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LOC646329 long non-coding RNA sponges miR-29b-1 and regulates TGFβ signaling in colorectal cancer

Abstract

Non-coding RNAs (ncRNAs) are reported to be regulators of signaling pathways that are involved in colorectal cancer (CRC) progression. Aiming at finding ncRNAs (miRNAs) that are differentially expressed in tumor versus normal colorectal tissue samples, online RNA-seq data were analyzed. Of between 18 candidate miRNAs, hsa-miR-29b-1 (miR-29b-1) represented the highest fold change of expression level. Hsa-miR-29b-1 is encoded from the third intron of LOC646329 long ncRNA gene. Surprisingly, two miR-29b sponging sites were predicted within exons of LOC646329 gene. Then, dual luciferase assay supported the interaction of miR-29b-1 with LOC646329-variant D transcript. Also, a direct indication of miR-29b-1 with 3′UTR sequence of SMAD3 gene was verified through dual luciferase assay and RT-qPCR analysis. Furthermore, a reverse pattern of expression was detected between miR-29b-1 and LOC646329-variant D transcript in about 25 pairs of CRC tumor samples, detected by RTqPCR. Consistently, overexpression of LOC646329-variant D transcript was followed by increased SMAD3 and p21 genes expression level and downregulation of CyclinD1 genes in HCT116 cells, detected by RT-qPCR, and western analysis. Also, overexpression of it was followed by increased G1 cell population of HCT-116 cells. All of these data suggested a tumor suppressor effect for LOC646329-variant D in CRC tumor tissue samples, consistent to its reduced expression level at late stages of CRC progression. Data also indicated that LOC646329-variant D exerts its suppression effect on CRC progression through sponging miR-29b, which in turn regulates Wnt and TGFB signaling pathways. This makes LOC646329-variant D transcript as a novel potential therapy target.

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Fig. 1

adopted from TCGA database. b Drawn heat map for the top 20 miRNAs which are differentially expressed in normal and tumor CRC samples using heatmap2 package in R programing. c Target predictions of 10 candidate miRNAs. The MAPK and TGFβ signaling were the major pathways to be targeted by candidate miRNAs

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Acknowledgements

Authors are thankful to the helps and advices of all lab mate in room 4402 at Genetics Dept. TMU, Tehran, Iran.

Funding

This work was supported by TMU and NIMAD (grant number 943387).

Author information

AJ performed the experiments and analyzed data, AJ and BS wrote manuscript; AJ and SD and BS designed experiments; BS supervised the study. SD advised the research. MB provided tumor/normal tissues and provided pathology information of them.

Correspondence to Bahram M. Soltani.

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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Javanmard, A., Dokanehiifard, S., Bohlooli, M. et al. LOC646329 long non-coding RNA sponges miR-29b-1 and regulates TGFβ signaling in colorectal cancer. J Cancer Res Clin Oncol (2020). https://doi.org/10.1007/s00432-020-03145-6

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Keywords

  • Colorectal cancer
  • lncRNA
  • miR-29b
  • Tgfβ signaling pathway
  • SMAD3