INTRODUCTION:

Many recent studies try to study the importance of non-coding RNAs, that includes microRNAs and lncRNAs, and their expression in detection of breast cancer in the suscetable women from this microRNA is the microRNA 145 in the paracancerous tissues and determination of its relationships with breast cancer incidences¹.

Breast cancer is one of the most dangerous malignant tumors that affect women globally, since it consisted of 8–12% of all malignancies². During 2015, around 1.4 million women are infected of breast cancer and it increased³. Also, the development of breast cancer usually occurs in Europe and North America, and it has enhanced more in the United States⁴. Also, according to the observation by⁵, the incidences of breast increased when it reached 50% within the upcoming 50 years and it will be the second most common cancers right after gastric cancer. Also, no obvious symptoms can be observed in the early stages of breast cancer, so it remains ignored which turns the mortality rate high⁶. observed that, the breast cancer patients reached to 62.4% in the 5 year period of the rate of the survival. Therefore, the study about breast cancer is important because of its high mortality and high rates of incidence.

Two miRNAs (mir-21 and miR-210) were consistently determined and there were six miRNAs (miR-145, miR-139-5p, miR-195, miR-99a, miR-497 and miR-205) that were verified and determined, regulated consistently in at least three inclusive studies. MiR-21 as the most consistently reported miRNA was verified in six study profiles. These miRNAs need to be investigated and validated, they could be potential process for the biomarkers of BC miRNA and used for early verification of breast cancer⁷.

The major indicator for the development of various kinds of tumors is; MicroRNAs (miRNAs)^8-10. miRNA-145 is generally used as indicators of breast cancer¹¹, to be associated with female breast and ovarian cancer.

Therefore, this study aimed to examine the importance of miRNA-145 as a biomarker for detecting the early stagesof breast cancer and its indications as prognostic value in breast cancer.

MATERIAL AND METHODS:

Patient Data:

The samples of breast cancer were consisted in para-cancerous tissues (which reached to 5 cm around the tumor) from 500 breast cancer patients who go surgical resection in the First Affiliated of governmental and private hospital from January 2015 to January 2018. The age of the patients ranged from 25–75 years with an average age of 43.44±4.25 years. The staging of the cancer and the pathological classification were based on the guidelines of the 2007 International Breast Cancer Typing ¹².

Inclusion and Exclusion Criteria:

The criteria for inclusion: Confirmed breast cancer patients through pathological biopsy. The samples of the cancer tissue were kept in liquid nitrogen and were stored at−90°C directly after surgical resection. The patients were not treated with chemotherapy or radiotherapy until they completed the medical record before the operation.

The criteria for exclusion: Combined with other cardiovascular and cerebrovascular diseases, respiratory tract and gastrointestinal disease patients, pregnant women, long-term bedridden patients, patients with physical disabilities, surgery-intolerant patients, patients transferred to other hospitals during treatment and patients who received certain treatment.

Main instruments and reagents:

· Total RNA extraction TRIzol kit.

· Real-time PCR instrument was used.

· M-MLV reverse transcriptase kit was from Vazyme Biotech Co., Ltd., (Nanjing, China).

· miR-145 and real-time PCR kit from Biomiga China (Shanghai, China).

· Primers of miR-145 and U6 (endogenous control) used in PCR reaction.

Table (1): Primers of miR-145 and U6.

	Primer sequences
miR-145	F	5′-ACACTCCAGCTGGGCAGGTCAAAA GGGTCC-3′
miR-145	R	5′-GGTGTCGTGGAGTCG-3′

F, forward; R, reverse

Methods of Detection:

· In liquid nitrogen, breast cancer tissue (80 mg) was grounded. Then the TRIzol reagent was added and mixed.

· The mixture was kept 30 minutes at room temperature.

· In accordance with the instructions of the manufacturer, total RNA was extracted.

· With ultraviolet spectrophotometer, the extracted RNA was examined.

· The purity and the concentration were determined by Electrophoresis.

· According to the instructions of the reverse transcription kit, total RNA was then reverse-transcribed, and cDNA samples were stored at −20°C.

· PCR reaction system was prepared according to the instructions from the manufacturer (10.5µl), and to obtain 20µl volume, DEPC water was added.

· Conditions of PCR reaction: 94°C for 10 min, followed by 40 cycles of 94°C for 45 sec, 60°C for 45 sec and 72°C for 45 sec.

Statistical Analysis:

Chi² –test and t-test (Student t-test) were used in statistical analysis for identification of the differences in the variables that affecting breast cancer incidences, while t-test was used for studying the differences among demographic characters and patients. The statistical analysis was conducted according to (SPSSPC+ computer Program, Version, 22).

RESULTS:

Demographic Data:

Our results observed in Table (2) cleared that the demographic characters of the patients under the study differ significantly (P < 0.01) among ages, body weight, residence, types of cancer and its degree, pathological staging, T staging, N staging and distant metastasis.

The results cleared that, the level of breast cancer increased with increasing age of patient till the age of 50 year of age, it level increased with increasing body weight and in individuals living in the city of a higher incidences than the countryside. Non-special type invasive cancer of a higher incidence, followed by early invasive cancer followed by Non-invasive cancer and the least incidences is the special type of invasive cancer.

The pathological staging of breast cancer differ significantly among individuals, as the higher incidences is the stage III, IV, stage II and the lowest incidences is the stage I. while, the distant metastasis observed in 70 5 of the studied patients.

Table (2): Basic information of the patients.

Cases (n=500)	No.	%	Chi2-test
Age (years)
<50	180	36	4.25**
≥50	320	64	4.25**
Body weight (kg)
<60	192	38.40	6.25**
≥60	308	61.60	6.25**
Residence
City	354	70.80	6.65**
Countryside	146	29.20	6.65**
Types
Non-invasive cancer	90	18	7.24**
Early invasive cancer	160	32
Special type invasive cancer	50	10
Non-special type invasive cancer	200	40
Pathological staging
I	70	14	8.25**
II	90	18
III	240	48
IV	100	20
Distant metastasis
Yes	350	70	5.22**
No	150	30	5.22**

** = significant at (P < 0.01)

miR-145 expression:

miR-145 expression level in breast cancer tissues was 3.25±2.26, and in para-cancerous tissues was 6.77±1.53. miR-145 expression level in breast cancer tissues was specifically lower than that in para-cancerous tissues (t=41.2., p<0.001).

Table (3): miR-145 expression

Origin	miR-145	t
Breast cancer tissues was	3.25±2.26 B	41.22**
Para-cancerous tissues was	6.77±1.53 A	41.22**

** = Significant at (P < 0.01)

Prognosis of Patients:

The prognosis of the breast cancer leads to the results among the studied patients and they were obtained by applying the median value of miR-145 patients that, were divided into miR-145 high expression group (≥2.24) their number reached to 350 cases, while, low expression group of miR-145 was (<2.24) that observed in (150 cases),. For 3 years, patients were followed up. Around 350 patients completed the follow-up, and the success rate of follow-up up to 95 %.

Table (4): Prognosis of patients

	miR-145
	High expression group	Low expression group
Number	350	150
Median	≥2.24	<2.24
Survival rates
After 1 year	90	83.1
After 2^nd year	79	71.20
After 3^rd year	70	59.80

Chi2 = 14.55 ** ** = significant at (P < 0.01)

DISCUSSION:

Breast cancer is one of the most major malignant tumors in the world affecting women. According to statistics, it is the top rank among malignancies affecting humans. Early stage breast cancer does not have any symptoms and only is detected at advanced stages and at that time, the treatment becomes useless. It leads to the poor prognosis with enhanced incidences of high mortalities among women.

According to Adhami et al.⁷, it was believed that the occurrences of breast cancer is generally caused by genetic factors, but the severity and the pathology of the disease is still not clearly understood. The occurrences of the breast cancer is affected by the cancer stem cells of miRNAs which are known as endogenous non-protein-coding RNA.

The regulation of cell proliferation, migration, invasion and apoptosis is influenced by miR-145. Gene expression of tumor is impacted by miR-145 also, with a massive effect on the occurrences of the effects of breast cancer.

Also, according to⁹the progression of breast cancer is generally associated to miRNA especially miRNA-145. The apoptosis and cell proliferation is usually controlled through miRNAs that have cell-specificity, are highly conserved, and its role¹¹.

The miR-145 is observed eukaryotic organisms, animals and humans, as it controls the gene expression that are related to the occurrences of cancer and modifications of the cancer features¹².

Therefore, in this study, miR-145 in para-cancerous tissues and breast cancer’s indicators when it was identified that was usually related to poor progression of human health and for treating breast cancer in the affected women.

That the results in the study indicated, that there is a down regulation of the miR-145in breast cancer tissues. It agreed with the study done by⁸ where it was reported that, the function of miR-145 in cancer in genital organs especially uterine and ovarian cancer, that indicating that miR-145 may be involved in the development of breast cancer.

Also, it was reported by¹³, the miR-145 may generally associated to the seriousness and the occurrences of breast cancer, also, the differentiation of the depth of invasion and the lymph node metastasis of miR-145 can be utilized as indicators and markers of tumor of its occurrences especially in the initial diagnosis of breast cancer as it generally controls the expression of mRNA of ERα and its coregulatory proteins are accountable for cell apoptosis, also it inhibits the breast cancer cell proliferation and enhances the development and the occurrences of breast cancer.

The initial verification of miR-145 is considered as standard indicators for enhancing the occurrences of breast cancers in women, also the results suggested that, the method used to detect miR-145 was PCR and it predicted the occurrences of breast cancer as the miR-145 gene expression is generally related to poor prognosis, this indicated that, miR-145 can be used as a prognostic indicator for patients with breast cancer and the effect of treatment for curing the breast cancer⁸.

The results suggested that the effect of mi-145 on the occurrences of breast cancer will be understood better if the sample size is increased, then accurate and high results will also be obtained.

The study concluded that, miR-145 is the main indicators for regulating the occurrences of breast cancer tissues and its affect in the development of cancer in patients. Also, miR-145 can be applied as a major prognostic indicator for breast cancer patients in the future and its detection gave indications on the occurrences and treatment of breast cancer.

ACKNOWLEDGEMENT:

The author is grateful to the Deanship of Southern Technical University, Thi-Qar and College of Medicine, University of Al-Qadisyiah, Iraq.to provide all facilities for the success of this work

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 31.05.2019 Modified on 18.06.2019

Research J. Pharm. and Tech. 2019; 12(12): 5923-5926.

DOI: 10.5958/0974-360X.2019.01027.8