (HIF1AN, TXNIP, MAP1B, SERF2, and LAPTM4A) was quantified using reverse transcription quantitative polymerase chain reaction. (B) The predicated binding area between TXNIP and miR-31-5p was obtained from ENCORI. (C) The knockdown efficiency of STARD7-AS1 in CC cells. (D) Luciferase reporter assay was conducted to measure the activity of TXNIP-Wt/Mut in the groups of NC inhibitor, miR-31-5p inhibitor, and miR-31-5p inhibitor + sh-STARD7-AS1#1. (E) RNA immunoprecipitation assays were conducted to quantify the enrichment of STARD7-AS1, miR-31-5p, and TXNIP in anti-IgG and anti-Ago2 groups.
CC, cervical cancer; IgG, immunoglobulin G; miR, microRNA; Mut, mutated; TXNIP, thioredoxin-interacting protein; Wt, wild type.
5. TXNIP is downregulated in CC cells and positively regulated by STARD7-AS1
Through bioinformatic analysis, TXNIP was predicated to be poorly expressed in CESC tumor tissues (n=306) relative to its expression in normal controls (n=13) (Fig. 5A). In line with the predication, our experimental results revealed that TXNIP was lowly expressed at RNA and protein levels in CC cell lines in comparison to its expression in HaCaT cells (Fig. 5B). Kaplan-Meier plotter analysis revealed that the poor prognosis of patients with cervical squamous cell carcinoma is strongly associated with low TXNIP expression (Fig. 5C). Upregulation of STARD7-AS1 or inhibition of miR-31-5p significantly increased TXNIP expression levels in both HeLa and CaSki cells (Fig. 5D and E).
Fig. 5
**p<0.01.
TXNIP is downregulated in CC cells. (A) The bioinformatics tool, GEPIA, was utilized to predict TXNIP expression in CESC tumor tissues and normal ones. (B) TXNIP mRNA and protein expression levels in CC cell lines and HaCaT cells were measured by RT-qPCR and western blotting. (C) The bioinformatics tool, Kaplan-Meier plotter, was adopted to analyze the correlation of TXNIP expression and the prognosis of patients with cervical squamous cell carcinoma. (D) TXNIP expression in CC cells overexpressing STARD7-AS1 was assessed by RT-qPCR. (E) Western blotting was conducted to quantify TXNIP protein level in response to STARD7-AS1 overexpression or miR-31-5p inhibition.
CC, cervical cancer; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; RT-qPCR, reverse transcription quantitative polymerase chain reaction; TXNIP, thioredoxin-interacting protein.
6. TXNIP deficiency reverses the impacts of STARD7-AS1 elevation on CC cell proliferation, autophagy and the activity of mTOR signaling
TXNIP expression was effectively reduced after sh-TXNIP#1/2 was transfected into CC cells (Fig. 6A). As illustrated in Fig. 6B and C, the viability and proliferation of CC cells were inhibited by overexpressed STARD7-AS1, and the changes were rescued by silenced TXNIP. Moreover, the promotive effects of STARD7-AS1 overexpression on ATF2 protein expression, LC3 I/II ratio, and MDC positive rate were partially reversed by TXNIP deficiency (Fig. 6D and F). Previous studies illustrate that TXNIP can regulate the mTOR pathway [19] and inhibition of the mTOR signaling induces autophagy in CC [20]. Hence, we speculated that STARD7-AS1 may regulate CC cell growth by mediating this signaling. Western blot analysis showed that upregulation of STARD7-AS1 weakened phosphorylated levels of mTOR and its downstream molecule S6K1 while upregulating TXNIP expression (Fig. 6G). In addition, the inhibitory impact of STARD7-AS1 overexpression on phosphorylated mTOR and S6K1 levels were rescued by knockdown of TXNIP (Fig. 6G). The finding showed that STARD7-AS1 inactivates the mTOR signaling pathway by upregulating TXNIP.
Fig. 6
*p<0.05, **p<0.01.
TXNIP deficiency reverses the impacts of STARD7-AS1 elevation on CC cell proliferation, autophagy and the activation of mTOR signaling. (A) TXNIP expression in CC cells transfected with sh-NC or sh-TXNIP#1/2 transfection was measured by reverse transcription quantitative polymerase chain reaction. (B-C) CCK-8 and colony formation assays were performed to measure the viability and proliferation of CC cells in the groups of pcDNA3.1, pcDNA3.1/STARD7-AS1, and pcDNA3.1/STARD7-AS1 + sh-TXNIP#1. (D) The levels of autophagy-related proteins (ATF2 and LC3) in abovementioned groups were quantified by western blot analysis. (E-F) MDC staining was conducted to evaluate cell autophagy. (G) The protein levels of TXNIP, p-mTOR, mTOR, p-S6K1, and S6K1 in pcDNA3.1, pcDNA3.1/STARD7-AS1, and pcDNA3.1/STARD7-AS1 + sh-TXNIP#1 groups were quantified by western blot analysis.
CC, cervical cancer; MDC, macrophage-derived chemokine; TXNIP, thioredoxin-interacting protein.
DISCUSSION
In this study, STARD7-AS1 was poorly expressed and predominantly located in cytoplasm. STARD7-AS1 overexpression hampered CC cell growth and promoted autophagy, implying the tumor-suppressive potential of STARD7-AS1 in CC.
According to ceRNA hypothesis, lncRNA can function as a ceRNA to exert key functions by sponging miRNAs and consequently modulating the expression of cancer-related genes [5, 21]. It has been reported by a previous study that lncRNA GAS5 plays as a ceRNA to modulate the miR-31-5p (previously named miR-31)/ARID1A axis in ovarian clear cell carcinoma [22]. Consistent with the published study, the current work demonstrated the involvement of miR-31-5p in lncRNA-mediated ceRNA network in CC. In addition, the oncogenic role of miR-31-5p has been validated in multiple human carcinomas such as papillary thyroid cancer [23], colon adenocarcinoma [24], and lung cancer [25]. Most importantly, miR-31-5p has been validated to be an oncogene in CC [26]. Here, miR-31-5p was upregulated in CC cell lines, which is in line with its expression status verified by previous studies [26]. In addition, upregulation of STARD7-AS1 decreased miR-31-5p level in CC cells, and the binding between STARD7-AS1 and miR-31-5p was also verified.
TXNIP, also named vitamin D3 up-regulated protein 1, is significantly upregulated in CC cells transfected with miR-31-5p inhibitor. It is noteworthy that downregulation of TXNIP is observed in various carcinomas, e.g., lymphoma and leukaemia, lacking genetic mutations [27]. The decrease and loss of TXNIP expression caused by hypermethylation of TXNIP promoter are found in carcinogenesis [28]. Previous studies illuminate that autophagy-related genes such as ATG10, ATG16L2, and ATG12 are correlated with TXNIP [29, 30]. Our current study validated that miR-31-5p targeted TXNIP, and TXNIP was poorly expressed in CC cells. Low expression of TXNIP predicated worse prognosis of CESC patients. TXNIP expression can be elevated by STARD7-AS1 upregulation or miR-31-5p inhibition, suggesting that STARD7-AS1 regulates TXNIP expression by interacting with miR-31-5p in CC cells. Rescue experiments showed that the repressive influence of STARD7-AS1 overexpression on CC cell growth were rescued by TXNIP silencing. Overactivation of autophagy in CC cells caused by STARD7-AS1 elevation was countervailed by TXNIP deficiency.
The mTOR signaling plays a vital role in maintaining cell homeostasis (cell survival and growth, cell metabolism) by phosphorylating its downstream substrates including 4E-binding protein 1, and S6 kinase [31]. Abnormally activated mTOR signaling has been found in various tumor types, including CC. For instance, rapamycin-inactivated mTOR signaling suppresses CC tumor growth in vivo, protein synthesis and cell proliferation in vitro [32]. LARP1 facilitates CC progression by regulating mTOR expression at post-transcriptional level [33]. These findings indicate that activation of mTOR pathway is essential for CC cell survival, proliferation, and growth. In the current investigation, overexpression of STARD7-AS1 reduced phosphorylation levels of mTOR and S6K1, and the alterations were partly rescued by the silencing of TXNIP.
The study reveals that STARD7-AS1 obstructs CC cell proliferation while inducing autophagy by targeting miR-31-5p/TXNIP to inhibit the mTOR signaling. The findings suggested that STARD7-AS1 might be a novel therapeutic target for CC treatment. The limitation of the study is that the anti-tumor effect of STARD7-AS1 was not further validated by establishing xenograft mouse models.
For future work, the upstream transcription factors that may alter STARD7-AS1 expression can be investigated. Other potential genes and signaling pathway that can be regulated by STARD7-AS1 in CC should be explored to clearly illustrate the complex underlying mechanisms. The influence of STARD7-AS1 on tumor growth and other cellular processes such as angiogenesis, ferroptosis, and epithelial-mesenchymal transition can also be explored since autophagy is closely associated with these cellular processes during cancer development [34, 35, 36].
SUPPLEMENTARY MATERIAL
Sequences of primers used for quantitative polymerase chain reactionTable S1
Conflict of Interest:No potential conflict of interest relevant to this article was reported.
Data Availability Statement:The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.
Author Contributions:
Conceptualization: Y.X.
Data curation: Y.X., C.Z.
Formal analysis: Y.X., C.Z.
Investigation: Y.X., C.Z.
Methodology: Y.X., C.Z.
Project administration: Y.X., L.X., C.Z.
Resources: Y.X., L.X., G.H., C.Z.
Software: Y.X., L.X., G.H., C.Z.
Supervision: Y.X., L.X., G.H., C.Z.
Validation: Y.X., C.Z.
Visualization: Y.X., C.Z.
Writing - original draft: Y.X., C.Z.
Writing - review & editing: Y.X., C.Z.
References
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