FOXP1-Induced Long Non-Coding RNA linc01003 Regulating Proliferation and Apoptosis of Colorectal Cancer Cells Through EZH2/HKDC1 and miR-106b-5p/DIC Regulation Axis

Abstract

Background: Long non-coding RNA with a length of more than 200 nucleotides, called long non-coding RNA (LncRNAs), lncRNAs can regulate its activity or change its location by interfering with the activity of mRNA and directly binding to proteins. It can also affect the expression of downstream genes by inhibiting RNA polymerase, And as a competitive endogenous RNAs to regulate gene expression, thus playing an important role in the biological process. Colorectal cancer is a malignant lesion of colorectal mucosa caused by environmental, genetic and epigenetic factors. The occurrence of colorectal cancer is a multi-stage, multi-gene process, but its pathogenesis is not fully understood. With the discovery of new molecular and epigenetic mechanisms in CRC, lncRNA has become a biological target for diagnostic, prognostic and therapeutic applications. Methods: Therefore, by screening the GEO database, we found that the expression of Linc01003 in cancer tissues was significantly different from that in paracancerous tissues, and then the expression was detected in 60 pairs of colorectal cancer tissues, five colorectal cancer cell lines and normal intestinal epithelial cell lines. In order to verify the phenotype of linc01003 by knockdown and overexpression experiments, and to explore the mechanism of linc01003 regulating mechanism of proliferation and apoptosis, the upstream transcription factors were predicted by database and verified by related experiments. The related downstream targets were screened by high throughput sequencing and verified by qRT-PCR, Western blot, RIP, ChIP, RNA-FISH and other experiments. Results: Highly expressed linc01003 is closely related to more advanced pathological stages and shorter life cycles.The results showed that the expression of LncRNA Linc01003 induced by transcription factor FOXP1 was significantly increased in colorectal cancer tissues and cells. Knockdown of Linc01003 in colorectal cells could significantly inhibit cell proliferation and induce apoptosis, while overexpression could promote cell proliferation. This can also be proved in animal experiments. After high throughput sequencing, it was found that the expression of crude oncogene HKDC1 was up-regulated and the expression of tumor suppressor gene DIC was down-regulated. Then through bioinformatics prediction and RIP experiments, it was proved that linc01003 binds to EZH2 and AGO2 proteins. CHIP experiments showed that Linc01003 could bind EZH2 to participate in the transcriptional regulation of HKDC1. Luciferase reporter gene confirmed that linc01003 competitive adsorption of miR-106b-5p promoted SLC25A10( DIC) expression. Therefore, we conclude that long non-coding RNA Linc01003 with high expression is associated with later stage and worse prognosis, and transcription factor FOXP1 can induce high expression of long non-coding RNA Linc01003. The high expression of linc01003 promotes the proliferation and inhibits the apoptosis of colorectal cancer cells. Lin01003 promotes the proliferation of colorectal cancer cells through EZH2/HKDC1 and miR-106b-5p/DIC regulatory axes. Conclusions: Therefore, we conclude that the transcription factor FOXP1 induced linc01003 promotes proliferation and inhibits apoptosis of colorectal cancer cells through EZH2/HKDC1 and miR-106b-5p/DIC regulatory axis . It is expected to be a diagnostic marker for CRC and a potential biotherapeutic target. Funding Statement: This study was supported by grants from the National Natural Sciences Foundation of China (grant no. 81772603), Key talents of young medicine in Jiangsu Province(grant no.QNRC2016662), Nanjing Health Science and Technology Development Medical Key Technology Development Project (grant no.YKK18190). Declaration of Interests: The authors declare no competing financial interests. Ethics Approval Statement: All patients provided written informed consent, and all the experiments were approved by the Research Ethics Committee of Nanjing Medical University. This study was conducted in strict accordance with the guidelines of the National Institutes of Health (NIH) on the use of laboratory animals. Our plan has been approved by the Animal Experimental Ethics Committee of Nanjing Medical University.