- Email: [email protected]
-3p inhibits proliferation, invasion and migration of clear cell renal cell carcinoma
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Blocking lncRNA MIR155HG/miR-155-5p/-3p inhibits proliferation, invasion and migration of clear cell renal cell carcinoma Meiman Taoa,b, Yang Zhoub, Yichen Jina, Jinxian Pua,* a b
Department of Urology, The First Affiliated Hospital of Soochow University, Suzhou, 215006, China Department of Urology, The Affiliated Hospital of Jiangsu University, Zhenjiang, 212001, Jiangsu, China
A R T I C LE I N FO
A B S T R A C T
Keywords: lncRNA MIR155HG miR-155-3p miR-155-5p Clear cell renal cell carcinoma
This study aimed to investigate the effect of blocking the MIR155HG/miR-155-5p/-3p axis on proliferation, invasion and migration of clear cell renal cell carcinoma. RT-qPCR was used to detect the expression of MIR155HG, miR-155-5p, miR-155-3p in clear cell renal cell carcinoma cell lines. To study the effects of blocking LncRNA MIR155HG and interfering with miR-155-5p and miR-155-3p on the biological function. The g proliferation of tumor was detected by CCK-8, and the cell invasion and migration abilities were detected by wound healing and transwell experiments. Western blot analyzed protein levels of KI67, PCNA, MMP2 and MMP9. Furthermore, TargetScan and miRDB were used to predict the co-target gene of miR-155-3p and miR-155-5p, and the functional analysis of co-target genes was performed using the DAVID. In the current research, the expression of MIR155HG was increased in ccRCC. Interference of MIR155HG inhibited the cellular functions of ccRCC cells, which was reversed by overexpression of miR-155-3p and miR-155-5p. In addition, MIR155HG interference repressed the expression of miR-155-5p and miR-155-3p in ccRCCs, while inhibition of miR-155-5p and miR-155-3p restrained the proliferation, invasion and migration of ccRCCs. Bioinformatics software analysis showed 13 co-targeting genes of miR-155-3p and miR-155-5p. Functional analysis presented that the target genes of miR-31-3p were involved in numerous of biochemical processes and pathways.Blocking lncRNA MIR155HG/miR-155-5p/-3p inhibits proliferation, invasion and migration of renal clear cell carcinoma, which provided a new method for early diagnosis and precise treatment of ccRCC.
1. Introduction Clear cell renal cell carcinoma (ccRCC) is the most common pathological type of renal cell carcinoma, accounting for about 70 % of renal cell carcinoma [1,2]. At present, surgery is still the first choice for the treatment of early and locally advanced renal cancer, but it is easy to relapse and about 30 % of patients will have metastasis [3]. The firstline treatment of advanced renal cancer is still based on anti-angiogenic targeted therapy. In recent years, the breakthrough of immunotherapy and targeted drugs has brought new hope for patients with advanced renal cancer. However, due to the limitation of drug efficacy and the lack of predictive indicators suitable for targeted therapy and immunotherapy, the overall survival rate of patients with renal cancer is still very low [4,5]. Therefore, it is of great significance to screen the differentially expressed genes related to ccRCC for elucidating the molecular typing and possible therapeutic targets of ccRCC, which can provide a theoretical basis for precise treatment of ccRCC. LncRNA is a kind of functional RNA with a length of more than 200
⁎
nucleotides. It has been found that the expression of lncRNA is closely related to many diseases, containing the occurrence of human tumors [6–9]. The high expression of lncRNA is closely related to the malignant degree of pancreatic cancer, liver cancer, breast cancer, prostate cancer, bladder cancer and other tumors [10–12]. Some of lncRNAs are very sensitive in tumors, which are likely to be tumor specific markers and may become new targets for tumor treatment [13]. In carcinomas, lncRNA can regulate gene expression at different levels through gene imprinting, chromatin remodeling, splicing regulation, mRNA degradation and translation regulation to affect cellular activities [7]. The results of the second-generation lateral sequence showed that MIR155HG is a precursor of miR-155 [14], and that blocking MIR155HG/miR-155 can inhibit the proliferation of glioma cells [14,15]. Recent studies have shown that lncRNA MIR155HG is highly expressed in tissues of renal clear cell carcinoma and is associated with prognosis, but its specific mechanism in renal clear cell carcinoma is not known [16,17]. Therefore, this study aims to investigate whether blocking the MIR155HG/miR-155-5p/-3p axis can suppress the
Corresponding author at: Department of Urology, The First Affiliated Hospital of Soochow University, 899 Pinghai Road, Suzhou 215006, Jiangsu, China. E-mail address: [email protected] (J. Pu).
https://doi.org/10.1016/j.prp.2019.152803 Received 31 October 2019; Received in revised form 12 December 2019; Accepted 23 December 2019 0344-0338/ © 2019 Published by Elsevier GmbH.
Please cite this article as: Meiman Tao, et al., Pathology - Research and Practice, https://doi.org/10.1016/j.prp.2019.152803
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Fig. 1. MIR155HG (A), miR155-3p (B) and miR155-5p (C) were significantly up-regulated in ccRCC as assessed by RT-qPCR. (D) Transfection efficiency of shRNAMIR155HG was detected using RT-qPCR 48 h post-transfection. (E) CCK-8 assay measured the OD value at 450 nm of 786-O cells in shRNA-NC and shRNAMIR155HG-1 groups at 24, 48 and 72 h. Western blotting analysis (F-H) was performed to detect protein expression of KI67 and PCNA. *p < 0.05, **p < 0.01, ***p < 0.001 vs. HKC-5 or Control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. HK-2 or shRNA-NC.
2. Materials and methods
using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s protocol. Further experiments were performed 48 h posttransfection.
2.1. Cell culture
2.3. Cell counting Kit-8 (CCK-8) assay
The human renal tubular epithelial cell line (HKC-5), the normal human renal cell line (HK-2), KIRC cell lines (LoMet-ccRCC and 786-O) as well as A498 and Caki-1 cells were obtained from the Institute of respiratory diseases, Guangzhou Medical University (Guangzhou, China). All cell lines were cultured in Dulbecco's modified Eagle's medium containing 10 % fetal bovine serum with 100ug/ml Streptomycin,100U/ml Penicillin, 1.5 mg/L Glutamine. Cells were cultured in a humidified incubator with 5 % CO2 at 37 °C.
The cell proliferation capacity of treated 786-O cell lines was assessed via the cell counting kit-8 (Dojindo, Rockville, MD, USA) according to manufacturer’s protocol. 786-O cell lines were cultured into 96-well plates and incubated for 24 h. Next, CCK-8 reagent (10 μl) was added into each well and incubated for 1 h. Finally, absorbance was measured at 450 nm and the percentage of viable cells was calculated (normalized to the control group).
proliferation, invasion and migration of renal clear cell carcinoma.
2.4. Migration and invasion assays 2.2. Transfection Cell migration and invasion were measured by wound healing and transwell assays according to the method described before [18]. The 786-O cells density was adjusted to 1*106/ cells/ml, 100 μl cell suspension of serum-free DMEM medium was added to the upper chamber
The 786-O cells were cultured in 6-well plates to 70–80 % confluence, and the MIR155HG inhibitor or the inhibitor, mimics and negative control (NC) RNAs of miR-155-5p or miR-155-3p was transfected 2
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Fig. 2. Wound-healing assays (A-B) and transwell (C-D) assays were used to assess the effects of shRNA-MIR155HG-1 on invasion, and migration capabilities of 786O cells on invasion, and migration capabilities. Western blotting analysis (E-G) was performed to detect protein expression of MMP2 and MMP9. ***p < 0.001 vs. Control; ###p < 0.001 vs. shRNA-NC.
2.6. Quantitative real-time PCR (qRT-PCR) analysis
of transwell cell, and added to the lower chamber was supplemented with 600 μl medium with 10 % FBS. Migrated cells were fixed and stained with 0.5 % crystal violet and counted with a microscope after 48 h.
The total RNA extracted was reverse transcribed into cDNA by TaqMan Reverse Transcription Kit (Takara Biotechnology Co., Ltd.), and then qPCR was performed by SYBR premix Ex Taq kit in fluorescence quantitative PCR to detect the relative expression of lncRNA. The reaction conditions of fluorescence quantitative PCR were: pre-denaturation at 95 ℃ for 30 s, 5 s at 95 ℃, and 40 cycles at 60 ℃ for 30 s. The following primers were used: MIR155HG-F: 5′-GTCCCAAATCTAGGTT CAAGTTCA-3′; MIR155HG-R: 5′-TCTCTATCTCATCTAAGCCTCA CAA-3′; U6-F: 5′-GCGCGTCGTGAAGCGTTC-3′; U6-R: 5′-GTGCAGGGT CCGAGGT-3′; GAPDH-F: 5′-CAGCCTCAAGATCATCAGCA-3′; GAPDH-R: 5′-TGTGGTCATGAGTCCTTCCA-3′
2.5. Western blot Total protein from 786-O cell lines was extracted using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) at 4 °C for 1 h. The concentration of protein sample was measured by the BCA method (Beyotime, Shanghai, China). The protein was separated by 10 % SDS-PAGE and transferred to membrane and blocked by 5 % skim milk at room temperature for 1 h followed by culture with primary antibody at 4 °C overnight. After washing with TBST, the corresponding horseradish peroxidase-labeled secondary antibody was incubated with the membrane for 2 h at room temperature. At last, the protein expression was detected by ECL (enhanced chemiluminescence). The primary antibody was used as followed: KI67 (1:1000; #ab15580; Abcam, USA) PCNA (1:1000, #163209; Cell signaling, USA) MMP-2 (1:1000, #40994; Cell signaling, USA); MMP-9 (1:1000, #13667; Cell signaling, USA); and GAPDH (1:2000; Abcam, USA).
2.7. Functional analysi TargetScan (http://www.targetscan.org) and miRDB (http:// miRdb.org/) was used to predict the co-target gene of miR-155-3p and miR-155-5p. To explore the functional role of miR-155-3p and miR155-5p co-target genes, the Database for Annotation, Visualization and Integrated Discovery software (DAVID)(www.david.ncifcrf.gov) combined the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were applied. The enrichment values of GO 3
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Fig. 3. Expression of miR-155-3p (A) and miR-155-5p (B) in 786-O cells transfected with shRNA-MIR155HG or NC control by RT-qPCR. (C)Expression of miR-155-5p in 786-O cells transfected with miR-155-5p-inhibitor or inhibitor-NC by RT-qPCR. (D) Expression of miR-155-3p in 786-O cells transfected with miR-155-3p-inhibitor or inhibitor-NC by RT-qPCR. (E) CCK-8 assay measured the OD value at 450 nm of 786-O cells in inhibitor-NC group miR-155-3p-inhibitor and miR-155-5p-inhibitor at 24, 48 and 72 h. (F) Western blotting analysis (F-H) was performed to detect protein expression of KI67 and PCNA. ***p < 0.001 vs. Control; ###p < 0.001 vs. inhibitor-NC.
compared with normal renal cells. So, we choose the 786-O cell line with highest MIR155HG /miR-155-5p/-3p expression for the next study.
and KEGG pathways were assessed through Fisher's exact test. 2.8. Statistical analysis All data were showed as mean ± SEM and all experiments were repeated three times. Data were analyzed by GraphPad 6.0 (GraphPad, Inc.). One-way analysis of variance followed by Tukey’s post hoc test or Student’s t-test, and p value less than 0.05 was considered as significant.
3.2. Interference of MIR155HG inhibited the cell proliferation in ccRCC To further investigate the role of lncRNA MIR155HG in ccRCC cell lines, shRNAs of MIR155HG (shRNA-MIR155HG-1 and shRNAMIR155HG-2) and the negative control (shRNA-NC) were transfected into 786-O cells. Transfection efficiency was detected using RT-qPCR 48 h post-transfection. The results demonstrated that the inhibitory effect of shRNA-MIR155HG-1 on MIR155HG expression level was superior to that of shRNA-MIR155HG-2 in 786-O cells (Fig. 1D). For CCK8 assay, cell viability of 786-O cells transfected with shRNAMIR155HG-1 was significantly inhibited compared to those cells transfected with shRNA-NC (Fig. 1E). Furthermore, the western blotting results indicated that interference with shRNA-MIR155HG-1 significantly decreased levels of proliferation-related proteins KI67 and PCNA in 786-O cells (Fig. 1F-H).
3. Results 3.1. MIR155HG was significantly up-regulated in ccRCC In the present study, the level of miR155HG, miR155-3p and miR155-5p expression were respectively detected in the human renal tubular epithelial cell line (HKC-5), the normal human renal cell line (HK-2), ccRCC cell lines (LoMet-ccRCC and 786-O), A498 and Caki-1 by RT-qPCR. As shown in Fig. 1A-C, miR155HG, miR155-3p and miR1555p expression level were significantly increased in ccRCC cell lines 4
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Fig. 4. Wound-healing assays (A-B) and transwell (C-D) were used to assess the effects of miR-155-5p-inhibitor and miR-155-3p-inhibitor in 786-O cells on invasion, and migration capabilities in 786-O cells. Western blotting analysis (E-G) for detecting protein expression of MMP2 and MMP9. ***p < 0.001 vs. Control; ### p < 0.001 vs. inhibitor-NC.
3.5. Silence of miR-155-5p and miR-155-3p repressed proliferation, invasion and migration of 786-O cells
3.3. Interference of MIR155HG inhibited invasion and migration of ccRCC We then studied the role of MIR155HG in invasion and migration of ccRCC cells by transwell invasion test and cell scratch test. As shown in Fig. 2A-D, the cell invasion test showed that the invasiveness of 786-O cells in the shRNA-MIR155HG-1 group was lower than that in the shRNA-NC group. Cell scratch test showed that the migration was slower in the shRNA-MIR155HG-1 group compared with shRNA-NC group. Furthermore, the western blotting results indicated that silencing of MIR155HG significantly decreased invasion and migration-related proteins MMP2 and MMP9 expression levels in 786-O cells (Fig. 2E-G).
As shown in Fig. 3E, for CCK-8 assay exhibited that cell proliferation of 786-O cells transfected with miR-155-5p-inhibitor or miR-155-3pinhibitor was significantly inhibited compared to those cells under treatment of inhibitor-NC. Furthermore, the western blotting results indicated that interference with miR-155-5p-inhibitor or miR-155-3pinhibitor significantly decreased expression levels of KI67 and PCNA in 786-O cells (Fig. 3F-H). Meanwhile, the cell invasion test showed that the invasiveness of 786-O cells in the miR-155-5p-inhibitor group or miR-155-3p-inhibitor group was lower than that inhibitor-NC group. Cell scratch test also found that in the miR-155-5p-inhibitor group and miR-155-3p-inhibitor group, the migration was hampered, compared with inhibitor-NC group (Fig. 4A-D). Furthermore, the western blotting results discovered that interference of miR-155-5p and miR-155-3p significantly decreased invasion and migration-related proteins MMP2 and MMP9 expression levels in 786-O cells (Fig. 4E-G).
3.4. Silence of MIR155HG decreased expression of miR-155-5p and miR155-3p in 786-O cells The results of the second-generation lateral sequence show that MIR155HG is a precursor of miR-155 [14]. As shown in Fig. 3A and B, the results of RT-qPCR showed that inhibition of miR-155-5p and miR155-3p expression in 786-O cells after inhibition of MIR155HG. To further investigate the role of miR-155-5p/3p in 786-O cell lines, miR155-5p-inhibitor, miR-155-3p-inhibitor and the negative control (inhibitor-NC) were transfected into 786-O cells. Transfection efficiency was detected using RT-qPCR, and the results determined that the inhibitory effect of miR-155-5p-inhibitor and miR-155-3p-inhibitor on miR155 expression level was superior to that of inhibitor-NC group in 786-O cells (Fig. 3C and D).
3.6. Down-regulation of MIR155HG hindered proliferation, invasion and migration of 786-O cells and this effect was reversed by overexpression of miR-155-3p and miR-155-5p To investigate the effect of blocking the MIR155HG/miR-155-5p/3p axis on proliferation, invasion and migration of ccRCC cell lines, shRNA-MIR155HG-1 and miR-155-3p-mimic, miR-155-5p-mimic or mimic-NC were co-transfected into 786-O cells. The results of RT-qPCR affirmed that the overexpression effects of miR-155-5p-mimic on miR155-5p expression level and miR-155-3p-mimic on miR-155-3p 5
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Fig. 5. Expression of miR-155-5p (A) in 786-O cells transfected with shRNA-MIR155HG + miR-155-5p-mimic or mimic-NC was measured by RT-qPCR. Expression of miR-155-3p (B) in 786-O cells transfected with shRNA-MIR155HG-1+miR-155-3p-mimic or NC control by RT-qPCR. (C) CCK-8 assay measured the OD value at 450 nm of 786-O cells, the negative control of shRNA-MIR155HG-1+mimic, shRNA-MIR155HG-1+miR-155-3p-mimic and shRNA-MIR155HG-1+miR-155-5pmimic at 24, 48 and 72 h. Western blotting analysis (D-F) was utilized to detect protein expression of KI67 and PCNA. ***p < 0.001 vs. shRNA-MIR155HG-1; ### p < 0.001 vs. or shRNA-MIR155HG-1+mimic-NC.
localization”. Under MF, the top GO terms were “negative transcription factor activity”, “negative sequence-specific DNA binding” and “negative metal ion binding”. Under CC, the top GO terms were “negative cytoplasm”. KEGG analysis revealed the top pathways, which include “Signaling pathways regulating pluripotency of stem cells”.
expression level were higher than that of mimic-NC and control group in 786-O cells (Fig. 5A and B). The results of CCK-8 assay indicated that MIR155HG silencing inhibited proliferation of 786-O cells, which was reversed by addition of miR-155-3p and miR-155-5p (Fig. 5C). Western blot was used to detect the expression of proliferation-related proteins KI67and PCNA, which revealed the similar results to CCK-8 (Fig. 5D-F). Meanwhile, the cell invasion test showed that the invasiveness of 786-O cells in the shRNAMIR155HG-1+miR-155-3p-mimic group and shRNA-MIR155HG1+miR-155-5p-mimic group was higher than that in the shRNAMIR155HG-1+mimic-NC group (Fig. 6A-D). Furthermore, the western blotting results indicated that elevation of miR-155-5p and miR-155-3p significantly increased the MIR155HG downregulation-reduced MMP2 and MMP9 expression levels in 786-O cells (Fig. 6E-G). The above results indicated that interference of MIR155HG hampered proliferation, invasion and migration of 786-O cells and this phenomenon was reversed by enhanced expression of miR-155-3p and miR-155-5p.
4. Discussions LncRNAs are closely associated with the occurrence of human tumors and can modulate gene expression at multiple levels via gene imprinting, chromatin remodeling, cell cycle regulation, splicing regulation, mRNA degradation and translation regulation. Highly expressed lncRNA is closely related to the malignancy of pancreatic cancer, liver cancer, breast cancer, prostate cancer, bladder cancer and other tumors. Some lncRNAs are very sensitive in tumors and are prone tumor-specific markers and are expected to become new targets for tumor therapy [13]. As an important regulator, MIR155HG is involved in the occurrence, development and various physiological and pathological processes of tumors. It has been reported that MIR155HG is positively correlated with tumor grade and inhibits the growth of glioma [19–23]. In the present study, MIR155HG was significantly upregulated in ccRCC, especially in 786-O cell line. The results were in consistent with the data of previous reports that lncRNA MIR155HG is increased in the tissues of clear cell carcinoma of the kidney and is associated with prognosis [16,17]. In the current study, to test the biological function of this lncRNA, shRNA-MIR155H was applied to transfect with 786Os. Cell viability of 786-O cells transfected with shRNA-MIR155HG-1 was significantly inhibited as illustrated by CCK-8. Concurrently, knockdown of MIR155HG suppressed the expression of proliferation-related proteins, including KI67 and PCNA. At the same time, notably inhibition of 786-
3.7. GO and KEGG analysis of co-target genes To determine the co-target genes of miR-155-3p and miR-155-5p, bioinformatics software analysis co-targeting genes for miR-155-3p and miR-155-5p in miRDB and TargetScan databases. The Venn showed that there were 13 co-targeting genes for miR-155-3p and miR-155-5p (Fig. 6H). The gene names are JARID2, TSHZ3, LSM14A, ANTXR2, LRRC59, PICALM, QKI, CREBRF, SERTAD2, AAK1, SMAD5, ZNF518B and BCORL1. Next, we explored the potential biological function and pathway of miR-155-3p and miR-155-5p in ccRCC. GO analysis was carried out to assess target gene enrichments in terms of biological process (BP), cellular component (CC) and molecular function (MF). Under BP, the top GO terms were “negative regulation of protein 6
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Fig. 6. Wound-healing assays (A-B) and transwell (C-D) assay assessed the effects of shRNA-MIR155HG-1+miR-155-3p-mimic and shRNA-MIR155HG-1+miR-1555p-mimic in 786-O cells on invasion, and migration capabilities. Western blotting analysis (E-G) conducted to detect protein expression of MMP2 and MMP9. (H) Venn diagram shows miR155-3p and miR155-5p co-targeting genes. ***p < 0.001 vs. shRNA-MIR155HG-1; ###p < 0.001 vs. shRNA-MIR155HG-1+mimic-NC.
After determining the MIR155HG/miR-155-5p/-3p axis in 786-O cells, we performed functional analysis to predict the molecular mechanisms underlying miR-155-5p and miR-155-3p. To identify the cotarget genes of miR-155-3p and miR-155-5p, bioinformatics software dissected the targets of miR-155-3p and miR-155-5p predicted by miRDB and TargetScan databases. The Venn diagram exhibited that there were 13 shared targets for miR-155-3p and miR-155-5p. GO analysis subsequently revealed that the 13 co-targeting genes participated in a variety of cellular processes potentially important to ccRCC progression in the categories of biological process, cellular component and molecular function. Regulation of protein localization, cytoplasm and sequence-specific DNA binding were selected as representatives of the three major categories, respectively. These results indicated that sequence-specific DNA binding might serve vital roles in the progression of ccRCC. KEGG pathway analysis determined that these target genes participated in signaling pathways regulating the pluripotency of stem cells as it was the most significantly enriched pathway. The results of the GO and KEGG analyses suggested that MIR155HG might function as a regulator in ccRCC.
O cells migration and invasion coupled with a marked decrease in MMP2 and MMP9 expression were observed after transfection with shRNA-MIR155H. These findings confirmed that interference with MIR155HG interference hindered proliferation, invasion and migration of 786-O cells. However, in vitro experiments have shown that blocking the expression of MIR155HG can significantly reduce the expression of miR-155-5p and miR-155-3p in glioma cells [24]. This correlation indicates that MIR155HG is a precursor of miR-155. Previous studies have shown that miR-155 is increased in clear cell renal cell carcinoma cells and can inhibit the expression of clear cell renal cell carcinoma after interference [25]. In previous studies, miR155-5p was considered to be the only functional miR-155, while studies have shown that miR155-3p may have similar functions to miR-155-5p in glioma [26,27]. MiR-1555p is related to the occurrence, development and prognosis of lung cancer, gastric cancer, breast cancer, cervical cancer, pancreatic cancer and other malignant tumors, and miR-155-3p is a typical multifunctional miRNA, which has been proved to play an important role in immune response, inflammation, tumorigenesis and other processes [23,28–31]. Therefore, we focused on the functions of miR-155-5p and miR-155-3p in ccRCC. Our data suggest that interference with miR-1555p and miR-155-3p inhibits proliferation, invasion and migration of 786-O cells, consistent with previous studies. The results further demonstrated that addition of miR-155-3p and miR-155-5p rescued the inhibitory impacts of MIR155HG decrease on proliferation, invasion and migration of 786-O cells and was reversed by miR-155-3p and 5p. In conclusion, this study confirmed the important correlation between MIR155HG and miR-155, as well as the important role of miR-155-5p/3p in the biological function of MIR155HG, and the role of miR-1555p/-3p in promoting the development and progress of ccRCC.
5. Conclusions In summary, we have presented evidence that blocking lncRNA MIR155HG/miR-155-5p/-3p inhibits proliferation, invasion and migration of renal clear cell carcinoma. These findings represent a novel method for combinatorial treatment of clear cell renal cell carcinoma through the MIR155HG/miR-155-5p/-3p axis.
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Authors’ contributions [9]
MMT carried out the study design and writing of the manuscript. YZ confirmed the completeness and validity of the data. YJ participated in the data collection and analysis. JXP conceived of the study, participated in its study design, and revised the manuscript. All authors read and approved the final manuscript.
[10]
[11]
Funding [12]
Not applicable.
[13]
Ethics approval and consent to participate Not applicable
[14]
Availability of data and materials [15]
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
[16]
Consent for publication [17]
Not applicable CRediT authorship contribution statement
[18]
Meiman Tao: Writing - original draft, Methodology, Software, Investigation, Formal analysis. Yang Zhou: Methodology, Software, Investigation, Validation, Formal analysis. Yichen Jin: Methodology, Software, Data curation. Jinxian Pu: Conceptualization, Writing - review & editing, Resources, Visualization.
[19]
[20]
Declaration of Competing Interest
[21]
The authors declare that there are no conflicts of interest. [22]
Acknowledgements Not applicable.
[23]
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Pathology - Research and Practice journal homepage: www.elsevier.com/locate/prp
Blocking lncRNA MIR155HG/miR-155-5p/-3p inhibits proliferation, invasion and migration of clear cell renal cell carcinoma Meiman Taoa,b, Yang Zhoub, Yichen Jina, Jinxian Pua,* a b
Department of Urology, The First Affiliated Hospital of Soochow University, Suzhou, 215006, China Department of Urology, The Affiliated Hospital of Jiangsu University, Zhenjiang, 212001, Jiangsu, China
A R T I C LE I N FO
A B S T R A C T
Keywords: lncRNA MIR155HG miR-155-3p miR-155-5p Clear cell renal cell carcinoma
This study aimed to investigate the effect of blocking the MIR155HG/miR-155-5p/-3p axis on proliferation, invasion and migration of clear cell renal cell carcinoma. RT-qPCR was used to detect the expression of MIR155HG, miR-155-5p, miR-155-3p in clear cell renal cell carcinoma cell lines. To study the effects of blocking LncRNA MIR155HG and interfering with miR-155-5p and miR-155-3p on the biological function. The g proliferation of tumor was detected by CCK-8, and the cell invasion and migration abilities were detected by wound healing and transwell experiments. Western blot analyzed protein levels of KI67, PCNA, MMP2 and MMP9. Furthermore, TargetScan and miRDB were used to predict the co-target gene of miR-155-3p and miR-155-5p, and the functional analysis of co-target genes was performed using the DAVID. In the current research, the expression of MIR155HG was increased in ccRCC. Interference of MIR155HG inhibited the cellular functions of ccRCC cells, which was reversed by overexpression of miR-155-3p and miR-155-5p. In addition, MIR155HG interference repressed the expression of miR-155-5p and miR-155-3p in ccRCCs, while inhibition of miR-155-5p and miR-155-3p restrained the proliferation, invasion and migration of ccRCCs. Bioinformatics software analysis showed 13 co-targeting genes of miR-155-3p and miR-155-5p. Functional analysis presented that the target genes of miR-31-3p were involved in numerous of biochemical processes and pathways.Blocking lncRNA MIR155HG/miR-155-5p/-3p inhibits proliferation, invasion and migration of renal clear cell carcinoma, which provided a new method for early diagnosis and precise treatment of ccRCC.
1. Introduction Clear cell renal cell carcinoma (ccRCC) is the most common pathological type of renal cell carcinoma, accounting for about 70 % of renal cell carcinoma [1,2]. At present, surgery is still the first choice for the treatment of early and locally advanced renal cancer, but it is easy to relapse and about 30 % of patients will have metastasis [3]. The firstline treatment of advanced renal cancer is still based on anti-angiogenic targeted therapy. In recent years, the breakthrough of immunotherapy and targeted drugs has brought new hope for patients with advanced renal cancer. However, due to the limitation of drug efficacy and the lack of predictive indicators suitable for targeted therapy and immunotherapy, the overall survival rate of patients with renal cancer is still very low [4,5]. Therefore, it is of great significance to screen the differentially expressed genes related to ccRCC for elucidating the molecular typing and possible therapeutic targets of ccRCC, which can provide a theoretical basis for precise treatment of ccRCC. LncRNA is a kind of functional RNA with a length of more than 200
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nucleotides. It has been found that the expression of lncRNA is closely related to many diseases, containing the occurrence of human tumors [6–9]. The high expression of lncRNA is closely related to the malignant degree of pancreatic cancer, liver cancer, breast cancer, prostate cancer, bladder cancer and other tumors [10–12]. Some of lncRNAs are very sensitive in tumors, which are likely to be tumor specific markers and may become new targets for tumor treatment [13]. In carcinomas, lncRNA can regulate gene expression at different levels through gene imprinting, chromatin remodeling, splicing regulation, mRNA degradation and translation regulation to affect cellular activities [7]. The results of the second-generation lateral sequence showed that MIR155HG is a precursor of miR-155 [14], and that blocking MIR155HG/miR-155 can inhibit the proliferation of glioma cells [14,15]. Recent studies have shown that lncRNA MIR155HG is highly expressed in tissues of renal clear cell carcinoma and is associated with prognosis, but its specific mechanism in renal clear cell carcinoma is not known [16,17]. Therefore, this study aims to investigate whether blocking the MIR155HG/miR-155-5p/-3p axis can suppress the
Corresponding author at: Department of Urology, The First Affiliated Hospital of Soochow University, 899 Pinghai Road, Suzhou 215006, Jiangsu, China. E-mail address: [email protected] (J. Pu).
https://doi.org/10.1016/j.prp.2019.152803 Received 31 October 2019; Received in revised form 12 December 2019; Accepted 23 December 2019 0344-0338/ © 2019 Published by Elsevier GmbH.
Please cite this article as: Meiman Tao, et al., Pathology - Research and Practice, https://doi.org/10.1016/j.prp.2019.152803
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Fig. 1. MIR155HG (A), miR155-3p (B) and miR155-5p (C) were significantly up-regulated in ccRCC as assessed by RT-qPCR. (D) Transfection efficiency of shRNAMIR155HG was detected using RT-qPCR 48 h post-transfection. (E) CCK-8 assay measured the OD value at 450 nm of 786-O cells in shRNA-NC and shRNAMIR155HG-1 groups at 24, 48 and 72 h. Western blotting analysis (F-H) was performed to detect protein expression of KI67 and PCNA. *p < 0.05, **p < 0.01, ***p < 0.001 vs. HKC-5 or Control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. HK-2 or shRNA-NC.
2. Materials and methods
using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s protocol. Further experiments were performed 48 h posttransfection.
2.1. Cell culture
2.3. Cell counting Kit-8 (CCK-8) assay
The human renal tubular epithelial cell line (HKC-5), the normal human renal cell line (HK-2), KIRC cell lines (LoMet-ccRCC and 786-O) as well as A498 and Caki-1 cells were obtained from the Institute of respiratory diseases, Guangzhou Medical University (Guangzhou, China). All cell lines were cultured in Dulbecco's modified Eagle's medium containing 10 % fetal bovine serum with 100ug/ml Streptomycin,100U/ml Penicillin, 1.5 mg/L Glutamine. Cells were cultured in a humidified incubator with 5 % CO2 at 37 °C.
The cell proliferation capacity of treated 786-O cell lines was assessed via the cell counting kit-8 (Dojindo, Rockville, MD, USA) according to manufacturer’s protocol. 786-O cell lines were cultured into 96-well plates and incubated for 24 h. Next, CCK-8 reagent (10 μl) was added into each well and incubated for 1 h. Finally, absorbance was measured at 450 nm and the percentage of viable cells was calculated (normalized to the control group).
proliferation, invasion and migration of renal clear cell carcinoma.
2.4. Migration and invasion assays 2.2. Transfection Cell migration and invasion were measured by wound healing and transwell assays according to the method described before [18]. The 786-O cells density was adjusted to 1*106/ cells/ml, 100 μl cell suspension of serum-free DMEM medium was added to the upper chamber
The 786-O cells were cultured in 6-well plates to 70–80 % confluence, and the MIR155HG inhibitor or the inhibitor, mimics and negative control (NC) RNAs of miR-155-5p or miR-155-3p was transfected 2
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Fig. 2. Wound-healing assays (A-B) and transwell (C-D) assays were used to assess the effects of shRNA-MIR155HG-1 on invasion, and migration capabilities of 786O cells on invasion, and migration capabilities. Western blotting analysis (E-G) was performed to detect protein expression of MMP2 and MMP9. ***p < 0.001 vs. Control; ###p < 0.001 vs. shRNA-NC.
2.6. Quantitative real-time PCR (qRT-PCR) analysis
of transwell cell, and added to the lower chamber was supplemented with 600 μl medium with 10 % FBS. Migrated cells were fixed and stained with 0.5 % crystal violet and counted with a microscope after 48 h.
The total RNA extracted was reverse transcribed into cDNA by TaqMan Reverse Transcription Kit (Takara Biotechnology Co., Ltd.), and then qPCR was performed by SYBR premix Ex Taq kit in fluorescence quantitative PCR to detect the relative expression of lncRNA. The reaction conditions of fluorescence quantitative PCR were: pre-denaturation at 95 ℃ for 30 s, 5 s at 95 ℃, and 40 cycles at 60 ℃ for 30 s. The following primers were used: MIR155HG-F: 5′-GTCCCAAATCTAGGTT CAAGTTCA-3′; MIR155HG-R: 5′-TCTCTATCTCATCTAAGCCTCA CAA-3′; U6-F: 5′-GCGCGTCGTGAAGCGTTC-3′; U6-R: 5′-GTGCAGGGT CCGAGGT-3′; GAPDH-F: 5′-CAGCCTCAAGATCATCAGCA-3′; GAPDH-R: 5′-TGTGGTCATGAGTCCTTCCA-3′
2.5. Western blot Total protein from 786-O cell lines was extracted using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) at 4 °C for 1 h. The concentration of protein sample was measured by the BCA method (Beyotime, Shanghai, China). The protein was separated by 10 % SDS-PAGE and transferred to membrane and blocked by 5 % skim milk at room temperature for 1 h followed by culture with primary antibody at 4 °C overnight. After washing with TBST, the corresponding horseradish peroxidase-labeled secondary antibody was incubated with the membrane for 2 h at room temperature. At last, the protein expression was detected by ECL (enhanced chemiluminescence). The primary antibody was used as followed: KI67 (1:1000; #ab15580; Abcam, USA) PCNA (1:1000, #163209; Cell signaling, USA) MMP-2 (1:1000, #40994; Cell signaling, USA); MMP-9 (1:1000, #13667; Cell signaling, USA); and GAPDH (1:2000; Abcam, USA).
2.7. Functional analysi TargetScan (http://www.targetscan.org) and miRDB (http:// miRdb.org/) was used to predict the co-target gene of miR-155-3p and miR-155-5p. To explore the functional role of miR-155-3p and miR155-5p co-target genes, the Database for Annotation, Visualization and Integrated Discovery software (DAVID)(www.david.ncifcrf.gov) combined the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were applied. The enrichment values of GO 3
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Fig. 3. Expression of miR-155-3p (A) and miR-155-5p (B) in 786-O cells transfected with shRNA-MIR155HG or NC control by RT-qPCR. (C)Expression of miR-155-5p in 786-O cells transfected with miR-155-5p-inhibitor or inhibitor-NC by RT-qPCR. (D) Expression of miR-155-3p in 786-O cells transfected with miR-155-3p-inhibitor or inhibitor-NC by RT-qPCR. (E) CCK-8 assay measured the OD value at 450 nm of 786-O cells in inhibitor-NC group miR-155-3p-inhibitor and miR-155-5p-inhibitor at 24, 48 and 72 h. (F) Western blotting analysis (F-H) was performed to detect protein expression of KI67 and PCNA. ***p < 0.001 vs. Control; ###p < 0.001 vs. inhibitor-NC.
compared with normal renal cells. So, we choose the 786-O cell line with highest MIR155HG /miR-155-5p/-3p expression for the next study.
and KEGG pathways were assessed through Fisher's exact test. 2.8. Statistical analysis All data were showed as mean ± SEM and all experiments were repeated three times. Data were analyzed by GraphPad 6.0 (GraphPad, Inc.). One-way analysis of variance followed by Tukey’s post hoc test or Student’s t-test, and p value less than 0.05 was considered as significant.
3.2. Interference of MIR155HG inhibited the cell proliferation in ccRCC To further investigate the role of lncRNA MIR155HG in ccRCC cell lines, shRNAs of MIR155HG (shRNA-MIR155HG-1 and shRNAMIR155HG-2) and the negative control (shRNA-NC) were transfected into 786-O cells. Transfection efficiency was detected using RT-qPCR 48 h post-transfection. The results demonstrated that the inhibitory effect of shRNA-MIR155HG-1 on MIR155HG expression level was superior to that of shRNA-MIR155HG-2 in 786-O cells (Fig. 1D). For CCK8 assay, cell viability of 786-O cells transfected with shRNAMIR155HG-1 was significantly inhibited compared to those cells transfected with shRNA-NC (Fig. 1E). Furthermore, the western blotting results indicated that interference with shRNA-MIR155HG-1 significantly decreased levels of proliferation-related proteins KI67 and PCNA in 786-O cells (Fig. 1F-H).
3. Results 3.1. MIR155HG was significantly up-regulated in ccRCC In the present study, the level of miR155HG, miR155-3p and miR155-5p expression were respectively detected in the human renal tubular epithelial cell line (HKC-5), the normal human renal cell line (HK-2), ccRCC cell lines (LoMet-ccRCC and 786-O), A498 and Caki-1 by RT-qPCR. As shown in Fig. 1A-C, miR155HG, miR155-3p and miR1555p expression level were significantly increased in ccRCC cell lines 4
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Fig. 4. Wound-healing assays (A-B) and transwell (C-D) were used to assess the effects of miR-155-5p-inhibitor and miR-155-3p-inhibitor in 786-O cells on invasion, and migration capabilities in 786-O cells. Western blotting analysis (E-G) for detecting protein expression of MMP2 and MMP9. ***p < 0.001 vs. Control; ### p < 0.001 vs. inhibitor-NC.
3.5. Silence of miR-155-5p and miR-155-3p repressed proliferation, invasion and migration of 786-O cells
3.3. Interference of MIR155HG inhibited invasion and migration of ccRCC We then studied the role of MIR155HG in invasion and migration of ccRCC cells by transwell invasion test and cell scratch test. As shown in Fig. 2A-D, the cell invasion test showed that the invasiveness of 786-O cells in the shRNA-MIR155HG-1 group was lower than that in the shRNA-NC group. Cell scratch test showed that the migration was slower in the shRNA-MIR155HG-1 group compared with shRNA-NC group. Furthermore, the western blotting results indicated that silencing of MIR155HG significantly decreased invasion and migration-related proteins MMP2 and MMP9 expression levels in 786-O cells (Fig. 2E-G).
As shown in Fig. 3E, for CCK-8 assay exhibited that cell proliferation of 786-O cells transfected with miR-155-5p-inhibitor or miR-155-3pinhibitor was significantly inhibited compared to those cells under treatment of inhibitor-NC. Furthermore, the western blotting results indicated that interference with miR-155-5p-inhibitor or miR-155-3pinhibitor significantly decreased expression levels of KI67 and PCNA in 786-O cells (Fig. 3F-H). Meanwhile, the cell invasion test showed that the invasiveness of 786-O cells in the miR-155-5p-inhibitor group or miR-155-3p-inhibitor group was lower than that inhibitor-NC group. Cell scratch test also found that in the miR-155-5p-inhibitor group and miR-155-3p-inhibitor group, the migration was hampered, compared with inhibitor-NC group (Fig. 4A-D). Furthermore, the western blotting results discovered that interference of miR-155-5p and miR-155-3p significantly decreased invasion and migration-related proteins MMP2 and MMP9 expression levels in 786-O cells (Fig. 4E-G).
3.4. Silence of MIR155HG decreased expression of miR-155-5p and miR155-3p in 786-O cells The results of the second-generation lateral sequence show that MIR155HG is a precursor of miR-155 [14]. As shown in Fig. 3A and B, the results of RT-qPCR showed that inhibition of miR-155-5p and miR155-3p expression in 786-O cells after inhibition of MIR155HG. To further investigate the role of miR-155-5p/3p in 786-O cell lines, miR155-5p-inhibitor, miR-155-3p-inhibitor and the negative control (inhibitor-NC) were transfected into 786-O cells. Transfection efficiency was detected using RT-qPCR, and the results determined that the inhibitory effect of miR-155-5p-inhibitor and miR-155-3p-inhibitor on miR155 expression level was superior to that of inhibitor-NC group in 786-O cells (Fig. 3C and D).
3.6. Down-regulation of MIR155HG hindered proliferation, invasion and migration of 786-O cells and this effect was reversed by overexpression of miR-155-3p and miR-155-5p To investigate the effect of blocking the MIR155HG/miR-155-5p/3p axis on proliferation, invasion and migration of ccRCC cell lines, shRNA-MIR155HG-1 and miR-155-3p-mimic, miR-155-5p-mimic or mimic-NC were co-transfected into 786-O cells. The results of RT-qPCR affirmed that the overexpression effects of miR-155-5p-mimic on miR155-5p expression level and miR-155-3p-mimic on miR-155-3p 5
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Fig. 5. Expression of miR-155-5p (A) in 786-O cells transfected with shRNA-MIR155HG + miR-155-5p-mimic or mimic-NC was measured by RT-qPCR. Expression of miR-155-3p (B) in 786-O cells transfected with shRNA-MIR155HG-1+miR-155-3p-mimic or NC control by RT-qPCR. (C) CCK-8 assay measured the OD value at 450 nm of 786-O cells, the negative control of shRNA-MIR155HG-1+mimic, shRNA-MIR155HG-1+miR-155-3p-mimic and shRNA-MIR155HG-1+miR-155-5pmimic at 24, 48 and 72 h. Western blotting analysis (D-F) was utilized to detect protein expression of KI67 and PCNA. ***p < 0.001 vs. shRNA-MIR155HG-1; ### p < 0.001 vs. or shRNA-MIR155HG-1+mimic-NC.
localization”. Under MF, the top GO terms were “negative transcription factor activity”, “negative sequence-specific DNA binding” and “negative metal ion binding”. Under CC, the top GO terms were “negative cytoplasm”. KEGG analysis revealed the top pathways, which include “Signaling pathways regulating pluripotency of stem cells”.
expression level were higher than that of mimic-NC and control group in 786-O cells (Fig. 5A and B). The results of CCK-8 assay indicated that MIR155HG silencing inhibited proliferation of 786-O cells, which was reversed by addition of miR-155-3p and miR-155-5p (Fig. 5C). Western blot was used to detect the expression of proliferation-related proteins KI67and PCNA, which revealed the similar results to CCK-8 (Fig. 5D-F). Meanwhile, the cell invasion test showed that the invasiveness of 786-O cells in the shRNAMIR155HG-1+miR-155-3p-mimic group and shRNA-MIR155HG1+miR-155-5p-mimic group was higher than that in the shRNAMIR155HG-1+mimic-NC group (Fig. 6A-D). Furthermore, the western blotting results indicated that elevation of miR-155-5p and miR-155-3p significantly increased the MIR155HG downregulation-reduced MMP2 and MMP9 expression levels in 786-O cells (Fig. 6E-G). The above results indicated that interference of MIR155HG hampered proliferation, invasion and migration of 786-O cells and this phenomenon was reversed by enhanced expression of miR-155-3p and miR-155-5p.
4. Discussions LncRNAs are closely associated with the occurrence of human tumors and can modulate gene expression at multiple levels via gene imprinting, chromatin remodeling, cell cycle regulation, splicing regulation, mRNA degradation and translation regulation. Highly expressed lncRNA is closely related to the malignancy of pancreatic cancer, liver cancer, breast cancer, prostate cancer, bladder cancer and other tumors. Some lncRNAs are very sensitive in tumors and are prone tumor-specific markers and are expected to become new targets for tumor therapy [13]. As an important regulator, MIR155HG is involved in the occurrence, development and various physiological and pathological processes of tumors. It has been reported that MIR155HG is positively correlated with tumor grade and inhibits the growth of glioma [19–23]. In the present study, MIR155HG was significantly upregulated in ccRCC, especially in 786-O cell line. The results were in consistent with the data of previous reports that lncRNA MIR155HG is increased in the tissues of clear cell carcinoma of the kidney and is associated with prognosis [16,17]. In the current study, to test the biological function of this lncRNA, shRNA-MIR155H was applied to transfect with 786Os. Cell viability of 786-O cells transfected with shRNA-MIR155HG-1 was significantly inhibited as illustrated by CCK-8. Concurrently, knockdown of MIR155HG suppressed the expression of proliferation-related proteins, including KI67 and PCNA. At the same time, notably inhibition of 786-
3.7. GO and KEGG analysis of co-target genes To determine the co-target genes of miR-155-3p and miR-155-5p, bioinformatics software analysis co-targeting genes for miR-155-3p and miR-155-5p in miRDB and TargetScan databases. The Venn showed that there were 13 co-targeting genes for miR-155-3p and miR-155-5p (Fig. 6H). The gene names are JARID2, TSHZ3, LSM14A, ANTXR2, LRRC59, PICALM, QKI, CREBRF, SERTAD2, AAK1, SMAD5, ZNF518B and BCORL1. Next, we explored the potential biological function and pathway of miR-155-3p and miR-155-5p in ccRCC. GO analysis was carried out to assess target gene enrichments in terms of biological process (BP), cellular component (CC) and molecular function (MF). Under BP, the top GO terms were “negative regulation of protein 6
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Fig. 6. Wound-healing assays (A-B) and transwell (C-D) assay assessed the effects of shRNA-MIR155HG-1+miR-155-3p-mimic and shRNA-MIR155HG-1+miR-1555p-mimic in 786-O cells on invasion, and migration capabilities. Western blotting analysis (E-G) conducted to detect protein expression of MMP2 and MMP9. (H) Venn diagram shows miR155-3p and miR155-5p co-targeting genes. ***p < 0.001 vs. shRNA-MIR155HG-1; ###p < 0.001 vs. shRNA-MIR155HG-1+mimic-NC.
After determining the MIR155HG/miR-155-5p/-3p axis in 786-O cells, we performed functional analysis to predict the molecular mechanisms underlying miR-155-5p and miR-155-3p. To identify the cotarget genes of miR-155-3p and miR-155-5p, bioinformatics software dissected the targets of miR-155-3p and miR-155-5p predicted by miRDB and TargetScan databases. The Venn diagram exhibited that there were 13 shared targets for miR-155-3p and miR-155-5p. GO analysis subsequently revealed that the 13 co-targeting genes participated in a variety of cellular processes potentially important to ccRCC progression in the categories of biological process, cellular component and molecular function. Regulation of protein localization, cytoplasm and sequence-specific DNA binding were selected as representatives of the three major categories, respectively. These results indicated that sequence-specific DNA binding might serve vital roles in the progression of ccRCC. KEGG pathway analysis determined that these target genes participated in signaling pathways regulating the pluripotency of stem cells as it was the most significantly enriched pathway. The results of the GO and KEGG analyses suggested that MIR155HG might function as a regulator in ccRCC.
O cells migration and invasion coupled with a marked decrease in MMP2 and MMP9 expression were observed after transfection with shRNA-MIR155H. These findings confirmed that interference with MIR155HG interference hindered proliferation, invasion and migration of 786-O cells. However, in vitro experiments have shown that blocking the expression of MIR155HG can significantly reduce the expression of miR-155-5p and miR-155-3p in glioma cells [24]. This correlation indicates that MIR155HG is a precursor of miR-155. Previous studies have shown that miR-155 is increased in clear cell renal cell carcinoma cells and can inhibit the expression of clear cell renal cell carcinoma after interference [25]. In previous studies, miR155-5p was considered to be the only functional miR-155, while studies have shown that miR155-3p may have similar functions to miR-155-5p in glioma [26,27]. MiR-1555p is related to the occurrence, development and prognosis of lung cancer, gastric cancer, breast cancer, cervical cancer, pancreatic cancer and other malignant tumors, and miR-155-3p is a typical multifunctional miRNA, which has been proved to play an important role in immune response, inflammation, tumorigenesis and other processes [23,28–31]. Therefore, we focused on the functions of miR-155-5p and miR-155-3p in ccRCC. Our data suggest that interference with miR-1555p and miR-155-3p inhibits proliferation, invasion and migration of 786-O cells, consistent with previous studies. The results further demonstrated that addition of miR-155-3p and miR-155-5p rescued the inhibitory impacts of MIR155HG decrease on proliferation, invasion and migration of 786-O cells and was reversed by miR-155-3p and 5p. In conclusion, this study confirmed the important correlation between MIR155HG and miR-155, as well as the important role of miR-155-5p/3p in the biological function of MIR155HG, and the role of miR-1555p/-3p in promoting the development and progress of ccRCC.
5. Conclusions In summary, we have presented evidence that blocking lncRNA MIR155HG/miR-155-5p/-3p inhibits proliferation, invasion and migration of renal clear cell carcinoma. These findings represent a novel method for combinatorial treatment of clear cell renal cell carcinoma through the MIR155HG/miR-155-5p/-3p axis.
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Authors’ contributions [9]
MMT carried out the study design and writing of the manuscript. YZ confirmed the completeness and validity of the data. YJ participated in the data collection and analysis. JXP conceived of the study, participated in its study design, and revised the manuscript. All authors read and approved the final manuscript.
[10]
[11]
Funding [12]
Not applicable.
[13]
Ethics approval and consent to participate Not applicable
[14]
Availability of data and materials [15]
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
[16]
Consent for publication [17]
Not applicable CRediT authorship contribution statement
[18]
Meiman Tao: Writing - original draft, Methodology, Software, Investigation, Formal analysis. Yang Zhou: Methodology, Software, Investigation, Validation, Formal analysis. Yichen Jin: Methodology, Software, Data curation. Jinxian Pu: Conceptualization, Writing - review & editing, Resources, Visualization.
[19]
[20]
Declaration of Competing Interest
[21]
The authors declare that there are no conflicts of interest. [22]
Acknowledgements Not applicable.
[23]
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