Changes of Three kinds of miRNAs, MALAT1 and PI3K/AKT/mTOR signaling pathway Protein Levels in Her-2 Positive Breast Cancer in neoadjuvant chemotherapy and their relationship with prognosis- A small sample study report CURRENT

Objective To investigate the roles of three kinds of miRNAs (MiR-99a, MiR-455 and MiR-143), Lnc MALAT1 and related proteins of PI3K/AKT/mTOR signaling pathway in neoadjuvant chemotherapy of patients with Her-2 positive breast cancer (BC) and analysed their related mechanisms. Method The frozen tumor tissue and serum samples before and after neoadjuvant chemotherapy of 14 cases patients with Her-2 positive BC who received neoadjuvant chemotherapy were collected. The relative expressions level of MiR-99a, MiR-455, MiR-143, MALAT1 and related proteins of PI3K/AKT/mTOR signaling pathway in tumor tissue and a part of serum samples were detected by qRT-PCR or western blot. Results MiR-99a level was negatively correlated with RFS and OS before and after chemotherapy. MiR-99a and MiR-455 levels after chemotherapy was negatively correlated with OS. The serum MALAT1 level was negatively correlated with RFS and OS in chemosensitivity group before and after chemotherapy. In chemotherapy resistance group, p-AKT level before chemotherapy was negatively correlated with T stage, p-mTOR level after chemotherapy was positively correlated with lymph node stage and negatively correlated with RFS and OS, PTEN level was negatively correlated with clinical TNM stage. Conclusion Serum MALAT1 and MiR-99a, MiR-455, MiR-143, p-AKT, p-mTOR and PTEN of tumor tissues in Her-2 positive BC patients can be used as markers to judge neoadjuvant chemotherapy sensitivity in Her-2 positive BC. Chemotherapy resistance is may be related to PI3K/AKT/mTOR signaling pathway. 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.


Introduction
Breast cancer (BC) is a highly heterogeneous malignant tumor, and the prognosis of different molecular subtypes of BC patients is quite different. BC has become the most common malignant tumor in women, and the incidence of BC is increasing [1] . Epidermal growth factor receptor 2 (Her-2)positive BC is a subtype of BC, and 20-30% of BC patients have Her-2 overexpression. High expression of Her-2 is closely related to the occurrence, development, invasion and migration of BC [2,3] . Her-2 positive BC patients are prone to metastasis and recurrence with poor prognosis. The median disease-free survival (DFS) and overall survival (OS) of patients are significantly shorter than those of Her-2 negative patients [4][5][6][7] .
The neoadjuvant chemotherapy has become the important routine treatments for BC. It is often used to treat patients with locally advanced BC or BC patients who need to reduce the extent of surgical resection. Although the prognosis of patients with different types of BC has been greatly improved through the various comprehensive treatments, about 15% of patients still have recurrence and distant metastasis. Among of them, chemotherapy resistance to chemotherapeutic drugs is one of the main causes of recurrence and metastasis of BC. Due to the high heterogeneity and individual differences of BC, about 20-30% of neoadjuvant chemotherapy patients show resistance to chemotherapeutic drugs [8] . The chemotherapy resistance of neoadjuvant chemotherapy not only prevents patients from receiving local surgery after chemotherapy, delays their condition, but also reduces the breast-conserving rate of patients. Neoadjuvant chemotherapy can be used to predict the in vivo sensitivity and chemotherapy resistance to chemotherapeutic drugs for the part of sub-types of BC with poor prognosis, such as Her-2 positive BC. The high heterogeneity of BC is the main cause of chemotherapy resistance. If there is resistance to chemotherapy, it will seriously affect the prognosis and survival rate of patients.
At present, miRNA (MicroRNAs) and lncRNA (Long non-coding RNA) are involved in the development of Her-2-positive BC and play an important role in neoadjuvant chemotherapy. In the human genome, only about 2% of the genes encode proteins, while up to 80% of the genomes are transcribed into non-coding RNA. Transcriptional studies based on second-generation sequencing have shown that a large number of miRNAs and lncRNA are abnormally expressed in cancer cells, and participate in the advance and onset of cancer and the process of chemotherapy resistance [9] .
MiRNAs may act on different pathways in various cancers and can serve as valuable sources for biomarker detections and optimal chemotherapeutic choices for BC patients [10][11][12] . Several studies have shown that some miRNAs are associated with prognosis in Her-2 positive BC [13,14] . The circulating miRNAs can be as chemotherapeutic response predictors, prognosticators or potential therapeutic targets [15] . Gu X et al found that the relative expression level of miR-451 was lower in the neoadjuvant chemotherapy resistant BC patients, suggesting the circulating miR-451 may have a functional significance in predicting the resistance to neoadjuvant chemotherapy in BC patients [16] . lncRNA MALAT1 (Metastasis-associated Lung Adenocarcinoma Transcript 1) is a conservative lncRNA that is highly expressed in many cancers, including BC [17] . MALAT1 levels are reported to be overexpressed in the serum or tissues of most tumors, and the MALAT1 levels often affect the tumor size, tumor stage, lymph node metastasis, and distant diffusion. Therefore, MALAT1 can be used as a new biomarker for early diagnosis, assessment of severity, or prognostic judgement. There are few reports on the relationship between MALAT1 and chemotherapy resistance in malignant tumors, and no literature on BC has been reported. Li P et al 's study showed that high MALAT1 level was associated with lower survival rate and bad response to oxaliplatin based chemotherapy in advanced colorectal cancer patients. Oxaliplatin-resistant colorectal cancer cells have high MALAT1 levels [18] .
We found that MiR-99a, MiR-455, MiR-143 and MALAT1 were associated with lymph node metastasis and prognosis in our previously experiment on Her-2 positive BC patients which has contributed to other magazines. In order to explore the role and mechanism of them in neoadjuvant chemotherapy of Her-2 positive BC, we further studied the three kind of miRNAs, MALAT1 and evaluated their correlation with clinical TNM stage, T stage, N stage of lymph node and survival time before and after neoadjuvant chemotherapy in Her-2 positive BC patients. We also detected the levels of related proteins of PI3K/AKT/mTOR signaling pathway (p-AKT, AKT, p-mTOR, mTOR and PTEN) in chemotherapy resistance groups, and explored the related mechanism of chemotherapy resistance.
Because of the lack of experimental funds, we only carried out a small sample experiment. This study is the first to be reported. 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

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.  (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.

Main steps of qRT-PCR detection for Tumor
(2) The statistical analysis of Real-time PCR gene expression: each sample detection was repeated three times. Co., Ltd (Shanghai, China) was responsible for the synthesis of primers. The sequence of primers was as follows (See Table 3). 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.

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.

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.

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 Xray film was placed in the dark box after exposure for 5-10 minutes.

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.

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.

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.

Comparison of molecular indicators before and after chemotherapy between different groups:
MiR-99a level after chemotherapy was significantly higher than that before chemotherapy (P<0.05). MiR-455 level in chemoresistance group was significantly lower than that before chemotherapy (P<0.01), and MiR-143 level in sensitive group was significantly higher than that before chemotherapy (P<0.01). There was no significant difference between OS and RFS in chemoresistance group and sensitive group (P>0.05). The relative expression level of MALAT1 in serum before and after chemotherapy was significantly higher than that in healthy control group (P<0.001). There was no significant difference in the MALAT1 level in tumor tissues before and after chemotherapy (P>0.05). There was no significant difference in MALAT1 between chemosensitivity group and chemotherapy resistance group before and after chemotherapy (P>0.05). There was no significant difference in OS and RFS between chemotherapy resistance group and chemosensitivity group after chemotherapy (P>0.05). The MALAT1 level in chemosensitivity group before chemotherapy was significantly higher than that after chemotherapy (P>0.05). In chemotherapy resistance group, the relative expression levels of p-AKT and p-mTOR after chemotherapy were significantly higher than those before chemotherapy (P<0.001, P<0.01). But, PTEN was significantly lower than before chemotherapy (P<0.05). See Table 6

Discussion
We tested three miRNAs, MALAT1 and some related proteins of PI3K/AKT/mTOR signaling pathway to find molecular markers that can predict neoadjuvant chemotherapy and prognosis of Her-2 positive BC patients. Because there was no project funding for this research, so we carried out a small sample study which is only an exploratory experiment.

MiR-99a
The roles of miR-99a in BC has been reported differently, one view is considered miR-99a to be a tumor suppressor and another view is considered miR-99a to be a cancer-promoting factor. Existing research data indicated that serum miR-99a expression was downregulated in BC patients, this downregulation was associated with poor prognosis, suggesting that serum miR-99a could function as a tumor suppressor in BC [19] . According to reports, miR-99a has been reported as a tumor suppressor gene in various cancers in humans. However, only limited information about the function of miR-99a in human BCs is available. Hu Y et al investigated the expression of miR-99a in BC tissue specimens and its antitumor activity in BC cells. They identified that the expression of miR-99a was significantly reduced in four kind of BC cell lines. More importantly, we found downregulation of miR-99a in BC specimens from ten different patients [20] . On the other hand, miR-99a was noticed to significantly inhibit cell proliferation and migration in triple-negative BC cells [21] . Another microarray study suggested that expression level of miR-99a was relatively low in BC tissues with lymph node metastasis [22] . In our study, the dynamic changes of miR-99a before and after neoadjuvant chemotherapy suggest that it may be a tumor suppressor.

MiR-143
MiR-143 has important physiological functions in BC cell proliferation, apoptosis, invasion, metastasis, angiogenesis and cell division. Its differential expression may be a molecular marker for clinical diagnosis and prognosis of BC. The role of miR-143 in BC is still controversial. Some literature suggests that miR-143 is high or low expression in BC [23][24][25] . Currently, more studies consider miR-143 as a tumor suppressor gene [26] . There's a study has shown that miR-143 is highly expressed in MCF-7 cells. Baradaran et al have found that miR-143 can inhibit the proliferation, invasion and migration of BC cells MDA-MB-468 [27] . Xia C et al results showed that the expression of miR-143-3p is significantly downregulated in BC cells. Upregulation of miR-143-3p inhibited the proliferation and migration of BC cells. Conversely, inhibition of miR-143-3p promoted the proliferation of cancer cells [28] . In our study, the dynamic changes of miR-143 before and after neoadjuvant chemotherapy suggest that it may be a tumor suppressor.

MiR-455-3p
The patients with infiltrating carcinoma or lymph node metastasis had a lower serum level of miR-455-3p than patients with the carcinoma in situ or patients without lymph node metastasis [29] . Wang B et al explored the effect of miR-455 on cell proliferation of BC, and found that miR-455 was downregulated in BC tissues and cells. Its overexpression inhibited cell proliferation, whereas its knockdown promoted cell proliferation of BC [30] . qRT-PCR analysis confirmed that the expression of miR-455-3p in TNBC cell lines MDA-MB-231 and MDA-MB-468 was higher than that in HR positive BC cell line MCF-7 (P<0.01) [31] .
Our study found that the relative expression level of MiR-99a after chemotherapy was significantly higher than that before chemotherapy. MiR-99a were negatively correlated with OS before and after chemotherapy. MiR-99a in the chemotherapy resistance group was negatively correlated with RFS and OS. MiR-455 after chemotherapy was negatively correlated with OS. MiR-455 in the chemotherapy resistance group was significantly lower than that before chemotherapy. MiR-143 in the chemotherapy sensitive was significantly higher than that before chemotherapy. In conclusion, three kinds of miRNAs in Her-2 positive BC tissues may become new kinds of molecular marker for predicting chemotherapy resistance of BC, and even become new kinds of potential molecular target.
It is of great significance to further explore the molecular mechanisms by which three miRNAs regulate chemoresistance in BC.

MALAT1 and PI3K/AKT/mTOR Signaling Pathway
MALAT1 is a typical lncRNA that is markedly up-regulated in BC. However, current understanding of the involvement of MALAT1 in BC development and prognosis remains unclear. In the Zheng L's study, the expression of MALAT1 in clinical samples of BC tissues was found to be significantly upregulated that was consistent with the result based on the dataset of the Cancer Genome Atlas (TCGA) at cBioportal. A negative correlation between overall survival and the expression of MALAT1 was statistically significant in the group of diagnosis age below 60 or in the group of infiltrating ductal carcinoma analyzed by TCGA database, which declared that MALAT1 might be a potentially useful prognostic factor [32] . Miao Y et al's research showed that an elevated MALAT1expression in BC tissue was significantly associated with lymph metastasis and adverse 5-year disease-free survival (DFS). They concluded that upregulation of MALAT1 plays an important role in BC development, serum MALAT1 level may be a potential tumor marker for BC diagnosis [33] . MALAT1 level was positively correlated with lymph node condition, estrogen receptor and tumor stage indicating its prognostic value. It can be used as amarker for diagnosis of BC [34] . Jadaliha M reported that assessment of the prognostic significance of MALAT1 in BC patients reveal elevated MALAT1 expression is associated with decreased disease-specific survival in ER negative, lymph node negative patients of the Her-2 and TNBC (triple negative breast cancer) molecular subtypes. They propose that MALAT1 can be used as a prognostic marker [35] . Wang Z et al found that BC patients with high expression of MALAT1 has a twofold increase in risk of relapse (p = 0.0083) compared to those with low expression. High level of MALAT1 is associated with BC relapse [36] . Our previous studies have shown that MALAT1 is the core signal molecule to promote the development and metastasis of Her-2-positive BC. MALAT1 is positively correlated with the numbers of lymph node metastasis of Her-2-positive BC. Knocking down the expression of MALAT1 in Her-2-positive BC cell lines can inhibit the proliferation and migration of BC cells [37] .
This study showed that the relative expression of MALAT1 in serum of sensitive patients before and after chemotherapy was negatively correlated with RFS and OS, which further indicated that MALAT1 might be a positive regulator of BC proliferation and metastasis. In this study, we found that the mechanism of Her-2 positive BC chemotherapy resistance may be related to PI3K/AKT/mTOR resistance signaling pathway.
The mechanism of chemotherapy resistance in cancer cells is very complex, involving genomic variation and changes in various signaling pathways. Among of them, PI3K/AKT/mTOR signaling pathway has been studied. The PI3K family is divided into three types.  [38] .
In BC, the activation of PI3K/AKT/mTOR signaling pathway is closely related to the chemotherapy resistance of endocrine therapy drugs, targeted drugs and chemotherapy drugs. Previous studies have shown that trastuzumab can reactivate PI3K/AKT/mTOR signaling pathway by reducing the stability of PTEN in the treatment of Her-2-positive BC targeting drugs, and promote the survival and chemotherapy resistance of BC cells [39] . Jin Y et al reported that MALAT1 can induce EMT via PI3K/AKT pathway [40] . Wang C et al [41] reported that the MALAT1effects on cholangiocarcinoma cells might be through activating the PI3K/AKT signaling pathway. Unfortunately, because there were only four cases in the chemotherapy resistance group, we found that there is no correlation between MALAT1 level and drug-resistant pathway proteins.
This study showed that PI3K/AKT/mTOR signaling pathway was significantly activated in tumor tissues of chemotherapy resistance group after neoadjuvant chemotherapy compared with chemosensitivity.
In chemotherapy resistance group, the relative expression levels of p-AKT and p-mTOR after chemotherapy were significantly higher than that before chemotherapy, but PTEN after chemotherapy was lower than that before chemotherapy.
The expression of MALAT1 in BC was positively correlated with the activation of PI3K/AKT/mTOR signaling pathway [42] . This study showed that in chemotherapy resistance group before chemotherapy the relative expression level of p-AKT was negatively correlated with T stage, AKT was positively correlated with OS, mTOR was negatively correlated with lymph node stage, PTEN was positively correlated with RFS and OS. The relative expression level of p-mTOR in chemotherapy resistance group after chemotherapy was positively correlated with lymph node stage, negatively correlated with RFS and OS, and negatively correlated with PTEN. These results suggest that MALAT1 may play an important role in the development of chemotherapy resistance in Her-2 positive BC by inducing the decline of PTEN protein and activation of PI3K/AKT/mTOR signaling pathway.
This study also showed that there was no significant difference in OS and RFS between chemotherapy resistance group and chemosensitivity group after chemotherapy, this result really surprised us. It may be caused small sample study. If there is funding in the future, we plan to increase the number of samples and carry on more in-depth research. Although there are few samples in this study, there are still many findings which can provide ideas for our future research.
In conclusion, Serum MALAT1 and p-AKT, p-mTOR and PTEN of tumor tissue can be used as markers to judge neoadjuvant chemotherapy sensitivity in Her-2 positive BC. Chemotherapy resistance is related to PI3K/AKT/mTOR signaling pathway. Serum MALAT1 may become a new molecular marker for predicting chemotherapeutic resistance in BC, and even a potential new molecular target. It is of great significance to further explore the molecular mechanism of MALAT1 regulating chemoresistance in BC cells.

Declarations
Ethics approval and consent to participate: The Ethics Committee of Zhejiang Cancer Hospital was secured for our research reported, and all authors abided the related rules of Ethics Committee when this study began. All authors abided the ethics in this clinical study. The Ethics Committee of Zhejiang Cancer Hospital approved to publish this paper. The research involving human subjects, human material, and human data have been performed in accordance with the Declaration of Helsinki and have been approved by an appropriate ethics committee of Zhejiang Cancer Hospital.

Consent for publication:
All authors declare that we consent for publication.
Disclosure of potential conflicts of interest: All authors declare that they have no conflict of interest with each other. Zhang Xiping has received research grants from Zhejiang Cancer Hospital and Zhejiang Provincial Health Department. Zhao Hongchan participated in the translation of papers and design of the project. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. All authors declare that they have no conflict of interest with each other. We claimed that this paper was original and would not have any financial interest in a company or its competitor, and that all authors meet criteria for authorship.
Authors' contributions: Zhao Hongcan participated in the writing and revision of this paper. Zhang Xiping provided the idea of this paper, wrote the part of this paper. All authors contributed to the intellectual context and approved the final version.    Compare to before chemotherapy, *P<0.05, **P<0.01, ***P<0.001 Figure 1 Comparison of expression levels of three miRNAs before and after chemotherapy in all patients Figure 2 Comparison of expression levels of three miRNAs before and after chemotherapy in sensitive and resistant groups  Relative expression level of MALAT1 in chemosensitive and chemoresistant patient groups before and after chemotherapy Note: compared to before chemotherapy, *P<0.05; NS means no significant Legend: There was no significant difference in MALAT1 expression in tumor tissuebetween chemosensitivity group and chemotherapy resistance group before and after chemotherapy (P > 0.05). MALAT1 in the sensitive group was significantly lower than that before chemotherapy (P<0.05). There was no significant difference in the expression level of MALAT1 in the chemotherapy resistance group before and after chemotherapy (P>0.05). There was no significant difference in the expression level of MALAT1 between the chemotherapy resistance and the chemosensitivity group before and after chemotherapy (P>0.05). (A: serum; B: tumor tissue) Figure 6 Expression of PI3K/AKT/mTOR pathway related proteins in chemotherapy resistance group

Figures
Note: compared to before chemotherapy, *P<0.05, * *P<0.01, *** P<0.001 Legend: The expression of p-AKT and p-MTOR after chemotherapy was significantly higher than that before chemotherapy (P<0.001, P<0.01), and PTEN before chemotherapy was significantly lower than that after chemotherapy (P<0.05). There was no significant difference between the other groups (P>0.05).

Figure 7
Expression levels of PI3K pathway related proteins by Western blot detection