International Journal of
Molecular Sciences Article
High Expression of XRCC6 Promotes Human Osteosarcoma Cell Proliferation through the β-Catenin/Wnt Signaling Pathway and Is Associated with Poor Prognosis Bin Zhu 1,† , Dongdong Cheng 1,† , Shijie Li 1 , Shumin Zhou 2 and Qingcheng Yang 1, * 1
2
* †
Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, No. 600, Yishan Road, Shanghai 200233, China; [email protected] (B.Z.); [email protected] (D.C.); [email protected] (S.L.) Institute of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, No. 600, Yishan Road, Shanghai 200233, China; [email protected] Correspondence: [email protected]; Tel.: +86-21-6436-9181 These authors contribute equally to this work.
Academic Editor: William Chi-shing Cho Received: 26 May 2016; Accepted: 14 July 2016; Published: 22 July 2016
Abstract: Increasing evidences show that XRCC6 (X-ray repair complementing defective repair in Chinese hamster cells 6) was upregulated and involved in tumor growth in several tumor types. However, the correlation of XRCC6 and human osteosarcoma (OS) is still unknown. This study was conducted with the aim to reveal the expression and biological function of XRCC6 in OS and elucidate the potential mechanism. The mRNA expression level of XRCC6 was measured in osteosarcoma cells and OS samples by quantitative transcription-PCR (qRT-PCR). The expression of XRCC6 protein was measured using Western blot and immunohistochemical staining in osteosarcoma cell lines and patient samples. Cell Counting Kit 8 (CCK8), colony-forming and cell cycle assays were used to test cell survival capacity. We found that XRCC6 was overexpressed in OS cells and OS samples compared with the adjacent non-tumorous samples. High expression of XRCC6 was correlated with clinical stage and tumor size in OS. Reduced expression of XRCC6 inhibits OS cell proliferation through G2/M phase arrest. Most importantly, further experiments demonstrated that XRCC6 might regulate OS growth through the β-catenin/Wnt signaling pathway. In conclusion, these findings indicate that XRCC6 exerts tumor-promoting effects for OS through β-catenin/Wnt signaling pathway. XRCC6 may serve as a novel therapeutic target for OS patients. Keywords: XRCC6; Ku70; osteosarcoma; β-catenin/Wnt signaling pathway; proliferation
1. Introduction The incidence of osteosarcoma (OS) ranks first among of all primary malignant bone tumors, predominantly affecting children and adolescence populations [1]. It often occurs in long bones, such as distal femur and proximal radius [2,3]. With the aid of effective chemotherapeutic drugs the survival rate of OS has been improved from 20% to 65% since the late 1970s [4,5]. However, treating metastatic and recurrent OS with current treatments is still limited [6]. In addition to this, the pathogenesis and etiology of osteosarcoma remain elusive [7]. X-ray repair complementing defective repair in Chinese hamster cells 6 (XRCC6) is a gene coding Ku70 protein [8]. It was reported to be involved in DNA recombination and repair, which plays a crucial role in genome stability and cell survival [9]. XRCC6 was reported as a key-element in the Non Homologous End Joining pathway and bound to DNA termini to protect DNA ends with high Int. J. Mol. Sci. 2016, 17, 1188; doi:10.3390/ijms17071188
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affinity from degradation [10]. It is hypothesized that the genetic polymorphism ofof XRCC6 might play an important role in tumorigenesis. The association between the expression XRCC6 and play the an important role in tumorigenesis. The association between the expression of XRCC6 and the risk risk of cancer, such as lung cancer [11], glioma [12,13], hepatocellular carcinoma [14,15] and so on,of cancer, as lung cancer experiments. [11], glioma [12,13], hepatocellular carcinoma [14,15] and so on, has been has beensuch studied by several However, the role of XRCC6 in OS is still unknown. studied by several experiments. However, the role of XRCC6 in OS is still unknown. The β-catenin/Wnt signaling pathway plays an important role in bone development, stem cell The β-catenin/Wnt pathwayactivation plays an has important role in development, cell biology and tumorigenesissignaling [16]. Its aberrant been linked tobone the pathogenesis ofstem various biologyin and tumorigenesis [16]. Its aberrant activation been linked to the pathogenesis of various tumors humans. Previous studies revealed that thehas β-catenin/Wnt pathway contributed to OS tumors in humans. Previous studies revealed that the β-catenin/Wnt pathway contributed OS development [17,18] and efficacy of OS chemotherapy [19]. Recently, several studies revealedtothat development [17,18] and efficacy of OS chemotherapy [19]. Recently, XRCC6 was a regulator of the β-catenin/Wnt signaling pathway [20,21]. several studies revealed that XRCC6 was a regulator of the β-catenin/Wnt signaling pathway [20,21]. In this paper, we reported, for the first time, that XRCC6 was overexpressed in human In this paper, we reported, for the first time, that XRCC6 was overexpressed in human osteosarcoma. Moreover, we found that knockdown of XRCC6 expression led to growth inhibition osteosarcoma. found that knockdown of XRCC6dysregulation, expression ledwhich to growth inhibition of OS cell lines Moreover, and causedwe β-catenin/Wnt signaling pathway indicated that of OS cell lines and caused β-catenin/Wnt signaling pathway dysregulation, which indicated that XRCC6 might play a crucial role in OS growth by influencing the β-catenin/Wnt signaling pathway. XRCC6 might play a crucial role in OS growth by influencing the β-catenin/Wnt signaling pathway.
2. Results 2. Results 2.1. 2.1.XRCC6 XRCC6IsIsOverexpressed OverexpressedininHuman HumanOsteosarcoma Osteosarcoma(OS) (OS)Samples Samplesand andCell CellLines Lines To To determine determine the the biological biologicalrole role of of XRCC6 XRCC6 in in human human OS OS cells, cells,we wefirst firstdetected detectedthe themRNA mRNA expression of XRCC6 using quantitative RT-PCR in human osteoblast cell line (hFOB) and expression of XRCC6 using quantitative RT-PCR in human osteoblast cell line (hFOB) andOS OScell cell lines (MNNG/HOS, MG63, U2OS). A higher expression of XRCC6 was observed in OS cell lines lines (MNNG/HOS, MG63, U2OS). A higher expression of XRCC6 was observed in OS cell lines compared (Figure 1A). 1A). Additionally, Additionally,the thesame sameresult resultwas wasalso alsofound foundat comparedwith withhuman human osteoblast osteoblast cell cell lines lines (Figure atthe the protein level using Western blot. Moreover, the expression of XRCC6 was detected in twenty protein level using Western blot. Moreover, the expression of XRCC6 was detected in twenty pairs pairs of human OS tissue samples and adjacent their adjacent non-tumorous controls. We that found that of human OS tissue samples and their non-tumorous tissue tissue controls. We found XRCC6 XRCC6 expression was significantly up-regulated in tumor tissue samples (60%) compared to the expression was significantly up-regulated in tumor tissue samples (60%) compared to the controls controls (Figure 1D,E). (Figure 1D,E).
Figure1.1. XRCC6 XRCC6expression expressionisisup-regulated up-regulatedininosteosarcoma osteosarcoma(OS) (OS)clinical clinicalsamples samplesand andcell celllines. lines. Figure (A–C)The ThemRNA mRNAand andprotein proteinexpression expressionlevel levelofofXRCC6 XRCC6were weremeasured measuredby byTaqMan TaqManreal-time real-timePCR PCR (A–C) andWestern Western blot blot assays assays in in OS OS cell cell lines lines (including (including MNNG/HOS, MNNG/HOS,MG63 MG63and andU2OS) U2OS)and andhuman human and osteoblasticcell cellline line(hFOB); (hFOB);(D,E) (D,E)relative relativeexpression expressionofofXRCC6 XRCC6was wasdetected detectedusing usingqRT-PCR qRT-PCRinin osteoblastic 20pairs pairsof ofOS OSsamples samplesand and their their corresponding corresponding noncancerous noncancerous samples. samples.The Theexpression expressionofofXRCC6 XRCC6 20 wasoverexpressed overexpressedin inOS OS tissues tissues compared compared with with the the noncancerous noncancerous tissues. tissues. Statistical Statisticalanalysis analysiswas was was performed using paired t test (D). performed using paired t test (D).
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2.2. 2.2. Knockdown of XRCC6 Expression Cell Proliferation Proliferationthrough through G2/M Phase Arrest in Vitro Knockdown of XRCC6 ExpressionInhibited Inhibited OS Cell G2/M Phase Arrest in Vitro In vitro experiments werecarried carriedout out to evaluate evaluate the role of of XRCC6 in tumorigenesis. In vitro experiments were thepotential potential role XRCC6 in tumorigenesis. MNNG/HOS U2OS transiently transfected a targeted to decrease MNNG/HOS andand U2OS cellscells werewere transiently transfected with with a targeted siRNAsiRNA to decrease expression expression of the gene. The relative expression of XRCC6 was shown in Figure 2A,B and Figure of the gene. The relative expression of XRCC6 was shown in Figure 2A,B and Figure S2. A S2. CCK-8 A was CCK-8 assay was used to determine cell proliferation. results showed that knockdown the of assay used to determine cell proliferation. The results The showed that knockdown the expression expression of XRCC6 inhibited MNNG/HOS and U2OS cells proliferation (Figure 2C,D). Cell cycle XRCC6 inhibited MNNG/HOS and U2OS cells proliferation (Figure 2C,D). Cell cycle changes were changes were analyzed by flow cytometry after transfection with si-XRCC6 or si-NC. Results of analyzed by flow cytometry after transfection with si-XRCC6 or si-NC. Results of cell cycle analysis cell cycle analysis revealed that inhibition of XRCC6 resulted in an elevated G2/M population in revealed that inhibition of XRCC6 resulted in an elevated G2/M population in both MNNG/HOS and both MNNG/HOS and U2OS cells (Figure 2E–H). Thus, knockdown of XRCC6 expression may U2OS cells (Figure 2E–H). Thus, knockdown of XRCC6 expression mayHowever, attenuatedata OS cell proliferation attenuate OS cell proliferation through G2/M phase arrest in vitro. showed that through G2/M phase arrest in vitro. However, data showed thatthe decreased expression XRCC6 in decreased expression of XRCC6 in MNNG/HOS did not influence ability of migration orof invasion MNNG/HOS did not influence the ability of migration or invasion (Figure S1A,B). (Figure S1A,B).
Figure 2. Knockdown XRCC6inhibited inhibited cell cell proliferation proliferation through phase arrest. (A,B) The The Figure 2. Knockdown ofof XRCC6 throughG2/M G2/M phase arrest. (A,B) expression of XRCC6 was downregulated by a targeted siRNA; (C,D) a CCK8 assay was used to detect expression of XRCC6 was downregulated by a targeted siRNA; (C,D) a CCK8 assay was used to detect the proliferation of MNNG/HOS cells transfection after transfection with targeted the proliferation of MNNG/HOS cellscells and and U2OSU2OS cells after with targeted siRNA.siRNA. Diagrams Diagrams showing the results of a CCK-8 assay that MNNG/HOS and U2OS proliferation were showing the results of a CCK-8 assay that MNNG/HOS and U2OS proliferation were inhibited by inhibited by downregulating XRCC6 expression; (E,G) cell cycle profiles determined by propidium downregulating XRCC6 expression; (E,G) cell cycle profiles determined by propidium iodide (PI) iodide (PI) staining and flow cytometry assays of MNNG/HOS transfected with si-XRCC6 or si-NC; staining and flow cytometry assays of MNNG/HOS transfected with si-XRCC6 or si-NC; (F,H) cell (F,H) cell cycle profiles determined by propidium iodide (PI) staining and flow cytometry assays of cycle profiles determined by propidium iodide (PI) staining and flow cytometry assays of U2OS U2OS transfected with si-XRCC6 or si-NC. The data are representative of three independent transfected withError si-XRCC6 or si-NC. data are representative of**three independent experiments. experiments. bars represent SDThe (Standard Deviation). * p < 0.05, p < 0.01 by Student’s t test. Error bars represent SD (Standard Deviation). * p < 0.05, ** p < 0.01 by Student’s t test.
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2.3. Decreased XRCC6 XRCC6 Expression 2.3. Decreased Expression Impaired Impaired Colony-Forming Colony-Forming Capacity Capacity of of OS OS Cells Cells Colony determine the colony-forming capacity after Colony forming forming assay assay was was carried carried out out to to determine the colony-forming capacity of of OS OS cells cells after knockdown of XRCC6 expression. It was found that the number and the size of the colonies were knockdown of XRCC6 expression. It was found that the number and the size of the colonies were both in the group in with the the control both obviously obviously decreased decreased in the XRCC6 XRCC6 knockdown knockdown group in comparison comparison with control group group (Figure 3A,C). The number of colonies was significantly reduced by 44% and 54.5% in MNNG/HOS (Figure 3A,C). The number of colonies was significantly reduced by 44% and 54.5% in MNNG/HOS and In In conclusion, these results demonstrated that that XRCC6 was and U2OS U2OS cells, cells,respectively respectively(Figure (Figure3B,D). 3B,D). conclusion, these results demonstrated XRCC6 important for OS was important forcell OSgrowth. cell growth.
Figure 3. Decreased XRCC6 impaired OS cell colony-forming capacity. (A,C) Colony formation assay of MNNG/HOS cells and and U2OS U2OS cells cells transfected transfected with with targeted targeted siRNA or si-NC. After two weeks, cells MNNG/HOS cells in each well wellwere werefixed fixedand andcounted. counted.Representative Representative photo micrographs of MNNG/HOS (A); photo micrographs of MNNG/HOS cellscells (A); and and U2OS cells (C), colonies in culture plates; (B,D) significant reduction in the colony-forming efficacy U2OS cells (C), colonies in culture plates; (B,D) significant reduction in the colony-forming efficacy in MNNG/HOS cells (B), (B), and and U2OS U2OS cells cells (D). Following XRCC6 knockdown. Data are expressed as MNNG/HOS cells mean ˘ SD.of ofthree three independent independent experiments. experiments. ** **pp
Molecular Sciences Article
High Expression of XRCC6 Promotes Human Osteosarcoma Cell Proliferation through the β-Catenin/Wnt Signaling Pathway and Is Associated with Poor Prognosis Bin Zhu 1,† , Dongdong Cheng 1,† , Shijie Li 1 , Shumin Zhou 2 and Qingcheng Yang 1, * 1
2
* †
Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, No. 600, Yishan Road, Shanghai 200233, China; [email protected] (B.Z.); [email protected] (D.C.); [email protected] (S.L.) Institute of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, No. 600, Yishan Road, Shanghai 200233, China; [email protected] Correspondence: [email protected]; Tel.: +86-21-6436-9181 These authors contribute equally to this work.
Academic Editor: William Chi-shing Cho Received: 26 May 2016; Accepted: 14 July 2016; Published: 22 July 2016
Abstract: Increasing evidences show that XRCC6 (X-ray repair complementing defective repair in Chinese hamster cells 6) was upregulated and involved in tumor growth in several tumor types. However, the correlation of XRCC6 and human osteosarcoma (OS) is still unknown. This study was conducted with the aim to reveal the expression and biological function of XRCC6 in OS and elucidate the potential mechanism. The mRNA expression level of XRCC6 was measured in osteosarcoma cells and OS samples by quantitative transcription-PCR (qRT-PCR). The expression of XRCC6 protein was measured using Western blot and immunohistochemical staining in osteosarcoma cell lines and patient samples. Cell Counting Kit 8 (CCK8), colony-forming and cell cycle assays were used to test cell survival capacity. We found that XRCC6 was overexpressed in OS cells and OS samples compared with the adjacent non-tumorous samples. High expression of XRCC6 was correlated with clinical stage and tumor size in OS. Reduced expression of XRCC6 inhibits OS cell proliferation through G2/M phase arrest. Most importantly, further experiments demonstrated that XRCC6 might regulate OS growth through the β-catenin/Wnt signaling pathway. In conclusion, these findings indicate that XRCC6 exerts tumor-promoting effects for OS through β-catenin/Wnt signaling pathway. XRCC6 may serve as a novel therapeutic target for OS patients. Keywords: XRCC6; Ku70; osteosarcoma; β-catenin/Wnt signaling pathway; proliferation
1. Introduction The incidence of osteosarcoma (OS) ranks first among of all primary malignant bone tumors, predominantly affecting children and adolescence populations [1]. It often occurs in long bones, such as distal femur and proximal radius [2,3]. With the aid of effective chemotherapeutic drugs the survival rate of OS has been improved from 20% to 65% since the late 1970s [4,5]. However, treating metastatic and recurrent OS with current treatments is still limited [6]. In addition to this, the pathogenesis and etiology of osteosarcoma remain elusive [7]. X-ray repair complementing defective repair in Chinese hamster cells 6 (XRCC6) is a gene coding Ku70 protein [8]. It was reported to be involved in DNA recombination and repair, which plays a crucial role in genome stability and cell survival [9]. XRCC6 was reported as a key-element in the Non Homologous End Joining pathway and bound to DNA termini to protect DNA ends with high Int. J. Mol. Sci. 2016, 17, 1188; doi:10.3390/ijms17071188
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2016, 17, 1188
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affinity from degradation [10]. It is hypothesized that the genetic polymorphism ofof XRCC6 might play an important role in tumorigenesis. The association between the expression XRCC6 and play the an important role in tumorigenesis. The association between the expression of XRCC6 and the risk risk of cancer, such as lung cancer [11], glioma [12,13], hepatocellular carcinoma [14,15] and so on,of cancer, as lung cancer experiments. [11], glioma [12,13], hepatocellular carcinoma [14,15] and so on, has been has beensuch studied by several However, the role of XRCC6 in OS is still unknown. studied by several experiments. However, the role of XRCC6 in OS is still unknown. The β-catenin/Wnt signaling pathway plays an important role in bone development, stem cell The β-catenin/Wnt pathwayactivation plays an has important role in development, cell biology and tumorigenesissignaling [16]. Its aberrant been linked tobone the pathogenesis ofstem various biologyin and tumorigenesis [16]. Its aberrant activation been linked to the pathogenesis of various tumors humans. Previous studies revealed that thehas β-catenin/Wnt pathway contributed to OS tumors in humans. Previous studies revealed that the β-catenin/Wnt pathway contributed OS development [17,18] and efficacy of OS chemotherapy [19]. Recently, several studies revealedtothat development [17,18] and efficacy of OS chemotherapy [19]. Recently, XRCC6 was a regulator of the β-catenin/Wnt signaling pathway [20,21]. several studies revealed that XRCC6 was a regulator of the β-catenin/Wnt signaling pathway [20,21]. In this paper, we reported, for the first time, that XRCC6 was overexpressed in human In this paper, we reported, for the first time, that XRCC6 was overexpressed in human osteosarcoma. Moreover, we found that knockdown of XRCC6 expression led to growth inhibition osteosarcoma. found that knockdown of XRCC6dysregulation, expression ledwhich to growth inhibition of OS cell lines Moreover, and causedwe β-catenin/Wnt signaling pathway indicated that of OS cell lines and caused β-catenin/Wnt signaling pathway dysregulation, which indicated that XRCC6 might play a crucial role in OS growth by influencing the β-catenin/Wnt signaling pathway. XRCC6 might play a crucial role in OS growth by influencing the β-catenin/Wnt signaling pathway.
2. Results 2. Results 2.1. 2.1.XRCC6 XRCC6IsIsOverexpressed OverexpressedininHuman HumanOsteosarcoma Osteosarcoma(OS) (OS)Samples Samplesand andCell CellLines Lines To To determine determine the the biological biologicalrole role of of XRCC6 XRCC6 in in human human OS OS cells, cells,we wefirst firstdetected detectedthe themRNA mRNA expression of XRCC6 using quantitative RT-PCR in human osteoblast cell line (hFOB) and expression of XRCC6 using quantitative RT-PCR in human osteoblast cell line (hFOB) andOS OScell cell lines (MNNG/HOS, MG63, U2OS). A higher expression of XRCC6 was observed in OS cell lines lines (MNNG/HOS, MG63, U2OS). A higher expression of XRCC6 was observed in OS cell lines compared (Figure 1A). 1A). Additionally, Additionally,the thesame sameresult resultwas wasalso alsofound foundat comparedwith withhuman human osteoblast osteoblast cell cell lines lines (Figure atthe the protein level using Western blot. Moreover, the expression of XRCC6 was detected in twenty protein level using Western blot. Moreover, the expression of XRCC6 was detected in twenty pairs pairs of human OS tissue samples and adjacent their adjacent non-tumorous controls. We that found that of human OS tissue samples and their non-tumorous tissue tissue controls. We found XRCC6 XRCC6 expression was significantly up-regulated in tumor tissue samples (60%) compared to the expression was significantly up-regulated in tumor tissue samples (60%) compared to the controls controls (Figure 1D,E). (Figure 1D,E).
Figure1.1. XRCC6 XRCC6expression expressionisisup-regulated up-regulatedininosteosarcoma osteosarcoma(OS) (OS)clinical clinicalsamples samplesand andcell celllines. lines. Figure (A–C)The ThemRNA mRNAand andprotein proteinexpression expressionlevel levelofofXRCC6 XRCC6were weremeasured measuredby byTaqMan TaqManreal-time real-timePCR PCR (A–C) andWestern Western blot blot assays assays in in OS OS cell cell lines lines (including (including MNNG/HOS, MNNG/HOS,MG63 MG63and andU2OS) U2OS)and andhuman human and osteoblasticcell cellline line(hFOB); (hFOB);(D,E) (D,E)relative relativeexpression expressionofofXRCC6 XRCC6was wasdetected detectedusing usingqRT-PCR qRT-PCRinin osteoblastic 20pairs pairsof ofOS OSsamples samplesand and their their corresponding corresponding noncancerous noncancerous samples. samples.The Theexpression expressionofofXRCC6 XRCC6 20 wasoverexpressed overexpressedin inOS OS tissues tissues compared compared with with the the noncancerous noncancerous tissues. tissues. Statistical Statisticalanalysis analysiswas was was performed using paired t test (D). performed using paired t test (D).
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2.2. 2.2. Knockdown of XRCC6 Expression Cell Proliferation Proliferationthrough through G2/M Phase Arrest in Vitro Knockdown of XRCC6 ExpressionInhibited Inhibited OS Cell G2/M Phase Arrest in Vitro In vitro experiments werecarried carriedout out to evaluate evaluate the role of of XRCC6 in tumorigenesis. In vitro experiments were thepotential potential role XRCC6 in tumorigenesis. MNNG/HOS U2OS transiently transfected a targeted to decrease MNNG/HOS andand U2OS cellscells werewere transiently transfected with with a targeted siRNAsiRNA to decrease expression expression of the gene. The relative expression of XRCC6 was shown in Figure 2A,B and Figure of the gene. The relative expression of XRCC6 was shown in Figure 2A,B and Figure S2. A S2. CCK-8 A was CCK-8 assay was used to determine cell proliferation. results showed that knockdown the of assay used to determine cell proliferation. The results The showed that knockdown the expression expression of XRCC6 inhibited MNNG/HOS and U2OS cells proliferation (Figure 2C,D). Cell cycle XRCC6 inhibited MNNG/HOS and U2OS cells proliferation (Figure 2C,D). Cell cycle changes were changes were analyzed by flow cytometry after transfection with si-XRCC6 or si-NC. Results of analyzed by flow cytometry after transfection with si-XRCC6 or si-NC. Results of cell cycle analysis cell cycle analysis revealed that inhibition of XRCC6 resulted in an elevated G2/M population in revealed that inhibition of XRCC6 resulted in an elevated G2/M population in both MNNG/HOS and both MNNG/HOS and U2OS cells (Figure 2E–H). Thus, knockdown of XRCC6 expression may U2OS cells (Figure 2E–H). Thus, knockdown of XRCC6 expression mayHowever, attenuatedata OS cell proliferation attenuate OS cell proliferation through G2/M phase arrest in vitro. showed that through G2/M phase arrest in vitro. However, data showed thatthe decreased expression XRCC6 in decreased expression of XRCC6 in MNNG/HOS did not influence ability of migration orof invasion MNNG/HOS did not influence the ability of migration or invasion (Figure S1A,B). (Figure S1A,B).
Figure 2. Knockdown XRCC6inhibited inhibited cell cell proliferation proliferation through phase arrest. (A,B) The The Figure 2. Knockdown ofof XRCC6 throughG2/M G2/M phase arrest. (A,B) expression of XRCC6 was downregulated by a targeted siRNA; (C,D) a CCK8 assay was used to detect expression of XRCC6 was downregulated by a targeted siRNA; (C,D) a CCK8 assay was used to detect the proliferation of MNNG/HOS cells transfection after transfection with targeted the proliferation of MNNG/HOS cellscells and and U2OSU2OS cells after with targeted siRNA.siRNA. Diagrams Diagrams showing the results of a CCK-8 assay that MNNG/HOS and U2OS proliferation were showing the results of a CCK-8 assay that MNNG/HOS and U2OS proliferation were inhibited by inhibited by downregulating XRCC6 expression; (E,G) cell cycle profiles determined by propidium downregulating XRCC6 expression; (E,G) cell cycle profiles determined by propidium iodide (PI) iodide (PI) staining and flow cytometry assays of MNNG/HOS transfected with si-XRCC6 or si-NC; staining and flow cytometry assays of MNNG/HOS transfected with si-XRCC6 or si-NC; (F,H) cell (F,H) cell cycle profiles determined by propidium iodide (PI) staining and flow cytometry assays of cycle profiles determined by propidium iodide (PI) staining and flow cytometry assays of U2OS U2OS transfected with si-XRCC6 or si-NC. The data are representative of three independent transfected withError si-XRCC6 or si-NC. data are representative of**three independent experiments. experiments. bars represent SDThe (Standard Deviation). * p < 0.05, p < 0.01 by Student’s t test. Error bars represent SD (Standard Deviation). * p < 0.05, ** p < 0.01 by Student’s t test.
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2.3. Decreased XRCC6 XRCC6 Expression 2.3. Decreased Expression Impaired Impaired Colony-Forming Colony-Forming Capacity Capacity of of OS OS Cells Cells Colony determine the colony-forming capacity after Colony forming forming assay assay was was carried carried out out to to determine the colony-forming capacity of of OS OS cells cells after knockdown of XRCC6 expression. It was found that the number and the size of the colonies were knockdown of XRCC6 expression. It was found that the number and the size of the colonies were both in the group in with the the control both obviously obviously decreased decreased in the XRCC6 XRCC6 knockdown knockdown group in comparison comparison with control group group (Figure 3A,C). The number of colonies was significantly reduced by 44% and 54.5% in MNNG/HOS (Figure 3A,C). The number of colonies was significantly reduced by 44% and 54.5% in MNNG/HOS and In In conclusion, these results demonstrated that that XRCC6 was and U2OS U2OS cells, cells,respectively respectively(Figure (Figure3B,D). 3B,D). conclusion, these results demonstrated XRCC6 important for OS was important forcell OSgrowth. cell growth.
Figure 3. Decreased XRCC6 impaired OS cell colony-forming capacity. (A,C) Colony formation assay of MNNG/HOS cells and and U2OS U2OS cells cells transfected transfected with with targeted targeted siRNA or si-NC. After two weeks, cells MNNG/HOS cells in each well wellwere werefixed fixedand andcounted. counted.Representative Representative photo micrographs of MNNG/HOS (A); photo micrographs of MNNG/HOS cellscells (A); and and U2OS cells (C), colonies in culture plates; (B,D) significant reduction in the colony-forming efficacy U2OS cells (C), colonies in culture plates; (B,D) significant reduction in the colony-forming efficacy in MNNG/HOS cells (B), (B), and and U2OS U2OS cells cells (D). Following XRCC6 knockdown. Data are expressed as MNNG/HOS cells mean ˘ SD.of ofthree three independent independent experiments. experiments. ** **pp
