Lai Yuan-Hui, Chen Jian, Wang Xiao-Ping, Wu Yan-Qing, Peng Hai-Tao, Lin Xiao-Hong, Wang Wen-Jian
Department of Thyroid and Breast Surgery, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, 510700, China.
Department of Organ Transplantation, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, 510700, China.
J Exp Clin Cancer Res. 2017 Jul 12;36(1):92. doi: 10.1186/s13046-017-0564-7.
Collagen triple helix repeat containing-1 (CTHRC1), which was firstly identified overexpressed in the adventitia and neointima of injured rat arteries, could inhibit collagen expression and increase cell migration. It was then found to be ubiquitously expressed in numerous cell types such as fibroblasts and smooth muscle cells, and aberrantly up-regulated in several malignant tumors. However, the functional role of CTHRC1 and its related mechanism in breast cancer still remains unclear.
CTHRC1 expressions in breast cancer tissues and cells were assessed by qRT-PCR, western blot and immunohistochemistry. The relative expression level of miR-134, miR-155, miR-30c and miR-630 in breast cancer cells respectively was detected by qRT-PCR. Wild type (Wt) and Mutant type (Mut) CTHRC1 3'UTR sequences were cloned into a psi-CHECK2 reporter vector, and the relative luciferase activity was detected by dual-luciferase reporter assay in indicated cells. The effect of ectopic expression of miR-30c or gain and loss of CTHRC1 on cell viability, cell proliferation, cell cycle progression and apoptosis, cell invasion and migration was respectively detected by CCK-8 assay, colony formation assay, flow cytometry analysis, transwell invasion/migration assay. Protein levels of β-catenin, active β-catenin, normal and phosphorylated form of GSK-3β were detected by western blot in indicated cells. Immunofluorescence staining of β-catenin was performed to observe nuclear localization.
We found CTHRC1 was frequently up-regulated in human breast cancer cells and tissues. Then our cohort study and further meta-analysis validated high expression of CTHRC1 was associated with aggressive clinicopathological features and poor clinical outcome of breast cancer patients. In addition, CTHRC1 promoted cell proliferation, invasion and migration and suppressed cell apoptosis in breast cancer, which might be by activating GSK-3β/β-catenin signaling and inhibiting Bax/Caspase-9/Caspase-3 signaling respectively; and these biological functions of CTHRC1 could be directly negatively regulated by miR-30c.
Taken together, we identified the role of miR-30c/CTHRC1 axis in breast cancer progression and demonstrated CTHRC1 may serve as a prognostic biomarker and therapeutic target for breast cancer.
含胶原蛋白三螺旋重复序列-1（CTHRC1）最初被鉴定为在损伤大鼠动脉的外膜和内膜中过表达，它可抑制胶原蛋白表达并促进细胞迁移。随后发现其在多种细胞类型如成纤维细胞和平滑肌细胞中广泛表达，并在几种恶性肿瘤中异常上调。然而，CTHRC1在乳腺癌中的功能作用及其相关机制仍不清楚。
通过qRT-PCR、蛋白质印迹法和免疫组织化学评估CTHRC1在乳腺癌组织和细胞中的表达。通过qRT-PCR检测乳腺癌细胞中miR-134、miR-155、miR-30c和miR-630的相对表达水平。将野生型（Wt）和突变型（Mut）CTHRC1 3'UTR序列克隆到psi-CHECK2报告载体中，并在指定细胞中通过双荧光素酶报告基因检测法检测相对荧光素酶活性。分别通过CCK-8法、集落形成试验、流式细胞术分析、Transwell侵袭/迁移试验检测miR-30c异位表达或CTHRC1的增减对细胞活力、细胞增殖、细胞周期进程和凋亡、细胞侵袭和迁移的影响。通过蛋白质印迹法检测指定细胞中β-连环蛋白、活性β-连环蛋白、GSK-3β的正常和磷酸化形式的蛋白质水平。进行β-连环蛋白的免疫荧光染色以观察其核定位。
我们发现CTHRC1在人乳腺癌细胞和组织中经常上调。然后我们的队列研究和进一步的荟萃分析证实，CTHRC1的高表达与乳腺癌患者的侵袭性临床病理特征和不良临床结局相关。此外，CTHRC1促进乳腺癌细胞增殖、侵袭和迁移并抑制细胞凋亡，这可能分别是通过激活GSK-3β/β-连环蛋白信号通路和抑制Bax/Caspase-9/Caspase-3信号通路实现的；而CTHRC1的这些生物学功能可被miR-30c直接负调控。
综上所述，我们确定了miR-30c/CTHRC1轴在乳腺癌进展中的作用，并证明CTHRC1可能作为乳腺癌的预后生物标志物和治疗靶点。
