<p>Knockdown of circ_0000512 Inhibits Cell Proliferation and Promotes Apoptosis in Colorectal Cancer by Regulating miR-296-5p/RUNX1 Axis</p>
Authors: Huili Wu
Li Zhang
Lihong Wang
Feifei Chu
Xingguo Xiao
Journal: OncoTargets and Therapy (2020)
Discipline: CLINICAL MEDICINE
Keywords: -miR-296-5p; RUNX1; apoptosis; circ_0000512; colorectal cancer
Affiliations: Department of Gastroenterology, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450000, Henan, People's Republic of China.

Abstract:  Background: Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. Increasing evidence showed that circular RNAs (circRNAs) played critical roles in the progression of CRC. However, the effects and underlying mechanisms of circ_0000512 in CRC progression remain unclear. Methods: The expression levels of circ_0000512, microRNA-296-5p (miR-296-5p) and runt-related transcription factor 1 (RUNX1) were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability, colony formation, cell cycle distribution and cell apoptosis were detected by Cell Counting Kit-8 (CCK-8) assay, colony formation assay and flow cytometry analysis, respectively. Western blot assay was utilized to measure the protein expression of Cyclin D1, Cleaved Caspase-3 and RUNX1. The interaction between miR-296-5p and circ_0000512 or RUNX1 was predicted by starBase and verified by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay. The mice xenograft model was established to explore the function of circ_0000512 in vivo. Results: The expression of circ_0000512 was increased in CRC tissues and cells. Knockdown of circ_0000512 suppressed cell viability and colony formation and arrested the cells at the G0/G1 phase while it accelerated apoptosis in CRC cells. Mechanistically, circ_0000512 could increase RUNX1 expression by acting as a molecular sponge of miR-296-5p in CRC cells. Furthermore, miR-296-5p downregulation or RUNX1 overexpression reversed the anti-proliferation and pro-apoptosis effects caused by circ_0000512 knockdown in CRC cells. In addition, circ_0000512 interference inhibited tumor growth by upregulating miR-296-5p and downregulating RUNX1 in vivo. Conclusion: Knockdown of circ_0000512 inhibited cell proliferation and induced apoptosis in CRC cell by regulating miR-296-5p/RUNX1 axis, which might provide a potential therapeutic target for CRC treatment. 

Retracted: No

PubPeer comments:
 This is one of several papers from different author teams that report using the same forward and reverse primers for quantitative PCR of miR-296-5p or miR-296-3p. Here, the  TaqMan MicroRNA Reverse Transcription Kit is said to have been used to reverse transcribe the microRNA. Then  qRT-PCR analysis was conducted using the SYBR Premix Ex Taq  with what are described as miR-296-5p specific primers  (Forward, 5u02b9-TGCCTAATTCAGAGGGTTGG-3u02b9; Reverse, 5u02b9-CTCCACTCCTGGCACACAG-3u02b9) . No reliable results for miR-296-5p would have been obtained with these methods. The TaqMan MicroRNA Reverse Transcription Kit cannot convert miRNAs into cDNA without a microRNA-specific RT primer that is sold separately along with proprietary qPCR primers and a TaqMan probe. When the correct RT primer is used, the product is DNA in which the mature miRNA sequence is attached to a longer proprietary sequence that was introduced by the RT primer. This sequence is needed for qPCR because the miRNA is otherwise not long enough for two primers and a probe to attach. The cDNA must then be amplified by the proprietary, miRNA-specific primer probe mixture sold along with the RT primer. Although the sequences are proprietary (or were), basically, there s the forward primer that covers the 5 part of the mature miRNA, a probe with several nucleotides corresponding to the 3 part of the mature miRNA and adjacent nucleotides in the RT-introduced sequence, and a reverse primer that recognizes another portion of the RT-introduced sequence. You could not amplify this cDNA with anything other than these primers. You could not just use another portion of the miR-296 precursor or primary transcript, for example, because it would not be part of the reverse-transcribed cDNA. What about the primers published in this paper? Here is the precursor sequence for miR-296, with the miR-296-5p arm (the one that is supposed to be amplified in this paper) in bold: AGGACCCUUCCAG**AGGGCCCCCCCUCAAUCCUGU**UGUGCCUAAUUCAGAGGGUUGGGUGGAGGCUCUCCUGAAGGGCUCU And here in bold is where the published  forward primer sequence would bind: AGGACCCUUCCAGAGGGCCCCCCCUCAAUCCUGUUG**UGCCUAAUUCAGAGGGUUGG**GUGGAGGCUCUCCUGAAGGGCUCU Obviously, this primer does not capture miR-296-5p at all. Also, the reverse primer recognizes a sequence adjacent to the miR-296 precursor in the genome, but this is not part of the precursor RNA. These forward and reverse primers might amplify genomic DNA, but they would not tell us anything about miR-296-5p and they would not make any product from the TaqMan MicroRNA Reverse Transcription Kit, even if the correct RT primer had been used. , 
 The papers that use these primers and methods incorrectly include the following: 1. [Zhang et al, 2019] 2. [Huang et al, 2021] 3. [Fan et al, 2020] 4. [Wang et al, 2020] 5. [Wang et al, 2020] 6. [Li et al, 2019] 7. [Zhang et al, 2021] 8. [Li et al, 2017] 9. [Ni et al, 2020] It appears to me that the first use of these primers in the literature was by a group that used them correctly, to examine the levels of the **primary** miR-296-containing transcript (Lee, et al, Mol Cells, 2017, and I want to stress that I find no problems with this paper.) It would seem that the methods and primers were subsequently copied without an understanding of the difference between primary, precursor, and mature miRNAs, or of the fact that precursor can give rise to different mature miRNAs. It is sobering for me to realize that entire author teams could be involved in putative microRNA research without understanding the fundamentals. ,