miR-449a and CDK6 in gastric carcinoma

Abstract

The present study aimed to identify the association between microRNA (miR/miRNA)-449a, the cyclin-dependent kinase (CDK)6 protein and gastric carcinoma, and discuss the effect of miR-449a on the expression of the CDK6 protein. Quantitative (q)PCR and western blot analysis were used to analyze the expression of the miR-449a and the CDK6 protein in gastric carcinoma and tumor-adjacent normal tissues. The real-time cell analyzer and the DAPI staining test were used to monitor the different miR-449a levels regulating the proliferation and apoptosis of the MGC-803 cell line. Immunofluorescence and western blot analyses were used to detect the expression level of the CDK6 protein in the cells of the miR-449a upregulation and downregulation groups, and a control group. A scratch test was used to study the effects of miR-449a expression on migration and invasion. It was found that the expression of miR-449a was downregulated and the expression of CDK6 protein was upregulated in gastric carcinoma tissue. The level of MGC-803 cell proliferation was decreased and the apoptosis level was increased by the upregulation of miR-449a expression, and the opposite effect was shown by the downregulation of expression. The expression of the CDK6 protein in the MGC-803 cells was downregulated by upregulating the expression of miR-449a. The distance of the scratch was shortened markedly after 12 h by downregulating the expression of miR-449a in the MGC-803 cells. The present study identified that a lower expression level of miR-449 and a higher expression level of CDK6 may contribute to the occurrence and development of gastric cancer. Furthermore, it was shown that miR-449a is able to regulate the expression of the CDK6 protein.

Keywords: MGC-803; cyclin-dependent kinase 6 protein; gastric carcinoma; microRNA-449; real-time cell analysis.

Figure 1

(A) Relative expression of miR-449a in gastric carcinoma and tumor-adjacent normal tissues, as determined by qPCR. ‘a’ represents the tumor-adjacent normal tissue; relative expression, 24.665±1.557. ‘b’ represents the gastric carcinoma tissue; relative expression, 49.207±13.433, P<0.001. (B) The relative expression of the CDK6 protein in the gastric carcinoma and tumor-adjacent normal tissues, as determined by western blot analysis. (C) ‘a’ represent the tumor-adjacent normal tissue; relative expression, 0.482±0.290. ‘b’ represents the gastric carcinoma tissue; relative expression, 0.792±0.165, P=0.003. (D) The relative expression of CDK6 protein was negatively correlated with the relative expression of miR-449a, as determined by Spearman’s correlation analysis, r(tumor-adjacent normal tissue)=-0.614; r(gastric carcinoma tissue)=-0.694, P<0.001. Relative expression is measured as a percentage and ^*P<0.05 vs. tumor-adjacent normal tissues.. miR, microRNA; qPCR, quantitative PCR; CDK6, cyclin-dependent kinase 6.

Figure 2

(A) Relative expression of the miR-449a. ‘a’ represents the miR-449a upregulation group; relative expression, 81.844±5.095, P<0.001. ‘b’ represents the control group; relative expression, 26.233±2.011. ‘c’ represents the miR-449a downregulation group; relative expression, 6.733±1.575. Relative expression is measured as a percentage and ^*P<0.05. (B) The experimental result of the scratch test (x100). (C) ‘a’ represents the miR-449a upregulation group; distance change between edges of scratch, 0.503±0.080. ‘b’ represents the control group; distance change between edges of scratch, 0.198±0.068. ‘c’ represents the miR-449a downregulation group; distance change between edges of scratch, 2.519±0.350, P<0.001. The unit is of measurement is μm and ^*P<0.05. miR, microRNA.

Figure 3

(A) Proliferation of the MGC-803 cells in 3 groups, as observed by RTCA. ‘a’ represents the miR-449a upregulation group; proliferation coefficient, 4.282±0.386, P<0.001. ‘b’ represents the control group; proliferation coefficient, 9.002±0.066. ‘c’ represents the miR-449a downregulation group; proliferation coefficient, 9.049±0.073. (B) The relative expression of the CDK6 protein in 3 groups. (C) ‘a’ represents the miR-449a upregulation group; relative expression of CDK6 protein, 21.243±4.097, P<0.001. ‘b’ represents the control group; relative expression, 41.831±1.647. ‘c’ represents the miR-449a downregulation group; relative expression, 67.418±1.369. Relative expression is measured as a percentage and ^*P<0.05. RTCA, real-time cell analysis; miR, microRNA; CDK6, cyclin-dependent kinase 6.

Figure 4

Experimental result of the immunofluorescence staining. The CDK6 protein is shown as bright green fluorescence, as observed by fluorescence microscopy using a wavelength of 475nm. (A) MGC-803 cells of the miR-449a upregulation group (magnification, ×400). (B) MGC-803 cells of the miR-449a downregulation group (magnification, ×400). (C and D) Cells of the control group. miR, microRNA; CDK6, cyclin-dependent kinase 6 (magnifications, ×400 for A and ×10 for D).

Figure 5

Experimental results of the DAPI staining. (A) The MGC-803 cells of the miR-449a upregulation group with nuclei stained and showing blue fluorescence (x400 magnification). (B) Cells of the miR-449a downregulation group (x400 magnification). (C) Cells of the control group (x400 magnification). (D) ‘a’ represents the MGC-803 cells of the miR-449a upregulation group; number of the apoptic cells, 68.909±7.698, P=0.002. ‘b’ represents the MGC-803 cells of the miR-449a downregulation group; number of apoptostic cells, 14.000±2.098 in 10 randomly selected fields of view. ‘c’ represents the cells of the control group; number of apoptotic cells, 21.000±3.606. ^*P<0.001 vs. control group.