ETS Proto-Oncogene 1-activated muskelin 1 antisense RNA drives the malignant progression of hepatocellular carcinoma by targeting miR-22-3p to upregulate ETS Proto-Oncogene 1
Authors: Qingzhong Yuan
Shuling Wang
Peng Luo
Guozheng Pan
Tao Cui
Faping You
Xiaomei Li
Jian Zhang
Journal: Bioengineered (2022)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: ETS1; MKLN1-AS; hepatocellular carcinoma; miR-22-3p.
Affiliations: Department of Hepatobiliary Sugery, Shengli Oilfield Central Hospital, Dongying, China, Department of Sales, Shanghai Topgen Biopharm Company Ltd, shanghai, china, Department of Medical Record, People Hospital of Dongying, Dongying, China

Abstract:  Long noncoding RNA muskelin 1 antisense RNA (MKLN1-AS) acted as an oncogenic regulator in hepatocellular carcinoma (HCC). This study was performed to investigate the functional mechanism of MKLN1-AS. MKLN1-AS, microRNA-22-3p (miR-22-3p) and ETS Proto-Oncogene 1 (ETS1) levels were examined using reverse transcription-quantitative polymerase-chain reaction. Protein expression was detected by Western blot. The target relation was analyzed by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. Cell proliferation ability was determined through cell counting kit-8 assay, colony formation assay and ethylenediurea assay. Angiogenesis was examined by tube formation assay. Cell migration and invasion were assessed via transwell assay. In vivo research was conducted by xenograft tumor model in nude mice. MKLN1-AS was upregulated in HCC tissues and cells. ETS1 promoted the ETS1 expression by binding to the 582-596 sites. Silence of MKLN1-AS suppressed cell growth, angiogenesis, migration, and invasion. MKLN1-AS interacted with miR-22-3p in HCC cells. The function of MKLN1-AS downregulation was relieved by miR-22-3p inhibition in HCC cells. ETS1 was validated as a target of miR-22-3p, and MKLN1-AS upregulated the ETS1 expression by sponging miR-22-3p. Overexpression of miR-22-3p retarded HCC progression by downregulating the level of ETS1. Tumor growth in vivo was also enhanced by MKLN1-AS through the regulation of miR-22-3p/ETS1 axis. These data demonstrated that ETS1-mediated MKLN1-AS contributed to the malignant phenotypes of HCC cells via depending on the miR-22-3p/ETS1 regulatory axis. 

Retracted: No

PubPeer comments:
 One of the qRT-PCR primers listed in Table 1 appears to be incorrect (NCBI blastn human G+T standard databases was used). Interestingly, one of the authors are affiliated to Dept. of sales (Shanghai Topgen Biopharm company), which is strange. The text is generally full of non-scientific and odd phrases. Furthermore, the last paragraph in the Discussion section is quite extraordinary, where the authors apparently seem to have been instructed to include references to other papers published in Bioengineered. There is no funding associated with this study, which is common for many papermill papers we have identified from China. ![file] , 
 #1 First of all, thank you for your questions about the article. 1u3001We have checked the primers, and the sequences were correct. When mirRNA was quantified, the specific stem-loop primers were used in reverse transcription instead of random primers or Oligo dT primers. So no other genes would be transcribed, and the primers were specific in the subsequent qPCR assay. 2u3001Luo Peng s affiliation to the sales department does not mean that he cannot participate in part of the experimental process. He helped to collect some clinical data. 3u3001Although the article is not supported by provincial and ministerial funds, there are corresponding scientific research support funds within the organization. , 
 #2 First of all, thank you for your questions about the article. 1u3001We have checked the primers, and the sequences were correct. When mirRNA was quantified, the specific stem-loop primers were used in reverse transcription instead of random primers or Oligo dT primers. So no other genes would be transcribed, and the primers were specific in the subsequent qPCR assay. 2u3001Luo Peng s affiliation to the sales department does not mean that he cannot participate in part of the experimental process. He helped to collect some clinical data. 3u3001Although the article is not supported by provincial and ministerial funds, there are corresponding scientific research support funds within the organization. , 
 I still don t get any relevant hits for humans: ![file] ![file] Additional questions: Do you have any approval number for the use of clinical specimens (HCC tissue) in this study? You explained in the paper that  protein band was analyzed via ImageJ software . How did you read the signal (instrument etc) before analysing)? , 
 Dear Indigofera tanganyikensis, I have concerns regarding the analysis of primers you have been doing. Because microRNAs are incredibly small (approximately 20-22 nts), it is not recommended to use standard PCR approaches to amplify them. Instead you will have 1 primer targeting HALF the microRNA. In this case the sequence AAGCUGCCAGUUGA (from the primer you flagged) will target the first half of the mature miR, and then you will have an RT primer (that doesn t appear to be shown) target the remainder of the miR. Furthermore, while I can get the same blastn result you did in one of the images you showed, if you scroll down a little you ll see that microRNA-22 is indeed a result. Please be more careful with your analysis. Errors in science absolutely exist, and pointing them out as you do on PubPeer is critically important. However claiming an error exists where there is none can do much more harm than good. ![file] ![file] ,