Yonsei Med J.  2019 Jul;60(7):611-618. 10.3349/ymj.2019.60.7.611.

Family with Sequence Similarity 83 Member H Promotes the Viability and Metastasis of Cervical Cancer Cells and Indicates a Poor Prognosis

• Chen C
• Li HF
• Hu YJ
• Jiang MJ
• Liu QS
• Zhou J

• KMID: 2450400
• DOI: http://doi.org/10.3349/ymj.2019.60.7.611

Abstract

PURPOSE
Family with sequence similarity 83 member H (FAM83H) plays key roles in tumorigenesis. However, the specific roles of FAM83H in cervical cancer (CC) have not been well studied.
MATERIALS AND METHODS
The RNA-seq data of 306 CC tissues and three normal samples downloaded from The Cancer Genome Atlas were used to analyze the expression of FAM83H. The Kaplan-Meier method was used to draw survival curves. Associations between FAM83H expression and clinicopathological factors were analyzed by chi-square test. Cox proportional hazards model was used to analyze prognostic factors. Loss-of-function assays were conducted to discover the biological functions of FAM83H in cell proliferation, colony formation, invasion, and migration. Real-time Quantitative Reverse Transcription PCR (qRT-PCR) and Western blotting were used to measure the expression levels of FAM83H in CC cell lines.
RESULTS
Our results demonstrated that FAM83H is overexpressed in CC tissues and that high FAM83H expression is associated with worse overall survival (OS). High FAM83H expression in CC was associated with clinical stage, pathologic tumor, and pathologic node. Univariate analysis suggested that FAM83H expression was significantly related to the OS of CC patients. Although multivariate analysis showed that FAM83H expression was not an independent prognostic factor for the OS of CC patients, the effects of FAM83H on CC cell growth and motility was significant. Loss-of-function experiments demonstrated that knockdown of FAM83H inhibited proliferation, colony formation, migration, and invasion of CC cells by inactivating PI3K/AKT pathway.
CONCLUSION
FAM83H might play a crucial role in CC progression and could act as a novel therapeutic target in CC.

Keyword

Cervical cancer; FAM83H; prognosis; proliferation; migration

MeSH Terms

Blotting, Western
Carcinogenesis
Cell Line
Cell Proliferation
Genome
Humans
Methods
Multivariate Analysis
Neoplasm Metastasis*
Polymerase Chain Reaction
Prognosis*
Proportional Hazards Models
Reverse Transcription
Uterine Cervical Neoplasms*

Figure

• 

  Fig. 1 FAM83H over-expression is associated with poor prognosis of CC. (A) FAM83H expression at mRNA level in CC tissues in TCGA database. (B) FAM83H expression at mRNA level in CC tissues in GEPIA database (match TCGA normal data). (C) FAM83H expression at mRNA level in CC tissues in GEPIA database (match TCGA normal and GTEx data). (D) Real-time Quantitative Reverse Transcription PCR used to measure the expression level of FAM83H in human CC cell lines (SiHa and HeLa). The expression of FAM83H was significantly higher in CC cells relative to control cells. (E) Kaplan-Meier curves of OS of CC patients in TCGA database. Patients were sub-grouped into high (n=152) or low (n=152) groups based on the median of FAM83H expression. p=0.027. (F) Kaplan-Meier curves of OS of CC patients in GEPIA database. Patients were sub-grouped into high (n=146) or low (n=146) groups based on the median of FAM83H expression. (G) Kaplan-Meier curves of OS of CC patients in the Human Protein Atlas database. Patients were sub-grouped into high (n=224) or low (n=67) groups based on best cut-off point (p=0.004). *p<0.05, **p<0.01. FAM83H, family with sequence similarity 83 member H; CC, cervical cancer; TCGA, The Cancer Genome Atlas; GEPIA, Gene Expression Profiling Interactive Analysis; OS, overall survival; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; TPM, transcripts per million; HR, hazard ratio.

• 

  Fig. 2 FAM83H expression in HeLa cells transfected with siRNA FAM83H 1# and siRNA FAM83H 2# were measured by Real-Time Quantitative Reverse Transcription PCR (A) and Western blotting (B and C). *p<0.01 compared to the si-con group. FAM83H, family with sequence similarity 83 member H; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

• 

  Fig. 3 Knockdown of FAM83H inhibits growth and viability of HeLa cells. CCK-8 assay showing viability of transfected HeLa cells at 24, 48, 72, and 96 h. *p<0.05 compared to control, **p<0.001 compared to control. CCK-8, Cell-Counting Kit 8; FAM83H, family with sequence similarity 83 member H; OD, optical density.

• 

  Fig. 4 Knockdown of FAM83H decreases colony formation of HeLa cells. (A) Representative images of colony formation assay (×2). The clones were dyed by 0.1% crystal violet. (B) Quantification of the colony numbers. *p<0.001 compared to control. FAM83H, family with sequence similarity 83 member H.

• 

  Fig. 5 Analysis of wound-healing (A; ×100) and transwell assay [B (×200) and C]. Cells were dyed by 0.1% crystal violet. In HeLa cells transfected with si-FAM83H, the invasion and migration were significantly decreased compared to the cells transfected with scrambled si-RNA (si-con). *p<0.001 compared to control. FAM83H, family with sequence similarity 83 member H.

• 

  Fig. 6 The expression of PI3K/AKT pathway related proteins. After FAM83H silencing, the expression of p-AKT and p-PI3K were all reduced significantly. (A) The expression of PI3K pathway related proteins were determined by Western blot. (B) Quantification of the protein expression levels in (A). *p<0.001 compared to control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FAM83H, family with sequence similarity 83 member H.

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