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MIR-147B regulated proliferation and apoptosis of gastric cancer cells by targeting CPEB2 via the PTEN pathway

Tao К
Tao К
, 
J-H. Dong
J-H. Dong
, 
D. Wang
D. Wang
, 
F. Li
F. Li
 et 
Z-T. Zhang
Z-T. Zhang
   | 01 mars 2023
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Article Category: Original Article
Publié en ligne: 01 mars 2023
Pages: 61 - 70
DOI: https://doi.org/10.2478/bjmg-2022-0007
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© 2022 Tao К., Dong J-H., Wang D., Li F., Zhang Z-T., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

(A) MiRNA expression in 3 GC tissues and adjacent normal tissues was analyzed by microarray analysis, (B) Quantification of microarray data showed that miR-147b was up-regulated in GC tissues. The data are expressed as mean multiple changes in miR-147b levels in GC tissues normalized to their corresponding controls.* P < 0.05, t test; (C) QPCR analysis showed that miR-147b was up-regulated in most GCs. The data shown are multiples of miR-147b expressions in GC cases normalized to paired adjacent normal tissues; (D) The expression of miR-147b in cultured GC cell lines and normal cells was analyzed by qPCR; (E) Wound healing analysis of BGC-823 and MGC-803 cells treated with miR-147b inhibitor or miR-147b NC.
(A) MiRNA expression in 3 GC tissues and adjacent normal tissues was analyzed by microarray analysis, (B) Quantification of microarray data showed that miR-147b was up-regulated in GC tissues. The data are expressed as mean multiple changes in miR-147b levels in GC tissues normalized to their corresponding controls.* P < 0.05, t test; (C) QPCR analysis showed that miR-147b was up-regulated in most GCs. The data shown are multiples of miR-147b expressions in GC cases normalized to paired adjacent normal tissues; (D) The expression of miR-147b in cultured GC cell lines and normal cells was analyzed by qPCR; (E) Wound healing analysis of BGC-823 and MGC-803 cells treated with miR-147b inhibitor or miR-147b NC.

Figure 2

(A) After treatment with miR-147b inhibitor or miR-147b NC and staining with apoptosis markers (FITC annexin V and PI), the apoptosis of BGC-823 and MGC-803 cells was examined by flow cytometry. In the apoptosis diagram, FITC Annexin V + / PI + represents late apoptosis, FITC Annexin V + / pi - represents early apoptosis, and FITC Annexin V- / pi - represents normal living cells; (B) Clonogenic assay of BGC-823 and MGC-803 cells treated with miR-147b inhibitor or miR-147b NC. For all qPCR results, the data are expressed as mean ± SEM, and the error line represents the standard deviation obtained from three independent experiments; (C) Cell proliferation assay of BGC-823 and MGC-803 cells treated with miR-147b inhibitor of CCK-8 or miR-147b NC. (D) MiR-147b was up-regulated in GC cells.
(A) After treatment with miR-147b inhibitor or miR-147b NC and staining with apoptosis markers (FITC annexin V and PI), the apoptosis of BGC-823 and MGC-803 cells was examined by flow cytometry. In the apoptosis diagram, FITC Annexin V + / PI + represents late apoptosis, FITC Annexin V + / pi - represents early apoptosis, and FITC Annexin V- / pi - represents normal living cells; (B) Clonogenic assay of BGC-823 and MGC-803 cells treated with miR-147b inhibitor or miR-147b NC. For all qPCR results, the data are expressed as mean ± SEM, and the error line represents the standard deviation obtained from three independent experiments; (C) Cell proliferation assay of BGC-823 and MGC-803 cells treated with miR-147b inhibitor of CCK-8 or miR-147b NC. (D) MiR-147b was up-regulated in GC cells.

Figure 3

(A) Double luciferase reporter vector of CPEB2 wild type and mutant, (B) Expression of miR-147b inhibitor accelerates CPEB2 3 ‘- UTR activity, (C) Expression of CPEB2 in MGC-803 cells of different groups.
(A) Double luciferase reporter vector of CPEB2 wild type and mutant, (B) Expression of miR-147b inhibitor accelerates CPEB2 3 ‘- UTR activity, (C) Expression of CPEB2 in MGC-803 cells of different groups.

Figure 4

(A)Expression of CPEB2 mRNA in GC tissues and adjacent normal tissues, (B) Nonparametric test results of the relationship between CPEB2 expression and clinicopathological factors, (C)Overall survival and disease-free survival in GC patients with lower CPEB2 expression, (D) immunohistochemical analysis results; (E) Results of immunoblotting.
(A)Expression of CPEB2 mRNA in GC tissues and adjacent normal tissues, (B) Nonparametric test results of the relationship between CPEB2 expression and clinicopathological factors, (C)Overall survival and disease-free survival in GC patients with lower CPEB2 expression, (D) immunohistochemical analysis results; (E) Results of immunoblotting.

Expression of transfected miRNA-147b in normal cells and different gastric cancer cells detected by RT-PCR (x±s)

Cell grouping Number of samples miR-147b T value Pvalue
Normal cell 3 0.99±0.01
SGC-7901 3 1.55±0.03 2.91 <0.05
BGC-823 3 1.74±0.12 2.91 <0.05
AGS 3 1.47±0.02 2.35 <0.05
MKN-45 3 1.44±0.03 2.91 <0.05
MGC-803 3 1.8±0.04 2.91 <0.05

Effect of transfection of the miR-147b inhibitor on the proliferation inhibition rate of gastric cancer cells by CCK-8 method (x±s)

grouping Cell Number samples of miR-147bnc miR-inhibitor 147b T value P value
MGC-803
0h 3 0.200±0.04 0.20±0.03 3.11 >0.05
24h 3 0.395±0.05 0.33±0.03 2.76 >0.05
48h 3 1.030±0.07 0.67±0.05 2.77 <0.05
60h 3 1.720±0.09 0.98±0.05 2.77 <0.05
72h 3 2.14±0.09 1.23±0.07 2.78 <0.05
BGC-823
0 h 3 0.20±0.04 0.20±0.03 3.07 >0.05
24h 3 0.49±0.05 0.33±0.03 2.78 >0.05
48h 3 1.13±0.07 0.62±0.05 2.18 <0.05
60h 3 1.56±0.09 0.85±0.05 2.77 <0.05
72h 3 1.84±0.1 1.02±0.07 2.78 <0.05

Effect of transfection of the miR-147b inhibitor on the number of clones of gastric cancer MGC-803 and BGC-823 cells

Group MGC-803 BGC-823
miR-147b nc 638±15 632±17
miR-147b inhibitor group 464±11 448±15
T value 2.35 2.35
P value < 0.05 < 0.05

Correlation between clinicopathological features and miR-147 expression in gastric cancer

Variance Number of samples miR-147b Low(n%) expression High(n%) χ2 P value
Age
<60 22 4 (18.1%) 18 (81.9%) 0.081 P >0.05
≥ 60 28 6 (21.4%) 22 (78.6%)
Gender
male 29 7 (24.1%) 22 (75.9%) 0.176 P >0.05
female 21 3 (14.3%) 17 (85.7%)
Tumor size
<4 cm 33 6 (18.2%) 27 (81.8%) 0.006 P >0.05
≥4 cm 17 4 (23.5%) 13 (76.5%)
TNM Stage
I-II 20 7 (35%) 13 (65%) 3.755 P =0.048
III-IV 30 3 (10%) 27 (90%)
Lymph node metastasis
No 23 8 (34.8%) 15 (65.2%) 4.232 P =0.040
Yes 27 2 (7.4%) 25 (92.6%)
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