FAM64A Promotes Osteosarcoma Cell Growth and Metastasis and Is Mediated by miR-493

Abstract

Aberrant expression of FAM64A was correlated with cell proliferation in various cancer types. We examined the expression of FAM64A and the upstream gene miR-493 in OS. The functions of miR-493 were revealed through extensive experiments. We found an increase of FAM64A gene and protein in OS tissues. Overexpression of FAM64A resulted in promoting tumor proliferation, migration, and invasion. The miR-493 targeted and negatively regulated FAM64A. Our data showed that upregulation of FAM64A in OS correlated with poor prognosis.

Figure 1

Expression of FAM64A in tumor tissues. (a) Differentially expressed genes were selected on GEO. (b) A total of 147 genes were obtained. (c) Gene expression of FAM64A in OS tissues and nontumor tissues using RT-RCR. (d) Protein expression of FAM64A in 5 pairs subject using western blot. (e) Kaplan plot for FAM64A in SARC. ^∗P < 0.05. OS, osteosarcoma.

Figure 2

Upregulation of FAM64A promoted the tumorigenicity of OS cells. (a) and (b) Protein expression of FAM64A was detected after vector and FAM64A plasmid transfection. (c) and (d) Proliferation of tumor cells was determined by CCK-8 assay. (e) and (f) Migration was measured by the wound healing assay. (g) and (h) Invasion of cells was analyzed using a Transwell assay. ^∗P < 0.05.

Figure 3

Functional effects of FAM64A siRNA knockdown on MG-63 and U-2 OS cells. (a) Protein expressions of FAM64A in si-control and si-FAM64A transfected cells. (g) and (h) migration assay of si-FAM64A transfectants. Representative photomicrographs are shown at 100 magnification.

Figure 4

Silencing FAM64A in mice. (a) Tumor volume of mice burden MG-63-Scramble/MG-63-siFAM64A cells was measured at 10, 20, and 30 days. (b) Mice were sacrificed after 30 days, and tumor weight of mice burden MG-63-Scramble/MG-63-siFAM64A cells was analyzed. (c) Survival rate of mice. ^∗P < 0.05.

Figure 5

FAM64A is the target of miR-493 in OS. (a) MiR493 binding sequence in 3′UTR of the FAM64A gene and the mutant sequence. (b) The pattern of FAM64A 3-UTR WT/MT inserted to psiCHECK2. (c) 293T cells were cotransfected with psiCHECK2-FAM64A 3′-UTR WT or psiCHECK2-FAM64A 3′-UTR MT and miR493 mimics or NC. The luciferase activity was measured. (d) and (e) mRNA levels of FAM64A in MG-63 and U-2 OS cells. (f) Detection of miR-493 expressions in OS tissues and nontumor tissues using RT-RCR; (f) correlation analysis of miR-493 levels with FAM64A mRNA levels. ^∗P < 0.05. UTR: untranslated region; WT: wild type; MT: mutant; NC: negative control.

Figure 6

miR-493 inhibits proliferation, migration, and invasion of OS cells via regulating FAM64A. (a–p) U-2 OS and MG-63 cells were transfected with miR-493 mimics and miR-493 inhibitors with FAM64A plasmids and FAM64A siRNAs. (a–d) Proliferation of tumor cells was determined by CCK-8 assay. (e–j) Migration was measured by wound healing assay. (k–p) Invasion of cells were analyzed using a Transwell assay. ^∗P < 0.05.