MIR-147B REGULATED PROLIFERATION AND APOPTOSIS OF GASTRIC CANCER CELLS BY TARGETING CPEB2 VIA THE PTEN PATHWAY
Tao K.1,2, Dong J-H.2, Wang D.1, Li F.2†#, Zhang Z-T.#1*
*Corresponding Author: Zhong-Tao Zhang, MD, Email: sxzhangzhongtao@sina.com, ORCID ID: 0000-0002-1184-2591 #: Zhong-Tao Zhang and Feng Li contributed equally to the article †: Co-corresponding author: Feng Li, Email: sxlifengwobuxin@sina.com, ORCID: 0000-0002-7322-422X
page: 10

RESULTS

miR-147b is up-regulated in GC cells and tissues The differentially expressed miR-147b was screened out in 3 pairs of samples by the miRNA chip (Fig. 1A), which was found three times higher than that of adjacent tissues in three random pairs of gastric cancer samples (Fig. 1B). QPCR further detected the expression of miR-147b in 50 pairs of gastric cancer and adjacent tissues, and miR-147bwasfound to be highly expressed in the gastric cancer tissues (0.53±0.02 vs 1.35±0.03, P<0.05) (Fig. 1C). The expression of miR-147b in each GC cell line was recorded as Normal cell0.99±0.01, SGC-7901: 1.55±0.03, BGC-8231.74±0.12, AGS: 1.47±0.02, MGC-803, 1.8±0.04, MKN-45, 1.44±0.03, P<0.05) (Fig. 1D, table 1). BGC-823 and MGC-803 cell lines with increased expression level of miR-147b were selected for further analysis. After transfection, the relative gene expression of miR-147b nc in the control group and miR-147b inhibitor group were found to be 1.00±0.09, and 0.23±0.07 respectively, with a significant difference (t = 3.07, P < 0.05), indicating successful transfection (Fig. 1D). miR-147b promotes tumor growth in human cell lines Scratch analysis showed that the miR-147b inhibitor group inhibited GC cell growth and reduced cell migration compared with miR-147b NC (Fig. 1E). In addition, by flow cytometry, we have also analyzed the apoptotic effect of miR-147b. As compared with miR-147b NC, miR-147b inhibitor increased the early apoptosis of MGC-803 cells [(61.8±0.2)% vs (55.4±0.1)% ]. It increased the early apoptosis of BGC-823 cells [(35.5 ± 0.2)% vs. (23.1 ± 0.2)%] (Fig. 2A). On the other hand, clone formation experiments showed that themiR-147b inhibitor drastically restricts the proliferation of BGC-823 and MGC-803 cells (Fig. 2B, table 2). CCK-8 proliferation experiment showed that themiR-147b inhibitor significantly inhibited the growth of BGC-823 and MGC-803 cells, and inhibition rate was more prominent as the time increases (t = 2.78, P < 0.05) (Fig. 2C and D, table 3). miR-147b affects the expression of CPEB2 in GC cells We searched the target gene CPEB2 that can bind to miR-147b through the target scan database, and we mapped the base pairs that miR-147b crosses with the 3 ‘untranslated region of the predicted target gene CPEB2 (Fig. 3A). We selected MGC-803 with relatively high endogenous CPEB2 content to detect whether the miR-147b inhibitor of miR-147b NC affects CPEB2 expression. The double luciferase reporter vectors of CPEB2 wild-type and mutant were further constructed (Fig.3A). The above vectors were co-transfected with NC and miR-147b inhibitor into MGC- 803 cells, respectively. It was found that CPEB2 was the target gene directly affected by miR-147b. In the presence of CPEB23 ‘- UTR, this activity produced about 20% (Fig. 3B), indicating that the expression of miR-147b inhibitor accelerated the activity of CPEB23’-UTR. Western blot showed that the expression of CPEB2 in MGC-803 cells in miR-147b inhibitor group and miR-147b NC+CPEB2 group showed higher concentration as compared to negative control group (Fig. 3C). These results suggest that CPEB2 is the target of miR-147b and is negatively regulated by it. B2 was highly expressed in GC cells and correlates with prognosis Quantitative PCR (qPCR) analysis of 50 pairs of clinical GC tissues and adjoining normal tissue samples showed that CPEB2 mRNA expression in tumor tissues was down regulated in 40 patients (80%) (Fig. 4A). The non-parametric test results of the relationship between CPEB2 expression and clinicopathological factors showed that the lower CPEB2 level was related to the GC pTNM stage (P < 0.05, stages I-II and III-IV, FIG. 4B). In addition, GC patients with lower CPEB2 expression showed lower survivability and improved prognosis (Fig. 4C). Immunohistochemical analysis showed that CPEB2 staining increased significantly in adjacent normal tissues (Fig. 4D). H & E staining showed more metastatic nodules in GC tissues compared with adjacent normal tissues (Fig. 4D). CPEB2 was found highly expressed in GC cells. CPEB2 promoted the PTEN pathway Western blot results showed that the expression of PTEN protein decreased significantly after reducing CPEB2 in 803 and 823 cell lines. At the same time, the expression of tumor suppressor genes p27 and E-cadherin was inhibited, while the expression of Ki67 and p21 reflecting tumor activity index increased significantly (Fig. 4E).



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