Knockdown of TRIM65 inhibits autophagy and cisplatin resistance in A549/DDP cells by regulating miR-138-5p/ATG7

Cisplatin resistance is the main cause of treatment failure in patients with non-small-cell lung cancer (NSCLC). Autophagy is a key mechanism of resistance to chemotherapy. Given that tripartite motif (TRIM)-containing proteins are involved in the regulation of autophagy and chemoresistance, we aimed to study the functions of TRIM protein members in autophagy-mediated chemoresistance of NSCLC. We found that TRIM65 was significantly increased in cisplatin-resistant NSCLC cell line (A549/DDP) as compared to the parental cell line (A549). Knockdown of TRIM65 can enhance cisplatin-induced apoptosis and inhibit autophagy in A549/DDP cells, as indicated by Annexin V/PI staining, caspase3 activity test, and LC3-II immunofluorescence staining. Additionally, knockdown of TRIM65 significantly decreased the expression of an important autophagy mediator, ATG7, which was a potential target of miR-138-5p. miR-138-5p inhibitor significantly abolished the effects of TRIM65 knockdown on autophagy and cisplatin-induced apoptosis. Moreover, TRIM65 induced the ubiquitination and degradation of TNRC6A, resulting in the suppressed expression of miR-138-5p. TRIM65 knockdown inhibited the growth of tumors derived from A549/DDP cells. Furthermore, cisplatin-resistant NSCLC tissues displayed higher expression of TRIM65 mRNA and lower expression of miR-138-5p as compared to cisplatin non-resistant ones. miR-138-5p expression was negatively correlated with TRIM65 mRNA in NSCLC tissues. Collectively, the present study indicates that TRIM65 knockdown attenuates autophagy and cisplatin resistance in A549/DDP cells via regulating miR-138-5p.


Introduction
Lung cancer ranks the first among all cancer-related deaths worldwide, and more than 1 million people die from lung cancer annually 1 . Approximately 80% of lung cancer cases are non-small-cell lung cancer (NSCLC) 2 . Surgery is the main approach to treat early-stage NSCLC. However, most of the patients have little opportunity to receive surgery because they have locally advanced disease or distant metastasis at diagnosis 3 . For these patients, the platinum-based drug, cisplatin, is currently the standard drug treatment. Unfortunately, acquired resistance causes treatment failure and the extremely poor prognosis of these individuals 4,5 . Therefore, a deeper understanding of the chemoresistance mechanisms may help to develop strategies for overcoming drug resistance and improving clinical outcomes 6 .
Autophagy, a evolutionally conserved process, is genetically regulated by a family of autophagy-related genes (ATGs), and allows the orderly degradation and recycling of intracellular organelles and cytoplasmic proteins 7,8 . Recently, autophagy has been identified as an important mechanism of resistance to chemotherapy 9,10 , and manupulation of autophagy has emerged as a promising strategy to overcome chemoresistance in cancer therapy 11 . For instance, hydroxychloroquine (CQ), an autophagy inhibitor, has been used to enhance the sensitivity to chemotherapy in NSCLC patients 12 . Micro-RNAs (miRNAs), a class of small endogenous non-coding RNAs, can regulate gene transcription by binding to the 3′-untranslated region (3′-UTR) of target mRNAs 13 . Increasing evidence has indicated that miRNAs may play a role in chemoresistance in some cancer cells. For example, upregulation of miR-21-3p increases resistance to cisplatin by targeting NAV3 in ovarian cancer cells 14 .
Overexpression of miR-15b and miR-16 reduces the resistance to vincristine by targeting Bcl-2 in gastric cancer cells 15 . miR-326 prevents multidrug resistance in breast cancer cells by targeting multidrug resistanceassociated protein (MRP-1) 16 . By a miRNA array, Wang et al. has identified 14 significantly differentiated expressed miRNAs in cisplatin-resistant NSCLC cell line (A549/ DDP) as compared to the parental cell line (A549) 17 . More interesting, several miRNAs have been found to enhance chemosensitivity and apoptosis of cancer cells by inhibiting autophagy, e.g. miR-21 18 , miR-26, 19 and miR-101 20 .
Tripartite motif (TRIM)-containing proteins have more than 80 members in humans, most of which could be defined as E3 ubiquitin ligase 21 . TRIM proteins are involved in the regulation of development 21 , immunity 22 , carcionogenesis 23 , autophagy 24 and chemoresistance [25][26][27][28][29][30] . It has been rarely explored whether TRIM proteins affect the chemoresistance and autophagy in NSCLC cells. In the current study, we found that TRIM65 was significantly increased in A549/DDP cells as compared to A549 cells. Knockdown of TRIM65 can inhibit autophagy and enhance cisplatin-induced apoptosis in A549/DDP cells. As TRIM65 is reported as a negative regulator of miRNA activity by forming stable complexes with trinucleotide repeat containing six (TNRC6) proteins 31 , we further explored the possible downstream miRNAs, which could target the autophagy mediator, ATG7. Our study may provide new insight into the role of TRIM65 in the autophagy-mediated chemoresistance of NSCLC.

TRIM65 expression was upregulated in A549/DDP cells and involved in cisplatin-induced apoptosis
To investigate the relationship between several TRIM family proteins and the cisplatin resistance in NSCLC, the mRNA expression of several TRIM family proteins was determined in A549 and A549/DDP cells. All the detected TRIM family proteins were more highly expressed in A549/DDP cells than in A549 cells (Fig. 1a), and TRIM65 showed the most significant rise at the transcriptional level (P < 0.0001). Western blotting results showed that TRIM65 protein level was also upregulated in A549/DDP cells as compared to that in A549 cells (Fig. 1b).
To explore the biological functions of TRIM65 in cisplatin-resistant human lung cancer cells, TRIM65 expression was knocked down in A549/DDP cells by lentivirus-mediated RNA interference. As shown in Fig.  1c, TRIM65 shRNA (shTRIM65-1, shTRIM65-2, and shTRIM65-3) transduction obviously decreased TRIM65 expression as compared to control shRNA (shNC). shTRIM65-1 and shTRIM65-2 were chosen for the following experiments. A549/DDP cells transduced with shTRIM65-1, shTRIM65-2 or shNC were exposed to DDP, and cell apoptosis was assessed by Annexin V/PI staining (Fig. 1d, e) and caspase3 activity test (Fig. 1f). Cisplatin at 10 and 20 μM significantly induced early apoptosis, and combined treatment with TRIM65 shRNA and cisplatin had a greater effect than cisplatin. It is noticeable that TRIM65 knockdown significantly increased the cell apoptotic rate in A549/DDP cells without cisplatin treatment.
The mRNA expression of autophagy-related proteins was then assessed. TRIM65 knockdown significantly suppressed the mRNA levels of ATG5, ATG7, and Beclin1, of which ATG7 changed the most (P < 0.001, Fig. 2c).

Knockdown of TRIM65 inhibited cisplatin resistance via miR-138-5p
To explore whether miR-138-5p mediated the function of TRIM65 on cisplatin resistance, cell apoptosis was determined in A549/DDP cells treated with shTRIM65-1 and miR-138-5p inhibitor in the presence of 10 μM cisplatin. As illustrated in Fig. 3, shTRIM65-1 caused a significant increased apoptosis in A549/DDP cells exposed to cisplatin, while miR-138-5p inhibitor significantly abolished the effects of TRIM65 knockdown. These data indicate that miR-138-5p is involved in TRIM65-mediated cisplatin resistance.

TRIM65 induced the ubiquitination and degradation of TNRC6A in A549 cells
TRIM65 is known to negatively regulate miRNAs by ubiquitinating TNRC6A 31 . Here, we found that TRIM65 overexpression had no effects on the mRNA expression of TNRC6A (Fig. 4a), but obviously reduced the protein expression of TNRC6A (Fig. 4b). Additional treatment of MG132, a proteasome inhibitor, abolished the effects of TRIM65 overexpression on TNRC6A protein, which indicated that TRIM65 downregulation of TNRC6A in A549 cells was mediated by a proteasome-related pathway. Immunoprecipitation experiments showed that TRIM65 bound with TNRC6A in A549 cells (Fig. 4c), and that TRIM65 overexpression induced the ubiquitination of TNRC6A (Fig. 4d). These data suggest that TRIM65 downregulates TNRC6A through a post-translational modification in A549 cells.

Knockdown of TRIM65 inhibited cisplatin-resistant tumor growth in vivo
To confirm the function of TRIM65 on cisplatin resistance in vivo, we established A549/DDP cell lines stably expressing TRIM65 shRNA (shTRIM65-1) or control shRNA (shNC), and these cells were injected subcutaneously to construct a mouse xenograft model. One week after subcutaneous implantation (Day 7), the mice were treated with cisplatin for 3 weeks. Slower tumor growth rates were detected in the shTRIM65-1 group as compared to the shNC group at the interval between Day 16 and Day 28 (Fig. 5a). On Day 28, the xenografts were recovered. The tumor size (Fig. 5b) and tumor weight (Fig. 5c) were also notably reduced, but cell apoptosis was remarkably increased in the shTRIM65-1 group in comparison to the shNC group. The shTRIM65-1 group displayed downregulated levels of TRIM65, ATG7, and LC3-II/LC3-I (Fig. 5e), and upregulated levels of TNRC6A, cleaved caspase3, (Fig. 5e) and miR-138-5p (Fig. 5f). These results indicate that TRIM65 knockdown inhibited the growth of tumors derived from A549/DDP cells. Further, we analyzed TRIM65 and miR-138-5p expression in the cisplatin non-resistant and resistant NSCLC tissues. Cisplatin-resistant NSCLC tissues displayed higher expression of TRIM65 mRNA (Fig. 6a) and lower expression of miR-138-5p (Fig. 6b) as compared to that in cisplatin non-resistant ones. A negative correlation was observed between miR-138-5p expression and TRIM65 mRNA in NSCLC tissues (Fig. 6c). These data clinically suggest the involvement of TRIM65 and miR-138-5p in cisplatin resistance. transduced with shTRIM65-1 and shNC, respectively (n = 6 per group). After 1 week (Day 7), the mice were intraperitoneally administered with cisplatin (5 mg/kg) every week for 3 weeks. a Tumor volume was estimated every 3 days. b-f On Day 28, the xenografts were resected (b) and weighed (c). TUNEL (d), western blotting (e) and real-time PCR (f) analyses were done to assess cell apoptosis rate, protein expression and miR-138-5p level, respectively. **P < 0.01, ****P < 0.0001 Fig. 6 Expression of TRIM65 and miR-138-5p in the cisplatin non-resistant (NR) and resistant (R) NSCLC tissues. Real-time PCR analysis of TRIM65 (a) and miR-138-5p (b) was performed in NR and R tissues (n = 30), and then correlation analysis between TRIM65 and miR-138-5p expression was carried out. ****P < 0.0001

Discussion
Evidence has shown that TRIM proteins promote chemoresistance of various human cancers, such as TRIM8 in colorectal cancer 26 , TRIM32 in breast cancer 28 , TRIM14 in gliomas 29 , and TRIM31 in pancreatic cancer 30 . TRIM65 is overexpressed in lung cancer tissues and may act as an oncogene in lung cancer by promoting lung cancer proliferation, migration, and invasion 34 . It is unclear whether TRIM65 is involved in chemotherapy resistance. Cisplatin resistance is the main cause of the treatment failure in patients with NSCLC. In the current study, we found the upregulation of TRIM65 in cisplatinresistant NSCLC tissues as compared to that in nonresistant ones. TRIM65 knockdown suppressed NSCLC chemoresistance in vitro as well as in vivo after cisplatin treatment, suggesting targeting TRIM65 may be a promising strategy to enhance chemotherapy response for NSCLC.
Autophagy is a highly regulated process that can disassemble unnecessary or dysfunctional components. In cancer, autophagy plays crucial roles in adaptive responses to stress, which promotes cell survival 35 . Autophagy is identified as a mechanism of resistance to chemotherapy 9,10 . Consistently with the previous findings 36 , a higher autophagy activity was observed in A549/DDP cells than in A549 cells (Fig S1). Autophagy-specific markers LC3-II and ATG7 were reduced in A549/DDP cells with TRIM65 knockdown, which indicated the role of TRIM65 in autophagy. Apoptosis, the best-described form of programmed cell death, is commonly activated during chemotherapy. Here, tumors derived from cells expressing TRIM65 shRNA had decreased growth rate, reduced levels of ATG7 and LC3-II/I, and increased cleaved cas-pase3 and apoptotic cell ratio after cisplatin treatment. TRIM65 may function in chemoresistance by connecting the autophagy and apoptosis process, although the precise mechanism is to be investigated.
TRIM65 is identified as an E3 ligase for TNRC6A, which is a component of RNA-induced silencing complex (RISC) and participates in miRNA-induced gene silencing 31 . In line with the previous findings, our immunoprecipitation experiments performed in A549 cells demonstrated that TRRIM65 interacted with TNRC6A and promoted the ubiquitination of the latter protein. miRNAs are important mediators for drug resistance and autophagy. Thus, we explored putative miRNAs targeting ATG7 and involving in cisplatin-resistance. By searching previous literatures 17,33 and web-accessible miRNA target databases, we found that miR-138-5p was the most likely miRNA. Further experiments showed that miR-138-5p directly bound to the 3′UTR of ATG7, which reduced ATG7 mRNA expression. The participation of miR-138-5p in chemosensitivity has been studied in NSCLC cells 37 and leukemia cells 38 . Here, cisplatin-resistant NSCLC cell lines and tissues had lower expression of miR-138-5p than cisplatin non-resistant controls, suggesting the involvement of miR-138-5p in cisplatin resistance. In a previous study on pancreatic cancer, miR-138-5p is shown to suppress serum starvation-induced autophagy by targeting SIRT1, but not by ATG7 39 . The inconsistency between the above study and ours may be ascribed to the different cell types. Moreover, miR-138-5p expression was negatively correlated with TRIM65 mRNA in NSCLC tissues. TRIM65 knockdown significantly increased miR-138-5p, and miR-138-5p inhibitor significantly abolished the effects of TRIM65 knockdown on autophagy and cisplatin-induced apoptosis. Collectively, these data suggest that miR-138-5p mediates the function of TRIM65 on autophagy and cisplatin resistance.
In summary, by studying cell lines, mouse models, and clinical samples, we have identified the novel function of TRIM65 on autophagy and cisplatin resistance in NSCLC through a miRNA-mediated pathway. TRIM65 may be used as a potential therapeutic target for the treatment of cisplatin-resistant NSCLC.

Real-time PCR analysis
Total RNA was isolated using TRIzol Reagent (Invitrogen) as per the manufacturer's instructions. To analyze mRNA levels of target genes, total RNA was reverse transcribed into cDNA with random primers using MMLV reverse transcriptase (Promega, Madison, WI, USA) according to the manufacturer's instructions. To quantify miR-138-5p level, total RNA was reverse transcribed with the specific reverse transcription (RT) primer. Real-time PCR was carried out in triplicate on an ABI 7500 instrument (Applied Biosystems, Foster City, CA, USA). GAPDH and U6 small nuclear RNA (snRNA) were amplified as an internal control for mRNA and miRNA expression, respectively. All the primers are listed in Table 1.

Cell apoptosis analysis
The percentages of cells undergoing apoptosis were measured by Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Beyotime). A549/DDP cells were plated in six-well plates (3.0 × 10 5 per well) and treated as indicated. Subsequently, the cells were collected, washed once with PBS, incubated with Annexin V and propidium iodide (PI) following the manufacturer's protocol, and analyzed by a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Caspase3 activity test
The activity of Caspase3 was measured by a colorimetric assay kit (KeyGEN Biotech, Nanjing, China) as per the manufacturer's protocol.

Luciferase assays
The wild type (WT, containing putative miR-138-5p binding sites) or mutate (mutant, putative binding sites were mutated) 3′UTR of ATG7 were constructed into the pGL3-Enhancer Vector (Promega). The constructs were co-transfected with pRL-TK (Promega), and miR-138-5p mimics or mimics control (Con). The pRL-TK plasmid was used as a transfection control. Luciferase activity was measured using a luciferase assay kit (Promega, Madison, WI, USA) at 48 h after transfection.

Immunoprecipitation (IP)
Cell lysate was prepared with RIPA buffer as mentioned above, incubated with antibody against TRIM65 (Abcam), TNRC6A (Abcam) or IgG (Santa Cruz Biotech., Santa Cruz, CA, USA) for 2 h, and then with protein A/G Plus agarose beads (Santa Cruz Biothech.) for 1 h. Immunoprecipitates were washed three times with RIPA buffer, fractionated by SDS-PAGE, and detected by western blotting with primary antibodies against TRIM65, TNRC6A, and ubiquitin (Abcam).

Xenograft experiments
To evaluate the in vivo effects of TRIM65, BALB/c athymic nude mice (4-5 week old) were randomly divided into two groups (n = 6) and injected with 5 × 10 6 A549/ DDP cells stably transduced with shTRIM65-1 and shNC, respectively. After 1 week (Day 7), the mice were intraperitoneally administered with cisplatin (5 mg/kg, Chinese medicine reagent, Beijing, China) every week for 3 weeks. Tumor volume was estimated every 3 days. On Day 28, mice were euthanized, and the xenografts were collected and weighed. The xenografts were subjected to real-time PCR, western blotting, and TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling) analyses (Roche, Indianapolis, IN, USA). All animal experiments were approved by the Committee on Animal Care and Use of Shanghai Chest Hospital.

Human lung cancer specimen collection
A total of 60 NSCLC subjects (30 cisplatin non-resistant and 30 cisplatin resistant) were collected at the Department of Thoracic Surgery, Shanghai Chest Hospital with written consent from the patients. The study was approved by the Institute Research Ethics Committee of Shanghai Chest Hospital.

Statistical analysis
Each experiment was repeated independently at least three times. Graphpad Prism Software (Graphpad Prism, San Diego, CA, USA) was used for the statistical analyses. Two-tailed Student's t-test was performed for statistical significance analysis between two groups, and one-way analysis of variance (ANOVA) was used for more than two groups. Pearson correlation analysis was done to identify the correlation between TRIM65 mRNA and miR-138-5p in NSCLC tissues. P < 0.05 was considered statistically significant.