RESUMO
Objective: To investigate the effects of maternally expressed gene 3 (MEG3) on endoplasmic reticulum stress-induced colon cancer cell apoptosis and the growth of transplanted human colonic carcinoma in nude mice. Methods:Colon cancer cell lines with the lowest expression level of MEG3 were screened by RT-PCR; negative control cell lines were constructed and stable transfectants were screened. RT-PCR was performed for measuring the lncRNA level of MEG3, cell growth was measured by CCK8, cell apoptosis was determined by Hoechst. Western blot was used to determine the protein levels of Bcl-2, Bax, cleaved Caspase-9, p-PERK, ATF-4, p-EIF2α, CHOP, cleaved Caspase-12, p-AMPK, AMPK, p-mTOR, and mTOR. The expression of CHOP was silenced by si-CHOP, and then cell apoptosis was determined by Hoechst. Transplanted human colonic carcinoma in nude mice was established and tumor volume was measured, tumor growth was measured by IHC Ki67, cell apoptosis was determined by TUNNEL, Western blot was used to determine the protein levels of CHOP, cleaved Caspase-12, and cleaved Caspase-3. Results: The SW480 cell line had the lowest expression level of MEG3 and was used in subsequent experiments. In cytological experiments, compared with those in control group, the cell growth, protein levels of Bcl-2 and ratio of p-mTOR/mTOR were decreased significantly; the apoptosis rate, protein levels of Bax, cleaved Caspase-9, p-PERK, ATF-4, p-eIFα, CHOP and cleaved Caspase-12 and ratio of p-AMPK/AMPK increased notably; the expression of CHOP was silenced; the apoptosis induced by pc-MEG3 could be significantly decreased. In animal experiments, compared with control group, the tumor volume and number of positive cells of Ki67 decreased significantly; the apoptosis rate, protein levels of CHOP, cleaved Caspase-12 and cleaved Caspase-3 increased markedly. Conclusion: MEG3 can promote colon cancer cell apoptosis by endoplasmic reticulum stress, and CHOP may play a crucial role in the process. Furthermore, AMPK/mTOR is also involved in the regulation of apoptosis by MEG3.
RESUMO
Traditional Chinese medicine believes that the occurrence and development of tumors is related to the body's Qi deficiency. " Invigorating Qi for consolidation of exterior" has became an effective way to treat tumors by traditional Chinese medicine. This study is based on the " invigorating Qi for consolidation of exterior" to explore the effect of flavonoid components in Qi-invigorating herbs Astragali Radix( AR) on the growth and immune function of mouse Lewis lung cancer xenografts,and further explore its mechanism of action. In the present study,high performance liquid chromatography was performed to analyze the flavonoid components in AR.The Lewis lung cancer model of C57 BL/6 mice was constructed,and the tumor volume of mice was determined by Visual Sonics Vevo2100 high frequency color ultrasound. The levels of IL~(-1)7 and RORγt in serum and tumor tissues were detected by ELISA and immunohistochemistry. The expression of IRE~(-1)/XBP~(-1) pathway-related proteins in tumor tissues was detected by Western blot. The results revealed that treatment of 5 and 10 g·kg~(-1)·d~(-1) of flavonoid components in AR significantly inhibited tumor growth of C57 BL/6 tumorbearing mice. The inhibition rates at the dose of 5 and 10 g·kg~(-1)·d~(-1) of flavonoid components in AR were( 29. 5±4. 4) % and( 43. 4±5. 2) %,respectively. The expression of IL~(-1)7 and RORγt in serum and tumor tissues of Lewis lung cancer mice were decreased,and the spleen index and thymus index were significantly enhanced by the flavonoid components in AR. Flavonoid components in AR could decrease the expression of X-box binding protein 1( XBP1),inositol-requiring enzyme( IRE1) and glucose regulated protein 78 k D( GRP78),and increase the expression of C/EBP homologous protein( CHOP),and the high-dose group is better,suggesting that the anti-lung cancer effect of flavonoid components in AR is related to the regulation of XBP1 mediated ERs. This study provides new evidence that the flavonoid components in AR could inhibit the tumor growth of C57 BL/6 tumor-bearing mice by regulating the body's immune function through " invigorating Qi for consolidation of exterior".