Open Access

miR-30c-2-3p suppresses the proliferation of human renal cell carcinoma cells by targeting TOP2A

Xiaoyong Huang

Xiaoyong Huang

Department of Clinical Medicine, Medical College of Yan’an UniversityYan’an, China
Search for this author on
Sciendo|Google Scholar
Huang, Xiaoyong
, 
Yuna Jia

Yuna Jia

Department of Clinical Medicine, Medical College of Yan’an UniversityYan’an, China
Search for this author on
Sciendo|Google Scholar
Jia, Yuna
, 
Haiyan Shi

Haiyan Shi

Department of Clinical Medicine, Medical College of Yan’an UniversityYan’an, China
Search for this author on
Sciendo|Google Scholar
Shi, Haiyan
, 
Haiyan Fan

Haiyan Fan

Department of Laboratory, The First Hospital of YulinYulin, China
Search for this author on
Sciendo|Google Scholar
Fan, Haiyan
, 
Lingbo Sun

Lingbo Sun

Department of Clinical Medicine, Medical College of Yan’an UniversityYan’an, China
Search for this author on
Sciendo|Google Scholar
Sun, Lingbo
, 
Huahua Zhang

Huahua Zhang

Department of Clinical Medicine, Medical College of Yan’an UniversityYan’an, China
Search for this author on
Sciendo|Google Scholar
Zhang, Huahua
, 
Yanfeng Wang

Yanfeng Wang

Clinical Laboratory of Affiliated Hospital of Yan’an UniversityYan’an, China
Search for this author on
Sciendo|Google Scholar
Wang, Yanfeng
, 
Jie Chen

Jie Chen

Department of Clinical Medicine, Medical College of Yan’an UniversityYan’an, China
Search for this author on
Sciendo|Google Scholar
Chen, Jie
, 
Jiaqi Han

Jiaqi Han

Department of Clinical Medicine, Medical College of Yan’an UniversityYan’an, China
Search for this author on
Sciendo|Google Scholar
Han, Jiaqi
, 
Mingming Wang

Mingming Wang

Department of Clinical Medicine, Medical College of Yan’an UniversityYan’an, China
Search for this author on
Sciendo|Google Scholar
Wang, Mingming
, 
Juan Du

Juan Du

Department of Clinical Medicine, Medical College of Yan’an UniversityYan’an, China
Search for this author on
Sciendo|Google Scholar
Du, Juan
 and 
Jing Zhang

Jing Zhang

  • Department of Clinical Medicine, Medical College of Yan’an UniversityYan’an, China
  • Yan’an Key Laboratory of Chronic Disease Prevention and ResearchYan’an, China
Search for this author on
Sciendo|Google Scholar
Zhang, Jing
  
Oct 09, 2023
miR-30c-2-3p suppresses the proliferation of human renal cell carcinoma cells by targeting TOP2A's Cover Image
About this article
Previous Article
Next Article
Cite
Download Cover
Article Category: Original article
Published Online: Oct 09, 2023
Page range: 124 - 135
DOI: https://doi.org/10.2478/abm-2023-0052
Keywords
© 2023 Xiaoyong Huang et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1.

miR-30c-2-3p expression in RCC cells. (A) The TCGA database was used to undertake an expression study of miR-30c-2-3p in RCC tissues (n = 241) and normal adjacent tissues (n = 70). (B) Analysis of the 5-year overall survival rate for RCC patients. (C) The expression of miR-30c-2-3p in RCC cell lines (786-O, Caki-1, and ACHN) and normal renal tubular epithelial cell line was examined using qRT-PCR (HK-2). ***P < 0.001.miR, microRNA; qRT-PCR, quantitative real-time polymerase chain reaction; RCC, renal cell carcinoma; TCGA, The Cancer Genome Atlas.
miR-30c-2-3p expression in RCC cells. (A) The TCGA database was used to undertake an expression study of miR-30c-2-3p in RCC tissues (n = 241) and normal adjacent tissues (n = 70). (B) Analysis of the 5-year overall survival rate for RCC patients. (C) The expression of miR-30c-2-3p in RCC cell lines (786-O, Caki-1, and ACHN) and normal renal tubular epithelial cell line was examined using qRT-PCR (HK-2). ***P < 0.001.miR, microRNA; qRT-PCR, quantitative real-time polymerase chain reaction; RCC, renal cell carcinoma; TCGA, The Cancer Genome Atlas.

Figure 2.

RCC cells experience reduced proliferation and increased apoptosis when miR-30c-2-3p is expressed more typically. (A) miR-30c-2-3p expression after transfection. (B) Cell proliferation at 24 h, 48 h, and 72 h posttransfection. (C) After transfection, 786-O, Caki-1, and ACHN cells form colonies. (D) Analysis of apoptosis by flow cytometry following transfection with NC or miR-30c-2-3p mimics. (E) Fas, FasL, caspase 3, and caspase 8 protein expression in 786-O, Caki-1, and ACHN cells after miR-30c-2-3p mimic transfection or NC. *P < 0.05, **P < 0.01, ***P < 0.001. miR, microRNA; NC, negative control; RCC, renal cell carcinoma; TOP2A, DNA topoisomerase II alpha; UTR, untranslated region.
RCC cells experience reduced proliferation and increased apoptosis when miR-30c-2-3p is expressed more typically. (A) miR-30c-2-3p expression after transfection. (B) Cell proliferation at 24 h, 48 h, and 72 h posttransfection. (C) After transfection, 786-O, Caki-1, and ACHN cells form colonies. (D) Analysis of apoptosis by flow cytometry following transfection with NC or miR-30c-2-3p mimics. (E) Fas, FasL, caspase 3, and caspase 8 protein expression in 786-O, Caki-1, and ACHN cells after miR-30c-2-3p mimic transfection or NC. *P < 0.05, **P < 0.01, ***P < 0.001. miR, microRNA; NC, negative control; RCC, renal cell carcinoma; TOP2A, DNA topoisomerase II alpha; UTR, untranslated region.

Figure 3.

TOP2A is the direct target of miR-30c-2-3p. (A) Predicted binding site of miR-30c-2-3p in TOP2A mRNA 3′-UTR. (B) The sequences of wild-type (TOP2A W) and mutant (TOP2A M) genes. (C) Dual-luciferase reporter assay to determine the influence of miR-30c-2-3p on wild-type and mutant miR-30c-2-3p binding site on TOP2A 3‘-UTR. Cells transfected with miR-30c-2-3p overexpression plasmid and GLO plasmids were used as controls. (D, E) TOP2A mRNA and protein expression in 786-O, Caki-1, and ACHN cells following transfection with miR-30c-2-3p mimics or NC. **P < 0.01.miR, microRNA; NC, negative control TOP2A, DNA topoisomerase II alpha; UTR, untranslated region.
TOP2A is the direct target of miR-30c-2-3p. (A) Predicted binding site of miR-30c-2-3p in TOP2A mRNA 3′-UTR. (B) The sequences of wild-type (TOP2A W) and mutant (TOP2A M) genes. (C) Dual-luciferase reporter assay to determine the influence of miR-30c-2-3p on wild-type and mutant miR-30c-2-3p binding site on TOP2A 3‘-UTR. Cells transfected with miR-30c-2-3p overexpression plasmid and GLO plasmids were used as controls. (D, E) TOP2A mRNA and protein expression in 786-O, Caki-1, and ACHN cells following transfection with miR-30c-2-3p mimics or NC. **P < 0.01.miR, microRNA; NC, negative control TOP2A, DNA topoisomerase II alpha; UTR, untranslated region.

Figure 4.

Expression of TOP2A in RCC tissues and cell lines. (A) Expression analysis of TOP2A in RCC tissues (n = 534) and normal adjacent tissues (n = 72) performed in TCGA database. (B) Five-year overall survival percentage analysis of patients with RCC. (C, D) TOP2A expression in 786-O, Caki-1, ACHN, and HE-2 cells. (E) TOP2A expression in RCC and adjacent tissues. **P < 0.01, ***P < 0.001.miR, microRNA; RCC, renal cell carcinoma; TCGA, The Cancer Genome Atlas; TOP2A, DNA topoisomerase II alpha; UTR, untranslated region.
Expression of TOP2A in RCC tissues and cell lines. (A) Expression analysis of TOP2A in RCC tissues (n = 534) and normal adjacent tissues (n = 72) performed in TCGA database. (B) Five-year overall survival percentage analysis of patients with RCC. (C, D) TOP2A expression in 786-O, Caki-1, ACHN, and HE-2 cells. (E) TOP2A expression in RCC and adjacent tissues. **P < 0.01, ***P < 0.001.miR, microRNA; RCC, renal cell carcinoma; TCGA, The Cancer Genome Atlas; TOP2A, DNA topoisomerase II alpha; UTR, untranslated region.

Figure 5.

TOP2A interference inhibits growth of RCC cells and promotes their apoptosis. ACHN, 786-O, and Caki-1 cells were transfected with shTOP2A-1, shTOP2A-2, or a negative control. (A, B) TOP2A mRNA and protein abundance after transfection. (C) Cell proliferation after transfection. (D) Colony formation in 786-O, Caki-1, and ACHN cells after transfection. (E) Study of apoptosis by flow cytometry following transfection with shTOP2A-1, shTOP2A-2, or a negative control. (F) Expression of Fas, FasL, caspase 8, and caspase 3 after transfection with shTOP2A-1, shTOP2A-2, or a negative control. *P < 0.05, **P < 0.01, ***P < 0.001.miR, microRNA; RCC, renal cell carcinoma; sh, short hairpin; TOP2A, DNA topoisomerase II alpha.
TOP2A interference inhibits growth of RCC cells and promotes their apoptosis. ACHN, 786-O, and Caki-1 cells were transfected with shTOP2A-1, shTOP2A-2, or a negative control. (A, B) TOP2A mRNA and protein abundance after transfection. (C) Cell proliferation after transfection. (D) Colony formation in 786-O, Caki-1, and ACHN cells after transfection. (E) Study of apoptosis by flow cytometry following transfection with shTOP2A-1, shTOP2A-2, or a negative control. (F) Expression of Fas, FasL, caspase 8, and caspase 3 after transfection with shTOP2A-1, shTOP2A-2, or a negative control. *P < 0.05, **P < 0.01, ***P < 0.001.miR, microRNA; RCC, renal cell carcinoma; sh, short hairpin; TOP2A, DNA topoisomerase II alpha.

Figure 6.

RCC development regulated by miR-30c-2-3p is caused by TOP2A. (A, B) TOP2A expression in 786-O, Caki-1, and ACHN cells treated with miR-30c-2-3p inhibitors (miR-30c-in) and/or TOP2A interference RNAs (shTOP2A). (C, D) Viability and colony formation results of 786-O, Caki-1, and ACHN cells treated with miR-30c-2-3p inhibitors (miR-30c-in) and/or TOP2A interference RNAs (shTOP2A). (E) Apoptosis changes of 786-O, Caki-1, and ACHN cells pre-transfected with different RNAs for 48 h. (F) Abundance of Fas/FasL/caspase 8/caspase 3/Bcl2/caspase 9 proteina in 786-O, Caki-1, and ACHN cells treated with miR-30c-2-3p inhibitors (miR-30c-in) and/or TOP2A interference RNAs (shTOP2A). *P < 0.05, **P < 0.01, ***P < 0.001.miR, microRNA; RCC, renal cell carcinoma; sh, short hairpin; TOP2A, DNA topoisomerase II alpha.
RCC development regulated by miR-30c-2-3p is caused by TOP2A. (A, B) TOP2A expression in 786-O, Caki-1, and ACHN cells treated with miR-30c-2-3p inhibitors (miR-30c-in) and/or TOP2A interference RNAs (shTOP2A). (C, D) Viability and colony formation results of 786-O, Caki-1, and ACHN cells treated with miR-30c-2-3p inhibitors (miR-30c-in) and/or TOP2A interference RNAs (shTOP2A). (E) Apoptosis changes of 786-O, Caki-1, and ACHN cells pre-transfected with different RNAs for 48 h. (F) Abundance of Fas/FasL/caspase 8/caspase 3/Bcl2/caspase 9 proteina in 786-O, Caki-1, and ACHN cells treated with miR-30c-2-3p inhibitors (miR-30c-in) and/or TOP2A interference RNAs (shTOP2A). *P < 0.05, **P < 0.01, ***P < 0.001.miR, microRNA; RCC, renal cell carcinoma; sh, short hairpin; TOP2A, DNA topoisomerase II alpha.

Figure S1.

Transfection efficiency verification of miR-30c-2-3p inhibitor. (A) miR-30c-2-3p expression after transfection with NC or miR-30c-2-3p inhibitor. (B) The mRNA expression of TOP2A after transfection with NC or miR-30c-2-3p inhibitor. (C) TOP2A protein expression in 786-O, Caki-1, and ACHN cells after miR-30c-2-3p mimic transfection or NC. **P < 0.01, ***P < 0.001.miR, microRNA; TOP2A, DNA topoisomerase II alpha.
Transfection efficiency verification of miR-30c-2-3p inhibitor. (A) miR-30c-2-3p expression after transfection with NC or miR-30c-2-3p inhibitor. (B) The mRNA expression of TOP2A after transfection with NC or miR-30c-2-3p inhibitor. (C) TOP2A protein expression in 786-O, Caki-1, and ACHN cells after miR-30c-2-3p mimic transfection or NC. **P < 0.01, ***P < 0.001.miR, microRNA; TOP2A, DNA topoisomerase II alpha.

Primers used in this work

Name Sequence (5′–3′)
TOP2A-R TTGGCATCATCGAGTTTGGGA
TOP2A-F TGGCTGTGGTATTGTAGAAAGC
GAPDH-F GACTTCAACAGCAACTCCCA
GAPDH-R TGGGTGGTCCAGGGTTTCTT
miR-30c-2-3p-RT GTCGTATCCAGTGCGTGTCGTGGA-GTCGGCAATTGCACTGGATACGACAGAGTAA
miR-30c-2-3p-F ATCCAGTGCGTGTCGTG
miR-30c-2-3p-R TGCTCTGGGAGAAGGCTGT
U6-RT CGCTTCACGAATTTGCGTGTCAT
U6-R CGCTTCACGAATTTGCGTGTCAT
U6-F GCTTCGGCAGCACATATACTAAAAT
Language:
English
Publication timeframe:
6 times per year
Journal Subjects:
Medicine, Assistive Professions, Nursing, Basic Medical Science, Basic Medical Science, other, Clinical Medicine, Clinical Medicine, other
Journal RSS Feed