Role and mechanism of miR-130a-3p in the chemosensitivity of retinoblastoma cells to vincristine


 Purpose

Chemoresistance remains the primary obstacle threatening the prognosis of retinoblastoma (RB). microRNAs (miRNAs) are acknowledged as critical regulator of drug resistance. This study explored the molecular mechanism of miR-130a-3p affecting the chemosensitivity of RB to vincristine (VCR).
Methods

miR-130a-3p expression of VCR-sensitive and VCR-resistant RB tissues was detected using RT-qPCR. VCR-resistant RB cell line Y79/VCR was induced. miR-130a-3p expression of Y79/VCR cell line and its corresponding parental cell line was detected. Y79/VCR cells were subjected to miR-130a-3p overexpression treatment. The cell proliferation was measured using MTT assay, and the IC50 value and drug resistance index were examined using CCK-8 assay. The targeting relationship between miR-130a-3p and PAX6 was predicted through bioinformatics analysis and verified using dual-luciferase assay. Functional rescue experiments were conducted to confirm the role of PAX6 in chemosensitivity of RB cells. The effect of miR-130a-3p on tumorigenesis and VCR sensitivity was observed in vivo.
Results

miR-130a-3p was downregulated in VCR-resistant RB tissues and cells. Overexpression of miR-130a-3p repressed the proliferation of VCR-resistant RB cells and enhanced chemosensitivity. miR-130a-3p targeted PAX6 expression. Overexpression of PAX6 reversed the effect of miR-130a-3p on chemosensitivity of RB. Overexpression of miR-130a-3p suppressed tumor growth and reduced VCR resistance in vivo.
Conclusion

miR-130a-3p enhanced the chemosensitivity of RB cells to VCR by targeting PAX6 expression.


Introduction
Retinoblastoma (RB) represents a primary intraocular malignancy commonly in children under 5 years old [1]. Most RB in one or both eyes is resulted from biallelic mutation of the RB suppressor gene (RB1) in immature retinal cells [2]. In clinical, RB is often manifested as leukocoria, and the patients may also present strabismus, misalignment of the eyes, and vision decline [3]. The available treatments include chemotherapy, radiotherapy, enucleation, as well as focal treatments including cryotherapy, transpupillary thermotherapy, and laser photocoagulation [4]. Chemotherapy is currently recognized as the rst-line treatment for RB in children and the chemotherapeutic agents can be delivered through intravenous, intra-arterial, periocular, and intravitreal injection [5]. At present, vincristine (VCR) constitutes one of the standard chemotherapeutic agents applied for RB [6]. However, the clinical application of chemotherapeutic agents is severely limited due to inherent or acquired resistance, eventually leading to unfavorable outcome and poor survival [7]. Thus, elucidating the mechanisms of chemoresistance and improving the therapy response have signi cant implications for RB patients. microRNAs (miRNAs) are a class of small endogenous non-coding RNAs (18-25 nt in length), participating in the modulation of gene expressions at the post-transcriptional level and negatively modulating the expression of tumor-associated molecules [8]. Dysregulated miRNAs are implicated in tumorigenesis, progression, and prognosis, and also miRNAs are prospective targets contributing to improving the diagnosis and prognosis prediction and enhancing the chemotherapy response [9]. It is well established that aberrant miRNA expression serves as the intervention target for the resistance of RB cells to chemotherapeutic agents [10][11][12]. miR-130a-3p is identi ed to be a tumor suppressor in various malignancies, such as breast cancer [13], lung cancer [14], and gastric cancer [15]. However, the role of miR-130a-3p in RB has not been studied before. Importantly, emerging evidence has revealed that miR-130a-3p plays a vital role in tumor chemoresistance [16][17][18][19]. For example, Hu [20]. Accordingly, we speculated that miR-130a-3p can regulate the sensitivity of RB to VCR through post-transcriptional regulation of target genes. This study herein sought to investigate the molecular mechanism of miR-130a-3p affecting the chemosensitivity of RB to VCR, which shall confer novel insights for the improvement of chemotherapeutic effect in RB patients.

Clinical sample
Tissue samples were collected from 34 RB patients for extirpation operation in Tianjin Medical University Eye Hospital from June 2019 to June 2020, aged 10-37 months (2.28 ± 0.58 years). All RB patients received VCR chemotherapy before operation, but did not receive radiotherapy. According to the recurrence time, they were allocated into two groups: 21 patients in the VCR-sensitive group (within 6 months) and 13 patients in the VCR-resistant group (over 6 months). The normal retinal tissues were collected from 13 patients with eyeball rupture as the control. The tissue samples were washed with phosphate-buffered saline (PBS), frozen overnight in liquid nitrogen, and then stored at -80℃.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted from tissues and cells using TRIzol (Invitrogen), and the concentration and purity of RNA were detected using ultraviolet spectrophotometer (1011U, NanoDrop Technologies Inc., Wilmington, DE, USA). The cDNA was generated according to the instructions of TaqMan MicroRNA Assays Reverse Transcription primer (4427975, Applied Biosystems, Inc., Carlsbad, CA, USA). The primers of miR-130a-3p and PAX6 were designed and synthesized by Takara (Dalian, China) ( Table 1). RT-qPCR was performed on ABI7500 qPCR instrument (ABI, Foster City, CA, USA). The reaction conditions were predenaturation at 95℃ for 10 min, denaturation at 95℃ for 10 s, annealing at 60℃ for 20 s, and extension at 72℃ for 34 s, a total of 40 cycles. The relative expression of genes was examined by 2-ΔΔCt method, with GAPDH or U6 as the internal reference. The experiment was repeated 3 times. Table 1 Primer sequence for RT-qPCR Name of primer Sequences

Western blot
The tissues were placed in centrifuge tubes and added with 500 µL radio-immunoprecipitation assay cell lysate (R0020, Solarbio, Beijing, China) (containing 1 mmol/L phenylmethylsulfonyl uoride). The tissues were homogenized at 1000 g until fully lysis. The homogenate was placed on ice for 30 min and then centrifuged at 4℃ and 12000 g for 4 min, and the supernatant was collected and stored at -80℃. The protein concentration was determined using bicinchoninic acid kit (AR0146, Boster, Wuhan, Hubei, China), and the concentration of each sample was adjusted to 3 µg/µL. The extracted protein was added with the loading buffer and boiled at 95℃ for 10 min. Equal amount of protein on each lane (30 µg) was separated on 10% SDS-polyacrylamide gel and transferred onto polyvinylidene uoride membranes (P2438, Sigma-Aldrich). The membranes were blocked with 5% bovine serum albumin (10-L16, Biopartner Science & Technology, Beijing, China) for 1 h and cultured with the primary antibody rabbit anti-PAX6 (ab195045, 1:1000, Abcam Inc., Cambridge, MA, USA) at 4℃ overnight. Following tris-buffered salinetween (TBST) buffer washing (3 times × 5 min), the membranes were cultured with the secondary antibody (ab6721, 1:2000, Abcam) for 1 h. Following TBST washing (3 times × 5 min), the bands were developed with enhanced chemiluminescence reagent. The gray level of each band was analyzed using Image J 1.48 (Media Cybernetics, Bethesda, MA, USA), with GADPH (ab181602, 1:10000, Abcam) as internal reference.

Dual-luciferase assay
The target site sequence (WT) of PAX6 mRNA 3'-UTR region and the sequence after the mutation of WT target site (MUT) were synthesized. The pmiR-RB-REPORT™ plasmid was subjected to restriction enzyme digestion, and then the synthetic target gene fragments WT and MUT were inserted into pmiR-RB-REPORT™, respectively. The constructed WT and MUT vectors were transfected with mimic-NC or miR-130a-3p mimic into 293T cells (CRL-3216, American Type Culture Collection, Manassas, Virginia, USA).
The cells were collected and lysed 48 h after transfection. The relative activity of luciferase was detected using dual-luciferase detection kit (RG005, Beyotime, Shanghai, China). The experiment was repeated 3 times.

Xenograft tumor in nude mice
Thirty-two BALB/c nude mice (aged 4-6 weeks and weighing 16-20g) were raised in laminar ow cabinet (speci c pathogen grade) and exposed to ultraviolet light at regular intervals. Cages, rubbish, drinking water, and feed were sterilized. The room temperature was maintained at 24℃-26℃, and the relative humidity was maintained at 40%-60%. Y79 or Y79/VCR cells were mixed with PBS respectively to prepare single cell suspension (1 × 10 6 cells/mL), and then 50 µL cell suspension was subcutaneously injected into the right side of each nude mouse. After 1 week, the mice were randomly allocated into four groups (N = 8): Y79 group, Y79/VCR group, Y79/VCR + agomiR-NC group, and Y79/VCR + agomiR-130a-3p group.
After that, agomiR-NC and miR-130a-3p agomiR were injected into the tumor site respectively. All the mice received intraperitoneal injection of VCR (0.5 mg/kg) for 5 weeks, once a week. The tumor growth was measured with vernier caliper every week. The tumor volume was calculated as follows: V = (L × W 2 )/2. After 5 weeks, the mice were euthanized with 100 mg/kg pentobarbital sodium (P3761, Sigma-Aldrich).
The xenograft tumors were resected and weighed.

Immunohistochemistry
The weighed tumor tissues were prepared into para n sections, dewaxed, dehydrated with gradient alcohol, and washed with tap water for 2 min. Then the sections were treated with 3% methanol

Statistical analysis
Data analysis was introduced using the SPSS 21.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). Data are expressed as mean ± standard deviation. The unpaired t test was used for comparison between two groups. One-way analysis of variance (ANOVA) was employed for the comparisons among multiple groups, followed by Tukey's multiple comparisons test. Repeated measure ANOVA was employed for the comparisons between groups at different time points. The p < 0.05 indicated a signi cant difference.
Results miR-130a-3p was downregulated in VCR-resistant RB tissues and cells miR-130a-3p is downregulated in renal cell carcinoma, breast cancer, and liver cancer [25][26][27], but its role in RB remains unknown. miR-130a-3p expression in RB tissues and normal retinal tissues was detected using RT-qPCR, and the results demonstrated that miR-130a-3p expression of RB tissues was signi cantly lower than that of normal retinal tissues (Fig. 1A). All RB patients received VCR chemotherapy before operation. According to the recurrence time, the RB patients were allocated into VCR-sensitive group and VCR-resistant group. RT-qPCR results showed that compared with the VCR-sensitive group, the VCRresistant group had reduced miR-130a-3p expression (Fig. 1B).
Moreover, compared with HRA cells, miR-130a-3p was poorly expressed in RB cell lines (Y79, WERI-Rb-1, SO-Rb50, and SO-Rb70), with Y79 cells showing the lowest expression (Fig. 1C). Then RB cell lines were subjected to VCR treatment, and the IC50 value was measured using CCK-8 assay to re ect the drug resistance of cells. The IC50 value of Y79 cells was the highest after VCR treatment (Fig. 1D), indicating that Y79 cells were the most resistant to VCR. Hence, we chose Y79 cell line for the follow-up experiments. We further detected miR-130a-3p expression of VCR-resistant cells (Y79/VCR) and its corresponding parental cells (Y79). The results revealed that miR-130a-3p expression in Y79/VCR cells was notably lower than that in parental cells (Fig. 1E) (all p < 0.05). Taken together, miR-130a-3p was downregulated in VCR-resistant RB tissues and cells.
Compared with that in the normal retinal tissues, PAX6 expression in RB tissues was signi cantly elevated (Fig. 3C). PAX6 expression in the VCR-resistant group was notably higher than that in the VCRsensitive group (Fig. 3D). PAX6 expression in Y79/VCR cells was also higher than that in parental cells (Fig. 3E). PAX6 expression in Y79/VCR cells after overexpression of miR-130a-3p was detected, and the results exhibited that compared with the mimic-NC group, the miR-130a-3p mimic group showed reduced PAX6 expression (Fig. 3F) (all p < 0.05). Brie y, miR-130a-3p targeted PAX6 expression in VCR-resistant RB cells.
Overexpression of PAX6 reversed the effect of miR-130a-3p on chemosensitivity of RB To further determine whether miR-130a-3p affected the proliferation and chemosensitivity of RB cells by targeting PAX6, we conducted a functional rescue experiment. Compared with the mimic NC + oe-NC group, the miR-130a-3p mimic + oe-NC group showed signi cantly reduced cell proliferation; compared with the miR-130a-3p mimic + oe-NC group, the miR-130a-3p mimic + oe-PAX6 group had enhanced cell proliferation (Fig. 4A) (p < 0.05). After overexpression of miR-130a-3p, the IC50 value and drug RI of cells were decreased notably, while on this basis, overexpression of PAX6 increased the IC50 value and drug RI of cells (Fig. 4B-C) (p < 0.05). Taken together, overexpression of PAX6 reversed the promoting effect of miR-130a-3p on chemosensitivity of RB.
Overexpression of miR-130a-3p suppressed tumor growth and reduced VCR resistance in vivo Y79 or Y79/VCR cells were injected subcutaneously into the right side of nude mice, and agomiR-NC and miR-130a-3p agomiR were injected into the tumor site respectively. When the tumor volume reached about 100 mm 3 , the nude mice were treated with VCR. Compared with those of the Y79 group, the tumor volume and weight of the Y79/VCR group were notably increased; compared with those of the Y79/VCR + agomiR-NC group, the tumor volume and weight of the Y79/VCR + agomiR-130a-3p group were decreased ( Fig. 5A-C) (p < 0.05). The positive expression rate of PAX6 was detected using immunohistochemistry. Compared with that of the Y79 group, the positive rate of PAX6 protein of the Y79/VCR group was reduced; compared with that of the Y79/VCR + agomiR-NC group, the positive rate of PAX6 protein of the Y79/VCR + agomiR-130a-3p group were decreased (Fig. 5D) (p < 0.05). In brief, overexpression of miR-130a-3p suppressed tumor growth and reduced VCR resistance in vivo by targeting PAX6 expression.

Discussion
Chemotherapeutic agents extensively used in the treatment of RB; however, one of the most salient problem is that patients are prone to develop drug resistance, resulting in chemotherapy failure [7]. miRNA has a role in gene silencing and translational repression by binding to target mRNAs, which is implicated in the chemoresistance of diverse malignancies [30]. This study demonstrated that overexpression of miR-130a-3p enhanced the chemosensitivity of RB to VCR by targeting PAX6 expression.
It is well established that dysregulation of miRNA contributes to the initiation and progression of tumors, and even the chemoresistance of tumors [9]. The used of miRNA mimics or antagomiRs to correct the expression of these dysregulated miRNAs can normalize the gene regulatory network and thereby make tumor cells sensitive to chemotherapy [31]. miR-130a-3p is identi ed as a tumor suppressor and frequently downregulated in variant malignancies, such as breast cancer [32], hepatocellular carcinoma [26], and renal cell carcinoma [27]. To our knowledge, we were the rst to demonstrate that miR-130a-3p expression was reduced in RB tissues and cells. Notably, the role of miR-130a-3p in tumor chemoresistance has been well documented in recent literature. For example, miR-130a-3p is downregulated in gemcitabine-resistant hepatocellular carcinoma cells, and activation of miR-130a-3p contributes to the chemosensitivity of hepatocellular carcinoma cells [33]. Similarly, overexpression of miR-130a-3p reverses cisplatin resistance of clear cell renal cell carcinoma [27]. We further detected miR-130a-3p expression of VCR-resistant cells and its corresponding parental cells. Our results revealed that miR-130a-3p was downregulated in VCR-resistant RB tissues and cells. Then, Y79/VCR cells were subjected to miR-130a-3p overexpression treatment. The proliferation, IC50 value, and RI of VCR-resistant RB cells transfected with miR-130a-3p mimic were measured. The results revealed that overexpression of miR-130a-3p repressed the proliferation of VCR-resistant RB cells and enhanced chemosensitivity.
Consistently, it is reported that upregulation of miR-130a-3p can repress the proliferation of breast cancer cells [32] and signi cantly strengthen the cell sensitivity to doxorubicin [34].
Subsequently, we sought to determine the target genes of miR-130a-3p in regulating the chemosensitivity of RB cells. Targetscan website (http://targetscan.org/vert_71/) predicted that miR-130a-3p had a binding site with PAX6, and dual-luciferase assay con rmed that miR-130a-3p could speci cally bind PAX6. PAX6 is a transcription factor belonging to the PAX family, which plays a vital role in the development of eyes, pancreas, and central nervous system [35]. PAX6 functions as a critical modulator in the coordination and pattern formation essential for retinogenesis, as well as the development of other ocular tissues [36].
PAX6 mutation causes eye defects and neurological abnormalities associated with structural alterations in the brain [35]. Importantly, aberrant upregulation of PAX6 has been observed in RB, leading to the onset and progression of RB [28]. PAX6 silencing represses growth and facilitates apoptosis of cultured human RB cells [37]. Also, PAX6 is reported to regulate the chemoresistance of glioblastoma stem cells to temozolomide [38]. Consequently, we speculated that miR-130a-3p affected the chemosensitivity of RB cells by targeting PAX6. This study exhibited that PAX6 expression was signi cantly elevated in RB tissues compared with that in the normal retinal tissues. PAX6 expression in the VCR-resistant tissues and cells was also notably upregulated. PAX6 expression was reduced in Y79/VCR cells transfected with miR-130a-3p mimic. Brie y, miR-130a-3p targeted PAX6 expression in VCR-resistant RB cells. Functional rescue experiments veri ed that overexpression of PAX6 attenuated the promoting effect of miR-130a-3p on the chemosensitivity of RB cells. Moreover, the murine model of RB was established and the model mice underwent VCR treatment. Our results con rmed that overexpression of miR-130a-3p suppressed tumor growth and reduced VCR resistance in vivo by targeting PAX6 expression.
To sum up, miR-130a-3p was downregulated in RB tissues and cells, and upregulation of miR-130a-3p contributed to enhancing the chemosensitivity of RB cells to VCR by targeting PAX6 expression. This study hinted the possibility of miR-130a-3p as a promising target for RB patients with chemoresistance and possessed certain signi cance for the improvement of clinical practice of chemotherapy. However, whether miR-130a-3p can be adopted as a breakthrough to improve sensitivity to VCR needs further veri cation. Additionally, whether other targets of miR-130a-3p participate in the mechanism of VCR resistance in RB remains unknown. The mechanism of other targets and downstream signaling pathway of miR-130a-3p in VCR resistance will be assessed in the future study.
Declarations miR-130a-3p was downregulated in VCR-resistant RB tissues and cells. A: miR-130a-3p expression of RB tissues and normal retinal tissues was detected using RT-qPCR, normal group N = 13, RB group N = 34. B: miR-130a-3p expression of patients in the VCR-sensitive group and VCR-resistant group was detected using RT-qPCR, VCR-sensitive N = 21, VCR-resistant group N = 13. C: miR-130a-3p expression of HRA cells and RB cell lines (Y79, WERI-Rb-1, SO-Rb50, and SO-Rb70) was detected using RT-qPCR. D: The IC50 value of RB cell lines after VCR treatment was detected using CCK-8 assay. E: miR-130a-3p expression of VCR-resistant cell line (Y79/VCR) and its corresponding parental cell line (Y79) was detected using RT-qPCR. The cell experiment was repeated 3 times independently. Data were presented as mean ± standard deviation. The unpaired t test was used for comparison between two groups. One-way ANOVA was employed for the comparison among multiple groups, followed by Tukey's multiple comparisons test, *p < 0. 05.

Figure 2
Overexpression of miR-130a-3p repressed the proliferation of VCR-resistant RB cells and enhanced chemosensitivity. Y79/VCR cells were transfected with miR-130a-3p mimic. A: The transfection e ciency was veri ed using RT-qPCR. B: The cell proliferation was measured using MTT assay. C: The IC50 value of cells in each group was detected using CCK-8 assay. D: The resistance index of cells in each group was detected using CCK-8 assay. The cell experiment was repeated 3 times independently. Data were presented as mean ± standard deviation. The unpaired t test was used for comparison between two groups, *p < 0. 05. Figure 3 miR-130a-3p targeted PAX6 expression. A: The binding site of miR-130a-3p and PAX6 was predicted through Targetscan. B: The targeting relationship between miR-130a-3p and PAX6 was veri ed using dual-luciferase assay. C: PAX6 expression of RB tissues and normal retinal tissues was detected using RT-qPCR, normal group N = 13, RB group N = 34. D: PAX6 expression of VCR-resistant RB tissues and VCRsensitive RB tissues was detected using RT-qPCR. E: PAX6 expression of VCR-resistant cell line (Y79/VCR) and its corresponding parental cell line (Y79) was detected using RT-qPCR. F: PAX6 expression of Y79/VCR cells after overexpression of miR-130a-3p was detected using RT-qPCR. The cell experiment was repeated 3 times independently. Data were presented as mean ± standard deviation. The unpaired t test was used for comparison between two groups. One-way ANOVA was employed for the comparison among multiple groups, followed by Tukey's multiple comparisons test, *p < 0. 05.

Figure 4
Overexpression of PAX6 reversed the effect of miR-130a-3p on chemosensitivity of RB. miR-130a-3p and PAX6 were overexpressed in Y79/VCR cells. A: The cell proliferation was measured using MTT assay. B: The IC50 value of cells in each group was detected using CCK-8 assay. C: The resistance index of cells in each group was detected using CCK-8 assay. The cell experiment was repeated 3 times independently. Data were presented as mean ± standard deviation. One-way ANOVA was employed for the comparison among multiple groups, followed by Tukey's multiple comparisons test, *p < 0. 05.