AIM:To investigate the roles of the ribonucleotide reductase M2 (RRM2) subunit in colorectal cancer (CRC) and ultraviolet (UV)-induced DNA damage repair. METHODS:Immunohistochemical staining of tissue microarray was p...AIM:To investigate the roles of the ribonucleotide reductase M2 (RRM2) subunit in colorectal cancer (CRC) and ultraviolet (UV)-induced DNA damage repair. METHODS:Immunohistochemical staining of tissue microarray was performed to detect the expression of RRM2. Seven CRC cell lines were cultured and three human colon cancer cell lines, i.e., HCT116, SW480 and SW620, were used. Reverse transcription polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels of RRM2, respectively. Cell proliferation assay, cell cycle analysis were performed. Cell apoptosis was evaluated by double staining with fluorescein isothiocyanate-conjugated Annexin Ⅴ and propidium iodide (PI) usingAnnexin Ⅴ/PI apoptosis kit. The motility and invasion of CRC cells were assessed by the Transwell chamber assay. Cells were irradiated with a 254 nm UV-C lamp to detect the UV sensitivity after RRM2 depletion. RESULTS:Immunohistochemical staining revealed elevated RRM2 levels in CRC tissues. RRM2 overexpression was positively correlated with invasion depth (P < 0.05), poorly differentiated type (P = 0.0051), and tumor node metastasis stage (P = 0.0015). The expression of RRM2 in HCT116 cells was downregulated after transfection, and HCT116 cell proliferation was obviously suppressed compared to control groups (P < 0.05). In the invasion test, the number of cells that passed through the chambers in the RRM2-siRNA group was 81 ± 3, which was lower than that in the negative control (289 ± 7) and blank control groups (301 ± 7.2). These differences were statistically significant (P < 0.01). Our data suggest that RRM2 overexpression may be associated with CRC progression. RRM2 silencing by siRNA may inhibit the hyperplasia and invasiveness of CRC cells, suggesting that RRM2 may play an important role in the infiltration and metastasis of CRC, which is a potential therapeutic strategy in CRC. In addition, RRM2 depletion increased UV sensitivity. CONCLUSION:These findings suggest that RRM2 may be a facilitating factor in colorectal tumorigenesis and UV-induced DNA damage repair.展开更多
Esophageal squamous cell carcinoma(ESCC),a malignancy of the digestive system,is highly prevalent and the primary cause of cancer-related deaths worldwide due to the lack of early diagnostic biomarkers and effective t...Esophageal squamous cell carcinoma(ESCC),a malignancy of the digestive system,is highly prevalent and the primary cause of cancer-related deaths worldwide due to the lack of early diagnostic biomarkers and effective therapeutic targets.Dysregulated ribonucleotide reductase(RNR)expression has been confirmed to be causally linked to tumorigenesis.This study demonstrated that ribonucleotide reductase small subunit M2(RRM2)is significantly upregulated in ESCC tissue and that its expression is negatively correlated with clinical outcomes.Mechanistically,HuR promotes RRM2 mRNA stabilization by binding to the adenine/uridine(AU)-rich elements(AREs)within the 3′UTR,resulting in persistent overexpression of RRM2.Furthermore,bifonazole is identified as an inhibitor of HuR via computational screening and molecular docking analysis.Bifonazole disrupts HuR-ARE interactions by competitively binding to HuR at F65,R97,I103,and R153 residues,resulting in reduced RRM2 expression.Furthermore,bifonazole exhibited antitumor effects on ESCC patient-derived xenograft(PDX)models by decreasing RRM2 expression and the dNTP pool.In summary,this study reveals the interaction network among HuR,RRM2,and bifonazole and demonstrated that bifonazole is a potential therapeutic compound for ESCC through inhibition of the HuR/RRM2 axis.展开更多
Cordycepin was the first adenosine analogue used as an anticancer and antiviral agent, which is extracted from Cordyceps militaris and hasn’t been biosynthesized until now. This study was first conducted to verify th...Cordycepin was the first adenosine analogue used as an anticancer and antiviral agent, which is extracted from Cordyceps militaris and hasn’t been biosynthesized until now. This study was first conducted to verify the role of ribonucleotide reductases(RNRs, the two RNR subunits, RNRL and RNRM) in the biosynthesis of cordycepin by over expressing RNRs genes in transformed C. militaris. Quantitative real-time PCR(qRT-PCR) and western blotting results showed that the m RNA and protein levels of RNR subunit genes were significantly upregulated in transformant C. militaris strains compared to the control strain. The results of the HPLC assay indicated that the cordycepin was significantly higher in the C. militaris transformants carrying RNRM than in the wildtype strain, whereas the RNRML was preferentially downregulated. For the C. militaris transformant carrying RNRL, the content of cordycepin wasn’t remarkably changed. Furthermore, we revealed that inhibiting RNRs with Triapine(3-AP) almost abrogated the upregulation of cordycepin. Therefore, our results suggested that RNRM can probably directly participate in cordycepin biosynthesis by hydrolyzing adenosine, which is useful for improving cordycepin synthesis and helps to satisfy the commercial demand of cordycepin in the field of medicine.展开更多
Fluorodeoxyglucose positron emission tomography/conlputed tomography （FDG PET/CT） is widely applied in non-small cell lung cancer （NSCLC）. The standardized uptake value （SUV）, a semi-quantitative index, plays an...Fluorodeoxyglucose positron emission tomography/conlputed tomography （FDG PET/CT） is widely applied in non-small cell lung cancer （NSCLC）. The standardized uptake value （SUV）, a semi-quantitative index, plays an essential role in NSCLC tbr diagnosis, staging, and efficacy evaklation. It has been px3posed that the SUV of tumors may correlate with the presence or absence of chemotherapy resistance-associated biomarkers based on studies that have displayed a close correlation between SUV and the expression levels of excision repair cross-complementary Group 1 （ERCC 1 ）1~1 and Tp53-induced glycolysis and apoptosis regulator.121 FDG avidity of NSCLC and ERCC 1 and ribonucleotide reductase subunit M 1 （RRM 1 ） levels have not been as extensively investigated. Based on these findings, we looked tbr correlations among metabolic parameters （SUVm,,. metabolic tumor volume [MTV], and total lesion glycolysis [TLG]） and ERCC1 and RRM1 expression in patients with NSCLC, to investigate whether FDG uptake reflects the presence or absence ofchemoresistance proteins （ERCC1 and RRM 1 ） within tumor cells.展开更多
背景与目的吉西他滨是目前治疗非小细胞肺癌(NSCLC)的一线药物。研究表明作为吉西他滨作用靶点的核苷酸还原酶亚单位M1(RRM1),其启动子区域的基因多态性可以影响吉西他滨对非小细胞肺癌患者的疗效。本研究旨在建立一种简便和高特异性的...背景与目的吉西他滨是目前治疗非小细胞肺癌(NSCLC)的一线药物。研究表明作为吉西他滨作用靶点的核苷酸还原酶亚单位M1(RRM1),其启动子区域的基因多态性可以影响吉西他滨对非小细胞肺癌患者的疗效。本研究旨在建立一种简便和高特异性的方法,检测RRM1(-37)位点的基因多态性,并以此方法分析RRM1(-37)位点A/C基因型与吉西他滨治疗NSCLC疗效的相关性。方法收集110例肺癌患者和40例健康人的外周血,提取基因组DNA,以RRM1基因的启动子区域为目的基因,合成野生型引物WF、突变型引物TF和共同的下游引物R。利用DNA荧光嵌入染料SYBR Green I进行荧光PCR扩增,对PCR产物进行熔解曲线分析以确定基因型。分析RRM1(-37)位点基因多态性与患者的一般状态、接受吉西他滨治疗的疗效及生存时间的关系。结果110例肺癌患者和40例健康人中A/A等位基因频率分别为32%和30%,两组人群在RRM1(-37)位点基因分布频率上无统计学差异,RRM1(-37)位点的基因多态性与肺癌患者的性别、病理类型及临床分期无相关性(P>0.05),且与肺癌患者接受吉西他滨治疗的疗效及生存时间无相关性(P>0.05)。结论本研究建立的引物特异性实时荧光PCR法可对RRM1(-37)位点进行准确的基因分型。展开更多
文摘AIM:To investigate the roles of the ribonucleotide reductase M2 (RRM2) subunit in colorectal cancer (CRC) and ultraviolet (UV)-induced DNA damage repair. METHODS:Immunohistochemical staining of tissue microarray was performed to detect the expression of RRM2. Seven CRC cell lines were cultured and three human colon cancer cell lines, i.e., HCT116, SW480 and SW620, were used. Reverse transcription polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels of RRM2, respectively. Cell proliferation assay, cell cycle analysis were performed. Cell apoptosis was evaluated by double staining with fluorescein isothiocyanate-conjugated Annexin Ⅴ and propidium iodide (PI) usingAnnexin Ⅴ/PI apoptosis kit. The motility and invasion of CRC cells were assessed by the Transwell chamber assay. Cells were irradiated with a 254 nm UV-C lamp to detect the UV sensitivity after RRM2 depletion. RESULTS:Immunohistochemical staining revealed elevated RRM2 levels in CRC tissues. RRM2 overexpression was positively correlated with invasion depth (P < 0.05), poorly differentiated type (P = 0.0051), and tumor node metastasis stage (P = 0.0015). The expression of RRM2 in HCT116 cells was downregulated after transfection, and HCT116 cell proliferation was obviously suppressed compared to control groups (P < 0.05). In the invasion test, the number of cells that passed through the chambers in the RRM2-siRNA group was 81 ± 3, which was lower than that in the negative control (289 ± 7) and blank control groups (301 ± 7.2). These differences were statistically significant (P < 0.01). Our data suggest that RRM2 overexpression may be associated with CRC progression. RRM2 silencing by siRNA may inhibit the hyperplasia and invasiveness of CRC cells, suggesting that RRM2 may play an important role in the infiltration and metastasis of CRC, which is a potential therapeutic strategy in CRC. In addition, RRM2 depletion increased UV sensitivity. CONCLUSION:These findings suggest that RRM2 may be a facilitating factor in colorectal tumorigenesis and UV-induced DNA damage repair.
基金funded by National Natural Science Foundation of China(grant numbers:81872335,82303891 and 82303119)The Central Plains Science and Technology Innovation Leading Talents(No.224200510015,China)+1 种基金Key scientific research project plan of colleges and universities in Henan Province(grant number:24A310025,China)Science and Technology Project of Henan Province(No.242102310414,China).
文摘Esophageal squamous cell carcinoma(ESCC),a malignancy of the digestive system,is highly prevalent and the primary cause of cancer-related deaths worldwide due to the lack of early diagnostic biomarkers and effective therapeutic targets.Dysregulated ribonucleotide reductase(RNR)expression has been confirmed to be causally linked to tumorigenesis.This study demonstrated that ribonucleotide reductase small subunit M2(RRM2)is significantly upregulated in ESCC tissue and that its expression is negatively correlated with clinical outcomes.Mechanistically,HuR promotes RRM2 mRNA stabilization by binding to the adenine/uridine(AU)-rich elements(AREs)within the 3′UTR,resulting in persistent overexpression of RRM2.Furthermore,bifonazole is identified as an inhibitor of HuR via computational screening and molecular docking analysis.Bifonazole disrupts HuR-ARE interactions by competitively binding to HuR at F65,R97,I103,and R153 residues,resulting in reduced RRM2 expression.Furthermore,bifonazole exhibited antitumor effects on ESCC patient-derived xenograft(PDX)models by decreasing RRM2 expression and the dNTP pool.In summary,this study reveals the interaction network among HuR,RRM2,and bifonazole and demonstrated that bifonazole is a potential therapeutic compound for ESCC through inhibition of the HuR/RRM2 axis.
基金the Natural Sciences Foundation of China Science (Nos.81872959, 81373920,30801522)Sichuan Province Youth Innovation Team Fund (No.19CXTD0055)China Scholarship Fund (No. 201708570027)。
文摘Cordycepin was the first adenosine analogue used as an anticancer and antiviral agent, which is extracted from Cordyceps militaris and hasn’t been biosynthesized until now. This study was first conducted to verify the role of ribonucleotide reductases(RNRs, the two RNR subunits, RNRL and RNRM) in the biosynthesis of cordycepin by over expressing RNRs genes in transformed C. militaris. Quantitative real-time PCR(qRT-PCR) and western blotting results showed that the m RNA and protein levels of RNR subunit genes were significantly upregulated in transformant C. militaris strains compared to the control strain. The results of the HPLC assay indicated that the cordycepin was significantly higher in the C. militaris transformants carrying RNRM than in the wildtype strain, whereas the RNRML was preferentially downregulated. For the C. militaris transformant carrying RNRL, the content of cordycepin wasn’t remarkably changed. Furthermore, we revealed that inhibiting RNRs with Triapine(3-AP) almost abrogated the upregulation of cordycepin. Therefore, our results suggested that RNRM can probably directly participate in cordycepin biosynthesis by hydrolyzing adenosine, which is useful for improving cordycepin synthesis and helps to satisfy the commercial demand of cordycepin in the field of medicine.
文摘Fluorodeoxyglucose positron emission tomography/conlputed tomography （FDG PET/CT） is widely applied in non-small cell lung cancer （NSCLC）. The standardized uptake value （SUV）, a semi-quantitative index, plays an essential role in NSCLC tbr diagnosis, staging, and efficacy evaklation. It has been px3posed that the SUV of tumors may correlate with the presence or absence of chemotherapy resistance-associated biomarkers based on studies that have displayed a close correlation between SUV and the expression levels of excision repair cross-complementary Group 1 （ERCC 1 ）1~1 and Tp53-induced glycolysis and apoptosis regulator.121 FDG avidity of NSCLC and ERCC 1 and ribonucleotide reductase subunit M 1 （RRM 1 ） levels have not been as extensively investigated. Based on these findings, we looked tbr correlations among metabolic parameters （SUVm,,. metabolic tumor volume [MTV], and total lesion glycolysis [TLG]） and ERCC1 and RRM1 expression in patients with NSCLC, to investigate whether FDG uptake reflects the presence or absence ofchemoresistance proteins （ERCC1 and RRM 1 ） within tumor cells.
文摘背景与目的吉西他滨是目前治疗非小细胞肺癌(NSCLC)的一线药物。研究表明作为吉西他滨作用靶点的核苷酸还原酶亚单位M1(RRM1),其启动子区域的基因多态性可以影响吉西他滨对非小细胞肺癌患者的疗效。本研究旨在建立一种简便和高特异性的方法,检测RRM1(-37)位点的基因多态性,并以此方法分析RRM1(-37)位点A/C基因型与吉西他滨治疗NSCLC疗效的相关性。方法收集110例肺癌患者和40例健康人的外周血,提取基因组DNA,以RRM1基因的启动子区域为目的基因,合成野生型引物WF、突变型引物TF和共同的下游引物R。利用DNA荧光嵌入染料SYBR Green I进行荧光PCR扩增,对PCR产物进行熔解曲线分析以确定基因型。分析RRM1(-37)位点基因多态性与患者的一般状态、接受吉西他滨治疗的疗效及生存时间的关系。结果110例肺癌患者和40例健康人中A/A等位基因频率分别为32%和30%,两组人群在RRM1(-37)位点基因分布频率上无统计学差异,RRM1(-37)位点的基因多态性与肺癌患者的性别、病理类型及临床分期无相关性(P>0.05),且与肺癌患者接受吉西他滨治疗的疗效及生存时间无相关性(P>0.05)。结论本研究建立的引物特异性实时荧光PCR法可对RRM1(-37)位点进行准确的基因分型。
