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The novel role of circular RNA ST3GAL6 on blocking gastric cancer malignant behaviors through autophagy regulated by the FOXP2/MET/mTOR axis

Penghui Xu, Xing Zhang, Jiacheng Cao, Jing Yang, Zetian Chen, Weizhi Wang, Sen Wang, Lu Zhang, Li Xie, Lang Fang, Yiwen Xia, Zhe Xuan, Jialun Lv, Zekuan Xu - Show less +10 more

17 Aug 2021-

TL;DR: It is revealed that circST3GAL6 functions as a tumour suppressor through the miR-300/FOXP2 axis in GC, regulates apoptosis and autophagy through FOXP2-mediated transcriptional inhibition of the MET axis and may be a biomarker for GC treatment.

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Abstract: Gastric cancer (GC) ranks third in motality among all cancers worldwide. Circular RNAs (circRNAs) play essential roles in the malignant progression and metastasis of gastric cancer. As a transcription factor, FOXP2 is involved in the progression of many tumours. However, the regulation and association between circRNAs and FOXP2 remain to be discovered. In our study, CircST3GAL6 was significantly depressed in GC tissues and cells. circST3GAL6 overexpression inhibited the proliferation, invasion and metastasis of GC cells in vitro and in vivo. Importantly, circST3GAL6 overexpression induced apoptosis and promote autophagy in GC cells. Furthermore, we found that circST3GAL6 sponged miR-300 and subsequently regulated FOXP2. We further revealed that FOXP2 suppressed the activation of the Met/AKT/mTOR axis, a classic pathway that regulates autophagy-mediated proliferation and migration. In summary, our ﬁndings revealed that circST3GAL6 functions as a tumour suppressor through the miR-300/FOXP2 axis in GC, regulates apoptosis and autophagy through FOXP2-mediated transcriptional inhibition of the MET axis and may be a biomarker for GC treatment.

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Summary (5 min read)

Jump to: [Introduction] – [Materials And Methods] – [Cell culture and treatment] – [RNA-seq analysis] – [Actinomycin D assay] – [Plasmid construction and stable transfection] – [Western blotting] – [CCK-8 assay] – [Colony formation assay] – [Transwell assay] – [Flow cytometric analysis] – [5-Ethynyl-2′-deoxyuridine (EdU) assay] – [Pull-down assay] – [Immunohistochemical (IHC) analysis of tissue samples] – [Transmission electron microscopy (TEM)] – [Nude mouse xenograft model] – [In vivo metastasis assay] – [Confocal microscopy] – [Chromatin immunoprecipitation assay] – [Statistics] – [CircST3GAL6 expression pro les in gastric cancer tissues and paired normal gastric tissues] – [Identi cation of the structure and characteristics of circST3GAL6] – [CircST3GAL6 inhibits tumour growth and metastasis in vivo] – [Knockdown of FOXP2 reverses the effect of miR-300 inhibitors on GC cells] – [FOXP2 transcriptionally inhibits MET and regulates autophagy] – [Discussion] and [Conclusion]

Introduction

Gastric cancer (GC) is the third most frequent cause of cancer-related deaths worldwide.
The overall survival of GC patients has remained unsatisfactory in recent years 6 .
Furthermore, circRNAs have been reported to play many important roles in regulating translation, sponging miRNA and proteins [10] [11] [12] .
FOXP2 has rarely been investigated 16, 17 .
CircST3GAL6 also regulates autophagy-mediated proliferation and migration through the FOXP2/Met/mTOR axis.

Materials And Methods

All the specimens were collected and snap-frozen in liquid nitrogen immediately after surgical resection.
TNM stage was based on the TNM classi cation system (American Joint Committee on Cancer classification, AJCC, 7 th edition).

Cell culture and treatment

The human GC cell lines BGC-823, SGC-7901, MGC-803, and MKN-45 and normal GES-1 stomach mucosa epithelium cells were cultured in RPMI 1640 (Wisent, Shanghai, China) supplemented with 10% foetal bovine serum (FBS) (Wisent, Biocenter, China) and 1% pencillin-streptomycin.
HGC-27 cells were cultured in RPMI 1640 (Wisent, Shanghai, China) supplemented with 20% FBS (Wisent, Biocenter, China) and 1% penicillinstreptomycin.

RNA-seq analysis

Total RNA was isolated from GC tissues and cells using TRIzol reagent .
Next, complementary DNA (cDNA) was reverse transcribed using PrimeScript RT Reagent (RR036A; TaKaRa, Japan).
Quantitative realtime polymerase chain reaction (qRT-PCR) was performed using the SYBR™ GREEN PCR Master Mix kit (4913914001; Roche, Shanghai, China).
The relative expression of RNA was normalized to the endogenous control glyceraldehyde 3-phosphate dehydrogenase and U6.
RIBOBIO Biotech (Guangzhou, China) provided all the speci c primers for circRNAs.

Actinomycin D assay

Cells were seeded at 5×10 4 cells per well in a 24-well plate overnight and then treated with 2 mg/L of actinomycin D (Sigma-Aldrich, USA).
Total RNA was harvested at the indicated time points (4 h, 8 h, 12 h, 24 h), and qRT-PCR was performed to analyse the stability of the circRNA and mRNA.

Plasmid construction and stable transfection

CircST3GAL6 cDNA was synthesized and cloned into the pcDNA3.1 vector (GenePharma, Shanghai, China).
Cells were transfected with plasmids according to the manufacturer's protocol.

Western blotting

Total protein from tissues and cells was extracted using RIPA lysis buffer (P0013B; Beyotime Biotechnology, China) containing PMSF.
The protein concentration was determined using the Bradford method.
Equal amounts of protein samples were resolved and separated by 10% SDS-PAGE using an electrophoresis apparatus (Bio-Rad, America) and transferred onto polyvinylidene di uoride (PVDF) membranes.
Finally, the membranes were washed and then incubated with secondary antibody for 2 h at room temperature.
The blots were then visualized by enhanced chemiluminescence detection.

CCK-8 assay

The authors plated BGC-823 and SGC-7901 cells in 96-well plates at ten thousand cells per well, and then added 10 μl of CCK-8 solution (Dojindo Laboratories, Kumamoto, Japan) to each well every other day according to the manufacturer's protocols.
After that, the cells were cultured for 2 hours at 37°C.
Two hours later, the authors recorded the absorbance of the cells at 450 nm using a microplate reader (BioTek, Winooski, VT, USA).

Colony formation assay

BGC-832 and SGC-7901 cells were seeded in different six-well plates.
Each well was inoculated with 1000 cells, and then the six-well plates were cultured in an incubator containing 5% CO 2 for 2 weeks.
Two weeks later, the cell proliferation state was observed after staining the cells.

Transwell assay

First, the authors inoculated speci c cells on the upper side of the Transwell compartments (Millipore, Billerica, MA, USA) and added 200 μl serum-free RPMI-1640 at the same time.
Next, the authors added 600μl of complete medium to the lower side of the Transwell compartments.
After incubation for 24 hours in an incubator containing 5% CO 2 , the authors rinsed the cells with PBS and then removed the cells that did not pass through the membrane with a cotton swab.
Finally, the authors xed and stained the cells with 75% alcohol and crystal violet.

Flow cytometric analysis

First, the authors inoculated the transfected cells in six-well plates.
Next, the authors collected all the cells and incubated them with the PE Annexin V Apoptosis Detection Kit reagents (BD Pharmingen, Franklin Lake, New Jersey, USA) for 2 days.
After 15 minutes of incubation, the authors observed the cells using CELL Quest software (BD Biosciences, San Jose, CA, USA).
The proportion of apoptosis is re ected by the corresponding quadrant on the apoptosis map.

5-Ethynyl-2′-deoxyuridine (EdU) assay

The authors inoculated the transfected cells in a 96-well plate at 10,000 per well and cultured them for 24 hours.
The next day, the authors added EdU solution (RiboBio, China) to each well of the 96-well plate for incubation.
After that, the cells were xed and then were subjected to Apollo staining and DNA staining using Apollo reaction solution and Hoechst 33342, respectively.
Finally, the authors observed the red and blue signals under the microscope to evaluate cell proliferation.
The cells were then washed with 4× sodium citrate buffer containing 0.1%.

Pull-down assay

A biotin-labelled probe for circST3GAL6 was designed by RiboBio (Guangzhou, China).
GC cells were harvested, lysed and incubated with the circST3GAL6 probe and oligo probe at 4°C overnight.
The RNA mixture was bound to the magnetic beads with washing buffer several times.
After that, the RNA was extracted using the RNeasy Mini Kit (QIAGEN, Germany) and detected by qRT-PCR.
Biotinylated miR-300 and biotinylated miR-NC were produced by GenePharma (Shanghai, China), and the methods were the same as those described above.

Immunohistochemical (IHC) analysis of tissue samples

The GC tissues were xed with 10% formalin and embedded in para n.
Next, the tissues were cut into sections and incubated with speci c primary antibody at 4°C overnight.
After washing twice, the sections were incubated with HRP-polymer-conjugated secondary antibody abcam, UK at 37°C for one hour.
These sections were then stained with 3,3-diaminobenzidine solution and haematoxylin.
Finally, the authors observed the slices via microscope.

Transmission electron microscopy (TEM)

The authors placed the cell pellet in a droplet of 2.5% glutaraldehyde in PBS buffer at pH 7.2 and xed the cells overnight at 4 °C.
The samples were then rinsed in PBS solution for 10 min three times and post xed in 1% osmium tetroxide for 60 min at room temperature.
Next, the authors exchanged alcohol with propylene oxide and in ltrated samples with increasing concentrations (25%, 50%, 75%, and 100%) of Quetol-812 epoxy resin mixed with propylene oxide.
The authors cut samples into sections (100 nm) using an ultramicrotome and poststained them with uranyl acetate for 10 min and lead citrate for 5 min at room temperature.
After that, the authors observed sections under a transmission electron microscope operated at 120 kV.

Nude mouse xenograft model

In total, 1×10 6 stably transfected GC cells or control cells were suspended in 100 µl of PBS and injected into each armpit of 4-week-old female BALB/c nude mice purchased from the Department of Laboratory Animal Center of Nanjing Medical University.
After 4 weeks, the mice were sacri ced, and the tumours were separated to measure their weights and volume.

In vivo metastasis assay

Stably transfected GC cells (1 × 10 6 ) were injected into the tail veins of 4-week-old female BALB/c nude mice.
After 28 days, the bioluminescent signals of lung metastasis were detected using an IVIS Imaging system (Caliper Life Sciences, Hopkinton, MA, USA).
After the mice were sacri ced, the lung metastatic lesions were assessed using haematoxylin and eosin (HE)-stained sections.
Luciferase reporter assay BGC-823 and SGC-7901 cells were seeded in 24-well plates and cotransfected with the corresponding plasmids and miRNA mimics.
The relative luciferase activity was normalized to Renilla luciferase activity.

Confocal microscopy

BGC-823 and SGC-7901 cells transfected with GFP-mRFP-LC3 lentivirus (GeneChem, China) were seeded into a 35-mm culture dish for confocal microscopy.
Red and yellow puncta representing autolysosomes and autophagosomes, respectively, were detected by confocal microscopy (Carl Zeiss, Germany).
Three random elds were selected for puncta quanti cation.

Chromatin immunoprecipitation assay

ChIP assays were performed using the Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (17-10085; Millipore Sigma, Burlington, Massachusetts, USA) according to the manufacturer's protocol.
Brie y, cells were fixed with 37% formaldehyde and collected in lysis buffer.
Chromatin fragments were sonicated on ice, and then the lysates were immunoprecipitated with normal rabbit IgG and FOXP2 antibodies (5337; Cell Signaling Technology).
Elution of the protein/DNA complexes was obtained after DNA purification using wash buffers and standard PCR.
DNA was extracted for PCR ampli cation of speci c DNA fragments.

Statistics

The data were presented as means ± SD in each experiment.
One-way analysis of variance and Student's t-test were applied.

CircST3GAL6 expression pro les in gastric cancer tissues and paired normal gastric tissues

To screen circRNAs involved in the progression of gastric cancer, circRNA-seq was used to analyse the expression of circRNAs in three pairs of gastric cancer tissues and adjacent normal tissues.
Next, the authors excluded circRNAs that were not included in circbase 22 and obtained 90 circRNAs.
The authors chose the top 20 circRNAs to generate a heat map according to the fold change (Figure 1A ).
To further verify the expression level of circST3GAL6 in gastric cancer tissues, the authors collected a cohort of 60 GC tissues and paired normal tissues, signi cantly downregulated expression of circST3GAL6 was detected via qRT-PCR (Figure 1C ).
Taken together, circST3GAL6 was signi cantly downregulated in GC tissues and GC cells, which deserved further study.

Identi cation of the structure and characteristics of circST3GAL6

CircST3GAL6 is located at chromosome 3 and connected to head-to-tail splicing by exons 2-5, as con rmed by Sanger sequencing (Figure 1E ).
The authors detected circST3GAL6 in cDNA with different primers and found that circST3GAL6 could not be ampli ed by divergent primers, con rming that circST3GAL6 was not attributable to genomic rearrangements or PCR artefacts (Figure 1H ).
Additionally, knockdown of circST3GAL6 obviously promoted cell proliferation and inhibited cell apoptosis compared with the negative control in BGC-823 and SGC-7901 cells (Figure 2B -D, Figure S1A-C ).
The authors observed reduced expression of vimentin, snail, slug and N-cadherin and upregulated E-cadherin expression in BGC-823 and SGC-7901 cells transfected with the circST3GAL6 overexpression plasmids.
Collectively, these results suggest that circST3GAL6 is a key factor that inhibits the proliferation, migration, and invasion of GC cells.

CircST3GAL6 inhibits tumour growth and metastasis in vivo

To investigate the potential role of circST3GAL6 in vivo, the authors subcutaneously injected 1×10 6 GC cells into nude mice transfected with circST3GAL6 overexpression or sh-circST3GAL6.
The lung tissues of mice were separated, and haematoxylin-eosin staining was performed to confirm the formation of lung metastases, the results of which were consistent with the luciferase signal (Figure 3E ).
In conclusion, these results indicate that circST3GAL6 inhibits tumorigenesis and metastasis in vivo.
Collectively, these results indicated that circST3GAL6 functions as a sponge of miR-300.
On the basis of the above results, the authors proved the potential regulation of FOXP2 by circST3GAL6 through sponging miR-300.

Knockdown of FOXP2 reverses the effect of miR-300 inhibitors on GC cells

Because the authors have proved that circST3GAL6 plays a tumour-suppressive role by sponging miR-300 and FOXP2 is the target gene of miR-300, they investigated how circST3GAL6 regulates FOXP2 via miR-300 and its effects on GC cells.
The transfection e ciency of FOXP2 siRNAs and overexpression plasmids was veri ed (Figure 7A ).
The promotion of proliferation and migration of GC cells caused by miR-300 knockdown could be restored via FOXP2 knockdown (Figure 7B -D, Figure S3B-D ).
The levels of cleaved caspase-3, bcl-2 and EMT-related proteins were further detected by western blotting (Figure 7G , Figure S3F ).
Combined with the above results, the authors proved that circST3GAL6 markedly regulates FOXP2 expression and regulate autophagy by sponging miR-300.

FOXP2 transcriptionally inhibits MET and regulates autophagy

The authors proved that circST3GAL6 promoted autophagy via the miR-300/FOXP2 axis, but the downstream mechanism of FOXP2 remains to be explored.
On the basis of above results, the authors tried to investigate whether FOXP2 regulated autophagy through Met.
Because most of the predicted sites were located between -1000 bp and +1 bp of the promoter sequence, two possible binding sites were con rmed according to the binding score (Figure 8A ).
These results showed that FOXP2 directly bound to binding site A on the promoter of Met.
These results were also con rmed by GFP/mRFP-LC3 dot analysis and TEM (Figure 8F , S4C).

Discussion

Gastric cancer (GC) ranks fth in incidence of all cancers worldwide, and its incidence is particularly high in East Asia, including China 1, 2 .
This is the rst report showing the circular RNA ST3GAL6 can inhibit the malignant progression of gastric cancer.
The authors tried to predict whether some RBPs could bind to circST3GAL6 via RBPmap (http://rbpmap.technion.ac.il/), and the results showed that KHDRBS1 might have a connection with circST3GAL6.
Because FOXP2 played a vital role in regulating gene transcription, the authors predicted the potential binding sites of the promoter of MET via JASPAR 19 .
Furthermore, the authors found that GC cell proliferation and migration were altered after applying the autophagy inhibitor 3-MA.

Conclusion

The authors con rmed that circST3GAL6 was signi cantly downregulated in GC cells and tissues.
CircST3GAL6 overexpression inhibits the malignant progression of gastric cancer through autophagy regulated by the FOXP2/MET/mTOR axis.
C. qRT-PCR showed that the expression of circST3GAL6 in GC tissues was signi cantly lower than that in adjacent normal tissues.
H. qRT-PCR products of linear and circular products ampli ed with convergent and divergent primers in cDNA and genomic DNA (gDNA) compared to GAPDH.
The proportion of circST3GAL6 in the nucleus and cytoplasm was con rmed by qRT-PCR.

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The novel role of circular RNA ST3GAL6 on blocking

gastric cancer malignant behaviors through

autophagy regulated by the FOXP2/MET/mTOR axis

Penghui Xu