Long non-coding RNAE330013P06 promotes progression of breast cancer with type 2 diabetes.

Abstract Background In previous research, we found diabetes rather than obesity was an independent risk factor of breast cancer. However, why diabetes could lead to increased risk of breast cancer patients remains elusive. Long non‐coding RNAE330013P06 has been shown to be upregulated in diabetes, and long non‐coding RNAs generally promote progression of cancer. Methods About 200 specimens of breast patients were obtained in previous clinical trial; 34 samples diagnosed as type 2 diabetes in breast cancer patient were enrolled in this research. Blood samples from 36 patients diagnosed as breast cancer without diabetes; 35 diabetic patients and 35 healthy peoples were obtained as control. All blood samples were measured by quantitative real‐time PCR (qRT‐PCR). Invasion and migration were tested by Transwell assay. Cell proliferation assay was tested by CCK‐8. Protein analysis was determined by Western blot. Results Compared with breast cancer patients without diabetes, diabetic patients without breast cancer and healthy peoples, LncRNAE330013P06 was upregulated in breast cancer patient with diabetes. Furthermore, of 34 breast patients, high LncRNAE330013P06 expression was significantly associated with family history, tumor‐node‐metastasis stage and lymph node metastasis. E33 promoted cancer cell growth in vitro via downregulation of P53. Conclusion Upregulation of LncRNAE330013P06 driven by type 2 diabetes is one of the factors which promoted progression of breast cancer.

higher body mass index (BMI). Our previous research showed that diabetes is an independent factor for risk of breast cancer rather than BMI. 3 Nevertheless, there were few breast patients with diabetes 1 that might be accounted for shorter life span of patients or its lower BMI. It has been demonstrated diabetic patients with breast cancer receiving metformin and neoadjuvant chemotherapy have a higher pCR rate than do diabetics not receiving metformin. 4 So, the underlying mechanism remains unknown. Upregulation of IGF-1 of plasm is an explanation why diabetes 2 would increase the risk of breast cancer. 5 But, some other researches seemed not to support this explanation.
Recently, long non-coding RNAs (LncRNA) of plasma, that were thought to promote cancer development, were found to take a significant role in diabetes 2 progression. 6 Long non-coding RNAs are non-coding RNAs that are longer than 200 nucleotides, and many of them were demonstrated to play a pivotal role in the development and progression of many diseases including cancer. 7,8 A series of LncRNAs have been demonstrated as oncogenes and/or tumor suppressor genes. 9 Furthermore, a number of recent studies suggested the involvement of LncRNAs in cancer development, including in breast cancer. 10,11 BC200 LncRNA plays an oncogenic role in estrogen-dependent breast cancer by targeting for attenuating deregulated cell proliferation and possibly serves as a prognostic marker. 12 Some of the candidate LncRNAs that are shown to be deregulated in breast cancer have the potential to be used as prognostic biomarkers. 13 For breast patients with diabetes 2, it is reported there are crosslink molecular factors in serum of patient plasma between these two diseases. So to find whether there is any kind of LncRNA in these two diseases and what role it takes in disease progression became very important to understand molecular mechanism of these two diseases. H19 (H19 Imprinted Maternally Expressed Transcript) is an RNA Gene, and is affiliated with the non-coding RNA class.
Diseases associated with H19 include Wilms tumor 2 and Beckwith-Wiedemann syndrome. 14 MT-LIPCAR (Mitochondrially Encoded Long Non-Coding Cardiac Associated RNA) is an RNA gene and is affiliated with the non-coding RNA class. Diseases associated with MT-LIPCAR include congestive heart failure and myocardial infarction. 15 GAS5 (growth arrest specific 5) is an RNA gene and is affiliated with the non-coding RNA class. Diseases associated with GAS5 include inflammatory bowel disease and autoimmune disease. 16 LncRNAs, E330013P06 (hereafter referred to as E33) showed that it is significantly upregulated in vivo and in vitro in macrophages under T2D conditions. 17 All these four types of non-coding RNA could be tested to upregulate in serum of some diabetic patients. So, we tested all these four types of RNA in serum of breast cancer patients with and without type 2 diabetes, and we found E33 is higher in breast cancer patients with type 2 diabetes than in those without it.
Further, E33 is higher in breast cancer patients with type 2 diabetes than those healthy ones or those patients only with type 2 diabetes without breast cancer. In vitro, T47D and MDA-MB-231 showed E33 transfection increased cell proliferation rather than migration and invasion. E33 downregulated P53, but not other protein factors in downstream pathways. All findings above showed E33 is a long non-coding RNA driven by diabetes that promotes development of breast cancer. Subsequently, the plasma samples were stored at −80°C.

| Breast cancer blood sample and cell culture
Clinical characteristics were collected, which was individuals including age, family history, tumor differentiation, tumor-node-metastasis stage (TNM), lymph node status, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (Her2). Tumors were staged according to the tumor-node-metastasis (TNM) staging system of the International Union Against Cancer. There was no radiotherapy, chemotherapy, or targeted therapy prior to the operation.

| Reverse transcription quantitative real-time PCR (qRT-PCR)
Total RNAs of plasma were extracted by TRIzol reagent (Invitrogen) according to the manufacturer's instructions and stored immediately at −80°C. Then, total RNA was quantified using a Nanovue (GE). Reverse transcription (RT) reaction was performed in the Bio-Rad C1000 PCR System (Bio-Rad). The E33 was determined using SYBR Mix (Tiangen) via Step-One System (Applied Biosystem). The processes of qRT-PCR reaction were conducted in a volume of 20 μL mixed with 30 ng cDNA templates and gene-specific primers for E33 18 (forward primer 5′-TCTTTCTCACAGGCCGCATT-3′,   reverse primer 5′-TGATTTCTCCACGGTCAGGC-

| Cell proliferation assay
The cells were cultured in 96-well plates. 10 μL CCK-8 solution (Dojindo) was added to each well and incubated for 2 hours at 37°C.
The absorbance was measured at 450 nm on a microplate reader according to the manufacturer's instructions. Three multiple holes were set in each hole to calculate the proliferation rate of the two groups of cells.

| Cell cycle assay
The cells were cultured in 24-well plates for 48 hours. The cells were collected and fixed with cold 70% ethanol at −20℃ overnight. Then, the cells were washed and resuspended in cold PBS and incubated at 37°C for 30 minutes with 10 mg/mL RNase and 1 mg/mL propidium iodide (Beyotime). RNA content was detected by flow cytometry (BD). The percentage of cells at G 0 /G 1 and S phases was analyzed using the Cell Quest acquisition software (BD Bioscience).

| Transwell invasion assay
The invasion assay was performed in Transwell (Corning   analysis was performed. Results were expressed as mean ± S.D (n = 3), and P < .05 was considered to be statistically significant.

| Upregulation of LncRNAE330013P06 in breast cancer specimens with diabetics
To confirm the higher E33 expression in breast cancer induced by diabetes, we selected four LncRNAs (H19, E33, LIPCAR, and GAS5) that have been shown to be associated with diabetes. These four was highly expressed just in breast cancer patients with diabetes rather than other groups (P < .001, Figure 2) ( Table 2).

| LncRNAE330013P06 high-expression was associated with poor prognosis of breast patients with diabetes
High E33 expression was remarkably correlated with TNM stage (P = .002), lymph node metastasis (P = .015), and family history (P = .013), but not correlated with patient's age and tumor grade, as well as ER, PR, and Her2 status (P > .05). Furthermore, Kaplan-Meier survival analysis revealed that high E33 expression was associated with low overall survival ( Figure 3). In 34 breast patients with diabetes, the median follow-up time was 51 months.
About 15% cases with high E33 expression got worse in breast cancer.

| Proliferation rate of breast cancer cells slows down after gene silencing
After transfection of E33, proliferation of the MDA-MB-231 and T47D cell cycle was measured; it was higher than that of the control.
The proliferation rate of the E33 overexpression MDA-MB-231 cells was also higher than that of the T47D cells ( Figure 4A).
These results showed E33 stimulated growth of breast cancer cells. Often, LncRNAs stimulated invasion or inhibited cell apoptosis according to past researches. But, direct transfection of E33 to breast cancer cell lines could promote cell growth.

| Invasion number of breast cancer cells, cell cycle, and apoptosis of breast cancer cells associated with E33
At same time, the invasion number of the E33 overexpression MDA-MB-231 and T47D cells were almost same with that control, and the invasion rate did not change too much after transfection ( Figure 4B).

| E33 promote cell growth via P53
E33 is miR143/145 like long non-coding RNA. According to past studies, expression of Myocdl, KLF4, ELK-1, and P53 might be downstream pathways of E33 ( Figure 6). So, we tested the expression of these four proteins. It is very clear with E33 transfection, only P53 was downregulated in these two cell lines ( Figure 6D). To confirm these cells transfection, we take photos in inverted fluorescent microscope to ascertain its transfection efficiency ( Figure 4C).
The results of Western blot showed that the expression levels of P53 could be downregulated by E33. P53 pathway is the major cause of cell growth by E33.  E33 expression was an independent factor of OS in breast cancer patients, and highly expressed LncRNA in diabetic patients without breast cancer may become a crucial factor in the development of breast cancer.

| D ISCUSS I ON
As we all know, breast cancer is a heterogeneous disease at the clinical characteristics and molecular level. 12,13 Long non-coding RNA is only one of factors in cancer progression. Even long non-coding RNA by itself is highly expressed in macrophage in blood. So, whether there is effect of long non-coding RNA from macrophage would promote cell growth of breast cancer cell is not elucidated.
But, long non-coding RNA is very important for breast cancer patients with type 2 diabetes.
Body Mass Index is an independent factor for diabetes. 3 And for Asians in United States, the risk of type 2 diabetes is very high when BMI is over 25, but for Whites, the same risk need BMI over 27. So, type 2 diabetes is extremely important factor for women with breast cancer in East Asia including Japan, Korea, Vietnam, and especially China.
E33 has been shown to upregulated in diabetes, and diabetes was a factor of induction of breast cancer. In this study, E33 expression was significantly correlated with TNM stage, lymph node metastasis, and family history, but not correlated with patient's age and tumor grade, as well as ER, PR, and Her2 status.
In conclusion, our data suggest that E33 could be serves as a biomarker and would be a crosslink in breast cancer patients and type 2 diabetes. And it needs to be researched more.