Silencing circPVT1 enhances radiosensitivity in non-small cell lung cancer by sponging microRNA-1208

Abstract

BACKGROUND:

Radiotherapy is one of main useful therapies in non-small cell lung cancer (NSCLC). Nevertheless, the underlying mechanism between NSCLC cell radiosensitivity and effective treatment remains unclear.

OBJECTIVE:

The aim is to explore the relationship between circular (circ) RNA and NSCLC cell radiosensitivity.

METHODS:

CircRNA plasmacytoma variant translocation 1 (PVT1) and microRNA (miR)-1208 expression in NSCLC cells were assessed using quantitative reverse transcriptase PCR (qRT-PCR). NSCLC cells were transfected with si-PVT1 or miR-1208 inhibitor and then exposed to irradiation. Cellular biology behaviors were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL), colony formation, invasion and western blot. Additionally, binding between circPVT1 and miR-1208 was testified by dual-luciferase reporter and RIP assay.

RESULTS:

CircPVT1 was upregulated in NSCLC cells after irradiation treatment. Silencing circPVT1 induced inhibition of NSCLC cell growth and invasion, accompanied by cell apoptosis and $\gamma$ -H2AX expression. Moreover, NSCLC cell proliferation and invasion was further inhibited by irradiation treatment in circPVT1-silenced cells, indicating a strong radiosensitivity of NSCLC cells. CircPVT1 functions as a competing endogenous RNA of miR-1208. Silencing miR-1208 reversed NSCLC cell sensitivity response to irradiation and activated PI3K/AKT/mTOR pathway in circPVT1-silenced cells.

CONCLUSIONS:

Silencing circPVT1 enhanced radiosensitivity of NSCLC cells by sponging miR-1208.

Keywords

NSCLC circPVT1 miR-1208 radiosensitivity PI3K/AKT/mTOR signaling

1. Introduction

Cancer is one of the most serious diseases with high incidence and mortality rate [1]. Thus, deep understanding mechanisms are of great significance for the treatment of lung cancer. Currently, cancer treatment strategies include radiotherapy, surgery, chemotherapy and immunotherapy [2, 3, 4]. Among them, radiotherapy is one of main means, and about 50% of cancer patients are treated with radiotherapy at their doctor’s recommendation [5]. In addition, patients who respond well to radiotherapy generally show better clinical outcomes in different cancers. Lung cancer is the primary cause of cancer-associated death worldwide [6]. Lung cancer includes two main types, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), and NSCLC accounts for approximately 85% [7, 8]. Radiotherapy is the firstly considered treatment for inoperable NSCLC patients who have no change of surgery. Unfortunately, clinical resistance of radiotherapy has been identified as a big obstacle during process of NSCLC treatment [9]. So, the improvement of radiosensitivity is extremely significant for effective treatment of NSCLC. A mass of evidence has elucidated that noncoding RNAs (ncRNAs), play significantly appreciated roles during regulating radiation responses, including, microRNAs (miRNAs) and circular RNAs (circRNAs) [10, 11, 12].

CircRNAs are circular ncRNAs that have no 5’-hat and 3’-tail structure [13]. CircRNAs are highly cell-specific and tissue-specific in eukaryotes [14]. A review has detailedly illustrated the relationship between circRNAs and the progression, deterioration, migration as well as recurrence of cancer [15]. CircPVT1 derives from the second exon of PVT1 gene, whose locus is embedded in lncRNA PVT1 and is highly expressed in a variety of cancers [16]. LncRNA PVT1 has been proved to modulate radiosensitivity of NSCLC, and knockdown of lncRNA PVT1 could enhance radiosensitivity [17]. Moreover, circPVT1 has been found to be high-expressed in NSCLC, and circPVT1 dramatically accelerate cell proliferation and invasion [18]. However, it was not well known whether circPVT1 modulated NSCLC radiosensitivity and how it exerted its functions.

MicroRNAs (miRNAs) have the length of 21–23 nucleotides [19] and are another a class of ncRNAs with line structure [20]. MiRNAs also have regulatory effects on cancer cell survival, including NSCLC [21]. For instance, miR-340-5p exhibited a lower expression level in NSCLC tissues and cells, and overexpressing miR-340-5p could limit cell proliferation [22]. Recently, it has been observed that circRNAs can work as miRNA sponges in several types of cancer [23]. For example, in NSCLC cells, circPVT1 could sponge miR-125b to facilitate cell proliferation and invasion [18]. Thus, we hypothesized that circRNA might be involved in radiosensitivity through specific downstream miRNA.

In this study, we for the first time exploited the function of circPVT1 on radiosensitivity of NSCLC cells. In addition, the potential mechanisms of circPVT1 regulating radiosensitivity of NSCLC were preliminarily identified.

2. Materials and methods

2.1 Cell lines and cell culture

Five kinds of human NSCLC cell lines (H226, H1650, A549, H292 and H1299) and normal cell line (bronchial epithelial cell, BEA-2B) were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’smedium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 100 U/ml penicillin and 100 $\mu$ g/ml streptomycinin (both from Sigma, St. Louis, MO, USA). All cells existed in a humidified condition, 5% CO ${}_{2}$ and 37 ${}^{\circ}$ C. Cell culture medium was replaced, when cells were cultured for 3–4 days.

2.2 Transfection assay

SiRNAs specific targeting circPVT1 (si-PVT1), miR-1208 mimic (miR-1208), miR-1208 inhibitor and their own negative controls were purchased from GenePharma company (Shanghai, China). Cells were cultured until logarithmic phase, and then mixed cell suspension was cultivated in 6-well plates (4 $\times$ 10 ${}^{5}$ /well) and maintained up to 24 h. 24 h later, cells were subjected to transfection with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the experimental needs [24].

2.3 Ionizing radiation treatment

5 $\times$ 10 ${}^{4}$ transfected cells suffered from irradiation with a linear accelerator (Varian Medical Systems, Palo Alto, CA, USA) in 25-T flasks. Cells were affected by various dose of radiation treatment (0, 2, 4, 6, and 8 Gy) with a 6-MV photon beam at a dose rate of 3.5 Gy/min and 160 kv X-ray energy for 24 h [25].

2.4 3-(4,5-Dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) assay

MTT assay was performed to detect cell proliferation as previously reported [26]. When stably transfected cells grew to logarithmic phase, they were planted into a 96-well plate and cultured overnight. Whereafter, 20 $\mu$ l of MTT (0.5 mg/ml, Invitrogen) was added into every well. After 4 h cultivation, culture medium in 96-well plates was moved lightly. In the next step, dimethyl sulfoxide (DMSO, Invitrogen) was utilized to dissolve purple precipitation. In the end, we made records of the absorbance value at a 490 nm on a microplate reader (BioTek Instruments Inc., Winooski, VT, USA).

2.5 TUNEL assay

To assess cell apoptosis, TUNEL staining was carried out [27]. Briefly, transfected cells were fixed with 4% formaldehyde. Then, the rest steps were accomplished, in accordance with specification of TUNEL kit (Roche, Mannheim, Germany). When viewing with a fluorescence microscope (DMI4000B, Leica, Wetzlar, Germany), TUNEL-tagged cells were computed.

2.6 Colony formation assay

For analysis of ability of cell survival, colony formation assay was performed as previously reported [28]. After cell transfection and radiation treatment, cells were seeded into 6-well plates (200 cells/well) and cultivated in incubator with 5% CO2 at 37 ${}^{\circ}$ C. At 14 days, cells were fixed, and stained with methanol (Sigma) and crystal violet (Sigma), respectively. The colonies ( $>$ 50 cells) were photographed and counted under a microscope.

2.7 Invasion assay

To assess the ability of invasion of cell, invasion experiment [29] was conducted with a 24-well transwell chamber plate (BD Biosciences, St Louis, MO, USA). A 24-well transwell chamber plate was divided into two chambers, upper and lower chamber, with an 8 $\mu$ m membrane in the middle. Before invasion experiment, Matrigel (100 $\mu$ l) (1:7 dilutions, BD Biosciences) was pre-coated onto membrane. Cell suspension (100 $\mu$ l) (5 $\times$ 10 ${}^{4}$ cells and free-medium) was seeded and cultivated onto the upper chamber, and the lower was full of complete culture medium. 36 h later, cells were fixed and stained with 4% paraformaldehyde (PFA, Sigma) and 1% crystal violet (Sigma), respectively. After the staining, invaded cells were viewed under a microscope and 5 random fields were selected to count average invasive cells.

2.8 Western blot

For the analysis of expression of related proteins, western blot assay was performed [30]. Proteins were prepared with RIPA (Vazyme, Nanjing, China) were applied to perform the detection of concentration with BCA Kit (Vazyme), equal protein specimens (40 $\mu$ g) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Then, the membranes carrying proteins were sealed with skim milk at room temperature for 2 h, incubated with corresponding primary antibodies at 4 ${}^{\circ}$ C overnight and maintained under secondary antibodies (1/2000, Abcam, Cambridge, UK) for 1 h. SuperSignal West Pico Plus Chemiluminescent Subatrate (Amersham Bioscience, Buckinghamshire, UK) was applied to achieve the visualization of protein blot. Moreover, intensity of band was analyzed adopting Quantity One 4.6.9 (Bio-Rad, Hercules, CA, USA). Primary antibodies included $\gamma$ -H2AX (1:1000, ab26350, Abcam), E-cadherin (1:5000, Cat: GTX124178, GeneTex, Inc., Irvine, CA, USA), N-cadherin (1:100, Cat: GTX11340, GeneTex), Vimentin (1:5000, Cat: GTX100619, GeneTex), p-p85 PI3K (phospho Tyr467, 1:1000, Cat: 79-505, roSci, Inc., Poway, CA, USA), p85 PI3K (1:1000, Cat: 20R-2152, Fitzgerald Industries International, Inc., Acton, MA, USA), p-mTOR (phospho Ser2448, 1:2000, Cat: GTX132804, GeneTex), mTOR (1:800, Cat: GTX50541, GeneTex), p-AKT (phospho Ser473, 1:2000, Cat: GTX128414, GeneTex), AKT (1:500, Cat: GTX13990, GeneTex) and $\beta$ -actin (1:2000, Cat: GTX629630, GeneTex).

Figure 1.

CircPVT1 was highly expressed in NSCLC cells. (A) CircPVT1 levels in human lung cancer cell lines (H226, H1650, A549, H292 and H1299) and normal bronchial epithelial cell line (BEAS-2B) were determined utilizing qRT-PCR. (B) CircPVT1 expression in (0, 2, 4, 6, and 8 Gy) irradiated human lung cancer cell lines H226, H1650, A549, H292 and H1299 cells was tested using qRT-PCR. Compared with BEAS-2B, $p<$ 0.05, $p<$ 0.01 or $p<$ 0.001, mean $\pm$ SD.

2.9 Dual-luciferase reporter assay

The potential downstream factor of circPVT1 was found out through circular RNA Interactome, namely miR-1208. For verifying targeted association between circPVT1 and miR-1208, the dual-luciferase reporter assay was performed as previously reported [31]. The sequence of circPVT1 untranslated region (3’-UTR) covering the predicted combining sites of miR-1208 or mutated sequences of combining sites were ligated into pGL4 Luciferase Reporter Vector (Promega, Madison, WI, USA), formed circPITX1-WT or circPITX1-MUT, severally. HEK-293T cells were co-transfected with recombinant vectors and miR-1208 (miR-NC). 48 h later, luciferase activity was evaluated with a Dual Luciferase Reporter Assay Kit (Vazyme).

2.10 RNA immunoprecipitation (RIP) assay

For investigating the interaction between circPVT1 and miR-1208, RIP was enforced with specific AGO2 antibody [32]. In brief, after HEK-293T cells were subjected to transfection with miR-1208/miR-NC or untreation, cells were lysed with RIRP, and then lysates of cellswere mixed with RIP immunoprecipitation buffer containing magnetic beads conjugated with human anti-Argonaute2 (AGO2) antibody (Abcam, ab32381), or NC mouse IgG (Millipore). After complexes underwent with washing and elution, RNAs were extracted and analyzed with qRT-PCR assay.

2.11 Quantitative reverse transcription PCR (qRT-PCR)

All RNAs in cells were collected with Trizol regent (Invitrogen). 1 $\mu$ g RNA was transcribed reversely with SuperScript First StrandcDNA System (Invitrogen). PCR amplification and circPVT1 expression as well as miR-1208 expression were accomplished with a LightCycler Brilliant SYBR GreenRT-qPCR kit (Roche Applied Science, Indianapolis, IN, USA) and TaqMan MicroRNA Assay Kit (Applied Biosystems, Foster City, CA, USA) on 7900 HT sequence detection system (Applied Biosystems), severally. Relative expression of circPVT1 and miR-1208 was normalized to GAPDH or U6, severally, using 2 ${}^{-\Delta\Delta CT}$ calculation method [33].

Figure 2.

Silencing circPVT1 repressed cell proliferation in NSCLC cells. (A) CircPVT1 expression in untreated, si-NC-transfected, si-PVT1-transfected NSCLC cell lines (A549 and H1299) was analyzed applying qRT-PCR. (B, C) Viability, (D, E) colony number in un-treated, si-NC-transfected, si-PVT1-transfected, si-NC-transfected plus 8 Gy-irradiated and si-PVT1-transfected plus 8 Gy-irradiated NSCLC cell lines (A549 and H1299) were explored through MTT and colony formation, respectively. $p<$ 0.01 or $p<$ 0.001, mean $\pm$ SD.

2.12 Statistical analysis

In the study, data was analyzed with Graphpad prism 5 (GraphPad Software, San Diego, CA, USA). The significance of two groups was assessed through one-way ANOVA or Student’s $t$ -test. If p was less than 0.05, the difference was significant.

Figure 3.

Silencing circPVT1 accelerated apoptosis in NSCLC cells. (A, B) apoptotic rate and (C) $\gamma$ -H2AX expression in un-treated, si-NC-transfected, si-PVT1-transfected, si-NC-transfected plus 8 Gy-irradiated and si-PVT1-transfected plus 8 Gy-irradiated NSCLC cell lines (A549 and H1299) were explored through apoptosis and western blot assays, respectively. The comparison between the two marked groups, $p<$ 0.05. $p<$ 0.01 or $p<$ 0.001, mean $\pm$ SD.

3. Result

3.1 CircPVT1 was highly expressed in NSCLC cells

For exploring expression level of circPVT1 in NSCLC cells, we utilized five kind of NSCLC cell lines (H226, H1650, A549, H292 and H1299) and a kind of normal lung cell line (BEAS-2B). qRT-PCR experiment was adopted to exploit circPVT1 expression level. As displayed in Fig. 1A, circPVT1 expression was significantly higher in all NSCLC cell lines than that in BEAS-2B cells ( $p<$ 0.05, $p<$ 0.01 or $p<$ 0.001). In addition, cells were treated with diverse doses of radiation (0, 2, 4, 6, and 8 Gy) and then circPVT1 expression was analyzed using qRT-PCR. It was observed that the expression of circPVT1 was elevated in NSCLC cells in a dose-dependent manner (Fig. 1B). Moreover, circPVT1 expression levels were higher in both A549 and H1299 cells than the other three NSCLC cell lines. Thus, A549 and H1299 cells were selected in subsequent experiments to explore the regulatory function of circPVT1 in NSCLC. Taken together, our data confirmed that circPVT1 was highly expressed in NSCLC cell lines and its level was enhanced by irradiation treatment, indicating that circPVT1 might potentially serve as a novel regulator in NSCLC cell radiosensitivity.

Figure 4.

Silencing circPVT1 suppressed the invasion of NSCLC cells. (A, B) Relative invasive rate and (C, D) epithelial-mesenchymal transitions (EMT)-related protein (E-cadherin, N-cadherin and Vimentin) expression in un-treated, si-NC-transfected, si-PVT1-transfected, si-NC-transfected plus 8 Gy-irradiated and si-PVT1-transfected plus 8 Gy-irradiated NSCLC cell lines (A549 and H1299) were investigated adopting invasion and western blot assays. The comparison between the two marked groups, $p<$ 0.05, $p<$ 0.01 or $p<$ 0.001, mean $\pm$ SD.

Figure 5.

CircPVT1 targeted and oppositely regulated miR-1208. (A) MiR-1208 expression in human lung cancer cell lines (A549 and H1299) and normal bronchial epithelial cell line (BEAS-2B) was determined utilizing qRT-PCR. Compared with BEAS-2B, $p<$ 0.01, mean $\pm$ SD. (B) MiR-1208 expression in (0, 2, 4, 6, and 8 Gy) irradiated human lung cancer cell lines (H226, H1650, A549, H292 and H1299) was tested using qRT-PCR. (C) Relative luciferase activity in PVT1-WT, PVT1-WT plus miR-NC, PVT1-WT plus miR-1208, PVT1-MUT, PVT1-MUT plus miR-NC, PVT1-MUT plus miR-1208-transfected HEK-293T cells were determined employing dual-luciferase reporter assay. Compared with miR-NC-transfected group, $p<$ 0.001, mean $\pm$ SD. (D) CircPVT1 expression in untreated plus AGO2 antibody, miR-NC-transfected plus AGO2 antibody, miR-1208-transfected plus AGO2 antibody, untreated plus IgG antibody, miR-NC-transfected plus IgG antibody and miR-1208-transfected plus IgG antibody groups was examined using RIP and qRT-PCR assays. Compared with miR-NC-transfected group, $p<$ 0.001, mean $\pm$ SD. (E) MiR-1208 expression in untreated, si-NC-transfected, si-PVT1-transfected NSCLC cell lines (A549 and H1299) was detected applying qRT-PCR. Compared with si-NC-transfected group, $p<$ 0.001, mean $\pm$ SD.

Figure 6.

Silencing circPVT1 repressed cell proliferation in NSCLC cells by regulating miR-1208. (A) MiR-1208 expression in untreated, NC inhibitor-transfected and miR-1208 inhibitor-transfected NSCLC cell lines (A549 and H1299) was tested utilizing qRT-PCR. (B, C) Viability and (D, E) colony number, in untreated, si-NC plus NC inhibitor-co-transfected, si-NC plus NC inhibitor-co-transfected plus 8 Gy-irradiated, si-PVT1 plus NC inhibitor-co-transfected plus 8 Gy-irradiated and si-PVT1 plus miR-1208 inhibitor-co-transfected plus 8 Gy-irradiated NSCLC cell lines (A549 and H1299) were explored through MTT and colony formation assay, respectively. The comparison between the two marked groups, $p<$ 0.01 or $p<$ 0.001, mean $\pm$ SD.

3.2 Silencing circPVT1 improved radiosensitivity in NSCLC cells

Since circPVT1 upregulation correlates to irradiation treatment, we asked whether circPVT1 knockdown could enhance radiosensitivity in NSCLC cells. To this end, A549 and H1299 cells were transfected with si-PVT1 or si-NC, respectively (both $p<$ 0.001, Fig. 2A). After 48 h of transfection, NSCLC cells were irradiated with/without 8 Gy. As exhibited in Fig. 2B–C, we found that circPVT1 knockdown significantly decreased the cell viability in A549 and H1299 cells compared to the si-NC group ( $p<$ 0.001). In addition, further inhibition of cell viability was observed in 8 Gy irradiation treatment group following circPVT1 knockdown ( $p<$ 0.001). To further evaluate the circPVT1 on cell survival, cell colony was applied in circPVT1-silenced cells treated with or without irradiation treatment. The results showed the number of colony formation in si-PVT1+8 Gy group was lower than that in si-NC+8 Gy group ( $p<$ 0.01, Fig. 2D–2E). TUNEL assay demonstrated that silencing circPVT1 remarkably enhanced NSCLC cell apoptosis ( $p<$ 0.01, $p<$ 0.001, Fig. 3A–B). Furthermore, the rate of apoptosis was significantly increased in si-PVT1 group when compared with si-NC group with irradiation treatment ( $p<$ 0.001). DNA double-strand break (DSB) is a leading manifestation of apoptosis except apoptotic rate. The $\gamma$ -H2AX protein acted as the biomarker of DSB and was detected by western blot. As displayed in Fig. 3C, $\gamma$ -H2AX protein expression was enhanced by irradiation treatment in circPVT1-knocked down cells ( $p<$ 0.001, Fig. 3C). Together, these data indicated that silencing circPVT1 enhanced radiosensitivity, facilitated cell apoptosis, and inhibited NSCLC cell growth.

3.3 Silencing circPVT1 inhibited NSCLC cell invasion

Subsequently, the influence of circPVT1 on NSCLC cell invasion was also studied. Figure 4A–B illustrated that circPVT1 knockdown impaired the migration ability of A549 and H1299 cells and 8 Gy radiation treatment further inhibited NSCLC cell invasive ability ( $p<$ 0.01 or $p<$ 0.001). Epithelial-mesenchymal transitions (EMT) process is involved in cell invasion. EMT-related proteins were evaluated by using western blot. In Fig. 4C–D, si-PVT1 transfection facilitated E-cadherin protein expression but decreased N-cadherin and Vimentin protein expression ( $p<$ 0.001). Interestingly, 8 Gy radiation treatment promoted EMT process in si-PVT1-transfected cells ( $p<$ 0.01 or $p<$ 0.001). These results suggested that both circPVT1 knockdown and radiation treatment had tumor-suppressive effects that impeded NSCLC cell invasion.

3.4 CircPVT1 was a potential sponge of miR-1208

To further explore the molecular mechanism of circPVT1 in regulating NSCLC cell radiosensitivity, the analysis between miRNAs and circPVT1 was conducted. The annlysis from circular RNA Interactome indicated that miR-1208 was a potential downstream miRNA of circPVT1. The results of manifested that miR-1208 was markedly downregulated in A549 and H1299 cells when compared with BEAS-2B cells in which it was identified by qRT-PCR ( $p<$ 0.001, Fig. 5A). In addition, with the increasing dose of radiation, miR-1208 was lowly expressed (Fig. 5B). For confirming the targeted relationship between circPVT1 and miR-1208, we found that circPVT1 might bind with miR-1208 (Fig. 5C). To further verify the interaction of circPVT1 with miR-1208, we constructed luciferase reporters containing wild-type circPVT1 or circPVT1 with mutation of the miR-1208 targeting site. Luciferase constructs were co-transfected with miR-1208 mimic/miR-NC into HEK293T cells. We found that miR-1208 mimic reduced the reporter activity of the construct with wild-type circPVT1 ( $p<$ 0.001, Fig. 5C). We then performed RIP assay to pull-down circRNA associated with miR-1208 via AGO2 antibody, followed by qRT-PCR. The results showed that circPVT1 was associated with miR-1208 via the targeting site ( $p<$ 0.001, Fig. 5D), indicating that there was the potential of the interaction between circPVT1 and miR-1208. Furthermore, circPVT1 down-regulated miR-1208 expression in NSCLC cells, as si-PVT1 transfection positively modulated the expression of miR-1208 in A549 and H1299 cells (both $p<$ 0.001, Fig. 5E). Above results unclosed that circPVT1 targeted and oppositely regulated miR-1208.

3.5 Silencing circPVT1 improved radiosensitivity in NSCLC cells by up-regulating miR-1208

To prove the function of miR-1208 on radiosensitivity regulated by circPVT1 in NSCLC cells, miR-1208 inhibitor was transfected to conduct the following experiments. Successful transfection was the key to the subsequent experiments. We found that our transfection assay was successful, as miR-1208 inhibitor transfection lowered miR-1208 expression in A549 and H1299 cells (both $p<$ 0.001, Fig. 6A). In addition, miR-1208 inhibitor improved cell viability (both $p<$ 0.01, Fig. 6B and C), increased the number of colony (both $p<$ 0.01, Fig. 6D and E), reduced apoptotic rate ( $p<$ 0.01 or $p<$ 0.001, Fig. 7A and B) and restrained the protein expression of $\gamma$ -H2AX (both $p<$ 0.001, Fig. 7C) in 8 Gy+si-circPVT1 group. Above results showed that silencing circPVT1 improved radiosensitivity in NSCLC cells by up-regulating miR-1208.

Figure 7.

Silencing circPVT1 accelerated apoptosis in NSCLC cells by regulating miR-1208. (A, B) apoptotic rate and (C) $\gamma$ -H2AX expression in untreated, si-NC plus NC inhibitor-co-transfected, si-NC plus NC inhibitor-co-transfected plus 8 Gy-irradiated, si-PVT1 plus NC inhibitor-co-transfected plus 8 Gy-irradiated and si-PVT1 plus miR-1208 inhibitor-co-transfected plus 8 Gy-irradiated NSCLC cell lines (A549 and H1299) were explored through apoptosis and western blot assays, respectively. The comparison between the two marked groups, $p<$ 0.01 or $p<$ 0.001, mean $\pm$ SD.

3.6 Silencing circPVT1 inhibited NSCLC cell invasion by up-regulating miR-1208

Next, NSCLC cell invasion was examined by transfecting miR-1208 inhibitor. It was seen that miR-1208 inhibitor transfection fortified invasive rate (both $p<$ 0.05, Fig. 8A and B). MiR-1208 inhibitor transfection could down-regulate E-cadherin, and up-regulate N-cadherin and Vimentin protein expression ( $p<$ 0.05, $p<$ 0.01 or $p<$ 0.001, Fig. 8C and D). Above results released that silencing circPVT1 suppressed the invasion of NSCLC cells by modulating the up-regulation of miR-1208.

Figure 8.

Silencing circPVT1 suppressed the invasion of NSCLC cells by regulating miR-1208. (A, B) Relative invasive rate and (C, D) epithelial-mesenchymal transitions (EMT)-related protein expression (E-cadherin, N-cadherin and Vimentin) in untreated, si-NC plus NC inhibitor-co-transfected, si-NC plus NC inhibitor-co-transfected plus 8 Gy-irradiated, si-PVT1 plus NC inhibitor-co-transfected plus 8 Gy-irradiated and si-PVT1 plus miR-1208 inhibitor-co-transfected plus 8 Gy-irradiated NSCLC cell lines (A549 and H1299) were investigated adopting invasion and western blot assays. The comparison between the two marked groups, $p<$ 0.05, $p<$ 0.01 or $p<$ 0.001, mean $\pm$ SD.

Figure 9.

Silencing circPVT1 blocked the PI3K/AKT/mTOR signaling by regulating miR-1208. (A, B) The p-p85 PI3K, p-mTOR and p-AKT expression in untreated, si-NC plus NC inhibitor-co-transfected, si-PVT1 plus NC inhibitor-co-transfected, si-NC plus NC inhibitor-co-transfected plus 8 Gy-irradiated, si-PVT1 plus NC inhibitor-co-transfected plus 8 Gy-irradiated and si-PVT1 plus miR-1208 inhibitor-co-transfected plus 8 Gy-irradiated NSCLC cell lines (A549 and H1299) was detected adopting western blot assay. The comparison between the two marked groups, $p<$ 0.05, $p<$ 0.01 or $p<$ 0.001, mean $\pm$ SD.

3.7 Silencing circPVT1 blocked PI3K/AKT/mTOR pathway by up-regulating miR-1208

To further exploit the deep mechanisms of circPVT1 and miR-1208 on radiosensitivity of NSCLC cells, we focused on PI3K/AKT/mTOR signaling pathway. The results in Fig. 9A and B indicated that si-PVT1 transfection or 8 Gy radiation decreased the phosphorylation level of PI3K, mTOR and AKT (all $p<$ 0.001), and si-PVT1 transfection further decreased above phosphorylation levels induced by 8 Gy radiation ( $p<$ 0.05, $p<$ 0.01 or $p<$ 0.001). However, transfecting miR-1208 inhibitor attenuated the changes of PI3K, mTOR and AKT phosphorylation triggered by circPVT1 knockdown ( $p<$ 0.05, $p<$ 0.01 or $p<$ 0.001). Above results demonstrated that silencing circPVT1 blocked the PI3K/AKT/mTOR signaling pathway by up-regulating miR-1208.

4. Discussion

Radiotherapy is one of the main methods to treat lung cancer in clinical practice. However, with the resistance of lung cancer cells to radiotherapy, the fact emerged that the survival rate of patients decreased and recurrence rate increased [34, 35]. This provides the impetus for us to look for ways to reduce the resistance of lung cancer to radiotherapy. So, our aim was to explore the possible mechanism of radiotherapy in NSCLC, thereby effectively improving the sensitivity of lung cancer cells to radiotherapy. In our study, we firstly found that circPVT1 was involved in radiosensitivity to NSCLC. Additionally, miR-1208 was observed to be a downstream target of circPVT1. Silencing circPVT1 could impede invasion and strengthen sensitivity of NSCLC to radiotherapy through up-regulating miR-1208. Furthermore, silencing circPVT1 also blocked PI3K/AKT/mTOR pathway through up-regulating miR-1208. This study laid the foundation for better clinical application of radiotherapy.

The improvement of radiosensitivity could availably inhibit cancer cell proliferation and motivate apoptosis, thereby enlarging the outcomes of radiotherapy [36, 37, 38]. CircRNAs are abnormally expressed in cancer cells [39]. It has been reported that circRNAs are involved in radiosensitivity in a mount of cancers. For example, circ_0000285 expression was abnormally increased in nasopharyngeal carcinoma (NPC) tissues with radioresistance, thus circ_0000285 acted as a biomarker for NPC and took part in NPC radiosensitivity [40]. Similarly, circ_0001313 was discovered to be up-regulated in colon cancer cells with low radiosensitivity, and knockdown of circ_0001313 improved radiosensitivity of colon cancer cells [41]. In addition, circMTDH.4 could reduce radioresistance and chemoresistance in NSCLC cells [42]. However, in our study, we discovered that circPVT1 was significantly elevated and silencing circPVT1 could heighten the sensitivity of lung cancer to radiotherapy and suppress invasion in NSCLC. We found a new circRNA associated with radiosensitivity in NSCLC. Our experimental results were similar to those of previous studies. CircPVT1 has been reported to be high-expressed and act as a proliferative factor in numerous cancers, such as NSCLC [24], gastric cancer [43], colorectal cancer (CRC) [44] and esophageal carcinoma [45]. Furthermore, in gastric cancer (GC), circPVT1 played an oncogenic effect, and downregulation of circPVT1 weakened chemotherapeutic resistance of paclitaxel [46]. In lung adenocarcinoma, overexpression of circPVT1 participated in resistance to chemotheraopy through miR-145-5p/ABCC1 axis [47]. Thus, circPVT1 may become a potential target of radiosensitivity enhancement in NSCLC.

In recent years, miRNAs have been identified to be involved in the regulation of radiosentivity in an increasing growing number of cancers, including NSCLC. For instance, miR-200a in NSCLC was low-expressed, and miR-200a could restrain invasion and enhance the sensitivity of NSCLC to radiotherapy through inhibition of HGF/cMet signal pathway [48]. Another article has reported that miR-9 expression levels were evidently downregulated in NSCLC cells, and miR-9 improved the radiosentivity to weaken NSCLC cell proliferative ability [49]. Our result was consistent with previous studied results. In NSCLC cells, miR-1208 was firstly recognized to be down-regulated. Moreover, we learnt about from previous researches that miRNAs acted as the sponge of circRNA and circRNA developed its regulatory effects on radiosentivity through certain miRNA. For example, circ_0073237 knockdown inhibited cancer development and enhanced radiosensitivity through sponging and increasing miR-1183 in glioma [50]. As well as, circMTDH.4 sponged miR-630 to inhibit radiosensitivity of NSCLC [42]. Through using circular RNA Interactome, miR-1208 was found to be a potential downstream target of circPVT1. Silencing circPVT1 enforced radiosensitivity and inhibited invasion in NSCLC.

Cancer process is closely related to a number of signal pathways, including PI3K/AKT/mTOR pathway [51]. Inhibiting PI3K/AKT/mTOR signaling pathway could effectively prevent NSCLC cell proliferation and initiate apoptosis [52, 53]. Furthermore, this signaling also took part in the regulation of radiosensitivity. For instance, in NSCLC, activating PI3K/AKT/mTOR signaling pathway affected radiosensitivity [54]. In addition, a study has shown that targeting PI3K/AKT/mTOR pathway become a new and potential mean of changing radiosensitivity of oral squamous cell carcinoma (OSCC) [55]. In this study, our experimental results revealed that silence of circPVT1 could block PI3K/AKT/mTOR pathway through up-regulating miR-1208. Up to now, although there were no articles reported that circPVT1 and miR-1208 could regulate PI3K/AKT/mTOR pathway, some miRNA was defined to modulate radiosensitivity through the regulation of PI3K/AKT/mTOR signaling pathway. For example, miR-21 could activate PI3K/AKT/mTOR signaling pathway, thereby affecting the sensitivity to radiotherapy in NSCLC cells [54]. Our results hinted that circPVT1 blocked PI3K/AKT/mTOR signaling pathway through up-regulating miR-1208, thereby improving NSCLC cell radiosensitivity.

In conclusion, this study demonstrated that inhibition of circPVT1 could impede NSCLC cell invasion and improve radiosensitivity through up-regulation of miR-1208, and this process was probably regulated by blocking PI3K/AKT/mTOR signaling pathway. These finding provided further insights into the mechanism of radiosensitivity of NSCLC.

Funding

This research was supported by Mechanism of Changes in Immune Status of High-incidence Lung Cancer in the Coal-burning Area of Eastern Yunnan on Comprehensive Treatment, Yunnan Provincial Department of Science and Technology-Kunming Medical University Joint Special General Project (No.2019FE001 (-106)), SPP1 Regulates Female Lung Cancer Cell Stem Resistance and its Mechanism of Drug Resistance in Xuanwei Area (No.2019J1313), Yunnan Provincial Department of Science and Technol-ogy-Kunming Medical University Applied Basic Research Joint Special Fund General Project and Project supported by the Ministry of Science and Technology: A Multi-Center Open Clinical Study of Stereotactic Body Radiotherapy Combined with Immune Checkpoint Inhibitors in the Treatment of Early Non-Small Cell Lung Cancer (No. 2018YFC1313202).

Author contributions

Conception: Meifang Huang, Tianqian Li and Bo Hou.

Interpretation or analysis of data: Qing Wang, Chongxin Li, Huahua Zhou and Bo Hou.

Preparation of the manuscript: Meifang Huang, Shengyi Deng and Zengbo Lv.

Revision for important intellectual content: Yongmei He, Guangying Zhu.

Supervision: Meifang Huang, Bo Hou and Guangying Zhu.

Footnotes

Acknowledgments

This research was supported by Mechanism of Changes in Immune Status of High-incidence Lung Cancer in the Coal-burning Area of Eastern Yunnan on Comprehensive Treatment, Yunnan Provincial Department of Science and Technology-Kunming Medical University Joint Special General Project (No.2019FE001 (-106)), SPP1 Regulates Female Lung Cancer Cell Stem Resistance and its Mechanism of Drug Resistance in Xuanwei Area (No. 2019J1313), Yunnan Provincial Department of Science and Technol-ogy-Kunming Medical University Applied Basic Research Joint Special Fund General Project and Project supported by the Ministry of Science and Technology: A Multi-Center Open Clinical Study of Stereotactic Body Radiotherapy Combined with Immune Checkpoint Inhibitors in the Treatment of Early Non-Small Cell Lung Cancer (No. 2018YFC1313202).

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