肿瘤相关成纤维细胞中环状RNA-CAFAC1上调促进肺腺癌侵袭转移的机制研究

Through high-throughput sequencing technologies, we identified CAFAC1 as a novel circular RNA (circRNA) which is highly over-expressed in cancer-associated fibroblasts. Further expression level validation of this circRNA was performed by real-time RT-PCR and we found that CAFAC1 is 6.81-fold upregulated in plasma of lung adenocarcinoma(LAC) patients compared to normal patients plasma. The expression level of CAFAC1 is positively correlated with the lymph node metastasis, and negatively correlated with the long-term survival time of patients significantly. Overexpressed of CAFAC1 in CAFs co-culture with A549 significantly promote the invasion and metastasis of A549. Furthermore, RNA-pulldown, RIP, Bio-Plex array experiment revealed that CAFAC1 could interact with hnRNPK, an RNA binding protein, to increase mRNA stability of CCL2, a known cytokine which promotes the invasion and metastasis of cancer cells. Based on the above clinical and experimental data, we hypothesized that “CAFAC1 could decoy hnRNPK to increase CCL2 expression at post-transcription level, which promotes the invasion and metastasis of LAC”. The current project aims to validate that CAFAC1 could promote invasion and metastasis of LAC in vitro and in vivo by using silencing and overexpressing strategy at first. Secondly, the mechanism of CAFAC1/hnRNPK/CCL2 axis to regulate the invasion and metastasis in LAC will be deeply validated by RNA pull down, FISH and rescue experiments. Finally, the mechanism will be confirmed in animal experiment and clinical LAC patients. To date, no data is available about the function and mechanisms of CAFAC1 promoting invasion and metastasis of LAC, therefore, our study is the original source of innovation and can provide new potential biomarkers for the prevention and treatment of LAC.

CAFAC1（简称CA）是项目组高通量筛选获得的一条在肺腺癌肿瘤相关成纤维细胞（CAF）中显著上调、功能未知、自主命名的环状RNA。前期发现：肺腺癌患者血浆中CA高表达与淋巴结转移呈正相关，水平越高预后越差；过表达CA的CAF促进共培养A549侵袭转移能力显著提升。生物信息学及预试验提示：CA可结合hnRNPK蛋白，转录后维持促转移因子CCL2的mRNA稳定性。故提出CAF中CA结合hnRNPK在转录后水平上调CCL2、促进肺腺癌侵袭转移的科学假说。本项目拟采用过表达/沉默策略，体内外证实CA高表达CAF可促进肺腺癌侵袭转移；以hnRNPK为切入点设计拯救实验，阐明CA转录后维持CCL2 mRNA稳定性的分子机制；结合临床大样本评价CA/hnRNPK/CCL2与肺腺癌侵袭转移的相关性。有关CA高表达CAF促进肺癌侵袭转移的功能机制未见报道，本研究具有源头创新性，可为肺腺癌防治提供新思路。

目前尚未有肺癌 CAFs 相关 circRNA 的功能及机制报道，故进一步探寻 circRNA 在 CAFs中介导肿瘤细胞侵袭转移能力的潜在功能及机制，这将有助于揭示肺腺癌侵袭转移的新机制。CAFAC1（简称CA）是项目组高通量筛选获得的一条在肺腺癌肿瘤相关成纤维细胞（CAF）中显著上调、功能未知、自主命名的环状RNA。进一步临床样本验证发现肺腺癌患者血浆中CA高表达与淋巴结转移呈正相关，水平越高预后越差；体内外功能研究上发现过表达CA的CAF促进共培养A549侵袭转移能力显著提升且使免疫缺陷小鼠成瘤能力显著增强。生物信息学、pulldown、质谱分析、western等支撑实验发现，CA可结合hnRNPK蛋白，在转录后水平维持促转移因子CCL2的mRNA稳定性。有关CA高表达CAF促进肺癌侵袭转移的功能机制未见报道，本研究具有源头创新性，可为肺腺癌防治提供新思路。