Limits...
CSE1L interaction with MSH6 promotes osteosarcoma progression and predicts poor patient survival

View Article: PubMed Central - PubMed

ABSTRACT

To discover tumor-associated proteins in osteosarcoma, a quantitative proteomic analysis was performed to identify proteins that were differentially expressed between osteosarcoma and human osteoblastic cells. Through clinical screening and a functional evaluation, chromosome segregation 1-like (CSE1L) protein was found to be related to the growth of osteosarcoma cells. To date, little is known about the function and underlying mechanism of CSE1L in osteosarcoma. In the present study, we show that knockdown of CSE1L inhibits osteosarcoma growth in vitro and in vivo. By co-immunoprecipitation and RNA-seq analysis, CSE1L was found to interact with mutS homolog 6 (MSH6) and function as a positive regulator of MSH6 protein in osteosarcoma cells. A rescue study showed that decreased growth of osteosarcoma cells by CSE1L knockdown was reversed by MSH6 overexpression, indicating that the activity of CSE1L was an MSH6-dependent function. In addition, depletion of MSH6 hindered cellular proliferation in vitro and in vivo. Notably, CSE1L expression was correlated with MSH6 expression in tumor samples and was associated with poor prognosis in patients with osteosarcoma. Taken together, our results demonstrate that the CSE1L-MSH6 axis has an important role in osteosarcoma progression.

No MeSH data available.


Related in: MedlinePlus

CSE1L knockdown inhibits osteosarcoma cell proliferation via MSH6.(A,C) Representative blots of MSH6 after transfection with pcDNA 3.1-MSH6, si-CSE1L or si-NC. β-actin was used as an internal control. (B,D) Cell Counting Kit-8 (CCK-8) assay was used to measure tumor cell proliferation after transfection with pcDNA 3.1-MSH6, si-CSE1L or si-NC. (E,G) Confirmation of protein expression levels following si-MSH6 transfection by western blotting in MNNG/HOS and U2OS cells. (F) (H) CCK-8 assay was performed to detect tumor cell proliferation after siRNA transfection. (I–L) Colony formation assays for MSH6-silenced osteosarcoma cells and control cells. (M) Representative blots displaying CSE1L protein expression in MNNG/HOS cells stably expressing sh-control or sh-MSH6. β-actin was used as an internal control. (N–P) Representative tumor-bearing mice, tumors isolated from nude mice, tumor volume, and tumor weights of an MNNG/HOS subcutaneous tumor model. Data are representative of results from three independent experiments. *P < 0.05. For western blotting, full-length gels are presented in Supplementary Figure S11.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5384328&req=5

f5: CSE1L knockdown inhibits osteosarcoma cell proliferation via MSH6.(A,C) Representative blots of MSH6 after transfection with pcDNA 3.1-MSH6, si-CSE1L or si-NC. β-actin was used as an internal control. (B,D) Cell Counting Kit-8 (CCK-8) assay was used to measure tumor cell proliferation after transfection with pcDNA 3.1-MSH6, si-CSE1L or si-NC. (E,G) Confirmation of protein expression levels following si-MSH6 transfection by western blotting in MNNG/HOS and U2OS cells. (F) (H) CCK-8 assay was performed to detect tumor cell proliferation after siRNA transfection. (I–L) Colony formation assays for MSH6-silenced osteosarcoma cells and control cells. (M) Representative blots displaying CSE1L protein expression in MNNG/HOS cells stably expressing sh-control or sh-MSH6. β-actin was used as an internal control. (N–P) Representative tumor-bearing mice, tumors isolated from nude mice, tumor volume, and tumor weights of an MNNG/HOS subcutaneous tumor model. Data are representative of results from three independent experiments. *P < 0.05. For western blotting, full-length gels are presented in Supplementary Figure S11.

Mentions: Our findings demonstrated that knockdown of CSE1L inhibited osteosarcoma cell proliferation. However, the role of MSH6 in osteosarcoma is unknown, and whether CSE1L functions through MSH6 is largely unknown. Since CSE1L interacts with MSH6 and affects its protein expression and stability in osteosarcoma cells, it is plausible that CSE1L functions through MSH6 and that MSH6 has an important role in osteosarcoma cell proliferation. To test this hypothesis, we overexpressed MSH6 in CSE1L-knockdown MNNG/HOS and U2OS cells. Western blotting confirmed that MSH6 protein levels were rescued in CSE1L knockdown cells after pcDNA 3.1-MSH6 plasmid transfection (Fig. 5A and C); as hypothesized, MSH6 overexpression rescued the CSE1L knockdown-induced cell growth inhibition (Fig. 5B and D). Then, we detected the expression of MSH6 in osteosarcoma cells and osteoblastic cells. We found that MSH6 was highly expressed in osteosarcoma cells (Supplementary Figure S5A and B). Next, MSH6-specific siRNA was used to knockdown MSH6 in osteosarcoma cells. MSH6 knockdown was validated using qRT-PCR and western blotting (Supplementary Figure S5C and D, Fig. 5E and G). CCK-8 and colony formation assays were used to measure cell proliferation. We found that knockdown of MSH6 significantly inhibited the growth of osteosarcoma cells (Fig. 5F,H–L). To determine the role of MSH6 in osteosarcoma in vivo, we generated a stable MSH6-knockdown MNNG/HOS cell line. Western blotting confirmed that MSH6 protein expression was significantly reduced (Fig. 5M). Transfected cells were subcutaneously injected into the left scapulae of nude mice. The results revealed that both tumor volume and weight were reduced in the MSH6 knockdown group than in the scramble control group (Fig. 5N–P).


CSE1L interaction with MSH6 promotes osteosarcoma progression and predicts poor patient survival
CSE1L knockdown inhibits osteosarcoma cell proliferation via MSH6.(A,C) Representative blots of MSH6 after transfection with pcDNA 3.1-MSH6, si-CSE1L or si-NC. β-actin was used as an internal control. (B,D) Cell Counting Kit-8 (CCK-8) assay was used to measure tumor cell proliferation after transfection with pcDNA 3.1-MSH6, si-CSE1L or si-NC. (E,G) Confirmation of protein expression levels following si-MSH6 transfection by western blotting in MNNG/HOS and U2OS cells. (F) (H) CCK-8 assay was performed to detect tumor cell proliferation after siRNA transfection. (I–L) Colony formation assays for MSH6-silenced osteosarcoma cells and control cells. (M) Representative blots displaying CSE1L protein expression in MNNG/HOS cells stably expressing sh-control or sh-MSH6. β-actin was used as an internal control. (N–P) Representative tumor-bearing mice, tumors isolated from nude mice, tumor volume, and tumor weights of an MNNG/HOS subcutaneous tumor model. Data are representative of results from three independent experiments. *P < 0.05. For western blotting, full-length gels are presented in Supplementary Figure S11.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384328&req=5

f5: CSE1L knockdown inhibits osteosarcoma cell proliferation via MSH6.(A,C) Representative blots of MSH6 after transfection with pcDNA 3.1-MSH6, si-CSE1L or si-NC. β-actin was used as an internal control. (B,D) Cell Counting Kit-8 (CCK-8) assay was used to measure tumor cell proliferation after transfection with pcDNA 3.1-MSH6, si-CSE1L or si-NC. (E,G) Confirmation of protein expression levels following si-MSH6 transfection by western blotting in MNNG/HOS and U2OS cells. (F) (H) CCK-8 assay was performed to detect tumor cell proliferation after siRNA transfection. (I–L) Colony formation assays for MSH6-silenced osteosarcoma cells and control cells. (M) Representative blots displaying CSE1L protein expression in MNNG/HOS cells stably expressing sh-control or sh-MSH6. β-actin was used as an internal control. (N–P) Representative tumor-bearing mice, tumors isolated from nude mice, tumor volume, and tumor weights of an MNNG/HOS subcutaneous tumor model. Data are representative of results from three independent experiments. *P < 0.05. For western blotting, full-length gels are presented in Supplementary Figure S11.
Mentions: Our findings demonstrated that knockdown of CSE1L inhibited osteosarcoma cell proliferation. However, the role of MSH6 in osteosarcoma is unknown, and whether CSE1L functions through MSH6 is largely unknown. Since CSE1L interacts with MSH6 and affects its protein expression and stability in osteosarcoma cells, it is plausible that CSE1L functions through MSH6 and that MSH6 has an important role in osteosarcoma cell proliferation. To test this hypothesis, we overexpressed MSH6 in CSE1L-knockdown MNNG/HOS and U2OS cells. Western blotting confirmed that MSH6 protein levels were rescued in CSE1L knockdown cells after pcDNA 3.1-MSH6 plasmid transfection (Fig. 5A and C); as hypothesized, MSH6 overexpression rescued the CSE1L knockdown-induced cell growth inhibition (Fig. 5B and D). Then, we detected the expression of MSH6 in osteosarcoma cells and osteoblastic cells. We found that MSH6 was highly expressed in osteosarcoma cells (Supplementary Figure S5A and B). Next, MSH6-specific siRNA was used to knockdown MSH6 in osteosarcoma cells. MSH6 knockdown was validated using qRT-PCR and western blotting (Supplementary Figure S5C and D, Fig. 5E and G). CCK-8 and colony formation assays were used to measure cell proliferation. We found that knockdown of MSH6 significantly inhibited the growth of osteosarcoma cells (Fig. 5F,H–L). To determine the role of MSH6 in osteosarcoma in vivo, we generated a stable MSH6-knockdown MNNG/HOS cell line. Western blotting confirmed that MSH6 protein expression was significantly reduced (Fig. 5M). Transfected cells were subcutaneously injected into the left scapulae of nude mice. The results revealed that both tumor volume and weight were reduced in the MSH6 knockdown group than in the scramble control group (Fig. 5N–P).

View Article: PubMed Central - PubMed

ABSTRACT

To discover tumor-associated proteins in osteosarcoma, a quantitative proteomic analysis was performed to identify proteins that were differentially expressed between osteosarcoma and human osteoblastic cells. Through clinical screening and a functional evaluation, chromosome segregation 1-like (CSE1L) protein was found to be related to the growth of osteosarcoma cells. To date, little is known about the function and underlying mechanism of CSE1L in osteosarcoma. In the present study, we show that knockdown of CSE1L inhibits osteosarcoma growth in vitro and in vivo. By co-immunoprecipitation and RNA-seq analysis, CSE1L was found to interact with mutS homolog 6 (MSH6) and function as a positive regulator of MSH6 protein in osteosarcoma cells. A rescue study showed that decreased growth of osteosarcoma cells by CSE1L knockdown was reversed by MSH6 overexpression, indicating that the activity of CSE1L was an MSH6-dependent function. In addition, depletion of MSH6 hindered cellular proliferation in vitro and in vivo. Notably, CSE1L expression was correlated with MSH6 expression in tumor samples and was associated with poor prognosis in patients with osteosarcoma. Taken together, our results demonstrate that the CSE1L-MSH6 axis has an important role in osteosarcoma progression.

No MeSH data available.


Related in: MedlinePlus