Correlation Study And Its Potentially Clinical Significance For Non-coding Small RNA Molecule MiR-34a And Twist1 In Breast Cancer

Objective:To compare a small non-coding RNA molecule, miR-34a, and Twistl, an important transcription factor which is correlated to tumor, expressive files in different types of breast cancer cell lines, investigate the relationship between miR-34a and Twistl and their new signal pathways of tumor metastasis for cancer diagnosis and treatment, and explore the possible clinical significance, as tumor repression gene miR-34a, for suppressing tumor metastasis. Methods:Firstly, the total RNAs, including small non-coding RNA were extracted by using miRNeasy Mini Kit, specific stem-loop reverse transcription real-time PCR was performed and analyzed for detecting miR-34a expression in five different types of breast cancer cell lines MDA-MB-231, MDA-MB-435, T47D, BT549 and MCF-7. Also, we examined the Twistl mRNA level by real time RT-RCR in all these cell lines. Then, to futher investgate the relationship of expression regulation between miR-34a and Twist1, pLKO-Tet-On-shTwistl contruct was established successfully by molecular cloning, and the lentivirus was produced, and then the inducible and stable knocking down expression shTwistl cell lines were established via transfection of Twistl lentivirus into MDA-MB-435 and 4T1 cell lines, respectively. We performed specific stem-loop reverse transcription real-time PCR and western blot analysis to measure miR-34a and Twistl levels in MDA-MB-435 and 4T1 cell lines. At same time, we applied the specific stem-loop reverse transcription real-time PCR to examine the Twistl effects on miR-34a after Doxycycline (DOX) induction. Results:The high quality of total RNAs were attained; The expression level of miR-34a is lower in breast tumor cells MDA-MB-231, MDA-MB-435 and BT-549 with high aggressiveness, whereas the expression level of miR-34a is higher in breast tumor cells MCF7 and T47D with low aggressiveness by the technique of specific stem-loop reverse transcription real-time PCR. We found the high expression level of Twistl mRNA in MDA-MB-435 and BT549 cell lines, and low expression in MCF7 and T47D cell lines.Thus, there exist an inverse corelation between miR-34a and Twistl expression.Then, we constructed a vector for the inducible and stable expression pLKO-Tet-On-shTwist1 successfully. We used shRNA interference and lentivirus cell transfection to knock down Twistl, and established the MDA-MB-435-shTwistl and 4T1-shTwistl cell lines and their control cell lines. We detected and analysized miR-34a and Twistl in these cell lines by specific stem-loop reverse transcription real-time PCR or reverse transcription real-time PCR. Real time PCR results showed that miR-34a is upregulated in both MDA-MB-435-shTwistl and 4T1-shTwistl cell lines when knocking down Twistl. And also we used the previous established HEK-293-Twistl for over-expression of Twistl and its control HEK-293 cell lines. The result by specific stem-loop reverse transcription real-time PCR indicates that miR-34a is downregulated after overexpression of Twistl by DOX induction in HEK-293-Twistl cell lines. Conclusion:We established the technique of specific stem-loop reverse transcription real-time PCR for detecting small non-coding RNA successfully; We established inducible and stable knockdown Twistl cell lines in MDA-MB-435 and 4T1 cancer cells; The expression level of miR-34a is reverse correlated with tumor cells’high aggressiveness; miR-34a expression and Twistl expression is negative correlated, indicating that miR-34a and Twistl might have relationship of regulation each other, and miR-34a, as tumor repress gene which plays important role in suppressing tumor metastasis, have great potential for breast cancer diagnosis, prevention and therapy. We should further investigate that whether miR-34a and Twist1 is regulated directly each other on the furture.