2.1. Patients information:We collected the frozen tissue samples (serum and tumor tissue) of 14 cases of patients with Her-2 positive BC who received neoadjuvant chemotherapy from June 2010 to May 2012 in our hospital sample bank, including 3 cases of stage IIB, 9 cases of stage IIIA and 2 cases of stage IIIC. The median age of 14 cases patients was 53.5 (38-65) years. All patients were confirmed as invasive ductal cancer by Mammotone biopsy before chemotherapy, and then received complete course of chemotherapy + targeted therapy. The chemotherapy regimens were EC*4→TH*4 (epirubicin+cyclophosphamide→docetaxel+herceptin).The therapeutic effects of neoadjuvant chemotherapy was evaluated by RECIST (version 1.1) after chemotherapy, which was divided into CR (complete response), PR (partial response), PD (progressive disease) and SD (stable disease). 10 cases of patients with CR or PR were enrolled in the chemosensitivity group, with a median age of 52 (38-65) years. 10 cases of chemosensitivity patients included 2 cases stage IIB, 6 cases stage IIIA and 2 cases stage IIIC. The efficacy of SD or PD was evaluated in the chemoresistance group. The median age of the chemotherapy resistance group was 56 (53-58) years old. 4 cases of chemoresistance patients including 1 cases stage IIB and 3 cases stage IIIA. Tumor inhibition rate after chemotherapy was evaluated in all patients and was calculated according to the change of the maximum diameter of the tumor before and after chemotherapy, it means Tumor inhibition rate = (Max diameter of pre-chemotherapy - Max diameter of post-chemotherapy)/ Max diameter of pre-chemotherapy x 100%. In addition, serum samples from 10 healthy persons were selected as healthy control group. The median age of the control group was 54.8 (39-69) years old.
2.2. Clinical monitoring indicators: The clinical TNM stage, tumor T stage, lymph node N stage, mortality rate, RFS (mom), OS (mon), and tumor inhibition rate were recorded. The deadline for follow-up was 31 December 2017. OS refers to the deadline for follow-up or the time of death during follow-up; RFS refers to the time of first recurrence and metastasis during follow-up.
2.3. Laboratory monitoring indicators: Quantitative RT-PCR was used to detect the relative expression level of three kinds of miRNAs and MALAT1 in tumor tissues of all BC patients, and also detect MALAT1 level in serum of healthy control group and all BC patients. Western blot was used to detect the relative expression levels of p-AKT, AKT, p-mTOR, mTOR and PTEN in 4 cases chemotherapy resistance patients. We studied the changes of the above molecular indicators levels before and after neoadjuvant chemotherapy in different groups, and compared the correlation between above molecular indicators and clinical monitoring indicators.
2.4 Experimental Detection Method of MiRNAs for Tumor Tissue
2.4.1 MiRNAs Extraction and Reverse Transcription: The main reagents are PureLink ® miRNA Isolation Kit (Thermo Fisher, NO: K1570-01) and SuperScript™ III Reverse Transcriptase (Thermo fisher, NO: 18080085). See Table 1.
2.4.2 Main steps of qRT-PCR detection for Tumor Tissue: The main reagents are PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, NO: A25779).
(1) Design and synthesis of RT-PCR primers: Primer Premier 6.0 and Beacon designer 7.8 were used to design quantitative PCR primers, which were synthesized by Bioengineering (Shanghai) Co., Ltd. The sequence of primers was as follows in Table 2.
(2) The statistical analysis of Real-time PCR gene expression: each sample detection was repeated three times.
2.5 Detection of Serum MALAT1 by qRT-PCR: The reagent used for extracting miRNA is MicroNeasy Serum/Plasma Kit (Qiagen NO:217184), and the reagent used for reverse transcription experiment is SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen NO:11752-050). Relevant experiments are carried out according to relevant operational requirements. Real-Time PCR was performed using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems NO: A25779). Primer Premier 6.0 and Beacon designer 7.8 were used to design quantitative PCR primers. Bioengineering Co., Ltd (Shanghai, China) was responsible for the synthesis of primers. The sequence of primers was as follows (See Table 3).
2.6 Detection of tissue MALAT1 by qRT-PCR: The reagents used for total RNA extraction are TRIzol® Plus RNA Purification Kit(Invitrogen NO:12183-555), RNase-Free DNase Set(Qiagen No. 79254), and the reagents used for reverse transcription experiment are SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen NO:11752-050). Real-Time PCR was performed using Power SYBR® Green PCR Master Mix (Applied Biosystems NO:4367659). Primer Premier 6.0 and Beacon designer 7.8 software were used to design quantitative PCR primers. Bioengineering Co., Ltd (Shanghai) was responsible for the synthesis of primers with the same primer sequence.
2.7 Western blot detection
2.7.1 Main detecting steps: We separated the tumor cells of drug resistant tumor tissue to do the Western blot detection. Total protein extraction kit (including Protease Inhibitor Cocktail) was used to extract total protein, and then BCA quantitative kit was used to quantify total protein detection. SDS-PAGE electrophoresis analysis, protein transfer membrane and transfer membrane sealing were performed according to relevant requirements.
2.7.2 Incubation of first antibody: First antibody is dissolved in T-TBS (containing 3% skimmed milk powder or BSA) in a certain proportion and incubated overnight at 4 ℃. Then T-TBS is rinsed for 5 min *4 times, see Table 4.
2.7.3 Second antibody incubation: Second antibody dissolves in T-TBS (containing 2% skimmed milk powder) at room temperature for 1 hour, then T-TBS is rinsed for 5 min *5 times, see Table 5.
2.7.4 Signal detection: Super Signal® West Dura Extended Duration Substrate was used, and follow the instructions to detect. To prepare 1 ml ECL, the transfer film was incubated at room temperature for 1 minute, then the redundant ECL reagent was removed, the fresh-keeping film was sealed, and X-ray film was placed in the dark box after exposure for 5-10 minutes.
2.7.5 Data analysis: Image J software was used to analyze the optical density of the bands. Each band was repeated three times. The relative expression of the target protein was expressed = {the objective protein (optical density)/internal parameter (optical density)} *10n, and the results were expressed as mean ± standard deviation.
2.8 Correlation analysis of experimental detection and clinical indicators: To evaluate the correlation of three miRNAs and MALAT1 levels in serum and tumor tissues before and after chemotherapy in different Her-2 positive group with clinical TNM stage, tumor T stage, lymph node stage, RFS, OS, and tumor inhibition rate (%).To evaluate the correlation between the levels of p-AKT, AKT, p-mTOR, mTOR and PTEN in tumor tissues of chemotherapy resistance groups with above clinical indicators.
2.9 Statistical methods: Paired T test was used to compare the different indicators of the same patient before and after chemotherapy. Pearson correlation analysis was used to analyze the correlation between the different indicators. Survival analysis (Mantel-Cox test) was used to compare the OS and RFS. P<0.05 was used to judge the statistical significance.